Targeting Cancer Cells with a Bisphosphonate Prodrug

Article abstract of DOI:10.1002/cmdc.201600465

Nitrogen-containing bisphosphonates have antitumor activity in certain breast cancer and myeloma patients. However, these drugs have limited oral absorption, tumor cell entry and activity, and cause bone side effects. The potencies of phosphorylated antiviral drugs have been increased by administering them as prodrugs, in which the negative charges on the phosphate moieties are masked to make them lipophilic. We synthesized heterocyclic bisphosphonate (BP) prodrugs in which the phosphonate moieties are derivatized with pivaloyloxymethyl (pivoxil) groups and that lack the hydroxy “bone hook” on the geminal carbon. When the lipophilic BP prodrugs enter tumor cells, they are converted into their active forms by intracellular esterases. The most active BP prodrug, tetrakispivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (7), was found to potently inhibit the in vitro growth of a variety of tumor cell lines, especially hematopoietic cells, at nanomolar concentrations. Consistent with this fact, compound 7 inhibited the prenylation of the RAP1A small GTPase signaling protein at concentrations as low as 1–10 nm. In preclinical studies, 7 slowed the growth of human bladder cancer cells in an immunodeficient mouse model. Thus, 7 is significantly more active than zoledronic acid, the most active FDA-approved BP, and a potential anticancer therapeutic.

Full text of DOI:10.1002/cmdc.201600465

DOI: 10.1002/cmdc.201600465
Full Papers
Targeting Cancer Cells with a Bisphosphonate Prodrug
+
[a]
+ [a]
[a]
[a]
Kenji Matsumoto , Kosuke Hayashi , Kaoru Murata-Hirai, Masashi Iwasaki,
[b]
[a]
[c]
[a, d]
Haruki Okamura, Nagahiro Minato, Craig T. Morita, and Yoshimasa Tanaka*
Nitrogen-containing bisphosphonates have antitumor activity
in certain breast cancer and myeloma patients. However, these
drugs have limited oral absorption, tumor cell entry and activi-
ty, and cause bone side effects. The potencies of phosphorylat-
ed antiviral drugs have been increased by administering them
as prodrugs, in which the negative charges on the phosphate
moieties are masked to make them lipophilic. We synthesized
heterocyclic bisphosphonate (BP) prodrugs in which the phos-
phonate moieties are derivatized with pivaloyloxymethyl (piv-
oxil) groups and that lack the hydroxy “bone hook” on the
geminal carbon. When the lipophilic BP prodrugs enter tumor
cells, they are converted into their active forms by intracellular
esterases. The most active BP prodrug, tetrakispivaloyloxy-
methyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate
(7), was found to potently inhibit the in vitro growth of a varie-
ty of tumor cell lines, especially hematopoietic cells, at nano-
molar concentrations. Consistent with this fact, compound 7
inhibited the prenylation of the RAP1A small GTPase signaling
protein at concentrations as low as 1–10 nm. In preclinical
studies, 7 slowed the growth of human bladder cancer cells in
an immunodeficient mouse model. Thus, 7 is significantly
more active than zoledronic acid, the most active FDA-ap-
proved BP, and a potential anticancer therapeutic.
Introduction
Geminal bisphosphonates (BPs) are synthetic compounds con-
taining P-C-P groups that are metabolically stable analogues of
[
1]
diphosphate. BPs were first synthesized more than 100 years
[
2]
ago and have a number of industrial uses. Because BPs bind
[
3]
[4]
to bone hydroxyapatite and inhibit bone resorption, a varie-
ty of BPs have been developed as therapeutics for the treat-
ment of bone-related diseases, such as Paget’s disease, osteo-
[
5]
porosis, and hypercalcemia of malignancy.
First-generation BPs are simple non-nitrogen-containing
compounds, such as etidronate and clodronate, that can be
metabolically incorporated into a b-g-methylene analogue of
ATP. The non-hydrolyzable ATP analogue inhibits ATP-depen-
dent intracellular enzymes like mitochondrial ADP/ATP translo-
[6]
case, leading to cell death. Second-generation BPs, such as
pamidronate and alendronate, contain alkylamine moieties and
function to inhibit farnesyl diphosphate synthase (FDPS) in the
mevalonate pathway, blocking the synthesis of downstream
farnesyl and geranylgeranyl diphosphate metabolites. Transfer
of the aliphatic chains of the diphosphates is required for the
activation of small guanosine triphosphate (GTP)-binding pro-
teins, such as Ras, Rho, Rac, Rab, and Rap, because the aliphat-
ic chains function to anchor the proteins to the cell membrane
for signaling. These GTP-binding signaling proteins are essen-
tial for the proliferation, survival, and migration of cells. Re-
lease of BPs from bone and uptake into osteoclasts causes os-
+
+
[
a] K. Matsumoto, K. Hayashi, K. Murata-Hirai, M. Iwasaki, Prof. N. Minato,
Prof. Y. Tanaka
Center for Innovation in Immunoregulative Technology and Therapeutics,
and Department of Immunology and Cell Biology, Graduate School of-
Medicine, Kyoto University, Kyoto, 606-8501 (Japan)
E-mail: ystanaka@nagasaki-u.ac.jp
[b] Prof. H. Okamura
Department of Tumor Immunology and Cell Therapy, Hyogo College of
Medicine, Nishinomiya, Hyogo, 663-8501 (Japan)
[7–9]
[c] Prof. C. T. Morita
teoclast apoptosis and decreases bone resorption.
Third-
Department of Internal Medicine and the Interdisciplinary Graduate-
Program in Immunology, University of Iowa Carver College of Medicine,
Iowa City Veterans Affairs Health Care System, 601 Highway 6 West, Re-
search (151), Iowa City, IA 52246 (USA)
generation BPs, including risedronate and zoledronic acid (Zol)
that have nitrogen-containing heterocyclic moieties, exhibit
more potent antiresorptive activity than second-generation
[10]
[
d] Prof. Y. Tanaka
BPs through stronger inhibition of FDPS.
Center for Bioinformatics and Molecular Medicine, Graduate School of-
Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-
The addition of Zol to standard treatments for breast cancer
and multiple myeloma improved disease-free survival and
8
523 (Japan)
[11–15]
overall survival, respectively.
These improvements ap-
+
[
] These authors contributed equally to this work.
peared to be independent of preventing bone-related events,
suggesting that BPs have antitumor activity besides preventing
bone metastases. Our goal was to develop BPs specifically de-
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600465.
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signed as cancer therapeutics for improved activity. However,
heterocyclic BPs, such as Zol and minodronate, fit well into the
[
16]
active site of FDPS making it difficult to design BPs with
higher activity.
An alternative approach to increase BP activity is to improve
BP cell entry. Cellular uptake of BPs through fluid-phase endo-
2
+
cytosis is enhanced by Ca , suggesting that the negatively
charged hydrophilic P-C-P structure limits BP entry into cellular
[
17]
vesicles. Oldfield and co-workers have reported several ap-
proaches to improving cellular uptake through the addition of
[
18,19]
a 9–12-carbon acyl chain to Zol and other BPs
or by mask-
ing the negatively charged P-C-P structure with pivoxil
[
20]
esters. For Zol, addition of a 9–12-carbon acyl chain decreas-
es FDPS inhibition by two- to three-fold, while increasing activ-
[
19]
ity in cellular assays by up to 22-fold. Similarly, the pivoxil
derivatives of mono- and dialkyl-BPs that inhibit FDPS or gera-
nylgeranyl diphosphate synthase (GGDPS) were consistently
more potent than their acid forms at inhibiting the prenylation
[
21]
of RAP1A in K562 cells and K562 proliferation. In this study,
we have increased the cellular uptake and activity of nitrogen-
containing BPs by masking the phosphonate charges with piv-
oxil groups resulting in novel BP prodrugs. These prodrugs
have greatly increased bioactivity against hematopoietic tumor
cells.
Results and Discussion
We synthesized BP prodrugs where the phosphonate charges
are masked by pivoxil groups, and evaluated the effect of piv-
oxil addition on their biological activity in vitro and in vivo. We
first synthesized seven BP prodrugs and their corresponding
active acid forms (Figure 1). Five of the pairs (2–6/9–13) were
Figure 1. Structures of pivoxil esters and their acid forms. Details of synthe-
sis are summarized in the Experimental Section.
[
22,23]
based on NE11809 (compound 8)
by varying the position
acid form, 14 (IC50 against U937 was 260 nm for 7, versus
26000 nm for 14). Compound 1 inhibited U937 cells 185-fold
more efficiently than its acid form, 8. However, the effect of
pivoxil derivatization of BPs was variable, ranging from 5- to
1618-fold (mean increase in potency: 291Æ588-fold). Similar
increases in activity of BP pivoxil derivatives compared with
their acid forms were noted with EJ-1 bladder carcinoma cells
(Table 2 and Supporting Information, Figure S3). Again, the
effect was variable depending on the BP, ranging from 1.2- to
422-fold (mean increase: 123Æ163-fold). For EJ-1, compound 7
of the methyl group on the heterocyclic ring, by the addition
of a halogen group, or by addition of a nitrogen atom to the
heterocyclic ring. A final compound substituted a thiazole for
the pyridine group. The synthesis of BP pivoxil derivatives is
detailed in Supporting Information, Figure S1. All of the BP
prodrugs inhibited the growth of U937, a human histiocytic
lymphoma cell line, more efficiently than their acid forms
(Table 1 and Supporting Information, Figure S2). For example,
compound 7 was found to be 100-fold more potent than its
Table 1. Comparison of pivoxil esters (PE) with their acid forms (H) in
growth inhibition of the U937 histiocytic lymphoma cell line.
Table 2. Comparison of pivoxil esters (PE) with their acid forms (H) in
growth inhibition of the EJ-1 bladder carcinoma cell line.
[
a]
[a]
PE/H
IC50 [mm]
Ratio IC50 (PE)/IC50 (H)
PE/H
IC50 [mm]
Ratio IC50 (PE)/IC50 (H)
PE
H
PE
H
1
2
:8
:9
5.3Æ0.4
0.68Æ0.05
5.6Æ0.7
3.6Æ0.3
5.3Æ0.4
2.4Æ0.0
0.26Æ0.01
980Æ80
1100Æ200
29Æ1
1:185
1:1618
1:5
1:56
1:13
1:8
2:9
11Æ0
13Æ2
1:1.2
1:422
1:1.5
1:37
1:3.9
1:256
1:142
2.3Æ0.1
5.4Æ0.6
7.8Æ0.2
5.7Æ1.3
970Æ30
3
4
5
6
7
:10
:11
:12
:13
:14
3:10
4:11
5:12
6:13
7:14
7.9Æ0.6
200Æ3
68Æ1
290Æ10
22Æ2
150Æ6
26Æ1
1:63
1:100
0.09Æ0.01
23Æ2
0.026Æ0.014
3.7Æ0.2
[
a] Experimental details are described in Supporting Information, Fig-
[a] Experimental details are described in Supporting Information, Fig-
ure S3.
ure S2.
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7
96-fold (Æ1442-fold) more sensitive to growth in-
Table 3. Comparison of 7 (pivoxil ester, PE) with 14 (acid form, H) in growth inhibition
of solid tumor cell lines.
[
a]
hibition with 7 than Zol. For example, HL60 (promye-
locytic leukemia) was 5679-fold and PEER (T-cell lym-
phoma) was 391-fold more sensitive to 7 than Zol.
The IC50 value for 7 averaged 515 nm for B-cell malig-
nancies, 116 nm for T-cell malignancies, and 107 nm
for myeloid malignancies versus 85100, 55000, and
Cell line
Origin
IC50 [mm]
(PE)
Ratio
IC50 (7)/IC50 (14)
7
14 (H)
7
86-0
Renal cell carcinoma
Gastric carcinoma
Osteosarcoma
0.88Æ0.15
1.2Æ0.8
25Æ1
100Æ60
66Æ5
1:28
1:83
1:81
1:455
1:324
1:44
1:167
1:300
1:284
1:142
MKN1
OST
PC-3
PK1
LK-2
G-361
TFK-1
0.81Æ0.20
0.22Æ0.06
0.34Æ0.06
0.80Æ0.33
0.12Æ0.02
0.10Æ0.05
0.74Æ0.03
0.026Æ0.014
85557 nm, respectively, for Zol (Figure 3 and Support-
Prostate carcinoma
Pancreatic cancer
100Æ20
110Æ20
35Æ1
ing Information, Figure S6 and Table S1). Thus, while
solid tumors were 27-fold more susceptible to 7 than
Zol, lymphoma and myeloid hematopoietic cell lines
were markedly more susceptible to 7 than to Zol.
To determine its mechanism of action, the effect of
[
b]
Squamous NSCLC
Melanoma
20Æ4
Cholangiocarcinoma
30Æ1
MRK-nu-1 Mammary carcinoma
EJ-1 Bladder carcinoma
210Æ10
3.7Æ0.2
7
on RAP1A prenylation in tumor cells was assessed.
[a] Experimental details are described in Supporting Information, Figure S4. [b] Non-
small-cell lung carcinoma.
Compound 7 strongly inhibited RAP1A geranylgera-
nylation in six tumor cell lines, with inhibition detect-
ed at concentrations as low as 1–10 nm (Figure 4). As
inhibited 142-fold more than 14. Given the high activity of 7,
growth inhibition by 7 and 14 were determined for cell lines
derived from solid tumors. Again, 7 was 28- to 455-fold more
potent than its acid form, 14 (Table 3 and Supporting Informa-
tion, Figure S4). Similarly, growth inhibition of hematopoietic
cell lines was enhanced by pivoxil protection, with 7 being
expected based on its inhibition of FDPS, Zol also inhibited
prenylation, but required concentrations of at least 10000–
100000 nm. For example, whereas unprenylated RAP1A was
detected in EJ-1 with 7 at 1 nm, Zol required a concentration
Table 5. Inhibition of U937 and EJ-1 cell proliferation by additional pivox-
100- to 3714-fold more active than 14 (Table 4 and Supporting
[
a]
il esters.
Information, Figure S5).
Based on these findings, we synthesized 28 additional BP
pivoxil and other esters (compounds 15–42, Figure 2) and as-
sessed their ability to inhibit the growth of the U937 and EJ-
Compd
IC50 [mm]
Compd
IC50 [mm]
U937 EJ-1
U937
EJ-1
7
39
0.26Æ0.01
0.65Æ0.11
1.1Æ0.3
2.2Æ0.1
2.5Æ0.2
2.6Æ0.1
3.0Æ0.2
3.3Æ0.2
3.4Æ0.1
3.5Æ0.6
5.0Æ0.5
6.1Æ0.5
6.2Æ0.5
6.3Æ0.2
6.6Æ0.1
0.026Æ0.014
0.059Æ0.006
3.5Æ0.5
1.7Æ0.2
1.1Æ0.4
4.3Æ0.5
6.3Æ0.5
4.8Æ0.5
3.7Æ0.4
4.5Æ0.4
4.2Æ0.4
9.0Æ0.6
8.8Æ0.7
12Æ2
30
19
16
20
26
15
28
36
37
27
38
42
41
40
6.6Æ0.3 10Æ1
1
cell lines (Table 5). However, again, 7 was the most potent in-
6.7Æ0.3
9.5Æ0.5
35
31
34
6.8Æ0.4 15Æ1
6.9Æ0.6 16Æ1
hibitor of U937 and EJ-1 cell growth. Therefore, we focused
further studies on 7. To determine its potential clinical useful-
ness, 7 was tested against a variety of tumor cell lines in com-
parison with Zol, the most potent FDA-approved BP (Figure 3
and Supporting Information, Figure S6 and Table S1). Com-
pound 7 was found to be, on average, 27-fold more active
than Zol for 52 solid tumor cell lines. Several tumor cell lines
such as the bladder cancer cell line EJ-1 and the gastric tumor
cell lines GCIY and MKN45 were highly sensitive to 7. Only two
solid tumor cell lines were more sensitive to Zol than 7. Com-
pound 7 was found to be even more active on average against
hematopoietic cell lines. Hematopoietic cell lines averaged
7.0Æ0.3
8.7Æ0.4
17
24
7.1Æ0.5 16Æ0
7.1Æ0.3
6.0Æ0.4
18
22
23
7.9Æ0.2 15Æ1
7.9Æ0.6 16Æ2
8.9Æ1.0 13Æ1
33
21
11 Æ2
17Æ1
50Æ3
70Æ1
8.5Æ0.3
16Æ1
56Æ2
79Æ7
25
32
29
12Æ3
[
a] Experimental details are described in the Supporting Information.
[
a]
Table 4. Comparison of 7 (pivoxil ester, PE) with 14 (acid form, H) in growth inhibition of lymphoma and leukemia hematopoietic cell lines.
Cell line
Origin
IC50 [mm]
Ratio
7
(PE)
14 (H)
IC50 (7)/IC50 (14)
MOLT-3
MOLT-4
PEER
T-cell acute lymphoblastic leukemia
T-cell acute lymphoblastic leukemia
T-cell acute lymphocytic leukemia
B-cell lymphoma
Non-Hodgkin’s lymphoma
Burkitt’s lymphoma
Burkitt’s lymphoma
Burkitt’s lymphoma
Acute monocytic leukemia
Histiocytic lymphoma
Erythroleukemia
0.13Æ0.09
0.16Æ0.12
180Æ20
120Æ10
82Æ6
1:1385
1:750
1:3565
1:1013
1:367
1:177
1:444
1:682
1:3714
1:100
1:552
0.023Æ0.009
0.078Æ0.018
0.18Æ0.08
0.43Æ0.52
0.27Æ0.01
0.11Æ0.07
C1R
79Æ6
SCC-3
Daudi
Raji
Ramos-RA1
THP-1
U937
66Æ3
76Æ5
120Æ10
75Æ5
0.035Æ0.061
0.26Æ0.01
0.029Æ0.006
130Æ10
26Æ1
K562
16Æ2
[a] Experimental details are described in Supporting Information, Figure S5.
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Figure 2. Structures of pivoxil esters. Details of synthesis are summarized in the Experimental Section and Supporting Information (chemistry and NMR
spectra).
of ~10000 nm. The differences between 7 and Zol are similar
to those noted with growth inhibition, albeit somewhat great-
er in magnitude. Thus, the activity of 7 is consistent with inhib-
ition of isoprenoid biosynthesis. Given that most strong inhibi-
growth of EJ-1 from weeks 4 through 7, such that at seven
weeks, EJ-1 tumor growth was decreased by 78% in mice
treated with compound 7 relative to control mice (Figure 5).
Thus, compound 7 has direct therapeutic benefits in the treat-
ment of human cancer in this preclinical mouse model.
[
24–26]
tors of GGDPS contain mono- or diacyl chains,
the activity
of 7 is likely due to inhibition of FDPS, although dual FDPS
The development of phosphate and phosphonate prodrugs
has been an important advance in anticancer and antiviral
[
27]
and GGDPS inhibition remains possible, as does direct or in-
direct inhibition of signaling kinases in addition to the inhibi-
[31–33]
therapy.
Esterification of phosphate- and phosphonate-
[
28,29]
tion of FDPS.
containing drugs can increase their gastrointestinal absorption,
survival in the systemic circulation, and cell penetration. A
number of different FDA-approved drugs contain the pivoxil
group used here or the related disoproxil group with accepta-
ble levels of toxicity. Adefovir dipivoxil and tenofovir disoproxil
fumarate have been used to treat hepatitis B virus and HIV
while cefditoren pivoxil is a broad-spectrum cephalosporin an-
To assess the activity of 7 against tumors in vivo, the effect
of 7 on the growth of EJ-1 bladder carcinoma cells was as-
sessed in immunodeficient NOG mice. Luciferase-expressing
EJ-1 bladder carcinoma cells were intraperitoneally inoculated
into NOG mice and the mice were then treated with com-
pound 7 twice a week. Compound 7 significantly inhibited the
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Figure 4. Inhibition of geranylgeranylation of RAP1A in tumor cells by com-
pound 7. Tumor cells were cultured with 7 or Zol for 16 h, lysed, and the
proteins were separated by SDS-PAGE, transferred onto PVDF membranes
and probed with anti-unprenylated RAP1A antibody. Data for inhibition of
[
30]
geranylgeranylation of RAP1A by Zol are from Idrees et al. and are includ-
ed for comparison. Note that the maximum concentration used for 7 was
100-fold less than that used for Zol.
Figure 3. Tumor cell growth inhibition by compound 7 in comparison to
Zol. A)–F) Dose-dependent inhibition of tumor cell growth by compound 7.
4
À1
Tumor cells were grown, harvested and resuspended at 1ꢁ10 cellsmL in
complete RPMI 1640 medium. A total of 0.05 mL of the cell suspension was
added to flat-bottomed 96-well plates, followed by 0.05 mL of three-fold
2
serial dilutions of compound 7. After incubation at 378C with 5% CO for
four days, 0.1 mL of CellTiter-Glo reagent (Promega, Madison, WI, USA) was
added and the luminescence resulting from ATP was measured using an
ARVO luminometer (PerkinElmer, Foster City, CA, USA). All experiments were
performed in triplicate: A) lymphomas: * MOLT-3, ~ J.RT3-T3.5,
Ramos (RA 1); B) myeloid leukemias: * HL60, ~ SCC-3,
P31/FUJ, ^ NOMO-
MCF-7, ^ MDA-MB-
31; D) renal cell carcinomas: * VMRC-RCW, ~ UOK121,
Caki-1, ^ A-704;
KP4-3, ^ MiaPaCa2; and
Colo320, ^ TAKAO. G) Comparison
&
Raji, ^
&
1
2
; C) mammary carcinomas: * YMB-1-E, ~ HMC-1-8,
&
&
E) pancreatic carcinomas: * KP4-1, ~ KP4-2,
F) other tumor cells: * 24TKB, ~ MKN1,
&
&
of compound 7 (left panel) with Zol (right panel) concentrations required
for half-maximal growth inhibition of various tumor cell lines. Ly=lympho-
mas, My=myeloid leukemia, Ma=mammary carcinomas, and Others=other
tumor cells. Cut-off values were arbitrarily set at 300 nm for 7 and 20000 nm
for Zol. Details of Zol growth inhibition are given in Supporting Information,
Figure S6.
Figure 5. Compound 7 inhibits EJ-1 bladder carcinoma growth in immuno-
deficient NOG mice. A) Whole-body bioluminescence imaging of tumor cells.
Immunodeficient NOG mice were intraperitoneally (i.p.) inoculated with
6
1
2
ꢁ10 EJ-1 cells transfected with pGL4.10[luc2] on day 0 and treated with
À1
mg/0.1 mL compound 7 (97.1 mgkg ) or 0.1 mL PBS i.p. on days 3 and 6.
À1
TM
On day 7, 0.1 mL of 15 mgmL VivoGlo luciferin (Xenogen, Alameda, CA,
USA) was injected i.p., and the mice were placed in the specimen chamber
and photon emissions transmitted from the mice were then measured. This
treatment regimen was repeated seven times, and the images from day 28
are shown. Luciferase units are photons per second per mouse. B) Inhibition
of tumor growth by compound 7 in vivo. The growth of EJ-1 was monitored
for weeks 2–7 as described in (A), and the average photon flux per second
for four to five mice is plotted ÆSEM. *p<0.05 by Mann–Whitney U test.
tibiotic. Besides pivoxil acyloxyalkyl esters, it may be possible
to further improve targeting of hematopoietic tumors using ar-
[
33–35]
yloxy phosphoramidate esters.
The tenofovir alafenamide
aryloxy phosphoramidate prodrug has increased levels in
plasma and lymphatic tissue and is 1000-fold more active
[
36]
against HIV than tenofovir and is more active in patients,
[
37,38]
with fewer side effects than its disoproxil derivative.
Unlike
creased dependence of hematopoietic tumors on prenylated
Ras or other GTPases for their growth. Whereas hematopoietic
cancer cells were more resistant to Zol than non-hematopoiet-
ic cancer cells (mean EC50 value of 77800 nm versus 9600 nm),
they were more sensitive to 7 (mean EC : 240 nm versus
nucleoside prodrugs, nitrogen-containing BP prodrugs would
be given intermittently and their predicted reduced deposition
into bone would minimize jaw osteonecrosis and atypical frac-
tures.
50
The mechanism for the high activity of 7 for hematopoietic
tumors may reflect increased uptake by 7 coupled with in-
770 nm). This suggests that BP uptake by hematopoietic
tumors is lower than with other tumors; therefore, the in-
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creased uptake of the 7 prodrug results in higher antiprolifera-
tive activity. To further improve the biological activity of nitro-
gen-containing BPs, it is necessary to examine and compare
their cell penetration, lipophilicity, and metabolic stability in
detail.
(230 mL) at room temperature and stirred at 658C for 30 min. A so-
lution of tetramethyl methylenediphosphonate (14 g, 60 mmol) in
methanol (10 mL) was added, and the reaction mixture was held at
reflux for 1.5 h. The resulting mixture was concentrated in vacuo to
give a crude product, which was used for the next reaction with-
out further purification. The crude product was dissolved in tolu-
ene (200 mL) and treated with p-toluenesulfonic acid monohydrate
(
114 mg, 0.6 mmol). The reaction mixture was held at reflux using
Conclusions
a Dean–Stark trap for 16 h and then diluted with CHCl . The result-
3
In conclusion, masking the negatively charged P-C-P structure
of BPs with pivoxil esters greatly increases their capacity to in-
hibit tumor cell growth. The most active BP pivoxil ester, com-
pound 7, was found to be particularly effective at inhibiting
the growth of hematopoietic cells, with IC50 values generally
between 20 and 200 nm, whereas the IC50 values for Zol were
up to 5679-fold higher, being generally greater than
ing mixture was washed with brine, dried over MgSO , filtered and
4
concentrated in vacuo to give a crude product that was purified
by silica gel column chromatography (MeOH/CHCl =10%) to yield
3
8
.9 g (60%) of the title compound as a colorless oil.
Tetrakispivaloyloxymethyl vinylidene-1,1-bisphosphonate (S2):
NaI (10.2 g, 68 mmol) and chloromethyl pivalate (POMCl, 12.4 mL,
8
5 mmol) were added to a solution of tetramethyl vinylidene-1,1-
bisphosphonate (4.2 g, 17 mmol) in MeCN (85 mL) and then held
20000 nm. Besides the direct effect of 7 on tumor growth, 7
at reflux for 14 h. After addition of H O, the resulting mixture was
2
also expands cytotoxic Vg2Vd2 Tcells in vitro and can be used
in combination with adoptively transferred Vg2Vd2 Tcells
in vivo to enhance tumor control in the NOG mouse model
extracted with EtOAc, and the organic layer was washed with
brine, dried over Na SO , filtered and concentrated in vacuo to
2
4
give a crude product that was purified by silica gel column chro-
matography (EtOAc/hexane=50%) to yield 3.8 g (35%) of the title
compound as a pale-yellow oil.
(Tanaka et al., unpublished results). Moreover, we speculate
that 7 may exhibit less bone deposition due to its lack of free
phosphonate moieties as well as the absence of the hydroxy
group (bone hook) on the germinal carbon of the P-C-P struc-
ture. Although further research is required, BP prodrugs could
increase the effectiveness of BP treatment for both hemato-
poietic and non-hematopoietic solid tumors.
Synthesis of 1,1-bisphosphonic acid tetrakispivoxil esters
Tetrakispivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-
bisphosphonate (7): 2-Aminothiazole (20 mg, 0.2 mmol) was
added to a solution of tetrakispivaloyloxymethyl vinylidene-1,1-bi-
sphosphonate (64 mg, 0.1 mmol) in CHCl (0.4 mL) and stirred at
room temperature for 1 h. The resulting mixture was concentrated
in vacuo to give a crude product, which was purified by silica gel
3
Experimental Section
General
column chromatography (EtOAc/CHCl =50%) to yield 69 mg
3
Thin-layer chromatography (TLC) was performed on pre-coated
plates (0.25 mm, silica gel plate 60 F245, Merck Millipore, MA, USA).
Column chromatography was conducted using silica gel (Kanto
Chemical Co. Inc., Chuo-ku, Tokyo, Japan). All reactions were con-
ducted under an atmosphere of air unless otherwise noted. Unless
otherwise stated, reagents were purchased from commercial sour-
(92%) of the title compound as colorless solid. The following com-
pounds were prepared using the same synthetic procedure: 1–6
and 15–26.
Tetrakispivaloyloxymethyl 2-(3-hydroxypyridine-2-ylamino) eth-
ylidene-1,1-bisphosphonate (27): To a solution of tetrakis-pivaloy-
1
13
loxymethyl
sphosphonate (15, 253 mg, 0.30 mmol) in EtOAc (15 mL) was
added 10% Pd/C (62 mg, 0.058 mmol) and stirred under H (1 atm)
2-(3-benzyloxypyridine-2-ylamino)ethylidene-1,1-bi-
ces and used without further purification. H and C NMR spectra
were obtained in CDCl3 solution on a JNM-AL-400 spectrometer
1
13
2
(
H NMR at 400 MHz, C NMR at 100 MHz) and a JNM-ECA-500
1
13
at room temperature for 1.5 h. The resulting mixture was filtered
and concentrated in vacuo to give a crude product, which was pu-
rified by silica gel column chromatography (EtOAc) to afford
spectrometer ( H NMR at 500 MHz, C NMR at 125 MHz) (JEOL Ltd.,
Akishima, Tokyo, Japan). H NMR chemical shifts were referenced to
tetramethylsilane (TMS) (0.00 ppm) and C NMR chemical shifts to
1
13
1
96 mg (87%) of the title compound. The following compounds
CDCl (77.0 ppm). Chemical shift values are expressed in parts per
3
were prepared using the same synthetic procedure: 28–39.
million (ppm). The following abbreviations were used for peak mul-
tiplicities: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet;
sept, septet; m, multiplet; and br, broad. IR spectra were recorded
on an FT/IR-4100 (JASCO Corp., Hachioji, Tokyo, Japan). Mass spec-
tra and high-resolution mass spectra were recorded on
JMS-HX/HX 110A (JEOL Ltd.). NMR spectral information
and physical data are provided in the Supporting Infor-
mation (chemistry and NMR spectral information).
Tetramethyl 2-(pyridine-2-ylamino)ethylidene-1,1-bisphospho-
nate (40): 2-Aminopyridine (47 mg, 0.50 mmol) was added to a so-
lution of tetramethyl vinylidene-1,1-bisphosphonate (122 mg,
General procedure for the synthesis of 1,1-bisphos-
phonic acid pivoxil esters (Scheme 1)
Tetramethyl vinylidene-1,1-bisphosphonate (S1): Fol-
[39]
lowing the protocol of Degenhardt and Burdsall, di-
ethylamine (6.3 mL, 60 mmol) was added to a solution of
paraformaldehyde (9.0 g, 300 mmol) in methanol Scheme 1. General procedure for the synthesis of 1,1-bisphosphonic acid pivoxil esters.
&
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Full Papers
À1
0
.50 mmol) in THF (1 mL) and stirred at room temperature for 34 h.
USA), 100 IUmL penicillin (Meiji Seika Kaisha, Ltd., Chuo-Ku,
À1
The resulting mixture was concentrated in vacuo to give a crude
product that was purified by silica gel column chromatography
Tokyo, Japan), and 100 mgmL streptomycin (Meiji Seika Kaisha)
and grown overnight at 378C with 5% CO in 225 cm flasks. Com-
2
2
(
MeOH/EtOAc=5–100%) to yield 171 mg (99%) of the title com-
pound 7 was then added to the flasks to the concentrations indi-
cated above. After incubation for 16 h, the cells were harvested
and resuspended in 100 mL lysis solution containing 1% NP-40
pound.
Tetraethyl 2-(pyridine-2-ylamino)ethylidene-1,1-bisphosphonate
(
of tetraethyl vinylidene-1,1-bisphosphonate (600 mg, 2.0 mmol) in
CHCl (8 mL) and stirred at room temperature for 3 h. The resulting
mixture was concentrated in vacuo to give a crude product, which
was purified by silica gel column chromatography (MeOH/CHCl =
1
(Wako Pure Chemical Industries Ltd., Chuo-ku, Osaka, Japan), 0.1%
41): 2-Aminopyridine (376 mg, 4.0 mmol) was added to a solution
sodium dodecyl sulfate (SDS; Tokyo Chemistry Industry Co., Ltd.,
Chuo-Ku, Tokyo, Japan), and 0.5% sodium deoxycholate (Wako) in
microcentrifuge tubes. After centrifugation at 15000 rpm for
3
1
0 min, the supernatants were transferred to new tubes and SDS-
3
urea buffer containing 6.7m urea (Wako), 5% SDS (Tokyo Chemistry
Industry), 100 mm Tris·HCl buffer, pH 7.4 (Wako), 0.25% bromophe-
nol blue (Wako), and 50 mm dithiothreitol (Wako) were added to
0%) to yield 772 mg (98%) of the title compound. Compound 42
was prepared by using the same synthetic procedure.
À1
give a protein concentration of 5 mgmL . The samples were
loaded on 15% polyacrylamide slab gels (Daiichi Pure Chemicals
General procedure for the synthesis of 1,1-bisphosphonic
acids (Scheme 2)
À1
Co., Ltd., Chuo-ku, Tokyo, Japan) at 50 mglane , and electrophor-
À1
esed at 120 mAh . The proteins were then transferred onto Poly-
screen (R) PVDF Transfer Membranes (PerkinElmer Inc., Waltham,
MA, USA) treated with goat anti-unprenylated RAP1A antisera (ꢁ
2
-(5-Methylpyridine-2-ylamino)ethylidene
1,1-bisphosphonic
acid (10). 2-Amino-5-methylpyridine (108 mg, 1.0 mmol) was
added to a solution of tetraethyl vinylidene-1,1-bisphosphonate
5
00, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and
horseradish peroxidase-conjugated anti-goat IgG antisera (ꢁ5000,
KPL Inc., Gaithersburg, MD, USA), followed by SuperSignal West
Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL,
USA). Although not shown, controls using goat anti-RAP1A and
anti-GAPDH antisera (Santa Cruz Biotechnology) were included in
this study. Chemiluminescence was detected on Amersham Hyper-
film MP (GE Healthcare Ltd., Little Chalfont, Buckinghamshire,
UK) using a Fuji Medical Film Processor FPM100 (Fuji Film Co.,
Ltd., Ashigara, Kanagawa, Japan).
(
150 mg, 0.50 mmol) in CHCl (2 mL) and stirred at room tempera-
3
ture for 1 h. The resulting mixture was concentrated in vacuo to
give a crude product that was purified by silica gel column chro-
matography (MeOH/CHCl =10%) to yield 212 mg (99%) of tet-
raethyl 2-aminoethylidene-1,1-bisphosphonate. Tetraethyl 2-amino-
ethylidene-1,1-bisphosphonate (102 mg, 0.25 mmol) was dissolved
in MeCN (2 mL) and treated with bromotrimethylsilane (TMSBr,
3
TM
0
.20 mL, 1.5 mmol). The resulting mixture was stirred at room tem-
perature for 4 h. The mixture was concentrated in vacuo to give
a crude product that was purified by recrystallization from ace-
tone/H O to yield 55 mg (74%) of the title compound. The follow-
Inhibition of EJ-1 bladder tumor cell growth by 7 in NOG immunode-
6
2
ficient mice: EJ-1 tumor cells (1ꢁ10 ) stably transfected with
ing compounds were prepared using the same synthetic proce-
pGL4.10[luc2] (Promega, Madison, WI, USA) were intraperitoneally
dure: 8–9 and 11–14.
(i.p.) inoculated into immunodeficient NOG mice (obtained from
the Central Institute for Experimental Animals, Kawasaki, Kanaga-
wa, Japan). Mice were treated i.p. with 2 mg compound 7 in 0.1 mL
À1
Biological assays
PBS (97.1 mgkg body weight for mice weighing 20.6 g), on days 3
À1
TM
and 6. On day 7, 0.1 mL of 15 mgmL VivoGlo luciferin (Xeno-
gen, Alameda, CA, USA) was i.p. injected, and the mice placed in
a specimen chamber mounted with a CCD camera cooled to
À1208C (In Vivo Imaging System, Waltham, MA, USA). Then, the
photon emission transmitted from the mice was measured. The
greyscale photographic images and bioluminescence color images
were superimposed. This treatment regimen was repeated for
seven weeks, and the images for week 4 are shown. Arbitrary luci-
ferase units were expressed as average flux (photons per second
per mouse), and the photon intensity values per mouse were plot-
ted with time. The growth of EJ-1 was monitored for weeks 2–7,
and the average photon flux per second for four to five mice is
plotted ÆSEM. Statistical significance was assess by the non-para-
metric Mann–Whitney U test. The animal studies were conducted
in accordance with the relevant laws and institutional guidelines,
and were approved by the local IACUC committee.
In vitro tumor cell growth inhibition: Tumor cells were grown, har-
4
À1
vested, and resuspended at 1ꢁ10 cellsmL in complete RPMI
640 medium. A total of 0.05 mL of the cell suspension was added
1
to flat-bottomed 96-well plates, followed by 0.05 mL of three-fold
serial dilutions of POM esters. After incubation at 378C with 5%
CO for 4 days, 0.1 mL of CellTiter-Glo reagent (Promega, Madison,
2
WI, USA) was added, and the luminescence resulting from ATP was
measured using an ARVO luminometer (PerkinElmer, Foster City,
CA, USA). All experiments were performed in triplicate. The con-
centrations required for 50% tumor cell growth inhibition (IC50
values) are shown. The sources for the cell lines are detailed in
[30]
Idrees et al.
Inhibition of geranylgeranylation of RAP1A in tumor cells: Tumor
cells were resuspended in 90 mL of complete RPMI 1640 medium
supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO,
À5
USA), 10 m 2-mercaptoethanol (Invitrogen Corp., Carlsbad, CA,
Scheme 2. General procedure for the synthesis of 1,1-bisphosphonic acids.
ChemMedChem 2016, 11, 1 – 9
www.chemmedchem.org
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&
These are not the final page numbers! ÞÞ

Full Papers
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Acknowledgements
The authors thank Ms. Chiyomi Inoue for technical assistance.
This work was supported by Grants-in-Aid for Scientific Research
from the Ministry of Education, Science, Culture, Sports, and Tech-
nology of Japan (MEXT), by “Special Coordination Funds for Pro-
moting Science and Technology” from MEXT and Astellas Pharma
Inc. through the “Formation of Center for Innovation by Fusion of
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AMED) (to Y.T.), and by the US Department of Veterans Affairs
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Keywords: anticancer agents · bisphosphonates · farnesyl
diphosphate synthase · lipophilicity · prodrugs
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Received: September 12, 2016
Revised: October 10, 2016
Published online on && &&, 0000
&
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FULL PAPERS
No bones about it: A novel bisphosph-
onate prodrug that has its P-C-P group
esterified with pivaloyloxymethyl
K. Matsumoto, K. Hayashi,
K. Murata-Hirai, M. Iwasaki, H. Okamura,
N. Minato, C. T. Morita, Y. Tanaka*
groups shows potent in vitro and in
vivo activity against tumor cells, espe-
cially lymphoma and myeloid leukemia
hematopoietic cells and certain solid
tumor cells. Bisphosphonate prodrugs
like this could increase the effectiveness
of treatment for both hematopoietic
and non-hematopoietic solid tumors.
&
& – &&
Targeting Cancer Cells with
a Bisphosphonate Prodrug
ChemMedChem 2016, 11, 1 – 9
www.chemmedchem.org
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