WO2012000322A1

WO2012000322A1 - Peptide deformylase inhibitors containing 4-methylene pyrrolidine

Info

Publication number: WO2012000322A1
Application number: PCT/CN2011/071367
Authority: WO
Inventors: 胡文浩; 石炜; 马海坤; 段月娇; 杨琍苹
Original assignee: 华东师范大学
Priority date: 2010-07-02
Filing date: 2011-02-28
Publication date: 2012-01-05
Also published as: CN101869563A; CN101869563B

Abstract

4-methylene pyrrolidine-containing peptide deformylase inhibitors having the following structure, wherein R₁=alkyl, R₂=H or alkyl, R₃=alkyl, aryl or heterocycle. The peptide deformylase inhibitors of the present invention can inhibit bacterial protein synthesis, have little chance of drug resistance, and can kill present antibiotics-resistant strains such as MRSA and PRSP.

Description

Peptide deformylase inhibitor containing 4-methylene pyrrole oxime

A peptide deformylase inhibitor containing 4-methylene pyrrolidine relates to a peptide deformylase (PDF) inhibitor. It belongs to the field of antibiotic antibiotics technology. Background technique

Antibiotic refers to a microbial product (secondary metabolite) that kills or inhibits certain specific microorganisms such as bacteria, fungi, rickettsia, mycoplasma, chlamydia and viruses at high dilution. In 1929, the British scholar Fleming first discovered penicillin in antibiotics. In 1940, Florey and Chain, based on the discovery of Fleming, invented penicillin for human injection. In 1944, Waksman isolated streptomycin, and it is exciting that this antibiotic is highly resistant to tuberculosis, making tuberculosis no longer a terminal illness. Since then, chloramphenicol (1947), neomycin (1949), oxytetracycline (1950), erythromycin (1952), tetracycline (1953), and semi-synthetic after 1959-1961 In the late 1980s, synthetic quinolone antibiotics (such as pipemidic acid, norfloxacin, ofloxacin, ciprofloxacin, etc.) were rapidly developed and widely used in clinical applications. infection. Nowadays, antibiotics have become a new force in the pharmaceutical market, occupying more than half of the drug market. At present, the global market for anti-infective drugs accounts for about 15% of pharmaceutical sales, ranking second in the global pharmaceutical market. But most bacteria are resistant to existing antibiotics.

The process of synthesizing proteins between human cells and pathogens is basically similar. The starting materials of both are methionine, but there is one biggest difference between them: Relative to human cells, the synthetic protein of the bacteria needs to be acylated with methionine. Finally, the peptide is subjected to deformylation by peptide deformylase, and then the N-terminal methionine is removed to complete the process of synthesizing the protein, and the human cell has no formylation-deformylation process. In view of this difference between the two, an inhibitor of the peptide deformylase (PDF) of a metalloproteinase containing Fe(Π) is chelated with Fe(II) to achieve peptide deformylase loss. The action of the activity inhibits the deformylation of the peptide deformylase, so that the bacteria cannot remove the N-terminal methionine, thereby selectively inhibiting the protein synthesis of the pathogen without affecting the protein synthesis process of the human cells.

Peptide deformylase inhibitors are a class of antibacterial drugs developed in recent years. These drugs have different mechanisms of action than known antibiotics, thus killing bacteria that are resistant to existing antibiotics. It has opened up a new path for the development of antibacterial drugs and has a good development prospect.

The first peptide deformylase inhibitor discovered was a natural product, actinoin, Actinonin.

( 1 ),

(I)

This substance was found to have excellent in vitro inhibitory peptide deformylase activity and antibacterial activity. However, this natural product does not have antibacterial activity in vivo and thus cannot be used as an antibacterial drug.

Peptide deformylase (PDF) is a formyl metalloproteinase that plays a key role in bacterial protein synthesis. Peptide deformylase (PDF) inhibitors inhibit PDF in this process. The action of the deformyl group, thereby inhibiting the process of bacterial synthesis of proteins. Therefore, this new mechanism of action for PDF inhibitors offers a new potential for antibiotic treatment without affecting the metabolism of eukaryotic organisms. As far as current research is concerned, the chances of bacteria developing resistance to PDF inhibitors are small. To date, two PDF inhibitors, BB-83698 and LBM-415, have entered Phase II clinical trials. In October 2001, the peptide deformylase developed by British Biotech inhibited U BB83698 ( II ) into one, and now the product has entered Phase II clinical trials.

BB83698

(II)

In October 2003, the peptide deformylase inhibitor LBM415 (III) developed by Vicuron

LBM415

(III) Summary of the invention

It is an object of the present invention to provide a peptide deformylase inhibitor which is an antibacterial agent which is capable of killing bacteria which are resistant to existing antibiotics.

In order to achieve the above object, the present invention provides a peptide formylase inhibitor comprising a 4-methylene pyrrolidine structure, and the 4-methylene pyrrolidine-containing peptide deformylase inhibitor has the following structure.

Wherein is an alkyl group; R ₂ is H or an alkyl group; and R ₃ is an alkyl group or an aromatic group or a heterocyclic ring.

The molecular weight of the 4-methylene pyrrolidine-containing peptide deformylase inhibitor of the present invention is from 300 to 500; soluble in dichloromethane, methanol, ethanol, hydrazine, hydrazine-dimethylformamide, two Methyl sulfoxide; slightly soluble in toluene, benzene, ether at room temperature, slightly soluble in water, insoluble in petroleum ether.

The 4-methylenepyrrolidine-containing peptide deformylase inhibitor of the present invention is determined by a DRX500 nuclear magnetic resonance spectrometer. The HH sample is prepared by using deuterated chloroform as a solvent, and the repetition period is 2 seconds, and the number of scans is 32 times. The structure of the product was shown to be a peptide deformylase inhibitor containing a formylhydroxylamine derivative of 4-methylene pyrrolidine.

Compared with the background art, the beneficial effects of the present invention are:

1. The peptide deformylase (PDF) of the peptide deformylase inhibitor of the 4-methylene pyrrolidine-containing formyl hydroxylamine derivative of the present invention is a formyl metalloproteinase capable of inhibiting bacteria The process of synthesizing proteins. Therefore, it not only provides a new possibility of antibiotic treatment, but also does not affect the metabolism of eukaryotes. At the same time, studies have shown that bacteria have little chance of developing resistance to PDF inhibitors. 2. The in vitro antibacterial activity of the peptide-formylase inhibitor of the 4-methylenepyrrolidine-containing formyl hydroxylamine derivative of the present invention shows that against Staphylococcus aureus, Staphylococcus epidermidis, a resistant strain such as methicillin-resistant gold The minimum inhibitory concentration (MIC) of Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae was 0.0625~8μ _§ /ιη1, showing good antibacterial properties against Gram-positive, negative and resistant bacteria, see Table 1.

Table 1 Minimum inhibitory concentration (MIC) of drug-resistant strains (in g/ml)

Awre^ is Staphylococcus aureus, MSSA is methicillin-sensitive Staphylococcus aureus, MRSA is methicillin-resistant Staphylococcus aureus, ^ ^r^Hifo is Staphylococcus epidermidis, penicillin and linezolid are commercially available bulk drugs, purity >99%. Linezolid for control was approved for marketing in 2000, and was developed by Pfizer. detailed description

Example 3: Example 4:

Example 5: Example 6:

Example 7

Obtaining the structure of the embodiment 1 is a total of 17 steps from a~q,

The conditions of each step are as follows:

Step a) BuBr, TBAB (tetrabutylammonium bromide), K ₂ C0 _{3 ;} Step b) NaOH, H ₂ 0;

Step c) HCHO(aq), (CH ₃ CH ₂ ) ₂ H, CH ₃ CH ₂ OH; Step d) (i) 4, Et ₃ N, t-BuCOCl (pivaloyl chloride), THF, -78 ° C (ii) BuLi, THF -78 °C;

Step e) PMBCl (p-methoxybenzyl chloride), Et ₃ N, DMF (Ν, Ν-dimethylformamide); Step f) N ₂ H ₄ .H ₂ 0, DMF/CH ₃ OH; Step g ) 50 ° C, 24 h;

Step h) LiOH, H ₂ O ₂ , THF/H ₂ 0; Step i) HCOOH, Ac ₂ 0, THF;

Step j) ClCOCOCl (oxalyl chloride), DMSO (dimethyl sulfoxide), Et ₃ N, CH ₂ C1 ₂ , -78 ° C _; step k) Ph ₃ PCH ₃ Br, t-BuOK, THF; Step 1) TFA (trifluoroacetic acid), CH ₂ C1 _{2 ;}

Step m) HOBT (1-hydroxybenzotriazole), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), ΜΜ (Ν-methylmorpholine) , CH ₂ Cl ₂ , 18h; Step n) LiOH, dioxane/H ₂ 0; Step o) ClCOOCH ₂ CH ₃ , Et ₃ N, THF; Step p) CO(NH ₂ ) ₂ 'H ₂ 0 ₂ (urea hydrogen peroxide), phthalic anhydride;

Step q) TFA/CH ₂ Cl _{2 ;}

The steps for obtaining the structural formula of Example 1 are as follows:



 Step a, Synthesis of diethyl n-butylmalonate (2):

(316 g, 1.98 mol) of commercially available diethyl malonate (1), (324 g, 2.38 mol) of n-bromobutane (BuBr), (22 g, 0.06 mol) of tetrabutylammonium bromide (TBAB), 330 g, 2.38 mol) potassium carbonate was added to a 1 L single-necked flask. Heat to reflux and react for 18 h. Heating was stopped and 500 ml of water was added to dissolve the solids. Add 200 ml of EA and dispense. The organic phase was dried over anhydrous sodium sulfate, suction filtered, dried, and then evaporated to dryness (eluent column) to obtain diethyl butyl malonate (2) 200 g. The yield was 47%.

¹ H NMR (500 MHz, CDCl ₃ ) δ 4.17-4.22 (m, 4H), 3.30-3.32 (m, lH), 1.87- 1.92 (m, 2H), 1.28-1.40 (m, 4H), 1.25-1.28 (t , J=7Hz, 6H), 0.89-0.92 (t, J=7Hz, 3H).

Step b, Synthesis of n-butylmalonic acid (3):

(120 g, 3 moi;) NaOH was added to a 500 mL single-necked flask and dissolved in 250 ml of water. The product (2) of the step a was added dropwise (118 g, 0.546 mol). After the dropwise addition was completed, the mixture was heated to reflux, and the reaction was stirred until clarified, and the oil-free layer was left for about 3 hours. Stop heating, add concentrated hydrochloric acid to the pH = 3~4. (A large amount of white solid precipitated) was filtered, and the filtrate was extracted twice with 200 ml of methyl ether. After drying over anhydrous sodium sulfate, it was dried and evaporated to dryness eluting with EtOAc EtOAc EtOAc EtOAc. The yield is 90%.

¹ H NMR (500 MHz , CDC1 ₃ ) δ 7.85-11.87 (br.s, 2H), 3.42-3.46 (t, J = 7 Hz, 1H), 1.94-1.98 (m, 2H), 1.35-1.41 (m, 4H) , 0.89-0.94 (t, J=7Hz, 3H).

Step c, Synthesis of 2-butylacrylic acid (4):

To a 2 L three-necked flask was added (78 g, 0.487 mol) of the product obtained in the step b (3), diethylamine (75 ml, 0.73 lmol), a mass concentration of 37% formaldehyde (78 ml, 0.974 mol), 1.5 L of ethanol. Heat to reflux and react for 16 h. The solvent was spun dry, and 4 mol/L of HC1 was added to mediate PH=3~4. It was extracted twice with 400 ml of methyl butyl ether. The organic layer was combined and dried over anhydrous sodium sulfate The yield was 84%.

1HNMR (500MHz, CDC1 ₃ ) δ 8.16~11.98(br.s,lH), 6.29(s,lH), 5.65 (s,lH), 2.30-2.33(t,J=8Hz,2H), 1.46-1.52( m, 2H), 1.34-1.4 (m, 2H), 0.92-0.95 (t, J = 7 Hz, 3H). Step d, (4S)-3-(2-Methylenehexanoyl)-4-benzyl Synthesis of 2-oxazolidone (5):

Add 90 mL of dry THF to a 250 mL dry three-necked flask equipped with a low temperature thermometer and nitrogen atmosphere, then add (3.9 g, 30.5 mmol) of the product from step c (4 chilled to -78 °. Add (5.5 mL, 38.0 mmol) Triethylamine, then slowly add C3.8mL, 31.7mmo) l pivaloyl chloride, control the drop rate, and keep the temperature of the reaction system below -60 degrees. The reaction system was reacted at -78 °C for 30 min and raised to room temperature. 2hr, then cool down to -78 degrees, stand by. Another 250mL single-necked flask, low temperature thermometer, nitrogen protection. Add 90 mL of anhydrous tetrahydrofuran, add (4.9 g, 27.6 mmoi; > (S)-4-benzyl-2-oxazolidinone, and cool to -78 °. Add 13.2 mL of 2.5 mol/L n-butyllithium. The drip acceleration was controlled, and the reaction was allowed to rise to room temperature for 30 min. The solution in the second reaction flask was added dropwise to the No. 1 reaction flask which had been cooled to -78 ° C. After the completion of the dropwise addition, the temperature was raised to room temperature and allowed to react overnight. The reaction was quenched by adding 40 mL of a 1 mol/L solution of KHC03. The majority of the tetrahydrofuran was removed by rotary evaporation, and 100 mL of ethyl acetate and 100 mL of water were added, and the organic phase was washed with 50 mL of brine, dried over anhydrous sodium sulfate and filtered. Dry, 7.8 g of an orange oil (yield: EtOAc (EtOAc: EtOAc): 4S)-3-(2-Methylenehexanoyl)-4-benzyl-2-oxazolidinone (5) 5.5 g, yield 63%.

¹ H NMR (500MHz, CDCl ₃ ) δ 7.29 (m, 5H), 5.40 (d, J = 7 Hz, 2H), 4.44 (m, lH), 4.22 (m, 2H), 3.37 (m, 1H), 2.82 (m, 1H), 2.39 (m, 2H), 1.43 (m, 4H), 0.93 (m, 3H).

Step e, Synthesis of N-((4-methoxy)benzyloxy)phthalimide (7):

To a 500 ml three-necked flask was added (26 g, 0.16 mol) of N-hydroxyphthalimide (6) and (25 g, 0.16 mol) of p-methoxybenzyl chloride (PMBC1). (53 ml, 0.37 mol) of triethylamine ( Et ₃ N) and 350 ml of DMF were added to give a dark red solution. Heat to 90 ° C, the reaction 40mins ~ lh. TLC monitoring. The reaction solution was poured into 1 L of ice water and stirred well for 15 mins. After suction filtration, the filter cake was washed with 400 ml of ice water. The obtained solid was air-dried at 60 ° C overnight to give a pale yellow solid, N-((4-methoxy)benzyloxy)phthalimide (7), 30 g, yield 68%.

lHNMR (CDC13, 500 MHz): δ 7.70-7.73 (m, 4H), 7.44 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 5.14 (s, 2H), 3.79 (s, 3H).

Step f, Synthesis of (4-methoxy)benzylhydroxylamine (8):

(30 g, 0.106 mol) of the product of step e (7), 140 ml of DMF and 300 ml of MeOH were placed in a 1 L three-necked flask to give a suspension. Heat to 60 ° C, completely dissolved in a pale yellow solution, dropwise (16ml, 0.330mol) N ₂ H ₄ .H ₂ O o lOmins, cooled to room temperature, solid appeared, added 100ml H ₂ O, floc suspension liquid. The MeOH was removed by rotary evaporation. It was extracted four times with 150 ml of ethyl acetate, dried over anhydrous Na ₂ SO ₄ , filtered, and evaporated to dryness. After adding 10 ml of MeOH, 6 ml of concentrated HCl was added dropwise, and a white solid was precipitated, which was filtered, and washed with a small ethyl acetate to give 15 g of PMBO H2.HCl. The obtained solid was dissolved in (8.45 g, 0.08 mol) of Na ₂ CO ₃ and 100 ml of aqueous solution, and extracted twice with 100 ml of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated. Viscous liquid (4-methoxy)benzylhydroxylamine (8), The yield was 75%.

1HNMR (CDC13, 500 MHz): δ 7.29-7.30 (d, J = 8.5 Hz, 2H), 6.89-6.91 (d, J = 8.5 Hz, 2H), 5.34 (s, 2H), 4.62 (s, 2H) , 3.81(s,3H).

Step g, Synthesis of (4S)-3-((2R)-3-p-methoxybenzyloxyamino-2-butylpropanoyl)-4-benzyl-2-oxazolone (9):

A lOOmL single-necked flask equipped with a condensing tube is protected by nitrogen. (7.8 g, 27.1 mmol) of the product (5) obtained in the step d and (10 g, 64.6 mmol) of the product (8) of the step f, warmed to 50 °, and reacted for 24 hr. After the reaction was completed, it was cooled to room temperature, diluted with 32 mL of ethyl acetate, and then added (20.9 g, 121.1 mmol) of p-toluenesulfonic acid dissolved in 26 mL of ethyl acetate solution, stirred for 1.5 hr, suction filtered, filter cake with a small amount of ethyl acetate wash. The organic phase was dried, 185 mL of butyl ether, EtOAc (EtOAc) EtOAc EtOAc (EtOAc)EtOAc. After dissolution, C0.81 g, 7.6 mmol of sodium carbonate was added and dissolved in 15 mL of water, and the reaction was stirred for 15 min. The liquid phase was extracted once with 15 mL of ethyl acetate. The organic phase was combined, washed with 15 mL of water, dried, filtered, and evaporated to give a colorless oil (4S)-3-((2R)-3-p-methoxy Benzobenzylamino-2-butylpropanoyl)-4-benzyl-2-oxazolidinone (9; >6.4 g. Yield: 58%.

1H NMR (CDC1 ₃ , 500 MHz): δ 7.23-7.32 (m, 5H), 7.17-7.19 (d, J = 8.5, 2H), 6.80-6.82 (d, J = 8.5, 2H), 5.75 (br, lH) ), 4.58-4.63 (m, 3H), 4.07-4.14 (m, 3H), 3.70 (s, 3H), 3.32-3.36 (m, lH), 3.12-3.20 (m, 2H), 2.38-2.43 (m , lH), 1.63-1.75 (m, lH), 1.35- 1.55 (m, lH), 1.26-1.3 l (m, 4H), 0.87-0.89 (t, J = 7 Hz, 3H).

Step h, Synthesis of (2R)-2-((4-methoxybenzyloxyamino)methyl)hexanoic acid (10):

In a 100 mL single-necked flask, (4.3 g, 9.8 mmoi; > product of step g (9) was dissolved in 26 mL of tetrahydrofuran and 6 mL of water, cooled to 0 °, slowly added (6.9 mL, 22.6 mmol 0% hydrogen peroxide and 9.5 mL1) .2mol/l lithium hydroxide solution, stirring for 1 hr under ice bath. Add 35 mL of 1 mol/1 sodium sulfite solution to the system and raise to room temperature for 30 min. The organic solvent was spun dry, and the aqueous phase was extracted 6 times with 10 mL of ethyl acetate. The aqueous phase was adjusted to pH 3-4 with 1 mol/1 hydrochloric acid, and extracted twice with 20 mL of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and filtered and evaporated to give the product (2R)-2- ((4-Methoxybenzyloxyamino)methyl)hexanoic acid (10) lg, Yield: 34%.

^{_{! HNMR (CDC1 3, 500 MHz}} ): δ 7.26-7.28 (d, J = 7Hz, 2H), 6.87-6.88 (d, J = 7Hz, 2H), 4.62-4.68 (m, 2H), 3.80 (s, 3H), 3.06-3.14(m,2H), 2.71-2.72(m,lH), 1.66-1.70(m,lH), 1.49-1.53(m,lH), 1.26-1.33(m,4H), 0.88- 0.91 (t, J = 7 Hz, 3H).

Step i, Synthesis of (2R)-2-(((4-methoxybenzyloxy)formylamino)methyl)hexanoic acid (11): A 500 mL single-necked flask was charged with 250 mL of anhydrous tetrahydrofuran under nitrogen atmosphere, and then C10.4 mL, 108 mmol) of acetic anhydride and P (25 mL, 540 mmol) of formic acid were added, and the mixture was heated to 50 °C for 2 hr, and then cooled to 0 °C. A solution of the product (10) of the step h and 25 mL of tetrahydrofuran was slowly added dropwise to the above system, and the dropping rate was controlled, and the mixture was dropped in 30 minutes, and the reaction was carried out for 30 minutes. The solvent was added to dryness, and 50 mL of dichloromethane and 50 mL of water was added. The organic phase was washed twice with 50 mL of brine, dried over anhydrous sodium sulfate and The crude product was purified by column chromatography (developing solvent: EtOAc _: EA=3:1~2:1) to give product (2R)-2-(((4-methoxybenzyloxy)carboxamido) Methyl)hexanoic acid (ll) 1.0 g. The yield was 60%. ^l HNMR (CDC1 ₃ , 500 MHz): δ 8.09 (br, lH), 7.26-7.35 (d, J = 8.5 Hz, 2H), 6.88-6.90 (d, J = 8.5 Hz, 2H), 4.72-4.76 ( m, 2H), 3.81 (s, 3H), 3.79-3.82 (m, 2H), 2.73-2.76 (m, lH) 1.56-1.66 (m, lH) 1.43-1.53 (m, 1H), 1.25-1.40 ( m, 4H), 0.87-0.90 (t, J=7Hz, 3H).

Step j, Synthesis of (2S)-N-tert-butoxycarbonyl-4-oxoproline methyl ester (13):

250 ml three-necked flask, add (9.5 ml, 133.97 mmol) dimethyl sulfoxide in 100 ml of dichloromethane, cool to -78 °C, add (9.0 ml, 102.7 mmol) oxalyl chloride, stir the reaction for 10 mins, maintain -78 ° , adding 20.0 g, 76.2 mmol) of the product of step i (12;) in 120 ml of dichloromethane (control temperature is not higher than -60 °;), adding -78 degree reaction for 2 h, -78 ° Add (28ml, 194mmoi;> triethylamine, naturally add to room temperature after adding dropwise, and quench the reaction by adding 60 ml of water. The organic phase is separated, the aqueous phase is extracted with 2×50 ml of dichloromethane, and the organic phase is combined and washed with saturated sodium chloride. Once, it was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (ethyl acetate: petroleum ether = 4:1) to give 18 g (2S)-N-tert-butoxycarbonyl-4-oxo- 13) 18 g, yield 91%.

1HNMR (CDC1 ₃ , 500 MHz): δ 4.69-4.81 (dd, J=9.7, 9.7 Hz, lH,), 3.88-3.90 (d, J = 15.4 Hz, 2H), 3.75 (s, lH), 2.88- 2.98(m, lH), 2.55-2.59(m, lH), 1.45, 1.47(2s,9H). Step k, (2S)-4-Methylenepyrrole-1,2-dicarbonate-1-tert Synthesis of butyl ester 2-methyl ester (14):

In a 50 ml three-necked flask, add (2.1 g, 6 mmol) of methyltriphenylphosphonium bromide, N ₂ protection, add 20 ml of anhydrous tetrahydrofuran solution, cool to 0 ° with ice bath, and add (0.71 g, 6.3 mmol) A solution of potassium butoxide in 10 ml of anhydrous tetrahydrofuran was allowed to react to room temperature for 1 h, cooled to 0 ° with ice-cooling, and then added (0.73 g, 3 mmol) of the product of step j (13), and allowed to react to room temperature, and the reaction was completed by TLC. It takes 3 hours. To the reaction system, 30 ml of water was added, and the tetrahydrofuran was removed by rotary evaporation, and extracted with 3×20 ml of ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, filtered, and the filtrate was concentrated and purified by column chromatography (mobile phase ethyl acetate: petroleum ether = 5: 1) 1.3 g of (2S)-4-methylenepyrrolidine-1,2-dicarbonate-1-tert-butyl ester 2-methyl ester (14) was obtained in a yield of 63%.

lHNMR (CDCI3, 500 MHz): δ 5.00 (m, 2H), 4.39-4.52 (m, lH), 4.06, 4.10 (2s, 2H), 3.73-3.74 (2s, 3H), 2.98 (m, lH), 2.65 (m, lH), 1.43, 1.48 (2s, 9H).

Step 1, (2S) -4-methylenepyrrole-2-methylcarbonate (15) Synthesis:

100 ml of a single-necked flask, adding (3.7 g, 16.3 mmol) of the product (14) obtained in step k, dissolved in 40 ml of dichloromethane, iced, dropwise (20 ml, 270 mmol) trifluoroacetic acid (TFA), dropwise The reaction was carried out at room temperature for 30 mins. The reaction solution was spun dry, and the pH was adjusted to 7-8 with triethylamine. The crude product (2S)-4-methylenepyrrolidine-2-methyl carbonate (15) was directly charged to the next step, yield was 100%.

Step m, (2S)-l-((2R)-2-(((4-methoxybenzyloxy)formylamino)methyl)hexanoyl)-4-methylenepyrrole-2-carboxylate Synthesis of methyl ester (16):

In a 50 ml three-necked flask, (0.6 g, 4.4 mmol) of 1-hydroxybenzotriazole (HOBT), (0.84 g, 4.4 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbamate The imine hydrochloride (EDCI), nitrogen-protected, was added (1.23 g, 3.96 mmol) to a solution of the product (11) obtained in step i and 10 ml of anhydrous dichloromethane. Add (0.97 ml, 8.8 mmol) of N-methylmorpholine (NMM), add a solution of the crude product of product (15) obtained from (1 g, 4.4 mmol) Step 1 and 5 ml of anhydrous dichloromethane. Overnight reaction. The reaction solution was washed once with 10 ml of a saturated aqueous solution of citric acid, and then washed once with 10 ml of saturated sodium carbonate. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give the product (2S)-l-((2R)-2 -((4-Methoxybenzyloxy;)formylamino)methyl)hexanoyl)-4-methylenepyrrolidin-2-methyl carbonate (16), yield 85%.

lHNMR (CDC1 ₃ , 500 MHz): δ 7.86, 8.07 (s, lH), 7.24-7.25 (d, J = 5 Hz, 2H), 6.88-6.89 (d, J = 5 Hz, 2H), 5.06-5.10 (m , 2H), 4.86-4.88 (m, lH), 4.48-4.50 (m, lH), 4.21-4.24 (m, lH), 3.83-3.88 (m, lH), 3.81 (s, 5H), 3.72 (s , 3H), 3.43-3.46(m, lH), 3.16-3.18(m, lH), 2.81-2.93(m,2H), 1.60-1.75 (m, lH), 1.42-1.58(m, lH), 1.20 -1.42 (m, 4H), 0.89-0.92 (t, J = 7 Hz, 3H).

Step _n , (2S)-l-((2R)-2-(((4-methoxybenzyloxy)formylamino)methyl)hexanoyl)-4-methylenepyrrole-2-carboxylate Synthesis of acid (17):

To a 50 ml single-necked flask was added (1.4 g, 3.4 mmol) of the product obtained in the step m (16), dissolved in 15 ml of tetrahydrofuran, and then added (0.14 g, 3.4 mmol) of a 15 ml aqueous solution of lithium hydroxide hydrate. After reacting at room temperature for 1 h, the reaction solution was extracted with 20 ml of ethyl acetate. The aqueous phase was adjusted to pH 4 to 5 with a saturated aqueous solution of citric acid, and then extracted twice with 20 ml of ethyl acetate, dried over anhydrous sodium sulfate and filtered. Dry, 1.35 g of product (2S)-l-((2R)-2-(((4-methoxybenzyloxy))carboxamido;)methyl)hexanoyl)-4-methylenepyrrole Alkane-2-carboxylic acid (17), yield 100%.

lHNMR (CDCI3, 500 MHz): δ 7.89, 7.92 (s, lH), 7.23-7.24 (d, J = 7 Hz, 2H), 6.88-6.89 (d, J = 7 Hz, 2H), 5.06-5.10 (m, 2H), 4.86-4.88 (m, lH), 4.48-4.50 (m, lH), 4.21-4.24 (m, lH), 3.83-3.88 (m, lH), 3.81 (s, 5H), 3.43-3.46 (m, lH), 3.16-3.18 (m, lH), 2.81-2.93 (m, 2H), 1.60 -1.75 (m, lH), 1.48-1.62 (m, lH), 1.20-1.30 (m, 4H), 0.86-0.89 (t, J = 7 Hz, 3H).

Step o, (2S)-N-(5-fluoro-2-pyridine)-l-((2R)-2-(((4-methoxybenzyloxy)formylamino)methyl)hexanoyl) Synthesis of -4-methylenepyrrole-2-amide (18):

A 50 ml three-necked flask was added with a solution of the product (17) obtained in the step n and 10 ml of anhydrous tetrahydrofuran under a nitrogen atmosphere, ice-cooled to 0 ° C, and added (0.36 ml, 2.5 mmol). Triethylamine was added dropwise (0.24 ml, 2.5 mmol) of ethyl chloroformate (C1C00CH ₂ CH ₃ ). A solution of (0.34 g, 3 mmol) of 2-amino-5-fluoropyridine in 5 ml of anhydrous tetrahydrofuran was added, and the mixture was reacted at room temperature overnight, and the reaction mixture was dried, diluted with 15 ml of ethyl acetate, and washed with 10 ml of saturated aqueous citric acid and then 10 ml. The organic phase was washed with anhydrous sodium sulfate, suction filtered, and dried to give the product (2S)-N-(5-fluoro-2-pyridine)-l-((2R)-2- (((4-Methoxybenzyloxy)formylamino)methyl)hexanoyl)-2,5-dihydropyrrole-2-amide (18), yield 81%.

1HNMR (CDC1 ₃ , 500 MHz): δ 8.36-8.50 (m, lH), 8.14-8.17 (m, lH), 7.86-8.07 (d, lH), 7.37-7.42 (m, lH), 7.24-7.25 ( d, J=5 Hz, 2H), 6.88-6.89 (d, J=5 Hz, 2H), 5.06-5.10 (m, 2H), 4.86-4.88 (m, lH), 4.48-4.50 (m, lH), 4.21 -4.24 (m,lH), 3.83-3.88(m,lH), 3.81(s,5H), 3.43-3.46(m,lH), 3.16-3.18(m,lH), 2.81-2.93(m,2H) , 1.60-1.75(m,lH),

1.42- 1.58 (m, lH), 1.20-1.42 (m, 4H), 0.89-0.92 (t, J = 7 Hz, 3H).

Step p, (2S)-N-(5-fluoro-1-oxo-2-pyridine)-l-((2R)-2-(((4-methoxybenzyloxy)formylamino)methyl Synthesis of hexanoyl)-4-methylenepyrrole-2-amide (19):

The product (18) obtained in the step o was added to a 25 ml single-necked flask, dissolved in 15 ml of ethyl acetate, and then added (0.57 g, 6 mm oi; > urea hydrogen peroxide complex (CO (NH) ₂ ;) H ₂ 0 ₂ ;), adding (0.89 g, 6 mmol) of phthalic anhydride in three portions, reacting at room temperature overnight, and quenching with a solution of (1.24 g, 10 mmol) of sodium sulfite in 10 ml, partition, organic The phase was washed once more with 10 ml of a saturated aqueous solution of sodium carbonate, and then dried over anhydrous sodium sulfate, filtered, and evaporated to give the compound (2.sup.. -l-((2R)-2-(((4-methoxybenzyloxy))carboxamido;)methyl)hexanoyl)-4-methylenepyrrolidin-2-amide (19), The yield was 90%.

1HNMR (CDC1 ₃ , 500 MHz): 58.36-8.50 (m, lH), 8.14-8.17 (m, lH), 7.86-8.07 (d, lH), 7.24-7.25 (d, J = 5 Hz, 2H), 7.10 -7.20(m,lH), 6.88-6.89 (d,J=5Hz,2H), 5.06-5.10(m,2H), 4.86-4.88(m,lH), 4.48-4.50 (m,lH),4.21- 4.24 (m,lH), 3.83-3.88(m,lH), 3.81(s,5H),

3.43- 3.46(m,lH), 3.16-3.18(m,lH), 2.81-2.93(m,2H), 1.60-1.75(m,lH), 1.42-1.58 (m, lH), 1.20-1.42 (m, 4H), 0.89-0.92 (t, J = 7 Hz, 3H).

Step q, (2S)-N-(5-fluoro-1-oxo-2-pyridine)-l-((2R)-2-((formylhydroxylamino)methyl)hexanoyl)-4-methyl Synthesis of pyryrrolidine-2-amide (20):

A solution of the product (19) obtained in the step p and 10 ml of dichloromethane was added to a 25 ml single-necked flask, and (35 ml, 67.5 mmol) of trifluoroacetic acid (TFA) was added dropwise, and the reaction was carried out at room temperature after completion of dropwise addition. Lh. The reaction solution was spun dry, diluted with 10 ml of dichloromethane, and then adjusted to a water phase with a saturated aqueous sodium carbonate. The mixture was separated and the aqueous phase was extracted with 10 ml of dichloromethane. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. g final product C2S)-N-0 fluoro-1-oxo-2-pyridine)-1 -((2R)-2-((formylhydroxylamino)methyl)hexanoyl)-4-methylenepyrrolidine 2-amide (20), yield 42%.

1HNMR (CDC1 ₃ , 500 MHz): δ 10.6 (s, lH), 8.42-8.46 (m, lH), 8.22 (m, lH), 7.70 (s, lH), 7.12-7.14 (m, lH), 5.10 (m, 3H), 4.60-4.63 (d, J = 13.5 Hz, lH), 4.20-4.23 (d, J = 13.5 Hz, lH), 3.91-3.96 (m, lH), 3.40-3.43 (d, J =11.5 Hz, lH), 3.15-3.16 (m, lH), 2.83-2.99 (m, 2H), 1.66-1.68 (m, lH), 1.47-1.50 (m, lH), 1.25-1.36 (m, 4H) ), 0.87-0.89 (t, J = 7Hz, 3H).

All raw materials and solvents involved in the above process are commercially available reagent grades.

Example 2

In addition to step o, the starting material was replaced by 2-amino-5-fluoropyridine to 2-amino-5methylthiazole. Other conditions were unchanged, and the product (2S)-N-(5-methyl-2-thiazole; )-l-((2R)-2-(((4-methoxybenzyloxy)formylamino)) Methyl)hexanoyl)-4-methylenepyrrolidine-2-amide. Then directly proceed to step q to obtain the final product (2S)-N-(5-methyl-2-thiazole)-WPR^-((formylhydroxylamino)methyl)hexanoyl)-4-methylenepyrrolidine- 2-amide. The rest is the same as in the first embodiment.

_{lHNMR (CDC1 3, 500 MHz)} : δ 10.5 (brs, lH), 7.68 (s, lH), 5.64 (brs, lH), 4.97-5.10 (m, 3H), 4.78 (m, lH), 4.23-4.26 (m, lH), 4.06-4.11 (m, lH), 3.40-3.42 (m, lH), 3.12-3.14 (m, 2H), 2.59-2.62 (m, lH), 2.27 (s, 3H), 1.91 (m,lH), 1.27-1.5 l(m,5H), 0.89-0.99(m,3H).

Example 3

In addition to step o, (2S)-N-(5-fluoro-2-pyridine;)-l 2R)-2-W4-methoxybenzyloxy;)formylamino;)methyl)hexanoyl)- After 4-methylenepyrrolidin-2-amide (18), without step p, step q is directly carried out to obtain the final product (2S)-N-(5-fluoro-2-pyridine)-1-((2R) )-2-((Formylhydroxylamino)methyl)hexanoyl)-4-methylenepyrrolidin-2-amide. The rest is the same as in the first embodiment.

1HNMR (CDC1 ₃ , 500 MHz): δ 9.82 (s, lH), 8.18-8.20 (m, lH), 8.07 (m, lH), 7.78 (s, lH), 7.39-7.41 (m, lH), 5.07-5.09 (d, J = 9.2 Hz „2H), 4.96 (m, lH), 4.52-4.55 (m, lH),

4.23-4.726(m,lH), 3.90-3.95(m,lH),3.43-3.45(m,lH), 3.19-3.20(m,lH),

2.88-2.90 (m, 2H), 1.67 (m, lH), 1.50 (m, lH), 1.29-1.32 (m, 4H), 0.84-0.87 (m, 3H).

Example 4

In addition to step o, the starting material was replaced by 2-amino-5-fluoropyridine to aniline. Other conditions were unchanged, and the product (2S)-N-phenyl-1-((2R)-2-(((4-methoxybenzyloxy))carboxamido;)methyl)hexanoyl)- 4-methylene pyrrolidine-2-amide. Then directly proceed to step q to obtain the final product (2S)-N-phenyl-l-((2R)-2-((formylhydroxylamino)methyl)hexanoyl)-4-methylenepyrrolidine-2- Amide. The rest is the same as in the first embodiment.

lHNMR (CDC1 ₃ , 500 MHz): δ 9.19 (s, lH), 7.76 (s, lH), 7.47-7.49 (m, 2H), 7.21-7.24 (m, 2H), 7.02-7.05 (m, lH) , 5.08-5.13 (m, 2H), 4.91-4.93 (m, lH), 4.51-4.54 (m, lH), 4.20-4.23 (m, lH), 3.86-3.91 (m, lH), 3.44-3.46 ( m,lH), 3.18-3.19(m,lH), 2.94-3.00(m,lH), 2.84(m,lH), 1.64-1.67(m,lH), 1.48-1.52(m,lH), 1.23- 1.32 (m, 4H), 0.82-0.85 (t, J = 6.7 Hz, 3H).

Example 5

In addition to step o, the starting material was replaced by 2-amino-5-fluoropyridine to 4-fluoro-aniline. Other conditions were unchanged, and the product (2S)-N-(4-fluoro-phenyl;)-l-((2R)-2-(((4-methoxybenzyloxy))carboxamido;) Methyl)hexanoyl)-4-methylenepyrrolidine-2-amide. Then directly proceed to step q to obtain the final product (2S)-N-(4-fluoro-phenyl;)-1-((2R)-2-((formylhydroxylamino))methyl)hexanoyl)-4- Methylene pyrrolidine-2-amide. The rest is the same as in the first embodiment.

lHNMR (CDCI3, 500 MHz): δ 9.32 (s, lH), 7.74 (s, lH), 7.36-7.39 (m, 2H),

6.84-6.87(t, J=8.4Hz„2H), 5.06-5.10(d, J=19Hz, 2H), 4.87-4.88(m,lH),

4.48-4.50(m,lH), 4.21-4.24(m,lH), 3.83-3.88(m,lH), 3.43-3.46(m,lH),

3.16-3.18(m,lH), 2.81-2.93(m,2H), 1.63-1.67(m,lH), 1.47-1.5 l(m,lH),

1.25-1.3 l(m,4H), 0.82-0.85 (t, J=6.75Hz, 3H).

Example 6

In addition to step o, the starting material was replaced by 2-amino-5-fluoropyridine to morpholine. Other conditions were unchanged, and the product (2S)-morpholine-1-((2R)-2-(((4-methoxybenzyloxy))carboxamido;)methyl)hexanoyl)-4- Methylene pyrrolidine-2-amide. Then, step q is directly carried out to obtain the final product (2S)-morpholine-1-((2R)-2-((formylhydroxylamino)methyl)hexanoyl)-4-methylenepyrrolidin-2-amide. The rest is the same as in the first embodiment.

1HNMR (CDC1 ₃ , 500 MHz): δ 7.70 (s, lH), 5.06-5.10 (m, 3H), 4.43-4.46 (m, lH), 4.30-4.32 (m, lH), 3.84-3.87 (m, lH), 3.42-3.78(m,10H), 3.18(m,lH), 2.92-2.94(m,2H), 1.60-1.75(m,lH), 1.25-1.46(m,5H), 0.90-0.93( t, J = 6.2 Hz, 3H). 91

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Claims

Claim

A peptide formylase inhibitor comprising 4-methylene pyrrolidine, characterized in that the peptide deformylase inhibitor has the following structure: