JP2857745B2 - Novel fluorine-containing compound for biological imaging using <19F> -NMR - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel fluorine-containing compound suitable for biological imaging using ¹⁹ F-NMR. More specifically, the present invention provides a compound of the general formula

[0002]

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[0003] (wherein R1 has the formula with one or two substituted amino group, an isothiocyanato group, or the terminal carboxyl group of the linear or branched alkyl group having a carbon number of from 1 8 - NHCO (CH ₂ ) nCOOH (n =
1-8) or _{-NH (CH 2) nCOOH (n} = 1-
A group represented by 8), R2 is a hydroxyl group, it was or is
A molecule represented by a straight-chain or branched-chain alkoxy group having 1 to 8 carbon atoms) has two rotation axes (C
₂ ) or a benzene derivative containing a mirror (m) symmetry element is used as a searcher for biological imaging using ¹⁹ F-NMR. In particular, for compounds having an isothiocyanato group, conjugates can be easily prepared with various proteins, so that the kinetics of the protein can be examined in vivo, or conjugated with a cancer-specific antibody, and then administered intravenously. perform imaging of cancer by examining the accumulation to at ^{19 F-MRI} is applicable for such.

[0004]

2. Description of the Related Art Conventionally, living body imaging using ¹⁹ F-NMR ( ¹⁹ F-MRI) has been carried out using an emulsion of a perfluoro compound developed as an oxygen-transporting infusion. For example, AV Ratner et al., A mouse or the emulsion of perfluorooctylbromide hamsters was administered intravenously, the liver and pancreas is an organ of the reticuloendothelial system ¹⁹ F-
Successful MRI (Magnetic Resonance in Medici
ne 5, 548-554, 1987). As an oxygen-carrying infusion, the best studied perfluorodecal called Fluosol-DA
Liver imaging using an emulsion of a 7: 3 mixture of in and perfluorotri-n-propylamine has been reported by E. McFarland et al. (Journal of Computer Assisted Tomog).
raphy, 9 (1), 8-15, 1985). HA Sloviter et al. Also developed perfluorotri-n-butylamin, which was developed for organ perfusion.
the heart of a mouse whose blood has been excreted and exchanged with 50% of the blood in the emulsion of e.
Successful imaging of liver, pancreas, lung, kidney, and major blood vessels (Journal of Computer Assisted Tom
ography, 9 (6), 1012-1019, 1985).

In addition, perfluorocarbon (perfluorocarbon)
tri-n-butylamine, perfluoro-2-n-butyltetrahydrofur
an, perflurooctylbromide or perfluorophenanthren
An attempt to perform lung imaging by inhaling the undiluted solution itself in e) has also been reported by SR Thomas et al. (Jou
rnal of Computer Assisted Tomography, 10 (1), 1-9,
1986). In addition to organ imaging, there are also reports on application examples of cancer imaging using the property that emulsions are phagocytosed by macrophages (RP Mason et al.
esonance Imaging, Vol 7, 475-485, 1989).

[0006] There are many related reports other than the examples given above, all of which use perfluorocarbons developed for oxygen-transporting infusions, so that the fluorine NM
The R signal was very complex and was not always suitable for biological imaging. From such a background, the appearance of a fluorine-containing compound suitable for biological imaging has been demanded.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a high S / N ratio which is difficult to achieve by conventional ¹⁹ F-MRI using an oxygen-transporting infusion. The purpose is to design and synthesize new fluorine-containing compounds suitable for imaging. Another object of the present invention is to
The novel fluorine-containing compound is to be administered to a living body after being bound to a living body-related molecule such as a protein, and used to examine the dynamics in the living body.

[0008]

In order to achieve the present invention, a compound is designed such that the signal of ¹⁹ F-NMR is singlet, strong, and there is no other signal near the signal. Must be synthesized. The present inventors have proposed NM as a compound satisfying such conditions.
The design of a compound having a large number of trifluoromethyl groups that cannot be distinguished by R was considered. For this purpose, there is no other fluorine atom at a position that can be coupled with the trifluoromethyl group, and a trifluoromethyl group equivalent to NMR is used.
Attention was paid to hexafluoroacetone having one. This substance is produced industrially, and it is considered that it is important to obtain it stably and inexpensively. Considering the economics of the molecule, elements other than fluorine are reduced as much as possible, and a fluorine-containing compound having only one type of fluorine signal and having C ₂ symmetry or mirror symmetry in the molecule is the most suitable. As a result, the present inventors have conducted intensive studies to find a compound that satisfies such conditions. As a result, a novel fluorine-containing compound has been found, and the present invention has been completed based on this finding.

That is, the present invention provides a compound represented by the general formula

[0010]

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[0011] (wherein R1 has the formula with one or two substituted amino group, an isothiocyanato group, or the terminal carboxyl group of the linear or branched alkyl group having a carbon number of from 1 8 - NHCO (CH ₂ ) nCOOH (n =
1-8) or _{-NH (CH 2) nCOOH (n} = 1-
8) wherein R2 is a hydroxyl group, or
A molecule represented by a straight-chain or branched-chain alkoxy group having 1 to 8 carbon atoms) has two rotation axes (C
₂ ) or a benzene derivative containing a mirror (m) symmetry element. Further, the present invention provides an emulsion or a liposome containing the above fluorine-containing compound as an active ingredient.
Used as a contrast agent for ¹⁹ F-NMR, or
The present invention relates to searching for the in vivo kinetics of a modified protein obtained by reacting a protein alone as a protein modifying agent with a protein by ¹⁹ F-NMR.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically. The compound represented by the above general formula of the present invention can be prepared by a known reaction (Fride I-Crafts reaction) using hexafluoroacetone and benzene, which are generally commercially available, in 1,3-Bis (hexafluoro-2).
-Hydroxy-2-propenyl) benzene
Is used as a starting material and is synthesized using a method represented by the following chemical formula.

[0013]

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That is, the starting material 1,3-Bis (hexafluor
O-2-hydroxy-2-propyl) benzene was nitrated with nitric acid in the presence of concentrated sulfuric acid according to the method described in Example 1 to be described later to give compound 1,3,5-Bis (hexafluoro-2- hydroxy-
2-propyl) nitrobenzene is obtained. The hydroxyl group of compound 1 can be converted to compound 2 by methylation by a general method (sodium hydride and dimethyl sulfate). That is, the compound 1 can be converted into sodium phenoxide in an appropriate solvent selected from dichloromethane, hexane, chloroform, benzene, and ether, and then dimethyl sulfate is added at room temperature or reflux temperature. further,
Other alkylation, for example, ethylation, can be performed in the same manner as described above using diethyl sulfate, but for the introduction of an alkyl group having a larger number of carbon atoms, a Williamson synthesis method using the corresponding alkyl halide is applied. It can be done by doing.

Compounds 1 and 2 can be prepared by catalytic reduction using palladium-carbon or reduction using iron chloride-iron.
And 4, but reduction with iron-hydrochloric acid can be performed in better yield. Further, the reduction of compound 2 needs to be performed by blowing hydrogen chloride gas into methanol, and the reaction hardly proceeds with hydrochloric acid. Next, derivatization of compounds 3 and 4 to isothiocyanate can be carried out using thiophosgene without a solvent or in the presence of a suitable solvent. As the solvent, dichloromethane, chloroform, dimethylformamide, benzene, toluene, or the like can be used. In this reaction, since hydrogen chloride is generated as a by-product, it is desirable to carry out the reaction in the presence of a base as an acid trapping agent. Compounds 7 and 8 can be more easily derived than compound 3. That is, compound 7 can be synthesized by reacting compound 3 with monoiodoacetic acid, and compound 8 can be synthesized by reacting compound 3 with an acid anhydride of succinic acid. The reaction solvent is not critical in any reaction, such as chloroform,
Common organic solvents such as dichloromethane, ether, tetrahydrofuran and dimethylformamide can be used. Each of the above compounds 1-8 gave a ¹⁹ F-NMR spectrum consisting of a fluorine signal of only one singlet derived from a trifluoromethyl group.

Compounds 1 and 2 are fat-soluble to the extent that they are convenient to use as emulsions or liposomes, but the degree can be controlled by the alkyl chain length of R2. Isothiocyanato derivatives 5 and 6
Can be easily reacted with a protein such as an enzyme or antibody to form a conjugate, and thus is a good searcher for investigating the in vivo kinetics of a target protein. Compounds 7 and 8 can also be prepared by a conventional method, for example, a method of biochemical experiment C protein research 1. A conjugate with a protein such as an enzyme or an antibody can be easily prepared by a method described in Kazuo Shibata, a chemical research method of higher-order structure, published by The University of Tokyo, pp. 98-103, 1974. When the antibody used here is a cancer-specific antibody, cancer imaging by ¹⁹ F-NMR becomes possible. In addition, by labeling the cells with the modified antibody, it becomes possible to follow the pharmacokinetics of immune T cells and a subset thereof, for example. Such ¹⁹ F-
The probe using NMR depends on the type of protein used.
It is easy to imagine that it can be applied to various applications both biochemically and medically.

[0017]

Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Example 1 3,5-Bis (hexafluoro-2-hydroxy-2-propyl) nitrobenzene (1) Bis (hexafluoro-2-hydroxy-2-propyl) benzene (4.1 g,
Concentrated nitric acid (4 ml) was added dropwise to a mixture of 20 mmol) and concentrated sulfuric acid (10 ml) over 30 minutes with good stirring. After the addition, the reaction solution was stirred at room temperature for 4 hours, poured into 100 g of ice, neutralized with a saturated aqueous solution of sodium bicarbonate, and extracted with dichloromethane (100 ml × 3). The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated with an evaporator. The obtained solid was further dried under reduced pressure using a pump to obtain pale yellow crystals (4.33 g, 95%). mp: 85-86 ° C, ¹ H-NMR (CDCl ₃ ) δ: 8.78 (2H, bs), 8.4
5 (1H, s), 4.13 (2H, bs), ¹⁹ F-NMR (CDCl ₃ ) δ: -76.08
(s), ¹³ C-NMR (CDCl ₃ ) δ: 148.86 (s), 132.58 (s), 131.
23 (d), 124.41 (d), 122.28 (q, J _CF = 288Hz), 76.9
6 (septet, J _CF = 30.8 Hz), GC-MS (m / z): 455 (M +,
5.0), 387 (10.0), 386 (M-CF ₃ ⁺ , 80.4), 317 (M-2CF ₃
⁺ , 11.7), 316 (M-CF ₃ ⁺ , 100), 270 (13.1), 242 (1
0.7), 145 (C ₆ H ₅ CF ₃ ⁺ , 11.0), 123 (10.9), 88.6 (14.
4), 76 (10.6), 75 (34.3), 69 (CF ₃ , 71.8), 51 (CF ₂ H,
10.3), 46 (NO ₂ ⁺ , 23.4) IR (KBr) ν _max cm ^-1 : 346
0 (brs, ν _OH ), 1545, 1356 (s, ν _NO2 )

Example 2 3,5-Bis (hexafluoro-2-methoxy-2-propyl) nitrobenzene (2) Suspend sodium hydride (2.4 g 60% in oil, 60 mmol) in dried THF (40 ml) The compound 1 (9.1 g, 20 mmol) in THF (40
ml) solution was added dropwise over 25 minutes. After completion of the dropwise addition, the mixture was further stirred at room temperature for 16 hours. Dimethyl sulfate (8.46 g, 64
mmol), and the mixture was stirred at room temperature for 6 hours, and then refluxed for 1 hour to complete the reaction. The obtained reaction solution was poured into 800 ml of water and extracted with ethyl acetate (300 ml × 3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure using an evaporator to obtain 13.9 g of a colored liquid. Purification by flash column chromatography (AcOEt: Hex = 2: 8) using silica gel (230-400 mesh, 200 g) gave quantitatively a colorless transparent liquid of Compound 2.
(9.7 g). ¹ H-NMR (CDCl ₃ ) δ: 8.62 (2H, bs), 8.19 (1H, bs), 3.
59 (6H, septet, J = 1.1Hz), ¹⁹ F-NMR (CDCl ₃ ) δ: -71.2
2 (s), ¹³ C-NMR (CDCl ₃ ) δ: 149.07 (s), 133.72 (d), 1
31.71 (s), 125.39 (d), 121.90 (q, J _CF = 289Hz), 8
2.31 (septet, J _CF = 28.9 Hz), 54.97 (q), GC-MS (m /
z): 483 (M ⁺ , 1.3), 464 (MF ⁺ , 6.9), 415 (14.3), 41
4 (M-CF ₃ , 100), 364 (7.7), 330 (23.1), 299 (6.9),
253 (5.4), 187 (5.9), 181 (5.7), 131 (5.1), 97 (5.
1), 81 (11.9), 75 (8.4), 69 (35.1), 46 (NO ₂ ⁺ , 5.2)

Example 3 3,5-Bis (hexafluoro-2-hydroxy-2-propyl) aniline (3) Compound 1 (4.55 g, 10 mmol) was added to a mixture of concentrated hydrochloric acid (8 ml) and methanol (5 ml). ) Was added, and iron powder (2.7 g, 48.3 mmol) was added over 1 hour while stirring well at room temperature. The reaction solution obtained by further stirring for 3 hours was poured into 150 ml of methanol, and the resulting precipitate was filtered using celite. The filtrate was concentrated with an evaporator, and the obtained residue was neutralized with a saturated aqueous sodium bicarbonate solution, and then adjusted to 100 ml with a saturated saline solution. The mixture was extracted with dichloromethane (200 ml × 2), and the organic layer was filtered again using celite. The filtrate was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed with an evaporator. The resulting powder was dried under reduced pressure using a pump to obtain white powdery compound 3 (3.71 g, 87%). mp: 143-143.5 ° C, ¹ H-NMR (CD ₃ CN) δ: 7.29 (1H, bs),
7.12 (2H, bs), 5.91 (2H, bs), 4.57 (2H, bs), ¹⁹ FN
MR (CD ₃ CN) δ: -74.47 (s), ¹³ C-NMR (CDCl ₃ ) δ: 149.79
(s), 132.80 (s), 123.94 (q, J _CF = 287.7Hz, CF ₃ ),
115.08 (d), 114.29 (d), 78.11 (septet, J _CF = 29.
72), DI-MS (m / z): 426 (14.3) 425 (M ⁺ , 100), 388 (1
9.3), 356 (16.2), 338 (43.2), 286 (21.3), 259 (74.
0), 241 (26.4), 190 (16.4), 120 (14.0), 93 (26.5),
92 (10.0), 74 (17.0), 69 (31.3), ¹⁹ F-NMR (CD ₃ CN)
δ: -74.47 (s), IR (KBr) ν _max cm ^-1 : 3440 (brs,
ν _OH ), 3422, 3344 (w, ν _NH2 )

Example 4 3,5-Bis (hexafluoro-2-methoxy-2-propyl) aniline (4) In a mixed solution of concentrated hydrochloric acid (0.8 ml) and methanol (11 ml),
Compound 3 (483 mg, 1 mmol) was added, and iron powder (270 m
g, 4.8 mmol). While blowing hydrochloric acid gas,
Stir well at room temperature for 40 minutes. The reaction solution was poured into about 50 ml of a saturated aqueous solution of sodium hydrogen carbonate, and extracted with dichloromethane (100 ml × 2). The mixture was filtered using Celite and dried over anhydrous magnesium sulfate. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure using an evaporator, and dried under reduced pressure using a vacuum pump to obtain colorless crystals (90.5%). mp: 109.5-110 ° C, ¹ H-NMR (CDCl ₃ ) δ: 7.17 (1H, bs),
6.96 (2H, bs), 4.00 (2H, bs), 3.48 (6H, septet, J =
1.1 Hz), ¹⁹ F-NMR (CD ₃ CN) δ: -71.37 (s), ¹³ C-NMR (CD
Cl ₃ ) δ: 147.21 (s), 129.72 (s), 122.28 (q, J _CF = 2
90.6Hz), 117.94 (d), 116.12 (d), 82.87 (septet, J
_CF = 28.3Hz), 54.34 (q), DI-MS (m / z): 454 (19.4),
453 (M ⁺ , 100), 423 (11.3), 393 (14.4), 392 (36.
0), 391 (91.8), 384 (39.8), 369 (36.4), 339 (24.
2), 322 (45.0), 300 (47.2), 257 (16.3), 253 (11.
0), 242 (11.5), 241 (25.4), 216 (17.6), 203 (14.
9), 188 (10.9), 181 (10.2), 172 (15.6), 134 (12.
3), 69 (46.4), 57 (10.5), 55 (11.0), IR (KBr) ν
_max cm ^-1 : 3503, 3420 (s, ν _NH2 )

Example 5 3,5-Bis (hexafluoro-2-hydroxy-2-propyl) isothiocyanatobenzene (5) Compound 3 (147 mg, 0.346 mmol) and trimethylamine (6
To a solution of 9.9 mg (0.692 mmol) in benzene (4 ml) was added thiophosgene (39.8 mg, 0.346 mmol) at room temperature under stirring. After stirring at room temperature for 1 hour, the formed salt was removed by filtration. The filtrate was concentrated by an evaporator and purified by flash column chromatography using silica gel.
A colorless compound 5 was obtained (120 mg, 74.5%). mp: 37.5-38.5 ℃, ¹ H-NMR (CDCl ₃ ) δ: 7.99 (s, 1H),
7.72 (s, 2H), 4.20 (s, 2H), ¹⁹ F-NMR (CDCl ₃ ) δ: -76.
06 (s), ¹³ C-NMR (CDCl ₃ ) δ: 138.53 (s), 133.63 (s),
131.19 (s), 127.34 (d), 126.54 (d), 122.04 (q, J
_CF = 290.9Hz), 76.80 (septet, J _CF = 30.5Hz), DI-
MS (m / z): 467 (M +, 82.4), 398 (M-CF 3 +, 26.6), 380
(30.7), 328 (M-CF ₃ -CF ₃ H ⁺ , 44.0), 301 (18.0), 232
(13.9), 162 (16.1), 135 (11.6), 132 (19.5), 100 (1
5.2), 97 (22.7), 94 (33.0), 72 (34.5), 69 (100), 55
(12.4), IR (KBr) ν _max cm ^-1 : 2090 (brs, ν _{N = C = S} )

Example 6 3,5-Bis (hexafluoro-2-methoxy-2-propyl) isothiocyanatobenzene (6) Compound 4 (5.0 g, 11 mmol) and trimethylamine (2.23
g, 22 mmol) in benzene (140 ml) was added thiophosgene (1.27 g, 11 mmol) at room temperature with stirring. After stirring at room temperature for 1.5 hours, the formed salt was removed by filtration.
The filtrate was concentrated with an evaporator and purified by flash column chromatography using silica gel to obtain colorless compound 6 (5.3 g, 97%). Colorless crystal mp: 56.5-56.8 ° C, ¹ H-NMR (CDCl ₃ ): 7.72
(s, 1H), 7.54 (s, 2H), 3.52 (s, 6H), ¹⁹ F-NMR (CDCl
₃ ) δ: -71.30 (s), ¹³ C-NMR (CDCl ₃ ) δ: 138.77 (s), 133.
56 (s), 131.12 (s), 127.27 (d), 126.46 (d), 121.97
(q, J _CF = 290.9Hz), 82.42 (septet, J _CF = 28.8H
z), 54.69 (q), DI-MS (m / z): 496 (M + H ⁺ , 16.3), 495
^{(M +, 89.9), 476} (MF +, 18.2), 426 (M-CF 3 +, 10
0), 364 (14.4), 342 (M-CF ₃ -CF ₃ CH ₃ ⁺ , 52.5), 258 (1
3.2), 181 (13.4), 147 (11.1), 70 (12.9), 69 (49.6),
57 (13.8), 55 (19.3), IR (KBr) ν _max cm ^-1 : 2071
(brs, ν _{N = C = S} )

Example 7 (3,5-bis (hexafluoro-2-hydroxy-2-propyl) phenylamino) acetic acid (7) Compound 3 (425 mg, 1 mmol), monoiodoacetic acid (186 m
g, 1 mmol) and ethyldiisopropyl (130 mg, 1 mmol)
Was refluxed for 8 hours.
NHCl (15 ml) was added and extracted with dichloromethane (50 ml) followed by ether (50 ml). The organic layers were combined, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated by an evaporator and purified by flash column chromatography using silica gel (230-400 mesh) to obtain a white powdery solid (170 mg) (35%). ¹ H-NMR (CDCl ₃ ) δ: 7.43 (s, 1H), 7.06 (s, 2H), 4.37
(bs, 3H), 4.03 (s, 2H), ¹⁹ F-NMR (CDCl ₃ ) δ: -76.08
(s), DI-MS (m / z): 483 (M ⁺ , 13.4), 439 (M-CO ₂ , 1
6.2), 438 (M-CO ₂ H, 100), 437 (11.8), 418 (10.2), 3
69 (24.3), 368 (24.8), 271 (11.3), 69 (22.7)

Example 8 3- (3,5-bis (hexafluoro-2-hydroxy-2-propyl) phenylamidoyl) propionic acid (8) Compound 3 (140 mg, 0.33 mmol) and succinic anhydride (33 mg,
(0.33 mmol) in methanol (5 ml) was stirred at room temperature for 1 hour, the solvent was distilled off with an evaporator, and the residue was purified by flash column chromatography using silica gel (230-400 mesh) to give a white powder. Was quantitatively obtained (111 mg). ¹ H-NMR (CD ₃ CN) δ: 8.80 (bs, 1H), 8.15 (s, 2H), 7.7
5 (s, 1H), 3.61 (s, 2H), 2.58 (bs, 2H), 2.52 (s, 2
H), ¹⁹ F-NMR (CD ₃ CN) δ: -74.47 (s), DI-MS (m / z): 508
(M-OH ⁺ , 15.9), 438 (18.6), 425 (8.1), 418 (10.
2), 369 (9.1), 69 (26.2), 56 (27.8), 55 (100)

[0025]

EFFECTS OF THE INVENTION The compounds represented by the above general formula according to the present invention all provide a single sharp signal with a high intensity, so that biological imaging by ¹⁹ F-NMR can be performed with extremely high sensitivity. I can do it. In addition, among the above-mentioned compounds, compounds having an isothiocyanato group which easily reacts with proteins and the like can be used for examining the distribution and dynamics of the body by binding to various proteins and cells. It is an important analytical tool in the fields of chemistry and medicine.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takashi Abe 7-6-4 Oshizawadai, Kasugai-shi, Aichi (56) References US Patent 3,879,430 (US, A) CHEMICAL ABSTRACT S (1975) 82: 58626t (58) Field surveyed (Int. Cl. ⁶ , DB name) C07C 229/18 C07C 233/25 C07C 331/28 C07C 205/32 C07C 205/19 C07C 215/68 C07C 217/76 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] 1. A compound of the general formula (Wherein R1 is one or two substituted amino group of a straight-chain or branched alkyl group having a carbon number of from 1 to 8, isothiocyanato group, the formula -NHCO (CH having a terminal carboxyl group was or ₂₎ nCOOH the (n = 1-8) or _{-NH (CH 2) nCOOH (n} = 1-8) a group represented by, R2 is a hydroxyl group or a linear or branched from 1 carbon atoms 8 A benzene derivative containing a symmetrical element with a _double rotation axis (C ₂ ) or a mirror surface (m) in the molecule represented by the following formula: 2. The benzene derivative according to claim 1, wherein R1 is an isothiocyanato group. 3. The benzene derivative according to claim 1, wherein R2 is a hydroxyl group.