WO2009145378A1 - Gd complex comprising dtpa-bis-amide ligand and method for preparing the same - Google Patents
Gd complex comprising dtpa-bis-amide ligand and method for preparing the same Download PDFInfo
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- WO2009145378A1 WO2009145378A1 PCT/KR2008/003144 KR2008003144W WO2009145378A1 WO 2009145378 A1 WO2009145378 A1 WO 2009145378A1 KR 2008003144 W KR2008003144 W KR 2008003144W WO 2009145378 A1 WO2009145378 A1 WO 2009145378A1
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- Prior art keywords
- ligand
- gadolinium
- dtpa
- bis
- complex
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- 239000003446 ligand Substances 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 37
- 229910052688 Gadolinium Inorganic materials 0.000 claims abstract description 44
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 claims abstract description 43
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000003756 stirring Methods 0.000 claims abstract description 15
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000002904 solvent Substances 0.000 claims abstract description 12
- 239000007787 solid Substances 0.000 claims abstract description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000012535 impurity Substances 0.000 claims abstract description 6
- 239000012153 distilled water Substances 0.000 claims abstract description 5
- CMIHHWBVHJVIGI-UHFFFAOYSA-N gadolinium(III) oxide Inorganic materials [O-2].[O-2].[O-2].[Gd+3].[Gd+3] CMIHHWBVHJVIGI-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 3
- 230000001376 precipitating effect Effects 0.000 claims abstract description 3
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 3
- 239000002872 contrast media Substances 0.000 claims description 25
- 230000007541 cellular toxicity Effects 0.000 claims description 17
- 230000005291 magnetic effect Effects 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 10
- 206010028980 Neoplasm Diseases 0.000 claims description 7
- 201000011510 cancer Diseases 0.000 claims description 7
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 3
- -1 2-hydroxyethyl- Chemical group 0.000 claims description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 abstract description 18
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 abstract description 3
- QTLPZFIGTRQFMC-UHFFFAOYSA-N cyclohexanecarboxylic acid;hydrochloride Chemical compound Cl.OC(=O)C1CCCCC1 QTLPZFIGTRQFMC-UHFFFAOYSA-N 0.000 abstract description 2
- 150000007942 carboxylates Chemical class 0.000 abstract 2
- 238000002360 preparation method Methods 0.000 description 23
- 150000000921 Gadolinium Chemical class 0.000 description 9
- HZHFFEYYPYZMNU-UHFFFAOYSA-K gadodiamide Chemical compound [Gd+3].CNC(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CCN(CC([O-])=O)CC(=O)NC HZHFFEYYPYZMNU-UHFFFAOYSA-K 0.000 description 9
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 7
- 230000006641 stabilisation Effects 0.000 description 7
- 238000011105 stabilization Methods 0.000 description 7
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 6
- 150000002500 ions Chemical class 0.000 description 6
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 230000002708 enhancing effect Effects 0.000 description 5
- FTZIQBGFCYJWKA-UHFFFAOYSA-N 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Chemical compound S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 FTZIQBGFCYJWKA-UHFFFAOYSA-N 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 239000011701 zinc Substances 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- ZQTGHRUFDUNWHF-UHFFFAOYSA-N 3-cyclohexylpropanoic acid;hydrochloride Chemical compound Cl.OC(=O)CCC1CCCCC1 ZQTGHRUFDUNWHF-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 230000005298 paramagnetic effect Effects 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- 238000004639 Schlenk technique Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- RMRFFCXPLWYOOY-UHFFFAOYSA-N allyl radical Chemical group [CH2]C=C RMRFFCXPLWYOOY-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- LGMLJQFQKXPRGA-VPVMAENOSA-K gadopentetate dimeglumine Chemical compound [Gd+3].CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.OC(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O LGMLJQFQKXPRGA-VPVMAENOSA-K 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 229960003330 pentetic acid Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000012857 radioactive material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
- C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/02—Formation of carboxyl groups in compounds containing amino groups, e.g. by oxidation of amino alcohols
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/38—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/04—Preparation of carboxylic acid amides from ketenes by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/14—The ring being saturated
Definitions
- the present invention relates to a new method of preparing a DTPA-bis- amide ligand having high molecular weight to increase magnetic relaxation ratio.
- the present invention relates to a new method of preparing a gadolinium (Gd) complex having high water-solubility by using the ligand.
- the present invention relates to an MR contrast medium for diagnosing cancer, which contains a gadolinium (Gd) complex synthesized by the above mentioned method, has a contrast enhancing effect, and has a reduced cell toxicity.
- Gd gadolinium
- Magnetic Resonance Image (MRI) method has been widely used owing to the imaging of a diagnosis.
- magnetic resonance image method can support images of weak skin tissue, the quality of images may be determined by high shading contrast enhancing action of the contrast medium used in shading contrast reaction.
- growth of contrast medium field for efficient magnetic resonance image method has attracted the skilled experts' sufficient attention recently.
- a contrast medium in such a magnetic resonance image includes thermodynamic stability, water-solubility, and a multidentate ligand structure being a cause of making paramagnetic Gd (III) ion. That is, it has to couple with at least one molecule of water to show high magnetic relaxation with water. Further, a magnetic resonance image contrast medium should not have a chemical activity, its cell toxicity in vivo should be low, and it should be exhausted completely after a diagnosis.
- An example of a contrast medium being approved for human body as a contrast medium in magnetic resonance image method includes an ionized Gd (III) complex such as diethylenetriamine-N,N,N' ,N' ' ,N 1 ' -pentaacetate, (N-Me- glucamine)2[Gd(DTPA)(H 2 O)] (Magnevist, Schering) having magnetic relaxation
- the above contrast medium has problems such as low water- solubility, low magnetic relaxation ratio, and relatively high cell toxicity in vivo. [Disclosure] [Technical Problem]
- embodiments of the present invention provide a gadolinium complex having high water-solubility by using the ligand prepared according to a method of preparing the ligand of the present invention.
- embodiments of the present invention provide an MR contrast medium capable of diagnosing cancer, which has higher contrast enhancing effect than the MR contrast medium of the prior art by using a complex prepared according to a method of preparing a gadolinium (Gd) complex of the present invention.
- embodiments of the present invention provide a contrast medium capable of diagnosing cancer, which can reduce cell toxicity more than the MR contrast medium of the prior art.
- a method of preparing a new DTPA-bis-amide ligand includes: first step of adding DTPA-bis-anhydride to N,N-demethylformamide with stirring; second step of adding 2-hydroxyethy ⁇ -trans ⁇ A- (aminomethyl)cyclohexylcarboxylatehydrochloride to the mixture with stirring", third step of conducting silica gel chromatography after removing all of solvent under low pressure and dissolving the mixture in methanol; and fourth step of drying the product obtained in the third step under vacuum state to obtain a ligand L4.
- 2-methoxyethyWr,affs ⁇ 4-(aminomethyl) cyclohexaneethyl carboxylatehydrochloride may be added instead of 2- hydroxyethyWra/7sH:-(aminomethyl) cyclohexyl carboxylatehydrochloride to obtain a ligand L5 in the fourth step.
- a ⁇ y ⁇ -trans-4-(minomethy ⁇ ) cyclohexylethylcarboxylate hydrochloride may be added instead of 2- hydroxyethyl- ⁇ ra/2_?-4-(aminomethyl ) cyclohexylethylcarboxylate hydrochloride to obtain a ligand L6 in the fourth step.
- the method may further include: fifth step of putting the DTPA-bis- amide ligand obtained in fourth step to the distilled water, and adding Gd 2 O 3 with stirring to prepare a mixture solution; sixth step of removing impurity and solvent from the mixture solution obtained in the fifth step; and seventh step of dissolving the material obtained in the sixth step in methanol and precipitating it with acetonitrile to obtain a solid.
- the ratio of molarity of the DTPA-bis-amide ligand to that of Gd 2 O 3 in the fifth step may be 1 to 1.
- the stirring of the fifth step may be conducted at a temperature ranging from 90 °C to 100 °C for the time ranging from 5 hours to 7 hours.
- the sixth step may include passing the mixture solution obtained in the fifth step through CeI ite to remove impurity and solvent.
- a DTPA-bis-amide ligand is prepared by the method as set forth above.
- the DTPA-bis-amide ligand may have an improved thermodynamic stability.
- gadolinium (Gd) complex is prepared by the method as set forth above. ⁇ 2i> The gadolinium (Gd) complex may be expressed by a chemical formula (1) be1ow: ⁇ 22> [Gd(L)(H 2 O)] -H 2 O (1),
- the gadolinium (Gd) complex may have an improved solubility and magnetic relaxation ratio.
- the gadolinium (Gd) complex may have a low cell toxicity.
- the MR contrast medium for diagnosing cancer may contain the gadolinium
- the ligand prepared by the method of preparing the present invention contains a transamic acid, a polar functional group, which makes the gadolinium complex have a high water-solubility.
- the gadolinium complex prepared by the method of preparing the present invention when used as an MR contrast medium for diagnosing cancer, it makes a contrast enhancing effect higher than the MR contrast medium of the prior art.
- the MR contrast medium of the present invention for diagnosing cancer contains a gadolinium (Gd) complex of the present invention, and it makes cell toxicity lower than the MR contrast medium of the prior art.
- FIG. 2 shows relaxation times (Ti and T 2 ) and corresponding relaxation ratios (Ri and R 2 ) of gadolinium complexes (Gd (L4) to Gd (L6)), Omniscan and pure water.
- FIG. 3 shows a relaxation time map (T 1 map) and a corresponding relaxation ratio map (Ri map) of gadolinium complexes (Gd (L4) to Gd (L6)),
- FIG. 4 compares cell toxicities of gadolinium complexes (Gd (L4) to Gd (L6)) and Omniscan by an MTT test. [Best Mode]
- the present invention relates to a new method of preparing a new DTPA- bis-amide ligand and a new gadolinium complex, wherein the DTPA-bis-amide ligand contains a transamic acid having polar functional groups and ester conjugates.
- This ligand contains polar functional groups and thus has a high water-solubility.
- DTPA of the present invention is an abbreviated term of DiethyleneTriamine PentaAcetic acid, a metal-affinitive chelate compound, which is a chemical protective agent against radiation hazard.
- the protective agent functions to remove a radioactive material out of it and to reduce cell toxicity.
- the complex of the present invention contains one or more atom(s) or ion(s) as a core and several ion molecules of other atoms or atomic groups, which have aromaticity, with being sterically coordinated to the core.
- An atomic ion molecule or an atomic group, which coordinates a core atom or an ion, is called "a ligand.”
- a bracket "[ ]” the bond between a core atom and a ligand is an ion bond or a covalent bond, the number of a ligand is included even though the generated atomic group is not electrified.
- FIG. 1 shows a method of preparing a gadolinium complex briefly.
- FIG. 1 shows a method of preparing a gadolinium complex briefly.
- FIG. 1 shows a method of preparing a gadolinium complex briefly.
- Table 1 shows proton adding equilibrium constant(K ⁇ ) of ligands (L4 to L6) prepared in a Preparation Example, and stabilization constant (ik), selection conditional stabilization constant(K s e i) of gadolinium complex es (Gd(L4) to Gd(L6)), and pM values of gadolinium (Gd(III)), calcium (Ca(II)), zinc (Zn(II)), and copper (Cu(II)) at pH 7.4 (the values represent bonding strength of ligands and metals).
- Proton adding equilibrium constant [K 1 ) can be obtained by the
- gadolinium complex When a gadolinium complex was used as a contrast medium, gadolinium could be separated from a ligand in human body, and cell toxicity might be caused by a toxic gadolinium. In order not to make this separation, the stronger the bond between a ligand and a gadolinium might be, the more stable it is.
- ligands (L4 to L6) use gadolinium (Gd) showing higher values than Omniscan (DTPABMA) in proton adding equilibrium constant ( ⁇ ), stabilization constant (4 L ), selection constant conditional stabilization constant (K'sei), and pM values, and it means that the bonding state between the ligand and the gadolinium is more stable, and it can be used as a good contrast medium.
- Gd gadolinium
- DTPABMA Omniscan
- FIG. 3 shows relaxation time map (Ti map) and relaxation ratio map (Ri map).
- relaxation time map (Ti map)
- the brighter signal strength means the longer relaxation time on the map.
- Gd (L4) to Gd (L6) shows brighter signal strength comparing with Omniscan. Since the longer relaxation time can be confirmed, the contrast medium of the present invention can keep its contrast effect longer, and thus it is proved to be more effective.
- Measurement of relaxation time Ti was progressed by a reverse recovery method of variable conducting time (TI) at 1.5T (64MHz). Magnetic resonance (MR) image needs another 35 TIs ranging from 50 to 1750 msec region. Relaxation time Ti was obtained from non-linear, square strokes of signal strengths measured at each TI values.
- CPMG Carr-Purcell-Meiboon-Gill
- continuous wave for measuring relaxation time T 2 can be obtained by measuring multi spinal echo.
- 34 images can acquire 34 other echo time (TE) ranging 10 to 1900 msec.
- Relaxation time T2 can be obtained from non-linear regular square forms for measuring multi spin-echo at each echo time.
- Relaxation ratio(Ri and R 2 ) was calculated as a reverse value of relaxation time(Ti and T 2 ) per mM.
- MTT searching method is to use an ability of mitochondria to reduce yellow soluble MTT tetrazolium to celadon green non-soluble MTT formazan (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) by dehydrogenase function. If alive cells are treated with MTT tetrazolium, MTT tetrazolium is reduced to form MTT formazan (3- (4,5 ⁇ dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) by reductase of mitochondria.
- MTT formazan when the cell was treated with a certain compound depending on its concentrations for predetermined hours to induce cell death, and treated with MTT tetrazolium, MTT formazan was formed at low concentration not representing cell toxicity, and MTT formazan was not formed at high concentration representing cell toxicity.
- Cell viability can be determined by measuring the formation of MTT formazan depending on the concentration gradient of such compound.
- FIG. 4 shows the result of comparing cell toxicities of gadolinium complexes (Gd (L4) to Gd (L6)) and Omniscan by using MTT test. That is, the higher cell viability a gadolinium complex might have, the lower the cell toxicity is. Cells which was not added anything are regarded as a control group. Cell viability of a control group was determined as 100% by measuring formation of MTT formazan. When various concentrations (0.5mM to 1OmM) of gadolinium complexes (Gd(L4) to Gd(L6)) and Omniscan were added to cells, the cells showed an amount of 60% or more of cell viability. It was identified that gadolinium complexes (Gd(L4) to Gd(L6)) having low cell toxicities can be effectively used a contrast medium through a graph of FIG. 4.
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Abstract
A method of preparing a new DTPA-bis-amide ligand includes the following: first step of adding DTPA-bis-anhydride to N,N-dimethylformamide to with stirring; second step of adding 2-hydroxyethyl-trans-4-(aminomethyl)cyclohexyl carboxylate hydrochloride, 2-methoxyethyl-trans-4-(aminomethyl) cyclohexyl carboxylate hydrochloride, or allyl-trans-4-(aminomethyl)cyclohexyl carboxylate hydrochloride with stirring; third step of conducting silica gel chromatography after removing all of solvent under low pressure and adding methanol to dissolve; and fourth step of drying the product under vacuum state. Further, a method of preparing a new Gadolinium complex further includes fifth step of putting the obtained DTPA-bis-amide ligand to the distilled water, and adding Gd2O3 with stirring; sixth step of removing impurity and solvent; and seventh step of dissolving it in methanol and precipitating it with acetonitrile to obtain a solid.
Description
[DESCRIPTION] [Invention Title]
GD COMPLEX COMPRISING DTPA-BIS-AMIDE LIGAND AND METHOD FOR PREPARING THE SAME [Technical Field]
<i> The present invention relates to a new method of preparing a DTPA-bis- amide ligand having high molecular weight to increase magnetic relaxation ratio.
<2> Further, the present invention relates to a new method of preparing a gadolinium (Gd) complex having high water-solubility by using the ligand.
<3> Further, the present invention relates to an MR contrast medium for diagnosing cancer, which contains a gadolinium (Gd) complex synthesized by the above mentioned method, has a contrast enhancing effect, and has a reduced cell toxicity. [Background Art]
<4> In general, Magnetic Resonance Image (MRI) method has been widely used owing to the imaging of a diagnosis. Although magnetic resonance image method can support images of weak skin tissue, the quality of images may be determined by high shading contrast enhancing action of the contrast medium used in shading contrast reaction. Thus, growth of contrast medium field for efficient magnetic resonance image method has attracted the skilled experts' sufficient attention recently.
<5> The necessary Properties of a contrast medium in such a magnetic resonance image include thermodynamic stability, water-solubility, and a multidentate ligand structure being a cause of making paramagnetic Gd (III) ion. That is, it has to couple with at least one molecule of water to show high magnetic relaxation with water. Further, a magnetic resonance image contrast medium should not have a chemical activity, its cell toxicity in vivo should be low, and it should be exhausted completely after a diagnosis.
<6> An example of a contrast medium being approved for human body as a contrast medium in magnetic resonance image method includes an ionized Gd
(III) complex such as diethylenetriamine-N,N,N' ,N' ' ,N1 ' -pentaacetate, (N-Me- glucamine)2[Gd(DTPA)(H2O)] (Magnevist, Schering) having magnetic relaxation
ratio of 4.7 mM'V1 (20MHz, 298K) and a neutral Gd(III) complex such as [Gd(DTPA-bismethylamide)(H2O)] (Qmniscan , Nycomed) having magnetic
relaxation ratio of 4.4InMV1 (20MHz, 298K).
<7> However, the above contrast medium has problems such as low water- solubility, low magnetic relaxation ratio, and relatively high cell toxicity in vivo. [Disclosure] [Technical Problem]
<8> The present invention has been made to solve the foregoing problems with the prior art, and embodiments of the present invention provide a gadolinium complex having high water-solubility by using the ligand prepared according to a method of preparing the ligand of the present invention. <9> Further, embodiments of the present invention provide an MR contrast medium capable of diagnosing cancer, which has higher contrast enhancing effect than the MR contrast medium of the prior art by using a complex prepared according to a method of preparing a gadolinium (Gd) complex of the present invention.
<io> Further, embodiments of the present invention provide a contrast medium capable of diagnosing cancer, which can reduce cell toxicity more than the MR contrast medium of the prior art.
[Technical Solution]
<π> According to an aspect of the present invention, there is provided a method of preparing a new DTPA-bis-amide ligand. The method includes: first step of adding DTPA-bis-anhydride to N,N-demethylformamide with stirring; second step of adding 2-hydroxyethy\-trans~A- (aminomethyl)cyclohexylcarboxylatehydrochloride to the mixture with stirring", third step of conducting silica gel chromatography after removing all of
solvent under low pressure and dissolving the mixture in methanol; and fourth step of drying the product obtained in the third step under vacuum state to obtain a ligand L4. <12> In the second step, 2-methoxyethyWr,affs^4-(aminomethyl) cyclohexaneethyl carboxylatehydrochloride may be added instead of 2- hydroxyethyWra/7sH:-(aminomethyl) cyclohexyl carboxylatehydrochloride to obtain a ligand L5 in the fourth step. <i3> In the second step, a\\y\-trans-4-(minomethy\) cyclohexylethylcarboxylate hydrochloride may be added instead of 2- hydroxyethyl-^ra/2_?-4-(aminomethyl ) cyclohexylethylcarboxylate hydrochloride to obtain a ligand L6 in the fourth step. <14> The method may further include: fifth step of putting the DTPA-bis- amide ligand obtained in fourth step to the distilled water, and adding Gd2O3 with stirring to prepare a mixture solution; sixth step of removing impurity and solvent from the mixture solution obtained in the fifth step; and seventh step of dissolving the material obtained in the sixth step in methanol and precipitating it with acetonitrile to obtain a solid. <15> The ratio of molarity of the DTPA-bis-amide ligand to that of Gd2O3 in the fifth step may be 1 to 1. <i6> The stirring of the fifth step may be conducted at a temperature ranging from 90 °C to 100 °C for the time ranging from 5 hours to 7 hours. <17> The sixth step may include passing the mixture solution obtained in the fifth step through CeI ite to remove impurity and solvent. <i8> According to another aspect of the present invention, a DTPA-bis-amide ligand is prepared by the method as set forth above.
<19> The DTPA-bis-amide ligand may have an improved thermodynamic stability. <20> According to a further aspect of the present invention, a gadolinium
(Gd) complex is prepared by the method as set forth above. <2i> The gadolinium (Gd) complex may be expressed by a chemical formula (1) be1ow:
<22> [Gd(L)(H2O)] -H2O (1),
<23> where L represents a ligand according to claim 8, and x represents 0 to
12. <24> The gadolinium (Gd) complex may have an improved solubility and magnetic relaxation ratio.
<25> The gadolinium (Gd) complex may have a low cell toxicity. <26> The MR contrast medium for diagnosing cancer may contain the gadolinium
(Gd) complex. [Advantageous Effects] <27> As set forth above, the ligand prepared by the method of preparing the present invention contains a transamic acid, a polar functional group, which makes the gadolinium complex have a high water-solubility. <28> Further, when the gadolinium complex prepared by the method of preparing the present invention is used as an MR contrast medium for diagnosing cancer, it makes a contrast enhancing effect higher than the MR contrast medium of the prior art. <29> Further, the MR contrast medium of the present invention for diagnosing cancer contains a gadolinium (Gd) complex of the present invention, and it makes cell toxicity lower than the MR contrast medium of the prior art.
[Description of Drawings] <30> FIG. 1 shows a process for synthesizing ligands (L4 to L6) and gadolinium complexes (Gd (L4) to Gd (L6)) of the present invention, wherein: <3i> When DTPA-bis-anhydride is added to DMF with stirring, and 2-hydroxy ethyWra/7.?-4-(aminomethyl) cyclohexylcarboxylate hydrochloride (R = (CH2)20H) is added, a ligand L4 is formed, and when GdA is added hereto, a gadolinium complex Gd (L4) is formed;
<32> When DTPA-bis-anhydride is added to DMF with stirring, 2-methoxy ethyl-
cyclohexylcarboxylate hydrochloride (R = (CH2)20Me) is added, a ligand L5 is formed, and when is added hereto, a gadolinium complex Gd (L5) is formed; and
<33> When DTPA-bis-anhydride is added to DMF with stirring and a\\yl-trans- 4-(aminofflethyl) cyclohexylcarboxylatehydrochloride (R=CH2CH=CH2) is added, a ligand L6 is formed, and when Gd2Os is added hereto, a gadolinium complex Gd
(L6) is formed. <34> FIG. 2 shows relaxation times (Ti and T2) and corresponding relaxation ratios (Ri and R2) of gadolinium complexes (Gd (L4) to Gd (L6)), Omniscan and pure water. <35> FIG. 3 shows a relaxation time map (T1 map) and a corresponding relaxation ratio map (Ri map) of gadolinium complexes (Gd (L4) to Gd (L6)),
Omniscan and pure water.
<36> FIG. 4 compares cell toxicities of gadolinium complexes (Gd (L4) to Gd (L6)) and Omniscan by an MTT test. [Best Mode]
<37> The present invention relates to a new method of preparing a new DTPA- bis-amide ligand and a new gadolinium complex, wherein the DTPA-bis-amide ligand contains a transamic acid having polar functional groups and ester conjugates. This ligand contains polar functional groups and thus has a high water-solubility.
<38> Further, DTPA of the present invention is an abbreviated term of DiethyleneTriamine PentaAcetic acid, a metal-affinitive chelate compound, which is a chemical protective agent against radiation hazard. The protective agent functions to remove a radioactive material out of it and to reduce cell toxicity.
<39> The complex of the present invention contains one or more atom(s) or ion(s) as a core and several ion molecules of other atoms or atomic groups, which have aromaticity, with being sterically coordinated to the core. An atomic ion molecule or an atomic group, which coordinates a core atom or an ion, is called "a ligand." To specify a complex, its chemical formula is represented in a bracket "[ ]," the bond between a core atom and a ligand is
an ion bond or a covalent bond, the number of a ligand is included even though the generated atomic group is not electrified.
<40>
<4i> Now, the following non-limiting examples are used to further describe or illustrate the invention. The examples are given for illustration of the invention and are not intended to be limiting thereof.
<42>
<43> EXAMPLES
<44>
<45> Preparation Example 1 (Ligand L4)
<46> 0.71g (2mmol) of DTPA-bis-anhydride was dissolved in 15mL N,N- dimethylformamide (DMF) with stirring, and 0.63g (4mmol) of 2-hydroxyethyl- £r,3/7S-4-(aminomethyl) eyelohexylcarboxylate hydrochloride was added. The reaction mixture was stirred at 65°C for 4 hours, all of solvent was removed under low pressure, and 1OmL of methanol was added to dissolve. Short silica gel (60 meshes) chromatography of the solution was conducted by passing methanol, and all of solvent was removed again. The obtained white solid was dried at 50°C for 8 hours and was kept under vacuum state.
<47> From the experimental data below, the resulting product of the preparation Example was identified as a ligand L4.
<48> Yield: 3.93 g (87%)
<49> 1H [J6-DMSO, 400 MHz] : δ 8.25 (s , 2H, CH2COM) , 4.17 (s , 2H, H2) , 3.54
(m, 8H, OCrøPD , 3.40 (m, 8H, H7, H5) , 3.07 (m, 4H, H3/H4) , 2.94 (m, 4H,
H3/H4) , 2.24 (m, 2H, H13) , 2.08 (m, 4H, H9) , 1.81 (m, 8H, H11/H12) , 1.37 (m, 2H, HlO) , 1.08 (m, 8H, H11/H12)
<50> 13C NMR (J6-DMSO, 100 MHz) : δ 175.46 (C1/C8) , 175.09 (C1/C8) , 172.96
(C14) , 172.26 (C6) , 65.83 (0Oi2CH2OH) , 65.26 (OCH2Ol2OH) , 63.99 (C2) , 59.25
(C7) , 56.49 (C5) , 54.83 (C3/C4) , 54.54 (C3/C4) , 44.84 (C13) , 43.02 (C9) , 42.76 (ClO) , 29.59 (CIl) , 28.52 (C12)
<5i> Calculated for C34H57N5OuSH2O: C: 45.17, H: 8.14, N: 7.75
<52> Measured for C34H57N5O14SH2O: C: 45.33, H: 7.86, N: 7.66
<53> FAB-MS im/z) :
<54> Calculated for C34H58N5Oi4: 760.85 ( [MH] +)
<55> Measured for C34H58N5O14: 760.65 ( [MH] +)
<56> Calculated for C34H57N5NaOi4: 782.83 ( [MNa] +)
<57> Measured for C34H57N5NaOi4: 782.60 ( [MNa] +)
<58>
<59> Preparation Example 2 (Ligand L5)
<60> This preparation method was progressed as the same way in Preparation Example 1, except that 2-methoxyethyl-fra/7Su-4-(aminomethyl) cyclohexylcarboxylatehydrochloride was used instead of
4-(aminomethyl) eyelohexylcarboxylate hydrochloride. The obtained white solid was dried at 50"C for 8 hours and was kept under vacuum state.
<6i> From the experimental data below, the resulting product of the preparation Example was identified as a ligand L5.
<62> Yield: 3.87 g (83%)
<63> 1H [ cfs-DMSO, 400 MHz] : δ 8.31 (s , 2H, CH2CON/?) , 4.14 (s , 2H, H2) , 4.09
(m, 4H, OC^2CH2OCH3) , 3.56 (m, 4H, OCH2CTy2OCH3) , 3.48 (m, 8H, H7, H5) , 3.36 (m, 4H, H3/H4) , 3.23 (s , 6H, OCH2CH2OCyY3) , 3.08 (m, 4H, H3/H4) , 2.93 (m, 4H, H9) ,
2.21 (m, 2H, H13) , 1.76 (m, 8H, H11/H12) , 1.37 Gn, 2H, HlO) , 1.08 (m, 8H, H11/H12)
<64> 13C U-DMSO, 100MHz]: δ 175.34 (C14), 172.88 (C1/C8), 172.05 (C1/C8), 170.09 (C6), 70.11 (OCH2OI2OCH3), 63.20 (OaI2CH2OCH3), 58.42 (OCH2CH2OOI3), 56.45
(C2), 54.83 (C7), 54.55 (C5), 51.85 (C3/C4), 49.72 (C3/C4), 44.84 (C13), 42.66 (C9), 37.07 (ClO), 29.53 (CIl), 28.52 (C12)
<65> Calculated for C36H61N5Oi4SH2O: C: 46.39, H: 8.33, N: 7.51
<66> Measured for C36H6IN5O14SH2O: C: 46.22 , H: 7.97 , N: 7.83
<67> FAB-MS {m/z) :
<68> Calculated for C36H62N5Oi4: 788.9 ( [MH] +)
<69> Measured for C36H62N5Oi4: 788.67 ( [MH] +)
+,
<70> Cal culated for C36H62N5NaOi4 : 810.88 ( [MNa] )
+.
<7i> Measured for C36H61N5NaOi4: 810.61 ( [MNa] )
<72>
<73> Preparation Example 3 (Ligand L6)
<74> This preparation method was progressed as the same way in Preparation Example 1, except that
cyclohexylcarboxylatehydrochloride was used instead of 2-hydroxyethyl-trans- 4-(aminomethyl) eyelohexylcarboxylate hydrochloride. The obtained white solid was dried at 50°C for 8 hours and was kept under vacuum state.
<75> From the experimental data below, the resulting product of the preparation Example was identified as a ligand L6.
<76> Yield: 3.6Og (82%)
<77> 1H NMR (oH)MS0, 400 MHz): 58.28 (s, 2H, CH2OM, 5.89 (m, 2H, OCH2C^=CH2), 5.22 (m, 4H, OCH2CH=CTy2), 4.52 (d, /= 4.52, 4H, OC#2CH=CH2), 4.15
(s, 2H, H2), 3.55 (m, 4H, H7), 3.48 (m, 4H, H5), 3.36 (m, 4H, H9), 3.08 (m, 4H, H3/H4), 2.94 (m, 4H, H3/H4), 2.25 (m, 2H, H13), 1.81 (m, 8H, H11/H12), 1.38 (m, 2H, HlO), 1.12 (m, 8H, H11/H12)
<78> 13C NMR U-DMSO, 100 MHz) : 5 174.94 (C14) , 172.93 (C1/C8) , 172.13
(C1/C8) , 170.24 (C6) , 133. 14 (OCH2Oi=CH2) , 117.75 (OCH2CH=OI2) , 64.41
(00I2CH=CH2) , 56.46 (C2) , 54.81 (C7) , 54.55 (C5) , 53.05 (C3/C4) , 52.27 (C3/C4) , 44.85 (C13) , 42.70 (C9) , 37.09 (ClO) , 29.58 (CIl) , 28.55 (C12)
<79> Calculated for C36H57N5O12TH2O: C: 49.25 , H: 8.15, N: 7.98
<80> Measured for C36H57N5O12TH2O: C: 49.13 , H: 7.80 , N: 8.17
<8i> FAB-MS Wz) :
<82> Calculated for C36H58N5O12: 752.41 ( [MH] +)
<83> Measured for C36H57N5O1^H2O: 752.55 ( [MH] +)
+.
<84> Calculated for C36H57N5NaO12: 774.39 ( [MNa] )
+,
<85> Measured for C36H57N50127H20: 774.50 ( [MNa] )
<86>
<87> Preparation Example 4 (Gadolinium complex Gd (L4))
<88> 0.73 g (1 mmol) of a ligand L4 was dissolved in 10 mL of the third distilled water and 0.18 g (0.5 mmol) of Gd2O3 was added. Suspension type reaction mixture was stirred at 100°C for 6 hours. A pale yellow solution was obtained at the end of the reaction. Dissoluble impurity and all of solvent were removed by passing the solution through CeI ite. The remained material was dissolved sufficiently by adding 5mL of methanol and was reprecipitated with 100 mL of acetonitrile to obtain the white solid. The obtained solid was filtered and then was dried. FIG. 1 shows a method of preparing a gadolinium complex briefly.
<89> From the experimental data below, the resulting product of the Preparation Example was identified as a gadolinium complex Gd (L4).
<90> Yield: 1.89 g (88%)
<9i> Calculated for C34H56GdN50158H20: C: 37.94, H: 6.74, N, 6.51
<92> Measured: C: 37.92 , H: 6.48 , N: 6.88
<93> FABMS WzY
<94> Calculated for C34H57GdN5O15: 933.09 ( [MH] +)
<95> Measured for C34H57GdN5O15: 932.77
<96> Cal culated for C34H55GdN5Oi4: 915.08 (MH - (H2O) )+
<97> Measured for C34H55GdN5O14: 914.84
<98>
<99> Preparation Example 5 (Gadolinium complex Gd (L5))
<ioo> This preparation method was progressed as the same way in Preparation Example 4, except that a ligand L5 was used instead of a ligand L4. FIG. 1 shows a method of preparing a gadolinium complex briefly.
<ioi> From the experimental data below, the resulting product of the preparation Example was identified as a gadolinium complex Gd (L5).
<iO2> Yield: 1.98 g (90%)
<i03> Calculated for C36H60GdN5O15SH2O: C: 39.16 , H: 6.94 , N: 6.34
<i04> Measured for C36H60GdN5Oi5SH2O: C: 39.02, H: 6.70, N: 6.65
<iO5> FABMS Wz) :
+
<iO6> Calculated for C36H59GdN5Oi4: 943.13(MH - (H2O) )
+
<iO7> Measured for C36H59GdN5Oi4: 942.78(MH - (H2O))
<108>
<iO9> Preparation Example 6 (Gadolinium complex Gd (L6))
<iio> This preparation method was progressed as the same way in Preparation Example 4, except that a ligand L6 was used instead of a ligand L4. FIG. 1 shows a method of preparing a gadolinium complex briefly.
<iii> From the experimental data below, the resulting product of the preparation Example was identified as a gadolinium complex Gd (L6).
<ii2> Yield: 1.80 g (87%)
<ii3> Cal culated for C36H56GdN50i36H20: C: 41.89, H: 6.64, N: 6.78
<ii4> Measured for C36H56GdN50i36H20: C: 42.09, H: 6.47 , N: 6.97
<ii5> FABMS im/z) :
<ii6> Calculated for C36H55GdN5Oi2: 907.1 (MH - (H2O) )+
<ii7> Measured for C36H55GdN5O12: 906.78
<118>
<ii9> The test in the Preparation Example was conducted by using standard Schlenk techniques under nitrogen atmosphere, and moisture was also previously removed from the required solvent. Reagents were purchased from Aldrich. The used water was third distilled water.
1 13
<i20> As H and C NMR measuring apparatus, Bruker Advance 400 or 500 in
Korea Basic Science Institute (RBSI) was used, and NMR measuring values were calculated on the basis of tetramethylsilane (TMS), which was preset to 0. Coupling constant (J) was measured by using Micromass QUATTRO II GC8000 series model, which has electronic energy ranging from 20 to 70 eV. The IR Spectrum was obtained by using Mattson FT-IR Galaxy 6030E in KBSI. Element analysis was conducted at the co-experimental laboratory in Kyungpook National University.
<i2i> In this experimental example below, in order to verify a remarkable effect of ligands (L4 to L6) and gadolinium complexes (Gd (L4) to Gd (L6)) of the present invention, they were compared with Omniscan, which is known as a paramagnetic contrast medium.
<122>
<123> Test Example 1 (Proton adding equilibrium constant, stabilization constant, selection constant, conditional stabilization constant, and pM value)
H
<124> Table 1 shows proton adding equilibrium constant(K\ ) of ligands (L4 to L6) prepared in a Preparation Example, and stabilization constant (ik), selection
conditional stabilization constant(K sei) of gadolinium complex es (Gd(L4) to Gd(L6)), and pM values of gadolinium (Gd(III)), calcium (Ca(II)), zinc (Zn(II)), and copper (Cu(II)) at pH 7.4 (the values represent bonding strength of ligands and metals).
<125> Proton adding equilibrium constant [K1 ) can be obtained by the
H 4" formula, Kx = [HjL]/ [HH1L] [H], wherein H;L means a proton added ligand, and i = 1, 2
<126> Stabilization constant iK&) can be obtained by the formula, 4L = [ML]/
[M] [L], wherein M is Gd, Ca, Zn, and Cu, L represents a ligand.
<i27> pM value can be obtained by the formula, pM = -log [M ], wherein pH = 7.4.
3 3
<i28> Further , the concentrat ions of [M]= l μ mol/dm and [L]= l . l u mol/dm were used. <129> [Table 1]
Constant log K (25t , μ, = 0.10 M (KCl))
L4 L5 L6 DTPABMA
[HL]Z[L][H] 9.94 9.95 9.81 9.37
[HzL]/[HLI[H] 5.15 5.18 4.81 4.38
[H3L]Z[H2L][H] 3.72 3.79 3.67 3.31
∑pAT, 18.81 18.92 18.29 17.06
[GdL]Z[Gd][L] 21.10 20.80 20.99 16.85
{logKcdL(pH7.4)} 18.56 18.25 18.58 14.84
[CaL]/[Ca][Ll 7.85 7.86 7.17 7.17
{logffcuføHM)} 5.31 5.31 4.76 5.11
[ZnL]Z[Zn][L] 11.78 11.80 1 1.03 12.04
(1OgA2nL(PH^)) 9.24 9.25 8.62 10.02
[CuL]ZlCu][L] 12.46 12.23 11.52 13.03
{logKc:uL(pH7.4)} 9.92 9.68 9.11 11.06
JlogK^Gd/Ca)] 13.25 12.94 13.82 9.68
pGd 17.56 17.25 17.58 13.88 pCa 4.31 4.31 3.76 4.19 pZn 8.24 8.25 7.61 9.06 pCu 8.92 8.68 8.11 10.05
<130>
<i3i> When a gadolinium complex was used as a contrast medium, gadolinium could be separated from a ligand in human body, and cell toxicity might be caused by a toxic gadolinium. In order not to make this separation, the stronger the bond between a ligand and a gadolinium might be, the more stable
it is. Thus, as can be seen at the Table 1, ligands (L4 to L6) use gadolinium (Gd) showing higher values than Omniscan (DTPABMA) in proton adding equilibrium constant (^ ), stabilization constant (4L), selection constant conditional stabilization constant (K'sei), and pM values, and it means that the bonding state between the ligand and the gadolinium is more stable, and it can be used as a good contrast medium.
<132>
<i33> Test Example 2 (Relaxation time and relaxation ratio)
<134> Relaxation time (Ti, T2) and relaxation ratio (Ri, R2) of gadolinium complexes (Gd (L4) to Gd (L6)) prepared in Preparation Example were measured. Even though the relatively less amount of contrast medium having high relaxation ratio was administrated, it showed a high contrast enhancing effect, and thus it was very important in magnetic resonance image. As can be seen in FIG. 2, Gd (L4) to Gd (L6) having higher molecular weights showed 1.6 times to 1.7 times higher relaxation ratio than that of Omniscan. Thus, the complex of the present invention can be considered as a contrast medium that can give signals more effectively. <i35> Further, FIG. 3 shows relaxation time map (Ti map) and relaxation ratio map (Ri map). In relaxation time map (Ti map), as relaxation time (Ti) is showed as a signal strength, the brighter signal strength means the longer relaxation time on the map. Thus, as can be seen in Fig. 3, Gd (L4) to Gd (L6) shows brighter signal strength comparing with Omniscan. Since the longer relaxation time can be confirmed, the contrast medium of the present invention can keep its contrast effect longer, and thus it is proved to be more effective. <136> Measurement of relaxation time Ti was progressed by a reverse recovery method of variable conducting time (TI) at 1.5T (64MHz). Magnetic resonance (MR) image needs another 35 TIs ranging from 50 to 1750 msec region. Relaxation time Ti was obtained from non-linear, square strokes of signal
strengths measured at each TI values.
<137> CPMG (Carr-Purcell-Meiboon-Gill) continuous wave for measuring relaxation time T2 can be obtained by measuring multi spinal echo. 34 images can acquire 34 other echo time (TE) ranging 10 to 1900 msec. Relaxation time T2 can be obtained from non-linear regular square forms for measuring multi spin-echo at each echo time. <138> Relaxation ratio(Ri and R2) was calculated as a reverse value of relaxation time(Ti and T2) per mM.
<139>
<i40> Test Example 3 (Cell toxicity)
<i4i> In order to investigate cell toxicity, MTT (Tetrazolium-based colorimetric) searching method was used. MTT searching method is to use an ability of mitochondria to reduce yellow soluble MTT tetrazolium to celadon green non-soluble MTT formazan (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide) by dehydrogenase function. If alive cells are treated with MTT tetrazolium, MTT tetrazolium is reduced to form MTT formazan (3- (4,5~dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) by reductase of mitochondria. That is, when the cell was treated with a certain compound depending on its concentrations for predetermined hours to induce cell death, and treated with MTT tetrazolium, MTT formazan was formed at low concentration not representing cell toxicity, and MTT formazan was not formed at high concentration representing cell toxicity. Cell viability can be determined by measuring the formation of MTT formazan depending on the concentration gradient of such compound.
<142> FIG. 4 shows the result of comparing cell toxicities of gadolinium complexes (Gd (L4) to Gd (L6)) and Omniscan by using MTT test. That is, the higher cell viability a gadolinium complex might have, the lower the cell toxicity is. Cells which was not added anything are regarded as a control group. Cell viability of a control group was determined as 100% by measuring formation of MTT formazan. When various concentrations (0.5mM to 1OmM) of
gadolinium complexes (Gd(L4) to Gd(L6)) and Omniscan were added to cells, the cells showed an amount of 60% or more of cell viability. It was identified that gadolinium complexes (Gd(L4) to Gd(L6)) having low cell toxicities can be effectively used a contrast medium through a graph of FIG. 4.
Claims
[CLAIMS] [Claim 1]
A method of preparing a new DTPA-bis-amide ligand, comprising: first step of adding DTPA-bis-anhydride to N,N-demethylformamide with stirring; second step of adding 2-hydroxyethyl-trans-A- (aminomethyl)cyclohexylcarboxylatehydrochloride to the mixture with stirring; third step of conducting silica gel chromatography after removing all of solvent under low pressure and dissolving the mixture in methanol; and fourth step of drying the product obtained in the third step under vacuum state to obtain a ligand L4. [Claim 2]
The method according to claim 1, wherein 2-methoxyethy1-trans-A- (aminomethyl)cyclohexylcarboxylatehydrochloride in the second step is added instead of 2-hydroxyethyl-£r,
2/?s^4-(aminomethyl )eyelohexylcarboxylate hydrochloride to obtain a ligand L5 in the fourth step.
[Claim 3]
The method according to claim 1, wherein allyWraπs-4- (aminomethyl)cyclohexylcarboxylatehydrochloride in the second step may be added instead of 2-hydroxyethyWra/7s-4- (aminomethyl)cyclohexylcarboxylatehydrochloride to obtain a ligand L6 in the fourth step.
[Claim 4]
The method according to any one of claims 1 to 3, further comprising: fifth step of putting the DTPA-bis-amide ligand obtained in fourth step to the distilled water, and adding Gd2C"3 with stirring to prepare a mixture solution; sixth step of removing impurity and solvent from the mixture solution obtained in the fifth step; and seventh step of dissolving the material obtained in the sixth step in methanol and precipitating it with acetonitrile to obtain a solid.
[Claim 5]
The method according to claim 4, wherein a ratio of molarity of the DTPA-bis-amide ligand to that of Gd2O3 in the fifth step is 1 to 1.
[Claim 6]
The method according to claim 4, wherein the stirring of the fifth step is conducted at a temperature ranging from 90 °C to 100 °C for the time ranging from 5 hours to 7 hours. [Claim 7]
The method according to claim 4, wherein the sixth step comprises passing the mixture solution obtained in the fifth step through CeI ite to remove impurity and solvent. [Claim 8]
A DTPA-bis-amide ligand prepared by the method according to any one of claims 1 to 3. [Claim 9]
The DTPA-bis-amide ligand according to claim 8, wherein the DTPA-bis- amide ligand has an improved thermodynamic stability. [Claim 10]
A gadolinium (Gd) complex prepared by the method according to any one of claims 4 to 7. [Claim 11]
The gadolinium (Gd) complex according to claim 10, wherein the gadolinium (Gd) complex is expressed by a chemical formula (1) below: [Gd(L)(H2O)] -H2O (1), where L represents a ligand according to claim 8, and x represents 0 to 12. [Claim 12]
The gadolinium (Gd) complex according to claim 11, wherein the gadolinium (Gd) complex has an improved solubility and magnetic relaxation ratio. [Claim 13]
The gadolinium (Gd) complex according to claim 11, wherein the gadolinium (Gd) complex has a low cell toxicity. [Claim 14]
An MR contrast medium for diagnosing cancer containing the gadolinium (Gd) complex according to any one of claims 10 to 13.
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KR1020080049115A KR20090123171A (en) | 2008-05-27 | 2008-05-27 | Gd complex comprising dtpa-bis-amide ligand and method for preparing the same |
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KR101236142B1 (en) * | 2010-09-30 | 2013-02-21 | 경북대학교 산학협력단 | MRI contrast agents comprising Gd-complexes |
KR101239129B1 (en) * | 2011-01-10 | 2013-03-05 | 한국원자력의학원 | Dual SPECT/MRI contrast agent and a process for the preparation thereof |
KR101239130B1 (en) * | 2011-01-10 | 2013-03-05 | 한국원자력의학원 | Magnetic resonance imaging contrast agent and a process for the preparation thereof |
KR101625656B1 (en) * | 2015-10-16 | 2016-05-30 | 최경석 | Process for preparing contrast agent for magnetic resonance imaging |
Citations (2)
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US5434287A (en) * | 1990-03-26 | 1995-07-18 | The United States Of America As Represented By The Department Of Health And Human Services | Bifunctional DTPA-type ligand |
US20010019709A1 (en) * | 1996-11-04 | 2001-09-06 | Werner Krause | Use of metal chelates as radiosensitizers |
-
2008
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US5434287A (en) * | 1990-03-26 | 1995-07-18 | The United States Of America As Represented By The Department Of Health And Human Services | Bifunctional DTPA-type ligand |
US20010019709A1 (en) * | 1996-11-04 | 2001-09-06 | Werner Krause | Use of metal chelates as radiosensitizers |
Non-Patent Citations (1)
Title |
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DUTTA, S. ET AL.: "Gd-complexes of DTPA-bis(amide) conjugates oftranexamic acid and its esters with high relaxivity and stability for magnetic resonance imaging", DALTON TRANSACTIONS, vol. 16, 13 March 2008 (2008-03-13), pages 2199 - 2206 * |
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