1. A process for preparation of DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetra acetic acid) -NHS ester, the process comprising the steps of: a. reacting cyclen with tert-butyl bromoacetate in the presence of a base in an organic solvent to obtain a tris-protected cyclen intermediate; b. reacting the obtained cyclen intermediate with benzyl bromoacetate in the presence of a base to obtain a benzyl-protected DOTA intermediate; c. subjecting the benzyl-protected intermediate to catalytic hydrogenation in the presence of palladium on carbon to obtain the corresponding carboxylic acid intermediate; d. activating the obtained acid intermediate with N-hydroxy succinimide in the presence of a coupling reagent to obtain an NHS ester intermediate; and e. removing tert-butyl protecting groups under acidic conditions to obtain DOTA-NHS ester.

2. The process as claimed in claim 1, wherein the base used in step (a) is any one of sodium acetate, Potassium Acetate, ammonium chloride, sodium chloride, Potassium orthophaosphate, sodium orthophosphate, sodium ortho silicate.

3. The process as claimed in claim 1, wherein the solvent used in step (a) is any one of N,N-dimethylacetamide, DMSO, CAN, Ethyl Acetate, isopropyl acetate, DMAC.

4. The process as claimed in claim 1, wherein the base used in step (b) is any one of potassium carbonate, sodium Carbonate, Sodium bicarbonate, Caesium Carbonate

5. The process as claimed in claim 1, wherein the hydrogenation catalyst used in step (c) is palladium on carbon.

6. The process as claimed in claim 1, wherein the coupling reagent used in step (d) is HBTU, HATU, PyBoc, T3P, Chloroformates, EDAC Hydrochloride.

7. The process as claimed in claim 1, wherein the deprotection in step (e) is carried out using acidic condition like TFA, HCl, Dioxane HCl, Methanolic HCl, IPA HCl,

8. The process as claimed in claim 1, wherein the synthesized DOTA-NHS ester is used for conjugation with biomolecules via amide bond formation.

Description:
[0001] The present invention relates to a process for preparation of DOTA-N-hydroxy succinimide (DOTA-NHS) ester derivatives and their applications in amide coupling reactions with biologically active molecules. More particularly, the present invention relates to scalable synthesis of tris-DOTA-NHS ester intermediates, which are useful for conjugation with amines present in peptides, proteins, antibodies, fluorescent probes, and other biomolecules, thereby facilitating the preparation of diagnostic and therapeutic agents.

BACKGROUND OF THE INVENTION

[0002] DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetra acetic acid) and its derivatives are widely used chelating agents for metal ions, particularly in the preparation of radiopharmaceuticals and molecular imaging probes. DOTA-based chelators exhibit excellent thermodynamic stability and kinetic inertness with metal ions such as Ga, Cu, Lu, and Y, making them highly suitable for biomedical applications.

[0003] To facilitate conjugation of DOTA derivatives to biomolecules, activated ester derivatives such as DOTA-NHS esters are commonly employed. NHS esters react readily with primary amines to form stable amide linkages, enabling efficient labelling of biomolecules.

[0004] Conventional process for the synthesis of DOTA-NHS derivatives often involve:
• Multi-step synthesis with low overall yields
• Difficult purification procedures
• Use of harsh reaction conditions
• Limited scalability for industrial production
• Formation of multiple side products due to uncontrolled alkylation steps.

[0005] Furthermore, selective functionalization of cyclin to obtain mono-functionalized or tris-substituted intermediates remains challenging because of the presence of four equivalent nitrogen atoms. Several literature methods describe the synthesis of DOTA derivatives; however, these approaches typically suffer from low efficiency, poor selectivity, and difficulties in large-scale preparation.
[0006]
[0007] Therefore, there exists a need to develop an improved and scalable synthetic method for preparing DOTA-NHS ester derivatives which overcomes abovementioned drawbacks

OBJECT OF THE INVENTION

[0008] An object of the present invention is to provide synthetic route for tris-DOTA-NHS ester and its applications in amide coupling reactions.

[0009] Another object of the present invention is to improved reaction efficiency and high purity intermediates, and scalable synthesis suitable for large-scale manufacturing of synthetic route for tris-DOTA-NHS ester

SUMMARY OF THE INVENTION

[00010] Accordingly, the present invention provides a process for preparation of DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetra acetic acid) -NHS ester. The process comprises reacting cyclen with tert-butyl bromoacetate in the presence of a base in an organic solvent to obtain a tris-protected cyclen intermediate, reacting the obtained cyclen intermediate with benzyl bromoacetate in the presence of a base to obtain a benzyl-protected DOTA intermediate, subjecting the benzyl-protected intermediate to catalytic hydrogenation in the presence of palladium on carbon to obtain the corresponding carboxylic acid intermediate, activating the obtained acid intermediate with N-hydroxy succinimide in the presence of a coupling reagent to obtain an NHS ester intermediate and removing tert-butyl protecting groups under acidic conditions to obtain DOTA-NHS ester
[00011] Typically, wherein the base used in step (a) is any one of sodium acetate, Potassium Acetate, ammonium chloride, sodium chloride, Potassium orthophaosphate, sodium orthophosphate, sodium ortho silicate.
[00012] Typically, wherein the solvent used in step (a) is any one of N,N-dimethylacetamide, DMSO, CAN, Ethyl Acetate, isopropyl acetate, DMAC.
[00013] Typically, wherein the base used in step (b) is any one of potassium carbonate, sodium Carbonate, Sodium bicarbonate, Caesium Carbonate.
[00014] Typically, wherein the hydrogenation catalyst used in step (c) is palladium on carbon.
[00015] Typically, wherein the coupling reagent used in step (d) is HBTU, HATU, PyBoc, T3P, Chloroformates, EDAC Hydrochloride.
[00016] Typically, wherein the deprotection in step (e) is carried out using acidic condition like TFA, HCl, Dioxane HCl, Methanolic HCl, IPA HCl,
[00017] Typically, wherein the synthesized DOTA-NHS ester is used for conjugation with biomolecules via amide bond formation.

DETAILED DESCRIPTION OF THE INVENTION

[00018] The foregoing objects of the present invention are accomplished and the problems and shortcomings associated with the prior art, techniques and approaches are overcome by the present invention as described below in the preferred embodiments.
[00019] The present invention provides a process for preparation of DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetra acetic acid) -NHS ester.

[00020] At first step, the process comprises reacting cyclen with tert-butyl bromoacetate in the presence of a first base solution in an organic solvent to obtain a tris-protected cyclen intermediate. In an embodiment, the first base solution is any one of sodium acetate, potassium Acetate, ammonium chloride, sodium chloride, Potassium orthophaosphate, sodium orthophosphate, sodium ortho silicate. Further, the solvent used is any one of N,N-dimethylacetamide, DMSO, CAN, Ethyl Acetate, isopropyl acetate. DMAC.

[00021] At second step, the process comprises reacting the obtained cyclen intermediate with benzyl bromoacetate in the presence of a second base solution to obtain a benzyl-protected DOTA intermediate. The second base solution used is any one of potassium carbonate, sodium Carbonate, Sodium bicarbonate, Cesium Carbonate.

[00022] At third step, the process comprises subjecting the benzyl-protected intermediate to catalytic hydrogenation in the presence of palladium on carbon to obtain the corresponding carboxylic acid intermediate. Specifically, the the hydrogenation catalyst used is palladium on carbon.

[00023] At fourth step, the process comprises activating the obtained acid intermediate with N-hydroxy succinimide in the presence of a coupling reagent to obtain an NHS ester intermediate. Specifically, the coupling agent used is HBTU, HATU, PyBoc, T3P, Chloroformates, EDAC Hydrochloride.

[00024] At fifth step, the process comprises removing tert-butyl protecting groups under acidic conditions to obtain DOTA-NHS ester. Specifically, the wherein the deprotection is carried out using acidic condition like TFA, HCl, Dioxane HCl, Methanolic HCl, IPA HCl, and the like.

[00025] In an embodiment, the synthesized DOTA-NHS ester is used for conjugation with biomolecules via amide bond formation.

Examples

Scheme-1: Shows a process for preparation of Tris-DOTA-NHS ester and its applications in amide coupling

1,4,7-Tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane hydrobromide (3).
To a flame-dried and cooled 2 L four neck round bottom flask, Cyclen (1,4,7,10-tetraazacyclododecane) (1) (30 g, 0.17 mol) was charged followed by addition of N,N-Dimethylacetamide (DMAC) (360 mL) at room temperature, under N2 atmosphere. The clear solution was then cooled to -20 ℃ and then added sodium acetate (NaOAc) (85 g, 1.02 mol) in one portion. The hazy reaction mixture was stirred for 30 min, before dropwise addition of diluted solution of t-butyl bromoacetate (101 g, 118 mL, 0.51 mol) in DMAc (120 mL) over a period of 1 h at same temperature. The reaction mixture was allowed to warm to room temperature and the vigorous stirring continued for 16 h. The completion of the reaction was confirmed by TLC. The reaction mixture was poured into water (1,800 mL) to get clear solution. The pH of the solution was adjusted to 8-9 by carefully portion wise addition of KHCO3, the product was precipitated out. The dichloromethane (DCM) (500 mL) was added to dissolve precipitate and layer was separated. The aqueous layer was extracted with DCM (2 x 300 mL) and the combined organic phase was washed with water (1 x 400 mL), brine (1 x 300 mL), dried over Na2SO4, filtered and concentrated in vacuo to afford crude solid product. The crude solid was triturated and stirred in diethyl ether (1 L) at 0-5 ℃ for 30 min. The free flow pure product was filtered and dried to afford 1,4,7-Tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane hydrobromide (3) (53 g, 59.13%) as a white solid. This was used as such for next step. Melting point (m.p.) 185–187 ℃ (lit.mp 179–181 ℃), TLC: Rf = 0.30 (MeOH/DCM 1:10 on silica gel 60 F254, stain-Draggendorf for visualization). 1H NMR (400 MHz, CDCl3) d 1.45 (s, 9H), 1.46 (s, 18H), 2.87 (m, 4H), 2.93 (m, 8H), 3.10 (m, 4H), 3.29 (s, 2H), 3.38 (s, 4H), 10.03 (br s, 1H); MS (ESI+): mlz 515 [M+H]+.

Synthesis of Tri-tert-butyl 2,2’,2"-(10-(2-(benzyloxy)-2-oxoethyl)-1,4,7,10 tetraazacyclododecane-1,4,7- triyl)triacetate (5).
To a flame-dried and cooled 2 L four neck round bottom flask, 1,4,7-Tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane hydrobromide (3) (10 g, 0.02 mol) was charged followed by addition of DCM (30 mL) under N2 atmosphere. To the clear solution was then added potassium carbonate (16 g, 0.12 mol) at room temperature. There was no exotherm observed. To the resulting suspension, benzyl bromoacetate (4) (6.67 g, 4.62 mL, 0.03 mol) was added dropwise over a period of 30 min. The reaction mixture was continued stirring for 16 h at room temperature. The completion of the reaction was confirmed by TLC. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The crude was triturated with fresh hexane and decanted (2-3 times, 50 mL) and concentrated in vacuo. The crude residue was bind on celite and triturated and decanted with hexane (5 x 50 mL) and finally filtered. This made sure to get rid-off excess benzyl bromoacetate. Subsequently, the celite bed was washed with DCM (3 x 100 mL) and the filtrate obtained was combined and concentrated in vacuo to afford tri-tert-butyl 2,2',2''-(10-(2-(benzyloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (5) (12.8 g, 99%) as a off-white s amorphous solid. This was used as such for next step. Melting point (mp): 193 °C (lit. mp 195 ℃), TLC: Rf = 0.8 (MeOH/DCM 1:10 on silica gel 60 F254, stain-Draggendorf & UV for visualization). 1H NMR (400 MHz, CDCI3) δ 7.37-7.36 (m, 5H), 5.15 (s, 2H), 3.35-2.00 (br m, 24 H), 1.45-1.41 (s, 27 H) ppm. MS (ESI+): m/z 663.4 [M+H]+, 685.3 [M+Na]+.

Synthesis of 2-(4,7,10-Tris(2-(ferf-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (6).
In a 700 mL autoclave, to a solution of tri-tert-butyl 2,2',2''-(10-(2-(benzyloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (5) (13 g, 0.02 mol) in MeOH (120 mL), palladium on carbon (wet 10% Pd, 1.3 g, 10%w/w loading) was added and the reaction mixture was stirred under hydrogen atmosphere (142.23 psi) at room temperature for 12 h. The completion of the reaction was confirmed by TLC. The reaction mixture was unloaded and the catalyst was removed by filtration through celite bed, washed with MeOH (2 x 100 mL). The combined filtrate was evaporated in vacuo to afford desired product 2-(4,7,10-Tris(2-(ferf-butoxy)-2-oxoethyl)- 1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (6) (11.20 g, 99 %) as a off-white amorphous solid. This was used as such for next step. Melting point (mp): 132 °C (lit. mp 130 °C), TLC: Rf = 0.2 (MeOH/DCM 1:10 on silica gel 60 F254, stain-Draggendorf & UV for visualization). 1H NMR (400 MHz, DMSO-d6): δ 3.40-3.06 (br m, 9H), 2.80-2.49 (m, 3H), 2.49-2.22 (m, 12H), 1.43-1.42 (s, 27H) ppm. MS (ESI): m/z 573 [M+H]+.

Synthesis of tri-tert-butyl 2,2’,2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (7).

To a flame-dried and cooled 250 mL single neck round bottom flask, 2-(4,7,10-Tris(2-(ferf-butoxy)-2-oxoethyl)- 1,4,7,10-tetraazacyclododecan-1-yl)acetic acid (6) (5 g, 0.01 mol) was charged followed by addition of acetonitrile (25 mL) under N2 atmosphere. To the reaction mixture was then added O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) (3.78 g, 0.01 mol), followed by addition of N-hydroxy succinimide (1.14 g, 0.01 mol) in one portion at room temperature. The reaction mixture was then continued stirring for 4 h. The completion of the reaction was confirmed by TLC. The reaction mixture was filtered and filtrate was concentrated in vacuo. The crude residue obtained was partitioned in EtOAc (100 mL) and water (50 mL), the organic phase washed with sat. NaHCO3 (2 x 50 mL), water (1 x 50 mL), brine (1 x 40 mL), dried over Na2SO4, filtered and concentrated in vacuo to afford tri-tert-butyl 2,2’,2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (7) (4.2 g, 71.83%) as a white solid. TLC: Rf = 0.3 (MeOH/DCM 15:85 on silica gel 60 F254, stain-Draggendorf & UV for visualization). 1H NMR (400 MHz, CDCI3) δ 3.41-3.00 (br m, 6H), 3.00-2.75 (br m, 8H), 2.75-1.75 (br m, 14 H), 1.44 (s, 27H) ppm. MS (ESI) m/z 692 [M + H]+

Synthesis of 2,2’,2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (8)

To a flame-dried and cooled 250 mL three neck round bottom flask, tri-tert-butyl 2,2’,2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane- 1,4,7-triyl)triacetate (1 g, 1.49 mmol) was suspended in 1,4-dioxane (10 mL) under N2 atmosphere. The reaction mixture was then cooled to 0 ℃ and then added 4 M HCl in 1,4-dioxane (2.5 mL). The reaction mixture was heated to 50 ℃ to get the homogenous clear solution and continued stirring for 2 h. The completion of the reaction was confirmed by TLC. The reaction mixture was concentrated in vacuo to afford 2,2’,2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (8) (331 mg, 44.25%) as a white solid. TLC: Rf : 0.01 (MeOH/ CH2CI2, 1:1). 1H NMR (400 MHz, DMSO-d6) δ 4.09-3.70 (br, 9 H), 3.50-2.85 (14 H), 2.69 (m, 4H), 2.49 (s, 4H) ppm.

Scheme-2: Shows a process of preparation (S)-2,2',2''-(10-(2-oxo-2-((1-phenylethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9a)

Synthesis of tri-tert-butyl,2,2',2''-(10-(2-oxo-2-((1-phenylethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(S)-triacetate (8a)

To a solution of tri-tert-butyl 2,2’,2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (7) (552 mg, 0.83 mmol) and (S)-1-phenylethan-1-amine (7a) (50 mg, 0.41 mmol) in DMF (2 mL), DIPEA (0.28 mL, 1.65 mmol) was added under N2 atmosphere at room temperature. The reaction mixture was stirred for 14 h at room temperature. The completion of the reaction was confirmed by TLC. The reaction mixture was poured into water (15 mL) and aqueous layer was extracted with ethyl acetate (3 x 15 mL). The combined organic phase was washed with ice-chilled brine (2 x 10 mL), dried over Na2SO4, filtered and concentrated in vacuo to get crude product. The crude was purified by silica gel (100-200 mesh) column chromatography using 2-4% MeOH/DCM as an eluent to afford tri-tert-butyl 2,2',2''-(10-(2-oxo-2-((1-phenylethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(S)-triacetate (8a) (150 mg, 53.96%) as a off-white solid. TLC: Rf : 0.3 (MeOH/ CH2CI2, 1:9). 1H NMR (400 MHz, CDCI3) δ 7.35-7.21 (m, 5H), 6.5 (br s, 1H), 5.03 (m, 1H), 3.38-2.5 (br m, 12 H), 2.50-2.00 (br m, 12 H), 1.62-1.22 (br m, 30 H) ppm. Direct Mass (ESI): m/z 677 [M+H].

Synthesis of (S)-2,2',2''-(10-(2-oxo-2-((1-phenylethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9a).

The compound tri-tert-butyl 2,2',2''-(10-(2-oxo-2-((1-phenylethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(S)-triacetate (8a) (50 mg, 0.07 mmol) was first dissolved in 1,4-dioxane (5 mL) under N2 atmosphere. The reaction mixture was then cooled to 0 ℃ and then added 4 M HCl in 1,4-dioxane (2.5 mL). The reaction mixture was allowed to reach room temperature and continued stirring for 4 h. The completion of the reaction was confirmed by TLC. The reaction mixture was concentrated in vacuo to afford (S)-2,2',2''-(10-(2-oxo-2-((1-phenylethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9a). (35 mg, 93.33%) as a white solid. TLC: Rf : 0.03 (MeOH/CH2CI2, 1:1). Direct Mass (MSI): m/z 508 [M+H]+.

Scheme-3: Shows a process of preparation of (S)-2,2',2''-(10-(2-((5-(((benzyloxy)carbonyl)amino)-1-carboxypentyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9b)

Synthesis of tri-tert-butyl 2,2',2''-(10-(2-((6-(((benzyloxy)carbonyl)amino)-1-(tert-butoxy)-1-oxohexan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(S)-triacetate (8b).

To a solution of tri-tert-butyl 2,2’,2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (7) (300 mg, 0.45 mmol) and tert-butyl n6-((benzyloxy)carbonyl)lysinate hydrochloride (7b) (167 mg, 0.44 mmol) in DMF (2 mL), DIPEA (0.19 mL, 1.12 mmol) was added under N2 atmosphere at room temperature. The reaction mixture was stirred for 14 h at room temperature. The completion of the reaction was confirmed by TLC. The reaction mixture was poured into water (10 mL) and aqueous layer was extracted with ethyl acetate (3 x 10 mL). The combined organic phase was washed with ice-chilled brine (2 x 15 mL), dried over Na2SO4, filtered and concentrated in vacuo to afford crude product. The crude was purified by silica gel (100-200 mesh) column chromatography using 2-5% MeOH/DCM as an eluent to afford tri-tert-butyl 2,2',2''-(10-(2-((6-(((benzyloxy)carbonyl)amino)-1-(tert-butoxy)-1-oxohexan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(S)-triacetate (8b) (250 mg, 62.66%) as a off-white solid. TLC: Rf : 0.35 (MeOH/ CH2CI2, 1:9). 1H NMR (400 MHz, CDCI3) δ 7.35-7.28 (m, 5H), 5.15 (s, 2H), 5.10-4.85 (m, 1 H), 3.50-2.5 (br m, 12 H), 2.50-1.65 (br m, 20 H), 1.65-1.20 (br m, 38 H) ppm. MS (ESI): m/z 891 [M+H]+, 913 [M+Na]+.

Synthesis of (S)-2,2',2''-(10-(2-((5-(((benzyloxy)carbonyl)amino)-1-carboxypentyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9b).
The compound tri-tert-butyl 2,2',2''-(10-(2-((6-(((benzyloxy)carbonyl)amino)-1-(tert-butoxy)-1-oxohexan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(S)-triacetate (8b) (50 mg, 0.06 mmol) was first dissolved in 1,4-dioxane (7 mL) under N2 atmosphere. The reaction mixture was then cooled to 0 ℃ and then added 4 M HCl in 1,4-dioxane (3.5 mL). The reaction mixture was allowed to reach room temperature and continued stirring for 4 h. The completion of the reaction was confirmed by TLC. The reaction mixture was concentrated in vacuo to afford (S)-2,2',2''-(10-(2-((5-(((benzyloxy)carbonyl)amino)-1-carboxypentyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9b). (30 mg, 81.08%) as a off-white solid. TLC: Rf : 0.04 (MeOH/CH2CI2, 1:1). Direct Mass (MSI): m/z 667 [M+H]+.

Scheme-4. Shows a process for preparation of of 2,2',2''-(10-(2-((2,6-difluorophenyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9c).

Synthesis of tri-tert-butyl 2,2',2''-(10-(2-((2,6-difluorophenyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (8c).

To a solution of tri-tert-butyl 2,2’,2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (7) (570 mg, 0.85 mmol) and 2,6-difluoroaniline (7c) (100 mg, 0.77 mmol) in DMF (2 mL), DIPEA (0.40 mL, 2.31 mmol) was added under N2 atmosphere at room temperature. The reaction mixture was stirred for 14 h at room temperature. The completion of the reaction was confirmed by TLC. The reaction mixture was poured into water (15 mL) and aqueous layer was extracted with ethyl acetate (3 x 15 mL). The combined organic phase was washed with ice-chilled brine (2 x 10 mL), dried over Na2SO4, filtered and concentrated in vacuo to afford crude product. The crude was purified by silica gel (100-200 mesh) column chromatography using 4-6% MeOH/DCM as an eluent to afford tri-tert-butyl 2,2',2''-(10-(2-((2,6-difluorophenyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (8c). (317 mg, 60%) as a off-white solid. TLC: Rf : 0.4 (MeOH/ CH2CI2, 1:9). MS (ESI): m/z 685 [M+H]+.

Synthesis of 2,2',2''-(10-(2-((2,6-difluorophenyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9c).

The compound tri-tert-butyl 2,2',2''-(10-(2-((2,6-difluorophenyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (8c) (100 mg, 0.15 mmol) was first dissolved in 1,4-dioxane (10 mL) under N2 atmosphere. The reaction mixture was then cooled to 0 ℃ and then added 4 M HCl in 1,4-dioxane (3.5 mL). The reaction mixture was allowed to reach room temperature and continued stirring for 4 h. The completion of the reaction was confirmed by TLC. The reaction mixture was concentrated in vacuo to afford 2,2',2''-(10-(2-((2,6-difluorophenyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (9c) (70 mg, 93.33%) as a off-white solid. TLC: Rf : 0.02 (MeOH/CH2CI2, 1:1). Direct Mass (MSI): m/z 516 [M+H]+.

Scheme-5. Shows a process for preparation of 2,2',2''-(10-(2-oxo-2-(piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid hydrogen chloride (9d).

Synthesis of tri-tert-butyl 2,2',2''-(10-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (8d).

To a solution of tri-tert-butyl 2,2’,2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (7) (395 mg, 0.59 mmol) and tert-butyl piperazine-1-carboxylate (7d) (100 mg, 0.54 mmol) in DMF (2 mL), DIPEA (0.28 mL, 1.61 mmol) was added under N2 atmosphere at room temperature. The reaction mixture was stirred for 14 h at room temperature. The completion of the reaction was confirmed by TLC. The reaction mixture was poured into water (10 mL) and aqueous layer was extracted with ethyl acetate (3 x 10 mL). The combined organic phase was washed with ice-chilled brine (2 x 15 mL), dried over Na2SO4, filtered and concentrated in vacuo to afford crude product. The crude was purified by silica gel (100-200 mesh) column chromatography using 5-8% MeOH/DCM as an eluent to afford tri-tert-butyl 2,2',2''-(10-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (8d) (300 mg, 75.37%) as a off-white solid. TLC: Rf : 0.3 (MeOH/ CH2CI2, 1:9). MS (ESI): m/z 741 [M+H]+.

Synthesis of 2,2',2''-(10-(2-oxo-2-(piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid hydrogen chloride (9d).

The compound tri-tert-butyl 2,2',2''-(10-(2-(4-(tert-butoxycarbonyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (8d) (100 mg, 0.13 mmol) was first dissolved in 1,4-dioxane (10 mL) under N2 atmosphere. The reaction mixture was then cooled to 0 ℃ and then added 4 M HCl in 1,4-dioxane (5 mL). The reaction mixture was allowed to reach room temperature and continued stirring for 4 h. The completion of the reaction was confirmed by TLC. The reaction mixture was concentrated in vacuo to afford 2,2',2''-(10-(2-oxo-2-(piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid hydrogen chloride (9d) (60 mg, 86.96%) as a off-white solid. TLC: Rf : 0.01 (MeOH/CH2CI2, 1:1). Direct Mass (MSI): m/z 509 [M+H]+.

[00036] In comparison with previously reported methods and commercially available DOTA-NHS esters, the DOTA-NHS ester obtained by the process of the present invention exhibits significantly improved chemical purity, typically greater than 98% as determined by HPLC, whereas conventional DOTA-NHS esters generally exhibit purity in the range of 90–95% and are often associated with degradation-related impurities.

[00037] The improved purity of the present DOTA-NHS ester is attributed to the controlled stepwise functionalization of cyclen, selective formation of tris-substituted intermediates, and minimized formation of over-alkylated and partially hydrolyzed by-products. In contrast, prior art processes often result in complex impurity profiles due to non-selective alkylation and harsh reaction conditions.

[00038] Further, the DOTA-NHS ester obtained by the present process demonstrates enhanced stability against hydrolysis, thereby retaining the active N-hydroxy succinimide ester functionality for a longer duration during storage and handling. Conventional DOTA-NHS esters are known to undergo rapid hydrolysis to corresponding carboxylic acids, leading to reduction in effective assay and performance.
[00039] The present DOTA-NHS ester also exhibits improved batch-to-batch consistency, owing to simplified work-up procedures and reduced dependence on chromatographic purification. In contrast, earlier processes often show variability due to incomplete reactions and inconsistent purification efficiency.
[00040] Additionally, the reduced level of hydrolytic and process-related impurities in the present DOTA-NHS ester results in enhanced reactivity towards amine-containing biomolecules, thereby providing higher conjugation efficiency as compared to conventional DOTA-NHS esters.
[00041] Thus, the DOTA-NHS ester prepared by the process of the present invention provides a combination of higher purity, improved stability, cleaner impurity profile, and superior functional performance, making it particularly advantageous for applications in bioconjugation, radiopharmaceutical synthesis, and diagnostic imaging.

Advantages of the invention

[00026] The process of preparation of the DOTA-NHS ester of the present invention can be used for amide coupling reactions with primary amines present in biomolecules, thereby enabling the synthesis of bioconjugates useful in diagnostics, imaging, and therapeutic applications.
[00027] The obtained activated ester readily reacts with primary amines, enabling efficient amide bond formation with biomolecules such as peptides, proteins, antibodies, imaging probes and small molecule ligands.
[00028] The process of preparation of the DOTA-NHS ester of the present invention is suitable for gram to kilogram scale production, making it highly practical for industrial manufacturing.
[00029] The process of preparation of the DOTA-NHS ester of the present invention enables selective formation of tris-substituted cyclen intermediates, thereby minimizing formation of undesired over-alkylated by-products.
[00030] The process of preparation of the DOTA-NHS ester of the present invention provides good yields at each step, resulting in improved overall yield of the final DOTA-NHS ester.
[00031] The process avoids complex purification procedures and can be performed using conventional laboratory equipment and reagents.
[00032] In the process of preparation of the DOTA-NHS ester of the present invention, most intermediates can be isolated through simple work-up and crystallization, reducing dependence on column chromatography and making the process suitable for scale-up.
[00033] The process of preparation of the DOTA-NHS ester of the present invention exhibits high chemical purity, making it suitable for sensitive applications such as bioconjugation and radiopharmaceutical synthesis.
[00034] The process of preparation of the DOTA-NHS ester of the present invention produces NHS ester reacts efficiently with amine-containing biomolecules, enabling the preparation of a wide range of DOTA-based bioconjugates.

[00035] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, and to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient, but such omissions and substitutions are intended to cover the application or implementation without departing from the spirit or scope of the claims of the present invention.
, Claims:We Claim

1. A process for preparation of DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetra acetic acid) -NHS ester, the process comprising the steps of:
a. reacting cyclen with tert-butyl bromoacetate in the presence of a base in an organic solvent to obtain a tris-protected cyclen intermediate;
b. reacting the obtained cyclen intermediate with benzyl bromoacetate in the presence of a base to obtain a benzyl-protected DOTA intermediate;
c. subjecting the benzyl-protected intermediate to catalytic hydrogenation in the presence of palladium on carbon to obtain the corresponding carboxylic acid intermediate;
d. activating the obtained acid intermediate with N-hydroxy succinimide in the presence of a coupling reagent to obtain an NHS ester intermediate; and
e. removing tert-butyl protecting groups under acidic conditions to obtain DOTA-NHS ester.

2. The process as claimed in claim 1, wherein the base used in step (a) is any one of sodium acetate, Potassium Acetate, ammonium chloride, sodium chloride, Potassium orthophaosphate, sodium orthophosphate, sodium ortho silicate.

3. The process as claimed in claim 1, wherein the solvent used in step (a) is any one of N,N-dimethylacetamide, DMSO, CAN, Ethyl Acetate, isopropyl acetate, DMAC.
4. The process as claimed in claim 1, wherein the base used in step (b) is any one of potassium carbonate, sodium Carbonate, Sodium bicarbonate, Caesium Carbonate
5. The process as claimed in claim 1, wherein the hydrogenation catalyst used in step (c) is palladium on carbon.
6. The process as claimed in claim 1, wherein the coupling reagent used in step (d) is HBTU, HATU, PyBoc, T3P, Chloroformates, EDAC Hydrochloride.
7. The process as claimed in claim 1, wherein the deprotection in step (e) is carried out using acidic condition like TFA, HCl, Dioxane HCl, Methanolic HCl, IPA HCl,
8. The process as claimed in claim 1, wherein the synthesized DOTA-NHS ester is used for conjugation with biomolecules via amide bond formation.