Hexa-lactoside-triazanonane triacetic acid (NOTA) derivative, method for radiolabeling hexa-lactoside positron emission tomography (PET) imaging agent for liver receptor with Ga-68, and hexa-lactoside PET imaging agent for liver receptor

Abstract

The present invention provides a hexa-lactoside-triazanonane triacetic acid (NOTA) derivative, a method for radiolabeling a hexa-lactoside positron emission tomography (PET) imaging agent for a liver receptor with Ga-68, and a hexa-lactoside PET imaging agent for a liver receptor. The hexa-lactoside-NOTA derivative is a conjugate of six chains of lactose with NOTA obtained by conjugating hexa-lactoside to a chelating agent p-thiocyanate-benzyl-triazanonane diacetic acid-glutamic acid in the presence of triethyl amine/dimethyl formamide as a solvent. The radiolabeling method comprises labeling with Ga-68 at room temperature. According to the present invention, the labeling effect is stable, the labeling efficiency of the labeled product is greater than 95%, the labeled product is highly stable and the radiochemical purity is still greater than 90% after 4 hours.

Claims

1. A hexa-lactoside-triazanonane triacetic acid (NOTA) derivative, having a structure of: ##STR00002##

2. A hexa-lactoside positron emission tomography (PET) imaging agent for a liver receptor, having a structure of: ##STR00003##

3. The imaging agent according to claim 2, which is prepared by the steps of: reacting 3-valent Ga-68 and a triazanonane triacetic acid (NOTA) conjugate of six chains of lactose with sodium acetate buffer in a freeze drying ampoule at room temperature.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a hexa-lactoside structure protected with a trifluoroacetate;

(2) FIG. 2 shows a method for conjugating HexaLac to diBoc-NOTA;

(3) FIG. 3 shows a method for removing the protecting group Boc from HexaLac-diBocNOTA;

(4) FIG. 4 shows a method for conjugating HexaLac to NHS-NOTA;

(5) FIG. 5 shows coordination bonding of Ga-68 to HexaLac-NCS-Bn-NODA-GA;

(6) FIG. 6 shows a method for conjugating HexaLac to p-NCS-Bn-NODAGA;

(7) FIG. 7A shows radiochemical purity analysis of Ga-68-HexaLac-NC S-Bn-NODA-GA;

(8) FIG. 7B shows radiochemical purity analysis of free Ga-68;

(9) FIG. 8 shows PET/CT imaging with Ga-68-HexaLac-NCS-Bn-NODA-GA;

(10) FIG. 9 shows the reproducibility of the labeling efficiency of HexaLac-NCS-Bn-NODA-GA with Ga-68; and

(11) FIG. 10 shows the stability of HexaLac-NCS-Bn-NODA-GA labeled with Ga-68 over times.

DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS

(12) Hereinafter, the detailed features and advantages of the present invention will be described in detail in the embodiments. The disclosure is sufficient to enable any person skilled in the art to understand and implement the technical contents of the present invention. Those skilled in the art can easily understand the related objects and advantages of the present invention from the disclosure of the specification, the claims and the drawings of the present invention. The following examples are provided for further illustrating the concept of the present invention in detail, instead of limiting the scope of the present invention in any way.

(13) In an embodiment of the present invention, Ga-68 is used as a nuclide and produced by using a Ga-68 generator. Therefore, with the Ga-68 generator, Ga-68 can be easily produced and used for labeling, which brings convenience in use. In addition, Ga-68 is a positron nuclide. The positron images obtained therewith are clearer than those obtained by single photon imaging. Ga-68 has a half-life of only 68 minutes, which is very suitable for use in targeted angiography and diagnosis. In an embodiment of the present invention, hexa-lactoside is conjugated to a suitable chelating agent that can be labeled with the radioactive isotope Ga-68, to develop a Ge-68-PET imaging agent for a liver receptor.

(14) For the selection of the metal chelating agent, DTPA and 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid (DOTA) are common metal chelating agent. Since Ga-68 is relatively small, a cyclic chelating agent such as 1,4,7-triazanonane triacetic acid (NOTA) can more tightly sequester Ga-68-sized metals, so the following series of NOTA chelating agents are used for conjugation with hexa-lactoside, including, diBocNOTA (di-tert-butyloxycarbonyl protected NOTA), NHS-NOTA (N-hydroxysucciniimide triazanonane), and p-NCS-Bn-NODA-GA (2,2-(7-(1-carboxy-4-((4-isothiocyanatobenzyl)amino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (e.g. p-SCN-benzyl-NODA-GA), and most suitable chelating agents are selected therefrom.

(15) The features and implementation of the present invention are described in detail below with reference to preferred examples.

Example 1. Preparation of Hexa-Lactoside

(16) The structure of the white solid hexa-lactoside protected with a trifluoroacetate is shown in FIG. 1. The analysis data of the compound:

(17) C.sub.140H.sub.247F.sub.3N.sub.12O.sub.76; TLC RP-18 (MeOH/1% TFA=5:5) R.sub.f=0.26; .sup.1H NMR (300 MHz, D.sub.2O) 4.46 (t, J=6.0 Hz), 4.30 (d, J=7.8 Hz), 4.29 (d, J=7.8 Hz), 3.83-3.06 (m), 2.53 (dd, J=14.7, 6.0 Hz), 2.41 (dd, J=14.7, 8.4 Hz), 2.12 (t, J=6.9 Hz), 1.46-1.19 (m); .sup.13C NMR (75 MHz, D.sub.2O) 176.50, 174.32, 173.28, 172.25, 171.35, 103.08, 102.22, 78.53, 75.50, 74.90, 74.61, 72.99, 72.66, 71.09, 70.66, 68.69, 66.43, 61.17, 60.26, 56.03, 51.07, 39.72, 39.33, 39.19, 35.46, 28.87, 28.49, 27.65, 26.05, 25.54, 24.92, 23.23; ESI-HRMS: calcd for 1124.5350, found: m/z 1124.5314 [M+3H].sup.+3.

(18) The deprotection step was as follows. The compound (626 mg, 0.186 mmol) was dissolved in triethylamine/ethanol/water (ratio by volume=1:1:8, 12 mL) and stirred overnight at room temperature (approximately 15 hrs). After the reaction was completed, the reaction solution was concentrated under reduced pressure and dried. Next, methanol (about 20 mL) was added and ultrasonicated (for 5 min). A white solid was precipitated out, which was pipetted into a centrifuge tube and centrifuged at 3000 rpm for two minutes. The methanolic supernatant was pipetted out and the lower solid was dried at a high vacuum level to obtain the deprotected hexa-lactoside compound (HexaLac, 503 mg) (yield 83%). The analysis data of the compound obtained: C.sub.138H.sub.248N.sub.12O.sub.75; TLC RP-18 (MeOH/1% TFA=5:5) R.sub.f=0.68; ESI-MS: calcd for 1092.53, found: m/z 1092.97 [M+3H].sup.+3. The hexa-lactoside is AHA-Asp[DCM-Lys(ah-Lac).sub.3].sub.2, that is, aminohexanoyl-aspartic acid[dicarboxymethyl-L-Lys(aminohexyl-lactose).sub.3].sub.2, referred to as HexaLac for short.

Example 2. Conjugation of HexaLac to diBoc-NOTA

(19) The route for conjugating HexaLac to diBoc-NOTA is shown in FIG. 2. A two-neck flask was purged with nitrogen. HexaLac-NH.sub.2 (1 eq., 10 mg, 0.003 mmol), HBTU (N,N,N,N-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate, 2.0 eq., 2 mg, 0.006 mmol), diBoc-NOTA (di-tert-butyloxycarbonyl protected NOTA, 2 eq, 2.5 mg, 0.006 mmol), and dimethyl formamide (DMF, 0.2 mL) were added to the flask. Then, DIEA (diethylamine, 4.0 eq, 1 L, 0.012 mmol) was added to the reaction flask and stirred for 1 hr. After the reaction was completed, ethyl acetate (EA) was added to precipitate the product. Next, the reaction system was centrifuged and the supernatant was removed. Then, EA was added again for washing, followed by centrifugation, and removal of the supernatant. The above procedure was repeated 3 times. Finally, the residue was purified by medium pressure liquid chromatography (MPLC) on a RP-18 column (MeOH/H.sub.2O=40%/60%.fwdarw.MeOH/H.sub.2O=100%/0%). The obtained product was HexaLac-diBocNOTA (10 mg in total, yield: 89%).

(20) Next, the Boc protecting group was removed. The route for removing the Boc protecting group from HexaLac-diBocNOTA was shown in FIG. 3. First, HexaLac-diBocNOTA (1.0 eq, 10 mg, 0.003 mmol), and methanol (0.5 mL) was added to a reaction flask. Next, sodium methoxide (20.0 eq, 3.2 mg, 12.5 mmol) was added to the reaction flask and stirred for 2 hrs. A white solid was precipitated out. The reaction system was centrifuged and the supernatant was removed. A small amount of methanol (0.5 mL) was added again for washing and the supernatant was removed. The above procedure was repeated 3 times. The residue was purified and separated by HPLC (acetonitrile/1% TFA=5%/95%.fwdarw.acetonitrile/1% TFA=70%/30%). The obtained product was HexaLac-NOTA (2 mg in total, yield: 25%). However, in the method for synthesizing HexaLac-diBocNOTA with diBocNOTA, in the step of removing the protecting group, a basic or acidic reaction condition is needed, which has an impact on the HexaLac, so the stability is less good and the product cannot be obtained successfully due to the susceptibility to decomposition. Therefore, in other embodiments, no NOTA derivative protected with tert-butyl is used.

Example 3: Conjugation of HexaLac to NHS-NOTA

(21) In order to avoid the failed reaction caused by a too strong reaction condition in the deprotection reaction, NHS-NOTA is used as a reactant. In particular, the HexaLac is linked in one step through reaction with the activated NHS end, so no additional deprotection reaction is needed. The route for conjugating HexaLac to NHS-NOTA is shown in FIG. 4. First, HexaLac-NH.sub.2 (1 eq, 10 mg, 0.003 mmol) was fed to a reaction flask and dissolved in dimethylformamide (1 mL) and triethylamine (0.002 mL). Next, N-hydroxysucciniimide triazanonane (i.e. NHS-NOTA) (2 eq., 0.004 mg, 0.006 mmol) was added. After stirring for 2 hrs, diethyl ether was added to precipitate the product. Next, the reaction system was centrifuged and the supernatant was removed. Diethyl ether was added again for washing, followed by centrifugation, and removal of the supernatant. The above procedure was repeated 3 times. Finally, the residue was purified by MPLC on a RP-18 column (MeOH/H.sub.2O=10%/90%.fwdarw.MeOH/H.sub.2O=100%/0%). The obtained product was HexaLac-NOTA (4 mg in total, yield: 30%). However, in this example, the synthesized HexaLac-NOTA is less stable under the labeling conditions in subsequent isotope labeling procedure.

(22) The isotope labeling experiment was further explained below. The method for radiolabeling HexaLac-NOTA with Ga-68 was as described by Yu (2015) (Yu H M, Chen J H, Lin K L, Lin W J. Synthesis of (68)Ga-labeled NOTA-RGD-GE11 heterodimeric peptide for dual integrin and epidermal growth factor receptor-targeted tumor imaging. J Labelled Comp Radiopharm 2015; 58(7): 299-303). 0.5 mL of Ga-68 (about 185 MBq) was added to 10 L of 0.1 M HEPES buffer (pH7-7.6). Then the solution was adjusted to pH 4.0-4.5 with 0.1 M HCl. Next, 50 g of NOTA-HexaLac was added, and ultrasonicated for 30 minutes (or heated at 90 C. for 30 min in other embodiments). The labeling efficiency in this embodiment is difficult to reach 90% or higher, and heating is needed. If the labeling is facilitated by microwave heating, although the labeling efficiency can be increased to 99% or higher, the labeled product is not stable and is prone to decomposition. In the mass spectrum, many peaks corresponding to the broken compounds are observed, suggesting that the labeled product is extremely unstable. In other words, the hexa-lactoside complex is not stable. However, if the labeling is performed at room temperature, labeling efficiency is very low because the chelating agent NOTA is in close proximity with the hexa-lactoside molecules.

Example 4: Conjugation of HexaLac to p-NCS-benzyl-NODA GA

(23) In order to overcome the problem of the isotope labeling experiment as described above, p-NCS-NODA-GA is used as a chelating agent in the following example. On one hand, the distance between NOTA and hexa-lactoside is relatively long, which can reduce the steric hindrance caused by hexa-lactoside when a metal is chelated. On the other hand, the amino groups of hexa-lactoside is bonded to and forms a thiourea linkage with the isothiocyanate of p-NCS-NODA-GA, which is click chemistry in one pot. The steps are simplified and no any deprotection step is involved. Furthermore, six-coordination binding can stabilize the chelation of NODA-GA with Ga-68 (FIG. 5 shows the coordination bonding of Ga-68 and HexaLac-NCS-Bn-NODA-GA).

(24) The route for conjugating HexaLac to p-NCS-Bn-NODA-GA is shown in FIG. 6. First, HexaLac-NH.sub.2 (39.7 mg, 12 mol) was dissolved in triethyl amine/dimethyl formamide (0.3 mL/3 mL). Next, p-thiocyanate-benzyl-triazanonane diacetic acid-glutamic acid (p-NCS-benzyl-NODA GA, 12.6 mg, 24 mol, Chematech, France. FW=521.59) was added, and reacted for 6 hrs with stirring. Then, diethyl ether (30 mL) was added to precipitate a solid. Next, the reaction system was centrifuged and the supernatant was removed. Diethyl ether (30 mL) was then added again, and ultrasonicated for 5 min, followed by centrifugation, and removal of the supernatant. A crude solid product was obtained. The obtained crude product was purified by MPLC, and then concentrated under reduced pressure and freeze dried to obtain HexaLac-NCS-benzyl-NODA GA (38.0 mg, 10 mol). In this embodiment, the yield was 82%. 20 g of Hexa-lactoside-NOTA was dissolved in 0.1% TFA/ddH.sub.2O (0.5 mL), and then analyzed by LC-MS (ABI 4000Q Trap LC/MS/MS). The peak signal determined is at 1266.6[M+3H].sup.3+, the molecular weight is 3797.

(25) A mass spectrum of HexaLac-NCS-Bn-NODA-GA was showed as following: .sup.1H NMR (400 MHz, D.sub.2O, 6): 7.34-7.32 (d, J=8.0, 2H, ArH), 7.23-7.21 (d, J=7.6, 2H, ArH), 4.41 (d, J=8.0, 6H, H.sub.Lac-1), 4.40 (d, J=7.6, 6H, H.sub.Lac-1), 2.81-3.94 (m, 137H), 1.22-2.54 (m, 72H). .sup.13C NMR (400 MHz, D.sub.2O, 6): 178.45 (CO), 176.42 (CO), 175.60 (CO), 174.65 (CO), 174.24 (CO), 173.23 (CO), 172.20 (CO), 171.36 (CO), 128.71 (CH, aromatic), 125.93 (CH, aromatic), 103.05 (CH, C.sub.Lac-1), 102.18 (CH, C.sub.Lac-1), 78.64 (CH), 75.44 (CH), 74.85 (CH), 74.59 (CH), 72.97 (CH), 72.67 (CH), 71.06 (CH), 70.58 (CH.sub.2, C.sub.Ah-1), 68.66 (CH), 66.95 (CH), 66.39 (CH), 61.09 (CH.sub.2), 60.29 (CH.sub.2), 57.83 (CH.sub.2), 56.03 (CH.sub.2), 51.48 (CH.sub.2), 51.01 (CH), 49.45 (CH.sub.2), 46.80 (CH.sub.2), 42.70 (CH.sub.2), 39.28 (CH.sub.2), 39.15 (CH.sub.2), 37.76 (CH.sub.2), 35.50 (CH.sub.2), 32.81 (CH.sub.2), 28.83 (CH.sub.2), 28.43 (CH.sub.2), 26.00 (CH.sub.2), 25.65 (CH.sub.2), 25.56 (CH.sub.2), 25.04 (CH.sub.2), 24.87 (CH.sub.2), 23.24 (CH.sub.2), 23.09 (CH.sub.2). HSQC: Correlation 128.71 (CH, aromatic) to 7.33 (d, J=8.0, 2H, ArH), 125.93 (CH, aromatic) to 7.22 (d, J=7.6, 2H, ArH), 103.05 (CH, C.sub.Lac-1) to 4.41 (d, J=8.0, 6H, H.sub.Lac-1), 102.18 (CH, C.sub.Lac-1) to 4.40 (d, J=7.6, 6H, H.sub.Lac-1).

(26) HexaLac-NCS-benzyl-NODA-GA was labeled with Ga-68. The labeling process was as described in Knetsch (2011) (Knetsch P A, Petrik M, Griessinger C M, Rangger C, Fani M, Kesenheimer C, et al. [.sup.68Ga]NODAGA-RGD for imaging alphavbeta3 integrin expression. Eur J Nucl Med Mol Imaging 2011; 38(7):1303-12). A gallium (Ga-68) chloride solution (0.5 mL, 101 mCi/mL) (NODAGA-RGD for imaging alphavbeta3 integrin expression. Eur J Nucl Med Mol Imaging 2011; 38(7):1303-12) was drawn by a syringe, added to and completely dissolved in HexaLac-NCS-benzyl-NODA-GA (40 g) and sodium acetate (13.61 mg) in a freeze drying ampoule in 1-2 minutes, and stood for 15 min. Then, 1.5 mL of normal saline was added and mixed uniformly, and then sampled for analysis and imaging. The radiochemical purity analysis of the labeled product is shown in FIGS. 7A and 7B. The imaging results are shown in FIG. 8. The labeling was performed five times consecutively, using 40 gNOTA-HL and Ga-68 with an activity of 50.5 mCi. The labeling effect was stable, and the radiochemical purity of the product was >95%. The reproducibility of the labeling efficiency is shown in FIG. 9. The stability over time is shown in FIG. 10. As shown, the labeled product has good stability. Regardless of the addition of stabilizers, the labeled product still has a radiochemical purity of 90% or greater after 4 hrs.

Example 5: Radiochemical Purity Test of Ga-68-HexaLac-NC S-benzyl-NODA-GA

(27) 10 mL of 0.1M EDTA was added to a developing tank, and an origin and a solvent front were marked at 1 cm and 5 cm of the RP-TLC plate. Then, a small amount of sample (1-2 L) was dotted on the origin of the RP-TLC plate, and then the plate was placed in the developing tank by using forceps. When the developing solution reached the solvent front, the plate was removed by using forceps and dried in an oven. The ITLC-SG plate was scanned by using a radio-TLC imaging scanner, and the spectrum was collected over 1 min. The result was calculated through a formula below:

(28) $\mathrm{Radiochemical}\mathrm{purity}\left(\%\right)=\frac{A}{B}100\%$

(29) A: The count area of the .sup.68Ga-hexa-lactoside peak (Rf=0.0-0.2)

(30) B: The count area of all peaks

Example 6: PET Imaging with Ga-68-HexaLac-NCS-benzyl-NODA-GA

(31) The Ga-68-HexaLac-NCS-benzyl-NODA GA (15 Ci/g) was injected into mice through the tail vein. After injection, PET/CT imaging was performed immediately for 15 min. During the imaging process, the test animals were anesthetized with isoflurane. After imaging, the PET/CT images were fused. The imaging results are shown in FIG. 8.

(32) Although embodiments of the present invention have been illustrated and described, various modifications and improvements will occur to those skilled in the art. The present invention is not intended to be limited to the particular forms illustrated, and the modifications made without departing from the spirit and scope of the present invention all fall within the scope as defined by the appended claims

(33) In view of the above, the present invention has not been found in similar products in terms of the characteristics and combination as a whole, and has not been disclosed before the application. Since the present invention meets the statutory requirements of the Patent Law, an application for invention patent is filed according to the Patent Law.

(34) Although the embodiments of the present invention have been disclosed above, they are not intended to limit the present invention. Any modifications made by persons skilled in the art to the shapes, structures, and features without departing from the spirit and scope of the present invention fall within the scope of the present invention as defined in appended claims.