HYDROXAMIC ACID TYPE CONTRAST AGENT CONTAINING RADIOISOTOPE FLUORIDE, PREPARATION METHOD AND APPLICATION THEREOF

Abstract

The present invention relates to a hydroxamic acid-based contrast agent containing an isotope of fluorine, which comprises a compound having a structure of Formula (III):

##STR00001## wherein R.sub.1 represents radioactive fluorine-18 (.sup.18F) or isotope fluorine-19 (.sup.19F), and R.sub.2 represents hydroxyamine (NH)OH. The hydroxamic acid-based contrast agent containing an isotope of fluorine provided in the present invention has the capability of selectively inhibiting histone deacetylase (HDAC) subtypes 8/6/3, and specifically targets to the focus of spinocerebellar ataxia with over-activation of HDAC. By labeling with the radioisotope fluorine-18, a positron emission tomography (PET) image is obtained with the hydroxamic acid-based contrast agent containing radioisotope fluorine-18, whereby spinocerebellar ataxia is effectively detected. Therefore, the hydroxamic acid-based contrast agent containing an isotope of fluorine provided in the present invention is potentially useful as a probe for early diagnosis and evaluation of the therapeutic effect of spinocerebellar ataxia.

Claims

1-4. (canceled)

5. A method for preparing a hydroxamic acid-based contrast agent containing an isotope of fluorine, comprising: Step I: preparing a precursor having a structure of Formula (I): ##STR00007## wherein R.sub.1 is nitro, and R.sub.2 is methoxy, by dissolving 2-methyl-4-nitrophenol, methyl 4-bromobutyrate, and potassium carbonate in dimethyl formamide, wherein the molar ratio of the 2-methyl-4-nitrophenol, the 4-methyl bromobutyrate and the potassium carbonate is 8.2:11:20, and reacting at 60-90 C. for 12-36 hrs; then adding ethyl acetate, and extracting with a saturated sodium bicarbonate solution, an aqueous hydrochloric acid solution, and a saturated saline; and collecting the organic layer, removing water, concentrating under reduced pressure, filtering, and purifying by column chromatography on silica gel, to obtain the precursor as a yellow solid; Step II: fluorinating the prepared precursor to produce an intermediate product wherein the intermediate product is a compound having a structure of Formula (II): ##STR00008## wherein R.sub.1 is radioactive fluorine-18 (.sup.18F) or isotope fluorine-19 (.sup.19F), and R.sub.2 is methoxy; and Step III: subjecting the intermediate product to a hydroxamic acid forming reaction, to form the final product which has a structure of Formula (III): ##STR00009## wherein R.sub.1 is radioactive fluorine-18 (.sup.18F) or isotope fluorine-19 (.sup.19F), and R.sub.2 is (NH)OH, and wherein the hydroxamic acid forming reaction comprises reacting the intermediate product, hydroxylamine, and sodium hydroxide for 5-20 min at 30-50 C.

6. (canceled)

7. The method for preparing a hydroxamic acid-based contrast agent containing an isotope of fluorine according to claim 5, wherein the fluorination is chemically labeling with the isotope Fluorine-18 or the isotope Fluorine-19.

8. The method for preparing a hydroxamic acid-based contrast agent containing an isotope of fluorine according to claim 5, wherein the fluorination comprises reacting absolute Fluorine-18 with the precursor for 10-30 min at 100-120 C.

9-10. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0018] FIG. 1 shows a reaction scheme for synthesizing a precursor of Formula (I) of a fluorine-18-hydroxamic acid-based compound according to the present invention;

[0019] FIG. 2 shows a reaction scheme for fluorinating a precursor of Formula (I) of a fluorine-18-hydroxamic acid-based compound according to the present invention and then producing a hydroxamic acid with the fluorinated product;

[0020] FIG. 3 shows TLC analysis of a fluorine-18-hydroxamic acid-based intermediate product of Formula (II) according to the present invention;

[0021] FIG. 4 shows HPLC analysis of a fluorine-18-hydroxamic acid-based compound of Formula (III) according to the present invention;

[0022] FIG. 5 shows test results of the inhibitory effect of a fluorine-19-hydroxamic acid-based compound of Formula (III) according to the present invention on histone deacetylases (HDAC);

[0023] FIG. 6 shows distribution by positron emission tomography (PET) imaging after a fluorine-18-hydroxamic acid-based compound of Formula (III) according to the present invention is injected to lateral ventricles of normal and sick mice (spinocerebellar ataxia); and

[0024] FIG. 7 shows MRI images of 6-week and 8-week old normal and sick mice used in the present invention.

DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS

[0025] Preferred embodiments or examples are provided in the present invention for illustrating the technical means adopted for solving the problems, instead of limiting the scope of the claims of the present invention. Equivalent changes and modifications made in accordance with and without departing from the scope of the present invention are covered in the present invention.

[0026] The present invention provides a hydroxamic acid-based contrast agent containing an isotope of fluorine, which comprises a compound having a structure of Formula (III):

##STR00004##

where R.sub.1 represents radioactive fluorine-18 (.sup.18F) or isotope fluorine-19 (.sup.19F), and R.sub.2 represents hydroxyamine (NH)OH.

[0027] When R.sub.1 is radioactive fluorine-18 (.sup.18F), the compound is a fluorine-18-hydroxamic acid-based compound having radioactivity; and when R.sub.1 is the isotope fluorine-19 (.sup.19F), the compound is a fluorine-19-hydroxamic acid-based compound having no radioactivity.

[0028] Based on the foregoing, the fluorine-18-hydroxamic acid-based compound or the fluorine-19-hydroxamic acid-based compound comprised in the hydroxamic acid-based contrast agent containing an isotope of fluorine provided in the present invention is obtained by fluorinating a compound having a structure of Formula (I)

##STR00005##

where R.sub.1 represents nitro, and R.sub.2 represents methoxy.

[0029] and then subjecting the fluorinated product to a hydroxamic acid forming reaction.

[0030] In other words, the compound having a structure of Formula (I) is a precursor of the hydroxamic acid-based contrast agent having an isotope of fluorine.

[0031] Methods for preparing the hydroxamic acid-based contrast agent having an isotope of fluorine and the precursor thereof are further described below. Herein, the hydroxamic acid-based contrast agent having an isotope of fluorine is exemplified with a fluorine-18-hydroxamic acid-based compound.

Example 1. Preparation of the Compound with a Structure of Formula (I)

[0032] FIG. 1 shows a reaction scheme for synthesizing a precursor of Formula (I) of a fluorine-18-hydroxamic acid-based compound according to the present invention. 4-nitro-2-methoxyphenol (1.53 g, 8.2 mmol, 1.0 eq), 4-methyl bromobutyrate (1.98 g, 11.0 mmol, 1.3 eq) and potassium carbonate (2.76 g, 20.0 mmol, 2.4 eq) were dissolved in dimethyl formamide (25 mL) and reacted overnight at 80 C. with stirring. Ethyl acetate (100 mL) was added, and extracted with a saturated sodium bicarbonate solution (100 mL), a 1 N aqueous hydrochloric acid solution (100 mL), and a saturated saline (100 mL). The organic layer was collected, removed of water with magnesium sulfate, concentrated under reduced pressure, filtered, and finally purified by column chromatography on silica gel using ethyl acetate/n-hexane (30:70) as a mobile phase, to obtain a precursor as a yellow solid of a fluorine-18-hydroxamic acid-based compound (2.26 g, yield 96%). .sup.1H NMR (300 MHz, D2O, ): 8.10-8.03 (m, 2H), 6.86-6.83 (d, J=8.7 Hz, 1H), 4.15-4.11 (t, J=6.3 Hz, 2H), 3.70 (s, 3H), 2.59-2.55 (t, J=7.2 Hz, 2H), 2.27 (s, 3H), 2.22-2.17 (m, 2H).

Example 2. Preparation of the Compound with a Structure of Formula (III)

[0033] The compound with a structure of Formula (III) is obtained by fluorinating the compound having a structure of Formula (I) and then subjecting the fluorinated product to a hydroxamic acid forming reaction. FIG. 2 shows a reaction scheme for fluorinating a precursor of Formula (I) of a fluorine-18-hydroxamic acid-based compound according to the present invention and then producing a hydroxamic acid with the fluorinated product. FIG. 2 shows a reaction scheme for fluorinating the precursor of Formula (I) of a fluorine-18-hydroxamic acid-based compound according to the present invention and then producing a hydroxamic acid with the fluorinated product.

[0034] An aqueous solution of fluorine-18 (200 mCi) was passed through QMA ion exchange resin, and then Krytofix2.2.2/potassium carbonate (1 ml, acetonitrile/water:85:15) was further passed through QMA. The Krytofix2.2.2[K.sup.18F] solution was collected in a reaction flask, sealed, and evaporated to dryness at 110 C. with introduction of nitrogen. Then, anhydrous acetonitrile (2.4 ml) was slowly added to the reaction flask in three portions, and azeotropically evaporated to dryness. The prepared precursor of Formula (I) (the compound with a structure of Formula (I)) of the fluorine-18-hydroxamic acid-based compound was dissolved in anhydrous acetonitrile (1 ml), added to the reaction flask and reacted for 20 min at 110 C., to obtain an intermediate product with a structure of Formula (II) below, which is a crude fluorine-18-product:

##STR00006##

[0035] where R.sub.1 represents radioactive fluorine-18 (.sup.18F) or isotope fluorine-19 (.sup.19F), and R.sub.2 represents methoxy.

[0036] The crude fluorine-18-product was cooled to room temperature and analyzed by thin layer chromatography (TLC) using ethyl acetate/hexane=2/4(v/v) as a developing phase, in which Rf is 0.7 (as shown in FIG. 3).

[0037] Then, the crude fluorine-18-product (the compound with a structure of Formula (II)) was evaporated to dryness, and then hydroxyamine (250 L, 4.0 M in MeOH) and sodium hydroxide (750 L, 1.0 M in MeOH) were added, and subjected to a hydroxamic acid forming reaction for 10 min at 40 C., to obtain the final product fluorine-18-hydroxamic acid-based compound.

[0038] The fluorine-18-hydroxamic acid-based compound was neutralized with 1.0 M hydrochloric acid, purified by running through a C18 cartridge, and then analyzed by radio-HPLC, where the flow rate is 2 ml/min, and the mobile phase is 80% water (with 0.1% trifluoroacetic acid)/20% acetonitrile. The experimental result shows that the fluorine-18-hydroxamic acid-based compound appears at about 16.6 min (as shown in FIG. 4).

Example 3. Assay of the Inhibitory Effect on Histone Deacetylases (HDAC)

[0039] To further investigate whether the fluorine-18-hydroxamic acid-based compound is useful as a HDAC inhibitor, the inhibitory effect on HDAC is assayed with the fluorine-19-hydroxamic acid-based compound. The fluorine-18-hydroxamic acid-based compound and the fluorine-19-hydroxamic acid-based compound have the same chemical properties, except that the fluorine-18-hydroxamic acid-based compound has radioactivity, and the fluorine-19-hydroxamic acid-based compound has no radioactivity.

[0040] The fluorine-19-hydroxamic acid-based compound was diluted in dimethyl sulfoxide (DMSO) to give 4 different concentrations. The enzyme activity of various subtypes of HDAC was assayed with a HDAC Fluorometric Assay Kit (available from Enzolifesciences). The fluorine-19-hydroxamic acid-based compound (10 L) and enzyme (15 L) were added to a white opaque 96-well plate, and then a substrate (25 L) was added and incubated for 30 min at room temperature. After incubation, the fluorescence intensity of the 96-well plate was measured on a microplate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm, and the fluorescence intensity measured was analyzed by Graph Pad Prism 6 to calculate IC50 (as shown in FIG. 5).

[0041] It can be seen from FIG. 5 that the fluorine-19-hydroxamic acid-based compound has an IC50 at which the HDAC8, HDAC6, and HDAC3 are selectively inhibited.

Example 4. Non-Invasive Imaging of Animal Model of Spinocerebellar Ataxia

[0042] The model mice (SCA17) of spinocerebellar ataxia were anaesthetized with a gas, and then the fluorine-18-hydroxamic acid-based compound (molecular imaging agent) (2 L, 0.75 MBq, 20 Ci) was injected by means of a stereotaxic apparatus to lateral ventricles of the mice. The distribution of the molecular imaging agent in the mice was scanned by high-resolution positron emission tomography/computed tomography (PET/CT) imaging for 1 hr, to observe the distribution of the agent in brain. The CT and PET images were overlapped and fused, and the ROI was circled by the Pmod software. The result shows that the agent is accumulated in a site in cerebellum of the mice with spinocerebellar ataxia, indicating that the agent has good targeting (as shown in FIG. 6). However, the MRI result shows that there is no significant difference between the cerebellum sizes of normal and sick mice, as shown in FIG. 7, in which TG represents sick mice and WT represents normal mice.

[0043] Based on the above experimental results, the hydroxamic acid-based contrast agent containing an isotope of fluorine-18 provided in the present invention is useful as a diagnostic contrast agent for targeting and imaging spinocerebellar ataxia.