TWI580434B - Imaging compounds for tracking histone deacetylase inhibitor and synthesis method thereof - Google Patents

Description

Contrast compound for tracking tissue protein deacetylase inhibitor and synthesis method thereof

The invention relates to a nuclear medical tracer synthesized by using a tissue protein deacetylase inhibitor and a synthetic method thereof, in particular to a combination with a tissue protein deacetylase in vivo for diagnosing a malignant tumor disease and A nuclear medicine imaging tracer for the response to zerheimer's disease and a method for its synthesis.

In the past, for many organs such as colon, rectum, cervix, stomach, prostate cancer and developmental disorders and other related diseases, the efficacy has not yet broken through. In addition to the above studies, which have not yet yielded satisfactory results, recent studies have also pointed out that brain atrophy occurs during natural aging, and it is an early feature of neurodegenerative diseases associated with impaired cognitive function and memory loss. Dementia-related diseases such as Alzheimer's disease cause significant loss of cognitive function, often causing the patient to deteriorate to a state where they cannot recover. It is known that there are no methods for treating Alzheimer's disease and related dementia, and the causes of these diseases are not well understood. In addition, preclinical studies have not found a strategy for early diagnosis of Alzheimer's disease.

Tissue protein deacetylase inhibitor (HDAC inhibitor, abbreviated as HDACi or HDAC inhibitor), is a drug class that inhibits the function of the tissue protein in the body to deacetylase. Currently, the medical community has studied through tissue protein deacetylase. Inhibitors to treat cancer and neurodegeneration Disease (neurodegenerative diseases). The specific mechanism of these inhibitors, the literature suggests possible characterization of genetic pathways, such as Richon et al. found that HDAC inhibitors can regulate p53 tumor suppressor function by directing p21 (WAF1).

Whether tumor diseases and Alzheimer's disease can be detected, diagnosed, and treated in a timely manner depends on the diagnostic method used. Recent advances in molecular biology have opened up several new avenues for the discovery, diagnosis, and treatment of malignant tumors. Although these new approaches are still in the process of research, the prospects are very promising. Many organs such as colon, rectum, cervix, stomach, prostate, etc., malignant tumors and developmental disorders of the organs have been found to have excessive HDAC-2 performance. In addition to the results of the above-mentioned organs, recent studies have also pointed out that chromatin modification in brain cells is related to the formation of memory. The research team pointed out that HDAC enzyme plays a key role, and mice with abnormal secretion of this enzyme will have memory loss. Similarly, a similar situation was found in the brain of patients with Alzheimer's disease. Therefore, the injection of tissue protein deacetylase inhibitor drugs (HDAC inhibitors) will be a new hope for the future treatment of malignant tumor diseases and Alzheimer's disease.

As a new generation of targeted anti-tumor drugs, Histone Deacetylase (HDAC) inhibitors have become a hot spot in drug research. The existing HDAC inhibitors can be mainly divided into: 1 hydroxamic acid: including Vorinostat; 2 ring tetrapeptides: including Romidepsin (FK228) and depsipeptide; 3 benzamide: including MS-275 And SC-027 and other related clinical trials in Alzheimer's disease; 4 short-chain fatty acids: including valproic acid, butyric acid and so on. Therapeutic effects of HDAC inhibitors on hematological tumors and solid tumors have been demonstrated in both in vivo and in vitro assays. In vitro tests confirmed HDAC inhibitors in a variety of tumor cells, including bladder, bone, breast, uterus, central nervous system, esophagus, lung, ovary, pancreas, prostate and other performance A good anti-tumor effect, which can cause obvious apoptosis, proliferation inhibition, and cell cycle arrest in these tumor cells. A variety of HDAC inhibitors have entered Phase I or Phase II or Phase III clinical studies of anti-tumor therapy due to their multi-channel efficient anti-cancer therapy.

At present, Vorinostat (suberoylanilide hydroxamic acid, SAHA) and Romidepsin (cyclic peptides) have been approved by the US FDA for the application of cutaneous T-cell lymphoma (CTCL) as an indication for solid tumors. Therapeutic applications are also in clinical trials. The benzotriamide HDAC inhibitor citabin has been approved by the microbial organisms in the mainland for SFDA approval. For the non-Hodgkin's lymphoma in clinical phase II/III studies, the FDA approved clinical research in the United States in 2010. The novel HDAC inhibitor can induce tumor cell differentiation and selective apoptosis at low dose and low concentration, and is non-toxic to normal cells, and its anti-tumor proliferation. Therefore, the clinical potential of HDAC inhibitors is considerable. These new assays can be used to detect subtle changes in cells and to detect malignant tumors earlier. Such new technologies also contribute to the development of treatment regimens that are tailored to individual differences in patients and move toward the scientific goals of personalized therapy.

The diagnosis of tumor is very important for whether or not the tumor, the nature of the tumor (benign malignant), the stage of the malignant tumor, and the presence or absence of metastasis. Tumors are often found late. At this point it has damaged one or more functions of vital vital organs and has even transferred to the body. Therefore, the key issue in the treatment of tumors is how to detect such tumors early, but early detection of malignant tumors is still very difficult.

Diagnosis of Alzheimer's disease (AD), basic examination of neuropsychological tests, blood routine, biochemical tests (liver and kidney function), vitamin B12 Concentration, thyroid function, syphilis serum test and brain computerized tomography or magnetic resonance imaging. High-order PET positron imaging diagnostic methods include: F-18-AV45 and F-18-PIB based on the theory of Amyloid hypothesis; based on the theory of microtubule-associated protein hypothesis (Tau hypothesis) The drug is not yet available.

Clinical imaging diagnosis includes X-ray examination, ultrasound examination, magnetic resonance imaging, X-ray tomography (CT) and radioisotope examination. The early diagnosis of tumors and Alzheimer's disease is of great significance, because only early diagnosis can obtain treatment, in order to obtain better therapeutic effects. However, due to various subjective and objective reasons, most patients have advanced tumors at the time of treatment or diagnosis, and the treatment effect is not satisfactory. Although the diagnosis of tumors is rapidly developing, many tumor examination methods are not practical enough. When the diameter is 1~1.5cm, the image map can be clearly displayed. General blood tests are not accurate enough. For example, prostate specific antigen (APA) is a glycoprotein. This antigen can only be produced by prostate cells. When there is a problem with the prostate (such as prostate cancer), the prostate cells will hyperplasia, producing excessive PSA, which increases the concentration of PSA in the blood. The doctor can analyze the patient's likelihood of developing prostate cancer based on the blood PSA concentration. However, there are several factors that lead to an increase in PSA, such as prostate infection, benign hyperplasia of the prostate. In addition, not all patients with prostate cancer have elevated PSA, resulting in a PSA test that cannot be diagnosed. The diagnosis of Alzheimer's disease, taking the latest F-18-AV45 and F-18-PIB PET drugs as an example, the image can only confirm the diagnosis of Alzheimer's disease, but can not confirm whether there is A Zhaimer's disease can also produce the same image response due to aging.

The invention is a continuation of the traditional nuclear medicine tracer angiography concept, combining a HDAC inhibitor SC-027 (C15H15N3O2) with a labeled radioactive nucleus, such as: F-18, I-123, I-125, I-131 , C-11, Ga-67, Ga-68, produce new nuclear medical tracers, such as F-18-SC-027, I-123-SC-027, I-124-SC-027, I-125- SC-027, I-131-SC-027, C-11-SC-027, Ga-67-DOTA-SC-027, Ga-68-DOTA-SC-027, used to bind tissue proteins that are overexpressed in vivo Acetylase, a novel group of compounds that produce a focused HDAC nuclear image.

The present invention aims to provide a novel diagnostic compound for malignant tumor diseases and Alzheimer's disease, using the positron decay characteristics of carbon-11, fluorine-18 or gallium-68 isotopes, and the positrons released by their decay, encountering cells The electrons produce an "annihilation reaction", forming a pair of 511 keV Gamma rays in opposite directions, obtaining images by positron emission tomography (PET); or using iodine-123, iodine-125, iodine-131 or Gama ray decay characteristics of isotopes such as gallium-67, the decay process is 100% electron capture, and the addition of horseradiation is performed by single photon emission tomography (SPECT).

According to an experimental data (such as an attachment) of the present invention, a Histone Deacetylase (HDAC) inhibitor (Inhibitor, SC-027, ie 钪-027) (C15H15N3O2) was traced at a dose of 0.1 cc of 300 μ Ci. Injection into the tail vein of the experimental mouse provides a novel nuclear medical tracer for the diagnosis of malignant tumor diseases and Alzheimer's disease.

Another object of the present invention is to provide a method for binding an HDAC inhibitor SC-027 to a marker radioactive nucleus Ga-67 or Ga-68, wherein the HDAC inhibitor SC-027 must be combined with the marker radionuclide Ga-67 or Ga-68. The marker precursor DOTA-SC-027 was synthesized first.

Another object of the present invention is to provide a marker precursor DOTA-SC-027, namely hydroxysuccinimide (DOTA-NHS-ester; N-hydroxysuccinimide, NHS) and a tissue protein deacetylase inhibitor (HDACi- Compound of SC027) for producing tissue protein deacetylation inhibitor Contrast compound. The synthesis method of the marker precursor DOTA-SC-027 comprises the following steps in sequence: Step A: taking DOTA-NHS-ester (N-hydroxysuccinimide, NHS, hydroxysuccinimide) and SC-027 respectively into two In the glass bottle; Step B: Drop the dimethylformamide (DMF) into the glass bottle of DOTA and SC-027 respectively. When dripping, the glass bottle should be rotated and dripped to completely dissolve it. Step C: Fix the round bottom bottle to the bottle. On the blender. After adding triethylamine (TEA) to the DOTA glass via a micropipette, transfer the solution in the DOTA bottle to the round bottom bottle with a micropipette; Step D: Adjust the stirrer speed to 500 rpm, and use a microdrop after 1 hour. The tube was placed in a round bottom bottle and the reaction was continued for 24 hours; Step E: 24 hours after the reaction, a freeze-drying procedure was performed; and Step F: a labeled precursor DOTA-SC-027 compound was produced.

A method for synthesizing the marker precursor DOTA-NHS-ester-HDACi-SC027 comprises the following steps:

1) Firstly, the electronic balance needs to be corrected. After the calibration, the weighing paper is placed on the scale to return to zero;

2) Take 165.5 mg of DOTA-NHS-ester (actual measurement value is 165.84 mg) and 106.6 mg of HDACi-SC027 (actual measurement value is 106.93 mg);

3) separately filled into two glass bottles, and after the medicine is used up, it should be sealed with parafilm;

4) Take 1ml of DMF with a micropipette, drop into DOTA glass bottle, and take 1ml to add SC027 In the glass bottle, when the glass bottle is dripped, the glass bottle is continuously dripped, so that it is completely dissolved, and it is not easy to dissolve, and the bottle can be sealed and dissolved vigorously;

5) Then the round bottom bottle is washed with acetone and dried;

6) Fix the round bottom bottle and reheat the stirrer;

7) Dissolve the two bottles, open with a bottle opener, add 4.34 ul of TEA in a glass vial of DOTA with a micropipette, and then transfer the solution in the DOTA bottle to a 25 ml round bottom bottle with a micropipette;

8) Turn on the heater at a speed of about 500 rpm and turn on the exhaust fan;

9) After one hour, put the SC027 into the round bottom bottle with a micro dropper, and also turn on the heater at a speed of about 500 rpm and place it for 24 hours;

10) After 24 hours, the color before and after the reaction is unchanged, all are pale yellow, the heating stirrer is turned off, and the two vials are divided into two vials, one vial, 0.5 ml, and the other, 1.6 ml.

11) Since DMF cannot be used in HPLC, it needs to be lyophilized and then remelted by other solvents. After placing the two glass bottles on one iron tray, use liquid nitrogen to pour into the tray to cool it, then put it into the freeze dryer and vacuum it. The bottle cap must be left with a gap to freeze. After lyophilization, the solution was analyzed by HPLC. A large number of samples were stored in a refrigerator at -20 ° C and used as a follow-up marker for radioactive nuclear species Ga-67 or Ga-68.

Since DMF cannot be placed on HPLC, it is lyophilized and dissolved. After placing the two glass bottles on one iron tray, use liquid nitrogen to pour into the tray to cool it, then put it into the freeze dryer and vacuum it. The bottle cap must be left with a gap to freeze. After lyophilization, the solution was analyzed by HPLC.

The binding of the HDAC inhibitor SC-027 to the marker radioactive nucleus Ga-67 or Ga-68 can be combined to produce a novel nuclear medical tracer Ga-67-DOTA-SC-027 and Ga-68-DOTA-SC-027, which The method comprises the following steps in sequence: 1) adding Ga-67 or Ga-68 to DOTA-SC-027, heating to 85-100 ° C for 15 to 30 minutes; 2) using Gat 67 or Ga for Kit form -68 mark. That is, the appropriate amount of DOTA-SC-027 liquid is firstly dispensed into a small glass bottle, and a freeze-concentration drying process is performed. After filling the inert gas, the small glass bottle is capped and sealed with a bottle sealer. Take the above-mentioned sealed and sealed Kit, take appropriate amount of Ga-67 or Ga-68 and pH buffer solution into the Kit, shake it and dissolve it, then place it in an autoclave, set the temperature at 85~100 °C to maintain 15 ~30 minutes; 3) Get two chemical structures: as well as

Claims (5)

A contrast-enhancing compound that traces the Histone Deacetylase (HDAC) inhibitor (SC-027, acetyl dinaline, molecular formula C ₁₅ H ₁₅ N ₃ O ₂ ), using the marker radionuclide and tissue protein to acetamidine The enzyme inhibitor, 4-acetamido-N-(2'-aminophenyl)benzamide, a combination of the formula C ₁₅ H ₁₅ N ₃ O ₂ A contrast compound is prepared for use in a nuclear medicine for the treatment of a tumor tracer. Before the HDAC inhibitor SC-027 is combined with a labeled radionuclide, the marker precursor DOTA-SC-027 must be synthesized, wherein the DOTA (referred to as 1, 4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) has the formula C ₁₆ H ₂₈ N ₄ O ₈ and has the following structure: Wherein the SC-27 (钪-027) (referred to as acetyl dinaline) has the formula C ₁₅ H ₁₅ N ₃ O ₂ and has the following structure: The structural formula of the contrast compound for tracking the tissue protein deacetylase inhibitor is as follows: . The contrast compound according to claim 1, wherein the labeled radioactive nucleus to which the contrast compound is combined comprises: gallium-67-hydroxysuccinimide-SC-027 (Ga-67-DOTA-SC027); gallium- 68-hydroxysuccinimide-SC-027 (Ga-68-DOTA-SC-027); carbon-11-SC-027 (C-11-SC-027); fluorine-18-SC-027 (F -18-SC-027); iodine-123-SC-027 (I-123-SC-027); iodine-125-SC-027 (I-125-SC-027); or iodine-131-SC-027 (I-131-SC-027) wherein the marker precursor, DOTA-SC-027, must be synthesized prior to binding of the HDAC inhibitor SC-027 to the marker radionuclide. The contrast compound according to claim 1, wherein the labeled precursor precursor DOTA-SC-027 must be synthesized before the binding of the HDAC inhibitor SC-027 to the labeled radioactive nucleus gallium-67 and gallium-68, the synthesis method of which comprises The following steps: Step A: Take DOTA-NHS-ester (N-hydroxysuccinimide, NHS, hydroxysuccinimide) and SC-027 into two glass bottles, step B: use dimethylformamide , referred to as DMF ) dripped into a glass bottle containing DOTA and SC-027, respectively, and dripped in from the periphery of the glass bottle to make it completely dissolved; Step C: Fix the round bottom bottle on the stirrer. After adding triethylamine (TEA) to the DOTA glass via a micropipette, transfer the solution in the DOTA bottle to the round bottom bottle with a micropipette; Step D: Adjust the stirrer speed to 500 rpm, and use a microdrop after 1 hour. Tube SC-027 was placed in a round bottom bottle for 24 hours; Step E: 24 hours reaction, freeze-drying procedure; and Step F: Chemical structure of the label precursor DOTA-SC-027: . The contrast compound of claim 1, wherein the HDAC inhibitor SC-027 is combined with the labeled radioactive nucleus gallium-67 or gallium-68 to produce a novel nuclear medical tracer Ga-67-DOTA-SC-027 and Ga-68-DOTA- SC-027, the method comprises the steps of: Step 1: Add Ga-67 or Ga-68 to DOTA-SC-027, heat to 85~100 °C for 15~30 minutes; Step 2: Use kit (Kit) In the form of gallium-67 or gallium-68 mark, the appropriate amount of DOTA-SC-027 liquid is first dispensed into a small glass bottle, and the freeze-concentration and drying process is carried out. After filling the inert gas, the small glass bottle is capped with a bottle sealer. Seal, take the above-mentioned cap and seal kit, take appropriate amount of Ga-67 or Ga-68 and pH buffer into the kit, shake it, put it in autoclave, set the temperature 85~100 °C is maintained for 15~30 minutes; Step 3: Two chemical structures of Ga-68-DOTA-HDACi-SC-027 and Ga-67-DOTA-HDACi-SC-027 are obtained: as well as . The contrast compound of claim 2, wherein the HDAC inhibitor SC-027 is combined with the marker radionuclide C-11 to produce a nuclear medical tracer charcoal-11-钪-027 (C-11-SC-027), the chemical structure thereof The formula is as follows: