NOVEL FLUORINE-CONTAINING BISPHOSPHONIC ACID DERIVATIVE AND USE THEREOF
20180022769 ยท 2018-01-25
Assignee
Inventors
- Yoshimasa TANAKA (Nagasaki-shi, Nagasaki, JP)
- Satoshi MIZUTA (Nagasaki-shi, Nagasaki, JP)
- Hiroshi UEDA (Nagasaki-shi, Nagasaki, JP)
Cpc classification
A61K39/4611
HUMAN NECESSITIES
C07F9/6506
CHEMISTRY; METALLURGY
A61K31/683
HUMAN NECESSITIES
C07F9/3873
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07F9/6506
CHEMISTRY; METALLURGY
A61K31/683
HUMAN NECESSITIES
Abstract
A series of fluorine-containing bisphosphonic acids in which an alkylamine side chain is added, a series of fluorine-containing bisphosphonic acids in which an amino group substituted by a heterocyclic group or a heterocyclic group containing a nitrogen atom is added, to the carbon atom of PC(F)P, and a series of fluorine-containing bisphosphonate derivatives in which the acid moiety thereof is esterified by an alkoxymethyl group such as POM group, n-butanoyloxymethyl (BuOM) group and the like, that is, the fluorine-containing bisphosphonic acid and fluorine-containing bisphosphonate derivative represented by the following formula (I):
##STR00001##
wherein each symbol is as defined in the DESCRIPTION, can efficiently induce proliferation of peripheral blood T cells that express V2V2 T cell receptor having superior cytotoxicity against tumor cells and virus infected cells, immunize tumor cells and virus infected cells, and can induce cytotoxicity by T cells.
Claims
1.-22. (canceled)
23. A compound represented by formula (I): ##STR00021## wherein Cy is an imidazolyl group, Y is a hydrogen atom, F is a fluorine atom, P is a phosphorus atom, R.sub.1 and R.sub.2 are the same or different from each other and each is an alkylcarbonyloxyalkyl group, j is a number 1, m is a number 0, and n is an integer of 1-6, or a pharmaceutically acceptable salt thereof.
24. The compound according to claim 23, wherein R.sub.1 and R.sub.2 are the same or different and each is a C.sub.2-7 alkylcarbonyloxy-C.sub.1-3 alkyl group, or a pharmaceutically acceptable salt thereof.
25. The compound according to claim 23, wherein R.sub.1 and R.sub.2 are the same or different and each is pivaloyloxymethyl (POM) group, or a pharmaceutically acceptable salt thereof.
26. The compound according to claim 23, which is a compound represented by formula (5) ##STR00022## or a pharmaceutically acceptable salt thereof:
27. A pharmaceutical composition comprising the compound according to claim 23, or a pharmaceutically acceptable salt thereof, as an active ingredient.
28. A pharmaceutical composition comprising the compound according to claim 24, or a pharmaceutically acceptable salt thereof, as an active ingredient.
29. A pharmaceutical composition comprising the compound according to claim 25, or a pharmaceutically acceptable salt thereof, as an active ingredient.
30. A pharmaceutical composition comprising the compound according to claim 26, or a pharmaceutically acceptable salt thereof, as an active ingredient.
31. A method of (i) treating a lymphocyte in a living body, (ii) proliferating and/or inducing a T cell, (iii) suppressing proliferation of a tumor cell, or (iv) treating cancer, comprising administering an effective amount of the compound according to claim 23, or a pharmaceutically acceptable salt thereof, to a living body.
32. A method of proliferating and/or inducing a T cell, comprising reacting ex vivo the compound according to claim 23, or a pharmaceutically acceptable salt thereof, with a sample containing T cells.
33. A method of suppressing proliferation of a tumor cell, comprising a step of reacting the compound according to claim 23, or a pharmaceutically acceptable salt thereof, with a sample containing T cells collected from a living body, and a step of returning the T cells to the living body.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0041]
[0042]
[0043]
DESCRIPTION OF EMBODIMENTS
[0044] One embodiment of the fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative to be used in the present invention is represented by the following formula (I):
##STR00004##
wherein Cy is a phenyl group or a heterocyclic group, Y is a hydrogen atom, an alkyl group, a halogen atom, an alkyl halide group, a hydroxyl group, an aryl group optionally substituted by a halogen atom or an alkoxy group, or an aralkyloxy group, F is a fluorine atom, P is a phosphorus atom, R is a hydrogen atom or an alkyl group, R.sub.1 and R.sub.2 are the same or different from each other and each is a hydrogen atom or an alkylcarbonyloxyalkyl group, j is a number 0 or 1, m is a number 0 or 1, and n is an integer of 1-6, provided that a compound wherein Cy is a 3-pyridyl group, m is 1, n is 1, Y is a hydrogen atom, and R.sub.1 and R.sub.2 are hydrogen atoms is excluded.
[0045] Cy is a phenyl group or a heterocyclic group, to which at least Y is bonded. The heterocyclic group is a 4- to 15-membered monocyclic heterocyclic group or condensed polycyclic heterocyclic group containing, as a ring-constituting atom besides carbon atom, 1-4 hetero atoms selected from a nitrogen atom, a sulfur atom and an oxygen atom. Examples of the heterocyclic group include furyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, quinolyl, isoquinolyl, quinazolyl, quinoxalyl, benzofuryl, benzothienyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzotriazole, indolyl, indazolyl, pyrrolopyrazinyl, imidazopyridinyl, imidazopyrazinyl, pyrazolopyridinyl, pyazolothienyl, pyrazolotriazinyl, oxetanyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, hexamethyleniminyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, oxazolinyl, thiazolinyl, imidazolinyl, dioxolanyl, dihydrooxadiazolyl, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrothiopyranyl, tetrahydrofuryl, pyrazolidinyl, pyrazolinyl, tetrahydropyrimidinyl, dihydroindolyl, dihydroisoindolyl, dihydrobenzofuranyl, dihydrobenzodioxinyl, dihydrobenzodioxepinyl, tetrahydrobenzofuranyl, chromenyl, dihydroquinolinyl, tetrahydroquinolinyl, dihydroisoquinolinyl, tetrahydroisoquinolinyl, dihydrophthalazinyl, 7-azaindolyl and the like.
[0046] Preferably, the above-mentioned heterocyclic group is a 5- to 10-membered heterocyclic group containing 1-3 hetero atoms selected from a nitrogen atom, a sulfur atom and an oxygen atom, more preferably a 5- or 6-membered heterocyclic group containing 1 or 2 hetero atoms selected from a nitrogen atom and a sulfur atom. Such heterocyclic group is specifically preferably imidazolyl, thiazolyl, pyridyl, pyrimidyl or 7-azaindolyl, more preferably imidazolyl or pyrimidyl, particularly preferably imidazolyl group.
[0047] In the above-mentioned heterocyclic group, Y is bonded at a substitutable position. Y is a hydrogen atom, an alkyl group (e.g., C.sub.1-10 alkyl group such as methyl, ethyl, hexyl, octyl and the like), a halogen atom, (e.g., chlorine atom, fluorine atom, bromine atom), an alkyl halide group (e.g., C.sub.1-3alkyl group (e.g., methyl, ethyl, propyl) substituted by 1 to 3 halogen atoms (as defined above), a hydroxyl group, an aryl group, or an aralkyloxy group. As used herein, the aforementioned aryl group is optionally substituted by a halogen atom (as defined above) or an alkoxy group (e.g., C.sub.1-3alkoxy group such as methoxy, ethoxy, propoxy and the like). Preferably, Y is a hydrogen atom, a C.sub.1-3 alkyl group (as defined above), a halogen atom, an alkyl halide group, an unsubstituted aryl group, more preferably, a hydrogen atom, a methyl group, a halogen atom, a trifluoromethyl group or a phenyl group, most preferably, a hydrogen atom or a bromine atom.
[0048] The aryl group encompasses a monocyclic aryl group and a condensed polycyclic aryl group, and specifically, phenyl, biphenyl, naphthyl, anthryl, phenanthryl and acenaphthylenyl can be mentioned. It is preferably a C.sub.6-18 aryl group, more preferably a C.sub.6-8 aryl group, particularly preferably a phenyl group.
[0049] The aralkyloxy group is preferably a C.sub.7-18 aralkyloxy group, specifically benzyloxy, phenethyloxy and the like, with preference given to benzyloxy.
[0050] R.sub.1 and R.sub.1 are the same or different from each other and each is a hydrogen atom or an alkylcarbonyloxyalkyl group, and at least one of R.sub.c and R.sub.2 is an alkylcarbonyloxyalkyl group. Preferably, both of R.sub.1 and R.sub.2 are alkylcarbonyloxyalkyl groups. Examples of the alkylcarbonyloxyalkyl group include a C.sub.2-7 alkylcarbonyloxy-C.sub.1-3 alkyl group, preferably, C.sub.3-4 alkylcarbonyloxy-methyl, particularly preferably, pivaloyloxymethyl or n-butanoyloxymethyl.
[0051] j is 0 or 1. m is 0 or 1, preferably 1. In the case wherein Cy is secondary amine such as pyrrolyl, pyrrolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, hexamethyleniminyl, oxazolidinyl, thiazolidinyl, imidazolidinyl and the like, m is 0, Cy is bonded to (CH.sub.2).sub.n group at the nitrogen atom. n is an integer of 1-6, preferably 1-3, particularly preferably 1.
[0052] The fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention can be efficiently produced by 3 synthesis steps when, for example, a fluorine atom is introduced into bisphosphonic acid (or a derivative thereof). First, (a) a reactive group such as an amino group and the like of bisphosphonic acid to be the starting material is protected, (b) a fluorine atom is introduced by a fluorinating agent, and (c) the protecting group introduced in step a is removed. Examples of the fluorinating agent to be used in step b include N-fluorosulfonimides such as N-fluorophenylsulfonimide, N-fluorotoluenesulfonimide, N-fluoromethanesulfonimide, N-fluorotrifluoromethanesulfonimide and the like, N-fluoropyridinium salts such as N-fluoro-2,4,6-trimethylpyridinium trifluoromethanesulfonate, N-fluoro-2,4,6-trimethylpyridinium tetrafluoroborate and the like, difluoroxenon, fluorine gas and the like. N-fluorosulfonimides are preferable in view of easy availability, easy handling, yield and the like.
[0053] Alternatively, when bisphosphonic acid (or a derivative thereof) into which a fluorine atom is introduced is obtainable, the fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention can also be produced by using the compound as a starting material and introducing a desired substituent. The reagent to be used, reaction conditions and the like can be selected and determined by a known method or by appropriately modifying or altering the method according to the kind of the starting material and substituent to be introduced.
[0054] The fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention can be specifically synthesized according to the synthesis procedure of the below-mentioned Examples.
[0055] The fluorine-containing bisphosphonic acid and fluorine-containing bisphosphonate derivative in the present invention may be pharmaceutically acceptable salts. In addition, when the fluorine-containing bisphosphonic acid and fluorine-containing bisphosphonate derivative of the present invention contain an isomer (e.g., optical isomer, geometric isomer and tautomer) and the like, the present invention encompasses such isomers and also encompasses solvate, hydrate and various shapes of crystals.
[0056] In the present invention, as a pharmaceutically acceptable salt, general salts pharmacologically and pharmaceutically acceptable salts can be mentioned. Specific examples of such salt include the following.
[0057] Examples of basic addition salt include alkali metal salts such as sodium salt, potassium salt and the like; alkaline earth metal salts such as calcium salt, magnesium salt and the like; ammonium salt; trimethylamine salt, triethylamine salt; aliphaticamine salts such as dicyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, procaine salt and the like; aralkylamine salts such as N,N-dibenzylethylenediamine and the like; heterocycle aromatic amine salts such as pyridine salt, picoline salt, quinoline salt, isoquinoline salt and the like; quaternary ammonium salts such as tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltriethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, tetrabutylammonium salt and the like; basic amino acid salts such as arginine salt, lysine salt and the like; and the like.
[0058] Examples of acid addition salt include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogencarbonate, perchlorate and the like; organic acid salts such as acetate, propionate, lactate, maleate, fumarate, tartrate, malate, citrate, ascorbate and the like; sulfonates such as methanesulfonate, isethionate, benzenesulfonate, p-toluenesulfonate and the like; acidic amino acid salts such as aspartate, glutamate and the like; and the like.
[0059] The novel fluorine-containing bisphosphonic acid and fluorine-containing bisphosphonate derivative of the present invention have a farnesyl diphosphate synthase inhibitory activity. As a result, it suppresses production of isoprenoid metabolites such as cholesterol, liposoluble vitamin, lipoprotein and the like, which are essential for cell survival and exhibits superior direct tumor damaging effect and virus-infected cell cytotoxicity effect. Therefore, the present invention provides direct or indirect antitumor drugs and antiviral agents containing the fluorine-containing bisphosphonic acid and/or a fluorine-containing bisphosphonate derivative as an active ingredient.
[0060] The antitumor and antiviral agent of the present invention can be used by administering to the living body, and preferably administered to mammals (human, mouse, rat, hamster, rabbit, cat, dog, bovine, sheep, monkey etc.).
[0061] The novel fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention specifically stimulates and proliferates and/or induces V2 positive T cells present in the blood such as peripheral blood in the living body, or lymph fluid, as well as can induce or potentiate an antitumor action of these cells. Therefore, the present invention provides lymphocyte-treating agent containing the fluorine-containing bisphosphonic acid and/or a fluorine-containing bisphosphonate derivative as an active ingredient.
[0062] As an antitumor action of the T cells, recognition of a molecule expressing in cancer cells, for example, MICA/B and IPP (isopentenyl pyrophosphate) via a T cell receptor thereof and injury of the cell by T cells can be mentioned. Furthermore, enhancement of antitumor activity by the action of cytokines such as TNF-, INF- and the like produced by T cells can be mentioned.
[0063] The lymphocyte-treating agent of the present invention has an action to proliferate and/or induce T cells in vivo and ex vivo. Therefore, the lymphocyte-treating agent of the present invention can be used by treating a sample containing T cells collected from a living body, or directly administering to a living body. Here, the living body means mammals (human, mouse, rat, hamster, rabbit, cat, dog, bovine, sheep, monkey etc.), and human is particularly preferable.
[0064] The present invention also includes a method of suppressing proliferation of tumor cells, comprising a step of proliferating and/or inducing T cells by reacting the lymphocyte-treating agent of the present invention on a sample containing T cells collected from a living body, and a step of returning the T cells to the living body.
[0065] As a sample containing T cells collected from a living body, blood such as peripheral blood and lymph fluid can be recited as examples. As a target of the lymphocyte-treating agent of the present invention, peripheral blood is preferable, and it is more preferable to use a mononuclear cell fraction separated from the peripheral blood by a specific gravity centrifugation method.
[0066] It is possible to stimulate T cells in a sample with the lymphocyte-treating agent of the present invention by culturing the lymphocyte-treating agent and the sample according to a conventional method. It is possible to induce and/or proliferate T cells by culturing in the presence of a fluorine-containing bisphosphonic acid and/or a fluorine-containing bisphosphonate derivative in a trace amount of 100 pM-100 M, preferably 100 pM-20 M, further preferably 100 pM-5 M.
[0067] Since the fluorine-containing bisphosphonic acid and a fluorine-containing bisphosphonate derivative as the active ingredient in the lymphocyte-treating agent of the present invention has a bisphosphonic acid skeleton, it shows resistance to alkaliphosphatase as compared to conventional pyrrophosphoric acid lymphocyte-treating agents (Biology Trace Element Research, 104, 131-140 (2005)). Therefore, as a culture medium of T cells to induce and/or proliferate T cells, one containing a serum can be used and, for example, human AB serum, fetal bovine serum and the like can be used. Since a medium containing a serum can be used, T cells can be advantageously provided in an amount sufficient for use in a cancer treatment, conveniently and in a short time.
[0068] As a constitution embodiment for use of the lymphocyte-treating agent of the present invention ex vivo for proliferating and/or inducing T cells, the fluorine-containing bisphosphonic acid and/or a fluorine-containing bisphosphonate derivative itself as the active ingredient may be used alone. In addition, it can also be produced as a solution of ethanol, DMSO and the like. Where necessary, other additive can also be added simultaneously. When the lymphocyte-treating agent is reacted with a sample, interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-18 (IL-18), and the like may also be added as an aid factor at a concentration of 0.1-150 IU/mL, preferably 1-100 IU/mL or 0.11-1000 ng/mL. Specific induction and/or proliferation of the T cells becomes remarkable by the addition of these.
[0069] Induction and/or proliferation of specific T cells by the lymphocyte-treating agent can be evaluated by measuring, after culturing, the IFN- amount and/or TNF- amount produced in the culture supernatant. For example, when the TNF- production amount is higher than that at the time of start of culture, T cells can be judged to have been induced. The IFN- amount and/or TNF- amount can be performed using a conventionally-known method by using an anti-IFN- antibody, an anti-TNF- antibody and the like.
[0070] The T cells treated with the lymphocyte-treating agent of the present invention as mentioned above can be used by administration as a medicament to a patient. For example, a mononuclear cell fraction derived from a patient having a tumor is treated with the lymphocyte-treating agent of the present invention, and a mononuclear cell fraction found to show proliferation and/or induction of T cells is administered as peripheral blood and the like to allow for exhibition of an antitumor activity. As an administration method, methods such as topical injection, intravenous injection, transdermal absorption and the like.
[0071] When the antitumor drug, antiviral agent and lymphocyte-treating agent of the present invention are used as pharmaceutical products, they are generally mixed with pharmaceutically acceptable carrier, excipient, diluent, filler, disintegrant, stabilizer, preservative, buffering agent, aromatic, colorant, sweetening agent, thickener, corrigent, solubilizing agents, and other additive known per se, specifically, water, vegetable oil, alcohol (e.g., ethanol, benzyl alcohol etc.), polyethylene glycol, glyceroltriacetate, gelatin, hydrocarbonate (e.g., lactose, starch etc.), magnesium stearate, talc, lanolin, petrolatum and the like, and a tablet, pill, powder, granule, suppository, injection, eye drop, liquid, capsule, troche, aerosol, elixir, suspension, emulsion, syrup and the like are formed by conventional methods, and they can be administered systemically or topically, orally or parenterally.
[0072] While the dose varies depending on the age, body weight, symptom, treatment effect, administration method and the like, it is generally 0.001 mg/kg-1000 mg/kg, preferably 0.01 mg/kg-100 mg/kg, per one time in the amount of active ingredient, for an adult, which is administered once to several times per day, orally or in the form of injection such as intravenous injection and the like.
[0073] The present invention encompasses direct and indirect antitumor drug and antiviral agent, and shows a treatment effect on benign and malignant tumor, and virus infected cells. In addition, the lymphocyte-treating agent of the present invention is useful for the prophylaxis and/or treatment of tumor. Examples of the tumor target include malignant tumors such as brain tumor (malignant astrocytoma, glioma having oligodendroglial tumor component etc.), esophagus cancer, gastric cancer, liver cancer, pancreatic cancer, large intestine cancer (colorectal cancer, rectal cancer etc.), urinary bladder cancer, lung cancer (non-small cell lung cancer, small cell lung cancer, primary and metastatic squamous cell carcinoma etc.), renal cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, neuroblastoma, sarcoma, bone and soft tissue tumor, bone tumor, osteosarcoma, testis tumor, extragonadal tumor, orchis tumor, uterine cancer (uterus cervix cancer, uterine body cancer etc.), head and neck tumor (maxilla cancer, laryngeal cancer, pharyngeal cancer, cancer of the tongue, mouth cavity cancer etc.), multiple myeloma, malignant lymphoma (reticulum cell sarcoma, lymphosarcoma, Hodgkin's disease etc.), polycythemia vera, leukemia (acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia etc.), thyroid cancer, renal pelvis cancer, ureter tumor, bladder tumor, gall bladder cancer, cholangiocarcinoma, choriocarcinoma, malignant melanoma, pediatric tumor (Ewing sarcoma family, Wilms' tumor, rhabdomyosarcoma, blood vessel sarcoma, testicular embryonal carcinoma, neuroblastoma, retinoblastoma, hepatoblastoma, nephroblastoma etc.) and the like and the like. As viral infectious disease to be the target, viral infectious disease such as HTLV-1 infections, HIV infections, influenza disease, herpes disease and the like, and the like can be mentioned. In the present invention, application to urinary bladder cancer, renal cancer, lung cancer, breast cancer, hematologic tumor such as leukemia and the like, and HTLV-1 infections is preferable.
EXAMPLES
[0074] The production method of the fluorine-containing bisphosphonic acid and a fluorine-containing bisphosphonate derivative of the present invention is specifically explained below, and shown below. The production method of the compound of the present invention is not limited to those specifically explained below.
[0075] Unless specifically indicated, all reactions were performed under air atmosphere. Unless specifically indicated, various reagents used were commercially available products.
(Measurement Method and Marking)
[0076] .sup.1H NMR, .sup.13C NMR and .sup.19F NMR spectra were measured by JNM-AL-400 spectrometer (.sup.1H NMR at 400 MHz, .sup.13C NMR at 100 MHz) and Varian-500PS spectrometer (.sup.1H NMR at 500 MHz, .sup.13C NMR at 125 MHz, .sup.19F NMR at 470 MHz) (JEOL Ltd., Akishima, Tokyo, Japan) in CDCl.sub.3 or D.sub.2O solution. .sup.1H NMR chemical shift refers to tetramethylsilane (TMS) (0.00 ppm) and .sup.13C NMR chemical shift refers to CDCl.sub.3 (77.0 ppm) and .sup.19F NMR chemical shift refers to CFCl.sub.3. The chemical shift is shown in one-millionth (ppm).
[0077] The multiplicity of the peak is abbreviated as follows. s, singlet; d, doublet; dt, doublet of triplets; ddd, doublet of doublet of doublets; dtt, doublet of triplet of triplets; t, triplet; tt, triplet of triplets; q, quartet; m, multiplet; br, broad; pent, pentet
[0078] Mass spectrum and high resolution mass spectrum were measured by JEOL IMS-T100TD (JEOL Ltd.).
[0079] Thin layer chromatography (TLC) was performed on a pre-coated plate (0.25 mm, silica gel plate 60F.sub.245, Merck Millipore, Mass.).
[0080] Column chromatography was performed on a silica gel plate (Kanto Chemical Co., Inc.).
LIST OF ABBREVIATIONS
[0081] Me: methyl,
Et: ethyl,
iPr: isopropyl,
Boc: t-butoxycarbonyl,
Boc.sub.2O: di-tert-butyl dicarbonate,
Et.sub.3N: triethylamine,
CH.sub.2Cl.sub.2: dichloromethane,
quant.: quantitatively obtained,
NFSI: N-fluorobenzenesulfonimide,
[0082] n-BuLi: n-butyllithium,
THF: tetrahydrofuran,
HCl: hydrochloric acid,
NaH: sodium hydride,
15-crown-5-ether: 15-crown-5-ether,
MeOH: methanol,
Ms: methanesulfonyl,
MsCl: methanesulfonyl chloride,
K.sub.2CO.sub.3: potassium carbonate,
DMF: N,N-dimethylformamide,
[0083] KH: potassium hydride,
18-crown-6-ether: 18-crown-6-ether,
select fluor: 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroboric acid)
Example 1
Synthesis of 3-amino-1-fluoro-propylidene-1,1-bisphosphonic acid (PAMF)
[0084] ##STR00005##
(1) tert-butyl(3,3-bis(diethoxyphosphoryl)propyl)carbamate
[0085] ##STR00006##
[0086] Tetraethyl-3-aminopropylidene-1,1-bisphosphonate.sup.[1] (460 mg, 1.5 mmol) dissolved in dichloromethane (20 mL) was reacted with Boc.sub.2O (334 L, 1.5 mmol) and triethylamine (205 L, 1.5 mmol) at room temperature. The reaction mixture was stirred for 9 hr, the solvent was removed under reduced pressure, and the solution was concentrated. As a result, colorless oily tert-butyl(3,3-bis(diethoxyphosphoryl)propyl)carbamate (575 mg, yield 99%) was obtained. .sup.[1] K. Ogawa, T. Mukai, Y. Arano, H. Hanaoka, K. Hashimot, H. Nishimura, H. Saji, J. Label. Compd Radiopharm. 2004, 47, 753-761.
[0087] .sup.1H NMR (500 MHz, CDCl.sub.3) 1.35 (t, J=7.1 Hz, 6H), 1.53 (s, 9H), 2.06-2.15 (m, 2H), 2.40 (tt, J=6.4, 23.9 Hz, 2H), 3.30-3.37 (m, 2H), 4.17-4.22 (m, 8H), 5.06 (br. s, NH);
[0088] .sup.13C NMR (125 MHz, CDCl.sub.3) 16.3-16.3 (m), 27.3, 28.3, 62.6-62.7 (m), 85.1, 146.6;
[0089] HRMS (ESI) m/z Calcd for C.sub.16H.sub.35NNaO.sub.8P.sub.2 [M].sup.+ 454.1736, found 454.1696.
(2) tert-butyl(3,3-bis(diethoxyphosphoryl)-3-fluoropropyl)carbamate
[0090] ##STR00007##
[0091] To tert-butyl(3,3-bis(diethoxyphosphoryl)propyl)carbamate (50 mg, 0.13 mmol) dissolved in THF (2.0 mL) was added dropwise n-BuLi (90 L, 1.6 M hexane solution, 0.14 mmol) at 78 C. under an argon atmosphere. After stirring for 10 min, N-fluorophenylsulfonimide (45 mg, 0.14 mmol) was added to the carbanion solution. The reaction mixture was allowed to reach room temperature over 1 hr. After stirring for 7 hr, the reaction was discontinued with saturated ammonium chloride (10 mL). The reaction product was extracted from the aqueous phase with ethyl acetate (210 mL), and the obtained organic phases were mixed. This was dehydrated over magnesium sulfate, and concentrated under reduced pressure. The reaction product was passed through a silica gel column by using acetone/n-hexane=1/1 solvent system. However, since the object product could not be obtained with high purity, 28 mg of a crude product containing tert-butyl(3,3-bis(diethoxyphosphoryl)-3-fluoropropyl)carbamate as the main component was used without further purification for the next reaction.
[0092] .sup.1H NMR (400 MHz, CDCl.sub.3) 1.36 (t, J=8.8 Hz, 12H), 1.42 (s, 9H), 2.33-2.43 (m, 2H), 3.45-3.48 (m, 2H), 4.22-4.31 (m, 8H), 5.18 (br. s, NH).
[0093] HRMS (ESI) m/z Calcd for C.sub.16H.sub.34FNNaO.sub.8P.sub.2 [M].sup.+ 472.1641, found 472.1646.
(3) 3-amino-1-fluoro-propylidene-1,1-bisphosphonic acid
[0094] ##STR00008##
[0095] tert-Butyl(3,3-bis(diethoxyphosphoryl)-3-fluoropropyl)carbamate (28 mg, 0.06 mmol) was dissolved in 1 mL of 6N hydrochloric acid, and the mixture was heated under reflux for 7 hr. The solvent was removed under reduced pressure and the reaction mixture was concentrated. The residue was recrystallized from water/methanol to give a yellow solid (14 mg, yield 45%).
[0096] .sup.1H NMR (500 MHz, D.sub.2O) 2.38-2.51 (m, 2H), 3.28-3.34 (m, 2H);
[0097] .sup.19F NMR (470 MHz, D.sub.2O) 183.4 (tt, J=23.1, 69.2 Hz);
[0098] HRMS (ESI) m/z Calcd for C.sub.3H.sub.9FNO.sub.6P.sub.2 [M].sup. 235.9889, found 235.9852.
Example 2
Synthesis of 1-fluoro-3-(methyl(pentyl)amino)propylidene-1,1-bisphosphonic acid (IBAF)
[0099] ##STR00009##
(1) tetraisopropylmonofluoromethylenediphosphonate.SUP.[2]
[0100] ##STR00010##
[0101] Tetraisopropyl methylenediphosphonate (2.5 g, 7.3 mmol) dissolved in 20 mL of DMF was cooled on ice to 0 C. NaH (386 mg, 60% in mineral oil, 16.6 mmol) dissolved in 20 mL of DMF was placed in a different flask, and cooled for 5 min at 0 C. This NaH solution was added dropwise to tetraisopropyl methylenediphosphonate. The reaction mixture was stirred at 0 C. for 10 min, and allowed to warm to room temperature. After stirring for 1 hr at room temperature, selectfluor (5.7 g, 16.6 mmol) dissolved in DMF was added, and the reaction mixture was stirred for 6 hr at room temperature. This was diluted with 50 mL of dichloromethane, and 50 mL of saturated ammonium chloride solution was added to discontinue the reaction. The aqueous phase was extracted with dichloromethane (250 mL) and the obtained organic phase was dried over magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and purified by silica gel column (eluent: gradient of ethyl acetate/n-hexane=1/2 to ethyl acetate 100%) to give monofluoro bisphosphonate (746 mg, yield 22%) and difluorobisphosphonic acid (615 mg, yield 28%). .sup.[2]V. Jo Davisson, Darrell R. Davis, Vyas M. Dixit, C. Dale Poulter, J. Org. Chem. 1987, 52, 1794-1801.
(2) tetraisopropyl-1-fluoro-3-hydroxypropylidene-1,1-bisphosphonate
[0102] ##STR00011##
[0103] To a suspension of NaH (96 mg, 60%, 2.4 mmol) in THF (15 mL) prepared under an argon atmosphere was added at 0 C. tetraisopropylmonofluoromethylenediphosphonate (724 mg, 2.0 mmol) dissolved in 5 mL of THF. After stirring for 30 min, 2-(2-iodoethoxy)tetrahydro-2H-pyran (615 mg, 2.4 mmol) and 15-crown-5-ether (88 mg, 0.4 mmol) dissolved in 2 mL of THF was added. The reaction mixture was stirred at room temperature for 24 hr, and the reaction was discontinued with saturated ammonium chloride solution. A compound was extracted from the aqueous phase with ethyl acetate (250 mL), and the obtained organic phases were mixed, dehydrated over magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure. A crude product of tetraisopropyl-1-fluoro-3-((tetrahydro-2H-pyran-2-yl)oxy)propylidene-1,1-bisphosphonate was treated with 2 mL of 1N hydrogen chloride methanol solution, and the mixture was stirred for 10 min. The reaction mixture was concentrated under reduced pressure and purified by silica gel column (eluate: acetone/n-hexane=1/1) to give tetraisopropyl-1-fluoro-3-hydroxypropylidene-1,1-bisphosphonate as a colorless oil (198 mg, yield 24%).
[0104] .sup.1H NMR (500 MHz, CDCl.sub.3) 1.37-1.38 (m, 24H), 2.42 (dtt, J=5.4, 15.2, 27.5 Hz, 2H), 3.88 (t, J=5.1 Hz, 2H), 4.11 (br. s, OH), 4.89-4.90 (m, 4H);
[0105] .sup.13C NMR (125 MHz, CDCl.sub.3) 23.7-23.8 (m), 24.2-24.3 (m), 36.8 (d, J=19.2 Hz), 73.1 (t, J=3.7 Hz), 73.2 (t, J=3.5 Hz);
[0106] .sup.19F NMR (470 MHz, CDCl.sub.3) 5-193.8 (tt, J=22.7, 78.0 Hz);
[0107] HRMS (ESI) m/z Calcd for C.sub.15H.sub.33FN.sub.2NaO.sub.7P.sub.2 [M].sup.+ 429.1583, found 429.1543.
(3) 2,2-bis(diisopropyloxyphosphoryl)2-fluoroethylmethanesulfonate
[0108] ##STR00012##
[0109] To tetraisopropyl-1-fluoro-3-hydroxypropylidene-1,1-bisphosphonate (190 mg, 0.47 mmol) dissolved in 5 mL of dichloromethane were added triethylamine (78 L, 0.56 mmol) and methanesulfonyl chloride (43 L, 0.56 mmol) at room temperature. The reaction mixture was stirred for 7 hr, and extracted with ethyl acetate (250 mL). The obtained organic phase was washed with water. Then, it was washed with salt water, dehydrated over magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column (solvent: acetone/n-hexane=1/1) to give 2,2-bis(diisopropyloxyphosphoryl)2-fluoroethylmethanesulfonate (209 mg, yield 92%) as a yellow oil.
[0110] .sup.1H NMR (500 MHz, CDCl.sub.3) 1.36-1.38 (m, 24H), 2.53-2.66 (m, 2H), 3.01 (s, 3H), 4.54 (t, J=7.8 Hz, 2H), 4.82-4.91 (m, 4H);
[0111] .sup.13C NMR (125 MHz, CDCl.sub.3) 23.7 (dt, J=2.8, 12.9 Hz), 24.2 (d, J=28.6 Hz), 32.9 (d, J=20.1 Hz), 37.3, 65.3 (q, J=6.9 Hz), 73.2 (t, J=3.7 Hz), 73.5 (t, J=3.7 Hz);
[0112] .sup.19F NMR (470 MHz, CDCl.sub.3) 5-195.0 (tt, J=23.1, 75.1 Hz);
[0113] HRMS (ESI) m/z Calcd for C.sub.16H.sub.35FNaO.sub.9P.sub.2S [M].sup.+ 507.1359, found 507.1353.
(4) tetraisopropyl-1-fluoro-3-(methyl(pentyl)amino)propylidene-1,1-bisphosphonate
[0114] ##STR00013##
[0115] To 2,2-bis(diisopropyloxyphosphoryl)2-fluoroethylmethanesulfonate (150 mg, 0.31 mmol) dissolved in 2.5 mL of DMF was added a solution of potassium carbonate (129 mg, 0.93 mmol) and N-hexylmethylamine (63 mg, 0.62 mmol). The reaction mixture was stirred at 80 C. for 19 hr, and water was added to discontinue the reaction. A compound was extracted from the aqueous phase with ethyl acetate (250 mL), and the obtained organic phase was washed with salt water, dehydrated over magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (solvent: acetone/n-hexane=1/1) to give tetraisopropyl-1-fluoro-3-(methyl(pentyl)amino)propylidene-1,1-bisphosphonate (31 mg, yield 21%) as a colorless oily substance.
[0116] .sup.1H NMR (500 MHz, CDCl.sub.3) 0.90 (t, J=7.4 Hz, 3H), 1.27-1.39 (m, 28H), 1.44-1.52 (m, 2H), 2.23-2.40 (m, 7H), 2.72-2.78 (m, 2H), 4.83-4.94 (m, 4H);
[0117] .sup.13C NMR (125 MHz, CDCl.sub.3) 14.0, 22.6, 23.7 (dt, J=3.0, 18.0 Hz), 24.3, (dt, J=1.4, 32.6 Hz), 27.0, 29.7, 30.3 (d, J=19.1 Hz), 42.0, 50.9 (q, J=6.2 Hz), 57.3, 72.6 (t, J=3.5 Hz), 72.9 (t, J=3.7 Hz);
[0118] .sup.19F NMR (470 MHz, CDCl.sub.3) 5-193.6 (tt, J=23.4, 76.2 Hz);
[0119] HRMS (ESI) m/z Calcd for C.sub.21H.sub.46FNNaO.sub.6P.sub.2 [M].sup.+ 512.2682, found 512.2686.
(5) 1-fluoro-3-(methyl(pentyl)amino)propylidene-1,1-bisphosphonic acid
[0120] ##STR00014##
[0121] Tetraisopropyl-1-fluoro-3-(methyl(pentyl)amino)propylidene-1,1-bisphosphonate (30 mg, 0.06 mmol) was dissolved in 1 mL of 6N hydrochloric acid, and heated under reflux for 7 hr. The reaction mixture was concentrated under reduced pressure to give 1-fluoro-3-(methyl(pentyl)amino)propylidene-1,1-bisphosphonic acid (20 mg, yield 99%) as a viscous oil.
[0122] .sup.1H NMR (500 MHz, D.sub.2O) 0.79 (t, J=7.1 Hz, 3H), 1.21-1.29 (m, 4H), 1.57-1.72 (m, 2H), 2.42-2.53 (m, 2H), 2.79 (s, 3H), 2.99-3.04 (m, 1H), 3.11-3.17 (m, 1H), 3.26-3.33 (m, 1H), 3.46 (m, 1H);
[0123] .sup.13C NMR (125 MHz, D.sub.2O) 12.9, 21.4, 23.1, 27.3 (d, J=19.9 Hz), 27.7, 39.4, 51.5-51.7 (m), 56.3;
[0124] .sup.19F NMR (470 MHz, D.sub.2O) 189.5 (tt, J=21.6, 69.9 Hz);
[0125] HRMS (ESI) m/z Calcd for C.sub.9H.sub.21FNNaO.sub.6P.sub.2 [M].sup. 320.0828, found 320.0843.
Example 3
Synthesis of 4-amino-1-fluoro-butylidene-1,1-bisphosphonic acid (ALEF)
[0126] ##STR00015##
(1) tert-butyl(4,4-bis(diethoxyphosphoryl)butyl)carbamate
[0127] ##STR00016##
[0128] To tetraethyl-4-aminobutylidene-1,1-bisphosphonate (345 mg, 1.0 mmol) dissolved in 10 mL of dichloromethane were added Boc.sub.2O (218 L, 1.0 mmol) and Et.sub.3N (139 L, 1.0 mmol) at room temperature. The reaction mixture was stirred for 20 hr, the solvent was removed under reduced pressure, and the solution was concentrated. The crude product was purified by silica gel column chromatography using acetone as a solvent to give tert-butyl(4,4-bis(diethoxyphosphoryl)butyl)carbamate (267 mg, yield 60%) as a colorless oil.
[0129] .sup.1H NMR (500 MHz, CDCl.sub.3) 1.29 (t, J=7.1 Hz, 12H), 1.38 (s, 9H), 1.71 (pent, J=7.1 Hz, 2H), 1.85-1.96 (m, 2H), 2.24 (tt, J=5.9, 23.9 Hz, 2H), 3.02-3.13 (m, 2H), 4.08-4.16 (m, 8H), 4.76 (br. s, NH);
[0130] .sup.13C NMR (125 MHz, CDCl.sub.3) 16.2 (d, J=2.8 Hz), 16.9 (d, J=2.5 Hz), 22.6 (t, J=5.1 Hz), 28.3, 28.9 (m), 30.1 (t, J=132.6 Hz), 39.7, 62.4 (d, J=6.5 Hz), 62.5 (d, J=6.7 Hz), 78.8, 155.8;
[0131] HRMS (ESI) m/z Calcd for C.sub.17H.sub.37NNaO.sub.8P.sub.2 [M].sup.+ 468.1892, found 468.1868.
(2) tert-butyl(4,4-bis(diethoxyphosphoryl)-4-fluorobutyl)carbamate
[0132] ##STR00017##
[0133] To tert-butyl(4,4-bis(diethoxyphosphoryl)butyl)carbamate (220 mg, 0.49 mmol) dissolved in 12 mL of THF was added dropwise n-BuLi (338 L, 1.6 M hexane solution, 0.54 mmol) at 78 C. under an argon atmosphere. After stirring for 10 min, N-fluorophenylsulfonimide was added to carbanion solution and the mixture was allowed to warm to room temperature over 1 hr. After stirring for 12 hr, the reaction was discontinued by adding 10 mL of ammonium chloride solution. A compound was extracted from the aqueous phase with ethyl acetate (210 mL), and the organic phase was blended, dehydrated over magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure and crudely purified by silica gel column chromatography (solvent: acetone/ethyl acetate=1/1). The crude product containing tert-butyl(4,4-bis(diethoxyphosphoryl)-4-fluorobutyl)carbamate as the main component was directly used in the next synthesis reaction (184 mg, yield <82%).
[0134] .sup.1H NMR (500 MHz, CDCl.sub.3) 1.36 (t, J=7.1 Hz, 12H), 1.43 (s, 9H), 1.82-1.88 (m, 2H), 2.12-2.26 (m, 2H), 3.09-3.20 (m, 2H), 4.20-4.33 (m, 8H), 4.62 (br. s, NH); HRMS (ESI) m/z Calcd for C.sub.17H.sub.36FNNaO.sub.8P.sub.2 [M].sup.+ 486.1798, found 486.1811.
(3) 4-amino-1-fluoro-butylidene-1,1-bisphosphonic acid
[0135] ##STR00018##
[0136] tert-Butyl (4,4-bis(diethoxyphosphoryl)-4-fluorobutyl)carbamate (100 mg, 0.22 mmol) was dissolved in 2 mL of 6N hydrochloric acid, and heated under reflux for 15 hr. The reaction mixture was concentrated under reduced pressure, whereby the solvent was removed. The residue was recrystallized from water/methanol to give 4-amino-1-fluoro-butylidene-1,1-bisphosphonic acid (15 mg, yield 74%) as a white solid.
[0137] .sup.1H NMR (500 MHz, D.sub.2O) 1.93-2.04 (m, 2H), 2.08-2.23 (m, 2H), 2.95-3.05 (m, 2H);
[0138] .sup.13C NMR (125 MHz, D.sub.2O) 21.7 (q, J=6.0 Hz), 29.3 (d, J=19.6 Hz), 39.5;
[0139] .sup.19F NMR (470 MHz, D.sub.2O) 189.5 (tt, J=23.8, 72.5 Hz);
[0140] HRMS (ESI) m/z Calcd for C.sub.9H.sub.19FNO.sub.8P.sub.2 [M].sup. 250.0046, found 250.0069.
Example 4
Synthesis of 1-fluoro-(2-imidazoyl-1-ethylidene)-1,1-bisphosphonic acid (ZOLF)
[0141] ##STR00019##
[0142] Tetrakisisopropyl-1-fluoro-(2-imidazoyl-1-ethylidene)-1,1-bisphosphonate (58 mg, 0.15 mmol) was dissolved in 1 mL of 6N hydrochloric acid, and heated under reflux for 12 hr. The reaction mixture was concentrated under reduced pressure, whereby the solvent was removed. The residue was recrystallized from water/methanol to give 1-fluoro-(2-imidazoyl-1-ethylidene)-1,1-bisphosphonic acid (42 mg, yield 99%) as a white solid.
[0143] .sup.1H NMR (500 MHz, D.sub.2O) 4.72-4.81 (m, 2H), 7.28 (s, 1H), 7.37 (s, 1H), 8.62 (s, 1H);
[0144] .sup.13C NMR (125 MHz, D.sub.2O) 51.2 (br. d, J=18.5 Hz), 118.7, 123.6, 135.9;
[0145] .sup.19F NMR (470 MHz, D.sub.2O) 189.8 (tt, J=25.7, 67.9 Hz, 1F);
[0146] HRMS (ESI) m/z Calcd for C.sub.5H.sub.8FN.sub.2O.sub.6P.sub.2 [M].sup. 272.9842, found 272.9807.
Example 5
Synthesis of tetrakispivaloyloxymethyl-1-fluoro-2-(1H-imidazoyl-1-ethylidene)-1,1-bisphosphonate (ZOLF-POM)
[0147] ##STR00020##
[0148] To potassium hydride (21 mg, 30%, 0.16 mmol) suspended in 2 mL of THF was added imidazole (11 mg, 0.16 mmol) at 0 C. under an argon atmosphere. After stirring at 0 C. for 1 hr, the mixture was stirred at room temperature for 30 min. This was cooled to 0 C., and 1.5 mL of a solution of tetrakispivaloyloxymethylvinylidene-1,1-bisphosphonate (100 mg, 0.16 mmol) in THF was added. This was stirred for 30 min, and 1.5 mL of a solution of 18-crown-6-ether (8.2 mg, 0.03 mmol) in THF was added. The reaction mixture was stirred for 15 min and selectfluor (82 mg, 0.23 mmol) was added. The reaction mixture was stirred for 17 hr, and the reaction was discontinued with 5 mL of aqueous ammonium chloride solution. A compound was extracted from the aqueous phase with ethyl acetate (210 mL), and the organic phase was mixed, dehydrated over magnesium sulfate. After filtration, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: acetone/n-hexane=l/1 to acetone/methanol=10/1). As a result, tetrakispivaloyloxymethyl-1-fluoro-2-(1H-imidazoyl-1-ethylidene)-1,1-bisphosphonate was obtained (30 mg, yield 26%).
[0149] .sup.1H NMR (500 MHz, CDCl.sub.3) 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);
[0150] .sup.13C NMR (125 MHz, CDCl.sub.3) 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;
[0151] .sup.19F NMR (470 MHz, CDCl.sub.3) 191.6 (tt, J=26.0, 71.8 Hz);
[0152] HRMS (ESI) m/z Calcd for C.sub.29H.sub.49FN.sub.2NaO.sub.14P.sub.2 [M].sup.+ 753.2541, found 753.2502.
[0153] Methods using the fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention as a lymphocyte-treating agent are specifically explained in Experimental Examples 1-5. The lymphocyte treatment method using the compound of the present invention is not limited to those specifically explained in the following.
[0154] The peripheral blood derived from each patient and used in the Experimental Examples was obtained from the patients hospitalized in the Nagasaki University Hospital and approved by the Nagasaki University Hospital clinical Research Ethics Committee.
Experimental Example 1 (FIG. 2)
[0155] Heparin blood samples (peripheral blood 10 mL) were collected from adult T cell leukemia patients (1), and diluted with 10 mL of PBS. This was overlaid on 20 mL of Ficoll-Paque and subjected to density gradient centrifugation at 600g for 30 min at room temperature. The layer directly above Ficoll-Paque was recovered, and washed 3 times with PBS to give peripheral blood mononuclear cells. The cells were suspended in 7 mL of Yssel medium, 1 mL therefrom was stained with PE-labeled anti-human CD3 monoclonal antibody and FITC-labeled anti-human V2 monoclonal antibody, and analyzed by flow cytometer. As a result, as shown in the panel on the left side in
Experimental Example 2 (FIG. 3)
[0156] Heparin blood samples (peripheral blood 10 mL) were collected from adult T cell leukemia patients (2), and diluted with 10 mL of PBS. This was overlaid on 20 mL of Ficoll-Paque and subjected to density gradient centrifugation at 600g for 30 min at room temperature. The layer directly above Ficoll-Paque was recovered, and washed 3 times with PBS to give peripheral blood mononuclear cells. The cells were suspended in 7 mL of Yssel medium, 1 mL therefrom was stained with PE-labeled anti-human CD3 monoclonal antibody and FITC-labeled anti-human V2 monoclonal antibody, and analyzed by flow cytometer. As a result, as shown in the panel on the left side in
Experimental Example 3 (FIG. 4)
[0157] Heparin blood samples (peripheral blood 10 mL) were collected from adult T cell leukemia patients (3), and diluted with 10 mL of PBS. This was overlaid on 20 mL of Ficoll-Paque and subjected to density gradient centrifugation at 600g for 30 min at room temperature. The layer directly above Ficoll-Paque was recovered, and washed 3 times with PBS to give peripheral blood mononuclear cells. The cells were suspended in 7 mL of Yssel medium, 1 mL therefrom was stained with PE-labeled anti-human CD3 monoclonal antibody and FITC-labeled anti-human V2 monoclonal antibody, and analyzed by flow cytometer. As a result, as shown in the panel on the left side in
Experimental Example 4 (FIG. 5)
[0158] Heparin blood samples (peripheral blood 10 mL) were collected from lung cancer patients (1), and diluted with 10 ml of PBS. This was overlaid on 20 mL of Ficoll-Paque and subjected to density gradient centrifugation at 600g for 30 min at room temperature. The layer directly above Ficoll-Paque was recovered, and washed 3 times with PBS to give peripheral blood mononuclear cells. The cells were suspended in 7 mL of Yssel medium, 1 mL therefrom was stained with PE-labeled anti-human CD3 monoclonal antibody and FITC-labeled anti-human V2 monoclonal antibody, and analyzed by flow cytometer. As a result, as shown in the panel on the left side in
Experimental Example 5 (FIG. 6)
[0159] Heparin blood samples (peripheral blood 10 mL) were collected from lung cancer patients (2), and diluted with 10 mL of PBS. This was overlaid on 20 mL of Ficoll-Paque and subjected to density gradient centrifugation at 600g for 30 min at room temperature. The layer directly above Ficoll-Paque was recovered, and washed 3 times with PBS to give peripheral blood mononuclear cells. The cells were suspended in 7 mL of Yssel medium, 1 mL therefrom was stained with PE-labeled anti-human CD3 monoclonal antibody and FITC-labeled anti-human V2 monoclonal antibody, and analyzed by flow cytometer. As a result, as shown in the panel on the left side in
[0160] Methods using the fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention as an antitumor immunostimulating agent are specifically explained in Experimental Examples 6-19. An antitumor immunostimulation method using the compound of the present invention is not limited to those specifically explained in the following.
[0161] In the Experimental Examples, the following tumor cell lines were used as the target of the detection of tumor cytotoxicity assay of V2 positive T cells. The number after the name of the cell indicates the source of supply.
[Source of Supply]
(1) Health Science Research Resources Bank
[0162] (2) supplied by Dr. Tatsufumi Nakamura, Nagasaki University
(3) supplied by Dr. Yoichi Nakamura, Nagasaki University monocyte tumor-derived U937 cells (U937)(1) monocyte tumor-derived P31/FUJ cells (P31/FUJ)(1) HCT-4 cells derived from HTLV-1 infected patients (HCT-4)(2) HCT-5 cells derived from HTLV-1 infected patients (HCT-5)(2) lung cancer-derived PC9 cells (PC9) (3) urinary bladder cancer-derived EJ-1 cells (EJ-1)(1)
Experimental Example 6 (FIG. 7)
[0163] Human histiocytic tumor cell line U937 cells were suspended in RPMI1640 medium at a cell concentration of 210.sup.5/200 L, and seeded by 200 L in a 96 well round bottom plate. The plate was centrifuged at 600g for 2 min, and the supernatant was removed. A dilution series of 100 nM, 1 M, 10 M, 100 M, 1000 M was prepared for PAMF, ALEF, ZOLF, and a dilution series of 1 nM, 10 nM, 100 nM, 1 M, 10 M was prepared for ZOLF-POM. The compound solutions of the dilution series were added by 200 L to the cell pellets after removal of the supernatant and incubated at 37 C. for 2 hr. The cells were washed 3 times with RPMI1640 medium, and 50 L of V2 positive T cells derived from normal adult (1) and having a cell concentration of 210.sup.5/50 L was added. Furthermore, PE-labeled anti-human CD107a monoclonal antibody (5 L) was added, and the mixture was incubated at 37 C. for 2 hr. Thereto was added FITC-labeled anti-human V2 monoclonal antibody (2 L), and the mixture was incubated on ice for 20 min. This was washed 3 times with 2% FCS-added PBS, and suspended in 200 L of 2% FCS-added PBS. This was analyzed by flow cytometer, the proportion of CD107a positive fractions in the V2 positive cells was calculated, and the compound concentration dependency was summarized in the graph of
Experimental Example 7 (FIG. 8)
[0164] Human histiocytic tumor cell line U937 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. A dilution series of 0 M, 1.25 M, 2.5 M, 5 M was prepared for ZOLF-POM. The compound solutions of the dilution series were added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (1) were reacted at an effector cell/target cell ratio of 0:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 8 (FIG. 9)
[0165] Human histiocytic tumor cell line U937 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. A dilution series of 0 M, 100 M, 300 M, 1000 M was prepared for ZOLF. The compound solutions of the dilution series were added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (1) were reacted at an effector cell/target cell ratio of 0:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 9 (FIG. 10)
[0166] Human histiocytic tumor cell line U937 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. A dilution series of 0 M, 300 M, 1000 M, 3000 M was prepared for ALEF. The compound solutions of the dilution series were added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (1) were reacted at an effector cell/target cell ratio of 0:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 10 (FIG. 11)
[0167] Human histiocytic tumor cell line U937 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. A dilution series of 0 M, 300 M, 1000 M, 3000 M was prepared for PAMF. The compound solutions of the dilution series were added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (1) were reacted at an effector cell/target cell ratio of 0:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 11 (FIG. 12)
[0168] Human histiocytic tumor cell line U937 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. A dilution series of 0 M, 300 M, 1000 M, 3000 M was prepared for IBAF. The compound solutions of the dilution series were added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (1) were reacted at an effector cell/target cell ratio of 0:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 12 (FIG. 13)
[0169] Human histiocytic tumor cell line U937 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. A dilution series of 0 M, 100 M, 300 M, 1000 M was prepared for ZOLF. The compound solutions of the dilution series were added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (2) were reacted at an effector cell/target cell ratio of 0:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 13 (FIG. 14)
[0170] Human histiocytic tumor cell line U937 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. The medium, ZOLF 500 M solution or ZOLF-POM 5 M solution was added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (3) were reacted at an effector cell/target cell ratio of 0.625:1, 1.25:1, 2.5:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 14 (FIG. 15)
[0171] Human histiocytic tumor cell line U937 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. The medium, ZOLF 500 M solution or ZOLF-POM 5 M solution was added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (4) were reacted at an effector cell/target cell ratio of 0.625:1, 1.25:1, 2.5:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 15 (FIG. 16)
[0172] Human monocyte tumor cell line P31/FUJ cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. The medium, ZOLF 500 M solution or ZOLF-POM 5 M solution was added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (3) were reacted at an effector cell/target cell ratio of 0.625:1, 1.25:1, 2.5:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 16 (FIG. 17)
[0173] Adult T cell leukemia cell line HCT-5 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. The medium, ZOLF 1 mM solution or ZOLF-POM 1 M solution was added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from adult T cell leukemia patients (4) were reacted at an effector cell/target cell ratio of 1.25:1, 2.5:1, 5:1, 10:1, 20:1, 40:1, 80:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 17 (FIG. 18)
[0174] Adult T cell leukemia cell line HCT-4 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. The medium, ZOLF-POM 1 M solution or ZOLF-POM 10 M solution was added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from adult T cell leukemia patients (4) were reacted at an effector cell/target cell ratio of 1.25:1, 2.5:1, 5:1, 10:1, 20:1, 40:1, 80:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 18 (FIG. 19)
[0175] Human lung cancer cell line PC9 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. A dilution series of 0 M, 1.25 M, 2.5 M, 5 M was prepared for ZOLF-POM. The compound solutions of the dilution series were added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from lung cancer patients (1) were reacted at an effector cell/target cell ratio of 0:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
Experimental Example 19 (FIG. 20)
[0176] Human bladder cancer cell line EJ-1 cells were suspended in RPMI1640 medium at a cell concentration of 110.sup.6/mL, and dispensed by 1 mL to a 15 mL conical tube. This was centrifuged at 600g for 5 min, and the supernatant was removed. A dilution series of 0 M, 1.25 M, 2.5 M, 5 M was prepared for ZOLF-POM. The compound solutions of the dilution series were added by 1 mL to the cell pellets after removal of the supernatant and incubated at 37 C. for 1 hr 45 min. Thereto was added 10 mM lanthanoid metal chelating agent by 2.5 L, and the mixture was further incubated for 15 min. These conical tubes were centrifuged at 600g for 5 min, and the cell pellets were washed 3 times with RPMI1640 medium. Then, the cell pellets were suspended in 5 mL of RPMI1640 medium, 2 mL therefrom was placed in a different conical tube, and 6 mL of RPMI1640 medium was further added. The cell suspension was seeded by 100 L in a 96 well round bottom plate. V2 positive T cells derived from normal adult (1) were reacted at an effector cell/target cell ratio of 0:1, 5:1, 10:1, 20:1, 40:1, and incubated for 40 min at 37 C. The plate was centrifuged at 600g for 2 min, 25 L of culture supernatant was taken, and diluted with 200 L of europium-added acetate buffer. The mixture was taken by 200 L, and time-resolved fluorescence was measured. The specific cytotoxicity rate was determined from the value of each sample, and the effector cell/target cell ratio dependency was graphically shown in
INDUSTRIAL APPLICABILITY
[0177] The novel fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention becomes a superior V2 positive T cell activator when it is reacted with the peripheral blood. When it is reacted with tumor cells or virus infected cells, it promotes sensitivity to a cytotoxicity action of V2 positive T cells, and functions as an antitumor or antiviral agent. From these findings, antitumor immunotherapy and antiviral infection treating method using the novel fluorine-containing bisphosphonic acid and/or a fluorine-containing bisphosphonate derivative of the present invention can be established. Specifically, peripheral blood mononuclear cells of cancer patients or virus infection patients are prepared, and cultured ex vivo in the presence of the novel fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention and IL-2 to induce proliferation of V2 positive T cells. The cells are intravenously or topically administered to the patients, whereby immunotherapy of cancer and virus infection, which utilizes V2 positive T cells, becomes possible. In addition, immunotherapy of cancer and virus infection, which utilizes V2 positive T cells, becomes possible by directly administering the novel fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention to cancer patients or virus infection patients. In this case, the novel fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention is incorporated into monocyte cells, and the fluorine-containing bisphosphonic acid directly inhibits farnesyl diphosphate synthase, and the fluorine-containing bisphosphonate derivative undergoes hydrolysis of the ester, is converted to fluorine-containing bisphosphonic acid and inhibits farnesyl diphosphate synthase. Due to the inhibitory action, isopentenyl diphosphate, which is a metabolite located directly upstream of the enzyme, is intracellularly accumulated. Isopentenyl diphosphate binds to an intracellular region of the butyrophilin 3A1 molecule present in the cellular membrane, and changes the conformation of the extracellular region or changes the degree of polymerization. The change is recognized by V2 positive T cells, and proliferation stimulation is produced. The proliferated T cells show high tumor cytotoxicity, and high virus infected cell toxicity. On the other hand, the novel fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention is incorporated into tumor cells and virus infected cells, during which a phenomenon similar to the changes in the monocytes occurs. That is, the fluorine-containing bisphosphonic acid directly inhibits farnesyl diphosphate synthase, and the fluorine-containing bisphosphonate derivative undergoes hydrolysis of the ester, is converted to the form of an acid and inhibits farnesyl diphosphate synthase. Due to the inhibitory action, isopentenyl diphosphate, which is a metabolite located directly upstream of the enzyme, is intracellularly accumulated. Isopentenyl diphosphate binds to an intracellular region of the butyrophilin 3A1 molecule present in the cellular membrane, and changes the conformation of the extracellular region or changes the degree of polymerization. The change is recognized by V2 positive T cells, and tumor cells and virus infected cells are efficiently injured. In this way, antitumor immunotherapy and antiinfection immunotherapy using the novel fluorine-containing bisphosphonic acid or fluorine-containing bisphosphonate derivative of the present invention become possible.
[0178] The compound of the present invention in an oil form is superior in solubility and preferably administered as a medicament.
[0179] This application is based on a patent application No. 2015-018260 filed in Japan (filing date: Feb. 2, 2015), the contents of which are incorporated in full herein.