JP2016065050A - Octreotide production method - Google Patents

Abstract

Provided is a method for producing octreotide having high yield and high purity in a liquid phase synthesis method. [MEANS FOR SOLVING PROBLEMS] Using an acetal compound represented by formula (3) or a salt thereof as an intermediate, R1a which is an amino protecting group is eliminated, and a condensation reaction with a protected amino acid and elimination of the protecting group are repeated. And a method for producing octreotide or a salt thereof by further conducting an oxidation reaction. [Selection figure] None

Description

  The present invention relates to a method for producing octreotide and a production intermediate thereof.

  Octreotide developed as a long-lasting somatostatin analog has the effect of suppressing excessive secretion of growth hormone, thyroid stimulating hormone and the like. Specifically, gastrointestinal hormone-producing tumors (carcinoid tumors, VIP-producing tumors, glucagonomas, insulinomas, gastrin-producing tumors) and various symptoms associated with acromegaly / pituitary giantism, as well as advanced / recurrent cancer It is used as a drug for the improvement of gastrointestinal symptoms associated with gastrointestinal obstruction in patient palliative medicine.

  Octreotide has the following formula (A)

As a method for producing the diol cyclic octapeptide represented by the formula (Patent Documents 1 to 3) and a liquid phase synthesis method (Patent Documents 4 and 5).

US Pat. No. 5,889,146 US Pat. No. 6,346,601 Canadian Patent Application Publication No. 2458084 US Pat. No. 4,395,403 International Publication No. 2007/110765

In the case of the solid-phase synthesis method, generally, the crude purity is low, mass synthesis is difficult, and expensive resins are used, which is not suitable for industrial production of pharmaceuticals.
On the other hand, in the liquid phase synthesis method, a reduction reaction is performed after peptide synthesis in order to diolify the C-terminal threonine residue. However, the yield of the reduction reaction is low and the industrially advantageous method is I could not say.
Accordingly, an object of the present invention is to provide a method for producing octreotide having high yield and high purity in a liquid phase synthesis method.

  Therefore, as a result of various studies on the synthesis of octreotide by a liquid phase synthesis method, the present inventor used a diolated threonine as a C-terminal residue raw material, and acetal protection with a dilong-chain alkoxybenzaldehyde as a protecting group for the diol moiety It was found that when the group was used, the crystallizing property of the acetal-protecting compound was high, and isolation by solid-liquid separation was easy. In other words, if the peptide elongation process by the elimination of the amino protecting group and the condensation reaction with the protected amino acid proceeds efficiently, and finally the acetal protecting group is eliminated and oxidized to form a disulfide bond, the liquid phase The present inventors have found that octreotide can be obtained in a high yield and high purity by a synthesis method.

  That is, the present invention provides the following [1] to [3].

[1] The following formula (B)

(Wherein R ¹ represents a hydrogen atom, an amino protecting group, an amino acid residue optionally having a protecting group or a peptide residue optionally having a protecting group;
R ² and R ³ each independently represents an alkyl group having 14 to 30 carbon atoms;
X, Y and Z are each independently a hydrogen atom, a halogen atom, an optionally substituted hydrocarbon group having 1 to 10 carbon atoms or an optionally substituted carbon atom having 1 to 1 carbon atoms. Represents 10 acyl groups)
Or an acetal compound represented by the formula:
[2] The following formula (1)

(In the formula, R ^1a represents an amino protecting group.)
And a diol compound represented by formula (2)

(Wherein R ² and R ³ each independently represents an alkyl group having 14 to 30 carbon atoms;
X, Y and Z are each independently a hydrogen atom, a halogen atom, an optionally substituted hydrocarbon group having 1 to 10 carbon atoms or an optionally substituted carbon atom having 1 to 1 carbon atoms. Represents 10 acyl groups)
And a benzaldehyde compound represented by the formula (3) is reacted in the presence of an acid:

(Wherein R ^1a , R ² , R ³ , X, Y and Z are the same as above)
The manufacturing method of the acetal compound represented by these.
[3] The amino protecting group R ^1a of the acetal compound (3) obtained by the production method described in [2] above is eliminated, the condensation reaction with the protected amino acid and the elimination of the protecting group are repeated, and the oxidation reaction (A) characterized in that

The manufacturing method of the octreotide represented by these, or its salt.

  Since the acetal compound of the formula (3) has high crystallinity and can be easily isolated by solid-liquid separation, each intermediate after the peptide chain formation reaction can be easily isolated and purified. Moreover, since the peptide elongation process is performed by a liquid phase synthesis method, it can be synthesized in a normal reaction vessel.

  Octreotide represented by the formula (A) of the present invention or a salt thereof is produced by a liquid phase synthesis method according to the following reaction formula.

(In the formula, R ⁴ represents a thiol protecting group, R ⁵ represents a hydroxyl protecting group, and X, Y, Z, R ^1a , R ² and R ³ are the same as above.)

  That is, octreotide or a salt thereof is obtained by reacting a diol compound represented by the formula (1) and a benzaldehyde compound represented by the formula (2) in the presence of an acid, and thereby representing an acetal compound represented by the formula (3). Prepared by removing the amino protecting group of the compound (3), repeating the condensation reaction with the protected amino acid and elimination of the protecting group to obtain the compound of formula (18), and then carrying out the oxidation reaction. The Here, the reaction of the protected amino acid is carried out by reacting in the order of protected Cys, protected Thr, protected Lys, protected Trp, protected Phe, protected Cys, and protected Phe as shown in the above reaction formula.

R ^1a represents an amino protecting group. As the amino protecting group, (1) a protecting group that can be removed with an acid (for example, t-butoxycarbonyl group (Boc), p-methoxybenzyloxycarbonyl group (Moz), formyl group (CHO), 2- (trimethylsilyl) (Ethoxycarbonyl group (Teoc), 1-adamantyloxycarbonyl group (Adoc), 2- (p-biphenyl) isopropyloxycarbonyl group (Bpoc), triphenylmethyl group (Tr), methoxymethyl group (MOM)); ) Protecting groups that can be eliminated by reduction (for example, benzyloxycarbonyl group (Cbz), allyl group, allyl group, N-benzyloxymethyl group (BOM)); (3) protecting groups that can be eliminated by a base (for example, 9 -Fluorenylmethyloxycarbonyl group (Fmoc), 2- (4-nitrophenyl) ethoxyca Bonyl group (Npeoc)); (4) Protecting groups that can be removed with zinc dust-acetic acid (for example, 2,2,2-trichloroethoxycarbonyl group (Troc), N-dithiasuccinoyl group (Dts), benzoic acid) Thiazole-2-sulfonyl group (Betsyl), 1,1-dimethyl-2,2,2-trichloroethoxycarbonyl group (TcBoc), N- (diphenyl-4-pyridyl) methyl group (Dppm)); (5) palladium Protecting groups that can be eliminated with an amine or the like under a catalyst (eg, allyloxycarbonyl group (Alloc)) and the like. Of these protecting groups, it is preferable to use a protecting group that can be eliminated with an acid, a protecting group that can be eliminated with a base, or a protecting group that can be eliminated by reduction. Further, R ^1a in the reaction formulas may be the same or different.

R ² and R ³ each represent an alkyl group having 14 to 30 carbon atoms. The alkyl group may be a linear or branched alkyl group, such as a tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group, heicosyl group, docosyl group, tricosyl group, tetracosyl group Groups and the like.

X, Y, and Z represent a hydrogen atom, a halogen atom, an optionally substituted hydrocarbon group having 1 to 10 carbon atoms, or an optionally substituted acyl group having 1 to 10 carbon atoms. Show. Here, examples of the halogen atom include a fluorine atom, a chlorine atom, and a bromine atom. As a C1-C10 hydrocarbon group which may have a substituent, a C1-C10 alkyl group is preferable, and C1-C10, such as a methyl group, an ethyl group, n-propyl group, an isopropyl group, is shown. An alkyl group of ˜6 is more preferred. As a C1-C10 acyl group which may have a substituent, a C2-C10 alkanoyl group is preferable and C2-C6 alkanoyl groups, such as an acetyl group and a propionyl group, are more preferable.
As these X, Y and Z, a hydrogen atom or a halogen atom is preferable, and a hydrogen atom is particularly preferable.

R ⁴ is a thiol protecting group, and specific examples include a trityl (triphenylmethyl) group, a trialkylsilyl group, a p-methoxybenzyl group, a t-butyl group, and an acetamidomethyl group.

R ⁵ is a hydroxyl-protecting group, and examples thereof include a benzyl group, a p-methoxybenzyl group, and a t-butyl group.

The reaction between the diol compound (1) and the benzaldehyde compound (2) is performed in the presence of an acid. Examples of the acid used include sulfonic acids such as camphorsulfonic acid and p-toluenesulfonic acid; and salts of sulfonic acids such as pyridinium p-toluenesulfonic acid.
0.3-0.7 mol, more preferably 0.5 mol of benzaldehyde compound (2) is used per mol of diol compound (1), 0.01-0.05 mol, more preferably 0.025 mol. It is preferable that the acid is added and the reaction is carried out by refluxing at 100 ° C. or higher in an organic solvent. As the organic solvent, hydrocarbon solvents such as toluene and cyclohexane are used. After completion of the reaction, the acetal compound (3) can be collected as a solid by adding an amine, concentrating, and adding acetonitrile to the concentrated residue. The reason why the acetal compound (3) can be easily separated as a solid is that the benzaldehyde compound has a long-chain alkoxy group having 14 to 30 carbon atoms at the ortho and para positions.

The elimination reaction of the amino protecting group of the acetal compound (3) depends on the type of the amino protecting group (R ^1a ). Depending on the type of amino protecting group, it may be eliminated with acid, reduction, base, zinc dust-acetic acid, amine under palladium catalyst, or the like. For example, when the amino protecting group is Fmoc, the amino protecting group may be eliminated using a base such as diethylamine.

  As the protection Cys, protection Thr, protection Lys, protection Trp, and protection Phe, Boc protection or Fmoc protection is preferable. The condensation reaction of these protected amino acids with each peptide [(4), (6), (8), (10), (12), (14), (16)]] is, for example, 1-ethyl-3- (3-Dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), N, N-dicyclohexylcarbodiimide (DCC), or O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyl Uronium hexafluorophosphate (HBTU), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride n hydrate (DMT-MM), etc. It can be carried out in the presence of a condensing agent. Preferably, it is desirable to add a condensation aid such as 1-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt), and a base such as diisopropylethylamine (DIPEA) and triethylamine. Specifically, the condensation reaction can be carried out by reacting at 0 to 40 ° C. for 1 to 48 hours in a halogen-based or ether-based solvent in the presence of a condensing agent.

  The deprotection reaction of each protected peptide [(5), (7), (9), (11), (13), (15)] is the same as the deprotection reaction of the acetal compound (3). It can be carried out.

The deprotection reaction of peptide (17) is a reaction for removing all protecting groups of R ^1a , R ⁴ , R ⁵ and the acetal group. In order to carry out the elimination reaction of these protecting groups in one step, it is desirable that all of R ^1a , R ⁴ , R ⁵ and the acetal group are protecting groups that can be eliminated by the same treatment. As such a protecting group, it is preferable to select a protecting group that can be ^{removed by} an acid as R ^1a , a trityl group as R ⁴ , and a t-butyl group as R ⁵ .
As a specific method for deprotecting peptide (17), deprotection with acid, for example, in the presence of an inorganic acid or trifluoroacetic acid, in an ester or ether solvent, 0 to 40 ° C., 1 to 48 hours. A deprotection reaction can be performed by reacting.

  The oxidation reaction of peptide (18) can be performed using, for example, air oxidation using ammonium acetate, activated carbon, iodine or the like. As the salt of octreotide (A), acid addition salts such as acetic acid, hydrochloric acid and sulfuric acid are preferable, and acetate is particularly preferable.

  Furthermore, the preferable chemical structure of octreotide (A) is as follows.

  The protected peptide (17) after completion of the above liquid phase synthesis reaction is acetal protected by benzaldehyde having a long-chain alkoxy group at the ortho-position and para-position, so that it can be easily crystallized and separated and purified. Very easy. Therefore, when the protected peptide (17) is synthesized, it can be purified with high purity by washing, crystallization, recrystallization or the like.

  In the above reaction scheme, the acetal protected intermediates are all novel compounds. When these are combined, the intermediate is represented by the formula (B).

(In the formula, R ¹ represents a hydrogen atom, an amino protecting group, an amino acid residue optionally having a protecting group or a peptide residue optionally having a protecting group; R ² , R ³ , X , Y and Z are the same as above.)

  Here, the amino acid residue which may have a protecting group and the peptide residue which may have a protecting group correspond to the groups in the above reaction formula. Specific examples of the compound of the formula (B) include (3), (4), (5), (6), (7), (8), (9), (10), ( 11), (12), (13), (14), (15), (16) and (17). Of these, the compounds (3), (4) and (17) are particularly important as synthetic intermediates.

According to the method of the present invention, the compounds (3), (4), and (17) have good crystallinity, and thus can be easily separated and purified by washing, crystallization, recrystallization, and the like. In addition, according to the method of the present invention (liquid phase synthesis method), difficult-to-remove impurities such as diastereomers and defective peptides are less likely to be produced than in the solid phase synthesis method, and if possible, they can be removed in an intermediate step. In addition, since intermediate compounds can be isolated compared to solid phase synthesis methods, it is possible to carry out filtration purification methods such as slurry methods and recrystallization methods that are relatively easy to operate and do not require expensive capital investment. It is easy to obtain an object having a purity.
In the solid phase synthesis method, the solvent to be used is limited, such as methylene chloride, but in the liquid phase synthesis method, a safe and inexpensive solvent suitable for industrialization can be used. Large-scale synthesis is easier than solid-phase synthesis. In the solid-phase synthesis method, the “preparative HPLC method” (generally expensive equipment and a large amount of organic solvent is required) is often used for purification of the final compound, but the purification yield is low due to low crude purity. Is low. In this liquid phase synthesis method, the purification yield is high due to the high purity of the crude product.

  EXAMPLES Next, an Example is given and this invention is demonstrated still in detail.

Abbreviations:
bD or bDCHO: bis (docosyloxy) benzaldehyde DIPEA: diisopropylethylamine THF: tetrahydrofuran DMF: dimethylformamide HOBt · H ₂ O: 1-hydroxybenzotriazole monohydrate HBTU: O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate DODT: 3,6-dioxa-1,8-octanedithiol TIPS: triisopropylsilane TFA: trifluoroacetic acid

Example (1) Synthesis of Fmoc-Thr-acetal-bD

bDCHO (30 g; 39.61 mmol), Fmoc-Thr-OL (26.08 g; 79.23 mmol), camphorsulfonic acid (0.46 g; 1.98 mmol) were dissolved in toluene (198 mL), and a Dean-Stark apparatus was used. At 130 ° C. After 6 hours, the reaction solution was cooled, DIPEA (0.51 g; 3.96 mmol) was added, and the mixture was concentrated under reduced pressure. Acetonitrile (1.8 L) was added to the concentrated residue, and the precipitated solid was isolated by filtration to obtain Fmoc-Thr-acetal-bD (43.59 g).
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.76 (d, J = 7.3 Hz, 2H), 7.63 (d, J = 7.3 Hz, 2H), 7.47 (d, J = 8.2 Hz, 1 H), 7.40 ( dd, J = 7.3, 7.3 Hz, 2H), 7.32 (dd, J = 7.3, 7.3Hz, 2H), 6.50 (dd, J = 8.2, 2.3 Hz, 1 H), 6.43 (d, J = 2.3 Hz, 1 H), 5.83 (s, 1H), 5.67 (d, J = 10.1 Hz, 1H), 4.47 (dd, J = 10.6, 6.9 Hz, 1H), 4.40 (dd, J = 106, 6.9 Hz, 1H) , 4.27 (dd, J = 6.9, 6.9 Hz, 1H), 4.18-4.03 (m, 3 H), 4.00-3.85 (m, 4 H), 3.65 (dd, J = 10.1, 1.8, 1H), 1.82- 1.70 (m, 4 H), 1.48-1.38 (m, 4H), 1.37-1.13 (m, 75H), 0.88 (dd, J = 6.8, 6.8 Hz, 6H)

(2) Synthesis of H-Thr-acetal-bD

Diethylamine (59.3 g; 810.78 mmol) was added to a solution of Fmoc-Thr-acetal-bD (43.16 g; 40.54 mmol), THF (405 mL), and DMF (405 mL), and the mixture was stirred at room temperature. After 1 hour, THF and excess diethylamine were distilled off by concentration under reduced pressure. Acetonitrile (1.7 L × 2) was added, and then crude H-Thr-acetal-bD (31.7 g) was isolated by filtration. The crude product was dissolved in a mixed solution (95 mL) of hexane / THF (3: 2) and purified by a silica gel column. The mobile phase of the silica gel column was carried out under the conditions of hexane / THF (3: 2) → (2: 3) → (1: 4) → 100% THF. After concentration, acetonitrile (400 mL) was added to the concentrated residue, and filtered to obtain purified H-Thr-acetal-bD (22.55 g) in a two-stage yield of 68%. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.48 (d, J = 8.2 Hz, 1 H), 6.47 (dd, J = 8.2, 2.3 Hz, 1 H), 6.41 (d, J = 2.3 Hz, 1 H ), 5.81 (s, 1 H), 4.13-4.04 (m, 3 H), 3.97-3.89 (m, 4 H), 2.53 (d, J = 1.4 Hz, 1 H), 1.82-1.70 (m, 4 H), 1.50-1.21 (m, 81 H), 0.88 (dd, J = 6.9, 6.9 Hz, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 160.8, 157.2, 127.7, 119.5, 105.3, 99.9, 97.7, 76.0, 74.2, 68.3, 68.1, 49.2, 31.9 (2C), 29.7 (28C), 29.40 (2C), 29.38 (2C), 29.23, 29.21, 26.1, 26.0, 22.7 (2C), 17.9, 14.1 (2C ).

(3) Peptide chain extension step a) Fmoc group deprotection step Fmoc-peptide-acetal-bD (1 equivalent) was dissolved in a THF / DMF mixture (volume of the substrate concentration 50 mM; 7: 3), and then diethylamine (20 eq) is added and stirred at room temperature until the reaction is complete. After concentration under reduced pressure, the remaining diethylamine is azeotroped with THF (2 × 5 v / w) to obtain H-peptide-acetal-bD. It is used for the next condensation reaction without further purification.

b) Condensation step After removal of excess diethylamine by THF azeotropy, Boc / Fmoc-AA-OH (1.2 equivalents), HOBt · H ₂ O with respect to H-peptide-acetal-bD prepared in the Fmoc deprotection step (1.2 eq.), HBTU (1.2 eq.), DIPEA (4.0 eq.), THF (amount of liquid that results in an H-peptide-acetal-bD concentration of 50 mM) are added. After completion of the reaction, the reaction solution is concentrated under reduced pressure and isolated by adding acetonitrile (1 × 60 v / w; 3 × 20 v / w).
a) Fmoc group deprotection step and b) Condensation step are repeated to sequentially bind amino acids, and all the protected Boc-D-Phe-Cys (Trt) -Phe-D-Trp (Boc) -Lys (Boc) -Thr (tBu) -Cys (Trt) -Thr-acetal-bD was synthesized.
¹ H NMR (400 MHz, CDCl ₃ ) δ 8.06 (m, 1H), 7.73 (m, 1H), 7.58-7.29 (m, 16H), 7.29-7.06 (m, 27H), 7.06-6.89 (m, 7H ), 6.70 (m, 1H), 6.64 (brs, 1H), 6.50 (m, 1H), 6.45-6.38 (m, 2H), 5.80 (s, 1H), 4.90 (m, 1H), 4.61 (m, 1H), 4.35 (m, 1H), 4.30 (m, 1H), 4.21-4.02 (m, 5H), 4.02-3.82 (m, 7H), 3.37-3.11 (m, 4H), 3.11-2.92 (m, 3H), 2.91-2.74 (m, 2H), 2.60-2.38 (m, 3H), 1.81-1.72 (m, 4H), 1.69 (s, 18H), 1.65 (s, 9H), 1.48-1.20 (m, 82H), 1.18 (s, 9H), 1.14 (d, J = 6.4 Hz, 3H), 1.02 (d, J = 6.0 Hz, 3H), 0.88 (dd, J = 6.6, 6.6 Hz, 6H).

(4) Synthesis of HD-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH
Boc-D-Phe-Cys (Trt) -Phe-D-Trp (Boc) -Lys (Boc) -Thr (tBu) -Cys (Trt) -Thr-acetal-bD → HD-Phe-Cys-Phe-D -Trp-Lys-Thr-Cys-Thr-OH
Boc-8mer-acetal-bD (20 g; 7.69 mmol) was added to 154 mL of a mixed solution of TFA / 1-dodecanethiol / DODT / TIPS / H ₂ O (80: 9: 9: 1: 1) cooled to 5 ° C. It was. After 5 minutes, the reaction solution was warmed to room temperature and stirred for 1.5 hours. TFA was azeotroped with dichloromethane (100 mL), acetonitrile (200 mL) was added, and the mixture was stirred for 30 minutes, and the bDCHO residue was removed by filtration. The filtrate was concentrated under reduced pressure, diisopropyl ether (2 × 200 mL) was added, and the solid was filtered to obtain HD Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH (11.71 g).

(4) Synthesis of octreotide
HD-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH → octreotide
HD-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH (7.0 g; 6.85 mmol) was added to a solution of THF (931 mL) and 0.1 N aqueous ammonium acetate (469 mL). . Activated carbon (0.7 g) was added thereto and stirred at room temperature. After completion of the reaction, the activated carbon was removed by filtration. THF was removed by concentration under reduced pressure, and the residual aqueous solution was lyophilized to obtain crude octreotide (7.5 g) with an HPLC purity of 85.6%. This was used for preparative HPLC purification.
ESI-MS: [M + H] ⁺ 1019

Claims (3)

The following formula (B)

(Wherein R ¹ represents a hydrogen atom, an amino protecting group, an amino acid residue optionally having a protecting group or a peptide residue optionally having a protecting group;
R ² and R ³ each independently represents an alkyl group having 14 to 30 carbon atoms;
X, Y and Z are each independently a hydrogen atom, a halogen atom, an optionally substituted hydrocarbon group having 1 to 10 carbon atoms or an optionally substituted carbon atom having 1 to 1 carbon atoms. Represents 10 acyl groups)
Or an acetal compound represented by the formula: The following formula (1)

(In the formula, R ^1a represents an amino protecting group.)
And a diol compound represented by formula (2)

(Wherein R ² and R ³ each independently represents an alkyl group having 14 to 30 carbon atoms;
X, Y and Z are each independently a hydrogen atom, a halogen atom, an optionally substituted hydrocarbon group having 1 to 10 carbon atoms or an optionally substituted carbon atom having 1 to 1 carbon atoms. Represents 10 acyl groups)
And a benzaldehyde compound represented by the formula (3), which is reacted in the presence of an acid:

(Wherein R ^1a , R ² , R ³ , X, Y and Z are the same as above)
The manufacturing method of the acetal compound represented by these. The amino-protecting group of the acetal compound (3) obtained by the production method according to claim 2 is eliminated, the condensation reaction with the protected amino acid and the elimination of the protecting group are repeated, and an oxidation reaction is further performed. Formula (A)

The manufacturing method of the octreotide represented by these, or its salt.