RU2574392C1 - METHOD FOR OBTAINING TREPEPTIDE OF GENERAL FORMULA (I) HPyr-His-TrpOH, APPLIED AS INTERMEDIATE COMPOUND (1-3 FRAGMENT) IN SYNTHESIS OF SYNTHETIC AGONISTS OF GONADOTROPIN-RELEASING-HORMONE (LH-RH), BY METHOD OF LIQUID-PHASE PEPTIDE SYNTHESIS WITHOUT PLACING AND REMOVAL OF PROTECTIVE GROUPS - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to method of obtaining HPyr-His-TrpOH

applied as main semi-product (1-3 fragment) in synthesis of synthetic antagonists of gonadotropine-releasing-hormone, LH-RH. Tripeptide (I) is obtained by method of liquid-phase peptide synthesis without application of stages of placement and removal of protective groups.

EFFECT: method makes it possible to reduce quantity of synthesis stages, is more economically effective due to absence of placement and removal of protective groups stages and makes it possible to obtain tripeptide with high purity.

1 ex

Description

The invention relates to the synthesis of peptides, in particular to the preparation of the tripeptide (I) used as an intermediate (1-3 fragment of LH-RH) in the synthesis of biologically active synthetic agonists of gonadotropin-releasing hormone LH-RH (LH-RH):

Gonadotropin-releasing hormone (GnRH, gonadoliberin), also known as luteinizing-releasing hormone (LH-RH), contains a sequence of 10 amino acids and is one of the representatives of the releasing hormones of the hypotolamus:

Due to the rapid destruction of GnRH in the body by endopeptidases (the half-life of endogenous GnRH is 2-8 minutes), synthetic modified GnRH agonists with a much longer period of action while maintaining (or enhancing) specific biological activity are widely used in pharmaceutical practice. As a rule, the modification of the gonadoliberin molecule occurs by substituting amino acids at positions 6 and 10, which, while maintaining affinity for pituitary receptors, slows down its cleavage by endopeptidases. It was found that, in addition to increased resistance to enzymes, GnRH agonists significantly exceed endogenous gonadoliberin in biological activity. Initially, GnRH agonists were developed to treat infertility due to hypogonadotropic hypogonadism, in accordance with the physiological value of gonadoliberin. However, subsequently it turned out that increased secretion of gonadotropins occurs only when the GnRH preparation is used in a pulsed mode, and long-term use of GnRH agonists leads to transient stimulation of gonadotropin secretion and, as a result, to a decrease in the level of secretion of sex steroids. In this regard, the indications for the use of GnRH agonists have expanded significantly. They are currently used for advanced prostate cancer, breast cancer, uterine fibroids, endometriosis, and in vitro fertilization (IVF).

Currently, 4 synthetic GnRH agonists are most widely used: buserelin, goserelin. leuprorelin and triptorelin, in the production of which tripeptide I is a key intermediate [1, 2, 3] upon receipt by the liquid-phase method.

In particular, a known method of producing nonapeptide leuprorelin [1]:

consisting in the condensation of tripeptide I and the hexapeptide HSerTyr-D-LeuLeuArgProNHEt.

The method of producing decapeptide triptorelin [2]:

according to scheme 3 + 7, where compound I is used as a tripeptide.

In RU No. 2444525, 03/10/2012, a method for producing a non-peptide of bushelerin is described [3]:

according to scheme 3 + 6 using tripeptide I hydrochloride as an intermediate.

From US No. 4024248, 03/17/1977, a method for producing an LH-RH analog (nonapeptide ethyl amide) by condensing peptide fragments according to the 3 + 6 scheme by the azide method, in which the tripeptide of the formula HPyr-His-Trp-NH-NH _{2 is} used as an intermediate, is known.

The closest in technical essence is known from DD No. 226895, 09/04/1985, a method for producing tripeptide I (HPyr-His-TrpOH), which includes 7 stages:

- statement benzyloxycarbonyl protection on L-pyroglutamic acid;

- production of histidine methyl ester;

- condensation of the protected L-pyroglutamic acid and L-histidine methyl ester in the presence of dicyclohexylcarbodiimide (DCC) to form the protected dipeptide methyl ester - Z-PyrHisOMe;

- removal of Z-protection using palladium on charcoal;

- gudrazinoliz dipeptide methyl ester to form N-PyrHisNHNH _2;

- obtaining the ethyl ester of the desired tripeptide H-PyrGluHisTrpOEt by the reaction of H-PyrHisNHNH ₂ and L-tryptophan ethyl ester hydrochloride using a standard azide condensation protocol;

- hydrolysis of H-PyrGluHisTrpOEt with sodium hydroxide in methanol to form a tripeptide.

The disadvantages of this method are:

- a relatively large number of stages (due to the stages of setting and removing protective groups),

- high cost of the process,

- the use of toxic reagents (benzyloxycarbonyl chloride, hydrazine hydrate, tert-butyl nitrite),

- stringent conditions of the process (when conducting condensation by the azide method, cooling to -25 - -30 ° C is necessary).

The objective of the invention is:

- simplification of the process of obtaining

- the exclusion of reagents for the setting and removal of protective groups,

- reduction in production costs I,

- reduction of the toxicity of the process.

The stated technical problem and the technical result achieved is achieved by the method of producing the tripeptide of formula (I) HPyr-His-TrpOH by liquid-phase peptide synthesis without using the stages of setting and removing protective groups, including the following stages:

1. Condensation of L-pyroglutamic acid (II) HPyrOH and N-hydroxysuccinimide (SuOH) in the presence of dicyclohexylcarbodiimide (DCC) in tetrahydrofuran to form activated ester (III);

2. The interaction of the obtained ester III with the sodium salt of L-histidine (IV) HHisOH in a solvent mixture of dimethylformamide-water with the formation of the HPyrHisOH (V) dipeptide;

3. The reaction of activated succinimide ester obtained without isolation by condensation of the dipeptide (V) and SuOH with L-tryptophan methyl ester in dimethylformamide to form the tripeptide methyl ester - HPyrHisTrpOMe (VI);

4. Hydrolysis of the methyl ester of formula (VI) in an aqueous methanol solution using lithium hydroxide and further precipitation of lithium ions with an equimolar amount of phosphoric acid gives the desired tripeptide I.

The developed synthesis method I, which allows us to solve the problem by 4-stage synthesis, is performed according to the following scheme:

Below is a General view of the method claimed as an invention, revealing the essence of this method.

The condensation of L-pyroglutamic acid (II) and N-hydroxysuccinimide (SuOH) in the presence of DCC is carried out in tetrahydrofuran for 48 hours. The precipitated dicyclohexylurea is filtered off and the solvent is removed in vacuo. The residue was dissolved in methylene chloride and allowed to crystallize at 5 ° C. After filtration, crystalline activated ether III is obtained in a yield of 69%.

The reaction of activated ester III and sodium salt of L-histidine obtained without isolation of neutralization IV with an aqueous solution of sodium hydroxide gives dipeptide V. Raw V obtained after distillation of the solvent in vacuo is dissolved in methanol, the pH is adjusted to 3 with concentrated hydrochloric acid and precipitated with tetrahydrofuran at temperature 5 ° С. Yield V (96% purity) is 82%.

Condensation of the dipeptide V and L-tryptophan methyl ester using SuOH and DCC in dimethylformamide over 24 hours leads to the tripeptide VI methyl ester. N-Methylmorpholine is used to convert the hydrochloride to the free base. The reaction product obtained after the removal of dimethylformamide in vacuo was dissolved in a 2-phase ethyl acetate-water system. Compound VI is precipitated from the aqueous layer with sodium bicarbonate solution at pH = 8. Further purification is carried out by washing raw VI with ethyl acetate. Yield VI (96.6% purity) is 65.2%.

The target tripeptide I is obtained by hydrolysis of VI with a water-methanol solution of lithium hydroxide for 1.5 hours. After the completion of the reaction, Li ^{+ is} bound with an equimolar amount of phosphoric acid, methanol is removed in vacuo, and aqueous solution I is lyophilized. Obtain I with a yield of 95% and a purity of 98.5% (HPLC).

The purity of the obtained HPyr-His-TrpOH tripeptide and its intermediates was confirmed by HPLC and NMR spectroscopy. Analytical HPLC was performed on a Shimadzu chromatograph. Column: Grom-Sil 12J ODS-4HE, 5 μm, 250 × 4.6 mm. Conditions: Gradient 20% B (0 min) 60% B (10 min) 70% B (15 min) 70% B (30 min). A — phosphate buffer pH 3 (20.4 g of KH ₂ PO _{4 was} dissolved in 3 L of distilled water and adjusted to pH 3 with concentrated phosphoric acid), B — acetonitrile. PMR spectra were obtained on a Bruker AM-360 instrument (operating frequency 360.13 MHz). ¹³ C NMR spectra were obtained on a Bruker AM-360 instrument (operating frequency 90.56 MHz). The specific rotation angle was measured on an OTAGO AR-300 automatic polarimeter. ESI-MS spectra were recorded on an Agilent LC / MS 1200 instrument during ionization of the sample by electrospray in the mode of registration of positive ions. Samples were prepared in an acetonitrile / water 1/1 system, concentration 2 mg / ml. Analysis conditions: flow 1 ml / min, pressure on the nibulizer 20 psi, temperature 360 ° C, flow rate of the drying gas 9 l / min, voltage 3500 V, target weight from 100 to 2000.

The described method is illustrated by the following example, reflecting the detailed implementation of each stage of the synthesis.

A. H-Pyr-OSu (III).

(2.5% р-р, тетрагидрофуран). Спектр ЯМР ¹Н (d₆-DMSO, δ, м.д.): 2.11-2.25 (m, 3Н, β-СН-Pyr+γ-СН₂-Pyr), 2.53-2.60 (m, 1Н, β-CH-Pyr), 2.84 (s, 4Н, 2*CH₂-OSu), 4.62-4.67 (m, 1Н, α-CH-Pyr), 8.23 (s, 1Н, NH-Pyr).30 g (0.23 mol) of pyroglutamic acid (II) are suspended in 0.5 L of tetrahydrofuran, and 26.7 g (0.23 mol) of N-hydroxysuccinimide are sprinkled with. Cool the suspension to 0 ° C and add 47.3 g (0.23 mol) of dicyclohexylcarbodiimide. The reaction mixture is warmed to room temperature and stirred for 48 hours. The precipitated urea is filtered off, the filtrate is evaporated in vacuo until the end of distillation (50 ° С, 12 mm Hg). Dissolve the oily residue in 200 ml of methylene chloride, seed the final product and leave at 5 ° C for 2 hours. The product is filtered off, washed with 50 ml of cold (5 ° C) methylene chloride and dried in vacuum (40 ° C, 12 mm Hg). Art.) to constant weight. 36 g (69.2%) of white crystalline III are obtained. Mp 136-138 ° C. $${\left[\alpha \right]}_D^{22}=+18.81$$

(2.5% solution, tetrahydrofuran). ¹ H NMR (d ₆ -DMSO, δ, ppm): 2.11-2.25 (m, 3H, β-CH-Pyr + γ-CH ₂ -Pyr), 2.53-2.60 (m, 1H, β- CH-Pyr), 2.84 (s, 4H, 2 * CH ₂ -OSu), 4.62-4.67 (m, 1H, α-CH-Pyr), 8.23 (s, 1H, NH-Pyr).

B. H-Pyr-His-OH (V).

(2% р-р, вода). Спектр ЯМР ¹Н (CDCl₃, δ, м.д.): 1.89-2.09 (m, 1Н, β-СН-Pyr), 2.46 (t, 2Н, γ-СН₂-Pyr), 2.51-2.66 (m, 1Н, β-CH-Pyr), 3.23-3.33 (m, 1Н, β-CH-His), 3.42-3.51 (m, 1Н, β-CH-His), 4.38-4.44 (m, 1H, α-CH-His), 4.81-4.87 (m, 1H, α-CH-Pyr), 7.40 (s, 1H, ar-His), 8.71 (s, 1H, ar-His). Спектр ЯМР ¹³C (CDCl₃, δ, м.д.): 181.41, 173.97, 173.05, 132.74, 128.31, 116.38, 56.11, 51.41, 28.52, 25.75, 24.62. ESMS, m/z 267.4 [M+H]⁺ (32.9), 533.4 [2M+H]⁺ (100), 799.4 [3M+H]⁺ (1.3).Dissolve 18.9 g (0.084 mol) of III in 35 ml of dimethylformamide. A solution of 9.3 g (0.06 mol) of histidine (IV) and 2.4 g (0.06 mol) of NaOH in 30 ml of water was added dropwise to the resulting solution at 0 ° C for 20 min. Stir the reaction mass for 30 minutes at 10 ° C and then 2 hours at room temperature until the reaction is complete. Evaporate the reaction mass in vacuo until the end of the distillation (50 ° C, 2 mm Hg). The oily residue is dissolved in 50 ml of methyl alcohol, acidified with conc. hydrochloric acid to pH = 3, add 100 ml of tetrahydrofuran and leave at 5 ° C for 12 hours. The precipitate is filtered, washed with 50 ml of tetrahydrofuran and dried in vacuum to constant weight (50 ° C, 2 mm Hg). Obtain 13.1 g (82%) of white amorphous V. The content of the basic substance 96% (HPLC). $${\left[\alpha \right]}_D^{24}=-6.67$$

(2% solution, water). ¹ H NMR Spectrum (CDCl ₃ , δ, ppm): 1.89-2.09 (m, 1H, β-CH-Pyr), 2.46 (t, 2H, γ-CH ₂ -Pyr), 2.51-2.66 (m , 1H, β-CH-Pyr), 3.23-3.33 (m, 1H, β-CH-His), 3.42-3.51 (m, 1H, β-CH-His), 4.38-4.44 (m, 1H, α- CH-His), 4.81-4.87 (m, 1H, α-CH-Pyr), 7.40 (s, 1H, ar-His), 8.71 (s, 1H, ar-His). ¹³ C NMR spectrum (CDCl ₃ , δ, ppm): 181.41, 173.97, 173.05, 132.74, 128.31, 116.38, 56.11, 51.41, 28.52, 25.75, 24.62. ESMS, m / z 267.4 [M + H] ⁺ (32.9), 533.4 [2M + H] ⁺ (100), 799.4 [3M + H] ⁺ (1.3).

B. H-Pyr-His-TrpOMe (VI).

(2% р-р, в 3% водном растворе уксусной кислоты). Спектр ЯМР ¹Н (d₆-DMSO, δ, м.д.): 1.70-1.83 (m, 1Н, β-CH-Pyr), 1.97-2.25 (m, 3Н, γ-СН₂-Pyr+β-СН-Pyr), 2.71-2.99 (m, 2Н, β-CΗ₂-Ηis), 3.04-3.21 (т, 2Н, β-СН₂-Trp), 3.56 (s, 3Н, СООМе), 3.96-4.03 (m, 1Н, α-CH-Trp), 4.49-4.58 (m, 2Н, α-СН- His+α-CH-Pyr), 6.77 (br. s, 1Н, CONH), 6.98 (t, 1H, ar-Trp), 7.06 (t, 1H, ar-Trp), 7.15 (s, 1H, ar-Trp), 7.34 (d, 1H, ar-Trp), 7.45-7.52 (m, 2H, ar-Trp+ar-His), 7.74 (s, 1H, ar-His), 8.06 (br. s, 1H, CONH), 8.32 (br. s, 1H, NH-Pyr), 10.88 (br. s, 1H, NH-His), 8.32 (br. s, 1H, NH-Trp). ESMS, m/z 467.4 [M+H]⁺ (100), 933.6 [2M+H]⁺ (6.3).Dissolve 6.42 g (0.024 mol) of V in 55 ml of dimethylformamide. 6.11 g (0.024 mol) of L-tryptophan methyl ester, 3.3 g (0.029 mol) of N-hydroxysuccinimide and 3.3 g (0.032 mol) of N-methylmorpholine are sprinkled with the resulting solution. After complete dissolution, the reaction mass is cooled to 10 ° C and at this temperature a solution of 6.5 g (0.032 mol) of dicyclohexylcarbodiimide in 20 ml of dimethylformamide is added dropwise over 1 h. Warm up to room temperature and stir for 24 hours. The precipitate of dicyclohexylurea is filtered off, and the filtrate is evaporated in vacuo until the end of distillation (50 ° С, 2 mm Hg). The remaining oil is dissolved in 50 ml of water, 30 ml of ethyl acetate are added, filtered and the aqueous layer is separated. Saturated sodium bicarbonate solution was added dropwise to the aqueous layer to pH 8, 50 ml of ethyl acetate were added, and it was left at 5 ° С for 3 h. Raw VI was filtered, stirred in ethyl acetate-water 30 ml of water and 30 ml of ethyl acetate, filtered, washed on a filter 20 ml of ethyl acetate and dried in vacuum to constant weight. 7.3 g (65.2%) of white powder VI are obtained. The content of the basic substance 96.6% (HPLC). $${\left[\alpha \right]}_D^{22}=-26.93$$

(2% solution, in a 3% aqueous solution of acetic acid). ¹ H NMR (d ₆ -DMSO, δ, ppm): 1.70-1.83 (m, 1H, β-CH-Pyr), 1.97-2.25 (m, 3H, γ-CH ₂ -Pyr + β- CH-Pyr), 2.71-2.99 (m, 2H, β-CΗ ₂ -is), 3.04-3.21 (t, 2H, β-CH ₂ -Trp), 3.56 (s, 3H, COOMe), 3.96-4.03 ( m, 1H, α-CH-Trp), 4.49-4.58 (m, 2H, α-CH- His + α-CH-Pyr), 6.77 (br.s, 1H, CONH), 6.98 (t, 1H, ar -Trp), 7.06 (t, 1H, ar-Trp), 7.15 (s, 1H, ar-Trp), 7.34 (d, 1H, ar-Trp), 7.45-7.52 (m, 2H, ar-Trp + ar -His), 7.74 (s, 1H, ar-His), 8.06 (br. S, 1H, CONH), 8.32 (br. S, 1H, NH-Pyr), 10.88 (br. S, 1H, NH-His ), 8.32 (br. S, 1H, NH-Trp). ESMS, m / z 467.4 [M + H] ⁺ (100), 933.6 [2M + H] ⁺ (6.3).

G. H-Pyr-His-TrpOH (I).

(2% р-р, вода). Спектр ЯМР ¹Н (D₂O, δ, м.д.): 1.59-1.72 (m, 1Н, β-CH-Pyr), 2.23-2.28 (m, 3Н, γ-СН₂-Pyr+β-СН-Pyr), 2.90-2.99 (m, 1Н, β-CH-His), 3.05-3.13 (m, 1Н, β-CH-His), 3.16-3.25 (m, 1Н, β-CH-Trp), 3.36-3.44 (m, 1Н, β-CH-Trp), 4.09-4.16 (m, 1Н, α-СН-His), 4.57-4.65 (m, 2Н, α-СН-Pyr+α-СН-Тгр), 7.01 (s, 1Н, ar-His), 7.16 (t, 1Н, ar-Trp), 7.23 (s, 1Н, ar-Trp), 7.25 (t, 1H, ar-Trp), 7.50 (d, 1H, ar-Trp), 7.67 (d, 1H, ar-Trp), 8.03 (s, 1H, ar-His). Спектр ЯМР ¹³C (D₂O, δ, м.д.): 181.29, 177.32, 173.46, 170.03, 135.30, 129.41, 126.54, 123.38, 120.99, 118.40 (2C), 117.77, 116.60, 110.99, 109.38, 55.90, 55.17, 52.00, 28.35, 26.66, 26.61, 24.30. ESMS, m/z 453.4 [M+H]⁺ (100), 905.5 [2M+H]⁺ (26.4).6.3 g (0.0135 mol) of VI are suspended in 47 ml of methyl alcohol. A solution of 0.44 g of lithium hydroxide (0.0184 mol) in 20 ml of water is added to the resulting suspension at room temperature. Stir the reaction mass for 1.5 hours until the end of the reaction (TLC). Orthophosphoric acid (85%) is added dropwise to a pH of 7-7.5 (equimolar amount). The precipitated lithium phosphate is filtered, and the filtrate is evaporated to a volume of 15 ml (distillation of methanol) and the residue is lyophilized. Obtain 5.8 g (94.9%) of white powder I. The content of the main substance of 98.5% (HPLC). $${\left[\alpha \right]}_D^{23}=-17.61$$

(2% solution, water). ¹ H NMR Spectrum (D ₂ O, δ, ppm): 1.59-1.72 (m, 1H, β-CH-Pyr), 2.23-2.28 (m, 3H, γ-CH ₂ -Pyr + β-CH -Pyr), 2.90-2.99 (m, 1H, β-CH-His), 3.05-3.13 (m, 1H, β-CH-His), 3.16-3.25 (m, 1H, β-CH-Trp), 3.36 -3.44 (m, 1H, β-CH-Trp), 4.09-4.16 (m, 1H, α-CH-His), 4.57-4.65 (m, 2H, α-CH-Pyr + α-CH-Tg), 7.01 (s, 1H, ar-His), 7.16 (t, 1H, ar-Trp), 7.23 (s, 1H, ar-Trp), 7.25 (t, 1H, ar-Trp), 7.50 (d, 1H, ar-Trp), 7.67 (d, 1H, ar-Trp), 8.03 (s, 1H, ar-His). ¹³ C NMR spectrum (D ₂ O, δ, ppm): 181.29, 177.32, 173.46, 170.03, 135.30, 129.41, 126.54, 123.38, 120.99, 118.40 (2C), 117.77, 116.60, 110.99, 109.38, 55.90, 55.17, 52.00, 28.35, 26.66, 26.61, 24.30. ESMS, m / z 453.4 [M + H] + ( 100), 905.5 [2M + H] + (26.4).

As can be seen from the above example, the described method greatly simplifies the process of obtaining the target compound by reducing the number of synthesis steps, makes it more cost-effective due to the absence of steps for setting and removing protective groups, and allows tripeptide I to be obtained with a purity of more than 98%.

Information sources

1. Chemical and pharmaceutical journal. 2014, 48 (3), 54-56.

2. Russian Biotherapeutic Journal 2013, 12 (4), 55-58.

3. RU No. 2444525. A method of producing nonapeptidethylamide and intermediates for its preparation, 03/10/2012.

Claims (1)

A method for producing a tripeptide of general formula (I) HPyr-His-TrpOH used as an intermediate compound (1-3 fragments) in the synthesis of synthetic gonadotropin-releasing hormone agonists (LH-RH), by the method of liquid-phase peptide synthesis without setting and removing protective groups, including following stages:
a) the condensation of L-pyroglutamic acid (II) HPyrOH and N-hydroxysuccinimide SuOH in the presence of dicyclohexylcarbodiimide (DCC) in tetrahydrofuran;
b) the interaction of the activated HPyrOSu ester (III) formed in step (a) with the sodium salt of L-histidine HHisOH (IV) in a dimethylformamide-water solvent mixture to form the HPyrHisOH (V) dipeptide;
c) the reaction of activated succinimide ester obtained without isolation by condensation of dipeptide (V) and SuOH with L-tryptophan methyl ester in dimethylformamide to form HPyr-His-TrpOMe tripeptide (VI) methyl ester;
g) hydrolysis of methyl ester of tripeptide (VI) in an aqueous methanol solution using lithium hydroxide;
e) precipitation of lithium ions with an equimolar amount of phosphoric acid after hydrolysis of the methyl ester of tripeptide (VI);
e) removal of methanol in vacuum and lyophilization of an aqueous solution of the target product of the tripeptide of the formula (I).