DESC:FIELD OF INVENTION

The present invention relates to an efficient solid-phase synthesis of Aviptadil represented by Formula-I.

H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2
Formula-I

BACKGROUND OF THE INVENTION

Aviptadil is an analog of vasoactive intestinal polypeptide (VIP) for the treatment of erectile dysfunction. Geneva-based Relief Therapeutics Holdings AG RFLB.S has a patent for Aviptadil, a synthetic form of a natural peptide that protects the lung.

NeuroRx Inc partnered with Relief to develop the drug in the United States. In June 2020, the U.S. Food and Drug Administration granted fast-track designation to Aviptadil for treatment of respiratory distress in COVID-19. In September 2020, NeuroRX submitted a request for an emergency use authorization to the US FDA for its use in patients in intensive care.

Aviptadil is first reported in US 3,880,826 A. The process for preparation of Aviptadil reported in this patent involves extraction and isolation of VIP from intestines of mammals.

U.S. Pat. No. US 3,862,927 A, Japan patents JPS52057174, JPS63179894 A, JPS63287798 A, JPS63179894 A & JPS63287798 A disclosed the preparation of Aviptadil by liquid fragment approach.

CN 103159845 B discloses process for preparation of Aviptadil using 1-9 amino acid fragments, 10-18 amino acid fragment, and 19 - 28 amino acid fragments and then coupled 3 fragments to obtain Aviptadil.

CN 104447963 B discloses process for preparation of Aviptadil using dipeptide fragment Fmoc-Leu-Asn-OH (23, 24 and 27, 28 amino acid) are synthesized by liquid phase method and solid phase synthesis using N-terminal Fmoc protected and side chain protected amino acids are coupled in presence of Rink Amide MBHA amino resins according to Aviptadil main chain.

Japan Patents JPS63179893 A, JPS63287797A & JP 01022897 A involves the process for preparation of Aviptadil, by deprotecttion of protected Aviptadil using strong acid such as HF, trifluoromethane sulfonic acid or by using triloweralkylsilyl halide; and further the protected Aviptadil prepared by liquid fragment approach.

However, Fragment synthesis has some drawbacks: the solubility of the protected fragments in the aqueous solvents used in the purification by HPLC and in the organic solvents used in the coupling reactions is usually low, reaction rates for the coupling of fragments are substantially lower than for the activated amino acid species in the conventional stepwise synthesis and, finally, the C terminal of each peptide fragment can be racemized during coupling.

Moreover the liquid phase synthesis is not suggestible on plant level synthesis, because we found great difficulty for isolating the products formed in the liquid phase synthesis. Further the usage and handling of HF, triloweralkylsilyl halide, trifluoromethane sulfonic acid is not suitable for commercial scale.

Therefore, due to increase the importance of the product in the current scenario, there is a need in the art to provide an alternate synthesis for Aviptadil, which circumvent the problems of prior art.

OBJECTIVES OF THE INVENTION

The objective of the present invention is to develop simple, robust, and commercially viable sequential process for the preparation of Aviptadil of the Formula 1.

Another object of the present invention is to provide a process for preparation of Aviptadil, with high yield and purity.

Another object of the present invention is to provide a process for preparation of Aviptadil continuous amino acid condensation, which minimizes the synthetic steps and analytical work, when compared to prior art process.

Another object of the present invention is to provide a process for preparation of Aviptadil, which minimizes the impurities.

Another object of the present invention is to provide a process for preparation of Aviptadil, in which the products obtained are easily isolated and purified.

BRIEF DESCRIPTION OF DRAWINGS:

Figure 1: Flow chart for process for solid-phase synthesis of Aviptadil (Formula-I) according to the present invention.

SUMMARY OF THE INVENTION

The main aspect of the present invention involves a process for the preparation of Aviptadil or its acetate salt, comprising the following steps:
a) anchoring of Na-protected C-terminal Asparagine to a resin;
b) selectively deprotecting the Na- amino-protecting group;
c) coupling the carboxyl terminus of the next Na-protected amino acid to the amine group obtained in step-b);
d) repeating steps b) and c) to obtain a peptide sequence;
e) crude Aviptadil is obtained by removal of protective groups and cleavage of peptide from the resin;
f) optionally, converting the Aviptadil to Aviptadil acetate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an improved process for preparation Aviptadil by sequential coupling employing solid phase approach. It involves sequential coupling of protected amino acid to prepare backbone of protected Aviptadil, which upon cleavage from resin and removal of protecting groups provides Aviptadil, which upon purification converts into Aviptadil acetate.

ABBREVIATIONS:

COMU: 1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium
Hexafluorophosphate
DIC: N,N'-diisopropylcarbodiimide
DEPBT: 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one
HBTU: O-Benzotriazole-N,N,N'N'-tetramethyl uronium hexafluorophosphate
HATU:1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate
PyBOP: benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate
HOBt: N-Hydroxybenzotriazole
HOAt; 1-Hydroxy-7-azabenzotriazole
Oxyma: ethyl cyano(hydroxyimino) acetate
DIPEA: Diisopropylethylamine
NNM: N-methyImorpholine
TMP : 2,2,6,6-Tetramethylpiperidine
DMF: N,N'-Dimethylformamide
DCM: dichloromethane
Fmoc: 9-fluorenylmethoxycarbonyl
t-Bu: tert-Butyl
Trt: triphenylmethyl.
Boc: t-butyloxycarbonyl
pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
EDT: ethanedithiol
TIS: Triisopropyl Silane
TFA: Trifluoroacetic acid

In one embodiment of the present invention is to provide a process for preparation of Aviptadil, comprising the steps of:
a) anchoring of Na-protected C-terminal Asparagine to a resin;
b) selectively deprotecting the Na-amino-protecting group;
c) coupling the carboxyl terminus of the next Na-protected amino acid to the amine group obtained in step-b);
d) repeating steps b) and c) to obtain a peptide sequence;
e) crude Aviptadil is obtained by removal of protective groups and cleavage of peptide from the resin;
f) optionally, converting the Aviptadil to Aviptadil acetate.

According to the present invention, the resin used for synthesis of peptide undergoes swelling in presence of a solvent selected from dichloromethane, N,N-dimethylformamide and N-methyl-2-pyrrolidone or mixtures. The resin used is selected from MBHA resin, Sieber Amide Resin.

The swelled resin is treated with N-terminus protected Asparagine in presence of coupling agents for a desired period of time to couple with resin. The solvent used is selected from dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone or mixtures thereof.

Optionally, before proceeding to the next step, the unreacted linkers on the resin are protected (capped) to avoid the undesired peptide chain formation. The capping is carried out in presence of acetic anhydride, pyridine and dichloromethane.

According to the present invention, the selective deprotection of Na-amino-protecting group of
Na-amino acid attached to the resin is done selectively in the presence of a nucleophilic base such as 20% piperidine in the presence of a solvent. The solvent used is selected from N, N-dimethylformamide, methylene chloride, N-methyl pyrrolidine or a mixture thereof.

The selectively deprotected amino group is then coupled with next N-protected amino acid in a solvent in the presence of a coupling reagent, coupling additive and optionally in presence of a base. The solvent used for the coupling reaction is selected from dichloromethane, dimethylformamide, N-methylpyrolidone, Dimethylsulfoxide or a mixture thereof.

The coupling agent was selected from the group consisting of HATU, HBTU, COMU, DEPBT, PyBOP, TBTU, or DIC; the Coupling additive was selected from the group consisting of oxyma pure or HOBt, HOAt; and the Base was selected from DIPEA, NMM or TMP.

According to the present invention, the Na-protected amino acids used are Na- protected and also side chain protected. The protecting group used for Na- protection is Fmoc; and the side chains protected with Trt, t-Bu, Boc, pbf. Further the Na-protected and side chain protected amino acids are as follow: Fmoc-His(Trt)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Asp(t-Bu)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmox-Asn(Trt)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Arg(pbf)-OH, Fmoc-Leu-OH, Fmoc-Arg(pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)OH, Fmoc-Met-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(t-bu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH.

According to the present invention, the cleavage from resin and global deprotection of the side chain protecting groups of peptide is carried out with a cocktail mixture.

The cleavage of the peptide from the resin involves treating the protected peptide anchored to the resin with an acid having at least one scavenger. The acid utilized in the cleavage reagent is TFA. The amount of TFA used for the purpose of cleavage of the peptide from the resin and global deprotection in the cocktail mixture may range from 80-90%. The scavengers used are selected from TIS, phenol, thioanisole, EDT, water or mixtures thereof.

In a preferred embodiment, the cocktail mixture used for the cleavage of the peptide from resin is TFA/EDT/TIS/water or TFA/EDT/phenol/thioanisole/TIS/water.

The resin after the completion of the reaction is optionally washed with solvents such as DMF and DCM to remove residual reagents and byproducts. The process is repeated if desired before proceeding to the next step.
The isolation of Aviptadil is carried out by precipitating with ether solvent to get Aviptadil as a solid. Ether solvents that are used for precipitation are selected from methyl tert-butyl ether, diethyl ether, t-butyl methyl ether, isopropyl ether or mixtures thereof.

The invention of the present application will be explained in more detail with reference to the following examples, which should not be construed as limiting the scope of the invention in any manner.
Examples
Example 1: Preparation of Aviptadil
Aviptadil was synthesized manually on Rink amide MBHA Resin by standard Fmoc solid phase synthesis strategy.

Stage-I: Anchoring of Fmoc-Asn(Trt)-OH to Rink amide MBHA Resin

The resin was soaked in DMF for swelling. Fmoc-Asn(Trt)-OH was treated with swelled Rink amide MBHA Resin in DMF in the presence of DIC (12 ml), HOBT (10.5 g) in DMF (180 ml); substitution level was determined by weight gain measurements and also by UV method.

Stage-II: Preparation of Fmoc-His(Trt)-Ser(tBu)-Asp(tBu)-Ala-Val-Phe-Thr(tBu)-Asp(tBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(pdf)-Leu-Arg(pdf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr-(tBu)-Leu-Asn(Trt)-Ser(tBu)-lle-Leu-Asn(Trt)-Rink amide MBHA Resin

Fmoc-Asn(Trt)-Rink amide MBHA resin (59.5 g) was swelled in DMF (300 ml) and deprotected with 20% piperidine in DMF (60 ml in 240 ml) at 25-30°C for 30 mins, after performing ninhydrin test, washing the reaction mixture with DCM followed by DMF.

The complete synthesis was achieved by stepwise coupling of Fmoc-Amino acids to the growing peptide chain on the resin. All the couplings were carried out using amino acids (17.7 g) in presence of HOBt (10.5 g), DIC (12 ml) and DMF (180 ml) as an solvent. The efficiency of coupling was monitored using Kaiser Ninhydrin test. The coupling step was repeated if Kaiser step was found positive. The sequence of addition for the synthesis of Aviptadil was Fmoc-Leu-OH, Fmoc-lle-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc- Val-OH, Fmoc-Ala-OH, Fmoc- Met-OH, Fmoc- Gln(Trt)-OH, Fmoc- Lys(Boc)-OH, FMoc- Arg(pdf)-OH, Fmoc-Leu-OH, Fmoc- Arg(pdf)-OH, Fmoc- Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Thr(tBu)-oH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc- Ala-OH, FMoc- Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, FMoc-His(Trt)-OH.

Stage-III: The stage-I resin was deprotected with 20% piperidine in DMF (60 ml in 240 ml) for 30 minutes, after washing with DMF (900 ml) , methanol (300 ml) and ether (600 ml) and then dried to get H-His(Trt)-Ser(tBu)-Asp(tBu)-Ala-Val-Phe-Thr(tBu)-Asp(tBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(pdf)-Leu-Arg(pdf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys (Boc)-Tyr-(tBu)-Leu-Asn(Trt)-Ser(tBu)-lle-Leu-Asn(Trt)-RAM Resin.

Yield: 174 g; yield percentage: 87%.

Stage-IV: The protected peptide resin of stage II along with global protection was cleaved by 1740 ml of cocktail mixture consisting of TFA/EDT/TIS/H2O (1522.5 ml/43.5 ml/87 ml/87 ml) to afford crude Aviptadil, and the obtained reaction mixture was stirred for 4 hours at 25°C. The reaction mixture was filtered and washed the resin with TFA. The obtained filtrate was partially distilled under reduced pressure and charged into pre-cooled diethyl ether (3480 ml) and stirred for 40 minutes at 25°C. Settled the reaction mixture and decanted the diethyl ether. Repeat the process for at least 4 times and then dried to get crude Aviptadil.

Yield: 79 g.

Stage V: Purification of crude Aviptadil using preparative HPLC.
First purification:
Aviptadil is purified by preparative HPLC using DAC column and following conditions:
Mobile Phase A: 0.1 % TFA in water (90 parts) + Mobile phase B (10 parts);
Mobile Phase B: 0.1% TFA in 80:20 ratio of Acetonitrile and Methanol;
Method: gradient; Flow rate (ml/Min): 200; Detection wavelength: 220 nm
Sample preparation: 20 grams of Aviptadil in mobile phase A
Table 1: Gradient program for first purification

Time (min) Mobile phase A (%) Mobile phase B (%)
0 80 20
30 80 20
31 76 24
67 76 24
68 50 50
100 50 50

The desired fractions are collected in the gradient range and the fractions whose purity = 90% are pooled. The pooled fractions are then subjected to further purification after rota evaporation.
Second purification:
Mobile Phase A: 0.3 % Acetic acid in purified water; Mobile Phase B: Acetonitrile; Flow rate: 200 ml/min; Detection wavelength: 220 nm;
Table 2: Gradient program for second purification
Time (min) Mobile phase A (%) Mobile phase B (%)
0 85 15
30 85 15
31 81 19
90 81 19
91 50 50
120 50 50

Fractions having purity = 99 % were pooled and concentrated by using rota vaporisation. Then concentrated solution used for acetate conversion:
Acetate conversion:
Mobile phase A: 20mM Ammonium acetate in purified water; Mobile phase B: Acetonitrile; Flow rate: 200 ml/min; Detection wavelength: 220 nm;
Table 3: Gradient program salt conversion
Time (min) Mobile phase A (%) Mobile phase B (%)
0 77 23
30 77 23
31 75 25
70 75 25
75 74 26
100 74 26
104 73 27
119 73 27
120 50 50
141 50 50

After HPLC analysis fractions having purity = 99 are pooled and concentrated using rotavaporisation. After concentration, the compound is lyophilized for 4-8 hours to provide pure Aviptadil acetate.

,CLAIMS:We Claim:

1. A process for the preparation of Aviptadil or its acetate salt, comprising the following steps:
a) anchoring of Na-protected C-terminal Asparagine to a resin;
b) selectively deprotecting the Na-amino-protecting group;
c) coupling the carboxyl terminus of the next Na-protected amino acid to the amine group obtained in step-b);
d) repeating steps b) and c) to obtain a peptide sequence;
e) crude Aviptadil is obtained by removal of protective groups and cleavage of peptide from the resin;
f) optionally, converting the Aviptadil to Aviptadil acetate.

2. The process as claimed in claim 1, resin used is selected from MBHA resin, Sieber Amide Resin.

3. The process as claimed in claims 1 and 2, the resin used for synthesis of peptide undergoes swelling in presence of a solvent selected from dichloromethane, N,N-dimethylformamide and N-methyl-2-pyrrolidone or mixtures; and swelled resin is treated with N-terminus protected Asparagine in presence of coupling agents for a desired period of time to couple with resin in solvent selected from dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone or mixtures thereof.

4. The process as claimed in claim 1, wherein deprotection of Na-amino-protecting group is carried out in presence of nucleophilic base such as 20% piperidine in the presence of a solvent. The solvent used is selected from N, N-dimethylformamide, methylene chloride, N-methyl pyrrolidine or a mixture thereof, preferably in N,N-dimethylformamide.

5. The process as claimed in claim 1, wherein coupling in step-c) is carried out in a solvent in the presence of a coupling reagent, coupling additive and optionally in presence of a base. The solvent used for the coupling reaction is selected from dichloromethane, dimethylformamide, N-methylpyrolidone, Dimethylsulfoxide or a mixture thereof, preferably in presence of DIC and HOBt in DMF.

6. The process as claimed in claim 1, wherein removal of protective groups and cleavage of peptide from the resin in step-e) is carried out in presence of an acid having at least one scavenger, wherein the scavengers used are selected from TIS, phenol, thioanisole, EDT, water or mixtures thereof.

7. A process for the preparation of Aviptadil, comprising the steps of:
a) anchoring of Fmoc-Asn(Trt)-OH to Rink amide MBHA Resin using DIC/HOBT in DMF;
b) deprotecting the Fmoc-protecting group to provide H-Asn(Trt)-Rink amide MBHA resin using 20% piperidine in DMF;
c) coupling the carboxyl terminus of the next Fmoc-Leu-OH to the amine group obtained in step-b) in presence of DIC and HOBT in DMF;
d) repeating steps b) and c) with Fmoc-amino acids selected from the group comprising of Fmoc-lle-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, FMoc-Arg(pdf)-OH, Fmoc-Leu-OH, Fmoc- Arg(pdf)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Thr(tBu)-oH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Ala-OH, FMoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, FMoc-His(Trt)-OH sequentially to obtain Fmoc-His(Trt)-Ser(tBu)-Asp(tBu)-Ala-Val-Phe-Thr(tBu)-Asp(tBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(pdf)-Leu-Arg(pdf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr(tBu)-Leu-Asn(Trt)-Ser(tBu)-lle-Leu-Asn(Trt)-Rink amide MBHA resin;
e) deprotecting the Fmoc-protecting group of resin obtained in step-d) with 20% piperidine in DMF to provide H-His(Trt)-Ser(tBu)-Asp(tBu)-Ala-Val-Phe-Thr(tBu)-Asp(tBu)-Asn(Trt)-Tyr(tBu)-Thr(tBu)-Arg(pdf)-Leu-Arg(pdf)-Lys(Boc)-Gln(Trt)-Met-Ala-Val-Lys(Boc)-Lys(Boc)-Tyr-(tBu)-Leu-Asn(Trt)-Ser(tBu)-lle-Leu-Asn(Trt)-Rink amide MBHA resin;
f) crude Aviptadil is obtained by removal of protective groups and cleavage of peptide of step-e) from the resin by using TFA/EDT/TIS/H2O;

g) converting the Aviptadil to Aviptadil acetate by using ammonium acetate in acetonitrile.