KR101931153B1 - Dtpa chelator specific for lysine and method for preparing the same - Google Patents

Abstract

The present invention relates to a DTPA chelator and to a manufacturing method thereof, and more particularly, to a DTPA chelator represented by chemical formula (I), a manufacturing method thereof, proteome-DTPA chelator conjugates conjugated to at least one of Lysine residues among amino sequences of the DTPA chelator, and a manufacturing method thereof. In the formula, l is an integer from 1 to 4, m is an integer from 1 to 10, and the -NCS is substituted at an ortho, meta or para position.

Description

TECHNICAL FIELD [0001] The present invention relates to a DTPA chelator having a thioether bond and an optionally bonded amine, and a method for producing the DTPA chelator.

The present invention relates to a DTPA chelator which specifically binds to an amine (NH ₂ ) present in a lysine residue of a protease and the like, and more particularly to a DTPA chelator which is excellent in structural flexibility and contains an amine NH ₂ ) and the amino acid residue (NH ₂ ) at the proximal end of the amino acid residues in the proteome and has a thioether bond, A DTPA chelator bound by a sieve, a novel synthetic method thereof, a chelator-bonded material and a radioisotope-labeled substance using the chelator.

In the case of a chelator used for labeling a peptide composed of a protein and an amino acid such as an antibody, an antibody fragment, a protein, etc., the amine (NH2) present in residues of lysine present in the protein, A method using a chelator having a reactor which is easy to react with an amine of an amine is used.

For example, the chelator can be used to treat therapeutic cancer cancers such as Pb-212, Bi-213, Th-232, and Sc-47, Y-90, Sm -153, Ho-166, Lu-177, Re-188 and the like, and positron emission radionuclides such as Sc-44, Ga-68 and Zr-89 which are diagnostic radioisotopes for diagnosis And gamma-emitting radionuclides such as Ga-67, Tc-99m, and In-111.

As a chelator that has been developed and used so far, for example, a chelator produced by using 4-amino-phenylalanine or 4-nitro-phenylalanine is used. However, such a calator has a problem that the binding site has a short length when bound to a radioactive labeling compound, and isotope binding may affect the activity. Thus, when the isotope is bound to a site that interferes with the binding of the active site or the active site, the target function of the physiologically active substance such as a protease may be lost.

Accordingly, a chelator having a linker space sufficient to reduce the adverse effect on the activity of the protease, having a certain degree of structural flexibility even when bound to a physiologically active substance, and binding the radioactive isotope of a metal to a coordinator is provided It is expected that it can be widely applied in related fields. In addition, binding of the element in the linker space to the radioactive isotope of the metal will improve the binding stability between the metal and the chelator.

Korea Publication No. 2004-0111640

One aspect of the present invention is to provide a DTPA chelator that is excellent in structural flexibility and specifically binds to a lysine (K) residue in an amino acid residue.

Another aspect of the present invention is to provide a low molecular weight compound-DTPA chelator conjugate wherein the DTPA chelator is conjugated to a low molecular weight compound.

Another aspect of the present invention is to provide a proteome-DTPA chelator conjugate conjugated to such a DTPA chelator.

Another aspect of the present invention is to provide a method for producing such a DTPA chelator conjugate.

Another aspect of the present invention is to provide a novel method of synthesizing such a DTPA chelator.

Another aspect of the present invention is to provide a diagnostic agent and a therapeutic agent to which the DTPA chelator is conjugated.

According to one aspect of the present invention, there is provided a compound represented by the following formula (I)

Wherein l is an integer from 1 to 4, m is an integer from 1 to 10,

Wherein the NCS is substituted at an ortho, meta or para position.

According to another aspect of the present invention, there is provided an amine-containing low-molecular compound-DTPA chelator conjugate wherein the DTPA chelator of the present invention is conjugated with an amine moiety of a low molecular weight compound having an amine moiety.

According to another aspect of the present invention, there is provided an amine-containing protease-DTPA chelator conjugate wherein the DTPA chelator of the present invention is conjugated to at least one Lysine residue in the amino acid sequence.

According to another aspect of the present invention, there is provided a method for preparing a protease-DTPA chelator conjugate comprising reacting an amine-containing or low-molecular-weight compound with the DTPA chelator of the present invention at a temperature of more than 0 to 50 for 10 minutes to 48 hours Method is provided.

According to still another aspect of the present invention, there is provided a process for preparing a compound represented by formula (a1), comprising reacting at least one of the compound represented by formula (a1) and the compound represented by formula (a2) ₂ and a cyclohexyl compound substituted with an -NH-Boc group,

In the formula (b1) and (b2), -NO _2, and -HN-Cbz is substituted on each of the ortho, meta or para position,

 (I), (a1) and (a2), l is an integer of 1 to 4, and in the above formulas (I), (b1) and (b2), m is an integer of 1 to 10 )

There is provided a process for preparing the DTPA chelator of formula (I).

According to another aspect of the present invention, there is provided a cancer diagnostic agent comprising a DTPA chelator conjugate conjugated with the DTPA chelator of the present invention to a low molecular compound or a proteome comprising at least one amine moiety.

According to another aspect of the present invention, there is provided a therapeutic agent for cancer comprising a DTPA chelator conjugate conjugated with the DTPA chelator of the present invention to a low molecular compound or a proteome comprising at least one amine moiety.

According to the present invention, it is possible to obtain a DTPA chelator excellent in structural flexibility, which specifically binds to an amine (NH2) and an amino acid residue, Lysine (K), and a novel synthesis method thereof. The synthesis method of the present invention can freely control the length of the linker, minimizing the degradation of physiological activity caused by binding of the chelator, and having thioether in the structure, 9 coordinator binding can be achieved and the stability of the metal-chelator binding is excellent. Thus, the DTPA chelator of the present invention can be used as a therapeutic agent for disease-target peptides including tumors and the like, therapeutic agents such as existing antibodies, E-Coli, It is expected that it will be widely used for the research and development to develop a new radiopharmaceutical using the modified protease.

Figure 1 shows the result of radioisotope labeling for the chelator of the present invention.
Figure 2 shows the result of radioisotope labeling (a) and post-5 day stability (b) for the chelator and rituximab conjugate of the present invention.
FIG. 3 shows HPLC results (b) and mass spectrometry results (c) of Compound 7 in the chelator synthesis process of the present invention in Examples.
FIG. 4 shows the structure (a), HPLC result (b) and mass spectrometry (c) of the compound (PSMA-DTPA) of the chelator and peptide conjugate of the present invention.
FIG. 5 shows an example of the chelator and proteome binding of the present invention.
Figure 6 shows the radioisotope binding structure (a) for the chelator of the present invention and the binding (b) between the radioisotope labeled chelator and the proteome.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, there is provided a DTPA chelator having a thioether moiety which can be easily bound to a radioactive isotope in a reaction in an aqueous solution at room temperature to improve label stability.

The DTPA chelator of the present invention has a property of specifically binding to an amine. More specifically, the DTPA chelator of the present invention, when bound to a protease, has an amino acid sequence of an alpha carbon amine or a protease Lysine, < / RTI > K) residues.

The DTPA chelator of the present invention is represented by the following formula (I).

Wherein l is an integer of 1 to 3, m is an integer of 1 to 10, and the -NCS may be substituted at an ortho, meta or para position.

When m is an integer of 11 or more, the hydrophobicity increases, and if it is not a salt form, it exists in an oil form. there is a problem.

Preferably, the DTPA chelator is represented by the following formula (II): wherein l is an integer of 1 to 2 and m is an integer of 1 to 5.

When the -NCS is present at the para-meta position as in the formula (II), it is preferable because it can be bonded to the amine present in the protease or the like without structural interference.

More preferably, the DTPA chelator of the present invention is represented by the following formula (III).

Since the DTPA chelator of the present invention includes a thioether moiety in particular, the bond stability with the metal can be greatly improved by forming a bond with a metal by a pair of non-covalent electrons existing in sulfur (S) of a thioether moiety.

Although not shown in the formula of the present invention, the unsubstituted hydrogen present in the hydrocarbon chain such as the backbone or the benzene ring of the DTPA chelator of the present invention may be added to the DTPA chelator of the present invention within a range that does not affect the structural characteristics of the DTPA chelator of the present invention. For example, a C _1-8 alkyl group or the like.

The DTPA chelator of the present invention can be coupled with a metal in an 8-valence or 9-valence form, wherein the metal can be a radioactive isotope.

The radioactive isotope that can be labeled on the DTPA chelator of the present invention is not particularly limited. For example, the radioactive isotope Sc-44, Ga-67, Ga-68, Zr-89, Tc- In-111 and the therapeutic radioisotope Sc-47, Y-90, Sm-153, Ho-166, Lu-177, Re-188, Pb-212, Bi-213 and Th- In particular, the DTPA chelator of the present invention has a property of binding to these radioactive isotopes at room temperature.

More specifically, the DTPA chelator of the present invention can stably bind a radioisotope by reacting the radioisotope with an aqueous solution of pH 3 to 7 at a temperature ranging from 4 to 45 캜 for about 5 minutes or more.

The DTPA chelator of the present invention may be labeled with a structure of formula (IV) or formula (V), for example, as follows in the radioactive isotope.

According to the present invention, there is provided a low molecular compound-DTPA chelator conjugate wherein the DTPA chelator of the present invention is conjugated with an amine moiety existing in a low-molecular compound having an amine moiety such as a peptide.

The low-molecular compound may be a peptide having an amine moiety, a hormone, or a combination thereof, but is not limited thereto, and may be any substance including an amine moiety. For example, the low-molecular compound may be at least one selected from the group consisting of PSMA, folate, RGD, bombesin and a-MSH.

According to the present invention, there is provided a protease-DTPA chelator conjugate wherein the DTPA chelator of the present invention is conjugated to at least one lysine residue in the amino acid sequence of the proteome.

The DTPA chelator of the present invention is specifically conjugated to an amine including a lysine residue, and in the case of a protein having an amino acid sequence comprising several lysine residues and an amine, an amine containing at least a portion of a lysine residue And the DTPA chelator of the present invention can be bonded.

Preferably, one to five DTPA chelators of the invention are conjugated to one proteome. When the number is less than 1, the labeling effect may be insufficient, and when it exceeds 6, there is a problem that the physiological activity is lowered.

The protein may be an antibody, a protein, an antibody-derived protein, a hormone, a peptide, or a combination thereof including an amino acid sequence including lysine. For example, the protein may be selected from the group consisting of rituximab, cetuximab and trastuzumab At least one antibody selected from the group or at least one fragment derived from said antibody, at least one selected from the group consisting of annexin V and afibibody.

The protein may be an antibody, a protein, an antibody-derived protein (ScFv, etc.), a hormone, a peptide, or a combination thereof containing an amino acid sequence including lysine. It does not.

For example, the protease may be selected from the group consisting of at least one antibody selected from the group consisting of rituximab, cetuximab, and trastuzumab, or at least one fragment derived from the antibody, annexin V and afibibody At least one, and the fragment may be (Fab) 2, Fab, ScFv, or the like.

The method for preparing the DTPA chelator conjugate with the protease or the low molecular weight compound may be carried out by mixing the protease or the low molecular weight compound with the DTPA chelator of the present invention at a pH of 5.5 to 8.5 on a buffer in the absence of amine, At a temperature of 4 to 45 캜, for example 4 to 30, for 10 minutes to 48 hours, for example 10 minutes to 3 hours.

The temperature and pH can be selected by selecting the DTPA chelator conjugate preparation and the condition that does not change the physiological activity properties of the low-molecular compound and the protein (protein) to be conjugated.

Preferably, the protease and the DTPA chelator of the present invention are mixed and reacted at a temperature of 4 to 30 ° C for 10 minutes to 3 hours in a HEPES buffer (50 mM HEPES Buffer, Ph 7.4).

In the method for preparing a DTPA chelator conjugate with the above-described proteolytic or low-molecular compound, the DTPA chelator is preferably mixed in a ratio of 1 to 10 equivalents based on 1 equivalent of a protease or a low molecular weight compound. When the amount of the DTPA chelator is less than 1 equivalent based on 1 equivalent of the protease or the low molecular weight compound, the strength may be insufficient when the DTPA chelator is used for radioisotope labeling, etc. When the DTPA chelator is more than 10 equivalents There is a possibility that a change in physiological activity characteristics may occur.

The DTPA chelator of the present invention is obtained by obtaining a DTPA chelator having thioether using cysteine among the amino acids constituting the protease. In particular, according to the present invention, a linker having a binding site Linker) can be freely adjusted by changing the length of the link.

More specifically, the method for preparing the DTPA chelator of formula (I) of the present invention comprises reacting at least one compound of formula (a1) and a compound of formula (a2) With a reaction compound (c) which is a cyclohexyl compound substituted with -NH ₂ and -NH-Boc groups, with at least one of them being a starting material:

In the formula (b1) and (b2), -NO _2, and -HN-Cbz is substituted on each of the ortho, meta or para position,

In the above formulas (I), (a1) and (a2), l is an integer of 1 to 4. In the above formulas (I), (b1) and (b2), m is an integer of 1 to 10.

Therefore, according to the present invention, the length of the linker can be easily controlled by controlling the number of carbon atoms of 1 and m.

The reaction compound (c) may be N-Boc-1,2-diaminocyclohexane.

Further, according to the present invention, there is provided a cancer diagnostic agent and a therapeutic agent comprising a DTPA chelator conjugate to which the DTPA chelator of the present invention is conjugated to a low molecular compound or a proteome comprising at least one amine moiety (-NH ₂ ).

For example, if the protease is at least one antibody selected from the group consisting of rituximab, cetuximab, bevacizumab and trastuzumab, or at least one fragment derived from the antibody, annexin V, etc., . ≪ / RTI >

The cancer may be, but is not limited to, non-Hodgkin's lymphoma, head and neck cancer, lung cancer, follicular lymphoma, sub-broad base lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, colon cancer, breast cancer, And may vary depending on the indications of the proteome that can be applied to the present invention.

That is, the DTPA chelator of the present invention can be used for treatment and diagnosis of cancer through conjugation with a low molecular weight compound and an antibody, but the application of the present invention is not limited thereto, and the use of E-Coli, And can be used for research and development to develop new radiopharmaceuticals using the developed proteomics.

It is also expected to be useful as a chelator for radioisotope labeling for the visualization of the behavior of new protein therapeutics or for various clinical applications.

Hereinafter, the present invention will be described more specifically by way of specific examples. The following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

1. The present invention DTPA Chelator  synthesis

The DTPA chelator of the present invention can be synthesized by the following Schemes 1-1, 1-2 and the like.

 [Scheme 1-1]

[Scheme 1-2]

In the following, a concrete synthesis procedure will be described based on Scheme 1-1.

(1) Preparation of Compound (1)

A solution containing N-Boc cysteine methyl ester (8 g, 34 mmol), cesium carbonate (22 g, 68 mmol) and acetonitrile (MeCN, 120 mL) was stirred in an ice- water bath). A solution of 4-nitrophenyl ethyl bromide (7.82 g, 34 mmol) in acetonitrile (30 mL) was slowly added dropwise while stirring the solution, and the mixture was reacted in an ice-water bath for 30 minutes. After the reaction, the reaction mixture was filtered using filter paper, and the solution was removed using a rotary evaporator. Further, EtOAc / H ₂ O was added and the organic layer was extracted using a separatory funnel, and the extracted organic layer was washed with NaHCO ₃ Washed with an aqueous solution, the organic layer over anhydrous Na ₂ SO ₄ After passing through the pad, the remaining water was removed, and the solvent was removed using a rotary evaporator. Further, the material was loaded onto silica gel flash column chromatography and separated by 20-40% EtOAc / n-Hx) to obtain 12 g (white solid form, 92%) of 1 Was obtained.

^{1 H NMR (400 MHz, CDCl} 3) d = 8.16 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H), 5.32 (d, J = 7.2 Hz, 1H), 4.58-4.50 (m, 1H), 3.05-2.92 (m, 4 H), 2.86 - 2.80 (m, 2 H), 1.44 (s, 9 H); MS (ESI +) m / z 285.5 [M-Boc] ⁺ , 407.5 [M + Na] < ⁺ >.

(2) Preparation of Compound 2

A solution of compound 1 (9.66 g, 25 mmol) in THF (20 mL) was slowly added dropwise to a solution of sodium borohydride (5.7 g, 15 mmol) in THF (80 mL) to obtain a reaction mixture. The mixture was heated to reflux for 15 minutes and then cooled to room temperature. Methanol (50 mL) was slowly added dropwise to the reaction mixture for 15 minutes and then heated to reflux for 30 minutes. The reaction was terminated by addition of aqueous NH ₄ Cl and concentrated under reduced pressure using a rotary evaporator.

The organic layer was extracted with EtOAc / H ₂ O using a separatory funnel. The extracted organic layer was washed with a saturated aqueous solution of NH ₄ Cl, washed with anhydrous Na ₂ SO ₄ After passing through the pad to remove the remaining water, the solvent was removed under reduced pressure using a rotary evaporator. Silica gel flash column chromatography (Silica-gel Flash Column Chromatography, 40% EtOAc / n-Hx) to yield 8.94 g (yellow solid form, 99% Was obtained.

^{1 H NMR (400 MHz, CDCl} 3) d = 8.16 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 4.99 (br s, 1H), 3.84-3.68 (m, 3H), 3.05-2.98 (m, 2H ), 2.92-2.70 (m, 2H), 2.70-2.58 (m, 2H), 2.15 (br s, 1H), 1.45 (s, 9H); MS (ESI +) m / z 257.4 [M-Boc] ⁺ , 379.3 [M + Na] < ⁺ >.

(3) Preparation of Compound 3

Dess-Martin periodinane (8 g, 18.8 mmol) was weighed in a well-dried round flask under Ar (g) conditions and dichloromethane (DCM, 90 mL) , tert-butanol (1.6 mL, 16.5 mmol) was added and the mixture was cooled with an ice-water bath. A solution obtained by dissolving Compound 2 (5.35 g, 15 mmol) in dichloromethane (20 mL) was slowly added dropwise thereto to prepare a reaction mixture. After stirring for 1 hour the reaction mixture under ice-water bath, Na ₂ S ₂ O ₃ An aqueous solution (15 mL, 13.3 g / H ₂ O 15 mL) was slowly added dropwise to terminate the reaction. Then, NaHCO ₃ The pH of the reaction solution was adjusted to about 7 to 8 while the aqueous solution was slowly added. The organic layer was extracted with dichloromethane using a separatory funnel, washed with anhydrous Na ₂ SO ₄ After passing through the pad, the remaining water was removed, and the solvent was removed under reduced pressure using a rotary evaporator. Silica gel flash column chromatography (Silica-gel Flash Column Chromatography, 40% EtOAc / n-Hx) to yield 4.62 g (yellow oil form, 87%) of 3 Was obtained.

^{1 H NMR (400 MHz, CDCl} 3) d = 9.64 (s, 1H) , 8.17 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 5.41 (br s, 1H), 4.40-4.22 (m, 1H), 3.06-2.94 (m, 4H), 2.92-2.80 (m, 2H), 1.46 (s, 9H); MS (ESI +) m / z 255.3 [M-Boc] ⁺ , 377.4 [M + Na] < ⁺ >.

(4) Preparation of Compound 4

The reaction mixture of compound 3 (4.57 g, 12.9 mmol), N-Boc-trans-1,2-diaminocyclohexane (2.76 g, 12.9 mmol) and dichloroethane (90 ml) under Ar (g) After stirring, sodium triacetoxyborohydride (3.83 g, 18.1 mmol) was added thereto, followed by stirring for an additional 1 hour. After cooling with ice water bath, NaHCO ₃ An aqueous solution was added to terminate the reaction. The organic layer was extracted with dichloromethane (DCM) using a separatory funnel, washed with anhydrous Na ₂ SO ₄ After passing through the pad, the remaining water was removed and the solvent was removed under reduced pressure using a rotary evaporator. Silica gel flash column chromatography (Silica-gel Flash Column Chromatography, 2-5% MeOH / DCM) to give 4.88 g (yellow oil form, 69%) of 4 Was obtained.

^{1 H NMR (400 MHz, CDCl} 3) d = 8.16 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 5.04 (br d, 1H), 4.45 (br s, 1H), 3.71 (br s, 1H), (M, 2H), 1.75-1.65 (m, 2H), 2.32-2.14 (m, , 2H), 1.44 (s, 9H), 1.43 (s, 9H), 1.35-1.10 (m, 5H); MS (ESI +) m / z 554.3 [M + H] < ⁺ >.

(5) Preparation of Compound 5

Compound 4 (4.82 g, 8.72 mmol) was stirred in a solution of 20% trifluoroacetic acid (TFA) / dichloromethane (DCM) (100 mL, v / v) for 1 hour and then the solvent was removed using a rotary evaporator ≪ / RTI > NaHCO ₃ The pH was adjusted to 8 with aqueous solution and extracted with dichloromethane (DCM) using a separatory funnel. The organic layer over anhydrous Na ₂ SO ₄ After passing through the pad, the remaining water was removed, and the solvent was removed under reduced pressure using a rotary evaporator. Potassium carbonate (9.63 g, 70 mmol) and tert-butyl bromoacetate (7.1 mL, 48 mmol) were added to the reaction mixture, which was then dissolved in acetonitrile (120 mL) under Ar Stir at room temperature. The resulting solid was removed using a filter, and the solvent was concentrated under reduced pressure using a rotary evaporator. Dichloromethane (DCM) was added to dissolve and the organic layer was extracted using a separatory funnel and anhydrous Na ₂ SO ₄ After passing through the pad to remove the remaining water, the solvent (dichloromethane, DCM) was removed under reduced pressure. Amino silica gel flash column chromatography (Amino Silica-gel Flash Column Chromatography, 5% EA / n-Hx) yielded 4.53 g (yellow oil form, 56%) of 5 Was obtained as an isomer mixture.

^{1 H NMR (400 MHz, CDCl} 3) d = 8.16-8.10 (m, 2H) , 7.45-7.39 (m, 2H), 3.64-3.26 (m, 10H), 3.10-2.50 (m, 12H), 2.10-1.86 (m, 2H), 1.76-1.62 (m, 2H), 1.50-1.38 (m, 45H), 1.20-1.00 (m, 4H); ¹³ CNMR (100 MHz, CDCl ₃ ) d = 172.78, 172.05, 171.78, 171.68, 171.58, 171.56, 171.39, 149.22, 148.91, 148.27, 146.63, 146.56, 129.70, 129.67, 129.56, 123.82, 123.70, 123.64, 80.57, 80.50, 32.62, 61.26, 54.99, 54.12, 53.25, 52.86, 52.76, 52.42, 51.91, 36.27, 36.21, 35.93, 33.82, 33.71, 33.53, 33.28, 30.63, 29.23, 28.89, 28.47, 28.28, 28.17, 28.10, 27.16, 26.21, 26.11, 26.00, 25.94, 25.89; MS (ESI +) m / z 924.3 [M + H] < ⁺ >.

(6) Preparation of Compound 6

Compound 5 (4.53 g, 4.9 mmol), dissolved in methanol (80 mL) and ethyl acetate (30 mL) under Ar (g), was treated with palladium on activated carbon (2.61 g, 1.23 mmol, 10% Pd, wet, Dugussa type) and purged with H ₂ (g). After stirring at room temperature for 18 hours, the solvent of the celite-filtered reaction mixture was concentrated under reduced pressure using a rotary evaporator. Amino silica gel flash column chromatography (Amino Silica-gel Flash Column Chromatography, 20-40% EA / n-Hx) yielded 3.16 g (colorless oil, 72%) of 6 Was obtained as an isomer mixture.

^{1 H NMR (400 MHz, CDCl} 3) d = 7.04-6.98 (m, 2H) , 6.64-6.58 (m, 2H), 3.70-3.24 (m, 10H), 3.12-2.50 (m, 12H), 2.10-1.90 (m, 2H), 1.70-1.52 (m, 2H), 1.50-1.38 (m, 45H), 1.20-1.00 (m, 4H); MS (ESI +) m / z 894.2 [M + H] < ⁺ >.

(7) Preparation of Compound 7

A solution of Compound 6 (3.16 g, 3.53 mmol) in HCl (150 mL, 6N (aq.)) Was stirred at room temperature for 2 hours and then concentrated under reduced pressure using a rotary evaporator. Water (H ₂ O, 30 mL) and chloroform (CHCl ₃ , 30 mL) were placed in an ice water bath and thiophosgene (479 ml, 6.28 mmol) was slowly added thereto. After stirring at room temperature for 2 hours, the water layer was separated using a partition wall funnel and wiped with dichloromethane (DCM), and the water layer was concentrated under reduced pressure. C18 Silica gel flash column chromatography (Amino Silica-gel Flash Column Chromatography, 20-40% acetonitrile / water) to give 1.58 g (white solid, 66%) of substance 7 as an isomeric mixture.

MS (ESI +) m / z 655.9 [M + H] < ⁺ >.

HPLC conditions: 30 to 100% B for 20 min (A: 0.1% TFA in water, B: Acetonitrile), R t = 10.04 min.

The HPLC results (b) and the mass spectrometry results (c) of the compound 7 are shown in FIG.

2. The present invention DTPA With chelator Protein  Assembly

 (1) Preparation of Rituximab (anti-CD 20 target antibody) conjugate

Rituximab is isolated and recovered from the antibody therapeutic vial composition using a 30 kDa molecular cut-off filter. At this time, it is recovered with 100 mM HEPES buffer (pH 7.5) or 100 mM PBS buffer (pH 7.4).

The compound 7 was reacted with 4 equivalents of the compound of the formula (1) based on 1 equivalent of rituximab for 2 hours at room temperature or for 24 hours or more at 4 캜 for 24 hours or more to obtain the compound 7 in which some of the amino acid residues of rituximab Lt; / RTI >

Thereafter, ultra-centrifugation was performed using a molecular cut-off spin column filter of 30 kDa to remove unreacted compound 7, and DTPA chelator (compound 7) conjugated rituximab Was recovered with 50 mM acetate buffer (pH 5).

(2) Preparation of cetuximab (anti-EGFR target antibody) conjugate

Cetuximab is isolated and recovered from the antibody therapeutic vial composition using a 30 kDa molecular cut-off filter. At this time, it is recovered with 100 mM HEPES buffer (pH 7.5) or 100 mM PBS buffer (pH 7.4).

The compound 7 was reacted with 4 equivalents of the compound 7 at room temperature for 2 hours or at 4 캜 for 24 hours or more on the basis of 1 equivalent of cetuximab to obtain the compound 7 with a portion of the lysine (K) residue of the cetuximab amino acid sequence .

Subsequently, ultracentrifugation was performed using a molecular cut-off spin column filter of 30 kDa to remove unreacted compound 7 and the DTPA chelator (compound 7) conjugated Cetuximab Was recovered with 50 mM acetate buffer (pH 5).

(3) Preparation of Bis-Annexin V conjugate

Compound 7 was reacted with 4 equivalents of Bis-Annexin V protease at room temperature for 2 hours or at 4 ° C for at least 24 hours based on 1 equivalent of Bis-Annexin V protein in 100 mM PBS buffer (pH 7.4) To bind to some of the amino acid sequences of the Bis-Annexin V protease with the Lysine [K] residue.

Thereafter, unreacted Compound 7 was removed by ultracentrifugation using a molecular cut-off spin column filter of 30 kDa and DTPA chelator (compound 7) conjugated Bis-Annexin V protease was recovered with 50 mM acetic acid buffer (pH 5).

(4) Preparation of low molecular compound PSMA conjugate

The compound 7 was reacted with 1.5 equivalents of the compound 7 at room temperature for 2 hours based on 1 equivalent of a PSMA derivative compound [Glu-Urea-Lys (Ahx)] contained in 100 mM PBS buffer (pH 7.4) A coupled DTPA chelator-PSMA conjugate was prepared.

MS (ESI +) m / z 1087.7 [M + H] < ^{+ &}

HPLC conditions: 30 to 100% B for 20 min (A: 0.1% TFA in water, B: Acetonitrile), R t = 11.94 min.

3. Radioisotope  sign

(1) radioisotope labeling of the chelator of the present invention

20 μg of DTPA chelator (Compound 7) is dissolved in acetic acid buffer (50 mM, pH 5, 100 μl) and placed in the vial. After injecting 1 mCi of radioactive isotope Y-90 contained in 50 μl of 0.05 N HCl solution using a syringe The reaction is carried out by slowly stirring at room temperature for 10 minutes.

For the labeling yield evaluation, 2 μl of the solution was added to the ITLC-SG plate and developed using a 75% methanol solution. The developed ITLC-SG plate was dried, and labeling yield was confirmed using a TLC scanner (Scan-RAM, RadioTLC Detector).

(Unlabeled Y-90, Rf = 0 to 0.1, Y-90-chelator, Rf = 0.8 to 1.0) as shown in Fig.

(2) Radioisotope labeling according to the concentration of the chelator of the present invention

1000 μg of the DTPA chelator (Compound 7) is dissolved in acetic acid buffer (50 mM, pH 5, 100 μl) and placed in the vial. A portion of this was taken to obtain the amounts of the DTPA chelator compounds of 0.1, 1, 5, 10 and 50 μg (0.13, 1.3, 6.5, 13, 65 nmol), respectively, followed by the addition of acetic acid buffer (50 mM, pH 5) The volume of the total solution was made up to 100 mu l. After injection of 0.1 mCi radioactive isotope Y-90 in 50 μl of 0.05 N HCl solution using a syringe The reaction is carried out by slowly stirring at room temperature for 10 minutes.

For the labeling yield evaluation, 2 μl of the solution was added to the ITLC-SG plate and developed using a 75% methanol solution. The developed ITLC-SG plate was dried, and the label yield was confirmed using a TLC scanner (Scan-RAM, RadioTLC Detector). The results are shown in Table 1.

Label yield according to DTPA chelator compound amount DTPA chelator compound amount Cover yield (%) G nmol 0.1 0.13 - One 1.3 10 5 6.5 95 10 13 98 20 26 98 50 65 98

(3) Radioisotope labeling of the chelator and rituximab conjugate of the present invention

500 μg of DTPA chelator (Compound 7) conjugated Acetate buffer (50 mM, pH 5, 500 μl) containing rituximab is loaded into the vial. After injecting 1 mCi of radioactive isotope Y-90 contained in 50 μl of 0.05 N HCl solution using a syringe The reaction is carried out by slowly stirring at room temperature for 10 minutes.

After the reaction, 2-5 μl of the solution was added to the ITLC-SG plate for evaluation of label yield, and developed using a sodium citrate solution (50 mM, sodium citrate solution).

The developed ITLC-SG plate was dried, and labeling yield was confirmed using a TLC scanner (Scan-RAM, RadioTLC Detector).

(Y-90, Rf = 0.8 ~ 1.0, Y-90-chelator conjugated rituximab, Rf = 0 ~ 0.1) was produced at a yield of 98% or more as shown in Figure 2 , And further showed excellent stability even after a lapse of 5 days (Fig. 2 (b)).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, It will be obvious to those of ordinary skill in the art.

(4) Radioisotope labeling of the chelator and PSMA target compound conjugate of the present invention

50 μg of DTPA chelator conjugated PSMA The target compound is dissolved in acetic acid buffer (50 mM, pH 5, 100 μl) and placed in a vial. After injection of 0.1 mCi of radioactive isotope Y-90 contained in 50 μl of 0.05 N HCl solution using a syringe The reaction is carried out by slowly stirring at room temperature for 10 minutes.

For label yield evaluation, 5 μl of the solution was added to the ITLC-SG plate and developed using a 75% methanol solution. The developed ITLC-SG plate was dried, and labeling yield was confirmed using a TLC scanner (Scan-RAM, RadioTLC Detector). (Unlabeled Y-90, Rf = 0 to 0.1, Y-90-chelator conjugated PSMA target compound conjugate, Rf = 0.8 to 1.0)

The compound (PSMA-DTPA) structure of the chelator-peptide conjugate of the present invention is shown in FIG. 4 (a).

Claims (18)

Is represented by the following formula (I)

Wherein l is an integer from 1 to 4, m is an integer from 1 to 10,
Wherein the NCS is substituted at an ortho, meta or para position.
The method of claim 1, wherein the DTPA chelator is represented by the following formula (II)

Wherein l is an integer from 1 to 2 and m is an integer from 1 to 5. 15. The DTPA chelator of claim < RTI ID = 0.0 > 1, < / RTI >
The DTPA chelator of claim 1, wherein the DTPA chelator is represented by the following formula (III).

The DTPA chelator of any one of claims 1 to 3, wherein the DTPA chelator is bonded to the metal in an 8-valence or 9-valence form.
5. The DTPA chelator of claim 4, wherein the metal is a radioactive isotope.
An amine-containing low-molecular compound-DTPA chelator conjugate, wherein the DTPA chelator of any one of claims 1 to 3 is conjugated with an amine moiety of a low molecular weight compound having an amine moiety (-NH ₂ ).
7. The method of claim 6, wherein the low molecular weight compound is a peptide having an amine moiety, a hormone or a combination thereof, an amine-containing low molecular weight compound-DTPA chelator conjugate
The amine-containing low molecular compound-DTPA chelator conjugate according to claim 6, wherein the low molecular compound is at least one selected from the group consisting of PSMA, folate, RGD, bombesin and a-MSH.
An amine-containing protease-DTPA chelator conjugate, wherein the DTPA chelator of any one of claims 1 to 3 is conjugated to at least one Lysine residue in the amino acid sequence of the proteome.
10. The conjugate of claim 9, wherein the protease is an antibody, protein, antibody-derived protease, hormone, peptide, or a combination thereof comprising an amino acid sequence comprising lysine.
10. The method of claim 9, wherein said protease is selected from the group consisting of at least one antibody selected from the group consisting of rituximab, cetuximab, and trastuzumab or at least one fragment derived from said antibody, At least one selected amine-containing protease-DTPA chelator conjugate.
Preparing a DTPA chelator conjugate by mixing an amine-containing protease or a low molecular compound with the DTPA chelator of any one of claims 1 to 3 and reacting at a temperature of more than 0 to 50 for 10 minutes to 48 hours; Way.
13. The method according to claim 12, further comprising the step of mixing the amine-containing protease or the low molecular compound with the DTPA chelator of any one of claims 1 to 3 and reacting at a temperature of 4 to 30 for 10 minutes to 3 hours. A method for producing a chelator conjugate.
The method for producing a DTPA chelator conjugate according to claim 12, wherein the DTPA chelator is mixed in an amount of 1 to 10 equivalents based on 1 equivalent of an amine-containing protease or a low molecular compound.
To the at least one of a general formula (a1) compound and the formula (a2) to the at least one compound of formula (b1) compound and the formula (b2) compounds as starting material, -NH ₂ and -NH-Boc group substituted With a reaction compound (c) which is a cyclohexyl compound,

In the formula (b1) and (b2), -NO _2, and -HN-Cbz is substituted on each of the ortho, meta or para position,

A method for preparing the DTPA chelator of formula (I)
In the above formulas (I), (a1) and (a2), l is an integer of 1 to 4. In the above formulas (I), (b1) and (b2), m is an integer of 1 to 10.
16. The method according to claim 15, wherein the reaction compound (c) is N-Boc-1,2-diaminocyclohexane.
delete A therapeutic agent for cancer comprising a DTPA chelator conjugate conjugated with 1 to 5 DTPA chelators according to any one of claims 1 to 3 to rituximab or cetuximab.

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