DESC:RELATED APPLICATION:
This application claims the benefit of priority of our Indian patent application
number IN 201621036256 dated Oct. 24, 2016, which is incorporated herein by
reference.
5 FIELD OF THE INVENTION:
The present invention relates to an improved process for the preparation of
Sincalide for Formula-I
NH
NH
NH
O
O
NH NH
O
S
O
CH3
NH
NH
O
OH
O
O
S
NH
CH3
NH2
O
O
H2N
O
OH
O
S
HO
O O
Formula-I
10 BACKGROUND OF THE INVENTION:
Sincalide is a synthetic cholecystokinin octapeptide with chemical name L-a-
aspartyl-O-sulfo-L-tyrosyl-L-methionylglycyl-L-tryptophyl-L-methionyl-L-a-
aspartyl-L-phenylalaninamide and represented as below.
15 Sincalide is approved with brand name Kinevac for parenteral administration, used
to (1) stimulate gallbladder contraction, as may be assessed by contrast agent
cholecystography or ultrasonography, or to obtain by duodenal aspiration a sample
of concentrated bile for analysis of cholesterol, bile salts, phospholipids, and
crystals; (2) to stimulate pancreatic secretion (especially in conjunction with
20 secretin) prior to obtaining a duodenal aspirate for analysis of enzyme activity,
3
composition, and cytology; (3) to accelerate the transit of a barium meal through the small bowel, thereby decreasing the time and-extent of radiation associated with fluoroscopy and x-ray examination of the intestinal tract.
US 3723406 disclosed Sincalide and its preparation. Further patents US 3714140, US 4102878, US 5117009 discloses process of preparation of Sincalide. 5
In drug substance synthesis there is always a continuing need for preparation process, which can obtain drug substance in high yields, more purity and has low impurities, and which is commercially feasible as well.
The inventors of the present invention surprisingly found that the process according to the present invention has many advantages compared to the known processes 10 such as desulfation is prevented (de-sulfation of the peptide occurs under acidic conditions required for side chain de-protection and cleavage of peptide from resin, resulting in lower yields), avoids the lengthy process as in fragment condensation method, avoids use synthesis of fragments in solution phase and then coupling on the resin. Thus, the overall yield was improved with low impurities. 15
SUMMARY OF THE INVENTION:
One aspect of the invention relates to an improved process of preparation of Sincalide comprising
a. amino acid chain assembling to solid support (Resin);
b. sulfation of tyrosine group; 20
c. cleavage of the peptide from solid support and de-protection of side chain;
d. removing a protecting group of sulfated ester in solution phase to obtain crude Sincalide; and
e. purification and lyophilization of crude Sincalide to obtain sincalide.
Another aspect of the present invention relates to an improved process of 25 preparation of Sincalide by combining solid phase and liquid phase method.
4
Another aspect of the present invention relates to an improved process of preparation of Sincalide by combining solid phase and liquid phase method comprises a step of sulfating tyrosine using a protected sulfate ester group.
BRIEF DESCRIPTION OF THE DRAWINGS:
In the following description, reference(s) is made to the accompanying drawings, in 5 which
Fig 1 shows process of preparation of Sincalide according to present invention.
DETAILED DESCRIPTION OF THE INVENTION:
The embodiments of the present invention described below are not intended to be exhaustive or to limit the invention to the precise forms disclosed in the following 10 detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices of the present invention.
In the following description, drawings and examples, Sulfation or sulphating or sulphonation are used interchangeably, and describe sulphonation of the tyrosine 15 residue.
The present invention relates to an improved process of preparation of Sincalide comprising
a) amino acid chain assembling to solid support (Resin);
b) sulfation of tyrosine group; 20
c) cleavage from solid support and deprotection of side chain;
d) removing a protected group of sulfated ester in solution phase to obtain crude Sincalide; and
e) purification and Lyophilization of crude sincalide.
In an embodiment the resin is selected from group consisting of chlorotrityl resin 25 (CTC), Sasrin, Wang Resin, 4-methytrityl 10 chloride, TentaGel S, TentaGel TGA, Rink acid resin, NovaSyn TGT resin,HMPB-AM resin, 4-(2-(amino methyl)-5-
5
methoxy)phenoxy butyric acid anchored to polymeric resin MBHA, 4-(4-(amino methyl)-3-methoxy)phenoxy butyric acid anchored to polymeric resin MBHA and 4-(2-(amino methyl)-3,3-dimethoxy)phenoxy butyric acid anchored to polymeric resin MBHA.
In an embodiment the step a) Amino acid chain assembling to solid support 5 comprises steps of
a. preconditioning or swelling a rink amide MBHA resin;
b. coupling first amino acid Fmoc-Phe-OH to the reactive group of resin to obtain Fmoc-Phe-MBHA resin and deprotection by removing Fmoc group; 10
c. coupling another amino acid [Fmoc-Asp(OtBu)-OH] free on one terminal group and protected on other terminal group and followed by deprotection;
d. repeating the above steps of coupling and deprotection with the amino acids to obtain the peptide chain assembled to solid support [represented 15 as Formula-I(a)]
Boc-Asp(O-tBu)-Tyr-Met-Gly-Trp(Boc)-Met-Asp(O-tBu)-Phe-Resin. formula-I(a)
In some embodiments the amino acid to be coupled is protected with protecting group. 20
The protecting group used for protecting Amino acid is selected from 4-tert-butyl ester, tert-butyloxy-carbonyl (Boc) or fluorenylmethoxycarbonyl (Fmoc) protective group.
The amino acids in step iv are Fmoc-Met-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly-OH, Fmoc-Tyr-OH, and these are consecutively coupled to obtain compound of 25 formula-I(a).
The coupling of amino acids with resin and subsequent amino acid assembling or coupling to obtain required peptide is carried out using coupling system comprising
6
a reaction solvent and a condensing agent, the condensing agent is selected from DIC / HOBt , PyBOP / HOBt / DIEA or HATU / HOBt / DIEA; said reaction solvent is selected from DMF, DCM, NMP or DMSO or combination thereof. The deprotection or splitting off of the Fmoc protective group was carried out using a solution of piperidine such as piperidine in DMF. 5 In another embodiment the step b) sulfation of tyrosine group comprises sulfation of tyrosine group of formula-1(a) using a protected sulfate ester to obtain peptide of formula-1(b).
Boc-Asp(O-tBu)-Tyr(SO3PG)-Met-Gly-Trp(Boc)-Met-Asp(O-tBu)-Phe-Resin Formula-1(b) 10
The PG is sulfate protecting group selected from trichloroethyl (TCE), isobutyl, neopentyl, DCV (dichlorovinyl), imidazole TCE.
The protected sulfate ester is a protected chlorosulfate selected from substituted alkyl chlorosulfate, 2,2,2-trichloroethyl chlorosulfate, isobutyl chlorosulfate, neopentyl chlorosulfate, DCV(dichlorovinyl) chlorosulfate, and imidazolium 15 derivative/ Imidazolium salt of trichloroethyl chlorosulfate (TCE)
In one embodiment the imidazolium salt of TCE is 2,3-Dimethyl-1-(2,2,2-trichloroethoxysulfonyl)-1H-imidazol-3-ium tetrafluoroborate, 2,3-dimethyl-1-((2,2,2-trichloroethoxy)sulfonyl)-1H-imidazol-3-ium trifluoromethanesulfonate.
The above protected sulfate ester are stable to the acidic conditions required for 20 side-chain deprotection and cleavage of the peptide from the resin, and can be readily removed at the end of the synthesis.
The sulfation step is carried in suitable conditions such as in presence of base.
The base is selected group consisting of trimethylamine (TEA), DMAP (Dimethylaminopyridine) or mixtures thereof. 25
7
In yet another embodiment step c) cleavage from solid support and deprotection of side chain comprises cleavage of peptide from resin and de-protection of side chain protecting groups of formula-1(b) to obtain a peptide of formula-1(c)
Asp-Tyr(SO3PG)-Met-Gly-Trp-Met-Asp-Phe-NH2 formula-1(c) 5
The cleavage of the peptide from resin and deprotection are performed using TFA (Trifluoroacetic acid) /TIPS or TIS (Triisopropylsilane)/water, preferably TFA:TIPS:H2O in ratio 95%: 2.5%: 2.5%.
In this step deprotection of side chain protecting groups will occur except the sulfate protecting group. 10
In still another embodiment step d) removing a protected group of sulfated ester in solution phase comprises removing the protecting group (PG) of the sulfate ester of tyrosine in presence of Zn and ammonium formate in presence of suitable conditions and a solvent to obtain crude sincalide of formula Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2. 15
The suitable conditions include but not limited to use of suitable solvent or mixture of solvents thereof that does not essentially obstruct the reaction, and conditions such as time and temperature.
In an embodiment the above step is carried out in presence of suitable solvent such as methanol solvent. 20
Step e) Purification and lyophilization of obtained crude Sincalide,
Purification and lyophilisation are preformed according to any known methods in the art.
In one embodiment the successful course of the coupling and de-protection can be monitored by means of the Kaiser test and bromophenol blue test. 25
8
Kaiser test is a qualitative test for the presence or absence of free primary amino groups, and it can be a useful indication about the completeness of a coupling step. The test is based on the reaction of ninhydrin with primary amines, which gives a characteristic dark blue colour. The test requires minimal amounts of analyte and is completed within a few minutes. 5
In one embodiment the kaiser test is performed as below
Kaiser Test Solutions:
Reagent A (100 % pyridine): Pour 50 ml of Pyridine into a small reagent bottle and label it “A”.
Reagent B (5 % ninhydrin solution): Dissolve 1.0 g of ninhydrin in 20 mL of 10 Ethanol and pour into a small reagent bottle and label it as “B”.
Reagent C (80% phenol): Dissolve 40 g of phenol in 10 mL of Ethanol and pour it into a small reagent bottle and label it “C”.
Kaiser test procedure: Take 10-15 beads of resin in a test tube and wash with ethanol, 2-3 times. To the tube add 2 to 3 drops of Reagent A, 2 to 3 drops of 15 Reagent B, 2 to 3 drops of Reagent C, followed by heating the tubes at 110°C for 5 minutes and then comparing the color with reference to check the completion of coupling reaction.
Advantages of the preparation process according to the present invention are: the process is simple, convenient and efficient for preparation of Sincalide, avoids the 20 lengthy process as in fragment condensation method, doesn’t use synthesis of fragments in solution phase and then coupling on the resin.
In one embodiment the process according to the present invention is shown in Fig-I.
25
9
The invention is further exemplified by the following non-limiting examples, which are illustrative representing the preferred modes of carrying out the invention. The invention's scope is not limited to these specific embodiments only but should be read in conjunction with what is disclosed anywhere else in the specification together with those information and knowledge which are within the 5 general understanding of the person skilled in the art.
Examples
Example-1:
Step-1: Amino acid chain assembling to solid support (Resin):
Swelling the Rink Amide MBHA resin (9.33 g) (0.536mmol/g) using glass reaction 10 vessel containing a sintered disk in DMF (50 ml) as solvent for 30 min to 1h.
1(a) Coupling of Fmoc-Phe-OH
Activated amino acid solution of Fmoc-Phe-OH (5.8 g, 3 eq) in 50 mL DMF, HOBt (2.03 g, 3 eq), DIC (2.32 ml, 3.0 eq) was added to above swelled resin and stirred for 3h at room temperature. Reaction completion was monitored by the 15 kaiser test. Reaction mixture was washed with DMF (3 x 50 ml). 20 % piperdine solution (2 x 10 ml) was added to the Fmoc-Phe-MBHA resin. Stirred for 10 min and drained. After completion of the Fmoc de-protection, reaction mixture was washed with DMF (3 x 50 ml), DCM (3 x 50 ml), and finally with DMF (3 x 50 ml). 20
1(b) Coupling of Fmoc-Asp(OtBu)-OH
Activated amino acid solution of Fmoc-Asp (OtBu)-OH (6.17 g, 3.0 eq) in 50 mL DMF, HOBt (2.03 g, 3 eq), DIC (2.32 ml, 3.0 eq) was added to above crude solution and stirred for 3h at room temperature. Reaction completion was confirmed by checking the Kaiser test. Reaction mass was washed with DMF (3 x 25 50ml). 20% Piperdine solution (2 x 10 ml) was added to the Fmoc-Asp(OtBu)-Phe-MBHA resin. Stirred for 10 min and drained. After completion of the Fmoc de-
10
protection, reaction mixture was washed with DMF (3 x 50 ml), DCM (3 x 50 ml), and finally with DMF (3 x 50 ml).
1(c) Coupling of Fmoc-Met-OH:
Activated amino acid solution of Fmoc-Met-OH (5.57 g, 3.0 eq) in 50 mL DMF, HOBt (2.03 g, 3 eq), DIC (2.32 ml, 3.0 eq) was added to above crude solution and 5 stirred for 3h at room temperature. Reaction completion was confirmed by checking the kaiser test. Reaction mass was washed with DMF (3 x 50 ml). 20 % Piperdine solution (2 x 10 ml) was added to the Fmoc-Met-Asp(OtBu)-Phe-MBHA resin. Stirred for 10 min and drained. After completion of the Fmoc de-protection, reaction mixture was washed with DMF (3 x 50 ml), DCM (3 x 50 ml), and finally 10 with DMF (3 x 50 ml).
1(d) Coupling Fmoc-Trp (Boc)-OH:
Activated amino acid solution of Fmoc-Trp (Boc)-OH (7.79 g, 3.0 eq) in 50 mL DMF, HOBt (2.03 g, 3 eq), DIC (2.32 ml, 3.0 eq) was added to above crude solution and stirred for 3h at room temperature. Reaction completion was 15 monitored by kaiser test. Reaction mass was washed with DMF (3 x 50 ml). 20% Piperdine solution (2 x 10 ml) was added to the Fmoc-Trp(Boc)-Met-Asp(OtBu)-Phe-MBHA resin. Stirred for 10 min and drained. After completion of the Fmoc de-protection, reaction mixture was washed with DMF (3 x 50 ml), DCM (3 x 50 ml), and finally with DMF (3 x 50 ml). 20
1(e) Fmoc-Gly-OH coupling:
Activated amino acid solution of Fmoc-Gly-OH (4.46 g, 3.0 eq) in 50 mL DMF, HOBt (2.03 g, 3 eq), DIC (2.32 ml, 3.0 eq) was added to above crude solution and stirred for 3h at room temperature. Reaction completion was monitored by checking the Kaiser test. Reaction mass was washed with DMF (3 x 50 ml). 20% 25 Piperdine solution (2 x 10 ml) was added to the Fmoc-Gly-Trp(Boc)-Met-Asp(OtBu)-Phe-MBHA resin. Stirred for 10 min and drained. After completion of the Fmoc de-protection, reaction mixture was washed with DMF (3 x 50 ml), DCM (3 x 50 ml), and finally with DMF (3 x 50 ml).
11
1(f) Fmoc-Met-OH coupling:
Activated amino acid solution of Fmoc-Met-OH (5.57 g, 3.0 eq) in 50 mL DMF, HOBt (2.03 g, 3 eq), DIC (2.32 ml, 3.0 eq) was added to above crude solution and stirred for 3h at room temperature. Reaction completion was confirmed by monitored the kaiser test. Reaction mass was washed with DMF (3 x 50 ml). 20% 5 Piperdine solution (2 x 10 ml) was added to the Fmoc-Met-Gly-Trp(Boc)-Met-Asp(OtBu)-Phe-MBHA resin. Stirred for 10min and drained. After completion of the Fmoc de-protection, reaction mixture was washed with DMF (3 x 50 ml), DCM (3 x 50 ml), and finally with DMF (3 x 50 ml).
1(g) Fmoc-Tyr-OH coupling: 10
Activated amino acid solution of Fmoc-Tyr-OH (6.05 g, 3.0 eq) in 50 mL DMF, HOBt (2.03 g, 3 eq), DIC (2.32 ml, 3.0 eq) was added to above crude solution and stirred for 3hr at room temperature. Reaction completion was confirmed by checking the Kaiser test. Reaction mass was washed with DMF (3 x 50 ml). 20% Piperdine solution (2 x 10 ml) was added to the Fmoc-Tyr-Met-Gly-Trp(Boc)-Met-15 Asp(OtBu)-Phe-MBHA resin. Stirred for 10 min and drained. After completion of the Fmoc de-protection, reaction mixture was washed with DMF (3 x 50 ml), DCM (3 x 50 ml), and finally with DMF (3 x 50 ml).
1(h) Boc-Asp (OtBu)-OH coupling:
Activated amino acid solution of Boc-Asp (OBu)-OH (4.34 g, 3.0 eq) in 50 mL 20 DMF, HOBt (2.03 g, 3 eq), DIC (2.32 ml, 3.0 eq) was added to above crude solution and stirred for 3h at room temperature. Reaction completion was confirmed by checking the Kaiser test. Reaction mass was washed with DMF (3 x 50 ml). 20% Piperdine solution (2 x 10 ml) was added to the Fmoc-Phe-MBHA resin. Stirred for 10 min and drained. After completion of the Fmoc de-protection, 25 reaction mixture was washed with DMF (3 x 50 ml), DCM (3 x 50 ml), and finally with DMF (3 x 50 ml) and get crude peptide of Boc-Asp(OtBu)-Tyr-Met-Gly-Trp (BOC)-Met-Asp(OtBu)-Phe-MBHA resin.
12
Step-2: Sulfation of tyrosine residue:
DCM was added to above crude peptide and cool to 0oC, added TEA (6.7 ml, 16 eq), TCESO3Cl (11.9 g, 16 eq) and DMAP (1.83g, 3eq). Reaction mass was stirred for overnight at room temperature. Reaction mass was washed with DMF (3 x 50 ml), DCM (3 x 50ml), and finally with MeOH (3x50ml) to obtain Boc-Asp(O-5 tBu)-Tyr(SO3C2H2Cl3)-Met-Gly-Trp(Boc)-Met-Asp(O-tBu)-Phe-Resin
Step-3: Cleavage from solid support and de-protection of side chains
Above obtained crude peptide was added to 80 ml of cocktail reagent (TFA:TIPS:H2O) 95%: 2.5%: 2.5% in 250 ml RBF at under ice bath and stirred for 2h. After 2h the solution was filtered and precipitated by the addition of 10 10 volumes of diethyl ether (800 ml). The obtained product was filtered and washed with diethyl ether and dried under vacuum to obtain Boc-Asp-Tyr (SO3 C2H2Cl3)-Met-Gly-Trp-Met-Asp-Phe-NH2
Step-4: Hydrolysis of Sulfate Ester (De-protection of sulfate protecting group) followed by purification 15
Hydrolysis of above crude peptide by using Zn/Ammonium formate, methanol, 2h,rt, After 2 h reaction mass was filtered through celite pad and crude sincalide was obtained with yield ,CLAIMS:We Claim:
1. A process for preparation of Sincalide using combination of solid phase and liquid phase synthesis comprising the steps of:
a. amino acid chain assembling to solid support or resin;
b. sulfation of tyrosine group; 5
c. cleavage from solid support and deprotection of side chain;
d. removing a protected group of sulfated ester in solution phase to obtain crude Sincalide; and
e. optionally, purification and lyophilization of crude Sincalide. wherein the sulfation is carried out using a protected sulfate ester selected 10 from substituted alkyl chlorosulfate, 2,2,2-trichloroethyl chlorosulfate, isobutyl chlorosulfate, neopentyl chlorosulfate, DCV(dichlorovinyl) chlorosulfate, and imidazolium derivative or Imidazolium salt of trichloroethyl chlorosulfate (TCE).
2. The process as claimed in claim 1, wherein the step a. comprising the steps of 15
a. preconditioning or swelling a rink amide MBHA resin;
b. coupling first amino acid Fmoc-Phe-OH to the reactive group of resin to obtain Fmoc-Phe-MBHA resin and deprotection by removing Fmoc group;
c. coupling another amino acid [Fmoc-Asp(OtBu)-OH] free on one terminal 20 group and protected on other terminal group and followed by deprotection;
d. repeating the above steps of coupling and deprotection with the amino acids to obtain the peptide chain assembled to solid support, represented as formula-I(a). 25
3. The process as claimed in claim 2, wherein coupling is carried out using coupling system comprising a reaction solvent and a condensing agent, the condensing agent is selected from DIC / HOBt , PyBOP / HOBt / DIEA or HATU / HOBt / DIEA; said reaction solvent is selected from DMF, DCM, NMP or DMSO or combination thereof. 30
14
4. The process as claimed in claim 2, wherein deprotection of the Fmoc protective group is carried out using piperidine in DMF.
5. The process as claimed in claim 1, wherein step b. is carried out in presence of DMAP and TEA and the peptide obtained after sulfation step b. is of formula-1(b). 5
6. The process as claimed in claim 1, wherein step c. is carried out using TFA:TIPS:H2O in the ratio of 95%: 2.5%: 2.5%, and the peptide obtained is formula-1(c).
7. The process as claimed in claim 1, wherein step d. is carried out in presence of Zn and ammonium formate.