RADIOLABELED MOEM TYPE OLIGONUCLEOTIDES AND PROCESS FOR THEIR PREPARATION

Abstract

The invention comprises radiolabeled MOEM type oligonucleotide of the formula (I), (I) wherein n, X.sup.1, X.sup.2, the linker (1), the linker (2), Q* and the receptor targeting moiety are as defined (I) the description. The radiolabeled oligonucleotides of the formula (I) can be used for the determination of the biodistribution and pharmacokinetics of the oligonucleotide in the tissue or body fluid.

##STR00001##

Claims

1. Radiolabeled oligonucleotide of the formula I ##STR00045## wherein, n is 0 or 1; X.sup.1 and X.sup.2 independently of each other are S or O; linker 1 is a C.sub.2-12-alkylene bridge, an ethylene glycol bridge containing 1 to 10 ethylene glycol units or a glycerol based bridge of the formula II ##STR00046##  wherein m is an integer of 1 to 6; linker 2 is an optionally amino group protected amino C.sub.2-12-alkylene bridge, an amino ethylene glycol bridge containing 1 to 10 ethylene glycol units; Q* stands for a residue of the formula III ##STR00047## wherein, n is an integer of 1 to 4, R.sup.1 and R.sup.2 independently of each other are hydrogen, CF.sub.3, C.sub.1-6-alkyl or R.sup.1 and R.sup.2 together with the carbon atom they are attached to form a C.sub.3-5-cycloalkane ring; Z* is a radiolabeled C.sub.1-C.sub.6 alkyl group; and the receptor targeting moiety is a moiety which adds additional functionality to the oligonucleotide.

2. The radiolabeled oligonucleotide of claim 1, wherein n is an integer of 1.

3. The radiolabeled oligonucleotide of claim 1, wherein R.sup.1 and R.sup.2 independently of each other are hydrogen, C.sub.1-2-alkyl or R.sup.1 and R.sup.2 together with the carbon atom they are attached to form a cyclopropyl ring.

4. The radiolabeled oligonucleotide of claim 1, wherein Z* is a radiolabeled methyl or ethyl group.

5. The radiolabeled oligonucleotide of claim 4, wherein the radiolabeling is a .sup.3H- or a .sup.14C-labeling, preferably a .sup.3H-labeling.

6. The radiolabeled oligonucleotide of claim 1, wherein the oligonucleotide comprises a contiguous nucleotide sequence of 7 to 30 nucleotides consisting of optionally modified DNA, PNA, RNA or LNA nucleoside monomers or combinations thereof.

7. The radiolabeled oligonucleotide of claim 1 of the formula Ib ##STR00048##

8. The radiolabeled oligonucleotide of claim 1 of the formula Ic ##STR00049##

9. The radiolabeled oligonucleotide of claim 1, wherein the receptor targeting moiety is a non-nucleotide moiety, preferably a asialglycoprotein receptor targeting moiety, more preferably a GalNAc moiety of formula IV ##STR00050## wherein R.sup.3 is hydrogen or a hydroxy protecting group and n is an integer from 0 to 10, preferably from 0 to 5, more preferably from 1 to 3, but most preferred is 2, corresponding salts, enantiomers and/or a stereoisomers thereof.

10. The radiolabeled oligonucleotide of claim 1 having a specific activity of 0.037 TBq/mmol (1 Ci/mmol) to 3.7 TBq/mmol (100 Ci/mmol), preferably of 0.111 TBq/mmol (3 Ci/mmol) to 1.85 TBq/mmol (50 Ci/mmol), more preferably of 0.185 TBq/mmol (5 Ci/mmol) to 0.925 TBq/mmol (25 Ci/mmol).

11. A process for the preparation of a radiolabeled oligonucleotide of the formula I, wherein Q* stands for the residue of the formula III, comprising conjugating a thiol of formula V ##STR00051## wherein, n is 0 or 1; X.sup.1 and X.sup.2 independently of each other are S or O; linker 1 is a C.sub.2-12-alkylene bridge, an ethylene glycol bridge containing 1 to 10 ethylene glycol units or a glycerol based bridge of the formula II ##STR00052##  wherein m is an integer of 1 to 6; linker 2 is an optionally amino group protected amino C.sub.2-12-alkylene bridge, an amino ethylene glycol bridge containing 1 to 10 ethylene glycol units; the receptor targeting moiety is a non-nucleotide moiety which adds additional functionality to the oligonucleotide, particularly an asialglycoprotein receptor targeting moiety, preferably a GalNAc moiety; with a radiolabeled maleinimide compound of formula VI ##STR00053## wherein R.sup.1 and R.sup.2, n and Z* are as above.

12. (canceled)

13. A method for the determination of the biodistribution and pharmacokinetics of an oligonucleotide in the tissue or body fluid the method comprising; a) administering an effective amount of radiolabeled oligonucleotide of claim 1 to the tissue or the body fluid to be examined; b) measuring the biodistribution and the pharmacokinetics of the radiolabeled oligonucleotide of claim 1 in the tissue or body fluid; and c) imaging the radiolabeled oligonucleotide of anyone of claim 1 in the tissue or the body fluid to be examined by autoradiography.

14. An oligonucleotide of the formula X ##STR00054## wherein, n is 0 or 1; X.sup.1 and X.sup.2 independently of each other are S or O; linker 1 is a C.sub.2-12-alkylene bridge, an ethylene glycol bridge containing 1 to 10 ethylene glycol units or a glycerol based bridge of the formula II ##STR00055##  wherein m is an integer of 1 to 6; linker 2 is an optionally amino group protected amino C.sub.2-12-alkylene bridge, an amino ethylene glycol bridge containing 1 to 10 ethylene glycol units; Q stands for a residue of the formula IIIa ##STR00056##  wherein n is an integer of 1 to 4,  R.sup.1 and R.sup.2 independently of each other are hydrogen, CF.sub.3, C.sub.1-6 alkyl or R.sup.1 and R.sup.2 together with the carbon atom they are attached to form a C.sub.3-5-cycloalkane ring;  Z is a C.sub.1-C.sub.6 alkyl group; and the receptor targeting moiety is a moiety which adds additional functionality to the oligonucleotide.

15. The oligonucleotide of claim 14, wherein Z is methyl or ethyl.

16. The oligonucleotide of claim 14 of the formula Xb ##STR00057## wherein R.sup.1, R.sup.2, n, Z, X.sup.2 and linker 1 are as above.

17. The oligonucleotide of claim 14 of the formula Xc ##STR00058## wherein R.sup.1, R.sup.2, n, Z, X.sup.1 and X.sup.2, linker 1 and linker 2 are as above.

18. The oligonucleotide of claim 14, wherein the receptor targeting moiety is a non-nucleotide moiety, preferably a asialglycoprotein receptor targeting moiety, more preferably a GalNAc moiety of formula VII ##STR00059## wherein R.sup.3 is hydrogen or a hydroxy protecting group and n is an integer from 0 to 10, preferably from 0 to 5, more preferably from 1 to 3, but most preferred is 2, corresponding salts, enantiomers and/or a stereoisomers thereof.

19. The oligonucleotide of claim 14, wherein the oligonucleotide comprises a contiguous nucleotide sequence of 7 to 30 nucleotides consisting of optionally modified DNA, PNA, RNA or LNA nucleoside monomers or combinations thereof.

Description

EXAMPLES

Abbreviations

Bq Becquerel

[0143] Ci curries

Da Dalton

[0144] DCM dichloromethane
DI deionized
DIPEA N,N-diisopropylethylamine (Hünig's base)

DMF N, N-dimethylformamide

[0145] DMSO dimethylsulfoxide
GBB glycerol based bridge
HV high vacuum
i iso
MeCN acetonitrile
MeOH methanol
min minutes
MOEM methoxyethylene maleimide
MOMCPM 1-(methoxymethyl)cyclopropyl maleimide
MOMEM 1-(2-methoxy-1-methyl-ethyl) maleimide
MS mass spectrometry
MTBE methyl tert-butyl ether
MW molecular weight
MWCO molecular weight cut of
n normal
NaOtBu sodium t-butoxide
PBS phosphate-buffered saline
p para
ppm parts per million
QWBA quantitative whole body autoradiography
rpm round per minutes
rt room temperature
SAX strong anion exchange
SCX strong cation exchange
t tertiary
TEA triethylamine
TFA trifluoroacetic acid
THF tetrahydrofurane

General Methods:

[0146] All oligonucleotides, which were used as starting materials, were synthesized from Roche Pharma research and early development. Tritium labeled [.sup.3H]methyl nosylate (tritritiomethyl 4-nitrobenzenesulfonate; molar activity: 3 TBq/mmol=80 Ci/mmol) was obtained from RC Tritec (Teufen, CH) as solution in toluene. PBS buffer was purchased from Thermo Fisher Scientific (Paisley, UK), in one (1×) and tenfold (10×) concentration. All other reagents and solvents were obtained from standard commercial sources and used without further purification. Liquid scintillation counting for tritium compounds was accomplished using a HIDEX 300 SL and ULTIMATE GOLD cocktail (PerkinElmer Inc., Waltham, Mass., USA). Analyzes for synthesis of maleimide derivatives were carried out by HPLC Agilent 1260 Infinity II at 220 nm wavelength, Waters XBridge C18, 4.6×150 mm, 3.5 μm column at 40° C. by elutents [A]=water+5% MeCN+0.05% TFA and [B]=MeCN+0.05% TFA by a flow of 1.0 mL/min with the following gradient: 0% [B] to 50% [B] in 10 min and to 80% after 12 min. Oligos 1-4 were determined by UPLC Agilent 1290 at 260 nm wavelength, ACQUITY UPLC Oligonucleotide BEH C18, 2.1×50 mm, 1.7 μm column at 80° C. ([A]=water/methanol/hexafluoro i-propanol/TEA: 950/25/21/2.3 mL; [B]=water/methanol/hexafluoro i-propanol/TEA: 175/800/21/2.3 mL) by a flow of 0.5 mL/min and the following gradient: 10% [B] to 25% [B] in 13 min. Large-scale purification was performed by TELEDYNE (Lincoln, Nebr., USA) Isco CombiFlash by the use of RediSep® normal-phase Silica Flash Columns (4 g). Solvent [A] was heptane and solvent [B] was methyl t-butyl ether. The column was initially equilibrated at 20% [B] using a flow rate of 18 mL/min, with the absorbance monitored at 214 nm. The elution gradient consisted of isocratic conditions at 20% [B] for 4 minutes, followed by liner gradients to 100% [B] in 14 minutes, and finally isocratic conditions at 100% [B] over 5 minutes. Mass spectrometry was performed by Waters Acquity UPLC H-class System equipped with Single Quadruple (SQ) and ESI Mass Detector. Radiochemical purity was measured using the β-radioactivity HPLC detector RAMONA Quattro with internal solid scintillator (Raytest, Straubenhardt, Germany). Preparative HPLC for MOEM* was performed by Gilson PLC 2020 with XBridge C18 column, 5 m, 10 mm×250 mm and using water+5% MeCN+0.05% TFA as mobile phase [A] and MeCN+0.05% TFA as mobile phase [B] as gradient with 0% [B] to 70% [B] in 18 min. .sup.1H NMR measurements were carried out on a Bruker Avance III 600 MHz spectrometer. The deuterium solvents used was dependent on the product solubility and has been detailed in each case. Chemical shifts are given in ppm with s for singulet, d for doublet, dd for double doublet, m for multiplet, J for indirect dipole-dipole coupling. Concentration was determined by Eppendorf BioSprectrometer® basic at 260 nm wavelength and the corresponding calculated molar extinction coefficient.

Example 1

Synthesis of [.SUP.3.H-methyl]N-methoxyethylene maleimide (MOEM*)

[0147] (Maleimide Compound of Formula VI, Wherein n=1, R.sup.1 and R.sup.2=H)

##STR00023##

a) Synthesis of exo-4-(2-hydroxyethyl)-1,7-dimethyl-10-oxa-4-aza-tricycle[5.2.1.0.SUP.2,6.]dec-8-en-3,5-dione (M3)

[0148] ##STR00024##

[0149] To a solution of commercially available N-(2-hydroxyethyl) maleimide M1 (200 mg, 1.42 mmol) in acetonitrile (2.0 mL) was added at rt 2,5-dimethylfuran M2 (722 mg, 802 μL, 7.51 mmol). The mixture was stirred at 65° C. in a sealed glass tube for 20 h. Removal of the solvent in vacuum and drying in HV gave the crude Diels-Alder adduct M3 as an exo/endo mixture in a 4:1 ratio as a light yellow oil. The endo/exo mixture was purified by Isco flash chromatography to separate the exo derivative in a high purity. Yield (exo): 185 mg (55%). MS (ESI): m/z=238.1 [M+H]+. .sup.1H NMR (DMSO-d.sub.6) δ ppm 6.36 (s, 2H), 4.69 (br s, 2H), 3.41 (s, 4H), 2.88 (s, 2H), 1.53 (s, 6H).

b) Synthesis of [.SUP.3.H-methoxy]-exo-4-(2-methoxyethyl)-1,7-dimethyl-10-oxa-4-aza-tricycle[5.2.1.0.SUP.2,6.]dec-8-en-3,5-dione (M5*)

[0150] ##STR00025##

[0151] 1.67 GBq (45 mCi) of [.sup.3H]-methyl nosylate M4* (125 μg, 0.561 μmol) as solution in toluene was diluted with cold (non-radioactive) methyl 4-nitrobenzenesulfonate M4 (122 μg, 0.561 μmol) in a 1:1 ratio to achieve a specific activity of approximately 40 Ci/mmol. Solution was evaporated, transferred into a sealed tube and concentrated to dryness under an argon flow. To the solid residue (M4*+M4) was added at rt a solution of Diels Alder adduct M3 (666 μg, 2.81 μmol) in 80 μL toluene followed by the addition of 2M sodium t-butoxide solution in THF (1.7 μL, 3.37 μmol). The mixture was stirred in a sealed tube at rt for 2.5 h. HPLC analysis showed the desired intermediate product M5* with a radiochemical purity of 50%.

[0152] The reaction mixture was diluted with DCM (1 mL) and directly purified by filtration through a SCX-2/SAX cartridge (Silycycle, 500 mg, pre-conditioned with DCM) to remove basic and acidic compounds. The cartridge was washed with DCM (5 mL) and the resulting solution was concentrated by evaporation to a volume of 100 μL to give the radiolabeled intermediate M5*.

[0153] The crude solution of M5* was used for the next step without further purification.

c) Synthesis of [.SUP.3.H-methyl]N-methoxyethylene maleimide (MOEM*)

[0154] ##STR00026##

[0155] The obtained crude solution of M5* was transferred into a sealed tube, diluted with toluene (70 μL) and heated at 90° C. for 2 h.

[0156] HPLC analysis showed full conversion to the deprotected product MOEM* and remaining unreacted [.sup.3H]methyl nosylate M4*. The reaction mixture was allowed to cool to rt and solvent concentrated to dryness under an argon flow. The residue was purified by preparative HPLC to give the desired product [.sup.3H-methyl]methoxyethylene maleimide (MOEM*) as a solution in the eluent mixture. The corresponding prep HPLC fraction, containing MOEM* in eluent mixture was directly used for the conjugation with Oligos 1, 2, 3 and 4. Radio yield: 253.5 MBq (6.85 mCi)=15.2%. radio concentration: 34.8 MBq/mL (0.94 mCi/mL), radiochemical purity: 99%. Specific activity could not be determined by MS due to low ionisation. The specific activity was assumed to be 40 Ci/mmol.

Example 2 (Non-Radioactive Conjugation

[0157] (Maleimide Compound of Formula VI, Wherein n=1, R.sup.1 and R.sup.2=H)

Oligonucleotides Used in the Examples

[0158]

TABLE-US-00003 (Oligo 1) 5′-GN2-C6-caG*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH; MW: 7709.5 g/mol; (Oligo 2) G*C*a*t*t*g*g*t*a*t*T*C*A*-C6SH; MW: 4537.6 g/mol; (Oligo 3) G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH; MW: 5491.5 g/mol; (Oligo 4) 5′-SH-C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; MW: 6742.3 g/mol;

General Procedure:

[0159] ##STR00027##

[0160] 1 equivalent of oligonucleotide with 5′ or 3′ end sulfhydryl linker was dissolved in PBS (volume factor: 250 mL/g). 1.3 equivalent of commercially available methoxyethylene maleimide (MOEM), dissolved in THF (volume factor: 200 mL/g), was added to the aqueous solution and stirred at room temperature for 1 h. UPLC analysis showed a complete addition of maleimide to oligo nucleotide. To exchange the buffer to water, the reaction mixture was transferred into an Amicon® Pro purification system (MWCO: 3.000 Da) and centrifuged at 4000 rpm. DI water was added and the process was repeated 4 times more to complete the exchange. The resulting aqueous solution was lyophilized to isolate the oligonucleotide as a colorless powder with a yield in range of 70%-95% and 90%-99% purity.

[0161] In accordance with the general procedure the oligonucleotides (Oligo 1 to 4) have been conjugated with MOEM.

TABLE-US-00004 a) Synthesis of conjugate 1 from Oligo 1 5′-GN2-C6-caG*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH-MOEM; Yield: 70%, purity: 90%, MS (m/z): 7859.4 [M-(H)].sup.- b) Synthesis of conjugate 2 from Oligo 2 G*C*a*t*t*g*g*t*a*t*T*C*A*-C6SH-MOEM; Yield: 93%, purity: 97%, MS (m/z): 4689.5 [M-(H)].sup.- c) Synthesis of conjugate 3 from Oligo 3 G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH-MOEM; Yield: 83%, purity: 95%, MS (m/z) 5642.6 [M-(H)].sup.- d) Synthesis of conjugate 4 from Oligo 4 5′-MOEM-SH-C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; Yield: 92%, purity: 99%, MS (m/z): 6892.7 [M-(H)].sup.-

Example 3 (Radioactive Conjugation)

[0162] (Maleimide Compound of Formula VI, Wherein R.sup.1 and R.sup.2=H)

Oligonucleotides Used in the Examples

[0163]

TABLE-US-00005 (Oligo 1) 5′-GN2-C6-caG*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH; MW: 7709.5 g/mol; (Oligo 3) G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH; MW: 5491.5 g/mol; (Oligo 4) 5′-SH-C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; MW: 6742.3 g/mol;

General Procedure:

[0164] ##STR00028##

[0165] 2 equivalents of oligonucleotide with 5′ or 3′ end sulfhydryl linker was dissolved in PBS (10×) (volume factor: 250 mL/g). 1 equivalent of MOEM*, directly used in prep HPLC eluent with a radio concentration of 35 MBq/mL (0.94 mCi/mL), was added to the aqueous oligonucleotide solution and stirred at room temperature for 1.5 h. UPLC analysis showed a conjugation of MOEM* to oligo nucleotide in range of 30% to 45%. 10 equivalent of cold (non-radioactive) MOEM, dissolved in THF (volume factor: 700 mL/g), was added and stirred at rt for 1 h. UPLC showed a complete conjugation. The reaction mixture was transferred into an Amicon® Pro purification system (MWCO: 3.000 Da) and centrifuged at 4000 rpm. PBS (1×) was added and the process was repeated 4 times to complete a solvent exchange and receive the purified product. The concentration and activity of resulting buffered solution were determined. Radiochemical yields were calculated in range of 69%-72%, specific molar activities could achieve from 0.61 TBq/mmol (16.5 Ci/mmol) to 0.74 TBq/mmol (20.1 Ci/mmol). Radiochemical purities were in range of 96.0% to 98.4%.

[0166] In accordance with the general procedure the oligonucleotides, Oligos 1, 3 and 4 have been conjugated.

a) Synthesis of Conjugate 1* [.SUP.3.H]—from Oligo 1

[0167] 5′-GN2-C6-caG*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH-[.sup.3H]-MOEM*; Yield 72%, radiochemical purity: 96.1%, activity: 14.1 MBq (0.38 mCi), specific molar activity: 0.74 TBq/mmol (20.1 Ci/mmol).

b) Synthesis of Conjugate 3* [.SUP.3.H]—from Oligo 3

[0168] G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH-[.sup.3H]-MOEM; Yield 69%, radiochemical purity: 96.0%, activity: 28.1 MBq (0.76 mCi), specific molar activity: 0.61 TBq/mmol (16.5 Ci/mmol).

c) Synthesis of Conjugate 4* [.SUP.3.H]—from Oligo 4

[0169] 5′-[.sup.3H]-MOEM-SH—C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; Yield 72%, radiochemical purity: 98.4%, activity: 29.2 MBq (0.79 mCi), specific molar activity: 0.68 TBq/mmol (18.3 Ci/mmol).

Example 4

Synthesis of [.SUP.1.H/.SUP.3.H-methyl]-1-(methoxymethyl)cyclopropyl maleimide (MOMCPM*)

[0170] (Maleimide Compound of Formular VI, Wherein R.sup.t and R.sup.2 Together are Cyclopropyl)

##STR00029##

a) Synthesis of exo-2-[1-(hydroxymethyl)cyclopropyl]-4,7-dimethyl-3a,7a-dihydro-4,7-epoxyisoindole-1,3-dione (M13)

[0171] ##STR00030##

[0172] To a solution of commercially available 1-[1-(hydroxymethyl)cyclopropyl] maleimide M11 (209 mg, 1.25 mmol) in acetonitrile (2.0 mL) was added at rt 2,5-dimethylfuran M2 (643 mg, 713 μL, 6.63 mmol). The mixture was stirred at 65° C. in a sealed glass tube for 22 h. Removal of the solvent in vacuum and drying in HV gave the crude Diels-Alder adduct M13 as an exo/endo mixture in a 4:1 ratio as a light yellow oil. The endo/exo mixture was purified by Isco flash chromatography to separate the exo derivative in a high purity. Yield (exo): 211 mg (64%). MS (ESI): m/z=264.1 [M+H]+. .sup.1H NMR (DMSO-d.sub.6) δ ppm 6.35 (s, 2H), 4.70 (br s, 1H), 3.38 (s, 2H), 2.78 (s, 2H), 1.50 (s, 6H), 0.89-0.93 (m, 2H), 0.63-0.67 (m, 2H).

b) Synthesis of exo-2-[1-(methoxymethyl)cyclopropyl]-4,7-dimethyl-3a,7a-dihydro-4,7-epoxyisoindole-1,3-dione (M15)

[0173] ##STR00031##

[0174] In a sealed tube, methyl nosylate M4 (50 mg, 0.23 mmol) and M13 (72.7 mg, 0.276 mmol) were dissolved in toluene (2.5 mL). The solution was cooled to 0° C. At this temperature, sodium t-butoxide solution (2 M in THF, 403 μL, 806 μmol) was dropped slowly to the reaction solution. The color immediately changed from colorless to dark brown. The ice bath was removed and the mixture was stirred at rt overnight. HPLC analysis showed the desired intermediate product M15. The reaction mixture was diluted with t-buthylmethyl ether (30 mL) and extracted with 2 M sodium carbonate (10 mL) and saturated sodium chloride solution (10 mL). The organic phase was dried with sodium sulfate, filtered and evaporated to dryness to get a light yellow liquid. HPLC analysis showed the desired intermediate M15 in a purity of 90%. The crude product was used for the next step without further purification.

b*) Synthesis of [.SUP.3.H-methoxy]-exo-2-[1-(methoxymethyl)cyclopropyl]-4,7-dimethyl-3a,7a-dihydro-4,7-epoxyisoindole-1,3-dione (M15*)

[0175] ##STR00032##

0.93 GBq (25 mCi) of [.sup.3H]-methyl nosylate M4* (70 μg, 0.313 μmol) as solution in toluene was diluted with cold (non-radioactive) methyl 4-nitrobenzenesulfonate M4 (68 μg, 0.313 μmol) in a 1:1 ratio to achieve a specific activity of approximately 40 Ci/mmol. Solution was evaporated, transferred into a sealed tube and concentrated to dryness under an argon flow. To the solid residue (M4*+M4) was added at rt a solution of Diels Alder adduct M13 (411 μg, 1.56 μmol) in 80 μL toluene followed by the addition of sodium t-butoxide solution (2 M in THF, 1.0 μL, 1.88 μmol). The mixture was stirred in a sealed tube at rt for 5.5 h. HPLC analysis showed the desired intermediate product M15* with a radiochemical purity of 66%.

[0176] The reaction mixture was diluted with DCM (1 mL) and directly purified by filtration through a SCX-2/SAX cartridge (Silycycle, 500 mg, pre-conditioned with DCM) to remove basic and acidic compounds. The cartridge was washed with DCM (5 mL) and the resulting solution was concentrated by evaporation to a volume of 100 μL to give the radiolabeled intermediate M15*.

[0177] The crude solution of M15* was used for the next step without further purification.

c) Synthesis of 1-[(1-methoxymethyl)-cyclopropyl] maleimide (MOMCPM)

[0178] ##STR00033##

[0179] Crude M15 (30 mg, 108 μmol) was transferred into a sealed tube, dissolved in toluene (4.4 mL) and heated at 110° C. for 2 h. HPLC analysis showed full conversion to the deprotected product MOMCPM. The reaction mixture was allowed to cool to rt and solvent concentrated to dryness under an argon flow. The residue was purified by preparative HPLC to give the desired product MOMCPM as a solution in the eluent mixture. The corresponding preparative HPLC fractions, containing MOMCPM in eluent mixture, were diluted with ethyl acetate (50 mL) and extracted 3× with sodium chloride (30 mL each). The organic phase was dried with sodium sulfate, filtered and evaporated to dryness to give 16 mg (yield 82%) in a purity of 98%.

[0180] .sup.1H NMR (CDCl.sub.3) δ ppm 6.64 (s, 2H), 3.41 (s, 2H), 3.36 (s, 3H), 1.03-1.05 (m, 1H), 0.98-1.00 (m, 2H), 0.92-1.08 (m, 1H)

c*) Synthesis of 1-[(1-methoxy-[.SUP.3.H]-methyl)-cyclopropyl] maleimide (MOMCPM*)

[0181] ##STR00034##

[0182] The obtained crude solution of M15* was transferred into a sealed tube, diluted with toluene (100 μL) and heated at 110° C. for 2 h.

[0183] HPLC analysis showed full conversion to the deprotected product MOMCOM* and remaining unreacted [.sup.3H]methyl nosylate M4*. The reaction mixture was allowed to cool to rt and solvent concentrated to dryness under an argon flow. The residue was purified by preparative HPLC to give the desired product 1-[(1-methoxy-[.sup.3H]-methyl)-cyclopropyl] maleimide (MOMCPM*) as a solution in the eluent mixture. The corresponding prep HPLC fraction, containing MOMCPM* in eluent mixture was directly used for the conjugation with Oligos 3 and 4. Radio yield: 140.6 MBq (3.80 mCi)=15.2%. radio concentration: 30.3 MBq/mL (0.82 mCi/mL), radiochemical purity: 99%. Specific activity could not be determined by MS due to low ionisation. The specific activity was assumed to be 40 Ci/mmol.

Example 5 (Non-Radioactive Conjugation)

[0184] (Maleimide Compound of Formula VI, Wherein n=1, R.sup.1 and R.sup.2 are Cyclopropyl)

Oligonucleotides Used in the Examples

[0185]

TABLE-US-00006 (Oligo 3) G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH; MW: 5491.5 g/mol; (Oligo 4) 5′-SH-C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; MW: 6742.3 g/mol;

General Procedure:

[0186] ##STR00035##

[0187] 1 equivalent of oligonucleotide with 5′ or 3′ end sulfhydryl linker was dissolved in PBS (volume factor: 250 mL/g). 1.3 equivalent of 1-[(1-methoxymethyl)-cyclopropyl] maleimide (MOMCPM), dissolved in THF (volume factor: 200 mL/g), was added to the aqueous solution and stirred at room temperature for 1 h. UPLC analysis showed a complete addition of maleimide to oligo nucleotide. To exchange the buffer to water, the reaction mixture was transferred into an Amicon® Pro purification system (MWCO: 3.000 Da) and centrifuged at 4000 rpm. DI water was added and the process was repeated 4 times more to complete the exchange. The resulting aqueous solution was lyophilized to isolate the oligonucleotide as a colorless powder with a yield in range of 86%-95% and 95%-98% purity.

[0188] In accordance with the general procedure the oligonucleotides (Oligo 3, 4) have been conjugated with MOMCPM.

TABLE-US-00007 a) Synthesis of conjugate 13 from Oligo 3 G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH-MOMCPM; Yield: 85%, purity: 95%, MS (m/z): 5668.6 [M-(H)].sup.- b) Synthesis of conjugate 14 from Oligo 4 5′-MOMCPM-SH-C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; Yield: 98%, purity: 98%, MS (m/z): 6818.7 [M-(H)].sup.-

Example 5 (Radioactive Conjugation)

[0189] (Maleimide Compound of Formula VI, Wherein n=1, R.sup.1 and R.sup.2 are Cyclopropyl)

Oligonucleotides Used in the Examples

[0190]

TABLE-US-00008 (Oligo 3) G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH; MW: 5491.5 g/mol; (Oligo 4) 5′-SH-C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; MW: 6742.3 g/mol;

General Procedure:

[0191] ##STR00036##

[0192] 1.2 equivalents of oligonucleotide with 5′ or 3′ end sulfhydryl linker was dissolved in PBS (10×) (volume factor: 250 mL/g). 1 equivalent of MOMCPM*, directly used in prep HPLC eluent with a radio concentration of 30.3 MBq/mL (0.82 mCi/mL), was added to the aqueous oligonucleotide solution and stirred at room temperature for 2 h. UPLC analysis showed a conjugation of MOMCPM* to oligo nucleotide in range of 26% to 44%. 10 equivalent of cold (non-radioactive) MOMCPM, dissolved in acetonitrile (volume factor: 700 mL/g), was added and stirred at rt for 2 h. UPLC showed a complete conjugation. The reaction mixture was transferred into an Amicon® Pro purification system (MWCO: 3.000 Da) and centrifuged at 4000 rpm. PBS (lx) was added and the process was repeated 4 times to complete a solvent exchange and receive the purified product. The concentration and activity of resulting buffered solution were determined. Radiochemical yields were calculated in range of 90%-799%, specific molar activities could achieve from 0.63 TBq/mmol (17.0 Ci/mmol) to 0.77 TBq/mmol (20.8 Ci/mmol). Radiochemical purities were in range of 97.3% to 98.1%.

[0193] In accordance with the general procedure the oligonucleotides, Oligos 3 and 4 have been conjugated with MOMCPM*

a) Synthesis of Conjugate 13* [.SUP.3.H]—from Oligo 3

[0194] G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH-[.sup.3H]-MOMCPM; Yield 90%, radiochemical purity: 98.1%, activity: 26.2 MBq (0.71 mCi), specific molar activity: 0.63 TBq/mmol (17.0 Ci/mmol).

b) Synthesis of Conjugate 14* [.SUP.3.H]—from Oligo 4

[0195] 5′-[.sup.3H]-MOMCPM-SH—C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; Yield 99%, radiochemical purity: 97.3%, activity: 34.8 MBq (094 mCi), specific molar activity: 0.77 TBq/mmol (20.8 Ci/mmol).

Example 6

Synthesis of [.SUP.1.H/.SUP.3.H-Methyl]-1-(2-Methoxy-1-Methyl-Ethyl) Maleimide (MOMEM.SUP.(*).)

[0196] (Maleimide Compound of Formula VI, Wherein n=1, R.sup.t=Methyl and R.sup.2=H)

##STR00037##

a) Synthesis of exo-4-(2-hydroxy-1-1methyl-ethyl)-4,7-dimethyl-3a,7a-dihydro-4,7-epoxyisoindole-1,3-dione (M23)

[0197] ##STR00038##

[0198] To a solution of commercially available 1-(2-hydroxy-1-methyl-ethyl) maleimide M21 (132 mg, 0.85 mmol) in acetonitrile (2.0 mL) was added at rt 2,5-dimethylfuran M2 (450 mg, 500 μL, 4.68 mmol). The mixture was stirred at 65° C. in a sealed glass tube for 20 h. Removal of the solvent in vacuum and drying in HV gave the crude Diels-Alder adduct M23 as an exo/endo mixture in a ratio of 4:1 as a light yellow oil with a purity of 98%. The endo/exo mixture was purified by Isco flash chromatography to separate the exo derivative in a high purity. Yield (exo): 123 mg (58%). MS (ESI): m/z=269.2 [M+NH.sub.4].sup.+. .sup.1H NMR (DMSO-d.sub.6) δ ppm 6.36 (d, J=1.6 Hz, 2H), 4.54-5.09 (m, 1H), 3.90-4.15 (m, 1H), 3.67 (dd, J=10.8, 8.1 Hz, 1H), 3.49 (dd, J=10.8, 6.4 Hz, 1H), 2.82-2.87 (m, 1H), 2.77-2.81 (m, 1H), 1.53 (d, J=4.0 Hz, 6H), 1.17 (d, J=7.0 Hz, 3H).

b) Synthesis of exo-2-(2-methoxy-1-methyl-ethyl)-4,7-dimethyl-3a,7a-dihydro-4,7-epoxyisoindole-1,3-dione (M25)

[0199] ##STR00039##

[0200] In a sealed tube, methyl nosylate M4 (49.8 mg, 0.23 mmol) and M23 (57.6 mg, 0.23 mmol), dissolved in toluene (2 mL) was cooled to 0° C. Sodium t-butoxide solution (2 M in THF, 138 μL, 0.28 mmol) was added dropwise. The color turned from colorless to brown. The mixture was stirred at room temperature for 3 h. HPLC analysis showed a conversion to the desired intermediate product. The reaction mixture was directly purified by filtration through two SAX-SCX cartridge (Sillicycle, 500 mg, pre-conditioned with toluene). The cartridges were washed with toluene (each by 5 mL) and the resulting solution was concentrated by evaporation to dryness to give a colorless oil. The crude product was purified by Isco flash chromatography by a gradient of heptane and MTBE from 0% to 60% MTBE in 15 minutes. Yield: 59 mg (97%). MS (ESI): m/z=266.1 [M+H].sup.+.

[0201] .sup.1H NMR (DMSO-d.sub.6) δ ppm 6.36 (s, 2H), 4.17-4.27 (m, 1H), 3.69 (dd, J=10.0, 8.9 Hz, 1H), 3.69 (dd, J=10.0, 8.9 Hz, 1H), 3.42 (dd, J=9.9, 5.9, 1H), 3.19 (s, 3H), 2.82-2.87 (m, 2H), 1.53 (d, J=3.6 Hz, 6H), 1.19 (d, J=7.1 Hz, 3H).

b*) Synthesis of [.SUP.3.H-methoxy]-exo-2-(2-methoxy-1-methyl-ethyl)-4,7-dimethyl-3a,7a-dihydro-4,7-epoxyisoindole-1,3-dione (M25*)

[0202] ##STR00040##

[0203] 0.93 GBq (25 mCi) of [.sup.3H]-methyl nosylate M4* (70 μg, 0.313 μmol) as solution in toluene was diluted with cold (non-radioactive) methyl 4-nitrobenzenesulfonate M4 (68 μg, 0.313 μmol) in a 1:1 ratio to achieve a specific activity of approximately 40 Ci/mmol. Solution was evaporated, transferred into a sealed tube and concentrated to dryness under an argon flow. To the solid residue (M4*+M4) was added at rt a solution of exo Diels Alder adduct M23 (393 μg, 1.56 μmol) in 80 μL toluene followed by the addition of sodium t-butoxide solution (2 M in THF, 1.0 μL, 1.88 μmol). The mixture was stirred in a sealed tube at rt for 2.5 h. HPLC analysis showed the desired intermediate product M25* with a radiochemical purity of 63%. The reaction mixture was diluted with DCM (1 mL) and directly purified by filtration through a SCX-2/SAX cartridge (Silycycle, 500 mg, pre-conditioned with DCM) to remove basic and acidic compounds. The cartridge was washed with DCM (5 mL) and the resulting solution was concentrated by evaporation to a volume of 100 μL to give the radiolabeled intermediate M25*.

[0204] The crude solution of M25* was used for the next step without further purification.

c) Synthesis of 1-(1-methoxy-1-methyl-ethyl) maleimide (MOMEM)

[0205] ##STR00041##

[0206] 55 mg (0.207 mmol) of M25 was transferred into a sealed tube, dissolved in toluene (500 μL) and heated at 90° C. for 16 h. HPLC analysis showed conversion to the deprotected product MOMEM. The reaction mixture was allowed to cool to rt and solvent concentrated to dryness. The residue was purified by flash chromatography to give the desired product methoxyethylene maleimide (MOMEM) in a purity of >96%. 19 mg (54%) could be isolated as a colorless oil.

[0207] MS (ESI): m/z=170.08 [M+H].sup.+.

[0208] .sup.1H NMR (DMSO-d.sub.6) δ ppm 6.98 (s, 2H), 4.23 (ddd, J=9.5, 7.1, 5.4 Hz, 1H), 3.67 (t, J=9.8 Hz, 1H), 3.39 (dd, J=10.0, 5.3 Hz, 1H), 3.19 (s, 3H), 1.24 (d, J=7.1 Hz, 3H).

c*) Synthesis of [.SUP.3.H-methyl]-1-(1-methoxy-1-methyl-ethyl) maleimide (MOMEM*)

[0209] ##STR00042##

[0210] The obtained crude solution of M25* was transferred into a sealed tube, diluted with toluene (100 μL) and heated at 110° C. for 2 h.

[0211] HPLC analysis showed full conversion to the deprotected product MOMEM* and remaining unreacted [.sup.3H]methyl nosylate M4*. The reaction mixture was allowed to cool to rt and solvent concentrated to dryness under an argon flow. The residue was purified by preparative HPLC to give the desired product [.sup.3H-methyl]methoxyethylene maleimide (MOMEM*) as a solution in the eluent mixture. The corresponding prep HPLC fraction, containing MOMEM* in eluent mixture was directly used for the conjugation with Oligos 3 and 4. Radio yield: 122.1 MBq (3.3 mCi)=13.2%. radio concentration: 30.0 MBq/mL (0.81 mCi/mL), radiochemical purity: 99%. Specific activity could not be determined by MS due to low ionisation. The specific activity was assumed to be 40 Ci/mmol.

Example 7 (Non-Radioactive Conjugation)

[0212] (Maleimide Compound of Formula VI, Wherein n=1, R.sup.1=Methyl and R.sup.2=H)

Oligonucleotides Used in the Examples

[0213]

TABLE-US-00009 (Oligo 3) G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH; MW: 5491.5 g/mol; (Oligo 4) 5′-SH-C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; MW: 6742.3 g/mol;

General Procedure:

[0214] ##STR00043##

[0215] 1 equivalent of oligonucleotide with 5′ or 3′ end sulfhydryl linker was dissolved in PBS (volume factor: 250 mL/g). 1.3 equivalent of 1-(1-methoxy-1-methyl-ethyl) maleimide (MOMEM), dissolved in THF (volume factor: 200 mL/g), was added to the aqueous solution and stirred at room temperature for 1 h. UPLC analysis showed a complete addition of maleimide to oligo nucleotide. To exchange the buffer to water, the reaction mixture was transferred into an Amicon® Pro purification system (MWCO: 3.000 Da) and centrifuged at 4000 rpm. DI water was added and the process was repeated 4 times more to complete the exchange. The resulting aqueous solution was lyophilized to isolate the oligonucleotide as a colorless powder with a yield in range of 83%-98% and 93%-98% purity.

[0216] In accordance with the general procedure the oligonucleotides (Oligo 3 and 4) have been conjugated with MOMEM.

TABLE-US-00010 a) Synthesis of conjugate 13 from Oligo 3 G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH-MOMCPM; Yield: 85%, purity: 95%, MS (m/z): 5668.6 [M-(H)].sup.- b) Synthesis of conjugate 14 from Oligo 4 5′-MOMCPM-SH-C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; Yield: 98%, purity: 98%, MS (m/z): 6818.7 [M-(H)].sup.-

Example 8 (Radioactive Conjugation)

[0217] (Maleimide Compound of Formula VI, Wherein n=1, R.sup.1=Methyl and R.sup.2=H)

Oligonucleotides Used in the Examples

[0218]

TABLE-US-00011 (Oligo 3) G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH; MW: 5491.5 g/mol; (Oligo 4) 5′-SH-C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; MW: 6742.3 g/mol;

General Procedure:

[0219] ##STR00044##

[0220] 1.2 equivalents of oligonucleotide with 5′ or 3′ end sulfhydryl linker was dissolved in PBS (10×) (volume factor: 250 mL/g). 1 equivalent of MOMEM*, directly used in prep HPLC eluent with a radio concentration of 30.0 MBq/mL (0.81 mCi/mL), was added to the aqueous oligonucleotide solution and stirred at room temperature for 2 h. UPLC analysis showed a conjugation of MOMEM* to oligo nucleotide in range of 62% to 66%. 10 equivalent of cold (non-radioactive) MOMEM, disolved in THF (volume factor: 700 mL/g), was added and stirred at rt for 2 h. UPLC showed a complete conjugation. The reaction mixture was transferred into an Amicon® Pro purification system (MWCO: 3.000 Da) and centrifuged at 4000 rpm. PBS (lx) was added and the process was repeated 4 times to complete a solvent exchange and receive the purified product. The concentration and activity of resulting buffered solution were determined. Radiochemical yields were calculated in range of 87%-89%, specific molar activities could achieve from 0.39 TBq/mmol (10.5 Ci/mmol) to 0.48 TBq/mmol (12.0 Ci/mmol). Radiochemical purities were in range of 93.4% to 94.3%

[0221] In accordance with the general procedure the oligonucleotides, Oligos 3 and 4 have been conjugated with MOMEM.

a) Synthesis of Conjugate 23* [.SUP.3.H]—from Oligo 3

[0222] G*A*G*t*t*a*c*t*t*g*c*c*a*A*C*T*-C6SH-[.sup.3H]-MOMEM; Yield 87%, radiochemical purity: 943%, activity: 16.7 MBq (0.45 mCi), specific molar activity: 0.48 TBq/mmol (12.0 Ci/mmol).

b) Synthesis of Conjugate 24* [.SUP.3.H]—from Oligo 4

[0223] 5′-[.sup.3H]-MOMEM-SH—C6*T*T*A*c*A*c*t*t*a*a*t*t*a*t*a*c*t*T*C*C; Yield 89%, radiochemical purity: 93.4%, activity: 14.8 MBq (0.40 mCi), specific molar activity: 0.39 TBq/mmol (10.5 Ci/mmol).