Method for the synthesis of trofinetide and its analogues

Abstract

The present invention relates to a method for the synthesis of Trofinetide (1) and its analogues The present invention further relates to a novel approach for the synthesis of Trofinetide (1) and its analogues through TAG approach by sequential amino acid coupling using solid-phase peptide synthesis. The patent also relates to a commercially viable purification method for the synthesis of highly pure Trofinetide (1) and its analogues. Formula (1)

Description

[0001]“METHOD FOR THE SYNTHESIS OF TROFINETIDE AND ITS ANALOGUES” FIELD OF THE INVENTION The present invention relates to a method for synthesizing Trofinetide (1) and its analogues. It further introduces a novel approach for the synthesis of Trofinetide (1) and its analogues using the TAG approach, involving sequential amino acid coupling through solid-phase peptide synthesis. Additionally, the invention provides a commercially viable purification method for producing high-purity Trofinetide (1) and its analogues. BACKGROUND OF THE INVENTION The tripeptides are used to treat the main symptoms of Rett syndrome (RTT). RTT is a genetically induced neurological disorder. Trofinetide is a tripeptide synthetic compound indicated for the treatment of Rett syndrome in people two years of age and older. Trofinetide is designated chemically as (2S)-2-{[(2S)-1-(2-aminoacetyl)-2- methylpyrrolidine-2-carbonyl] amino} pentane dioic acid (IUPAC). Its empirical formula is C13H21N3O6and its molecular weight is 315.33 g / mol. The chemical structure is: Trofinetide is the first drug approved by the US Food and Drug Administration in March 2023. It is an oral medication sold under the brand name Daybue. Like its parent tripeptide Glypromate, Trofinetide is also tripeptide in which proline has been modified. Tripeptide Glypromate (glycine-proline glutamate) is a naturally occurring small- molecule neuroprotectant derived from IGF-1 which inhibits caspase III dependent apoptosis, for the potential treatment of neurodegenerative diseases by IV infusion. Tetrahedron 61 (2005) 10018–10035, Neuren Pharmaceuticals Medicinal Chemistry Group, provides a method for the synthesis of ten analogues of GPE modified at proline residue. US 2003 / 0055004 A1 patent application reported the synthesis of GPE by modifying glutamic acid and glycine residue on solid-phase peptide synthesis. US 2023023114 A1 patent application provided the pharmaceutical composition of crystalline forms of Trofinetide and Trofinetide hydrates. The synthetic strategies for the preparation of Trofinetide and its analogues adopted in the prior art includes the use of a variety of coupling reagents linking different amino acids and different protecting groups for the amino acids. Still, there is a long-felt need in the art to develop a simple method for the synthesis of Trofinetide and its analogues which are economical, commercially scalable, cost- efficient, have better yield, less impurities, can be readily purified by well-known techniques and use safe starting materials. The prior art discloses the traditional solid phase synthesis which involves use of polymeric materials and non-solubility issues. The solution phase synthesis involves removal of impurities as a major issue for the preparation of Trofinetide and its analogues. Hence, the present inventors found a novel approach for the synthesis of Trofinetide and its analogues via peptide tagging which ameliorates the drawbacks of the prior art. Tagging approach in the peptide synthesis is economical, advantageous better than the traditional solid phase synthesis and solution phase synthesis. OBJECTS OF THE INVENTION One objective of the present invention is to provide a method for synthesizing Trofinetide (1) and its analogues. Another objective is to provide a method for synthesizing Trofinetide, and its analogues derived from insulin-like growth factor-1 (IGF-1). A further objective is to provide a method for synthesizing Trofinetide and its analogues through sequential amino acid couplings using the TAG approach. Another objective is to provide a simple, commercially feasible purification method utilizing safe starting materials, reagents, and solvents. Yet another objective is to provide an environmentally friendly, cost-effective, and efficient method for synthesizing Trofinetide (1) and its analogues with high yield and purity. SUMMARY OF THE INVENTION One aspect of the present invention provides an improved method for the synthesis of Trofinetide and its analogues. Another aspect of the present invention provides a method for the synthesis of Trofinetide and its analogues using sequential amino acid coupling through the TAG approach. Yet another aspect of the present invention provides a method for the synthesis of Trofinetide (1) and its analogues. wherein the method comprises the following steps: a) coupling the N-protected C-terminal of the first amino acid (6) with TAG-OH in the presence of a coupling reagent, base, and solvent to obtain the TAG-linked amino acid (5); b) deprotecting the Fmoc group in the TAG-linked amino acid (5) using a deprotecting agent in the presence of a base and solvent, followed by coupling with the N- protected second amino acid (4) in the presence of a coupling reagent, base, and solvent to obtain the dipeptide compound in situ; c) deprotecting the Fmoc group of the dipeptide obtained in step (b), followed by coupling with the N-protected third amino acid (3) in the presence of a coupling reagent, base, and solvent to obtain the TAG-linked tripeptide (2); d) cleaving the N-protecting group and TAG from the tripeptide compound of formula (2) using a cocktail mixture to obtain crude Trofinetide (1a); e) optionally, purifying the crude Trofinetide (1a) to obtain pure Trofinetide (1). In another aspect of the present invention provides a facile purification method for the synthesis of high pure Trofinetide and its analogues. BRIEF DESCRIPTION OF DRAWINGS Figure 1 illustrates High Performance liquid Chromatogram of Trofinetide (1) Figure 2 illustrates (1H) Proton nuclear magnetic resonance spectrum of Trofinetide (1) Figure 3 illustrates Liquid chromatography–mass spectrometry of Trofinetide (1) Figure 4 illustrates (1H) Proton nuclear magnetic resonance spectrum of compound (5) Figure 5 illustrates (1H) Proton nuclear magnetic resonance spectrum of compound (2) Figure 6 illustrates (1H) Proton nuclear magnetic resonance spectrum of compound (8) Figure 7 illustrates mass spectrometry of compound (8) Figure 8 illustrates (1H) Proton nuclear magnetic resonance spectrum of compound (9) Figure 9 illustrates mass spectrometry of compound (9) DETAILED DESCRIPTION OF THE INVENTION The term “suitable solvent” used in the present invention until unless specified is selected from, but are not limited to “alcoholic solvents” such as methanol, ethanol, isopropyl alcohol, n-propanol, butanol and the like; “ester solvents” such as ethyl acetate, methyl acetate, n-butyl acetate, isobutyl acetate, sec-butyl acetate, isopropyl acetate and the like; “ether solvents” such as tetrahydrofuran, diethyl ether, methyl tert-butyl ether, 2-methyl tetrahydrofuran, 1,4-dioxane and the like; “hydrocarbon solvents” such as toluene, xylene, cyclohexane, hexane, heptane, n-pentane, petroleum ether and the like; “chloro solvents” such as dichloromethane, ethylene dichloride, carbon tetrachloride, chloroform and the like; “polar aprotic solvents” such as dimethylformamide, dimethylacetamide, dimethylsulfoxide and the like; “nitrile solvents” such as acetonitrile and the like; “ketone solvents” such as acetone, methyl isobutyl ketone, methyl ethyl ketone and the like; and water and / or mixtures thereof. The term “suitable base” used herein the present invention until unless specified is selected from inorganic bases like “alkali metal carbonates" such as sodium carbonate, potassium carbonate, lithium carbonate, cesium carbonate and the like; "alkali metal bicarbonates" such as sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, cesium bicarbonate and the like; "alkali metal hydroxides" such as sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide and the like; "alkali metal alkoxides" such as sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, lithium methoxide, lithium ethoxide, sodium tert-butoxide, potassium tert- butoxide, lithium tert-butoxide and the like; organic bases like dimethylamine, diethylamine, diisopropyl amine, diisopropylethylamine (DIPEA), diisobutylamine, trimethylamine, triethylamine, triisopropylamine, tributylamine, tert.butyl amine, pyridine, piperidine, 4-dimethylamino pyridine (DMAP) or mixtures thereof. The term “coupling agent” used herein the present invention until unless specified is selected from the group consisting of Benzotriazole- 1 -yl-oxy-tris- (dimethylamino)- phosphonium hexafluorophosphate (BOP), Benzotriazole- 1-yl-oxy-tris-pyrrolidino- phosphonium hexafluorophosphate (PyBOP), O-(IH-Benzotriazol-l-yl)-N,N, N,N- tetramethyluronium tetrafluoroborate (TBTU), O-(7-Azabenzotriazole- 1 -yl)-N,N,N,N- tetramethyluronium tetrafluoroborate (TATU), O-(lH-Benzotriazole-l-yl)-N, N, N, N- tetramethyluronium hexafluorophosphate,(HBTU), 2-(7- Aza-lH-benzotriazole-l-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), N,N- dicyclohexylcarbodiimide (DCC), Ν,Ν'-Diisopropylcarbodiimide (DIC), 1-[(1-(Cyano- 2-ethoxy-2-oxoethylideneaminooxy) dimethylaminomorpholino)]uronium hexafluorophosphate (COMU) or l-Ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC.HC1). The following description is provided to assist in a comprehensive understanding of exemplary embodiments of the invention. The present invention provides a novel method for the synthesis of Trofinetide and its analogues using tagging approach. The tagging approach is cost-efficient compared to solid and solution phase peptide synthesis methods. The preparation method provides a commercially scalable, cost-efficient method with improved yield and impurities are controlled within the ICH limits. In another embodiment, the present invention provides a method for synthesis of Trofinetide (1) or its analogues as illustrated in Scheme 1: In another embodiment, the steps involved in the preparation of Trofinetide (1) or its analogues as shown in scheme 1 are as follows: Step a) of the foregoing process involves coupling the N-protected C-terminal of the first amino acid (6) with TAG-OH in the presence of a coupling reagent, a base, and solvent under appropriate reaction condition to obtain the TAG-linked amino acid (5). The coupling agent used in step a) is selected from the list as defined above, preferably DIC. The base used in stap a) is selected from the list as defined above, preferably DMAP. The solvent used in stap a) is selected from the list as defined above, preferably THF. The step a) reaction is carried out at a suitable temperature of about 0°C to about 5°C for a sufficient period till completion of the reaction. Step b) of the foregoing process involves deprotecting the Fmoc group in the TAG-linked amino acid (5) using a deprotecting agent in the presence of a base and solvent, followed by coupling with the N-protected second amino acid (4) in the presence of a coupling reagent, base, and solvent under appropriate reaction condition to obtain the dipeptide compound in situ. The deprotecting agent used in step b) is piperidine. The coupling agent used in step b) is selected from the list as defined above, preferably COMU. The base used in stap b) is selected from the list as defined above, preferably DBU and DIPEA. The solvent used in stap b) is selected from the list as defined above, preferably THF. The step a) reaction is carried out at a suitable temperature of about 0°C to about 5°C for a sufficient period till completion of the reaction. Step c) of the foregoing process involves deprotecting the Fmoc group of the dipeptide obtained in step (b), followed by coupling with the N-protected third amino acid (3) in the presence of a coupling reagent, base, and solvent under appropriate reaction condition to obtain the TAG-linked tripeptide (2). The deprotecting agent used in step c) is piperidine / DBU. The coupling agent used in step c) is selected from the list as defined above, preferably COMU. The base used in stap b) is selected from the list as defined above, preferably DIPEA. The solvent used in stap b) is selected from the list as defined above, preferably THF. Step d) of the foregoing process involves cleaving the N-protecting group and TAG from the tripeptide compound of formula (2) using a cocktail mixture under appropriate reaction condition to obtain crude Trofinetide (1a). The cocktail mixture is a mixture of TFA (95%), TIPS (2.5%) and H2O (2.5%). In another embodiment of the present invention, the N-protected first amino acid is having a structure of Fmoc-γ-Glu-OtBu (6); N-protected second amino acid is having a structure of Fmoc-α-CH3-Pro-OH (4); and N-protected third amino acid is having a structure of Boc-Gly-OH (3). In yet another embodiment of the present invention, the synthesis of linear peptide backbone is carried out on TAG-OH. The term TAG-OH referred herein is (2,4- bis(octadecyloxy)phenyl) methanol (7). TAG-OH (7) In another embodiment of the present invention, the TAG linked amino acid is Fmoc- Glu-(OCH2-TAG)-OtBu (5) and the TAG linked tripeptide is Boc-Gly-α-CH3-Pro-Glu- (OCH2-TAG)-OtBu (2). In a preferred embodiment, the present synthetic approach allows the introduction of TAG ((2,4-bis-octadecyl oxy-phenyl)-methanol) to the C- C-terminus end of γ-glutamic acid at the first step. In yet another embodiment of the present invention, according to step e) the purification of peptides is carried out by reverse phase HPLC using a mixture of solvents selected from TFA in water, acetic acid, acetonitrile, orthophosphoric acid in water, triethyl amine in water, ammonium acetate in water and ammonium bicarbonate in water. In yet another embodiment of the present invention, the final isolation of crude peptide after prep HPLC purification is carried out by lyophilization. In yet another embodiment of the present invention, the side modification is done either in the early stage of synthesis or after complete peptide backbone synthesis. Alternatively, the above-mentioned compounds are prepared by liquid phase easily by using the Tagging technique on an industrial scale without any difficulty with good purity and in high yield. The compounds were prepared with an acid-labile Tagging technique to provide the desired protected tripeptide and subsequently desired final Trofinetide with good yield and purity. The present invention provides a preparation method for fragment synthesis on less expensive Tags instead of costly resins. This provides better yield and less impurity wherein the peptides can be easily purified using simple non-expensive techniques. The method described herein in the present invention can provide Trofinetide and its analogues with good yield and high purity on an industrial scale. In another embodiment, the present invention provides a facile purification method for the preparation of high pure tripeptide and its analogues. The purity of Trofinetide (1) obtained by the current method is greater than 97% by HPLC and impurities are controlled less than 1.0% (w / w) and preferably less than 0.5%(w / w). A flow chart for the sequential amino acids coupling for the synthesis of Trofinetide and its analogues using TAG approach is represented in scheme 2. DEFINITIONS: A list of Abbreviations used in the specification is given in Table 1: Sr. No Abbreviation Name 1 Fmoc 9- fluorenylmethoxycarbonyl 2 TAG (2, 4-Bis-octadecyl oxy-phenyl)-methanol 3 DMAP 4-Dimethyl aminopyridine 4 HBTU Hexafluorophosphate Benzotriazole Tetramethyl Uronium 5 COMU (1-Cyano-2-ethoxy-3-oxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate 6 HATU O-(7-Aza-benzotriazole- l-oxy)-N, N, N', N'- tetramethyluronium hexafluorophosphate 7 DIC N, N’- Diisopropylcarbodiimide 8 HOBt 1-hydroxybenzotrizole 9 HOAt 1 -hydroxy-7-azabenzotriazole 10 DIPEA N, N’-diisopropylethylamine 11 Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulphonyl chloride 12 DMF N, N’- dimethyl formamide 13 DCM Dichloromethane 14 THF Tetrahydrofuran 15 TFA Trifluoroacetic acid 16 NMP N-methyl pyrrolidone 17 DMSO Dimethyl sulfoxide 18 EDT Ethanedithiol 19 DODT 3,6-dioxa-1 ,8-octanedithiol 20 TIPS Tri isopropyl silane 21 DBU 1,8-diazabicyclo [5.4.0] undec-7-ene The following examples are meant to illustrate the present invention. The examples are presented to exemplify the invention and are not to be considered as limiting the scope of the invention. EXAMPLES Example 1: Synthesis of Trofinetide (1) Step 1: Preparation of Fmoc-Glu-(OCH2-TAG)-OtBu (5). (Attachment of TAG to the 1stamino acid) To a stirred solution of TAG-OH (7) (5.0 g) in Tetrahydrofuran (50.0 mL) Fmoc-γ-Glu- OtBu (6) (4.9 g), DMAP (0.047 g) and DIC (1.80 mL) were added at 0-5oC. The reaction mixture was stirred for 20 minutes. The reaction progress was monitored by TLC (mobile phase: 20% EA in hexane). After completion of the reaction, the reaction mixture was filtered through celite bed. The filtrate was concentrated under reduced pressure by maintaining a temperature below 35 °C; then the precooled acetonitrile (60 mL) was added to the residue at 5-10 °C and stirred for 10 minutes. The solid residue was filtered, washed with acetonitrile, and dried for 4-6 hrs to get titled compound. Yield: 7.7 g. Step 2: Preparation of H-Glu-(O-TAG)-OtBu (Fmoc deprotection of the TAG attached amino acid) To a stirred solution of Fmoc-Glu-(OCH2-TAG)-OtBu (5) (2 g) in Tetrahydrofuran (20.0 mL), DBU (0.26 mL) and piperidine (0.26 mL) was charged at 0-5°C and stirred for 15 minutes at 25-30 °C. After the completion of deprotection of Fmoc group, the reaction mass was cooled to 0-5 °C, and the mixture pH was adjusted with 1 M aq. HCl to ~7.0. The reaction mass was washed with purified water (5V x 2). The obtained organic layers were dried over anhydrous sodium sulphate to get titled compound, which was taken for next step without purification. Step 3: Preparation of Boc-Gly-α-CH3-Pro-γ-Glu-(OCH2-TAG)-OtBu (2) [2ndand 3rdamino acids coupling]: To the step 2 solution having H-Glu-(O-TAG)-OtBu (0.5 g), Fmoc-α-CH3-Pro-OH (4) (0.232 g), COMU (0.35 g) and DIPEA (0.31 mL) in THF (5.0 mL) were added at 0-5°C and stirred for 1 hour. After completion of the reaction, the reaction mixture was concentrated under reduced pressure by maintaining a temperature below 35°C. The pre- cooled acetonitrile was added to the residue and stirred for 10 minutes. The obtained solid was filtered, washed with acetonitrile and dried to get the coupled product of Fmoc-α- CH3-Pro-γ-Glu-(OCH2-TAG)-OtBu. Yield: 0.56g Fmoc deprotection and coupling procedure were followed for the following amino acids as per the sequence of amino acids. The solid Fmoc-α-CH3-Pro-γ-Glu-(OCH2-TAG)- OtBu (0.56 g) is deprotected using piperidine (1.5 eq.) / DBU (1.0 eq.) and coupled with Boc-Gly-OH (3) (0.113 g) using COMU (0.31 g) and DIPEA (0.31 mL) as described in the above procedure to get titled compound (2). Yield: 0.35g Step 4: Preparation of Trofinetide (1) [Cleavage of TAG]: The tagged fragment Boc-Gly-α-CH3-Pro-Glu-(OCH2-TAG)-OtBu (2) (0.35 g) was deprotected by stirring for 30 mins in a in a mixture of TFA (95%), TIPS (2.5%) and H2O (2.5%) while monitoring the progress by TLC. After completion of the reaction, the solution was filtered and neutralized with DIPEA. The reaction mass was concentrated under reduced pressure at 30- 35oC. The reaction mass solidified on trituration with n- heptane (l060 mL l 0V). Solid filtered over Buchner funnel, washed with n-heptane (265 mL, 2 x 2.5 V) and dried for 4-6 hrs in VTD at 35 - 40 °C to afford Trofinetide as a white solid. Structure and purity of the Trofinetide (1) was confirmed by NMR, LCMS; m / z = 429.62 with TFA salt and by HPLC. Yield: 150 mg Example 2: By following the above example 1 Tag procedure, two more analogues H- Gly-Aib-Asp-OH 8 (LCMS; m / z = 276.2 [M+H]1) and H-Gly-Aib-Glu-OH 9 (LCMS; m / z = 290.1 [M+H]1) can be prepared at commercial scale.

Claims

We claim:

1. A method for the synthesis of Trofinetide (1) and its analogues,which comprises: a) coupling N-protected C-terminal of first amino acid (6)with TAG-OH (7)in the presence of a coupling reagent, base and a solvent to obtain TAG linked amino acid (5).b) deprotecting Fmoc group in TAG linked amino acid (5) with deprotecting agent in the presence of a base in a solvent; followed by coupling with second N-protected methyl substituted amino acid (4)in the presence of a coupling reagent, base and a solvent to obtain dipeptide compound of in-situ; c) deprotecting Fmoc group of dipeptide obtained in step (b) followed by coupling with third N-protected amino acid (3)in the presence of a coupling reagent, base and a solvent to obtain TAG linked tripeptide compound of Formula (2);d) cleaving the N-protecting group and TAG from the tripeptide compound of Formula (2) with a cocktail mixture to obtain Trofinetide (1);e) optionally purifying the Trofinetide (1) to obtain pure Trofinetide (1).

2. The process as claimed in claim 1, wherein the coupling agent used in the reaction is selected from the group consisting of Benzotriazole- 1 -yl-oxy-tris- (dimethylamino)- phosphonium hexafluorophosphate (BOP), Benzotriazole- 1-yl-oxy-tris-pyrrolidino- phosphonium hexafluorophosphate (PyBOP), O-(IH-Benzotriazol-l-yl)-N,N, N,N- tetramethyluronium tetrafluoroborate (TBTU), O-(7-Azabenzotriazole- 1 -yl)- N,N,N,N-tetramethyluronium tetrafluoroborate (TATU), O-(lH-Benzotriazole-l-yl)- N, N, N, N-tetramethyluronium hexafluorophosphate,(HBTU), 2-(7- Aza-lH- benzotriazole-l-yl)- 1,1,3,3 -tetramethyluronium hexafluorophosphate (HATU), N,N- dicyclohexylcarbodiimide (DCC), Ν,Ν'-Diisopropylcarbodiimide (DIC), 1-[(1- (Cyano-2-ethoxy-2-oxoethylideneaminooxy) dimethylaminomorpholino)]uroniumhexafluorophosphate (COMU) or l-Ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC.HC1).

3. The process as claimed in claim 1, wherein the base used in the reaction is selected from the group consisting of 4-Dimethylaminopyridine (DMAP), 1,8-Diazabicyclo [5.4.0] undec-7-ene (DBU) 4. The process as claimed in claim 1, wherein the solvent used in the reaction is selected from the group consisting of “ether solvents” such as tetrahydrofuran, diethyl ether, methyl tert-butyl ether, 2-methyl tetrahydrofuran, 1,4-dioxane and the like.

5. The process as claimed in claim 1, wherein the cocktail mixture consisting of (TFA TIPS / Water 95% / 2.5% / 2.5%).

6. A process for the preparation of compound 8 and compound 9by sequential coupling following the Tagg approach, as described in claim 1.

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