Synthesis method of Fmoc-Phe (4-Boc2-guanidino)-OH

Abstract

The invention provides a synthesis method of Fmoc-Phe (4-Boc2-guanidino)-OH, and relates to the technical field of amino acid derivative synthesis, 4-amino-L-phenylalanine is used as a raw material, and the Fmoc-Phe (4-Boc2-guanidino)-OH is prepared through guanidination reaction, Fmoc protection and Boc protection in sequence. The method has the advantages of easily available initial raw materials, mild chemical reaction conditions in each step, easy post-treatment and purification of each intermediate, facilitation of the improvement of the product purity, facilitation of large-scale production, and reduction of the synthesis cost; a new thought and possibility are provided for the synthesis of the Fmoc-Phe (4-Boc2-guanidyl)-OH.

Description

Technical Field

[0001] This invention relates to the field of amino acid derivative synthesis technology, specifically a method for synthesizing Fmoc-Phe(4-Boc2-guanidino)-OH. Background Technology

[0002] Fmoc-Phe(4-Boc2-guanidinyl)-OH is an important amino acid derivative, chemically named fluorenylmethoxycarbonyl-4-(N,N-di-tert-butyloxycarbonyl-guanidinyl)-L-phenylalanine, with CAS number 187283-25-6 and molecular formula C2. 35 H 40 N4O8.

[0003] The molecular structure contains three key protecting groups: Fmoc (9-fluorenylmethoxycarbonyl) protecting the α-amino group, two Boc (tert-butyloxycarbonyl) protecting the guanidinium group, and the carboxyl group remaining in a free state. This multi-protection strategy makes it valuable in peptide synthesis, serving as a key protected amino acid monomer in solid-phase peptide synthesis (SPPS) for introducing phenylalanine residues with guanidinium functional groups into the solid-phase synthesis process. Simultaneously, the 4-Boc2-guanidinium side chain protecting group in its molecular structure can be selectively deprotected after peptide synthesis, thereby preparing functionalized peptides with antibacterial or bioactive properties. For example, N-trans-cinnamyl-p-fluorophenylalanine-p-guanidinylphenylalanine-leucine-arginine-NH2 (BMS-197525), synthesized from this compound, can block the activation of protease-activated receptors, thus inhibiting human thrombin receptor (PAR-1). Furthermore, Fmoc-Phe (4-Boc2-guanidinium) protects the α-amino group. 2- Guanidinyl guanidine (-OH) has applications and promising prospects in functionalized polymer materials, feed additives, and other fields.

[0004] Currently, the main synthetic routes for Fmoc-Phe(4-Boc2-guanidino)-OH are as follows:

[0005]

[0006] This route uses 4-nitro-L-phenylalanine (compound 1) as a starting material, reacting it with Fmoc-Cl under alkaline conditions to generate Fmoc-4-nitro-L-phenylalanine (compound 2). The nitro group of compound 2 undergoes a reduction reaction under metal-protonated acid catalysis, reducing the nitro group to an amino group to give Fmoc-4-amino-L-phenylalanine (compound 3). Compound 3 then condenses with compound 4 under acid catalysis to generate the product. Although 4-nitro-L-phenylalanine is an intermediate of Zolmitriptan and is readily available, this route presents the following challenges:

[0007] 1. Process safety and compliance risks

[0008] ① Use of metal powder: Using zinc powder for nitro reduction will produce hydrogen gas, which poses a risk of combustion and explosion. This places extremely high demands on the explosion-proof level, ventilation and exhaust gas treatment of the production site, and significantly increases the investment in safety facilities and the complexity of operation.

[0009] ② Byproduct treatment: The reaction produces zinc-containing wastewater, which increases the cost of environmental treatment and the complexity of "three wastes" declaration;

[0010] ③ Nitro compounds: These involve nitro intermediates, and their storage, transportation, and production are typically subject to more stringent scrutiny in safety assessments and regulatory approvals.

[0011] 2. Process stability and yield issues

[0012] ① Stability of Fmoc group: The acid-catalyzed condensation step in the second half of the route, as well as the acidic environment that may not have been completely removed in the previous reduction step, both pose a threat to the acid-sensitive Fmoc protecting group, which may lead to partial deprotection, generation of impurities, reduction of the purity and yield of the final product, and increase of purification difficulty and cost.

[0013] ② Operational complexity: The multi-step reaction, the involvement of air / moisture sensitive reagents (such as zinc powder and acid catalysts), and the harsh post-processing conditions (quenching, extraction, drying, etc.) result in poor process scale-up reproducibility and high requirements for equipment and control precision.

[0014] 3. Economic efficiency and "atom economy"

[0015] ① The process is lengthy: it requires three reaction steps (Fmoc protection → nitro reduction → guanidinolation condensation);

[0016] ② Reagent cost: Using N,N'-di-Bocthiourea as a guanidinization reagent has low atom economy and the reagent itself may be expensive;

[0017] ③ Limited overall yield: The cumulative yield of multi-step reactions is affected, and purification losses may further increase costs. Summary of the Invention

[0018] To address the technical shortcomings of existing synthetic routes for Fmoc-Phe(4-Boc2-guanidino)-OH, this invention provides a method for preparing Fmoc-Phe(4-Boc2-guanidino)-OH. This route uses readily available starting materials, employs mild chemical reaction conditions in each step, and facilitates easy post-processing and purification of intermediates, which is beneficial for improving product purity, promoting large-scale production, and reducing synthesis costs.

[0019] A method for synthesizing Fmoc-Phe(4-Boc2-guanidino)-OH, using 4-amino-L-phenylalanine (compound II) as a raw material, wherein the carbon atom attached to the amino group of 4-amino-L-phenylalanine is S-chiral.

[0020] The first step is to perform a guanidinolation reaction: 4-amino-L-phenylalanine reacts with a guanidinolation reagent to generate (2S)-2-amino-3-(4-guanidinophenyl)propionic acid (intermediate III); the enantiomers of 4-amino-L-phenylalanine can also be guanidinolated in the same way.

[0021] The second step involves Fmoc protection: Under alkaline conditions, the α-amino group of (2S)-2-amino-3-(4-guanidinylphenyl)propionic acid is protected with Fmoc to obtain Fmoc-(S)-4-guanidinylphenylalanine (intermediate IV).

[0022] The third step involves Boc protection: Under acidic conditions, Fmoc-(S)-4-guanidinophenylalanine undergoes a substitution reaction with di-tert-butyl dicarbonate, and the two nitrogen atoms of the guanidino group are protected with Boc to obtain Fmoc-Phe(4-Boc2-guanidino)-OH (compound I).

[0023] Boc and Fmoc are orthogonal protecting groups. Boc is sensitive to acids but stable to bases, while Fmoc is sensitive to bases but stable to acids. Since guanidinolation occurs under acidic conditions, this invention first protects Fmoc and then Boc.

[0024] Further, the guanidinolation reaction process: 4-amino-L-phenylalanine is condensed with thiourea at 30~60℃ under acid catalysis to generate (2S)-2-amino-3-(4-guanidinophenyl)propionic acid; more specifically, thiourea, 4-amino-L-phenylalanine and organic acid are added to a reaction flask in sequence and reacted at 55~60℃ for 22~24h. After the reaction is complete as detected by liquid phase, the reaction solution is concentrated to dryness under reduced pressure at 50~55℃. The crude product is crystallized with water to obtain (2S)-2-amino-3-(4-guanidinophenyl)propionic acid.

[0025] Furthermore, the solvent for the guanidinolation reaction is selected from at least one of tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide, methanol, acetic acid, trifluoroacetic acid, and propionic acid; the organic acid used in the guanidinolation reaction is one of acetic acid, trifluoroacetic acid, propionic acid, and butyric acid, and the pH of the reaction system is controlled at 4.5.

[0026] Furthermore, the reagent for the Fmoc protecting group is selected from Fmoc-OSu and FmocCl; the base providing the alkaline conditions is selected from sodium carbonate, potassium carbonate, and sodium bicarbonate; and the solvent for Fmoc protection is selected from tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide, and water, or a mixture of two of them.

[0027] Furthermore, the Fmoc protection process: (2S)-2-amino-3-(4-guanidinophenyl)propionic acid, 1,4-dioxane, and water are added sequentially to the reaction flask. Sodium bicarbonate is then added to the reaction flask and stirred until dissolved, noting the generation of bubbles. The temperature of the reaction solution is lowered to 0-5℃. Solid FmocCl is added to the reaction flask in batches. After the liquid phase is checked and the reaction is complete, diethyl ether is added to the reaction flask to extract impurities. Ethyl acetate is then added to the aqueous phase of the reaction flask to adjust the pH to 5-6. The product is extracted by separation. The solution is concentrated to dryness under reduced pressure at 40-45℃. The crude product is crystallized with ethyl acetate / n-heptane = 1:1 to obtain Fmoc-(S)-4-guanidinophenylalanine.

[0028] Furthermore, the acid protected by Boc is selected from acetic acid, methanesulfonic acid, p-toluenesulfonic acid, and aminosulfonic acid; the solvent protected by Boc is selected from dichloromethane, ethyl acetate, tetrahydrofuran, dichloroethane, and carbon tetrachloride.

[0029] Furthermore, the Boc protection process: Fmoc-(S)-4-guanidinophenylalanine, ditert-butyl dicarbonate, toluene, and aminosulfonic acid were added sequentially to a single-necked flask and reacted at 20-25°C. The product gradually precipitated out. After the reaction was confirmed to be complete by liquid chromatography, the crude product obtained by filtration was slurryed with ethyl acetate / n-heptane = 1:2 to obtain pure Fmoc-Phe(4-Boc2-guanidino)-OH.

[0030] Beneficial effects of this invention:

[0031] 1. A novel route for synthesizing Fmoc-Phe(4-Boc2-guanidino)-OH is provided, offering new ideas and possibilities for the synthesis of Formula I.

[0032] 2. The starting material 4-amino-L-phenylalanine is readily available, the chemical reaction conditions in each step are mild, and the post-processing and purification of each intermediate are easy, which is conducive to improving the purity of the product, large-scale production and reducing costs.

[0033] 3. A novel synthetic method is provided for both the Formula III and Formula IV compounds, which are two key intermediates. Attached Figure Description

[0034] Figure 1 For compound III 1 HNMR spectrum;

[0035] Figure 2 This is the LC-MS positive ion spectrum of compound III;

[0036] Figure 3 The LC-MS negative ion spectrum of compound III;

[0037] Figure 4 For compound IV 1 HNMR spectrum;

[0038] Figure 5 This is the LC-MS positive ion spectrum of compound IV;

[0039] Figure 6 The LC-MS negative ion spectrum of compound IV is shown below.

[0040] Figure 7 For compound I 1 HNMR spectrum;

[0041] Figure 8 This is the LC-MS positive ion spectrum of compound I;

[0042] Figure 9 This is the LC-MS negative ion spectrum of compound I. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and design various embodiments with various modifications suitable for a particular purpose.

[0044] First, the English abbreviations involved in this invention will be explained.

[0045] Boc: tert-butyloxycarbonyl;

[0046] Cbz: benzyloxycarbonyl;

[0047] Fmoc: 9-fluorenylmethoxycarbonyl;

[0048] Phe: Phenylalanine

[0049] Fmoc-OSu: 9-fluorenylmethyl-N-succinimide carbonate;

[0050] FmocCl: 9-fluorenyl chloroformate;

[0051] SPPS: Solid-phase peptide synthesis;

[0052] PAR-1: Human thrombin receptor;

[0053] BMS-197525: N-trans-cinnamoyl-p-fluorophenylalanine-p-guanidinophenylalanine-leucine-arginine-NH2.

[0054] Example 1

[0055] A synthetic method for Fmoc-Phe(4-Boc2-guanidino)-OH, the synthetic route of which is as follows:

[0056] .

[0057] I. Guanidinization reaction: Thiourea, 4-amino-L-phenylalanine (compound II), and acetic acid were added sequentially to a 250 mL reaction flask and reacted at 55-60 °C for 22-24 h. The proportions are shown in Table 1.

[0058] The 4-amino-L-phenylalanine molecule contains both aromatic amine and aliphatic amine structural segments.

[0059] Aliphatic amines: pKa = 9.3 (strongly basic), exist in protonated form (RNH3) at pH = 4.5. + (It is mainly composed of ) and has low reactivity with thiourea;

[0060] Aromatic amines: pKa=4.25 (strong acidity), also exist as free base (RNH2) at pH=4.5, preferentially react with thiourea to form a single condensation product.

[0061] This embodiment uses acetic acid to provide precise pH control. The phosphate / citrate buffer system is not superior to acetic acid, and its excessive buffering capacity may cause the pH to deviate from the target value.

[0062] This embodiment precisely controls the protonation (passivation) of aliphatic amines at pH=4.5, supported by thermodynamic calculations.

[0063] Fatty amine paperization rate:

[0064] ;

[0065] Aromatic amine protonation rate:

[0066] .

[0067] After the reaction was confirmed to be complete by liquid chromatography (HPLC), the reaction solution was concentrated to dryness under reduced pressure (20-50 Pa) at 50-55℃. The crude product was crystallized by water precipitation to obtain 27.1 g of (2S)-2-amino-3-(4-guanidinylphenyl)propionic acid (intermediate III), with a yield of 92.8%, a pale yellow solid, and a purity of 98.5%.

[0068] LCMS: [M+1]=223 [M-1]=221;

[0069] 1 H NMR (400 MHz, DMSO) δ 9.91 (s, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.6 Hz, 3H), 3.47 (dd, J = 7.9, 4.6 Hz, 1H), 3.07 (dd, J = 14.5, 4.8Hz, 1H), 2.83 (dd, J = 14.3, 8.3 Hz, 1H), 2.02 (s, 3H).

[0070] Table 1

[0071] Raw material name molecular weight (MW) Molar quantity (MOL) Equivalent EQ Quality WEIGHT Thiourea 76.12 0.1314 1.00 10.0g 4-Amino-L-phenylalanine (Compound of Formula II) 180.20 0.1314 1.00 23.7g Acetic acid 60.05 1.6652 12.67 100.0g

[0072] II. Fmoc protection: Add (2S)-2-amino-3-(4-guanidinylphenyl)propionic acid (intermediate III), dioxane, and water to a 500mL reaction flask in sequence. Add sodium bicarbonate to the reaction flask and stir until dissolved, noting that bubbles are generated. Cool the reaction solution to 0~5℃, and add FmocCl solid to the reaction flask in batches, according to the ratio in Table 2.

[0073] The reaction was confirmed to be complete by high-performance liquid chromatography (HPLC). 250 mL of diethyl ether was added to the reaction flask to extract impurities. Then, 250 mL of ethyl acetate was added to the aqueous phase in the reaction flask to adjust the pH to 5-6. The product was extracted by separation. The solution was concentrated to dryness under reduced pressure (20-50 Pa) at 40-45°C. The crude product was crystallized with ethyl acetate / n-heptane (1:1) to give 18.1 g of Fmoc-(S)-4-guanidinophenylalanine (intermediate IV), yield 90.6%, as an off-white solid with a purity of 98.3%.

[0074] LCMS: [M+1]=445 [M+18]=462 [M-1]=443 [2M+1]=889 [M / 2-1]=887;

[0075] 1H NMR (400 MHz, DMSO) δ 9.87 (s, 1H), 7.88 (d, J = 7.6 Hz, 2H), 7.69- 7.62 (m, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.45 - 7.37 (m, 2H), 7.35 - 7.25(m, 2H), 7.18 (d, J = 8.6 Hz, 2H), 4.26 - 4.05 (m, 4H), 3.57 (s, 1H), 3.02(dd, J = 13.7, 4.4 Hz, 1H), 2.82 (dd, J = 13.9, 10.3 Hz, 1H), 2.02 (s, 3H).

[0076] Table 2

[0077] Raw material name molecular weight (MW) Molar quantity (MOL) Equivalent EQ Quality WEIGHT (2S)-2-amino-3-(4-guanidinophenyl)propionic acid (59574-11-7) 222.24 0.0450 1.00 10.0g water —— —— —— 100.0g Dioxane —— —— —— 100.0g Sodium bicarbonate 84.01 0.1350 3.00 11.3g FmocCl (9-fluorenyl chloroformate) 258.70 0.0450 1.00 11.6g

[0078] III. Boc Protection: Fmoc-(S)-4-guanidinophenylalanine (intermediate IV), Boc anhydride, toluene, and aminosulfonic acid were added sequentially to a 250 mL single-necked flask, with the proportions shown in Table 3. The reaction was carried out at 20-25 °C. The product gradually precipitated out. After the reaction was confirmed to be complete by liquid chromatography (HPLC), the crude product was obtained by filtration. Ethyl acetate / n-heptane = 1:2 was used to slurry Fmoc-Phe(4-Boc2-guanidino)-OH (compound of formula I) in 13.1 g pure product, with a yield of 90.3%, white solid and a purity of 99.2%.

[0079] LCMS: [M+1]=445 [M+18]=462 [M-1]=443 [2M+1]=889 [M / 2-1]=887;

[0080] 1 H NMR (400 MHz, DMSO) δ 9.87 (s, 1H), 7.88 (d, J = 7.6 Hz, 2H), 7.69- 7.62 (m, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.45 - 7.37 (m, 2H), 7.35 - 7.25(m, 2H), 7.18 (d, J = 8.6 Hz, 2H), 4.26 - 4.05 (m, 4H), 3.57 (s, 1H), 3.02(dd, J = 13.7, 4.4 Hz, 1H), 2.82 (dd, J = 13.9, 10.3 Hz, 1H), 2.02 (s, 3H).

[0081] Table 3

[0082] Raw material name molecular weight (MW) Molar quantity (MOL) Equivalent EQ Quality WEIGHT Fmoc-(S)-4-guanidinophenylalanine (351518-30-4) 444.48 0.02250 1.0 10.0g Boc anhydride (di-tert-butyl dicarbonate) 218.25 0.04725 2.1 10.3g Toluene —— —— —— 50.0g Aminosulfonic acid 97.09 0.00225 0.1 0.3g

[0083] Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art and related fields based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

Claims

1. A method for synthesizing Fmoc-Phe(4-Boc2-guanidino)-OH, characterized in that, Using 4-amino-L-phenylalanine as a raw material, The first step involves guanidinization: 4-amino-L-phenylalanine reacts with a guanidinizing agent to generate (2S)-2-amino-3-(4-guanidinylphenyl)propionic acid; The second step involves Fmoc protection: Under alkaline conditions, the α-amino group of (2S)-2-amino-3-(4-guanidinylphenyl)propionic acid is protected with Fmoc to obtain Fmoc-(S)-4-guanidinylphenylalanine. The third step involves Boc protection: Under acidic conditions, Fmoc-(S)-4-guanidinophenylalanine undergoes a substitution reaction with di-tert-butyl dicarbonate, and the two nitrogen atoms of the guanidino group are protected with Boc to obtain Fmoc-Phe(4-Boc2-guanidino)-OH.

2. The method for synthesizing Fmoc-Phe(4-Boc2-guanidino)-OH according to claim 1, characterized in that, The acid protected by Boc is selected from one of acetic acid, methanesulfonic acid, p-toluenesulfonic acid, and aminosulfonic acid.

3. The method for synthesizing Fmoc-Phe(4-Boc2-guanidino)-OH according to claim 1, characterized in that, The solvent for Boc protection is selected from one of dichloromethane, ethyl acetate, tetrahydrofuran, dichloroethane, and carbon tetrachloride.

4. The method for synthesizing Fmoc-Phe(4-Boc2-guanidino)-OH according to claim 2 or 3, characterized in that, Boc protection process: Fmoc-(S)-4-guanidinophenylalanine, ditert-butyl dicarbonate, toluene, and aminosulfonic acid were added sequentially to a single-necked flask and reacted at 20-25°C. The product gradually precipitated out. After the reaction was confirmed to be complete by liquid chromatography, the crude product obtained by filtration was slurryed with ethyl acetate / n-heptane = 1:2 to obtain pure Fmoc-Phe(4-Boc2-guanidino)-OH.

5. A method for synthesizing (2S)-2-amino-3-(4-guanidinophenyl)propionic acid, characterized in that, It is generated by the reaction of 4-amino-L-phenylalanine with a guanidinizing agent. The guanidinization reaction process is as follows: 4-amino-L-phenylalanine is condensed with thiourea at 30~60℃ under acid catalysis to generate (2S)-2-amino-3-(4-guanidinylphenyl)propionic acid.

6. The method for synthesizing (2S)-2-amino-3-(4-guanidinophenyl)propionic acid according to claim 5, characterized in that, Thiourea, 4-amino-L-phenylalanine, and organic acid were added sequentially to a reaction flask and reacted at 55-60°C for 22-24 hours. After the reaction was confirmed to be complete by liquid phase detection, the reaction solution was concentrated to dryness under reduced pressure at 50-55°C. The crude product was crystallized by water precipitation to obtain (2S)-2-amino-3-(4-guanidinylphenyl)propionic acid.

7. The method for synthesizing (2S)-2-amino-3-(4-guanidinophenyl)propionic acid according to claim 6, characterized in that, The solvent for the guanidinolation reaction is selected from at least one of tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide, methanol, acetic acid, trifluoroacetic acid, and propionic acid; the organic acid used in the guanidinolation reaction is one of acetic acid, trifluoroacetic acid, propionic acid, and butyric acid, and the pH of the reaction system is controlled at 4.

5.

8. A method for synthesizing Fmoc-(S)-4-guanidinophenylalanine, characterized in that, Under alkaline conditions, the α-amino group of (2S)-2-amino-3-(4-guanidinophenyl)propionic acid is protected with Fmoc to obtain Fmoc-(S)-4-guanidinophenylalanine; the reagent for applying the Fmoc protecting group is selected from Fmoc-OSu and FmocCl.

9. The method for synthesizing Fmoc-(S)-4-guanidinophenylalanine according to claim 8, characterized in that, The solvent protected by Fmoc is selected from one or a mixture of two of tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, dimethyl sulfoxide, and water; the base providing the basic conditions is one of sodium carbonate, potassium carbonate, and sodium bicarbonate.

10. The method for synthesizing Fmoc-(S)-4-guanidinophenylalanine according to claim 9, characterized in that, Fmoc protection process: (2S)-2-amino-3-(4-guanidinylphenyl)propionic acid, 1,4-dioxane, and water are added to the reaction flask in sequence. Then sodium bicarbonate is added to the reaction flask and stirred until dissolved. Note that bubbles are generated. The temperature of the reaction solution was lowered to 0-5℃, and FmocCl solid was added to the reaction flask in batches. After the reaction was confirmed to be complete by liquid phase detection, diethyl ether was added to the reaction flask to extract impurities. Then, ethyl acetate was added to the aqueous phase of the reaction flask to adjust the pH to 5-6, and the product was extracted by separation. The solution was concentrated to dryness under reduced pressure at 40-45℃. The crude product was crystallized with ethyl acetate / n-heptane = 1:1 to obtain Fmoc-(S)-4-guanidinophenylalanine.

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