CN116808229A - Polyethylene glycol-nemaltevir prodrug as well as preparation method and application thereof - Google Patents

Polyethylene glycol-nemaltevir prodrug as well as preparation method and application thereof Download PDF

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CN116808229A
CN116808229A CN202210284778.4A CN202210284778A CN116808229A CN 116808229 A CN116808229 A CN 116808229A CN 202210284778 A CN202210284778 A CN 202210284778A CN 116808229 A CN116808229 A CN 116808229A
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reaction
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polyethylene glycol
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张径
卓林胜
肖凯
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Changsha Innovation Pharmaceutical Industrial Technology Research Institute Co ltd
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Changsha Innovation Pharmaceutical Industrial Technology Research Institute Co ltd
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Abstract

The invention relates to a polyethylene glycol-Nemactetvir prodrug, and a preparation method and application thereof, belonging to the technical field of biological pharmacy. The polyethylene glycol-Nemactetvir prodrug does not need to be taken together with the CYP3A4 inhibitor ritonavir, reduces the risk of drug-drug interaction, is beneficial to the crowd who has chronic diseases and needs to take other drugs, provides a new drug selection, and has good application prospect. The preparation method of the polyethylene glycol-nemaltevir prodrug compound I has the advantages of mild reaction conditions, high yield, simple and convenient operation and environmental friendliness.

Description

Polyethylene glycol-nemaltevir prodrug as well as preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a polyethylene glycol-nemaltevir prodrug, and a preparation method and application thereof.
Background
Nirmatrelvir/Ritonavir is a complex formulation developed by pyroxene and consists of 2 tablets of Nirmatrelvir (PF-07321332) and 1 tablet of Ritonavir, trade name Paxlovid. Nirmatrelvir blocks viral replication by blocking SARS-CoV-2-3CL protease activity, ritonavir helps slow down Nirmatrelvir metabolism. The 3CL protease is the main protease of coronaviruses and plays a critical role in the translation of polyproteins.
Polyethylene glycol (PEG) is a synthetic polymer with excellent biocompatibility and has been approved for clinical use by the us FDA. Polyethylene glycol has good solubility in water and most organic solvents, is widely applied to surface modification of various drug carriers, so as to avoid conditioning effects of macrophages and immune systems in vivo on the drug carriers, improve the circulation time in vivo, prolong the half-life period of the drug, increase accumulation of the drug at tumor positions through EPR (Enhanced Permeability and Retention) effect, exert passive targeting effect and improve the treatment effect. At present, there are multiple PEG-modified small molecule drugs on the market or into clinical stages such as PEG-naloxone, PEG-irinotecan, and PEG-paclitaxel, etc. (org. Process Res. Dev.2020,24, 1364-1372).
Prodrugs refer to compounds that are less or inactive in vitro and which release the active substance in vivo by enzymatic or non-enzymatic action to exert their pharmacological effect, often referred to as novel chemical entities, also called prodrugs, by covalent bonding of the active drug, i.e., the prodrug, to some non-toxic compound. The prodrug has the advantages of improving the physical and chemical properties of the original drug, improving the selectivity of the drug on a target site, improving the absorption, distribution, transportation, metabolism and other pharmacokinetic processes of the drug in vivo, prolonging the acting time, improving the bioavailability, eliminating bad smell, reducing the toxic and side effects of the drug and the like, and particularly, the antitumor prodrug obtained by combining the prodrug with tumor treatment is designed, thereby greatly improving the defects of large toxicity, no selectivity, low bioavailability and the like of most chemotherapeutic drugs (Eur.J.Pharm.Biopharm.2021, 165, 219).
Small molecule drugs are generally less directly linked to PEG, but rather are linked by various types of linker arms. The proper connecting arm is selected to achieve the purposes of slow release, targeted release, drug loading increase and the like. Typical linker arms include hydrazides, amino acids, polypeptides, and N-Mannich bases. According to the design concept of the prodrug, the invention carries out drug design aiming at the structural transformation site of the nemaltevir.
In the prior art, polyethylene glycol-nemaltevir prodrug compounds, their preparation, drug release behavior and anti-novel coronavirus activity (anti-SARS-CoV-2) are not disclosed.
Therefore, research on polyethylene glycol-Nemactetvir prodrug compounds and a preparation method thereof is still needed, a new drug selection is provided, and the application prospect is good.
Disclosure of Invention
Aiming at the technical problems that the ritonavir needs to be taken together with the CYP3A4 inhibitor ritonavir, the risk of drug-drug interaction is increased, chronic diseases are not benefited, and people needing to take other drugs are not benefited, the invention provides a novel polyethylene glycol-Nemacetavir prodrug compound and a preparation method thereof according to the design concept of a prodrug, the prodrug does not need to be taken together with the CYP3A4 inhibitor ritonavir, the risk of drug-drug interaction is reduced, the crowd who is benefited for chronic diseases and needs to take other drugs is benefited, and the prodrug and the preparation method thereof have good application prospects.
To solve the above technical problems and achieve the above object, a first aspect of the present invention provides a novel polyethylene glycol-nemaltevir prodrug compound, or a pharmaceutically acceptable salt thereof, having a structure represented by formula I:
wherein R is 1 ，R 2 ，R 3 Each independently is hydrogen or a PEG modifier, and R 1 ，R 2 ，R 3 Are not hydrogen at the same time;
the PEG modifier is L-D; wherein L is a linking group, D is a linear, Y-type or multi-branched polyethylene glycol;
or a pharmaceutically acceptable salt thereof.
In some embodiments, L is a linking group selected from:
wherein R is 1-5 、R 10 、R 19 、R 24 、R 25 、R 30 And R is 31 Independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-6 alkenyl, C6-12 aryl, C6-12 aralkyl;
R 6-9 、R 11-18 、R 20-23 、R 26-29 independently selected from: -H, -F, -Cl, -Br, -I, nitro, amino, hydroxy, mercapto, cyano, alkynyl, alkenyl, C1-C10 acyloxy, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 alkyl substituted amino;
x is independently selected from:
n and m are each independently integers of 1 to 30.
In some embodiments, wherein D is a linear, Y-type, or multi-branched polyethylene glycol;
the linear polyethylene glycol has a structure shown in a general formula IIa or IIb:
wherein p and q are each independently integers of 1 to 500.
The Y-type polyethylene glycol has a structure shown in a general formula IIIa or IIIb:
wherein i and h are each independently integers of 1 to 500.
The multi-branched polyethylene glycol has a structure shown in a general formula IVa, IVb, IVc or IVd:
wherein k and y are each independently integers of 1 to 500;
j. each w is independently an integer of 1 to 10.
In some embodiments, the compound is selected from:
or a pharmaceutically acceptable salt thereof.
An embodiment relates to a pharmaceutical composition comprising a compound of any one of embodiments of formulas Ia-1 to Ia-25, formulas Ib-1 to Ib-5, or formulas Ic-1 to Ic-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
An embodiment is directed to a pharmaceutical composition comprising a compound of any one of embodiments of formulas Ia-1 to Ia-25, formulas Ib-1 to Ib-5, or formulas Ic-1 to Ic-4, or a pharmaceutically acceptable salt thereof, and an inhibitor of CYP3A4 for use in the treatment of a disease associated with a viral infection. In some embodiments, it is used to treat new coronatine. In another aspect of the invention, a method for preparing the polyethylene glycol-nemaltevir prodrug compound I is provided.
In general, the compounds described herein may be prepared by methods known in the chemical arts, particularly in view of the description contained herein. Certain methods for preparing the compounds described herein are provided as additional features of embodiments and exemplified in the reaction schemes and experimental sections provided below.
Variables in schemes a through C have the same meaning as defined herein unless otherwise indicated. Scheme a:
as exemplified in scheme a,obtaining a PEG derivative L-D from a linear, Y-type or multi-branched polyethylene glycol D (which may be obtained from commercial sources) reacted with a linking group L; PEG derivative L-D reacts with thionyl chloride to obtain D-L-Cl, then is coupled with intermediate M-3 under the action of strong alkali, and the compound Ia is obtained after hydrolysis, coupling and substitution reaction. Wherein intermediate M-3 can be prepared from a suitable commercial starting material using methods known in the chemical arts. Reference is made in detail to embodiments herein.
Compared with the prior art, the preparation method of the compound Ia has the following advantages: 1) Raw materialsIs cheap and easy to obtain; 2) The synthetic route is short, and four steps of reactions are performed; 3) The reaction condition is mild, and the reaction can be carried out at room temperature; 4) Post-treatment does not need column chromatography purification, and the product with high yield and high purity can be obtained through conventional filtration and drying, thereby being convenient and quick; 5) The substrate has strong adaptability; 6) The yield is higher, the yield of most steps is more than 95%, and the yield of some steps is about 90%; 7) Because no column purification is used, only conventional filtration is adopted, and the generated waste water is less and the environment is friendly.
Scheme B:
as exemplified in scheme B, compound M-41 can be prepared from Nemactetvir (which is commercially available) after the addition of a protecting group, or from intermediate M-3 via an addition of a protecting group, hydrolysis, coupling, substitution reaction to give compound M-41; the compound M-41 is reacted with D-L-Cl and then deprotected to obtain a compound Ib.
Compared with the prior art, the preparation method of the compound Ib has the following advantages: 1) Raw materialsIs cheap and easy to obtain; 2) The synthetic route is shorter, and the two steps of reactions are carried out; 3) The reaction condition is mild, and the reaction is carried out at room temperature; 4) Post-treatment does not need column chromatography purification, and is convenient and quick; 5) The substrate has strong adaptability; 6) The yield is higher; 7) The three wastes are less discharged.
Scheme C:
intermediate M-3 is hydrolyzed and reacted with compound M-49 to give compound Ic, as exemplified in scheme C.
Compared with the prior art, the preparation method of the compound Ic has the following advantages: 1) The raw material compound M-49 is cheap and easy to obtain; 2) The synthetic route is shorter, and the reaction is performed in one step; 3) The reaction condition is mild, and the reaction is carried out at room temperature; 4) Post-treatment does not need column chromatography purification, and is convenient and quick; 5) The substrate has strong adaptability; 6) The yield is higher; 7) The three wastes are less discharged.
For some of the steps of the process described hereinabove for preparing the compounds of the invention, it is necessary to protect the potentially reactive functional groups which are not desired to react and thus cleave the protecting groups. In such cases, any compatible protecting group may be used.
All of the above-described reactions and preparations of the novel starting materials used in the foregoing methods are conventional and appropriate reagents and reaction conditions for their manifestations or preparations, and procedures for isolating the desired product will be well known to those skilled in the art from the precedent of the reference, as well as examples and preparations thereof.
The preparation method of the polyethylene glycol-nemaltevir prodrug compound I has the advantages of mild reaction conditions, high yield, simple and convenient operation and environmental friendliness.
The invention also provides application of the polyethylene glycol-nemaltevir prodrug compound I in preparing medicines for preventing and/or treating new coronaries pneumonia.
The invention also provides a medicament which is a reagent prepared by taking the polyethylene glycol-nemortevir prodrug compound I as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
In the technical scheme of the invention, a novel molecule is creatively constructed by adopting the design principle of prodrugs: PEG derivative is used for modifying polyethylene glycol of Nemactetvir-Nemactetvir prodrug compound I, and the compound I does not need to be taken together with CYP3A4 inhibitor ritonavir, so that the risk of drug-drug interaction is reduced, and the compound I is beneficial to people with chronic diseases and other drugs.
Therefore, the polyethylene glycol-Nemactetvir prodrug compound I modified by polyethylene glycol has good application prospect in treating new coronatine pneumonia. Meanwhile, the preparation method of the compound I has the advantages of mild reaction conditions, high yield, simple and convenient operation and environmental friendliness.
In summary, the invention has the following beneficial technical effects:
1. the polyethylene glycol-Nemactetvir prodrug compound I provided by the invention does not need to be taken together with the CYP3A4 inhibitor ritonavir, reduces the risk of drug-drug interaction, is beneficial to the crowd taking other drugs for chronic diseases, provides a new drug selection, and has good application prospect.
2. According to the preparation method of the polyethylene glycol-nemortevir prodrug compound I, provided by the invention, the optimal process condition is determined by examining the influence of factors such as reaction temperature, substrate molar ratio, catalyst and the like on the yield and purity, and the preparation method has the characteristics of mild reaction condition, high yield, simplicity and convenience in operation and environmental friendliness.
In the description of the present invention, it should be understood that the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present invention, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
In the present invention, the expressions "compound a" and "compound represented by formula a" and "formula a" mean the same compound.
Detailed Description
In order to better understand the technical solution of the present invention, the following further discloses some non-limiting examples, which are further described in detail.
The reagents used in the present invention are all commercially available or can be prepared by the methods described herein.
Basic example 1: preparation of intermediate M-3
Step 1: methyl (1R, 2S, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carboxylate hydrochloride (50 g,243 mmol), N- (t-butoxycarbonyl) -3-methyl-L-valine (1.2 eq) and 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU, 1.2 eq) were added to the reaction flask, and N, N-dimethylformamide (DMF, 100 mL) and acetonitrile (900 mL) were added to the flask in an ice bath and stirred. N, N-diisopropylethylamine (3 eq) was added to the reaction system under ice bath. After the addition, naturally heating to room temperature and stirring overnight. The reaction solvent was removed under reduced pressure to give a crude product, which was dissolved in ethyl acetate (400 mL), washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, followed by purification by column chromatography to give oily compound M-1 (88 g, 95%).
Step 2: 1 (80 g,1 eq), TFA (20 eq) and DCM (200 mL) were added sequentially to a round bottom flask and reacted overnight at room temperature. TLC checked the reaction was complete and stopped. The reaction solution was poured into 300mL of water, naHCO 3 The pH was adjusted to weakly basic, extracted with DCM (200 mL. Times.3), the combined organic phases washed with NaCl solution, dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to give crude M-2 (55 g, 93%). 1 H NMR(400 MHz,DMSO-d6)d 6.76(d,J＝9.3Hz,1H),4.24(s,1H),4.06(d,J＝9.4Hz,1H), 3.99(d,J＝10.4Hz,1H),3.73(dd,J＝10.3,5.3Hz,1H),3.65(s,3H),1.55–1.49(m, 1H),1.41(d,J＝7.5Hz,1H),1.12(s,3H),0.98(s,9H),0.87(s,3H).
Step 3: triethylamine (151 mL,1.08 mol) and then ethyl trifluoroacetate (64.1 g, 457 mmol) were added to a methanol solution (180 mL) of compound 2 (55 g,280 mmol) under ice-bath, and the reaction mixture was then warmed to 50 ℃ and stirred for 16 hours. The reaction solvent was removed under reduced pressure to give a crude product, which was dissolved in ethyl acetate (400 mL), washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, followed by purification by column chromatography to give oily compound M-3 (70 g, 94%). 1 H NMR(400MHz,CDCl 3 )δ8.05(d, J＝12.6Hz,1H),4.60(d,J＝12.6Hz,1H),4.45(d,J＝7.3Hz,1H),3.72–3.64(m, 4H),3.57–3.46(m,1H),2.17–1.93(m,1H),1.09–1.04(m,3H),1.03–1.00(m, 3H),0.95(s,9H).
Basic example 2: preparation of intermediate M-5
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Step 1: a methanolic solution of ammonia (7M; 0.53L,3.68 mol) and methyl (S) -2- ((tert-butoxycarbonyl) amino) -3- ((S) -2-oxopyrrolidin-3-yl) propanoate (62 g,0.22 mol) was added to the reaction flask and the reaction mixture was stirred at 25℃for 48 hours. Concentration gave M-4 (57.5 g, 98%) as a yellow solid. 1 H NMR(400MHz,Methanol-d4)d 4.10(dd,J＝11.0,3.8Hz,1H),3.40– 3.31(m,2H),2.48(dh,J＝9.8,4.5Hz,1H),2.41–2.28(m,1H),2.04(ddd,J＝14.1, 11.1,4.4Hz,1H),1.87(dq,J＝12.5,8.8Hz,1H),1.74(ddd,J＝14.0,10.3,4.1Hz, 1H),1.45(s,9H).
Step 2: to a solution of M-4 (57.5 g,0.18 mol) in isopropanol (500 mL) was added HCl isopropanol (5.5M; 163mL,895 mmol) under ice-bath conditions. The reaction mixture was stirred at 50 ℃ for 4 hours, cooled to room temperature, stirred at room temperature overnight, and then concentrated to give the hydrochloride salt of M-5 as a white solid (37 g, 99%). 1 H NMR(400MHz,Methanol-d4)d 4.04(dd,J＝9.2,4.4Hz, 1H),3.39(dd,J＝9.0,4.5Hz,2H),2.90–2.68(m,1H),2.43(ddt,J＝12.8,8.9,4.6 Hz,1H),2.21–1.96(m,2H),1.88(dq,J＝12.6,9.1Hz,1H).
Example 1 (scheme a):
step 1: mPEG-5K-COOH (5 g,1 eq), L-alanine tert-butyl ester (1.5 eq), HOBt (2 eq) and DMF (40 mL) were added sequentially to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. Cold methyl tertiary butyl ether (300 mL) was added to the reaction system, a large amount of solid was precipitated, suction filtration and drying were carried out to obtain white solid M-6 (4.8 g) with a yield of 94%. 1 H NMR(400 MHz,CDCl 3 )δ4.50–4.38(m,1H),4.02(s,2H),3.80–3.48(m,448H),3.37(s,3H), 1.40(s,9H),1.37(d,J＝6.3Hz,3H).
Step 2: m-6 (4 g,1 eq), TFA (15 eq) and DCM (30 mL) were added sequentially toIn a round bottom flask, the reaction was carried out overnight at room temperature. TLC checked the reaction was complete and stopped. The reaction solution was poured into 100mL of water, pH was adjusted to weak base with NaHCO3, extracted with DCM (100 ml×3), the organic phases were combined, washed with NaCl solution, dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to give crude product. The crude product was dissolved in a small amount of DCM, cold methyl tert-butyl ether (300 mL) was added, a large amount of solid was precipitated, suction filtration was carried out, and the white target compound M-7 (3.6 g) was obtained in 91% yield. 1 H NMR(400MHz,CDCl 3 )δ4.50–4.38(m,1H),4.17 (s,2H),4.04–3.39(m,456H),3.35(s,3H),1.33(d,J＝6.3Hz,3H).
Step 3: m-7 (3.5 g,1 eq) and DCM (30 mL) were added sequentially to the reaction flask, thionyl chloride (15 eq) was added at room temperature and the reaction was refluxed for 5h. The reaction system was desolventized under reduced pressure to give a crude product (3.4 g) of Compound M-8 in 97% yield. 1 H NMR(400MHz,CDCl 3 )δ4.50–4.38(m,1H),4.16(s,2H), 4.02–3.36(m,456H),3.34(s,3H),1.36(d,J＝6.3Hz,3H).
Step 4: compound M-3 (440 mg,1 eq) and DCM (30 mL) were added to the reaction flask and stirred. And (5) protecting the reaction bottle by nitrogen. NaH (3 eq) was added in portions to the above flask under ice bath. After stirring under nitrogen for 10 minutes, compound M-8 (3.0 g,0.5 eq) was added to the reaction system. The reaction was continued for 8 hours in ice bath and was complete by TLC. The reaction was slowly poured into ice water, extracted with DCM (60 mL. Times.3), the organic phases combined, washed with NaCl solution, dried over anhydrous sodium sulfate and the solvent removed under reduced pressure to give the crude product. The crude product was dissolved in a small amount of DCM, cold methyl tert-butyl ether (300 mL) was added, a large amount of solid was precipitated, suction filtration was carried out, and the white solid compound M-9 (2.8 g) was obtained in 88% yield. 1 H NMR(400MHz,CDCl 3 ) δ8.05(d,J＝12.6Hz,1H),4.60(d,J＝12.6Hz,1H),4.50–4.38(m,2H),4.16(s, 2H),4.02–3.36(m,460H),3.31(s,3H),2.17–1.93(m,1H),1.36(d,J＝6.3Hz,3H)， 1.09–1.04(m,3H),1.04–0.99(m,3H),0.94(s,9H).
Step 5: compound M-9 (2.5 g,1 eq), liOH (3 eq), meOH (150 mL) and water (15 mL) were added to a round bottom flask and the reaction was stirred overnight at room temperature. TLC checked the reaction was complete and stopped. The reaction mixture was poured into 100mL of water, pH was adjusted to weak acidity with 2N HCl, and DCM was extractedThe organic phases were combined, washed with NaCl solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude product. The crude product was dissolved in a small amount of DCM, cold methyl tert-butyl ether (300 mL) was added, a large amount of solid was precipitated, suction filtration was carried out, and the white target compound M-10 (2.36 g) was obtained in 95% yield. 1 H NMR(400MHz,CDCl 3 )δ 8.06(d,J＝12.6Hz,1H),4.61(d,J＝12.6Hz,1H),4.50–4.39(m,2H),4.17(s,2H), 4.02–3.37(m,455H),3.30(s,3H),2.16–1.91(m,1H),1.35(d,J＝6.3Hz,3H)，1.09 –1.03(m,3H),1.01–0.96(m,3H),0.92(s,9H).
Step 6: m-10 (2 g,1 eq), M-5 (1.2 eq), HOBt (2 eq) and DMF (20 mL) were added sequentially to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. Cold methyl tert-butyl ether (300 mL) was added to the reaction system, a large amount of solids was precipitated, and the mixture was suction-filtered and dried to give the desired compound M-11 (1.97 g) in 91% yield. 1 H NMR(400MHz,DMSO-d6)d 9.41(d,J＝8.6Hz,1H),8.29(d,J＝8.9Hz,1H),7.55(s,1H),7.31(br s,1H),7.03 (br s,1H),4.51–4.37(m,2H),4.34–4.22(m,2H),4.16(s,2H),4.02–3.35(m, 460H),3.33(s,3H),3.12(t,J＝9.0Hz,1H),3.07–2.98(m,1H),2.44–2.35(m,1H), 2.16(dt,J＝10.5,7.4Hz,1H),1.98–1.88(m,1H),1.71–1.59(m,1H),1.53–1.44 (m,2H),1.41(d,J＝7.7Hz,1H),1.37(d,J＝6.3Hz,3H),1.11(s,3H),0.99(s,9H), 0.83(s,3H).
Step 7: methyl N- (triethylammonium sulfonyl) carbamate (Prague reagent; 3 eq) was added to a solution of M-11 (1.5 g,1 eq) in DCM (20 mL) at room temperature and stirring was continued for 2h. TLC monitored reaction was complete. The dichloromethane was removed under reduced pressure, the residue was dissolved in ethyl acetate (200 mL), washed twice with a mixture of saturated aqueous sodium bicarbonate (200 mL) and saturated aqueous sodium chloride (100 mL), and dried over anhydrous sodium sulfate. The ethyl acetate was removed under reduced pressure, the residue was dissolved in a small amount of methylene chloride, cold methyl t-butyl ether (300 mL) was added, a large amount of solid was precipitated, and the mixture was suction-filtered and dried to give the white solid compound Ia-1 (1.1 g) in 75% yield. 1 H NMR(400MHz,CDCl 3 )δ9.43(d,J＝8.4Hz,1H),9.03(d,J＝8.6Hz, 1H),7.68(s,1H),4.99–4.91(m,1H),4.50–4.36(m,2H),4.16–4.13(m,2H),4.03 –3.35(m,461H),3.31(s,3H),3.17–3.11(m,1H),3.04(td,J＝9.4,7.1Hz,1H), 2.40(tdd,J＝10.4,8.4,4.4Hz,1H),2.14(ddd,J＝13.4,10.9,4.4Hz,16 1H),2.11– 2.03(m,1H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.36(d,J＝6.3Hz, 3H),1.32(d,J＝7.6Hz,1H),1.03(s,3H),0.99(s,9H),0.84(s,3H).
Example 2:
the substrate in the step 1 is changed into tert-butyl glycinate for reaction, and the preparation method is prepared by the same method as in the example 1. 1 H NMR(400MHz,CDCl 3 )δ9.45(d,J＝8.4Hz,1H),9.02(d,J＝8.6Hz,1H), 7.68(s,1H),4.99–4.91(m,1H),4.50–4.36(m,1H),4.16–4.13(m,2H),3.99–3.35(m,464H),3.31(s,3H),3.17–3.11(m,1H),3.04(td,J＝9.4,7.1Hz,1H),2.40 (tdd,J＝10.4,8.4,4.4Hz,1H),2.14(ddd,J＝13.4,10.9,4.4Hz,16 1H),2.11–2.03 (m,1H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.32(d,J＝7.6Hz,1H), 1.03(s,3H),0.99(s,9H),0.83(s,3H).
Example 3:
the substrate in the step 1 was changed to L-valine t-butyl ester and reacted, and the same preparation method as in example 1 was used. .1H NMR (400 MHz, CDCl 3) δ9.43 (d, J=8.4 Hz, 1H), 9.03 (d, J=8.6 Hz, 1H), 7.68 (s, 1H), 4.99-4.91 (m, 1H), 4.50-4.36 (m, 2H), 4.16-4.13 (m, 2H), 4.03-3.35 (m, 461H), 3.31 (s, 3H), 3.17-3.11 (m, 1H), 3.04 (td, J=9.4, 7.1Hz, 1H), 2.40 (tdd, J=10.4, 8.4,4.4Hz, 1H), 2.14 (ddd, J= 13.4,10.9,4.4Hz,16 1H), 2.11-2.03 (m, 2H), 1.76-1.65 (m, 2H), 1.57 (ddd, J=7.6, 5.5Hz, 1.32 Hz), 1.7.6 Hz (d, 1.6 Hz), 1.0.96 (s, 1H), 2.11 (tdd, J=10.4, 8.4Hz, 1H).
Example 4:
the substrate in the step 1 was changed to (S) -2-amino-3, 3-dimethylbutyrate to react, and the preparation method was the same as that of example 1. . 1 H NMR(400MHz,CDCl 3 ) δ9.45 (d, j=8.4 hz, 1H), 9.02 (d, j=8.6 hz, 1H), 7.68 (s, 1H), 4.99-4.91 (m, 1H), 4.50-4.36 (m, 2H), 4.16-4.13 (m, 2H), 4.03-3.35 (m, 461H), 3.31 (s, 3H), 3.17-3.11 (m, 1H), 3.04 (td, j=9.4, 7.1hz, 1H), 2.40 (tdd, j=10.4, 8.4,4.4hz, 1H), 2.14 (ddd, j= 13.4,10.9,4.4Hz,16 1H), 2.11-2.03 (m, 1H), 1.76-1.65 (m, 2H), 1.57 (dd, j=7.6, 5.5hz, 1H), 1.32 (d, j=7.1 hz, 1H), 2.14 (tdd, j=7.6 hz, 1H), 1.3.3.8, 1H), 3.3 (3 s, 3.8, 1H).The substrate in the step 1 was changed to L-alanyl-L-alanine tert-butyl ester and reacted, and the same procedure as in example 1 was followed. 1 H NMR(400MHz,CDCl 3 ) δ9.46 (d, j=8.4 hz, 1H), 9.03 (d, j=8.6 hz, 1H), 7.68 (s, 1H), 4.99-4.91 (m, 1H), 4.50-4.32 (m, 3H), 4.16-4.13 (m, 2H), 4.03-3.35 (m, 461H), 3.31 (s, 3H), 3.17-3.11 (m, 1H), 3.04 (td, j=9.4, 7.1hz, 1H), 2.40 (tdd, j=10.4, 8.4,4.4hz, 1H), 2.14 (ddd, j= 13.4,10.9,4.4Hz,16 1H), 2.11-2.03 (m, 1H), 1.76-1.65 (m, 2H), 1.57 (dd, j=7.6, 5.5hz, 1H), 1.39 (d, j=6.1 hz, 1.33, 3H), 1.3 s (d, 3H), 1.3.4, 4hz, 1.3H).
Step 1: (S) -2- (((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-methoxy-5-oxopentanoic acid (50 g,1 eq), dimethyl glutamate (1.1 eq), HOBt (2 eq) and DMF (100 mL) were added sequentially to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. Purifying the reaction system by column chromatography to obtain white solidBody M-12 (79.8 g) was obtained in 83% yield. 1 H NMR(400 MHz,CDCl 3 )δ7.82(dd,J＝8.2,1.3Hz,2H),7.73(d,J＝9.0Hz,1H),7.62(dd,J＝ 7.5,1.4Hz,2H),7.50(td,J＝7.7,1.2Hz,2H),7.41(td,J＝7.9,1.5Hz,2H),6.06(d, J＝8.4Hz,1H),5.45–5.38(m,1H),4.42(d,J＝4.9Hz,2H),4.37–4.24(m,2H), 3.64(s,3H),2.47–2.26(m,4H),2.21–2.07(m,2H),1.98–1.83(m,2H),1.44– 1.38(m,18H).
Step 2: m-12 (75 g,1 eq), TFA (20 eq) and DCM (150 mL) were added sequentially to a round bottom flask and reacted overnight at room temperature. TLC checked the reaction was complete and stopped. The reaction solution was poured into 200mL of water, naHCO 3 The pH was adjusted to weakly alkaline, extracted with DCM (100 mL. Times.3), the combined organic phases were washed with NaCl solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give M-13 (59 g) as a white solid in 96% yield. 1 H NMR(400MHz,CDCl 3 )δ7.82(dd,J＝8.2,1.3Hz,2H),7.73(d,J＝9.0Hz, 1H),7.62(dd,J＝7.5,1.4Hz,2H),7.50(td,J＝7.7,1.2Hz,2H),7.41(td,J＝7.9,1.5 Hz,2H),6.06(d,J＝8.4Hz,1H),5.45–5.38(m,1H),4.42(d,J＝4.9Hz,2H),4.39 –4.24(m,2H),3.66(s,3H),2.48–2.25(m,4H),2.23–2.09(m,2H),1.99–1.81(m, 2H).
Step 3: m-13 (50 g,1 eq), methyl 3-phthalimidopropionate (1.2 eq), HOBt (2 eq) and DMF (100 mL) were added sequentially to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. The above reaction system was purified by column chromatography to give M-14 (61 g) as a white solid in 91% yield. 1 H NMR(400MHz,CDCl 3 )δ7.84–7.77(m,3H),7.64–7.58(m,3H),7.54– 7.47(m,3H),7.41(td,J＝7.8,1.5Hz,2H),6.06(d,J＝8.4Hz,1H),5.43–5.39(m, 1H),4.50–4.39(m,4H),4.36–4.30(m,1H),4.17–4.11(m,1H),3.70(s,6H),3.64 (s,1H),2.45–2.36(m,4H),2.07–1.85(m,4H),1.41–1.34(m,6H).
Step 4: m-14 (60 g) was dissolved in DMF (120 mL), piperidine (40 mL) was added, stirred at room temperature for 2 hours, 500mL methyl tert-butyl ether was added to precipitate a large amount of white solid, filtration was performed, the cake was slurried with 100mL methyl tert-butyl ether for 30 minutes, filtration was performed, and the cake was flushed with methyl tert-butyl etherWashing (80 ml x 3), draining the filter cake under reduced pressure gave M-15 (37 g) as a white solid in 92% yield. 1 H NMR(400MHz,CDCl 3 )δ7.64 –7.50(m,2H),4.50–4.37(m,2H),4.17–4.10(m,1H),3.73–3.60(m,10H),2.48 –2.32(m,4H),2.09–1.92(m,4H),1.42–1.34(m,6H).
Step 5: mPEG-5K-COOH (20 g,1 eq), M-15 (1.2 eq), HOBt (2 eq) and DMF (100 mL) were added sequentially to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. Cold methyl tertiary butyl ether (500 mL) is added into the reaction system, a large amount of solid is separated out, suction filtration and drying are carried out, and the white target compound M-16 (20 g) is obtained, and the yield is 93%. 1 H NMR(400MHz,CDCl 3 ) δ7.64–7.50(m,2H),4.50–4.37(m,2H),4.17–4.10(m,3H),3.87–3.41(m, 505H),3.38(s,3H),2.48–2.32(m,4H),2.09–1.92(m,4H),1.42–1.34(m,6H).
Step 6: m-16 (20 g,1 eq), liOH (3 eq), meOH (10 mL) and water (10 mL) were added to a round bottom flask and the reaction was stirred overnight at room temperature. TLC checked the reaction was complete and stopped. The reaction solution was poured into 100mL of water, pH was adjusted to weak acidity with 2N HCl, extracted with DCM (100 mL. Times.3), the organic phases were combined, washed with NaCl solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude product. The crude product was dissolved in a small amount of DCM, cold methyl tert-butyl ether (300 mL) was added, a large amount of solid was precipitated, suction filtration was carried out, and the white target compound M-17 (18 g) was obtained in 90% yield. 1 H NMR(400MHz,CDCl 3 )δ7.66– 7.51(m,2H),4.51–4.36(m,2H),4.15–4.08(m,3H),3.87–3.41(m,480H),3.38 (s,3H),2.48–2.32(m,4H),2.09–1.91(m,4H),1.43–1.35(m,6H).
Step 7, step 8, step 9, step 10 and step 11 were referred to in this order to step 3, step 4, step 5, step 6 and step 7, respectively, in example 1, to finally obtain the target compound Ia-6 (3.2 g) as a white solid. 1 H NMR(400MHz,CDCl 3 )δ9.43(d,J＝8.4Hz,3H),9.03(d,J＝8.6Hz,3H),7.70(s, 3H),7.66–7.51(m,2H),4.99–4.92(m,3H),4.51–4.36(m,5H),4.15–4.08(m, 6H),3.93–3.41(m,488H),3.38(s,3H),3.17–3.11(m,3H),3.04(td,J＝9.4,7.1Hz, 3H),2.48–2.36(m,7H),2.17–2.11(m,3H),2.11–1.95(m,7H),1.76–1.65(m, 6H),1.60–1.54(m,3H),1.46–1.37(m,6H),1.32(d,J＝7.6Hz,3H),1.03(s,9H), 0.98(s,27H),0.85(s,9H).
Example 7:
step 1: compound M-3 (3 g,1 eq) and DCM (30 mL) were added to the reaction flask and stirred. And (5) protecting the reaction bottle by nitrogen. NaH (3 eq) was added in portions to the above flask under ice bath. After stirring for 10 minutes under nitrogen, p-nitrobenzyl chloroformate (1.1 eq) was added to the reaction system. The reaction was continued for 8 hours in ice bath and was complete by TLC. The reaction was slowly poured into ice water, extracted with DCM (60 mL. Times.3), the organic phases combined, washed with NaCl solution, dried over anhydrous sodium sulfate and the solvent removed under reduced pressure to give the crude product. The crude product was purified by column chromatography to give Compound M-22 (4.1 g) as a white solid in 92% yield. 1 H NMR(400 MHz,DMSO-d6)δ8.11(d,J＝8.4Hz,2H),7.62(d,J＝8.2Hz,2H),5.19(s,2H), 4.25(d,J＝8.5Hz,1H),4.15(s,1H),3.85(dd,J＝10.5,5.3Hz,1H),3.72(d,J＝10.5 Hz,1H),3.64(s,3H),1.53(dd,J＝7.4,5.2Hz,1H),1.43(d,J＝7.6Hz,1H),1.01(d, J＝3.3Hz,12H),0.82(s,3H).
Step 2: m-22 (4 g,1 eq), ammonium chloride (3 eq), ethanol (30 mL) and water (10 mL) were added sequentially to the reaction flask and stirred at room temperature. Iron powder (3 eq) was added in portions to the above reactor flask and reacted under reflux for 2h. TLC monitored reaction was complete. The reaction system was filtered through celite to remove iron powder and inorganic salts. The filtrate was poured into 200mL of ethyl acetate, and washed with saturated brine for 2 times, dried over anhydrous sodium sulfate, and purified by column chromatography to give Compound M-23 (3.8 g) as a white solid in 96% yield. 1 H NMR(400 MHz,DMSO-d6)δ7.27–7.22(m,2H),6.60–6.56(m,2H),5.17(s,2H),4.25(d,J＝ 8.5Hz,1H),4.22–3.96(m,3H),3.85(dd,J＝10.5,5.3Hz,1H),3.72(d,J＝10.5Hz, 1H),3.64(s,3H),1.53(dd,J＝7.4,5.2Hz,1H),1.43(d,J＝7.6Hz,1H),1.02(d,J＝ 3.3Hz,12H),0.83(s,3H).
Step 3: mPEG-5KCOOH (3 g,1 eq), M-23 (1.2 eq), HOBt (2 eq) and DMF (100 mL) were added in sequence to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. Cold methyl tertiary butyl ether (500 mL) is added into the reaction system, a large amount of solid is separated out, suction filtration and drying are carried out, and the white target compound M-24 (2.9 g) is obtained, and the yield is 89%. 1 H NMR(400MHz, DMSO-d6)δ7.88–7.85(m,2H),7.27–7.23(m,2H),5.18(s,2H),4.25(d,J＝8.5Hz, 1H),4.22–3.96(m,5H),3.88–3.41(m,495H),3.38(s,3H),1.53(dd,J＝7.4,5.2 Hz,1H),1.43(d,J＝7.6Hz,1H),1.02(d,J＝3.3Hz,12H),0.83(s,3H).
Step 4, step 5 and step 6 were referred to in sequence in step 5, step 6 and step 7, respectively, in example 1, to finally obtain the target compound Ia-7 (1.8 g) as a white solid. 1 H NMR(600MHz,DMSO-d6)δ9.03 (d,J＝8.6Hz,1H),7.88–7.85(m,2H),7.68(s,1H),7.27–7.23(m,2H),5.19(s, 2H),5.01–4.96(m,1H),4.22–3.96(m,5H),3.92–3.41(m,497H),3.38(s,3H), 3.17–3.11(m,1H),3.05–3.02(m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H), 2.11–2.03(m,1H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.32(d,J＝ 7.6Hz,1H),1.02(s,3H),0.99(s,9H),0.84(s,3H).
Example 8:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 2-methyl-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝8.6Hz, 1H),7.68(s,1H),7.55–7.51(m,1H),7.39–7.35(m,1H),7.18–7.15(m,1H),5.23(s,2H), 5.01–4.96(m,1H),4.22–3.96(m,5H),3.92–3.41(m,497H),3.38(s,3H),3.17–3.11(m, 1H),3.05–3.02(m,1H),2.41–2.38(m,1H),2.31(s,3H),2.15–2.12(m,1H),2.11–2.03(m, 1H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.32(d,J＝7.6Hz,1H),1.02(s,3H), 0.99(s,9H),0.84(s,3H).
Example 9:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 3-methyl-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝8.6Hz, 1H),7.68(s,1H),7.29–7.26(m,1H),7.22–7.20(m,1H),7.17–7.14(m,1H),5.16(s,2H),5.01– 4.96(m,1H),4.22–3.96(m,5H),3.92–3.41(m,497H),3.38(s,3H),3.17–3.11(m,1H),3.05 –3.02(m,1H),2.41–2.38(m,1H),2.24(s,3H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76– 1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.99(s,9H), 0.84(s,3H).
Example 10:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 2, 5-dimethyl-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d, J＝8.6Hz,1H),7.68(s,1H),7.57(s,1H),7.11(s,1H),5.20(s,2H),5.01–4.96(m,1H),4.22– 3.96(m,5H),3.92–3.41(m,497H),3.38(s,3H),3.17–3.11(m,1H),3.05–3.02(m,1H),2.41 –2.38(m,1H),2.33(s,3H),2.24(s,3H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76–1.65(m, 2H),1.57(dd,J＝7.6,5.5Hz,1H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.99(s,9H),0.84(s, 3H).
Example 11:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 3, 5-dimethyl-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝ 8.6Hz,1H),7.68(s,1H),7.14(s,2H),5.11(s,2H),5.01–4.96(m,1H),4.22–3.96(m,5H),3.92 –3.41(m,497H),3.38(s,3H),3.17–3.11(m,1H),3.05–3.02(m,1H),2.41–2.38(m,1H), 2.18(s,6H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5 Hz,1H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.99(s,9H),0.84(s,3H).
Example 12:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 3-methoxy-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝8.6 Hz,1H),7.68(s,1H),7.55–7.51(m,1H),7.04–7.01(m,1H),6.92–6.89(m,1H),5.18(s,2H), 5.01–4.96(m,1H),4.22–3.96(m,5H),3.92–3.41(m,501H),3.38(s,3H),3.17–3.11(m, 1H),3.05–3.02(m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76– 1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.98(s,9H), 0.83(s,3H).
Example 13:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 2-methoxy-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝8.6 Hz,1H),7.68(s,1H),7.53–7.48(m,1H),7.30–7.26(m,1H),7.10–7.07(m,1H),5.24(s,2H), 5.01–4.96(m,1H),4.22–3.96(m,5H),3.92–3.41(m,500H),3.38(s,3H),3.17–3.11(m, 1H),3.05–3.02(m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76– 1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.98(s,9H), 0.84(s,3H).
Example 14:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 3-chloro-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝8.6Hz, 1H),7.68(s,1H),7.54–7.51(m,1H),7.47–7.45(m,1H),7.24–7.21(m,1H),5.22(s,2H),5.01– 4.96(m,1H),4.22–3.96(m,5H),3.92–3.41(m,498H),3.38(s,3H),3.17–3.11(m,1H),3.05 –3.02(m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76–1.65(m,2H), 1.57(dd,J＝7.6,5.5Hz,1H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.98(s,9H),0.83(s,3H).
Example 15:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 3-fluoro-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝8.6Hz, 1H),7.68(s,1H),7.56–7.47(m,1H),7.16–7.10(m,2H),5.20(s,2H),5.01–4.96(m,1H),4.22– 3.95(m,5H),3.92–3.41(m,498H),3.38(s,3H),3.17–3.11(m,1H),3.05–3.02(m,1H),2.41 –2.38(m,1H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76–1.65(m,2H),1.57(dd,J＝7.6, 5.5Hz,1H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.98(s,9H),0.83(s,3H).
Example 16:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 3, 5-difluoro-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝8.6 Hz,1H),7.68(s,1H),7.03–6.99(m,2H),5.22(s,2H),5.01–4.96(m,1H),4.22–3.95(m,5H), 3.92–3.41(m,498H),3.38(s,3H),3.17–3.11(m,1H),3.05–3.02(m,1H),2.41–2.38(m, 1H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H), 1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.98(s,9H),0.83(s,3H).
Example 17:
the substrate p-nitrobenzyl chloroformate in step 1 was converted to 3-cyano-4-nitrobenzyl carbonyl chloride and reacted by the same method as in example 7. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝8.6Hz, 1H),7.68(s,1H),7.63(s,1H),7.51–7.46(m,1H),7.36–7.31(m,1H),5.19(s,2H),5.01–4.96(m, 1H),4.22–3.95(m,5H),3.92–3.41(m,498H),3.38(s,3H),3.17–3.11(m,1H),3.05–3.02 (m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76–1.65(m,2H),1.57 (dd,J＝7.6,5.5Hz,1H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.98(s,9H),0.83(s,3H).
Example 18:
step 1: m-23 (10 g,1 eq), N-t-butoxycarbonyl-L-alanine (1.2 eq), HOBt (2 eq) and DMF (100 mL) were added sequentially to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. The above reaction system was purified by column chromatography to give M-27 (10.7 g) as a white solid in 81% yield. 1 H NMR(400MHz,DMSO-d6)δ9.23(s,1H),7.60–7.51(m,2H),7.36–7.29 (m,2H),6.08(d,J＝7.3Hz,1H),5.17(s,2H),4.38–4.29(m,1H),4.25(d,J＝8.5 Hz,1H),4.22–3.96(m,1H),3.85(dd,J＝10.5,5.3Hz,1H),3.72(d,J＝10.5Hz, 1H),3.64(s,3H),1.53(dd,J＝7.4,5.2Hz,1H),1.47(d,J＝7.6Hz,1H),1.42(s,9H), 1.38(d,J＝5.7Hz,3H),1.02(d,J＝3.3Hz,12H),0.83(s,3H).
Step 2:m-27 (10 g,1 eq), TFA (15 eq) and DCM (150 mL) were added sequentially to a round bottom flask and reacted overnight at room temperature. TLC checked the reaction was complete and stopped. The reaction solution was poured into 200mL of water, naHCO 3 The pH was adjusted to weakly alkaline, extracted with DCM (100 mL. Times.3), the combined organic phases were washed with NaCl solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give M-28 (8.1 g) as a white solid in 94% yield. 1 H NMR(400MHz,DMSO-d6)δ9.45(s,1H),7.60–7.54(m,2H),7.35–7.30(m,2H), 5.17(s,2H),4.20–4.08(m,1H),4.25(d,J＝8.5Hz,1H),4.22–3.96(m,1H),3.85 (dd,J＝10.5,5.3Hz,1H),3.72(d,J＝10.5Hz,1H),3.66(s,3H),3.60–3.25(m,2H), 1.53(dd,J＝7.4,5.2Hz,1H),1.43(d,J＝7.6Hz,1H),1.35(d,J＝5.1Hz,3H),1.02 (d,J＝3.3Hz,12H),0.83(s,3H).
Step 3, step 4, step 5 and step 6 were referred to in order from step 3, step 4, step 5 and step 6 in example 7, respectively, to finally obtain the target compound Ia-18 (3.1 g) as a white solid. 1 H NMR(600MHz, DMSO-d6)δ9.48(s,1H),9.03(d,J＝8.6Hz,1H),7.88–7.85(m,2H),7.68(s,1H), 7.57(d,J＝8.6Hz,1H),7.27–7.23(m,2H),5.19(s,2H),5.01–4.96(m,1H),4.42– 4.32(m,1H),4.22–3.96(m,5H),3.92–3.41(m,497H),3.38(s,3H),3.17–3.11(m, 1H),3.05–3.02(m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H),2.11–2.03(m, 1H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.38(d,J＝6.5Hz,3H),1.32 (d,J＝7.6Hz,1H),1.02(s,3H),0.99(s,9H),0.84(s,3H).
Example 19:
the substrate N-t-butoxycarbonyl-L-alanine in step 1 was changed to N-t-butoxycarbonyl-L-valine and reacted by the same manner as in example 18. 1 H NMR(600MHz,DMSO-d6)δ9.48 (s,1H),9.05(d,J＝8.6Hz,1H),7.88–7.85(m,2H),7.68(s,1H),7.57(d,J＝8.6Hz, 1H),7.27–7.23(m,2H),5.19(s,2H),5.01–4.96(m,1H),4.33–4.26(m,1H),4.22 –3.96(m,5H),3.92–3.41(m,497H),3.38(s,3H),3.17–3.11(m,1H),3.05–3.02 (m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H),2.11–2.03(m,2H),1.76–1.65 (m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.38(d,J＝6.5Hz,3H),1.32(d,J＝7.6Hz, 1H),1.02(s,3H),0.99(s,9H),0.94(d,J＝6.7,1.8Hz,6H),0.84(s,3H).
Example 20:
the substrate N-t-butoxycarbonyl-L-alanine in step 1 was changed to N-t-butoxycarbonyl-L-leucine and reacted by the same manner as in example 18. 1 H NMR(600MHz,DMSO-d6)δ9.48 (s,1H),9.03(d,J＝8.6Hz,1H),7.88–7.85(m,2H),7.68(s,1H),7.57(d,J＝8.6Hz, 1H),7.27–7.23(m,2H),5.19(s,2H),5.01–4.96(m,1H),4.42–4.32(m,1H),4.22 –3.96(m,5H),3.92–3.41(m,497H),3.38(s,3H),3.17–3.11(m,1H),3.05–3.02 (m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76–1.67 (m,2H),1.65–1.50(m,3H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),0.99(s,9H),0.88 (d,J＝6.1Hz,6H),0.84(s,3H).
Example 21:
the substrate N-t-butoxycarbonyl-L-alanine in step 1 was changed to N-t-butoxycarbonyl-L-isoleucine and reacted by the same manner as in example 18. 1 H NMR(600MHz,DMSO-d6)δ9.48(s, 1H),9.03(d,J＝8.6Hz,1H),7.88–7.85(m,2H),7.68(s,1H),7.57(d,J＝8.6Hz, 1H),7.27–7.23(m,2H),5.19(s,2H),5.01–4.96(m,1H),4.42–4.32(m,1H),4.22 –3.96(m,5H),3.92–3.41(m,497H),3.38(s,3H),3.17–3.11(m,1H),3.05–3.02 (m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H),2.11–2.06(m,1H),2.03–1.93 (m,1H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.35–1.25(m,3H),, 1.02(s,3H),0.99(s,9H),0.94–0.82(m,6H),0.84(s,3H).
Example 22:
step 1: compound M-3 (10 g,1 eq) and DCM (30 mL) were added to the reaction flask and stirred. And (5) protecting the reaction bottle by nitrogen. NaH (3 eq) was added in portions to the above flask under ice bath. After stirring for 10 minutes under nitrogen, chloromethyl chloroformate (1.1 eq) was added to the reaction system. The reaction was continued for 8 hours in ice bath and was complete by TLC. The reaction was slowly poured into ice water, extracted with DCM (60 mL. Times.3), the organic phases combined, washed with NaCl solution, dried over anhydrous sodium sulfate and the solvent removed under reduced pressure to give the crude product. The crude product was purified by column chromatography to give Compound M-32 (10.1 g) as a white solid in 82% yield. 1 H NMR(400MHz, DMSO-d6)δ5.64–5.58(m,1H),4.25(d,J＝8.5Hz,1H),4.15(s,1H),3.85(dd,J＝ 10.5,5.3Hz,1H),3.72(d,J＝10.5Hz,1H),3.64(s,3H),1.53(dd,J＝7.4,5.2Hz, 1H),1.43(d,J＝7.6Hz,1H),1.01(d,J＝3.3Hz,12H),0.82(s,3H).
Step 2: m-32 (10 g,1 eq), cesium carbonate (3 eq) of N-t-butoxycarbonyl-L-alanine (1.1 eq) and DMF were added to a reaction flask and reacted overnight at 85 ℃. TLC monitored reaction was complete. The reaction system was poured into 300mL of ethyl acetate, and washed with a saturated sodium chloride solution for 2 times, dried over anhydrous sodium sulfate, and ethyl acetate was removed under reduced pressure to give a crude product, which was purified by column chromatography to give Compound M-33 (11 g) as a white solid in 83% yield. 1 H NMR(400MHz,DMSO-d6)δ5.88–5.82(m,1H),5.49(d,J＝8.2Hz,1H), 4.37–4.31(m,1H),4.25(d,J＝8.5Hz,1H),4.15(s,1H),3.85(dd,J＝10.5,5.3Hz, 1H),3.72(d,J＝10.5Hz,1H),3.64(s,3H),1.53(dd,J＝7.4,5.2Hz,1H),1.48(d,J ＝7.6Hz,1H),1.42(s,9H),1.33(d,J＝6.6Hz,3H),0.99(d,J＝3.3Hz,12H),0.83(s, 3H).
Step 3, step 4, step 5, step 6 and step 7 were referred to in order from step 2, step 3, step 4, step 5 and step 6, respectively, in example 18, to finally obtain the target compound Ia-22 (2.6 g) as a white solid. 1 H NMR(600MHz,DMSO-d6)δ9.03(d,J＝8.6Hz,1H),5.83(s,2H),5.01–4.96(m, 1H),4.52–4.44(m,1H),4.41(d,J＝8.4Hz,1H),4.15(s,1H),4.02(s,1H),3.91(dd, J＝10.4,5.5Hz,1H),3.92–3.41(m,497H),3.37(s,3H),3.17–3.11(m,1H),3.05 –3.02(m,1H),2.41–2.38(m,1H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.76– 1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.37(d,J＝6.8Hz,3H),1.32(d,J＝7.6 Hz,1H),1.02(s,3H),0.99(s,9H),0.84(s,3H).
Example 23:
the substrate N-t-butoxycarbonyl-L-alanine in step 2 was changed to N-t-butoxycarbonyl-L-valine and reacted by the same manner as in example 22. 1 H NMR(400MHz,DMSO-d6)δ9.03 (d,J＝8.6Hz,1H),5.83(s,2H),5.01–4.96(m,1H),4.52–4.44(m,1H),4.41(d,J ＝8.4Hz,1H),4.15(s,1H),4.02(s,1H),3.91(dd,J＝10.4,5.5Hz,1H),3.92–3.41 (m,497H),3.37(s,3H),3.17–3.11(m,1H),3.05–3.02(m,1H),2.41–2.38(m, 1H),2.18–2.03(m,3H),,1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.32(d, J＝7.6Hz,1H),1.09(s,3H),1.02–0.87(m,15H),0.85(s,3H).
Example 24:
the substrate N-t-butoxycarbonyl-L-alanine in step 2 was changed to N-t-butoxycarbonyl-L-isoleucine and reacted by the same manner as in example 22. 1 H NMR(600MHz,DMSO-d6)δ 9.03(d,J＝8.6Hz,1H),5.83(s,2H),5.01–4.96(m,1H),4.52–4.44(m,1H),4.41 (d,J＝8.4Hz,1H),4.15(s,1H),4.02(s,1H),3.91(dd,J＝10.4,5.5Hz,1H),3.92– 3.41(m,497H),3.37(s,3H),3.17–3.11(m,1H),3.05–3.02(m,1H),2.41–2.38 (m,1H),2.15–1.98(m,3H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.34 –1.25(m,3H),1.02(s,3H),0.99(s,9H),0.92–0.83(m,9H).
Example 25:
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the substrate N-t-butoxycarbonyl-L-alanine in step 2 was changed to N-t-butoxycarbonyl-L-leucine and reacted by the same manner as in example 22. 1 H NMR(400MHz,DMSO-d6)δ9.03 (d,J＝8.6Hz,1H),5.83(s,2H),5.01–4.96(m,1H),4.52–4.44(m,1H),4.41(d,J ＝8.4Hz,1H),4.15(s,1H),4.02(s,1H),3.91(dd,J＝10.4,5.5Hz,1H),3.92–3.41 (m,497H),3.37(s,3H),3.17–3.11(m,1H),3.05–3.02(m,1H),2.41–2.38(m, 1H),2.15–2.12(m,1H),2.11–2.03(m,1H),1.78–1.69(m,2H),1.64–1.57(m, 4H),1.32(d,J＝7.6Hz,1H),1.02(s,3H),1.00(s,9H),0.92–0.84(m,9H).
Example 26 (scheme B):
step 1: m-3 (10 g,1 eq), isopropanol (75 mL), water (25 mL) and di-tert-butyl dicarbonate (1.1 eq) were each added sequentially to the reaction flask and stirred at room temperature. Potassium carbonate (3 eq) was added in small portions at the same temperature and allowed to react at 75℃with stirring overnight. TLC monitored reaction was complete. The reaction system was poured into 300mL of ethyl acetate, washed with a saturated sodium chloride solution for 2 times, dried over anhydrous sodium sulfate, and the ethyl acetate was removed under reduced pressure to give crude product, which was purified by column chromatography to give Compound M-38 (12.1 g) as a white solid in 96% yield. 1 H NMR(400MHz,DMSO-d6)δ4.52(s,1H),4.21(s,1H),3.93(d,J＝ 10.4Hz,1H),3.79(dd,J＝10.3,5.3Hz,1H),3.65(s,3H),1.55(dd,J＝7.4,5.2Hz, 1H),1.42(d,J＝7.5Hz,1H),1.36(s,9H),1.01(s,3H),0.93(s,9H),0.84(s,3H).
Step 2: m-38 (12 g,1 eq), liOH (3 eq), meOH (10 mL) and water (10 mL) were added to a round bottom flask and the reaction was stirred overnight at room temperature. TLC checked the reaction was complete and stopped. The reaction mixture was poured into 100mL of water, pH was adjusted to weak acid with 2N HCl, a large amount of white solid was precipitated, and the mixture was suction-filtered and dried to give the desired white compound M-39 (10.2 g) in 88% yield. 1 H NMR(400MHz,DMSO-d6) δ12.96–12.43(m,1H),4.47(s,1H),4.16(s,1H),3.91(d,J＝10.4Hz,1H),3.75(dd, J＝10.3,5.3Hz,1H),1.55(dd,J＝7.4,5.2Hz,1H),1.43(d,J＝7.5Hz,1H),1.37(s, 9H),1.01–0.96(m,12H),0.83(s,3H).
Step 3: m-39 (10 g,1 eq), M-5 (1.2 eq), HOBt (2 eq) and DMF (20 mL) were added sequentially to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. The reaction system was poured into 300mL of ethyl acetate, washed with a saturated sodium chloride solution for 2 times, dried over anhydrous sodium sulfate, and the ethyl acetate was removed under reduced pressure to give a crude product, which was purified by column chromatography to give Compound M-40 (10.8 g) as a white solid in 78% yield. 1 H NMR(400MHz,Methanol-d4)δ4.47(s,1H),4.16(s,1H), 4.10(dd,J＝11.0,3.8Hz,1H),3.40–3.31(m,2H),3.91(d,J＝10.4Hz,1H),3.75 (dd,J＝10.3,5.3Hz,1H),2.48(dh,J＝9.8,4.5Hz,1H),2.41–2.28(m,1H),2.04 (ddd,J＝14.1,11.1,4.4Hz,1H),1.87(dq,J＝12.5,8.8Hz,1H),1.74(ddd,J＝14.0, 10.3,4.1Hz,1H),1.55(dd,J＝7.4,5.2Hz,1H),1.43(d,J＝7.5Hz,1H),1.36(s,9H), 1.01(s,3H),0.93(s,9H),0.84(s,3H).
Step 4: methyl N- (triethylammonium sulfonyl) carbamate (Bogis reagent; 3 eq) was added to a solution of M-40 (10 g,1 eq) in DCM (20 mL) and left to stir at room temperature for 2 hours. TLC monitored reaction was complete. The dichloromethane was removed under reduced pressure, the residue was dissolved in ethyl acetate (200 mL), washed twice with a mixture of saturated aqueous sodium bicarbonate (200 mL) and saturated aqueous sodium chloride (100 mL), and dried over anhydrous sodium sulfate. The ethyl acetate was removed under reduced pressure, and the residue was purified by column chromatography to give Compound M-41 (7.3 g) as a white solid in 75% yield. 1 H NMR(400MHz,DMSO-d6)δ4.52–4.46(m,1H),4.39 (s,1H),4.18(s,1H),3.91(d,J＝10.4Hz,1H),3.75(dd,J＝10.3,5.3Hz,1H),3.31– 3.22(m,2H),2.51–2.45(m,1H),2.08–1.94(m,2H),1.91–1.79(m,2H),1.55(dd, J＝7.4,5.2Hz,1H),1.43(d,J＝7.5Hz,1H),1.36(s,9H),1.05–0.99(m,12H),0.87 (s,3H).
Step 5: m-41 (7 g,1 eq), (R) - (1-chloro-1-oxopropan-2-yl) carbamic acid tert-butyl ester (1.2 eq) and DCM (30 mL) were added to the reaction flask and stirred at room temperature. N, N-diisopropylethylamine (3 eq) was added to the reaction system, and stirring was continued at room temperature overnight.The dichloromethane was removed under reduced pressure, the residue was dissolved in ethyl acetate (200 mL), washed twice with a mixture of saturated aqueous sodium bicarbonate (200 mL) and saturated aqueous sodium chloride (100 mL), and dried over anhydrous sodium sulfate. The ethyl acetate was removed under reduced pressure, and the residue was purified by column chromatography to give compound M-42 (7.1 g) as a white solid in 79% yield. 1 H NMR(400MHz,DMSO-d6)δ4.54 –4.46(m,1H),4.27(s,1H),4.15(s,1H),3.88(d,J＝10.4Hz,1H),3.71(dd,J＝10.3, 5.3Hz,1H),3.32–3.18(m,2H),2.53–2.48(m,1H),2.09–1.94(m,2H),1.91– 1.75(m,2H),1.56(dd,J＝7.4,5.2Hz,1H),1.46(s,9H),1.41(d,J＝7.5Hz,1H),1.37 (s,9H),1.28(d,J＝6.2Hz,3H),1.05–0.99(m,12H),0.85(s,3H).
Step 6: m-42 (7 g,1 eq), TFA (15 eq) and DCM (30 mL) were added sequentially to a round bottom flask and reacted overnight at room temperature. TLC checked the reaction was complete and stopped. The reaction was poured into 100mL of water, pH was adjusted to weakly basic with NaHCO3, the mixture was extracted with DCM (100 mL. Times.3), the organic phases were combined, washed with NaCl solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude M-43 (5.1 g) as pale yellow product in 97% yield. The next reaction was directly carried out without further purification. 1 H NMR(400MHz,DMSO-d6)δ4.54– 4.46(m,1H),4.27(s,1H),4.15(s,1H),3.88(d,J＝10.4Hz,1H),3.71(dd,J＝10.3, 5.3Hz,1H),3.32–3.18(m,2H),2.53–2.48(m,1H),2.09–1.94(m,2H),1.91– 1.75(m,2H),1.56(dd,J＝7.4,5.2Hz,1H),1.42(d,J＝7.5Hz,1H),1.28(d,J＝6.2 Hz,3H),1.01(s,3H),0.93(s,9H),0.82(s,3H).
Step 7: mPEG-5K-COOH (5 g,1 eq), M-43 (1.5 eq), HOBt (2 eq) and DMF (40 mL) were added sequentially to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. Cold methyl tertiary butyl ether (300 mL) is added into the reaction system, a large amount of solid is separated out, suction filtration and drying are carried out, and white solid Ib-1 (5.4 g) is obtained, and the yield is 97%. 1 H NMR(400MHz,DMSO-d6)δ 4.54–4.46(m,1H),4.27(s,1H),4.21(s,2H),4.15(s,2H),3.89–3.42(m,496H), 3.38(s,3H),3.32–3.18(m,2H),2.53–2.48(m,1H),2.09–1.94(m,2H),1.91– 1.75(m,2H),1.56(dd,J＝7.4,5.2Hz,1H),1.44(d,J＝7.5Hz,1H),1.27(d,J＝6.2 Hz,3H),1.02(s,3H),0.98(s,9H),0.85(s,3H).
Example 27:
a reaction was carried out by changing the substrate t-butyl (R) - (1-chloro-1-oxopropan-2-yl) carbamate in step 5 to t-butyl (R) - (1-chloro-3-methyl-1-oxobutan-2-yl) carbamate, and the preparation method of example 26 was otherwise followed. 1 H NMR(400MHz,DMSO-d6)δ4.54–4.46(m,1H),4.27(s,1H),4.21(s, 2H),4.15(s,2H),3.89–3.42(m,496H),3.38(s,3H),3.32–3.18(m,2H),2.53– 2.48(m,1H),2.13–2.04(m,1H),2.09–1.94(m,2H),1.91–1.75(m,2H),1.56(dd, J＝7.4,5.2Hz,1H),1.44(d,J＝7.5Hz,1H),1.02(s,3H),0.99(s,9H),0.93–0.89(m, 6H),0.85(s,3H).
Example 28:
a reaction was carried out by changing the substrate t-butyl (R) - (1-chloro-1-oxopropan-2-yl) carbamate in step 5 to t-butyl (R) - (1-chloro-4-methyl-1-oxopentan-2-yl) carbamate, and the preparation method of example 26 was otherwise followed. 1 H NMR(400MHz,DMSO-d6)δ4.55–4.47(m,1H),4.26(s,1H),4.21(s,2H),4.16(s, 2H),3.88–3.43(m,496H),3.38(s,3H),3.32–3.18(m,2H),2.53–2.48(m,1H),2.09–1.94(m, 2H),1.91–1.75(m,2H),1.62–1.57(m,3H),1.56(dd,J＝7.4,5.2Hz,1H),1.44(d,J＝7.5Hz, 1H),1.02(s,3H),0.98(s,9H),0.89(d,J＝6.1Hz,6H),0.83(s,3H).
Example 29:
a reaction was carried out by changing the substrate (R) - (1-chloro-1-oxopropan-2-yl) carbamic acid tert-butyl ester in step 5 to 4-aminobenzyl carbonyl chloride, otherwise prepared as in example 26The preparation method is as follows. 1 H NMR(400MHz, DMSO-d6)δ7.62–7.57(m,2H),7.35–7.31(m,2H),5.16(s,2H),4.52–4.46(m, 1H),4.39(s,1H),4.18(s,1H),4.09(s,2H),3.88–3.43(m,496H),3.38(s,3H),3.31– 3.22(m,2H),2.51–2.45(m,1H),2.08–1.94(m,2H),1.91–1.79(m,2H),1.55(dd, J＝7.4,5.2Hz,1H),1.43(d,J＝7.5Hz,1H),1.36(s,9H),1.05–0.99(m,12H),0.85 (s,3H).
Example 30:
the substrate (R) - (1-chloro-1-oxopropan-2-yl) carbamic acid tert-butyl ester in step 5 was changed to (S) -4- (2-aminopropionamido) benzyl carbonyl chloride and reacted by the same way as in example 26. 1 H NMR (400MHz,DMSO-d6)δ7.56(d,J＝7.8Hz,2H),7.33(d,J＝7.9Hz,2H),5.16(s, 2H),4.51–4.42(m,2H),4.36(s,1H),4.19(s,1H),4.02(s,2H),3.91–3.43(m, 496H),3.39(s,3H),3.31–3.22(m,2H),2.51–2.45(m,1H),2.08–1.94(m,2H), 1.91–1.79(m,2H),1.55(dd,J＝7.4,5.2Hz,1H),1.43(d,J＝7.5Hz,1H),1.39(s, 9H),1.32(d,J＝5.8Hz,3H),1.05(s,3H),0.97(s,9H),0.84(s,3H).
Example 31:
step 1: a methanolic solution of ammonia (7M; 0.53L,3.68 mol) and methyl (S) -2- ((N, N-di-tert-butoxycarbonyl) amino) -3- ((S) -2-oxopyrrolidin-3-yl) propionate (20 g,0.22 mol) were added to the reaction flask and the reaction mixture was stirred at 25℃for 48 hours. Concentration gave M-44 (18.4 g, 96%) as a yellow solid. 1 H NMR(400MHz,Methanol-d4)d 4.10(dd,J＝11.0,3.8Hz,1H),3.40– 3.31(m,2H),2.48(dh,J＝9.8,4.5Hz,1H),2.41–2.28(m,1H),2.04(ddd,J＝14.1, 11.1,4.4Hz,1H),1.87(dq,J＝12.5,8.8Hz,1H),1.74(ddd,J＝14.0,10.3,4.1Hz, 1H),1.45(s,18H).
Step 2: methyl N- (triethylammonium sulfonyl) carbamate (primary)A Gibbs reagent; 3 eq) was added to a solution of M-44 (18 g,1 eq) in DCM (20 mL) and left to stir at room temperature for 2 hours. TLC monitored reaction was complete. The dichloromethane was removed under reduced pressure, the residue was dissolved in ethyl acetate (200 mL), washed twice with a mixture of saturated aqueous sodium bicarbonate (200 mL) and saturated aqueous sodium chloride (100 mL), and dried over anhydrous sodium sulfate. The ethyl acetate was removed under reduced pressure, and the residue was purified by column chromatography to give compound M-45 (13.3 g) as a white solid in 78% yield. 1 H NMR(400MHz,Methanol-d4)δ4.72–4.61(m,1H), 3.33–3.22(m,2H),2.59–2.53(m,1H),2.38–2.31(m,1H),2.24–2.16(m,1H), 1.98–1.92(m,1H),1.88–1.82(m,1H),1.46(s,18H).
Step 3: compound M-45 (10 g,1 eq) and DCM (30 mL) were added to the reaction flask and stirred. And (5) protecting the reaction bottle by nitrogen. NaH (3 eq) was added in portions to the above flask under ice bath. After stirring for 10 minutes under nitrogen, the compound (9H-fluoren-9-yl) methyl- (1-chloro-1-oxypropane-2-yl) carbamate (1.2 eq) was added to the reaction system. The reaction was continued for 8 hours in ice bath and was complete by TLC. The reaction was slowly poured into ice water, extracted with DCM (60 ml×3), the organic phases were combined, washed with NaCl solution, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure to give crude product, which was purified by column chromatography to give compound M-46 (12.4 g) as a white solid in 68% yield. 1 H NMR(400MHz,Methanol-d4)δ7.84–7.80(m,2H),7.64 –7.60(m,2H),7.50(td,J＝7.7,1.2Hz,2H),7.41(td,J＝7.8,1.4Hz,2H),5.45– 5.38(m,1H),4.59–4.49(m,1H),4.42(s,2H),3.93–3.85(m,1H),3.76–3.69(m, 1H),3.67–3.60(m,1H),2.73–2.67(m,1H),2.18–2.10(m,1H),1.98–1.90(m, 2H),1.85–1.78(m,1H),1.45(s,18H),1.36(d,J＝6.6Hz,2H).
Step 4: m-46 (12 g) was dissolved in DMF (40 mL), piperidine (15 mL) was added, and the mixture was stirred at room temperature for 2 hours. TLC checked the reaction was complete and stopped. The reaction was slowly poured into ice water, extracted with DCM (100 ml×3), the organic phases were combined, washed 3 times with saturated NaCl solution, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure to give crude product, which was purified by column chromatography to give compound M-47 (7.5 g) as a white solid in 95% yield. 1 H NMR(400MHz,Methanol-d4)δ4.59–4.49(m,1H),3.93–3.85(m,1H), 3.76–3.69(m,1H),3.67–3.60(m,1H),2.73–2.67(m,1H),2.18–2.10(m,1H), 1.98–1.90(m,2H),1.85–1.78(m,1H),1.45(s,18H),1.36(d,J＝6.6Hz,2H).
Step 5: mPEG-5K-COOH (5 g,1 eq), M-47 (1.2 eq), HOBt (2 eq) and DMF (100 mL) were added sequentially to a round bottom flask and stirred at room temperature for 15min. N, N' -diisopropylcarbodiimide (2 eq) was added to the reaction system, followed by a reaction at room temperature overnight. TLC checked the reaction was complete and stopped. Cold methyl tert-butyl ether (500 mL) was added to the reaction system, a large amount of solids were precipitated, and the mixture was suction-filtered and dried to give the desired compound M-48 (4.9 g) in 91% yield. 1 H NMR(400MHz,CDCl 3 ) δ4.60–4.48(m,1H),4.17(s,2H),3.92–3.42(m,499H),3.38(s,3H),2.73–2.67(m, 1H),2.18–2.10(m,1H),1.99–1.92(m,2H),1.88–1.80(m,1H),1.46(s,18H), 1.35(d,J＝6.6Hz,2H).
Step 6: m-48 (4 g,1 eq), TFA (15 eq) and DCM (30 mL) were added sequentially to a round bottom flask and reacted overnight at room temperature. TLC checked the reaction was complete and stopped. The reaction solution was poured into 100mL of water, naHCO 3 The pH was adjusted to weakly alkaline, the mixture was extracted with DCM (100 mL. Times.3), the organic phases were combined, washed with NaCl solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give crude M-49 (3.7 g) in 97% yield, which was directly taken to the next reaction without further purification. 1 H NMR(400MHz,CDCl 3 )δ4.71–4.65(m, 1H),4.16(s,2H),3.91–3.41(m,499H),3.37(s,3H),2.73–2.67(m,1H),2.18–2.11 (m,1H),2.03–1.94(m,2H),1.81–1.74(m,1H),1.32(d,J＝6.6Hz,2H).
Step 7: m-3 (0.2 g,1 eq), M-49 (1 eq) and isopropanol were added to the reaction flask and left to stir at room temperature. 1d hydrochloric acid is added into the reaction system, and reflux reaction is carried out for 3h. TLC checked the reaction was complete and stopped. The reaction solution was poured into 100mL of water, naHCO 3 The pH was adjusted to weakly alkaline, the mixture was extracted with DCM (100 mL. Times.3), the organic phases were combined, washed with NaCl solution, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure to give crude product, and the crude product was purified by column chromatography to give Ic-1 (2.8 g) as a pale yellow solid in 95% yield. 1 H NMR(400MHz, CDCl 3 )δ8.05(d,J＝12.6Hz,1H),7.36(d,J＝8.6Hz,1H),4.64–4.50(m,3H), 4.16(s,2H),4.10–4.02(m,1H),3.89–3.42(m,496H),3.38(s,3H),2.80–2.73(m, 1H),2.19–2.12(m,1H),2.07–1.98(m,2H),1.86–1.78(m,1H),1.46–1.41(m, 1H),1.34(d,J＝6.0Hz,3H),1.10(s,3H),1.02(s,4H),0.94(s,9H).
Example 32:
the substrate (9H-fluoren-9-yl) methyl- (1-chloro-1-oxypropane-2-yl) carbamate in step 3 was converted to (9H-fluoren-9-yl) methyl- (1-chloro-3-methyl-1-oxybutan-2-yl) carbamate for reaction, and the preparation method was otherwise the same as that of example 31. 1 H NMR(400MHz,CDCl 3 )δ8.04(d,J＝12.6 Hz,1H),7.35(d,J＝8.6Hz,1H),4.64–4.50(m,3H),4.17(s,2H),4.10–4.01(m, 1H),3.89–3.41(m,496H),3.38(s,3H),2.80–2.73(m,1H),2.19–2.12(m,1H), 2.07–1.98(m,3H),1.86–1.78(m,1H),1.46–1.41(m,1H),1.10(s,3H),1.02(s, 4H),0.94(s,9H),0.87(d,J＝7.1,6H).
Example 33:
the substrate (9H-fluoren-9-yl) methyl- (1-chloro-1-oxypropane-2-yl) carbamate in step 3 was converted to (9H-fluoren-9-yl) methyl- (1-chloro-4-methyl-1-oxolan-2-yl) carbamate and reacted by the same method as in example 31. 1 H NMR(400MHz,CDCl 3 )δ8.06(d,J＝12.6 Hz,1H),7.36(d,J＝8.6Hz,1H),4.64–4.50(m,3H),4.16(s,2H),4.10–4.02(m, 1H),3.89–3.42(m,496H),3.38(s,3H),2.80–2.73(m,1H),2.19–2.12(m,1H), 2.07–1.98(m,2H),1.86–1.78(m,1H),1.72–1.56(m,3H),1.46–1.41(m,1H), 1.10(s,3H),1.02(s,4H),0.93(s,9H),0.87(d,J＝6.4Hz,6H).
Example 34:
the substrate (9H-fluoren-9-yl) methyl- (1-chloro-1-oxypropane-2-yl) carbamate in step 3 was converted to 4- ((9H-fluoren-9-yl) methoxy) carbonyl) amino) benzylcarbonyl chloride for reaction, and the preparation method of example 31 was otherwise followed. 1 H NMR(600MHz,DMSO-d6)δ9.43(d,J＝8.4Hz,1H), 9.03(d,J＝8.6Hz,1H),7.60(d,J＝7.9Hz,2H),7.35–7.31(m,2H),5.01–4.96(m, 1H),4.41(d,J＝8.4Hz,1H),4.17–4.13(m,3H),3.89–3.42(m,497H),3.39–3.35 (m,5H),3.17–3.11(m,1H),3.05–3.02(m,1H),2.41–2.38(m,1H),2.15–2.12 (m,1H),2.11–2.03(m,1H),1.76–1.65(m,2H),1.57(dd,J＝7.6,5.5Hz,1H),1.32 (d,J＝7.6Hz,1H),1.02(s,3H),0.99(s,9H),0.83(s,3H)。

Claims (10)

1. A polyethylene glycol-nemaltevir prodrug compound characterized by having the structure shown in formula I:
wherein R is 1 ，R 2 ，R 3 Each independently is hydrogen or a PEG modifier, and R 1 ，R 2 ，R 3 Are not hydrogen at the same time;
the PEG modifier is L-D; wherein L is a linking group, D is a linear, Y-type or multi-branched polyethylene glycol;
or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein L is selected from the group consisting of:
wherein R is 1-5 、R 10 、R 19 、R 24 、R 25 、R 30 And R is 31 Independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-6 alkenyl, C6-12 aryl, C6-12 aralkyl;
R 6-9 、R 11-18 、R 20-23 、R 26-29 independently selected from: -H, -F, -Cl, -Br, -I, nitro, amino, hydroxy, mercapto, cyano, alkynyl, alkenyl, C1-C10 acyloxy, C1-C10 alkyl, C1-C10 alkoxy, C1-C10 alkyl substituted amino;
x is independently selected from:
wherein n and m are each independently integers of 1 to 30.
3. The compound of claim 1, wherein D is a linear polyethylene glycol having a structure of formula IIa or IIb:
wherein p and q are each independently integers of 1 to 500.
4. The compound of claim 1, wherein D is a Y-type polyethylene glycol having a structure of formula IIIa or IIIb:
wherein i and h are each independently integers of 1 to 500.
5. The compound of claim 1, wherein D is a branched polyethylene glycol having a structure according to formula IVa, IVb, IVc or IVd:
wherein k and y are each independently integers of 1 to 500;
j. each w is independently an integer of 1 to 10.
6. A compound according to claim 1, selected from:
or a pharmaceutically acceptable salt thereof.
7. A pharmaceutical composition comprising a compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
8. A process for the preparation of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, comprising the steps of:
wherein D, L are as defined in claim 1.
9. A process for the preparation of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, comprising the steps of:
wherein D, L are as defined in claim 1.
10. Use of a compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 7 in the manufacture of a medicament for the prevention and/or treatment of new coronapneumonitis.
CN202210284778.4A 2022-03-22 2022-03-22 Polyethylene glycol-nemaltevir prodrug as well as preparation method and application thereof Pending CN116808229A (en)

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