Multi-target-directed design, syntheses, and characterization of fluorescent bisphosphonate derivatives as multifunctional enzyme inhibitors in mevalonate pathway

Article abstract of DOI:10.1016/j.bbagen.2013.02.011

Background Mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. Four enzymes in mevalonate pathway, including MVK, PMK, MDD, and FPPS, play important regulatory roles in cholesterol biosynthesis and cell proliferation. Methods The following methods were used: cloning, expression and purification of enzymes in mevalonate pathway, organic syntheses of multifunctional enzyme inhibitors, measurement of their IC₅₀ values for above four enzymes, kinetic studies of enzyme inhibitions, molecular modeling studies, cell viability tests, and fluorescence microscopy. Results and conclusions We report our multi-target-directed design, syntheses, and characterization of two blue fluorescent bisphosphonate derivatives compounds 15 and 16 as multifunctional enzyme inhibitors in mevalonate pathway. These two compounds had good inhibition to all these four enzymes with their IC₅₀ values at nanomolar to micromolar range. Kinetic and molecular modeling studies showed that these two compounds could bind to the active sites of all these four enzymes. The fluorescence microscopy indicated that these two compounds could easily get into cancer cells. General significance Multifunctional enzyme inhibitors are generally more effective than single enzyme inhibitors, with fewer side effects. Our results showed that these multifunctional inhibitors could become lead compounds for further development for the treatment of soft-tissue tumors and hypercholesteremia.

Full text of DOI:10.1016/j.bbagen.2013.02.011

BBAGEN-27456; No. of pages: 8; 4C: 6, 7
Biochimica et Biophysica Acta xxx (2013) xxx–xxx
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journal homepage: www.elsevier.com/locate/bbagen
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Multi-target-directed design, syntheses, and characterization of
fluorescent bisphosphonate derivatives as multifunctional enzyme
inhibitors in mevalonate pathway
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Q14
Jinbo Gao b, Jinggong Liu a, Yongge Qiu b, Xiusheng Chu b, Yuqin Qiao b, Ding Li a,
a
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School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou University City, 132 Waihuan East Road, Guangzhou 510006, PR China
b
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, PR China
7
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a r t i c l e i n f o
a b s t r a c t
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Article history:
Background: Mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes 24 Q2
and many bacteria. Four enzymes in mevalonate pathway, including MVK, PMK, MDD, and FPPS, play important 25
Received 13 December 2012
Received in revised form 30 January 2013
Accepted 12 February 2013
Available online xxxx
regulatory roles in cholesterol biosynthesis and cell proliferation.
26
Methods: The following methods were used: cloning, expression and purification of enzymes in mevalonate 27
pathway, organic syntheses of multifunctional enzyme inhibitors, measurement of their IC50 values for above 28
four enzymes, kinetic studies of enzyme inhibitions, molecular modeling studies, cell viability tests, and fluores- 29
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Keywords:
Bisphosphonate
cence microscopy.
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Results and conclusions: We report our multi-target-directed design, syntheses, and characterization of two blue 31
fluorescent bisphosphonate derivatives compounds 15 and 16 as multifunctional enzyme inhibitors in 32
mevalonate pathway. These two compounds had good inhibition to all these four enzymes with their IC50 33
values at nanomolar to micromolar range. Kinetic and molecular modeling studies showed that these two com- 34
pounds could bind to the active sites of all these four enzymes. The fluorescence microscopy indicated that 35
Multifunctional inhibitor
Mevalonate kinase
Phosphomevalonate kinase
Mevalonate 5-diphosphate decarboxylase
Farnesyl pyrophosphate synthase
these two compounds could easily get into cancer cells.
36
General significance: Multifunctional enzyme inhibitors are generally more effective than single enzyme inhibi- 37
tors, with fewer side effects. Our results showed that these multifunctional inhibitors could become lead com- 38
pounds for further development for the treatment of soft-tissue tumors and hypercholesteremia.
39
© 2013 Published by Elsevier B.V. 40
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1. Introduction
kinase (MVK), phosphomevalonate kinase (PMK), and mevalonate 56
5-diphosphate decarboxylase (MDD). These three enzymes catalyze 57
consecutive steps downstream from HMG-CoA reductase in the 58
mevalonate pathway, and are responsive to cholesterol in-take in ani- 59
mals. The blockade of the mevalonate pathway is a concept that has 60
found widespread clinical use, with statins as drugs that inhibit 61
HMG-CoA reductase and reduce cholesterol biosynthesis [5], and 62
nitrogen-containing bisphosphonates (N-BPs) as drugs for osteoporosis 63
therapy that target FPPS and inhibit protein prenylation [6]. Some mul- 64
tiple inhibitory compounds, including statins [7], bisphosphonates [8], 65
farnesyl transferase inhibitors (FTIs), and geranyl geranyltransferase 66
inhibitors (GGTIs) [9], have been developed previously targeting 67
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The mevalonate pathway is an important cellular metabolic path-
way present in all higher eukaryotes and many bacteria, and has so far
been exploited in the design of drugs treating and preventing human
diseases, including cardiovascular diseases and osteoporosis [1]. This
pathway is also a potentially important target for the treatment of
tumors especially bearing mutations in protein p53 [2,3]. Farnesyl
pyrophosphate synthase (FPPS) is a key regulatory enzyme in the
mevalonate pathway [4]. Besides, mevalonate pathway also contains
a unique series of three sequential ATP-dependent enzymes that
convert mevalonate to isopentenyl diphosphate (IPP): mevalonate
UNCORRECTED PR
mevalonate pathway. Statin's side effects have been the subject of 68
much controversy over the past few decades, since more and more re- 69
search is revealing serious potential adverse reactions from statin med- 70
Abbreviations: FPP, farnesyl pyrophosphate; FPPS, farnesyl pyrophosphate synthase;
GPP, geranyl diphosphate; HMG-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA
reductase; IPP, isopentenyl diphosphate; MDD, mevalonate 5-diphosphate decarbox-
ylase, also known as mevalonate 5-pyrophosphate decarboxylase or MPD; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; MVAPP, mevalonate
5-diphosphate; MVK, mevalonate kinase; N-BPs, nitrogen-containing bisphosphonates;
PMK, phosphomevalonate kinase; PP, pyrophosphate or diphosphate; TsCl, toluene sulfo-
nyl chloride
ications [10–12].
71
Bisphosphonate therapy has been considered as standard therapy 72
in the management and care of cancer patients with metastatic bone 73
disease and patients with osteoporosis with fewer side effects 74
[13–15]. In addition, bisphosphonates can be easily modified in vari- 75
ous positions of their P-C-P structure. Its central carbon has great 76
metabolic stability, and also provides a scaffold that can be modified 77
⁎
Corresponding author. Tel.: +86 13 5337 65290 fax: +86 20 3994 3058.
E-mail address: liding@mail.sysu.edu.cn (D. Li).
0304-4165/$ – see front matter © 2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.bbagen.2013.02.011
Please cite this article as: J. Gao, et al., Multi-target-directed design, syntheses, and characterization of fluorescent bisphosphonate derivatives as
multifunctional enzyme inhibitors in mevalonate pathway, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.02.011

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with additional substituents, such as the hydroxyl group and
heteroaromatic rings found in risedronate and zoledronate [16,17].
However, these bisphosphonates all bear substantial negative charge
at physiological pH, which limit the compounds' ability to penetrate
the cell membrane. In an effort to circumvent this perceived limita-
tion, some biodegradable protecting groups have been used to mask
a negative charge until after penetration of the cell membrane
[18,19]. Further investigation and improvement of bisphosphonates
are still required.
Our strategy in the design of novel multifunctional inhibitors
based on bisphosphonates is to link some known enzyme inhibitors
with covalent chemical bond in the P-C-P backbone, which targets
on four different enzymes in the mevalonate pathway, including
MVK, PMK, MDD, and FPPS. In the present paper, we report our
multi-target-directed design, syntheses, and characterization of two
bisphosphonate derivatives as multifunctional enzyme inhibitors in
the mevalonate pathway, which mainly contain the following func-
tional groups: the first one is bisphosphonate targeting on FPPS; the
second one is geranyl group with aromatic substitution targeting on
ATP binding site of MVK, PMK, and MDD; and the third one is
mevalonate group with fluorine substitution targeting on mevalonate
binding site of MVK, PMK, and MDD.
WX2-100 cation-exchange resin was purchased from Aldrich. All 137
other reagents were of research grade or better, and were obtained 138
from commercial sources. Organic syntheses of bisphosphonate de- 139
rivatives were shown in Scheme 1.
140
Synthesis of tetraethyl ethenylidenebisphosphonate (3). The com- 141
pound was synthesized following a reported procedure with minor mod- 142
ification [24]. Paraformaldehyde (10.4 g, 0.35 mol) and diethylamine 143
(5.08 g, 0.069 mol) were combined in 0.2 L of methanol, and the mixture 144
was warmed until clear. Compound 1 (20.0 g, 0.069 mol) was added, 145
and the resulting mixture was heated under reflux for 24 h. Then addi- 146
tional 0.2 L of methanol was added, and the solution was concentrated 147
under vacuum at 35 °C. Toluene (0.1 L) was added, and the solution 148
was again concentrated. This last step was repeated to ensure complete 149
removal of methanol to give the product 2 as a clear liquid. 1H NMR 150
(CDCl3) δ 4.02 (m, 8 H, OCH2CH3, J=7.3 Hz), 3.63 (overlapping m, 2H, 151
CH3OCH2, J=5.4 Hz and 15.6 Hz), 3.20 (s, 3H, CH3O), 2.52 (tt, 1H, 152
PCHP, J=6.0 Hz and 24.01 Hz), 1.18 (t, 12H, CH2CH3, J=7.1 Hz).
153
p-Toluenesulfonic acid monohydrate (0.50 g) was added, and the 154
mixture was heated under reflux. Methanol was removed from the 155
reaction mixture either by collection in a Dean-Stark trap or by ad- 156
sorption into 4 Å molecular sieves contained in a Soxhlet extractor. 157
After 14 h, the solution was concentrated. The crude product was 158
diluted with 1 L of chloroform, and washed with water (2×150 mL). 159
The solution was dried over MgSO4 and concentrated to give the prod- 160
uct 3. 1H NMR (CDCl3) δ 6.98 (distorted dd, 2H, H2C=, J=33.8 Hz and 161
37.71 Hz), 4.32–4.00 (m, 8H, OCH2CH3), 1.32 (t, 12H, OCH2CH3, J= 162
7.1 Hz). This NMR data is consistent with that reported previously [24]. 163
Synthesis of 2-(3-chloropropyl)-2-methyl-1,3-dioxolane (4). A 164
mixture of 5-chloro-2-pentanone (19.6 g, 158 mmol), ethylene glycol 165
(49.7 g, 800 mmol), and p-toluenesulfonic acid monohydrate 166
(300 mg, 1.6 mmol) was heated in 500 mL of toluene under reflux 167
with a Dean–Stark trap for 24 h. The mixture was then washed with 168
10% aq. NaHCO3 solution (3×30 mL), followed by brine (3×30 mL). 169
The organic phase was dried over anhydrous Na2SO4. After filtration, 170
the solvent was removed under reduced pressure, and the residual 171
liquid was distilled to give 24 g (92%) of compound 4 as a colorless 172
liquid. 1H NMR (300 MHz, CDCl3) δ 3.95 (4 H, m, OCH2CH2O), 3.60 173
(2 H, t, J=7.0 Hz, CH2Cl), 1.85 (4 H, m, CH2-CH2), 1.35 (3 H, s, CH3). 174
Synthesis of tetraethyl 5-(2-methyl-1,3-dioxolan-2-yl) pentane-1, 175
1-diyldiphosphonate (6). A titrated solution containing 1.66 mmol of 176
the Grignard reactant of the compound 4 (0.062–2.44 M) in THF 177
was slowly added to a magnetically stirred solution of tetraethyl 178
100 2. Materials and Methods
101 2.1. Materials
102
Lactate dehydrogenase, pyruvate kinase, phosphoenol pyruvate,
103 NADH, (RS)-mevalonic acid lactone, and ATP were purchased from
104 Sigma. Taq DNA polymerase, HB101 competent cells, and E. coli strain
105 BL21(DE3) competent cells came from Invitrogen Life Technologies.
106 Synthesized oligonucleotides were obtained from Tech Dragon Com-
107 pany of Hong Kong. T4 DNA ligase and restriction enzymes came
108 from MBI Fermentas of Germany. All other reagents were of research
109 grade or better and were obtained from commercial sources. The rat
110 liver MVK, MDD, and FPPS were obtained and assayed as previously
111 described [20–22]. Tris buffer was used instead of phosphate buffer
112 in enzyme storage and enzyme assay.
113 2.2. Cloning, expression, and purification of rat PMK
114
A rat liver Quick-Clone cDNA library was purchased from Clontech
115 (Palo Alto, CA). Standard cloning technology was used to clone the
116 gene of rat PMK. DNA sequencing of the cloned rat PMK gene was
117 performed, and the inserted gene sequence was identified to be the
118 same as previously deposited in NCBI without any mutation.
119 Established methods were used to prepare rat PMK [20], and the pro-
120 tein was purified to apparent homogeneity as shown in SDS-PAGE in
121 supporting information, which was stored at −80 °C in 50 mM po-
122 tassium phosphate buffer, pH 7.5, 0.1 mM EDTA, 5% glycerol, and
123 5 mM β-mercaptoethanol. The activity of the enzyme was assayed
124 spectrophotometrically following the decrease in absorbance at
125 340 nm as reported previously [23].
ethenylidenebisphosphonate 3 (500 mg, 1.66 mmol) in dry THF 179
(10 mL) at −15 °C under Ar. The reaction progress was followed by 180
using TLC, and the reaction was usually complete at the end of the addi- 181
tion. The mixture was warmed to room temperature, and then slowly 182
poured into a saturated solution of NH4Cl (20 mL). The mixture was 183
extracted with ether (2×20 mL), and the combined organic layers 184
were dried and concentrated under reduced pressure. The crude resi- 185
due was purified by using flash chromatography with appropriate elu- 186
ent to give the product 6 as an oil. MS (ESI): m/z 431 (M+H)+
.
187
Synthesis of (E)-3,7-dimethyl-octa-2,6-dienyl acetate (7). To a 188
mixture of alcohol/phenol/amine (1 mmol) and acetic anhydride 189
(1.2 mmol), La(NO3)3·6H2O (10 mol%) was added. After completion 190
of the reaction as monitored by using TLC, water was added to the re- 191
action mixture, and the product was extracted into ethyl acetate 192
(3×20 mL). The combined organic layers were washed with brine 193
and concentrated in vacuum, which was purified by using silica gel 194
column chromatography to afford the acetylated product 7. 1H NMR 195
(300 MHz, CDCl3) δ 5.32 (br t, 1H), 5.06 (m, 1H), 4.57 (d, 2H, J= 196
7.5Hz), 2.03 (s, 3H), 2.03–2.09 (m, 4H), 1.68 (s, 3H), 1.66 (s, 3H), 197
126 2.3. Organic syntheses of blue fluorescent bisphosphonate derivatives
127
1H, 13C, and 31P NMR spectra were recorded on a Varian Mercury
128 300 MHz NMR spectrometer at room temperature. Chemical shifts
129 are reported in ppm on the δ scale relative to the internal standard
130 TMS. Flash chromatography was performed in columns of various di-
131 ameters with silica gel by elution with appropriate solvents. Analyti-
132 cal thin layer chromatography (TLC) was carried out on Merck silica
133 gel 60G254 plates (25 mm), and developed with appropriate sol-
134 vents, and was visualized either with UV light or by dipping into a
135 staining solution of potassium permanganate and then heating. AG
136 MP-1M 100–200 resin, chloride form was from Bio-Rad. Dowex 50
1.58 (s, 3H).
198
Synthesis of (2E, 6E)-3,7-dimethyl-8-oxoocta-2,6-dienyl acetate (8). 199
Geranyl acetate (7) (6.84 g, 34.9 mmol) pre-dissolved in 100 mL of 95% 200
ethanol was added dropwise over 40 min to a refluxing solution of SeO2 201
(5.8 g, 52 mmol) in 300 mL of 95% ethanol. The mixture was heated 202
Please cite this article as: J. Gao, et al., Multi-target-directed design, syntheses, and characterization of fluorescent bisphosphonate derivatives as
multifunctional enzyme inhibitors in mevalonate pathway, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.02.011

J. Gao et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx
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Scheme 1. Organic syntheses of blue fluorescent bisphosphonate derivatives.
203 under reflux for 22 h. The black precipitate was removed by vacuum fil-
204 tration over a pad of silica-gel, and washed with 95% ethanol. Solvent
205 was removed at reduced pressure, and 400 mL of ethyl ether was
206 added. Organic layer was washed with brine (4×80 mL), dried over
207 Na2SO4, and concentrated under vacuum. The residue was purified by
208 using silica gel column chromatography to give a light yellow oil
209 (2.8 g) with a yield of 41%. Rf 0.75 (7:3 v/v hexanes: EtOAc); 1H NMR
210 (300 MHz, CDCl3) δ 9.37 (s, 1H), 6.46 (d, J=6.0 Hz, 1H), 5.39 (d, J=
211 6.0 Hz, 1H), 4.60 (d, J=7.2 Hz, 2H), 2.50 (q, J=7.5 Hz, 2H), 2.24
212 (t, J=7.5 Hz, 2H), 2.06 (s, 3H), 1.75 (s, 6H).
at reduced pressure. The aqueous residue was saturated with solid 228
NaCl, and the solution was extracted with ethyl acetate (4×50 mL). 229
The combined organic extracts were dried over MgSO4 and concen- 230
trated to give 2.95 g of compound 9 as a yellowish oil. 1H NMR δ 231
7.89 (d, 1H), 7.82 (br s, 1H), 7.31 (dd, 1H), 6.63 (d, 1H), 6.58 (dd, 232
1H), 5.42–5.36 (m, 2H), 4.12 (d, 2H, J=7.5 Hz), 3.85 (s, 3H), 3.72 233
(d, 2H, J=6.5 Hz), 2.22–2.15 (m, 2H), 2.08–2.03 (m, 2H), 1.68 (s, 3H), 234
1.66 (s, 3H).
235
Synthesis of methyl 2-((2E, 6E)-8-bromo-2,6-dimethylocta-2,6- 236
dienylamino) benzoate (10). The compound was synthesized following 237
a reported procedure with minor modification [25]. To a solution of 238
compound 9 (2.8 g, 12 mmol) in THF at −15 °C were added PBr3 239
(15 mmol). The reaction flask was transferred to an ice bath and 240
allowed to stir for 1 h, and then water was added. The mixture was 241
extracted with ether, washed with ice cold brine, dried with MgSO4, 242
and filtered. The solvent was removed in vacuo to give 10 as a yellow 243
oil. 1H NMR δ 7.9 (dd, 1H), 7.32 (ddd, 1H), 6.63 (dd, 1H), 6.59 (ddd, 244
1H), 5.40 (m, 2H), 4.07 (d, 2H, J=7.0 Hz), 3.83 (s, 3H), 3.74 (d, 2H, 245
J=7.5 Hz), 2.17 (m, 2H), 2.08 (m, 2H), 1.72 (s, 3H), 1.68 (s, 3H). This 246
213
214 2,6-dienylamino) benzoate (9). Methyl anthranilate hydrochloride
215 (1.00 g, 5.33 mmol) and aldehyde (4.39 g, 15.5 mmol) were
Synthesis of methyl 2-((2E, 6E)-8-hydroxy-2,6-dimethyl-octa-
8
UNCORRECTED PR
216 dissolved in ClCH2CH2Cl (21 mL). Acetic acid (2.50 mL, 43.2 mmol)
217 and 4 Å molecular sieves were added, and the reaction solution was
218 stirred for 5 min at room temperature. After NaBH(OAc)3 (6.89 g,
219 32.5 mmol) was added, the solution was stirred for 2.7 h at room
220 temperature, and then quenched by addition of 5% NaHCO3 dropwise
221 at 0 °C. The product was extracted with ether, and the combined ex-
222 tracts were washed with brine, dried with MgSO4, and concentrated
223 to afford a colorless oil. Final purification by using flash column chro-
224 matography gave compound 9-OAc as a colorless oil. K2CO3 (7.12 g,
225 51.6 mmol) was added to 9-OAc (3.61 g, 17.2 mmol) dissolved in
226 methanol, and the mixture was allowed to stir for 3 h at room tem-
227 perature. Water was added (300 mL), and methanol was removed
NMR data is consistent with that reported previously [25].
247
Synthesis of methyl 2-((2E, 6E)-9,9-bis(diethoxyphosphoryl)-2,6- 248
dimethyl-13-(2-methyl-1,3-dioxolan-2-yl)-trideca-2,6-dienylamino)- 249
benzoate (11). To a suspension of NaH (0.94 g, 60% dispersion in min- 250
eral oil, 24 mmol) in anhydrous THF at 0 °C was added 15-crown-5 251
(0.23 mL, 1.2 mmol) followed by compound 6 (5.8 mmol). After 1 h, 252
Please cite this article as: J. Gao, et al., Multi-target-directed design, syntheses, and characterization of fluorescent bisphosphonate derivatives as
multifunctional enzyme inhibitors in mevalonate pathway, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.02.011

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J. Gao et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx
253 the allylic bromide 10 was added via cannula as a solution in THF. The
254 reaction mixture was allowed to warm to room temperature overnight,
255 and then quenched by addition of water. The mixture was extracted
256 with ether, and the combined organic extracts were dried with MgSO4
257 and filtered. The filtrate was concentrated in vacuo, and the resulting
258 residue was purified by using flash chromatography to give product
259 11. 1H NMR (300 MHz, CDCl3) δ 8.01–7.79 (m, 1H), 7.38–7.25 (t, 1H,
260 J=7.0 Hz), 6.72–6.51 (q, 2H), 5.51–5.31 (m, 2H), 4.22–3.41 (m, 17H),
(m, 4H), 2.16–1.25 (m, 23H). MS (ESI): m/z 624 (M+H)+. HRMS 319
(m/z): Calcd for (C26H40FNO11P2 +H)+ 624.2133, found 624.2136. 320
Synthesis of 2-((2E, 6E)-15-carboxy-15,15-difluoro-14-hydroxy- 321
2,6,14-trimethyl-9,9-diphosphonopentadeca-2,6-dienylamino) benzoic 322
acid (16). To a solution of compound 14 (0.45 mmol) in anhydrous 323
CH2Cl2 at 0 °C were added 2,4,6-collidine (0.59 mL, 4.5 mmol) and 324
TMSI (0.58 mL, 4.5 mmol), and the reaction mixture was allowed to 325
warm to room temperature over a period of 2 h. Toluene was then 326
added, and the volatiles were removed in vacuo to afford a white solid. 327
This material was dissolved in aqueous LiOH (5 mL, 1 N) at room tem- 328
perature. After 24 h, the mixture was lyophilized to afford a gray solid 329
16. 1H NMR (300 MHz, D2O) δ 7.96–7.82 (m, 1H), 7.39–7.25 (t, 1H, J= 330
7.2 Hz), 6.75–6.51 (q, 2H, J=7.0 Hz), 5.51–5.32 (m, 2H), 4.45–3.75 331
(m, 3H), 2.16–1.25 (m, 23H). 13C NMR δ 176.8, 176.6, 150.5, 140.3, 332
135.2, 133.3, 132.3, 131.5, 128.6, 127.7, 120.8, 117.6, 114.4, 77.9, 51.3, 333
39.8, 28.7, 27.4, 27.2, 24.7, 16.4, 14.7, 13.5. MS (ESI): m/z 624 334
(M+H)+. HRMS (m/z): Calcd for (C26H39F2NO11P2+H)+ 642.2039, 335
261 2.22–1.21 (m, 35H). MS (ESI): m/z 431 (M+H)+
262
Synthesis of methyl 2-((2E, 6E)-9,9-bis-(diethoxyphosphoryl)-2,6-
.
263 dimethyl-14-oxopentadeca-2,6-dienylamino)-benzoate (12). A solu-
264 tion of compound 11 (1.21 mmol) in 80% acetic acid (10 mL) was
265 heated at 65 °C for 1.5 h. The progress of the reaction was followed by
266 TLC (CH2Cl2–MeOH, 95:5 v/v). The mixture was cooled to room tem-
267 perature, and then was concentrated under reduced pressure. The resi-
268 due was dissolved in CH2Cl2 (15 mL), and the resulting solution was
269 washed with a saturated solution of NaHCO3 (4×5 mL), dried and
270 then concentrated under reduced pressure. The crude material was pu-
271 rified by using flash chromatography to give compound 12 as a colorless
272 oil. 1H NMR (300 MHz, CDCl3) δ 7.91–7.81 (m, 1H), 7.41–7.22 (t, 1H),
273 6.72–6.50 (q, 2H, J=7.5 Hz), 5.52–5.29 (m, 2H), 4.25–3.75 (m, 13H),
274 2.46–1.22 (m, 35H).
found 642.2042.
336
2.4. Cell viability and fluorescence imaging assay
337
Cells were seeded in 96-well plates in serum-containing media, and 338
allowed to attach for 24 h. The medium was then removed and replaced 339
with serum-free medium containing 0.2% BSA with or without bisphos- 340
phonates at various concentrations. The cells were incubated for 72 h. 341
Following incubation, the medium was removed, the cells were washed, 342
275
Synthesis of methyl 2-((2E, 6E)-9,9-bis-(diethoxyphosphoryl)-16-
276 ethoxy-15-fluoro-14-hydroxy-2,6,14-trimethyl-16-oxohexadeca-2,6-
277 dienylamino) benzoate (13). To a suspension of zinc dust (1.5 equiv)
278 in THF (0.5 mL/mmol) was added dibromoethane (20 μL/mmol). The
279 mixture was heated under refluxed for a few minutes. A solution of
280 compound 12 (1 equiv.) in THF (1 M) was added dropwise. The reac-
281 tion was stirred at room temperature for 15 min, and a solution of
282 bromofluoroacetate was added. The reaction mixture was stirred at
283 room temperature for 1.5 h, quenched with saturated solution of
284 NH4Cl, and extracted with ether. The combined organic layers were
285 washed with saturated aqueous NaHCO3 and saturated aqueous
286 NH4Cl, and dried with MgSO4. After evaporation of the solvent, the
287 crude product was purified by using flash column chromatography to
288 give compound 13 with a yield of 70%. 1H NMR (300 MHz, CDCl3) δ
289 7.96–7.82 (m, 1H), 7.39–7.25 (t, 1H, J=7.5 Hz), 6.75–6.51 (q, 2H, J=
290 7.2 Hz), 5.51–5.32 (m, 2H), 4.45–3.75 (m, 17H), 2.16–1.25 (m, 38H).
and cell viability was measured by using the standard MTT assay.
343
344
345
3. Results and discussion
3.1. Organic syntheses of bisphosphonate derivatives
Compounds 15 and 16 were prepared as potential multifunctional 346
enzyme inhibitors in mevalonate pathway, as shown in Scheme 1. First- 347
ly, 5-chloro-2-pentanone, ethylene glycol and p-toluenesulfonic acid 348
monohydrate were heated under reflux in toluene to give protected 349
compound 4. Tetraethyl ethylidene bisphosphonate 3 is a well know 350
important intermediate [24], which can undergo efficient Michael addi- 351
tion reactions with various nucleophiles due to its electrophilic proper- 352
ty. Then, tetraethyl ethenylidenbisphosphonate 3 was reacted with the 353
corresponding organomagnesium reagent 5 (from compound 4) to af- 354
ford compound 6. The reaction was run by the dropwise addition of a 355
THF solution of organomagnesium reagent 5 to a stirred solution of 356
compound 3 in THF under N2 at −15 °C. Compound 10 was then pre- 357
pared from the geraniol [25]. The geraniol was first converted to the 358
corresponding acetate 7 by treatment with acetic anhydride in the pres- 359
ence of catalytic amount of La(NO3)3·6H2O. The resulting compound 7 360
was oxidized with selenium dioxide to give the aldehyde 8. Treatment 361
of the aldehyde with methyl anthranilate under reductive amination 362
condition, followed with the removal of acetate group afforded com- 363
pound 9. The treatment of alcohol 9 with PBr3 gave the corresponding 364
bromide 10[25]. The allylic bromide 10 was coupled with compound 6 365
to afford compound 11 in modest yield. The hydrolysis of 11 in 80% 366
acetic acid at 65 °C provided the corresponding ketone 12, which was 367
then reacted with ethyl bromofluoroacetate and bromodifluoroacetate 368
through Reformatsky reaction yielding corresponding compounds 13 369
and 14, respectively. Finally, the corresponding fluorescently tagged 370
bisphosphonate salts 15 and 16 were obtained using standard hydroly- 371
sis conditions by treatment with trimethylsilyl iodide, followed by a 372
291
Synthesis of methyl 2-((2E, 6E)-9,9-bis-(diethoxyphosphoryl)-
292 16-ethoxy-15,15-difluoro-14-hydroxyl-2,6,14-trimethyl-16-oxohex-
293 adeca-2,6-dienylamino) benzoate (14). To a suspension of zinc dust
294 (1.5 equiv.) in THF (0.5 mL/mmol) was added dibromoethane
295 (20 μL/mmol). The mixture was heated under refluxed for a few
296 minutes. A solution of compound 12 (1 equiv.) in THF (1 M) was
297 added dropwise. The reaction was stirred at room temperature for
298 15 min, and the solution of bromodifluoroacetate was added. The re-
299 action mixture was stirred at room temperature for 1.5 h, quenched
300 with saturated solution of NH4Cl, and extracted with ether. The com-
301 bined organic layers were washed with saturated aqueous NaHCO3
302 and saturated aqueous NH4Cl, and dried with MgSO4. After evapora-
303 tion of the solvent, the crude product was purified by using flash col-
304 umn chromatography to give compound 14 with a yield of 78%. 1H
305 NMR (300 MHz, CDCl3) δ 7.96–7.82 (m, 1H), 7.39–7.25 (t, 1H, J=
306 7.2 Hz), 6.75–6.51 (q, 2H, J=6.0 Hz), 5.51–5.32 (m, 2H), 4.45–3.75
307 (m, 16H), 2.16–1.25 (m, 38H).
308
Synthesis of 2-((2E, 6E)-15-carboxy-15-fluoro-14-hydroxy-2,6,14-
309 trimethyl-9,9-diphosphonopentadeca-2,6-dienylamino) benzoic acid
310 (15). To a solution of compound 13 (0.45 mmol) in anhydrous CH2Cl2
311 at 0 °C were added 2,4,6-collidine (0.59 mL, 4.5 mmol) and TMSI
312 (0.58 mL, 4.5 mmol), and the reaction mixture was allowed to warm
313 to room temperature over a period of 2 h. Toluene was then added,
314 and the volatiles were removed in vacuo to afford a white solid. This
315 material was dissolved in aqueous LiOH (5 mL, 1 N) at room tempera-
316 ture. After 24 h, the mixture was lyophilized to afford a gray solid 15.
basic work-up procedure.
373
3.2. Inactivation studies of compounds 15 and 16 for enzymes MVK, 374
PMK, MDD, and FPPS 375
317
318 7.2 Hz), 6.75–6.51 (q, 2H, J=7.0 Hz), 5.51–5.32 (m, 2H), 4.45–3.75
1H NMR (300 MHz, D2O) δ 7.96–7.82 (m, 1H), 7.39–7.25 (t, 1H, J=
Three ATP-dependent enzymes, MVK, PMK, and MDD, were assayed 376
spectrophotometrically following a continuous enzyme-coupled assay 377
Please cite this article as: J. Gao, et al., Multi-target-directed design, syntheses, and characterization of fluorescent bisphosphonate derivatives as
multifunctional enzyme inhibitors in mevalonate pathway, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.02.011

J. Gao et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx
5
Inhibitor
t1:1
t1:2
Table 1
IC50 values of compounds 15 and 16 for rat MVK, PMK, MDD, and FPPS.
0.12
0 µM
t1:3
t1:4
Compound
IC50 (μM)
0.09
0.06
0.03
0.00
Rat MVK
Rat PMK
Rat MDD
Rat FPPS
12.5 µM
t1:5
t1:6
15
16
3.4
2.7
5.9
4.2
1.9
0.8
0.035
0.029
25 µM
50 µM
378 method reported previously [26]. FPPS was assayed following our previ-
379 ously established method [20]. The inhibitory effects of bisphosphonate
380 derivatives 15 and 16 on the activities of these four enzymes were stud-
381 ied by using Dixon plot [27], and the IC50 values were determined by a
382 reciprocal plot of a series of remaining velocity versus corresponding in-
383 hibitor concentration, as shown in Table 1. Bisphosphonate derivatives
384 15 and 16 both exhibited good inhibitory activity simultaneously
385 against rat MVK, PMK, MDD, and FPPS with their IC50 values at from
386 nanomolar to micromolar range. The structural difference between
387 these two compounds is that compound 16 has two fluorine substitu-
388 ents while compound 15 has only one fluorine substituent. Compound
389 16 showed slightly better activity than compound 15.
0.0
0.1
0.2
0.3
0.4
0.5
0.6
ATP (mM)
80
60
40
20
0
Inhibitor
50 µM
390
Because active sites of these enzymes all have two distinct binding
25 µM
OOF
391 sites that specifically bind two kinds of substrates, we performed in-
392 hibition kinetic studies of each compound with varying concentra-
393 tions of one substrate while fixing another substrate concentration.
394 Our results showed that compound 16 is competitive with both sub-
395 strates of the enzymes MVK, PMK, and MDD (with example of MDD
396 as shown in Figs. 1 and 2), indicating that compound 16 can bind si-
397 multaneously to both substrate binding sites of these enzymes. Com-
398 pound 16 is composed of a hydrophilic part (mevalonate) and a
399 hydrophobic part (geranyl group), which is similar to that of the sub-
400 strates for ATP-dependent enzymes (MVK, PMK, and MDD) and FPPS.
12.5 µM
0 µM
0
2
4
6
8
10
1/ [ATP] (mM-1)
Fig. 2. Competitive inhibition of compound 16 against rat MDD with varying concen-
tration of ATP. The concentration dependence of initial rates is shown in double-
reciprocal plots, and was determined in the absence and presence of compound 16 at
different concentrations.
0.25
Inhibitor (µM)
0.20
0 µM
This similarity of the property between the inhibitors and the sub- 401
strates, together with their occupation of both binding sites, could 402
all enhance their interactions with the enzymes, and therefore lead- 403
25 µM
0.15
ing to good enzyme inhibitions.
404
50 µM
0.10
3.3. Modeling studies on three-dimensional structure–activity relationship 405
100 µM
0.05
In order to further study the binding modes of compounds 15 and 406
16 in the active sites of these four enzymes, we calculated the mini- 407
mum energy conformations of compounds 15 and 16 docked into 408
the models based on the crystal structures of rat MVK-ATP complex 409
(PDB ID: 1KVK), human PMK (PDB ID: 3CH4), human MDD (PDB ID: 410
3D4J), and human FPPS–zoledronate–IPP complex (PDB ID: 2F8Z). 411
MolDock scoring function was used to calculate score grids for rapid 412
dock evaluation. Potential binding sites (cavities) were detected 413
using the grid-based cavity prediction algorithm. The best docked 414
interactive conformations of these two potent inhibitors in these 415
four enzymes are shown in Fig. 3. The more active compound 16 416
gave lower total interactive energy and higher MolDock scores of 417
0.00
0
20
40
60
80
100
MVAPP (µM)
Inhibitor (µM)
100 µM
50
40
30
20
10
0
50 µM
UNCORRECTED PR
−292.36, −136.21, −171.53 and −247.25 kcal/mol, respectively, 418
25 µM
0 µM
indicating that compound 16 may has a higher binding affinity with res- 419
idues in the active sites. Compounds 15 and 16 all bind to both ATP and 420
mevalonate site of three ATP-dependent enzymes (MVK, PMK, and 421
MDD). Molecular docking results are quite consistent with our enzyme 422
kinetic analysis results. Compounds 15 and 16 also occupied both 423
Zoledronic acid and IPP binding site in human FPPS X-ray crystal struc- 424
ture with Zoledronic acid inhibitor. Zoledronic acid is a bisphosphonate 425
that has been developed for prevention and treatment of osteoporosis 426
[28]. It has been reported that Zoledronic acid bind to FPPS through oc- 427
cupying DMAPP or GPP site of the enzyme [29]. Our results indicate that 428
compounds 15 and 16 may occupy not only the GPP substrate binding 429
0.00
0.01
0.02
0.03
0.04
0.05
0.06
1/[MVAPP] (µM-1)
Fig. 1. Competitive inhibition of compound 16 against rat MDD with varying concen-
tration of MVAPP. The concentration dependence of initial rates is shown in double-
reciprocal plots, and was determined in the absence and presence of compound 16 at
different concentrations.
Please cite this article as: J. Gao, et al., Multi-target-directed design, syntheses, and characterization of fluorescent bisphosphonate derivatives as
multifunctional enzyme inhibitors in mevalonate pathway, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.02.011

6
J. Gao et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx
A: Rat MVK MV
Mg2+
Compound 15
ATP
Compound 16
B: Human PMK
Compound 16
ATP
MVAP
Compound 15
C: Human MDD
Fig. 4. Uptake of bisphosphonate analogue 15 by human Hela cells. Cells were incubated
with compound 15 (1 μM) for 48 h, and were subsequently examined via fluorescent mi-
croscopy. Control cells under phase-contrast microscopy and under fluorescent microsco-
py are also shown.
Compound 16
ATP
site but also the IPP substrate binding site. The inhibitions of the en- 430
zymes are significantly enhanced by the binding of the inhibitors to 431
both substrates binding sites of these four enzymes.
432
MVAP
The detailed interactions, including hydrogen-bonding, hydropho- 433
bic and electrostatic interactions, between the inhibitors and the 434
enzymes, were examined as shown in supporting information. Com- 435
pounds 15 and 16 have similar structures, and therefore, similar 436
enzyme-inhibitor interactions were observed. Our docking results in- 437
dicated that these two compounds have good interactions with the 438
enzyme active sites via hydrogen bonds, metal ions, hydrophobic in- 439
teractions, and electrostatic interactions. Overall, hydrogen bonding 440
and electrostatic interactions among the bisphosphonates, carboxyl- 441
ate, Mg2+, hydroxyl group, and the side chains of amino acids help 442
to hold the inhibitors in the active sites, and play important roles in 443
Compound 15
IPP
D: Human FPPS
Fig. 3. The binding modes of compounds 15 and 16 to the models of crystal structure of
rat MVK-ATP complex (PDB ID: 1KVK), human PMK (PDB ID: 3CH4), human MDD (PDB
ID: 3D4J), and human FPPS–zoledronate–IPP complex (PDB ID: 2F8Z). MolDock scoring
function was used to calculate score grids for rapid dock evaluation. Potential binding
sites (cavities) were detected using the grid-based cavity prediction algorithm. Com-
pounds 15 and 16 are represented as stick. The substrates ATP, MVA, MVAP, MVAPP,
zoledronate or GPP, and IPP are represented in stick format (green). Mg2+ ions are rep-
resented in ball and stick format (pink). Crystal structure of the proteins without the
substrate, and the positions of the corresponding substrates were predicted by using
the MolDock software algorithm. Compounds 15 and 16 were found to fully overlap
with two substrates of all these proteins.
Compound 16
Compound 15
Zoledronate (or GPP)
Mg2+
Please cite this article as: J. Gao, et al., Multi-target-directed design, syntheses, and characterization of fluorescent bisphosphonate derivatives as
multifunctional enzyme inhibitors in mevalonate pathway, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.02.011

J. Gao et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx
7
444 the binding of the inhibitors with the enzymes. The geranyl group
445 moiety shows strong hydrophobic interactions with nearby impor-
446 tant amino acids, and the orientations of these amino acid side chains
447 play important roles for enhancing the potency of the inhibitors.
some hydrophilic parts (carboxylate and bisphosphonate) and hydro- 462
phobic parts (geranyl group and benzene ring), which may facilitate 463
their penetration of lipid membranes to reach the target enzymes, 464
therefore increasing their effectiveness.
465
It should be noted that compounds 15 and 16 are fluorescent due to 466
their incorporation of an anthranilate fluorophore, which may be useful 467
in studies of bisphosphonate localization both in cultured cells and in 468
whole organisms. Fluorescence imaging is a very important technique 469
for biological studies and clinical applications due to high temporal 470
and spatial resolutions, and fluorescent bisphosphonates could be po- 471
tentially used as biological probes. In the present study, fluorescent mi- 472
croscopy was performed to assess the cellular uptake of the fluorescent 473
compounds 15 and 16. Hela cells were incubated with compound 15 or 474
16 (1 μM) for 48 h, and were subsequently examined via confocal fluo- 475
rescent microscopy. As shown in Figs. 4 and 5, the fluorescence micros- 476
copy clearly shows that fluorescent compounds 15 and 16 can easily 477
448 3.4. Effects of blue bisphosphonate derivatives on cell viability and cell
449 localization
450
Some previous studies have shown that bisphosphonates exert di-
451 rect cytostatic and proapoptotic effects in the ranges of several to thou-
452 sands micromolar on a variety of human tumor cell lines (myeloma,
453 breast, prostate, pancreas) in a concentration- and time-dependent
454 manner [30–33]. However, these bisphosphonates are rapidly adsorbed
455 by bone, therefore, mainly used in treating bone-related diseases. So, it
456 is desirable to have some relatively lipophilic bisphosphonates, which
457 could be generally used as potential anticancer or antiparasitic agents
458 [34]. In the present study, compounds 15 and 16 showed relatively
459 good cell viability to Hela cells with their IC50 values of 32.8 8.5 μM
460 and 18.7 3.1 μM, respectively, which could serve as lead compounds
461 for further improvement. Compounds 15 and 16 are composed of
penetrate Hela cells.
478
In summary, two multi-target-directed inhibitors 15 and 16 based on 479
bisphosphonate were synthesized and evaluated against rat mevalonate 480
kinase, phosphomevalonate kinase, mevalonate 5-diphosphate decar- 481
boxylase, and farnesyl pyrophosphate synthase. These two compounds 482
OOF
showed good inhibition for all these enzymes in mevalonate pathway 483
with high inhibitory activities. Multifunctional enzyme inhibitors are 484
generally more effective than single enzyme inhibitors, with fewer side 485
effects. The potential therapeutic applications of these multifunctional 486
inhibitors could extend beyond the treatment of metastatic bone 487
disease to encompass soft-tissue tumors and other diseases such as 488
hypercholesteremia, in which targeting the mevalonate pathway has 489
been shown to be effective. These results suggest a valuable role for 490
such compounds as metabolic probes in cell cultures, and encourage fur- 491
ther efforts to design bisphosphonate-based inhibitors.
492
Acknowledgements
493
We thank City University of Hong Kong and Sun Yat-sen Universi- 494
ty for financial support of this study.
495
Appendix A. Supplementary data
496
Cloning, expression, and purification of His-tagged rat 497
phosphomevalonate kinase. The detailed interactions including 498
hydrogen-bonding, hydrophobic and electrostatic interactions between 499
the inhibitors and the enzymes in docking analysis. The above informa- 500
tion can be found in the online version at http://dx.doi.org/10.1016/j. 501
bbagen.2013.02.011.
502
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Please cite this article as: J. Gao, et al., Multi-target-directed design, syntheses, and characterization of fluorescent bisphosphonate derivatives as
multifunctional enzyme inhibitors in mevalonate pathway, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbagen.2013.02.011