Synthesis and Immunomodulatory Activity of Fluorine-Containing Bisphosphonates

Article abstract of DOI:10.1002/cmdc.201800764

Immune checkpoint blockade using anti-PD-1/PD-L1 or anti-CTLA-4 monoclonal antibodies (mAbs) has revolutionized cancer treatment. However, many types of cancer do not respond and for those that do, only a minority of patients achieve durable remissions. Therefore, oncoimmunologists are working to develop adoptive cell therapies for non-hematopoietic tumors by harnessing immune effector cells such as αβ T cells and γδ T cells. In contrast to conventional αβ T cells that recognize peptides in the context of MHC class I or II molecules, γδ T cells expressing Vγ2Vδ2 T cell receptors (also termed Vγ9Vδ2) are stimulated by isoprenoid metabolites (phosphoantigens) such as isopentenyl diphosphate in a butyrophilin-3A1-dependent manner. Vγ2Vδ2 T cells kill almost all types of tumor cells that have been treated with bisphosphonates. In this study, we synthesized a series of fluorine-containing bisphosphonates based on current drugs and found that they stimulated Vγ2Vδ2 T cell killing of tumor cells. A fluorine-containing prodrug analogue of zoledronate where phosphonate moieties were masked with pivaloyloxymethyl groups markedly enhanced Vγ2Vδ2 T-cell-mediated cytotoxicity, and also promoted the expansion of peripheral blood Vγ2Vδ2 T cells. These results demonstrate that a prodrug of a fluorine-containing zoledronate analogue can sensitize tumor cells for killing as well as expand Vγ2Vδ2 T cells for adoptive cell therapy.

Full text of DOI:10.1002/cmdc.201800764

Accepted Article
Title: Synthesis and immunomodulatory activity of fluorine-containing
bisphosphonates
Authors: Yoshimasa Tanaka, Satoshi Mizuta, Mohammed S. O. Tagod,
Masashi Iwasaki, Yoichi Nakamura, Hiroaki Senju, Hiroshi
Mukae, and Craig T. Morita
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To be cited as: ChemMedChem 10.1002/cmdc.201800764
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A Journal of
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Synthesis and immunomodulatory activity of fluorine
bisphosphonates
‒
containing
Satoshi Mizuta+,[a] Mohammed S. O. Tagod+,[a,b] Masashi Iwasaki,[c] Yoichi Nakamura,[d] Hiroaki
Senju,[a,d] Hiroshi Mukae,[d] Craig T. Morita,[e] and Yoshimasa Tanaka*[a,b,c,f]
[a]
[b]
[c]
Dr. S. Mizuta, M. S. O. Tagod, Dr. H. Senju, Prof. Y. Tanaka
Center for Bioinformatics and Molecular Medicine, Graduate School of Biomedical Sciences, Nagasaki University,
1-12-4 Sakamoto, Nagasaki, 852-8523, Japan
M. S. O. Tagod, Prof. Y. Tanaka
Program for Nurturing Global Leaders in Tropical and Emerging Infectious Diseases, Graduate School of Biomedical Sciences, Nagasaki University,
1-12-4 Sakamoto, Nagasaki, 852-8523, Japan
Dr. M, Iwasaki, Prof. Y. Tanaka
Center for Innovation in Immunoregulative Technology and Therapeutics, Gradualte School of Medicine, Kyoto University,
Yoshidakonoe-cho, Kyoto, 606-8501, Japan
[d]
[e]
Prof. Y, Nakamura, Dr. H. Senju, Prof. H. Mukae
Department of Respiratory Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan
Prof. C. T. Morita
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, Iowa City, IA 52246 U. S. A.
Prof. Y. Tanaka
[f]
Hyogo College of Medicine, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501, Japan
These authors contributed equally to this work.
[+]
Supporting information for this article is given via a link at the end of the document.
and CTLA-4 immune checkpoint therapies.[1] CTLA-4 was
identified as a negative regulator of T cells,[2-3] and it was
subsequently shown that blocking CTLA-4 signaling using a
monoclonal antibody (mAb) could cure established tumors in
mice.[4] PD-1 was identified as another negative regulator
expressed by T cells[5-6] that interacts with its ligands, PD-L1 and
PD-L2. We derived an anti-murine PD-L1 mAb and
demonstrated that mAb treatment augmented T cell mediated
anti-tumor activity in vitro as well as in vivo in preclinical mouse
models.[7] We also showed that anti-human PD-L1 mAbs
augmented the anti-tumor activity of human Vγ2Vδ2 T cells in
vitro.[8] PD-1 checkpoint blockade has been shown to be
effective in the treatment of patients with a number of different
cancers including melanoma, non-small cell lung cancer, kidney
cancer, urotherlial carcinoma, head and neck carcinoma,
hepatocellular carcinoma, and Hodgkin lymphoma.[9-10] However,
alternatives to immune checkpoint blockade are needed
because the overall response rates for immune checkpoint
monotherapy range from ~20-65% with a minority of durable
remissions. Moreover, a significant number of patients develop
treatment‒related autoimmune diseases such as pneumonitis,
inflammatory arthritis, thyroid disease, inflammatory bowel
disease, and hepatitis.
Abstract: Immune checkpoint blockade using anti-PD-1/PD-L1 or
anti-CTLA-4 monoclonal antibodies (mAbs) has revolutionized
cancer treatment. However, many types of cancer do not respond
and for those that do, only a minority of patients achieve durable
remissions. Therefore, oncoimmunologists are working to develop
adoptive cell therapies for non-hematopoietic tumors by harnessing
immune effector cells such as αβ T cells and γδ T cells. In contrast
to conventional αβ T cells that recognize peptides in the context of
MHC class I or II molecules, γδ T cells expressing Vγ2Vδ2 T cell
receptors (also termed Vγ9Vδ2) are stimulated by isoprenoid
metabolites (phosphoantigens) such as isopentenyl diphosphate in a
butyrophilin 3A1‒dependent manner. Vγ2Vδ2 T cells kill almost all
types of tumor cells that have been treated with bisphosphonates. In
this study, we synthesized
a
series of fluorine‒containing
bisphosphonates based on current drugs and found that they
stimulated Vγ2Vδ2 T cell killing of tumor cells. A fluorine‒containing
prodrug analog of zoledronate where phosphonate moities were
masked with pivaloyloxymethyl groups markedly enhanced Vγ2Vδ2
T cell‒mediated cytotoxicity, and also promoted the expansion of
peripheral blood Vγ2Vδ2 T cells. These results demonstrate that a
prodrug of a fluorine‒containing zoledronate analog can sensitize
tumor cells for killing as well as expand Vγ2Vδ2 T cells for adoptive
cell therapy.
One of the most promising cancer immunotherapies is the
adoptive transfer of immune effector cells that specifically
recognize malignant tumors naturally or through expression of
chimeric antigen receptors. Both T cells and NK cells can kill
human tumor cells.[11] T cells are divided into αβ and γδ T cells.
Whereas conventional αβ T cells recognize peptides in the
Introduction
Standard cancer treatments comprise surgery, radiotherapy,
chemotherapy, hormone therapy, and targeted therapy.
Recently, immunotherapy has become a clinically validated
intervention for many cancer patients with the advent of PD-1
1
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context of MHC class I, Ib, or II molecules, human γδ T cells do
not recognize peptides and are not restricted by MHC molecules.
The most abundant human γδ T cell subset in the peripheral
blood expresses Vγ2Vδ2 T cell receptors (TCR) (also termed
Results and Discussion
Geminal bisphosphonates have a P-C-P backbone and are
analogs of inorganic pyrophosphoric acid. The second and third
generation
geminal
bisphosphonates
are
termed
Vγ9Vδ2). Vγ2Vδ2
T cells are stimulated by isoprenoid
nitrogen‒containing bisphosphonates (N-BPs) and have
a
metabolites such as the (E)-4-hydroxy-3-methylbut-2-enyl-1-
diphosphate (HMBPP) metabolite in the 2-C-methyl-D-erythritol-
hydroxyl group at the R1 position and a nitrogen‒containing alkyl
or aryl side chain at the R2 position (Fig. 1A).[29] The second
generation N-BPs pamidronate (1), alendronate (2), and
4
phosphate isoprenoid pathway used by bacteria and
apicomplexan parasites or isopentenyl diphosphate (IPP) and
dimethylallyl diphosphate (DMAPP) metabolites in the
mevalonate isoprenoid pathway of mammalian cells.[12-16]
Stimulation requires the immunoglobulin superfamily protein,
butyrophilin 3A1 (BTN3A1), that is expressed on all tumor cells,
and the Vγ2Vδ2 TCR expressed by γδ T cells.[17-19]
ibandronate (3) contain
a nitrogen‒containing alkyl group
whereas the third generation N-BPs risedronate (4) and
zoledronate (5) have a nitrogen-containing heterocyclic group at
the R2 position (Fig. 1B).[30]
We first replaced the hydroxyl group by a fluorine atom at the
R1 position of second and third generation N-BPs (1-5). The
details of the synthesis of fluorine‒containing N-BPs (6-8) (Fig.
1C) are described in the Supporting Information.
When tumor cells are treated with nitrogen‒containing
bisphosphonates (N-BPs), N-BPs are internalized via
fluid‒phase endocytosis where they inhibit cytoplasmic farnesyl
diphosphate synthase (FDPS) resulting in the accumulation of
upstream IPP and DMAPP metabolites (Supporting Information,
Fig. S1).[20-24] IPP and DMAPP then bind to the B30.2
intracellular domain of BTN3A1. Although the precise
mechanism remains unknown, isoprenoid‒metabolite binding to
the B30.2 domain of BTN3A1 can be sensed at the cell surface
by Vγ2Vδ2 T cells through their TCRs (Supporting Information,
Fig. S2).[25] Because N-BPs were designed as therapeutics for
bone‒related diseases such as Paget’s disease and
osteoporosis, they mimic inorganic pyrophosphate and have
high affinity for hydroxyapatite in bone.[26] The backbone
structure of N-BPs is P-C(OH)-P, with the hydroxyl group (the
"bone hook") increasing N-BPs bone binding.[27] However,
because bone binding is not required for stimulation of Vγ2Vδ2
T cells, it may be possible to further optimize N-BPs for their use
in cancer immunotherapy.
Fluorine atoms are present in a number of drugs because
they can improve bioactivity by altering physicochemical and/or
pharmacokinetic properties.[28] In the present study, we sought to
determine the effect of substituting a fluorine atom for the
hydroxyl group in N-BP on their activity for cancer
immunotherapy. We therefore synthesized a series of N-BP
analogs to bisphosphonate drugs where a fluorine atom was
substituted for the hydroxyl group. The activity of the analogs
was compared to that of the parent compounds by treating
tumor cells and measuring their killing by Vγ2Vδ2 T cells. To
increase activity, a pivaloyloxymethyl derivative of one of the
fluorine‒containing N-BPs was synthesized and the effect of this
modification on cell‒mediated cytotoxicity and expansion of
Vγ2Vδ2 T cells was determined.
Fig. 1. Structures of bisphosphonates. (A) General structure of geminal
bisphosphonates. (B) Structures of second and third generation
nitrogen‒containing
fluorine‒containing
bisphosphonates
bisphosphonate
(N-BPs).
analogs.
(C)
Structures
of
Fluorine‒containing
bisphosphonates have a fluorine atom in place of the hydroxyl group at the R1
position.
In brief, the amino group of tetraethyl-3-aminopropylidene-1,1-
bisphosphonate was protected with a Boc group and fluorine
was introduced into the R1 position. Boc and ethyl groups were
then removed to yield a pamidronate analog (6). For the
synthesis of an alendronate analog (7), tetraethyl-4-
aminobutylidene-1,1-bisphosphonate was used as a starting
material and essentially the same synthetic approach was
2
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employed. To prepare an ibandronate analog (8), tetraisopropyl
[31]
monofluoromethylenediphosphonate was first synthesized
and reacted with 2-(2-iodoethoxy)tetrahydro-2H-pyran. After the
protective group was removed under acidic conditions, the
nascent hydroxyl group was exposed to methanesulfonyl
chloride and the resulting compound was reacted with N-
methylpentylamine. Finally, isopropyl groups were removed
under acidic conditions.
A risedronate analog (9) was
synthesized as described.[32] The synthetic scheme for a
fluorine‒analog of zoledronate (10) (Fig. 1C) is shown in
Scheme 1 and the procedure is detailed in the chemistry part of
the Experimental Section.
Scheme 1. Procedure for the synthesis of 1-fluoro-2-(imidazol-1-yl)ethylidene-
1,1-bisphosphonic acid (10).
The activity of the fluorine‒containing N-BP analogs was
compared to that of their parent compounds by measuring
Vγ2Vδ2 T cell‒mediated cytotoxicity against 786-0 human renal
carcinoma cells pretreated with the compounds using a non-
radioactive cellular cytotoxicity assay (Supporting Information,
Fig. S3).[33] As shown in Fig. 2, Vγ2Vδ2 T cells from a healthy
adult donor failed to kill untreated 786-0 cells. When the tumor
cells were pretreated with compound 1 (pamidronate), however,
Vγ2Vδ2 T cells exhibited potent lytic activity against the N-
BP‒sensitized tumor cells in a dose‒dependent manner. The
replacement of the hydroxyl group by a fluorine atom at the R1
position significantly reduced the lytic activity of Vγ2Vδ2 T cells
against 786-0 cells (compare compound 1 with compound 6).
Similar decreases in activity were observed for the other second
generation N-BPs (compare compound 2 (alendronate) with 7
and compounds 3 (ibandronate) with 8). Thus, the substitution of
a fluorine atom for the hydroxyl group at the R1 position of
second generation N-BPs reduced their ability to stimulate killing
by Vγ2Vδ2 T cells.
Fig. 2. Comparison of Vγ2Vδ2
T
cell‒mediated cytotoxicity induced by
conventional N-BPs (left panels) or their fluorine-containing analogs (right
panels). 786-0 renal carcinoma cells were either not treated (∆) or pretreated
with second generation N-BPs (1, 2, or 3) at 1000 μM (○) or 3000 μM (●) or
their fluorine-containing analogs (6, 7 or 8) at 1000 μM (○) or 3000 μM (●) or
third generation N-BPs (4 or 5) at 300 μM (○) or 1000 μM (●) or their
fluorine‒containing analogs (9 or 10) at 1000 μM (○) or 3000 μM (●). 786-0
renal carcinoma cells were pretreated with the N-BPs at 37oC for 2 h. The
chelate-forming BM-HT prodrug reagent was then added to 25 μM and the
cells incubated at 37oC for an additional 15 min. After being washed with
RPMI1640 medium, the cells were cultured with Vγ2Vδ2 T cells for 40 min.
Supernatants from the cultures were then harvested and mixed with Eu3+
solution. Specific lysis was determined by measuring time-resolved
fluorescence using a PHERAStar multiplate reader. Data show mean ± SD
and are representative of three independent experiments.
The immunomodulatory activity of third generation N-BPs is
more potent than that of second generation N-BPs. When 786-0
cells were pretreated with third generation N-BPs (compounds 4
and 5), Vγ2Vδ2 T cells exhibited potent lytic activity against the
N-BP‒sensitized tumor cells. Substitution of a fluorine atom for
3
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the hydroxyl group at the R1 position slightly diminished the
cytolytic activity of Vγ2Vδ2 T cells (compare 4 with 9 and 5 with
10), maintaining levels of specific lysis well above 40% at the
effector‒to‒target ratio of 40:1. The difference in the effect of
fluorine substitution between second and third generation N-BPs
is clearly evident when the levels of specific lysis are compared
under identical conditions (effector‒to‒target ratio of 20:1 and
treatment with 1,000 μM N-BP). Whereas second generation N-
BPs showed large decreases in specific lysis (compounds 1/6
(14.7% versus 1.7%), 2/7 (30.1% versus 7.7%), and 3/8 (58.8%
versus 14.1%)), third generation N-BPs showed only moderate
decreases in specific lysis (compounds 4/9 (50.6% versus
41.9%), and 5/10 (46.0% versus 35.2%)) (Supporting
Information, Table S1). The results clearly demonstrate that the
incorporation of a fluorine atom into N-BPs does not improve
their in vitro immunomodulatory activity.
compound 10. Given that the zoledronate (5)‒based prodrug is
unstable, the incorporation of a fluorine atom at the R1 position is
pivotal for the synthesis of a POM derivative.
Scheme 2. Procedure for the synthesis of tetrakispivaloxylmethyl-1-fluoro-2-
(1H-imidazol-1-yl)ethylidene-1,1-bisphosphonate (11).
Previously, we and others reported that some prodrugs of N-
BPs have greatly increased immunomodulatory activity for
Vγ2Vδ2 T cells as compared to their acid forms.[24, 34] Oldfield
and
co-workers
earlier
reported
the
synthesis
of
tetrakispyvaloyloxylmethyl 2-(pyridine-2-ylamino)ethyliene-1,1-
bisphosphonate and found that the pyvaloyloxylmethyl (POM)
ester compound was one of the most active N-BP‒derivatives
that they tested.[34] Based on this finding, we synthesized a
series of N-BP POM esters and their corresponding acid forms
and compared their biologic acitivities. The immunomodulatory
activity of the N-BP POM esters ranged from 20-fold to 1,100-
fold more potent than that of their acid forms. Because
zoledronate is the most active compound among approved N-
BPs drugs, we attempted to synthesize a prodrug of zoledronate
(5). However, a POM‒derivative of zoledronate was unstable
and decomposed immediately after synthesis.
Fig.
3.
Vγ2Vδ2
T
cell‒mediated
cytotoxicity
induced
by
tetrakispivaloxylmethyl-1-fluoro-2-(1H-imidazol-1-yl)ethylidene-1,1-
bisphosphonate (11). 786-0 and UOK121 renal carcinoma cells were
pretreated with 0 μM (∆), 1 μM (○) or 5 μM (●) of 11 at 37oC for 2 h. Similarly,
EJ-1 bladder carcinoma cells and U937 histiocytic leukemia cells were
pretreated with 0 μM (∆), 1.25 μM (▲), 2.5 μM (○) or 5 μM (●) of 11. The cells
were further treated with 25 μM of BM-HT at 37oC for 15 min and specific lysis
by Vγ2Vδ2 T cells determined as in Fig. 2. Data show mean ± SD and are
representative of three independent experiments.
We therefore attempted to synthesize a prodrug (11) of the
fluorine‒containing zoledronate analog (10) (Supporting
Information, Fig. S4). The substitution of a fluorine atom for the
hydroxyl group at the R1 position of zoledronate stabilized its
POM derivative and allowed its isolation. The synthesis is
detailed in the chemistry part of the Experimental Section
(Scheme 2). When compound 11‒pretreated 786-0 renal
carcinoma cells were incubated with Vγ2Vδ2 T cells, the N-
BP‒sensitized tumor cells were killed in a dose‒dependent as
well as effector‒to‒target ratio‒dependent manner (Fig. 3). 40%
of the 786-0 cells were specifically lysed by Vγ2Vδ2 T cells after
pretreatment with compound 10 at 1,000 μM and an
effector‒to‒target ratio of 40:1. Essentially the same
immunomodulatory effect was achieved with 1 μM compound 11,
demonstrating that its activity is ~1,000-fold higher than
To determine if compound 11 was active with other tumor cell
types, UOK121 human renal carcinoma cells, EJ-1 human
bladder carcinoma cells, and U937 human pro-monocytic
histiocytic lymphoma cells were treated with compound 11 and
their killing by Vγ2Vδ2 T cells determined. Vγ2Vδ2 T cells
efficiently killed each of the cell lines in a dose‒dependent as
well as effector‒to‒target ratio‒dependent manner. The specific
lysis of the cell lines by Vγ2Vδ2 T cells ranged from 20.7% to
48.2% at a compound 11 concentration of 5 μM and an
4
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effector‒to‒target ratio of 20:1 (Supporting Information, Table
S2). Note that, as expected, compound 11‒induced cell kiling is
specific for human cells given that compound 11‒treated murine
tumor cells were not killed by Vγ2Vδ2 T cells whereas human
tumor cell lines and virus‒transformed cell lines were all killed to
varying extents (Supporting Information, Table S3). These
results show that fluorine‒containing N-BP prodrugs such as
compound 11 combined with the adoptive transfer of Vγ2Vδ2 T
cells could be useful for the treatment of a wide variety of tumors.
We next examined whether compound 11 could induce the
expansion of Vγ2Vδ2 T cells from a cancer patient. PBMC from
a lung cancer patient were incubated with compound 11 in the
presence of IL-2. Small clusters of cells were noted on day 4/5
leading to large cell clumps on day 7 (Fig. 4A) indicating that
compound 11 was internalized in monocytes and dendritic cells
and stimulated Vγ2Vδ2 T cell proliferation. The proportion of
Vγ2Vδ2 T cells increased from 4.1% on day 0 to 98.6% on day
11 (Fig. 4B). The total number of Vγ2Vδ2 T cells increased from
2.03 × 105 cells on day 0 to 1.47 × 108 cells on day 11 (724-fold
expansion). To determine if the expanded Vγ2Vδ2 T cells from
the lung cancer patient were functional, expanded Vγ2Vδ2 T
cells were incubated with compound 11‒pretreated PC-9 lung
carcinoma cells and cytotoxicity measured. Vγ2Vδ2 T cells
expanded by compound 11, lysed pretreated PC-9 lung
carcinoma cells in a concentration‒dependent as well as
effector‒to‒target ratio‒dependent manner (Fig. 4C). Similar
results were obtained using Vγ2Vδ2 T cells drived from PBMCs
from a healthy adult donor where the allogeneic lung cancer cell
line, H520, was efficiently killed by Vγ2Vδ2 T cells suggesting
that adoptive cancer immunotherapy using allogenic Vγ2Vδ2 T
cells is possible (Supporting Information, Fig. S5). Thus,
compound 11 efficiently expanded Vγ2Vδ2 T cells and the
expanded cells specifically killed compound 11‒sensitized lung
cancer cells.
decreased tumor cell growth and the triggering of tumor
immunity by Vγ2Vδ2 T cells.
Fig. 4. Immunomodulatory effects of tetrakispivaloxylmethyl-1-fluoro-2-(1H-
imidazol-1-yl)ethylidene-1,1-bisphosphonate (11) on Vγ2Vδ2 T cells derived
from a lung cancer patient. (A) Expansion by 11 of Vγ2Vδ2 T cells from
PBMCs derived from a patient with lung cancer. PBMCs were stimulated with
11 and cell clustering was assessed on days 4, 5, 6, and 7. (B) Flow
cytometric analysis of Vγ2Vδ2 T cells before (day 0) and after (day 11)
expansion with 11. Cells on days 0 and 11 were stained with FITC‒conjugated
anti-Vδ2 mAb and PE‒conjugated anti-CD3 mAb and analyzed for expression
of Vδ2 and CD3 through a FACS Verse flow cytometer. (C) Specific lysis of
PC-9 lung carcinoma cells by compound 11‒expanded Vγ2Vδ2 T cells. (Left
panel) Spontaneous and maximal time‒resolved fluorescence of PC-9 cells.
PC-9 cells were treated with 25 μM of BM-HT at 37oC for 15 min. After
washing, the cells were incubated in the absence (spontaneous) or presence
(maximum) of 0.125% detergent in DMSO for 40 min. Supernatants from the
cultures were then mixed with Eu3+ solution and the spontaneous and
maximum release determined by measuring time‒resolved fluorescence using
a PHERAStar multiplate spectrophotometer. The spontaneous release rate
was 18% (Right panel) Specific lysis of PC-9 cells by Vγ2Vδ2 T cells. PC-9
cells were pretreated with 0 μM (), 1.25 μM (■), 2.5 μM (▲), or 5 μM (●) of
11 at 37oC for 2 h. The cells were further treated with 25 μM of BM-HT at 37oC
for 15 min. The specific lysis of pretreated‒PC-9 cells by Vγ2Vδ2 T cells
expanded by compound 11 was determined as in Fig. 2. Data show mean ±
SD and are representative of three independent experiments.
Phosphorus‒containing therapeutics are receiving increasing
attention.[35] Because the cell permeability of BPs is relatively
low, prodrugs of BPs have been designed and synthesized.[36]
Esterification of BPs increased their direct cytotoxic activity
compared with the corresponding BP acid.[37] This demonstrates
the importance of the cell membrane permeability of BPs in
Conclusions
A series of fluorine‒containing N-BPs induced anti-tumor
activity in Vγ2Vδ2 T cells upon treatment of tumor cells.
Although the substitution of a fluorine atom for the hydroxyl
group at the R1 position of N-BPs reduced their
immunomodulartory activity, the introduction of a fluorine atom
was essential for the synthesis and stabilization of compound 11,
determining
their
biological
activity.
Moreover,
a
bisphosphonamidate BP prodrug exhibits selectivity in tumor
entry.[38] Similar approaches have been used to develop new
immunomodulatory prenyl pyrophosphates with increased cell
entry and stability.[39-41] Taken together, the development of BP
prodrugs could open a new avenue for cancer therapy through
more efficient blocking of isoprenoid biosynthesis leading to
a
prodrug
of
the
zoledronate
analog
10.
The
immunomoduolatory activity of the pivaloyloxymethylated
prodrug 11 was ~1,000-fold more potent than its corresponding
5
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Synthesis of 1-fluoro-2-(imidazol-1-yl)ethylidene-1,1-bisphosphonic
acid (10): A solution of tetraethyl-1-fluoro-[2-(imidazol-1-yl)ethylidene]-
1,1-bisphosphonate (58 mg, 0.1 5 mmol) in 6 N HCl (1.0 mL) was stirred
under reflux for 12 h, and then the reaction mixture was concentrated in
vacuo to remove volatiles. The residue was recrystallized from
H2O/MeOH to afford the title compound as a white solid (42 mg, 99%
yield).
acid form 10. By using this synthetic strategy, it may be possible
to synthesize N-BP prodrugs optimized for cancer
immunotherapy.
Experimental Section
1H NMR (500 MHz, D2O) δ 4.72−4.81 (m, 2H), 7.28 (s, 1H), 7.37 (s, 1H),
8.62 (s, 1H); 13C NMR (120 MHz, D2O) δ 51.2 (br. d, J = 18.5 Hz), 118.7,
123.6, 135.9; 19F NMR (471 MHz, D2O) δ −189.8 (tt, J = 25.7, 67.9 Hz,
1F); HRMS (ESI) m/z Calcd. for C5H8FN2O6P2 [M]- 272.9842, found
272.9807.
Chemistry
All NMR spectra were obtained through an AL400 NMR spectrometer
(JEOL Ltd., Akishima, Tokyo, Japan) and a 500PS NMR spectrometer
(Varian Medical Systems Inc., Palo Alto, CA) and processed using an
ACD/SpecManager Enterprise platform (ACD/Labs, Tronto, Ontario,
Canada). 1H, 13C, and 19F NMR spectra were recorded as chemical shifts
(δ) in parts per million (ppm) relative to the solvent peak using
tetramethylsilane, (2, 2, 3, 3-d4) trimethylsilyl-3-propanoic acid, sodium
salt (Thermo Fisher Scientific, Waltham, MA) for 1H and 13C and
trichlorofluoromethane (Merck, Darmstadt, Germany) for 19F as internal
standards. Chemical shifts (δ) were quoted in parts per million (ppm) and
coupling constants (J) were measured in hertz (Hz). The following
abbreviations were used to describe multiplicities; s: singlet, d: doublet, t:
triplet, q: quartet, quint.: quintet, sext.: sextet, sept.: septet, br: broad, and
m: multiplet. High resolution mass spectra (HRMS, m/z) were obtained
using a JMS-700N spectrometer (JEOL Ltd.) for electron ionization or a
JMS-T100TD spectrometer (JEOL Ltd.) for electrospray ionization (ESI+).
All reactions were performed in apparatuses with magnetic stirring under
an inert atmosphere. Flash column chromatography was performed over
Silica Gel C60 (50-200 μm) (Fuji Silysia Chemical Ltd., Kasugai, Aichi,
Japan) using an eluent system as described in each individual
experiment. Thin‒layer chromatography was performed using TLC Silica
Gel 60 F254 aluminum sheets (Merck).
Synthesis of tetrakispivaloxylmethyl-1-fluoro-2-(1H-imidazol-1-yl)
ethylidene-1,1-bisphosphonate (11): The title compound was
synthesized according to a slightly modified literature procedure.[15] To a
suspension of KH (21 mg, 30%, 0.16 mmol) in THF (2.0 mL) was added
imidazole (11 mg, 0.16 mmol) at 0 ˚C under argon atmosphere. After
stirring for 1 h at 0oC and then for 30 min at room temperature, a solution
of tetrakispivaloyloxymethyl vinylidene-1,1-bisphosphonate (100 mg, 0.16
mmol) in THF (1.5 mL) was added to the cooled mixture at 0oC. After
stirring for 30 min, a solution of 18-crown-6-ether (8.2 mg, 0.03 mmol) in
THF (1.5 mL) was added. The reaction mixture was stirred for 30 min,
and then Selectfluor (82 mg, 0.23 mmol) was added. The mixture was
stirred for 17 h, and the reaction was quenched with by the addition of
aqueous sat. NH4Cl (5.0 mL). The aqueous layer was extracted with
AcOEt (2 × 10 mL). The combined organic layers were dried over MgSO4,
concentrated in vacuo. The residue was purified by silica-gel column
chromatography (eluent; Acetone:n-hexane = 1/1 to Acetone /MeOH =
10/1) to give the title compound (30 mg, 26% yield).
1H NMR (500 MHz, CDCl3) δ 1.23 (s, 36H), 4.67 (ddd, J = 9.1, 10.3, 25.9
Hz, 2H), 5.60−5.71 (m, 8H), 6.96 (s, 1H), 7.02 (s, 1H), 7.51 (s, 1H); 13C
NMR (125 MHz, CDCl3) δ 26.8, 26.8, 38.7, 28.7, 48.1 (m), 82.8 (dt, J =
3.2, 70.2 Hz), 120.7 (d, J = 1.6 Hz), 129.2, 138.5 (d, J = 1.2 Hz), 176.5,
176.6; 19F NMR (470 MHz, CDCl3) δ −191.6 (tt, J = 26.0, 71.8 Hz);
HRMS (ESI) m/z Calcd for C29H49FN2NaO14P2 [M]+ 753.2541, found
753.2502.
Tetraethyl-1-fluoro-[2-(imidazol-1-yl)ethylidene]-1,1-
bisphosphonate:
Tetraethyl-1-fluoro-[2-(imidazol-1-yl)ethylidene]-1,1-
bisphosphonate was synthesized according to a literature procedure. In
brief, to a suspension of KH (137 mg, 30%, 1.0 mmol) in THF (7.0 mL)
was added imidazole (68 mg, 1.0 mmol) at 0oC under argon atmosphere.
After stirring for 1 h at 0oC and then for 30 min at room temperature, a
solution of 1,1-bis(diethylphosphono)ethylene (300 mg, 1.0 mmol) in THF
(2.0 mL) was added to the cooled mixture at 0oC. After stirring for 30 min,
a solution of 18-crown-6-ether (54.2 mg, 0.2 mmol) in THF (2.0 mL) was
added. The reaction mixture was stirred for 30 min, to which was added
Selectfluor (530 mg, 1.5 mmol). The mixture was stirred for 10 h, and the
reaction was quenched by the addition of aqueous sat. NH4Cl (10 mL).
The aqueous layer was extracted with AcOEt (2 × 10 mL). The combined
organic layers were dried over MgSO4, concentrated in vacuo. The
residue was purified by silica-gel column chromatography (eluent;
Acetone:n-hexane = 1/1 to Acetone /MeOH = 10/1) to give the title
compound (58 mg, 55% yield).
Biological assays
Derivation of Vγ2Vδ2 T cells: Peripheral blood samples were obtained
from a healthy adult volunteer and a lung cancer patient after approval of
the institutional review board of Nagasaki University Hospital with written
informed consent. The peripheral blood samples were heparinized and
diluted with an equal volume of phosphate-buffered saline (PBS (-),
Nissui Pharmaceutical Co., Ltd. Taito-ku, Tokyo, Japan) and peripheral
blood mononuclear cells (PBMCs) were purified by density gradient
centrifugation at 600 × g for 30 min at ambient temperature. PBMCs were
washed two times with PBS (-) and resuspended in Yssel’s medium
containing human AB serum (Cosmo Bio Co., Ltd., Koto-ku, Tokyo,
Japan) at a cell concentration of 1.6 × 107 cells/mL. The cell suspension
(1.5 mL each) was placed into wells of a 24-well plate, to which was
added 1.5 μL each of 1 mM tetrakis-pivaloyloxymethyl 2-(thiazole-
2ylamino)ethylidene-1,1-bisphosphonate (PTA in dimethylsulfoxide,
1H NMR (500 MHz, CDCl3) δ 1.33 (dt, J = 7.1, 11.7 Hz, 12H), 4.17‒4.27
(m, 8H), 4.67 (ddd, J = 9.6, 18.9, 25.9 Hz, 2H), 7.01 (s, 1H), 7.03 (s, 1H),
7.56 (s, 1H). 1H NMR spectrum corresponds to that of reference.[15]
6
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10.1002/cmdc.201800764
ChemMedChem
FULL PAPER
Techno Suzuta Co., Ltd., Heiwa-machi, Nagasaki, Japan) or 7.5 μL each
of 1 mM compound 11, tetrakispivaloxylmethyl-1-fluoro-2-(1H-imidazol-1-
yl)ethylidene-1,1-bisphosphonate. After the plate was incubated at 37oC
with 5% CO2 overnight, 15 μL of 10,000 U/ml interleukin-2 (IL-2, Shionogi
Pharmaceutical Co., Ltd., Chuo-ku, Osaka, Japan) were added to each
well. After the plate was incubated at 37oC with 5% CO2 for 24 h, the
medium was replaced with new Yssel’s medium containing 100 U/mL of
IL-2. IL-2 was added to each well every day until day 10. On day 11, the
cell suspension was transferred into 50 mL conical tubes, which were
centrifuged at 600 × g at 4oC for 5 min. After the culture supernatant was
removed, the cell pellets were dispersed by tapping, and
cryopreservation medium added (Cell Banker I, Nippon Zenyaku Kogyo.
Co., Ltd., Koriyama, Fukushima, Japan) to give a cell concentration of 1
× 107 cells/mL. The cell suspension (1 mL each) was dispensed into
cryopreservation vials and placed at -80oC overnight and then stored in
liquid nitrogen.
fluorescence was then determined using an ARVO (PerkinElmer Inc.,
Waltham, MA) or a PHERAstar (BMG Labtech Ltd., Allmendgruen,
Ortenberg, Germany) multiplate reader. All experiments were performed
in triplicate. The maximum release was calculated as [maximum release
(counts)
–
background (counts)] and the spontaneous release as
background (counts)]. Then, the
[spontaneous release (counts)
–
spontaneous release rate (%) was calculated as [(100 x spontaneous
release)/maximum release].
Microscopic analysis: During the expansion of Vγ2Vδ2 T cells with
compound 11, cell images were captured under a BZ-X700 fluorescence
microscope (Keyence Corp., Higashi Yodogawa-Ku, Osaka, Japan).
Flow cytometric analysis: Freshly isolated PBMCs and N-
BP‒expanded cells were plated out at 2 × 105 cells/50 μL in a round-
bottom 96-well plate (Corning Inc.). The cells were incubated with 3 μL of
fluorescein isothiocyanate (FITC)‒conjugated TCR Vδ2 mAb (BD
Biosciences, San Diego, CA) and 3 μL of phycoerythrin (PE)‒conjugated
anti-cluster of differentiation 3 (CD3) mAb on ice for 15 min. After being
washed three times with 200 μL of PBS, the cells were resuspended in
600 μL of PBS and analyzed using a FACSCalibur flow cytometer
(Becton Dickinson, Franklin Lakes, NJ). The stained cells were analyzed
using FlowJo ver. 10 (FlowJo LLC, Ashland, Oregon).
Tumor cell lines: The human renal carcinoma cell line 786-0 was
purchased from American Type Culture Collection (Manassas, VA). The
human renal cell carcinoma cell line UOK121 was a kind gift from Dr.
Hirohito Kobayashi (Tokyo Women’s Medical University, Shinjuku, Tokyo,
Japan). The human bladder carcinoma cell line EJ-1 and the human
histiocytic lymphoma cell line U937 were purchased from Health Science
Research Resources Bank (Sennan, Osaka, Japan). The human lung
carcinoma cell line PC-9 was obtained from RIKEN BioResource Center
(Tsukuba, Ibaraki, Japan).
Acknowledgements
This work was supported by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science, Culture,
Sports, and Technology of Japan 16K08844 (MEXT) (to Y.T.),
by the Japan Agency for Medical Research and Development
12723070 and DNW-17004 (to Y.T.), by the SUNBOR grant
(Suntory Foundation) (to S.M.), by the Department of Veterans
Affairs (Veterans Health Administration, Office of Research and
Non-radioactive cellular cytotoxicity assay: Tumor cells were
pretreated with 0 – 3,000 μM of conventional or fluorine-containing N-
BPs at 37oC with 5% CO2 for 2 h and treated with 25 μM of
bis(butyryloxymethyl)
4’-hydroxymethyl-2,2':6',2''-terpyridine-6,6''-
dicarboxylate (BM-HT,
a
non-radioactive chelate-forming proligand,
Techno Suzuta Co., Ltd.) at 37oC with 5% CO2 for 15 mim. The cells
were washed three times with RPMI1640 medium (Merck & Co., Inc.,
Kenilworth, NJ) supplemented with 10% fetal calf serum (FCS) (Merck &
Co., Inc.), 10-5 M 2-mercaptoethanol (Wako Pure Chemical Industries,
Ltd., Chuo-ku, Osaka, Japan), 100 U/mL of penicillin (Meiji Seika Pharma
Co., Ltd., Chuo-ku, Tokyo, Japan), 100 μg/mL of streptomycin (Meiji
Seika Pharma Co., Ltd.) and resuspended in the RPMI1640 medium at a
cell concentration of 5 × 105 cells/mL. To wells of a round-bottom 96-well
plate (Corning Inc., Corning, NY) 100 μL of the tumor cell suspension
were added. Cryopreserved Vγ2Vδ2 T cells were thawed and the labeled
tumor cells were mixed with 100 μL of the thawed Vγ2Vδ2 T cells at
effector‒to‒target ratios of 0, 0.625, 1.25, 2.5, 5, 10, 20, and 40:1. The
plate was then incubated at 37oC with 5% CO2 for 40 min. For
determination of the maximum release rate, the labeled cells were lysed
with 0.125% digitonin (Merck & Co., Inc.) in 19% dimethylsulfoxide in
H2O. After the cell suspensions were mixed well by pipetting, the plate
was centrifuged at 300 × g for 2 min at room temperature and 25 μL of
the supernatants were transferred into a new round-bottom 96-well plate.
To each well, 250 μL of europium solution were added (Techno Suzuta
Co., Ltd.), 200 μL of which were transferred into wells of a 96-well optical
plate (ThermoFisher Scientific Inc., Waltham, MA). Time‒resolved
Development,
Biomedical
Laboratory
Research
and
Development) Grant 1 I01 BX000972-01A1 (to C.T.M.), National
Cancer Institute Grant P30CA086862 (Core Support) (to C.T.M.).
C.T.M. is the Kelting Family Scholar in Rheumatology.
Conflict of interest
Y.T. is a co-inventor of novel terpyridine‒derivative proligands
for measuring cytotoxicity: PCT/JP2015/059838. C.T.M. is a co-
inventor of US Patent 8,012,466 on the development of live
bacterial vaccines for activating γδ T cells and has no other
financial or non-financial conflict of interest. The other authors
declare no financial or non-financial conflicts of interest.
Keywords: cancer immunotherapy • Vγ2Vδ2 γδ T cells • fluorine
• nitrogen-containing bisphosphonate • non-radioactive cellular
cytotoxicity assay
7
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10.1002/cmdc.201800764
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Entry for the Table of Contents
Cancer immunotherapy has become a clinically validated intervention. Although much attention is focused on mAb-based
immunotherapies, the efficacy of immune checkpoint therapy is limited. To develop more effective treatments, we synthesized
fluorine-containing N-BPs and a prodrug 11 for use with Vγ2Vδ2 T cell-based immunotherapies.
9
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