RU2303602C2 - Method for tripeptide production - Google Patents

Abstract

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to method for production of tripeptides of general formula I: A-Pro-GlY-Pro-OX, wherein A is H, Bzl, t-Bu. Synthesis is carried out by liquid phase method by condensation of C-terminal proline derivatives of general formula H-Pro-OX¹, wherein X¹ is Bzl, t-Bu with protected dipeptide of general formula Y-Pro-Gly-OH, wherein Y is Z, Boc, and obtained protected tripeptide of general formula Y-Pro-Gly-Pro-X¹ is treated with deprotecting reagents and optionally obtained product is acetylated and optionally hydrochlorides are obtained.

EFFECT: improved method for tripeptide production.

2 cl, 7 ex

Description

The invention relates to pharmaceutical chemistry, specifically to a method for producing tripeptides of the general formula:

A-Pro-Gly-Pro-OX,

where A is H, Ac; X - H, Bzl, Bu ^t , or their hydrochlorides, and may find application in medicine to obtain physiologically active peptides.

Tripeptides containing the Pro-Gly-Pro sequence are part of the known regulatory peptides (β-casomorphine, calcitonin, gastrin) and a number of neurotropic peptide preparations, such as semax and selank [1]. In addition, the tripeptide Pro-Gly-Pro has antiulcer activity [2].

A known method of producing H-Pro-Gly-Pro-OH bromide hydrate by acylating the C-terminal dipeptide with N-benzyloxycarbonyl-L-proline acid chloride followed by removal of the N-terminal protecting group by treatment with a solution of hydrogen bromide in acetic acid [3].

The disadvantage of this method is the low yield of the target product and the necessity of using N-benzyloxycarbonyl-L-proline chloride, which is an unstable compound; in addition, highly toxic reagents thionyl chloride or phosphorus pentachloride are used in its preparation [4].

A known method of producing the tripeptide Ac-Pro-Gly-Pro-OH, solid-phase method by condensation of the C-terminal proline associated with the polymer substrate, with Boc-Pro-Gly-OH using N, N'-dicyclohexylcarbodiimide as a condensing agent, followed by cleavage from the tripeptidylpolymer of the N-terminal protective group, acylation with acetic anhydride, and cleavage from the polymer position by treatment with anhydrous HF [5]. The disadvantages of this method are the need to use special hardware (solid-state synthesizer and a device for working with liquid hydrogen fluoride) and the use of highly toxic hydrogen fluoride in the synthesis, which makes this method unsuitable for preparative production of the target compounds.

The closest in technical essence to the described method is a method for producing H-Pro-Gly-Pro-OH, based on the condensation of methyl ester of L-proline and N-carbobenzoxy-L-prolylglycine, followed by treatment of the protected tripeptide with release reagents [6]. The disadvantages of this method is the multi-stage and low yield of the target product.

The objective of the invention is to simplify the process of obtaining and increasing the yield of target tripeptides.

The solution to this problem is achieved by the claimed method, which consists in the fact that the synthesis of tripeptides of the general formula (I) is carried out in solution by condensation of a proline derivative of the general formula: H-Pro-OX ¹ , where X ¹ is benzyl (Bzl), tert.-butyl (Bu ^t ), with a protected dipeptide of the general formula: Y-Pro-Gly-OH, where Y is benzyloxyacarbonyl (Z), tert-butyloxycarbonyl (Boc), and the obtained protected tripeptide of the general formula: Y-Pro-Gly-Pro- OX ¹ , where Y and X ¹ have the meanings indicated above, are treated with release reagents and the resulting product, if necessary jerk acetylation.

List of abbreviations

AcOH - acetic acid

Ac - Acetyl

AcONp - para-nitrophenyl acetate

Vos - tert.-butyloxycarbonyl

Bu ^t - tert.-butyl

Bzl - benzyl

DCC - N, N'-Dicyclohexylcarbodiimide

DCHU - N, N'-Dicyclohexylurea

DMF - N, N-dimethylformamide

HOBt - 1-hydroxybenzotriazole

HONp - para-nitrophenol

HONSu - N-hydroxysuccinimide

TFA - trifluoroacetic acid

Z - benzyloxycarbonyl

HPLC - High Performance Liquid Chromatography

TLC - thin layer chromatography

experimental part

In the synthesis, derivatives of L-amino acids from Bachem (Switzerland) were used. DCC, TFA, HONSu, HONp from Fluka (Switzerland). DMF was purified by distillation over ninhydrin and barium oxide. Analytical HPLC was performed on a Gilson chromatograph (France).

TLC was performed on glass chromatographic plates Merck-Kiselgel 60 (Germany) in the following solvent systems:

A - chloroform: methanol: AcOH 9: 1: 0.5 B - chloroform: methanol: 32% AcOH 15: 4: 1 B - chloroform: methanol: 32% AcOH 5: 3: 1 G - chloroform: methanol: ethyl acetate 6: 3: 1

¹ H-NMR spectra were recorded on a WH-500 Bruker 500 MHz spectrometer (Germany) in DMSO-d ₆ at 300 K. Chemical shifts (δ, ppm) were measured relative to tetramethylsilane.

Mass spectra were recorded on an Analytical Compact MALDI 4 mass spectrometer from Kratos (Great Britain).

The described method is illustrated by the following examples.

Example 1

one.

4.1 g (55 mmol) of glycine is dissolved in 55 ml of 1N. NaOH solution, add a solution of 15.6 g (50 mmol) of Boc-Pro-ONSu in 100 ml of DMF with cooling and stir for 24 h, monitoring the progress of the reaction using TLC in system B. The solution is evaporated, the residue is dissolved in water, the aqueous solution is extracted ether (2 × 20 ml), acidified to pH 2-3 12 N. HCl solution and extracted with a mixture of ethyl acetate with butanol in a ratio of 2: 1 (2 × 150 ml). The combined organic extracts were washed with saturated NaCl and water until neutral and evaporated. The residue was crystallized from ether-hexane 1: 1, filtered, dried in vacuo. As a result, 11.6 g (85%) of Ia are obtained with R _f 0.57 (A), 0.71 (B), 0.86 (C).

2.

To a solution of 9.0 g (44 mmol) of H-Pro-OBzl in 100 ml of DMF cooled to -10 ° C, a solution of 10.9 g (40 mmol) of Ia, 6.7 g (44 mmol) of HOBt, cooled to the same temperature, is added with vigorous stirring and 9.2 g (44 mmol) of DCC in DMF. The reaction mixture was stirred for 10 min at -10 ° C and left at room temperature for 24 hours. The condensation was monitored by TLC in systems A and D. After the reaction was complete, the precipitated DCHU was filtered off, the filtrate was evaporated, the residue was dissolved in ethyl acetate and washed successively with 2 % H ₂ SO ₄ (3 × 20 ml), water, 2% NaHCO ₃ solution, water until neutral. The solution was evaporated, the residue was triturated with hexane, filtered off, dried in vacuo. As a result, 17.1 g (93%) of IB with R _f 0.80 (A), 0.88 (B), 0.50 (D) are obtained.

3.

6 g (13.1 mmol) of tripeptide Ib are dissolved in 3 N. a solution of HCl in AcOH for 1 h. The complete release is controlled by TLC in system B. After 1 h, the reaction mixture is evaporated in vacuo. The residue was triturated with dry cold ether, filtered, dried in vacuo. As a result, 5.0 g (97%) of I are obtained. The homogeneity of the obtained product is confirmed by analytical HPLC under gradient elution with buffer B in buffer A (A — 0.1% TFA, B — 80% acetonitrile in buffer A) from 10 to 70% of the buffer B in 30 min on an Ultrasphere ODS column 4.6 × 250 mm, grain size 5 μm, detection at 220 nm. Purity according to HPLC 95%, R _t 17.5 min (under the indicated conditions). R _f 0.19 (A), 0.52 (B), 0.76 (C). ¹ H-NMR spectrum (DMSO-d ₆ ), δ, ppm): 7.36 (arom. 5H), 5.11 (CH ₂ -Ph, 2H);

Pro: 4.24 (α-CH, 1H); 2.32, 1.88 (β-CH ₂ , 2H); 1.92 (γ-CH ₂ , 2H); 3.58, 3.54 (δ-CH ₂ , 2H);

Gly: 8.72 (NH, 1H); 4.11, 3.98 (α-CH ₂ , 2H);

Pro: 4.39 (α-CH, 1H); 2.20, 1.86 (β-CH ₂ , 2H); 1.92, 1.86 (γ-CH ₂ , 2H); 3.41, 3.25 (δ-CH ₂ , 2H).

Example 2

one.

19.5 g (0.260 M) of glycine are dissolved in 260 ml of 1 N. NaOH, the resulting solution was added to a solution of 96.0 g (0.260 M) Z-Pro-ONp in 600 ml of DMF cooled to 0 ° C. The reaction mixture was stirred at room temperature for 16 hours, evaporated, the residue was dissolved in 300 ml of water and extracted three times with ether. The ether layer was separated, 150 ml of 2N was added to the aqueous layer. HCl solution, the precipitated oil is extracted with 600 ml of ethyl acetate. The organic layer was separated and washed with water until neutral, dried over Na ₂ SO ₄ , evaporated, the residue was recrystallized from ether. The result is: 67.5 g (85%) IIa with So pl. 122-123 ° C; R _f 0.67 (A); R _f 0.81 (B), R _f 0.65 (C).

2.

0.920 g (3 mmol) of compound IIa was dissolved in 40 ml of DMF, 0.33 ml (3 mmol) of N-methylmorpholine was added to the resulting solution, cooled to -15 ° C, and 0.39 ml (3 mmol) of isobutyl chloroformate was added with stirring. The reaction mixture was stirred for 5 minutes at -10 ° C, then a solution of 0.73 g (3 mM) HCl · H-Pro-OBzl and 0.33 ml (3 mM) N-methylmorpholine in 20 ml DMF was added to it. After an hour, the reaction mixture was evaporated, the resulting oil was dissolved in 50 ml of ethyl acetate and washed successively with 2% H ₂ SO ₄ , water, 5% NaHCO ₃ , water. The organic layer was evaporated. 1.48 g (100%) of compound IIb are obtained in the form of an oil. R _f 0.87 (B).

3.

1.48 g (3 mmol) of compound IIb are dissolved in 60 ml of ethanol and hydrogenated over 5% Pd / C. The completeness of the hydrogenation process is monitored by TLC in system B. The catalyst is filtered off, washed three times with ethanol, the filtrate is evaporated, the resulting oil is triturated in ether, and the resulting precipitate is filtered off. Obtain 0.78 g (96%) of compound II with a mp of 151-155 ° C, R _{t of} 5.44 min and a purity of 96.6% according to analytical HPLC. Column Ultropac TSK ODS-120T 4.6 × 250 mm, 5 μm. Buffer A: 0.05 M KN ₂ PO ₄ , pH 3.0; buffer B: 70% acetonitrile + 30% H ₂ O; gradient: 10-70% B in 30 minutes R _f 0.16 (B).

MALDI-MS m / z, found: 270.23 [M + H] ⁺ , calculated: 269.3

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm): the presence of cis-, trans-conformers in a ratio of 1: 6 is observed:

for trans-conformer:

Pro - 4.24 (α-CH, 1H); 2.30 / 1.88 (β-CH, 2H)

Gly - 8.70 (NH, 1H); 4.07 / 3.96 (α-CH, 1H)

Pro - 4.24 (α-CH, 1H); 2.13 / 1.85 (β-CH, 2H)

Example 3

0.79 g (2 mmol) of compound I was dissolved in 40 ml of water and hydrogenated over 5% Pd / C. The completeness of the hydrogenation process is monitored by TLC in system B. The catalyst is filtered off, washed on the filter with water, the filtrate is evaporated, the remaining oil is evaporated three times with isopropyl alcohol, triturated in ether, and the resulting precipitate is filtered off. Obtain 0.57 g (93%) of compound III with So pl. 172-176 ° C, R _t 5.44 min. and a purity of 96.6% according to analytical HPLC. Column Ultropac TSK ODS-120T 4.6 × 250 mm, 5 μm. Buffer A: 0.05 M KN ₂ PO ₄ , pH 3.0; buffer B: 70% acetonitrile + 30% H ₂ O; gradient: 10-70% B in 30 minutes R _f 0.16 (B).

MALDI-MS m / r, found: 270.23 [M + H] ⁺ , calculated: 269.3

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm): the presence of cis-, trans-conformers in a ratio of 1: 6 is observed:

for trans-conformer:

Pro - 4.24 (α-CH, 1H); 2.30 / 1.88 (β-CH, 2H)

Gly - 8.70 (NH, 1H); 4.07 / 3.96 (α-CH, 1H)

Pro - 4.24 (α-CH, 1H); 2.13 / 1.85 (β-CH, 2H)

Example 4

0.61 g (2.25 mmol) of compound II was dissolved in 5 ml of DMF, 0.25 ml (2.25 mmol) of N-methylmorpholine and 0.45 g (2.5 mmol) of AcONp were added to the resulting solution. The reaction mixture was kept at room temperature for 8 hours (the completeness of the reaction was monitored by TLC in system B). Then the reaction mixture was evaporated, the resulting oil was dissolved in water, extracted twice with ether, the ether layer was discarded, and the aqueous was purified by HPLC on a 24 × 250 mm, 5 μm Diasorb column. Buffer A: 0.1% TFA; buffer B: acetonitrile; gradient: 0-40% B in 20 minutes Obtain 0.67 g (95%) of compound IV with a mp of 91-99 ° C, R _t 8.99 min and a purity of 98.7% according to analytical HPLC. Column Ultropac TSK ODS-120T 4.6 × 250 mm, 5 μm. Buffer A: 0.05 M KH ₂ PO ₄ , pH 3.0; buffer B: 70% acetonitrile + 30% H ₂ O; gradient: 10-70% B in 30 minutes R _f 0.25 (B).

MALDI-MS m / z, found: 312.4 [M + H] ⁺ , calculated: 311.3.

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm): cis- is observed; trans-conformers in a 1: 1 ratio:

for cis-conformer:

Pro 4.37 (α-CH, 1H); 2.19 / 2.15 (β-CH, 2H)

Gly - 8.20 (NH, 1H); 4.06 / 3.79 (α-CH, 1H)

Pro - 4.56 (α-CH, 1H); 2.20 (β-CH, 2H)

Ac - 1.89 (-CH ₃ , 3H)

for trans-conformer:

Pro - 4.329 (α-CH, 1H); 1.97 (β-CH, 2H)

Gly 7.88 (NH, 1H); 3.93 / 3.81 (α-CH, 1H)

Pro - 4.23 (α-CH, 1H); 2.13 / 2.09 (β-CH, 2H)

Ac - 1.96 (-CH ₃ , 3H)

Example 5

0.61 g (2.25 mmol) of compound II was dissolved in 5 ml of DMF, 0.25 ml (2.25 mmol) of N-methylmorpholine and 0.26 g (2.5 mmol) of acetic anhydride were added to the resulting solution. The reaction mixture was kept at room temperature for 1 hour (the completeness of the reaction was monitored by TLC in system B). Then the reaction mixture is evaporated, the resulting oil is dissolved in water and subjected to purification by HPLC on a Diasorb column 24 × 250 mm, 5 μm. Buffer A: 0.1% TFA; buffer B: acetonitrile; gradient: 0-40% B in 20 minutes Obtain 0.57 g (81%) of compound IV with T. pl. 92-95 ° C, R _t 8.99 min and a purity of 96.6% according to analytical HPLC. Column Ultropac TSK ODS-120T 4.6 × 250 mm, 5 μm. Buffer A: 0.05 M KH ₂ PO ₄ , pH 3.0; buffer B: 70% acetonitrile + 30% H ₂ O; gradient: 10-70% B in 30 minutes R _f 0.25 (B).

MALDI-MS m / z, found: 312.4 [M + H] ⁺ , calculated: 311.3.

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm): the presence of cis-, trans-conformers in a ratio of 1: 1:

for cis-conformer:

Pro 4.37 (α-CH, 1H); 2.19 / 2.15 (β-CH, 2H)

Gly - 8.20 (NH, 1H); 4.06 / 3.79 (α-CH, 1H)

Pro - 4.56 (α-CH, 1H); 2.20 (β-CH, 2H)

Ac - 1.89 (-CH ₃ , 3H)

for trans-conformer:

Pro - 4.329 (α-CH, 1H); 1.97 (β-CH, 2H)

Gly 7.88 (NH, 1H); 3.93 / 3.81 (α-CH, 1H)

Pro - 4.23 (α-CH, 1H); 2.13 / 2.09 (β-CH, 2H)

Ac - 1.96 (-CH ₃ , 3H)

Example 6

0.89 g (2.25 mmol) of compound I was dissolved in 10 ml of DMF, 0.25 ml (2.25 mmol) of N-methylmorpholine and 0.26 g (2.5 mmol) of acetic anhydride were added to the resulting solution. The reaction mixture was kept at room temperature for 1 hour (the completeness of the reaction was monitored by TLC in system B). Then the reaction mixture was evaporated, the residue was dissolved in water and hydrogenated over 5% Pd / C. After completion of the hydrogenation process (control by TLC in system A), the catalyst is filtered off, washed on the filter three times with water, the aqueous solution of the target product is evaporated to 1/3 of the volume and subjected to HPLC purification on a 24 × 250 mm, 5 μm Diasorb column. Buffer A: 0.1% TFA; buffer B: acetonitrile; gradient: 0-40% B in 20 minutes Obtain 0.63 g (91%) of compound IV with So pl. 90-94 ° C, R _t 8.99 min and a purity of 96.2% according to analytical HPLC. Column Ultropac TSK ODS-120T 4.6 × 250 mm, 5 μm. Buffer A: 0.05 M KH ₂ PO ₄ , pH 3.0; buffer B: 70% acetonitrile + 30% H ₂ O; gradient: 10-70% B in 30 minutes R _f 0.25 (B).

MALDI-MS m / z, found: 312.4 [M + H] ⁺ , calculated: 311.3.

¹ H-NMR spectrum (DMSO-d ₆ , δ, ppm): the presence of cis-, trans-conformers in a ratio of 1: 1:

for cis-conformer:

Pro 4.37 (α-CH, 1H); 2.19 / 2.15 (β-CH, 2H)

Gly - 8.20 (NH, 1H); 4.06 / 3.79 (α-CH, 1H)

Pro - 4.56 (α-CH, 1H); 2.20 (β-CH, 2H)

Ac - 1.89 (-CH ₃ , 3H)

for trans-conformer:

Pro 4.329 (α-CH, 1H); 1.97 (β-CH, 2H)

Gly 7.88 (NH, 1H); 3.93 / 3.81 (α-CH, 1H)

Pro 4.23 (α-CH, 1H); 2.13 / 2.09 (β-CH, 2H)

Ac - 1.96 (-CH ₃ , 3H)

Example 7. Prolyl-glycyl-proline tert-butyl ester hydrochloride

one.

19.5 g (260 mmol) of glycine are dissolved in 260 ml of 1 N. NaOH, the resulting solution was added to a solution of 96.0 g (260 mmol) of Z-Pro-ONp in 600 ml of DMF cooled to 0 ° C. The reaction mixture was stirred at room temperature for 16 hours, evaporated, the residue was dissolved in 300 ml of water and extracted three times with ether. The ether layer was separated, 150 ml of 2N was added to the aqueous layer. HCl solution, the precipitated oil is extracted with 600 ml of ethyl acetate. The organic layer was separated and washed with water until neutral, dried over Na ₂ SO ₄ , evaporated, the residue was recrystallized from ether. The result is: 67.5 g (85%) of the product with T pl. 122-123 ° C; R _f 0.81 (B), R _f 0.65 (D).

2.

To a solution of 45.7 g (220 mmol) of H-Pro-Obu ^t HCl in 500 ml of DMF, cooled to -10 ° С, a solution of 67.5 g (220 mmol) of Va, 33.4 g (220 mmol) cooled to the same temperature is added with vigorous stirring ) HOBt and 45.3 g (220 mmol) of DCC in 100 ml of DMF. The reaction mixture was stirred for 10 min at -10 ° C and left at room temperature for 24 h. Condensation was monitored by TLC in systems A and D. After the reaction, the precipitated DCHU was filtered off, the filtrate was evaporated, the residue was dissolved in ethyl acetate (800 ml) and washed successively with 2% H ₂ SO ₄ (3 × 250 ml), water (3 × 250 ml), 2% NaHCO ₃ solution (3 × 250 ml), water until neutral. The solution was evaporated, the residue was triturated with hexane, filtered off, dried in vacuo. As a result, 90.9 g (90%) of Vb are obtained, with R _f 0.82 (A), 0.91 (B), 0.6 (D).

3.

90.9 g (198 mmol) of Vb was dissolved in 1000 ml of EtOH and hydrogenated over 5% Pd / C until the starting compound disappeared by TLC in system B. Then the catalyst was filtered off, 198 ml of 1N was added to the filtrate. an aqueous solution of hydrochloric acid and evaporated to dryness, the oily residue is evaporated with isopropyl alcohol, the residue is treated with ether. The precipitate formed is filtered off, washed on the filter with ether and dried. The result is 68.1 g (95%) of V with R _f 0.15 (A), 0.45 (B), 0.70 (C). The homogeneity of the obtained product is also confirmed by analytical HPLC under gradient elution with buffer B in buffer A (A - 0.1% TFA, B - 80% acetonitrile in buffer A) from 10 to 70% buffer B in 30 min on an Ultrasphere ODS 4.6 × column 250 mm, grain size 5 μm, detection at 220 nm. Purity according to HPLC, 96%, R _t 15.9 min (under the indicated conditions). ¹ H-NMR spectrum (DMSO-d ₆ ), δ, ppm: 1.37 (-C (CH ₃ ) ₃ , 9H);

Pro: 4.23 (α-CH, 1H); 2.32, 1.87 (β-CH ₂ , 2H); 1.91 (γ-CH ₂ , 2H); 3.58, 3.54 (δ-CH ₂ , 2H);

Gly: 8.72 (NH, 1H); 4.11, 3.97 (α-CH ₂ , 2H);

Pro: 4.37 (α-CH, 1H); 2.22, 1.86 (β-CH ₂ , 2H); 1.92, 1.86 (γ-CH ₂ , 2H); 3.40, 3.25 (δ-CH ₂ , 2H).

As can be seen from the above examples, the described method is simple and allows to obtain the target products on a preparative scale in high yield.

Literature

1. Advances in physiological sciences. T.34, No. 1, p.14-19 (2003).

2. RF patent No. 2252779, cl. A61K 38/07 (2005).

3. J. Mol. Biol. V.17, p. 255-272 (1966).

4. Greenstein J., Vinitz M. Chemistry of amino acids and peptides. -M: World (1965).

5. US patent 6310041, CL 514/12 (2001).

6. Izv. USSR Academy of Sciences, Dep. Chem. Sciences, 1959, No. 4, pp. 736-738.

Claims (2)

1. The method of obtaining tripeptides of the General formula A-Pro-Gly-Pro-OX, where A is H, Ac; X - H, benzyl (Bzl), tert.-butyl (Bu ^t ), or their hydrochlorides, characterized in that the synthesis is carried out by the liquid-phase method by condensation of the C-terminal derivative of proline of the general formula H-Pro-OX ¹ , where X ¹ - Bzl, Bu ^t with an N-protected dipeptide of general formula Y-Pro-Gly-OH, where Y is benzyloxycarbonyl (Z), tert-butyloxycarbonyl (Boc), and the resulting protected tripeptide of general formula Y-Pro-Gly-Pro-OX ^1, where Y and X ¹ are as defined above, is treated with a deprotecting reagent, and the resulting product, if necessary, and optionally subjected to acetylation floor ayut hydrochlorides. 2. The method according to claim 1, characterized in that X ¹ is benzyl (Bzl), Y is benzyloxycarbonyl (Z), and the release of the protected tripeptide is carried out by catalytic hydrogenolysis.