阅读说明：本技术 一种硝基咪唑类衍生物及其制备方法和应用 (Nitroimidazole derivative and preparation method and application thereof ) 是由 吉训明 吴泽辉 孙雨丽 程雪波 陈华龙 于 2021-06-16 设计创作，主要内容包括：本发明提供了一种硝基咪唑类衍生物,其结构式为式I所示；其中,R-(1)为R-(4)选自H或者C1-8的烷基、饱和烷氧基或酰胺基中的任一种,R-(5)选自C1-8的烷基、饱和烷氧基或酰胺基,取代基选自-(CH-(2))n、-(OCH-(2)CH-(2))n-,n＝1-4的整数；R-(2)选自C1-8的烷基、饱和烷氧基或酰胺基,取代基选自-(CH-(2))n、-(OCH-(2)CH-(2))n-中的任一种,其中n＝1-4的整数；R-(3)选自-(CH-(2))-(m)、-(CH-(2))-(m)-CH-COOH、m＝1-7的整数。本发明还提供了上述硝基咪唑衍生物的制备方法和应用。本发明提供的硝基咪唑衍生物改善了显像剂的药代动力学。(The invention provides a nitroimidazole derivative, the structural formula of which is shown in formula I; wherein R is 1 Is composed of R 4 Selected from H or any one of alkyl, saturated alkoxy or amido of C1-8, R 5 Selected from C1-8 alkyl, saturated alkoxy or amido, and the substituent is selected from- (CH) 2 )n、‑(OCH 2 CH 2 ) n-, n-is an integer of 1 to 4; r 2 Selected from C1-8 alkyl, saturated alkoxy or amido, and the substituent is selected from- (CH) 2 )n、‑(OCH 2 CH 2 ) n-is any one ofWherein n is an integer of 1 to 4; r 3 Is selected from- (CH) 2 ) m 、‑(CH 2 ) m ‑CH‑COOH、 m is an integer of 1 to 7. The invention also provides a preparation method and application of the nitroimidazole derivative. The nitroimidazole derivatives provided by the invention improve the pharmacokinetics of the imaging agent.)

1. A nitroimidazole derivative is characterized in that the structural formula is shown as formula I,

wherein the content of the first and second substances,

R₁is composed ofR₄Selected from H or any one of alkyl, saturated alkoxy or amido of C1-8, R₅Selected from C1-8 alkyl, saturated alkoxy or amido, and the substituent is selected from- (CH)₂)_n、-(OCH₂CH₂)_n-n is an integer from 1 to 4;

R₂selected from C1-8 alkyl, saturated alkoxy or amido, and the substituent is selected from- (CH)₂)_n、-(OCH₂CH₂)_n-any one of (a) and (b), wherein n is an integer from 1 to 4;

R₃is selected from- (CH)₂)_m、-(CH₂)_m-CH-COOH、m is an integer of 1 to 7.

2. The nitroimidazole derivative of claim 1, selected from any one of the compounds of formula II, III or IV,

wherein R is C1-8 alkyl, saturated alkoxy or amido, and the substituent is selected from- (CH)₂)_n、-(OCH₂CH₂)_nAnd n is an integer of 1 to 4.

3. As in claimThe nitroimidazole derivative according to claim 1 or 2, which is selected from the group consisting of nitroimidazole derivatives having a structural formulaOrAny one of the compounds of (1).

4. The nitroimidazole derivative of any one of claims 1-3, wherein F is¹⁹F or¹⁸F。

5. The process for the preparation of nitroimidazole derivatives according to any one of claims 1 to 4, comprising:

providing a precursor compound with a structural formula V, VI or VII, carrying out fluorination reaction to obtain an intermediate compound, and hydrolyzing the intermediate compound to obtain a nitroimidazole derivative;

wherein R is₆And R₇The amino protecting groups are respectively selected independently and are the same or different, and the amino protecting group is preferably any one of 2,4, 6-trimethoxybenzyl, 4-methoxybenzyl or tert-butylcarbonyl;

R₈is any one of tert-butyloxycarbonyl (Boc), Trityl (Trityl), 2,4, 6-trimethoxybenzyl, 2, 4-dimethoxybenzyl or 4-methoxybenzyl;

LG is a leaving group, preferably a sulfonate leaving group or a halogen.

6. The method according to claim 5, wherein when the nitroimidazole is a labeled compound, the method further comprises:

a) will be dried¹⁸F]F^-18-crown-6/KHCO₃Adding the complex into a solution containing a precursor compound to obtain a reaction solution; the solvent of the solution is prepared by mixing the following components in a volume ratio of 9: 1 mixed t-amyl alcohol/acetonitrile solution;

b) reacting the reaction solution for 5-20min at the temperature of 80-120 ℃;

c) after the reaction is finished and cooled, adding proper amount of high-purity water, and passing through an activated Oasis HLB solid-phase extraction column; preferably, the Oasis column is activated by a method comprising the steps of: eluting the Oasis column with 10mL of ethanol, then drying the column with 10mL of air, eluting the column with 10mL of high-purity water, and finally drying the column with 10mL of air;

d) washing the Oasis HLB solid phase extraction column with high-purity water, pushing air to dry the column, and leaching the intermediate adsorbed on the column with 1mL of ethanol into a clean container;

e) removing ethanol in a container containing the intermediate, adding 1mL of trifluoroacetic acid, uniformly mixing by vortex, placing in an oil bath at 50 ℃ for reacting for 5min, removing the solvent after the reaction is finished, and adjusting the pH to be neutral; preferably, the ethanol is removed by nitrogen sparging;

f) adding 1mL of physiological saline, uniformly mixing by vortex, and filtering by using a sterile filter membrane of 0.22 mu m to obtain the composition.

7. The method of claim 6, wherein the [ alpha ], [ beta ] -is¹⁸F]F^-18-crown-6/KHCO₃The complex is prepared by a method comprising the following steps:

1) will accelerator produced¹⁸F]F^-The solution is passed through a QMA chromatography column to cause the solution to pass through¹⁸F]F^-Is adsorbed on a chromatographic column;

preferably, the QMA chromatography column is activated by a method comprising the steps of: 10mL of 0.5M sodium bicarbonate solution is used for leaching the QMA column, 10mL of air is used for drying, 10mL of high-purity water is used for leaching, and 10mL of air is used for drying;

2) 1mL of 18-crown-6/KHCO₃The leacheate will¹⁸F]F^-Eluting from QMA column to obtain eluate;

preferably, the leacheate is formulated by a method comprising the steps of:

uniformly mixing an 18-crown-6/acetonitrile solution with the concentration of 20-35mg/mL and a potassium bicarbonate aqueous solution with the concentration of 20-35mg/mL according to the volume ratio of 4: 1-7: 1 to obtain the compound;

3) the eluent is put in an oil bath at 100 ℃ and is added with N₂Blow drying to obtain the product¹⁸F]F^-18-crown-6/KHCO₃A crude complex;

preferably, the method further comprises 4) adding anhydrous acetonitrile into the crude complex product for azeotropic dehydration, and repeating the steps for a plurality of times to obtain dried [ 2 ]¹⁸F]F^-18-crown-6/KHCO₃A complex compound.

8. Use of nitroimidazole derivatives according to any one of claims 1 to 4 for the preparation of imaging agents for the diagnosis of hypoxia-related diseases.

9. The use of claim 8, wherein the hypoxia-related disease is selected from one or more of a tumor, stroke, and atherosclerosis.

10. A PET imaging agent for diagnosing hypoxia-related diseases, comprising the nitroimidazole derivative according to any one of claims 1 to 4.

Technical Field

The invention relates to the field of nuclear medicine PET imaging agents, in particular to nitroimidazole derivatives capable of being used for preparing imaging agents, and a preparation method and application thereof.

Background

Hypoxia, also known as hypoxia, is a state in which the intracellular concentration of oxygen is too low to meet metabolic demand, and is caused by an imbalance between oxygen supply and consumption. Hypoxia can be generally classified into perfusion-related (acute) hypoxia caused by insufficient blood flow, diffusion-related (chronic) hypoxia caused as the diffusion distance increases for tumor growth, and anemic hypoxia caused by decreased oxygen transport capacity. Hypoxia is a common feature of most solid malignancies, directly affecting clinical response to therapy by affecting tumor growth, metastatic ability and resistance to cell death. Hypoxia is an important effect phenomenon of ischemia, is a main factor of diseases such as stroke and myocardial infarction, and plays a role in structural and functional changes of chronic cardiovascular and cerebrovascular diseases. Treatment of stroke is critical by identifying hypoxic tissue and determining the extent of the penumbra. Therefore, the development of noninvasive modern medical imaging PET imaging agent for early diagnosis and treatment of diseases characterized by hypoxia is beneficial to reducing morbidity and mortality.

Hypoxic PET imaging agents can selectively reside in hypoxic cells. The degree of hypoxia is diagnosed by detecting the radioactivity intensity of the imaging agent concentrated in the lesion tissue by PET. The present hypoxia imaging agent mainly aims at the identification and diagnosis of tumor hypoxia area, and the PET hypoxia imaging medicine with the most extensive clinical research is' 2¹⁸F]FMISO, which is a¹⁸Analogs of the F-labeled cell sensitizer MISO, which bind selectively to hypoxic cells. But because of the low absolute uptake of the tumor tissue in vivo, the liver uptake is too highThe clearance rate of high, non-target tissue is slower, resulting in longer injection and imaging intervals (above 90 min), and a low target/non-target ratio. On the basis of the tumor hypoxia imaging agent, tumor hypoxia imaging agents with excellent properties are developed at home and abroad, such as: preparation of nitroimidazole N-linked arabinoside¹⁸F]FAZA, preparation of 2, 3-butanediol by changing N-linked 2-propanol¹⁸F]FETNIM, nitroimidazole derivative [ 2 ] modified with a triazole ring¹⁸F]HX4, branched chain using amide¹⁸F]EF5，⁶⁴Cu-labeled bis (thiosemicarbazide) metal chelate complex [ 2 ]⁶⁴Cu]ATSM and [ 2 ]⁶⁴Cu]ATSE, all achieved relatively satisfactory results in tumor hypoxia imaging. However, the hypoxic probe is developed for tumor hypoxia diagnosis, so that the hypoxic probe has certain limitation when being applied to brain hypoxia imaging. For example: [¹⁸F]Due to the fact that FMISO mainly passively diffuses, PET data acquisition can be carried out only after 2 hours after injection, signal-to-noise ratio is too small, and image contrast is not obvious, so that PET imaging agents with targeting property, high brain intake, high pharmacokinetics and high signal-to-noise ratio still need to be further broken through.

Disclosure of Invention

In order to overcome the above [ 2 ]¹⁸F]The invention relates to a series of poor pharmacokinetics problems caused by FMISO passive diffusion, in particular to nitroimidazole derivatives containing amino acid characteristic groups, which are obtained by introducing different types of amino acids by taking nitroimidazole as a basic framework and aims to provide a method suitable for being used¹⁸Novel F-labelled nitroimidazole derivatives and corresponding derivatives¹⁸F-labeled nitroimidazole derivatives, their preparation and use¹⁹F-labeled analogs and other uses as reference standards, methods of making such compounds, compositions comprising such compounds, kits comprising such compounds or compositions, and uses of such compounds, compositions, or kits for Positron Emission Tomography (PET) imaging of hypoxia-related critical diseases such as tumors, stroke, atherosclerosis, and the like.

One of the purposes of the invention is to provide nitroimidazole derivatives containing amino acid characteristic groups. The compound has a hypoxia response characteristic group and an amino acid transmembrane transport characteristic group, belongs to nitroimidazole derivatives for diagnosing hypoxia-related diseases, and has a structural formula shown in formula I,

wherein the content of the first and second substances,

R₁is composed ofR₄Selected from H or any one of alkyl, saturated alkoxy or amido of C1-8, R₅Selected from C1-8 alkyl, saturated alkoxy or amido, and the substituent is selected from- (CH)₂)n、-(OCH₂CH₂)_n-n is an integer from 1 to 4;

R₂selected from C1-8 alkyl, saturated alkoxy or amido, and the substituent is selected from- (CH)₂)_n、-(OCH₂CH₂)_n-any one of (a) and (b), wherein n is an integer from 1 to 4;

R₃is selected from- (CH)₂)_m、-(CH₂)_m-CH-COOH、Wherein m is an integer of 1 to 7.

In one embodiment according to the invention, the nitroimidazole derivative is selected from any one of the compounds with the structural formula II, III or IV,

wherein R is C1-8 alkyl, saturated alkoxy or amido, and the substituent is selected from- (CH)₂)n、-(OCH₂CH₂) n-1-4.

In one embodiment according to the present invention, the nitroimidazoles are selected from those of the formula

Any one of the compounds of (1).

In one embodiment according to the invention, F is¹⁹F or¹⁸F。

The invention also provides a preparation method of the nitroimidazole derivative, which comprises the following steps:

providing a precursor compound with a structural formula V, VI or VII, carrying out fluorination reaction to obtain an intermediate compound, and hydrolyzing the intermediate compound to obtain a nitroimidazole derivative;

wherein R is₆And R₇The amino protecting groups are respectively selected independently and are the same or different, and the amino protecting group is preferably any one of 2,4, 6-trimethoxybenzyl, 4-methoxybenzyl or tert-butylcarbonyl;

R₈is any one of tert-butyloxycarbonyl (Boc), Trityl (Trityl), 2,4, 6-trimethoxybenzyl, 2, 4-dimethoxybenzyl or 4-methoxybenzyl;

LG is a leaving group, preferably a sulfonate leaving group or a halogen.

In one embodiment according to the present invention, when the nitroimidazole is a labeling compound, further comprising:

a) will be dried¹⁸F]F^-18-crown-6/KHCO₃Adding the complex into a solution containing a precursor compound to obtain a reaction solution; the solvent of the solution is prepared by mixing the following components in a volume ratio of 9: 1 mixed t-amyl alcohol/acetonitrile solution;

b) reacting the reaction solution for 5-20min at the temperature of 80-120 ℃;

c) after the reaction is finished and cooled, adding proper amount of high-purity water, and passing through an activated Oasis HLB solid-phase extraction column; preferably, the Oasis column is activated by a method comprising the steps of: eluting the Oasis column with 10mL of ethanol, then drying the column with 10mL of air, eluting the column with 10mL of high-purity water, and finally drying the column with 10mL of air;

d) washing the Oasis HLB solid phase extraction column with high-purity water, pushing air to dry the column, and leaching the intermediate adsorbed on the column with 1mL of ethanol into a clean container;

e) removing ethanol in a container containing the intermediate, adding 1mL of trifluoroacetic acid, uniformly mixing by vortex, placing in an oil bath at 50 ℃ for reacting for 5min, removing the solvent after the reaction is finished, and adjusting the pH to be neutral; preferably, the ethanol is removed by nitrogen sparging;

f) adding 1mL of physiological saline, uniformly mixing by vortex, and filtering by using a sterile filter membrane of 0.22 mu m to obtain the composition.

In one embodiment according to the invention, the [ alpha ], [ beta ] or a¹⁸F]F^-18-crown-6/KHCO₃The complex is prepared by a method comprising the following steps:

1) will accelerator produced¹⁸F]F^-The solution is passed through a QMA chromatography column to cause the solution to pass through¹⁸F]F^-Is adsorbed on a chromatographic column;

preferably, the QMA chromatography column is activated by a method comprising the steps of: 10mL of 0.5M sodium bicarbonate solution is used for leaching the QMA column, 10mL of air is used for drying, 10mL of high-purity water is used for leaching, and 10mL of air is used for drying;

2) 1mL of 18-crown-6/KHCO₃The leacheate will¹⁸F]F^-Eluting from QMA column to obtain eluate;

preferably, the leacheate is formulated by a method comprising the steps of:

uniformly mixing an 18-crown-6/acetonitrile solution with the concentration of 20-35mg/mL and a potassium bicarbonate aqueous solution with the concentration of 20-35mg/mL according to the volume ratio of 4: 1-7: 1 to obtain the compound;

3) the eluent is put in an oil bath at 100 ℃ and is added with N₂Blow drying to obtain the product¹⁸F]F^-18-crown-6/KHCO₃A crude complex;

preferably, the method further comprises 4) adding anhydrous acetonitrile into the crude complex product for azeotropic dehydration, and repeating the steps for a plurality of times to obtain dried [ 2 ]¹⁸F]F^-18-crown-6/KHCO₃A complex compound.

The invention provides application of the nitroimidazole derivative in preparing an imaging agent for diagnosing hypoxia-related diseases.

Preferably, the hypoxia-related disease is selected from one or more of a tumor, stroke, and atherosclerosis.

The invention further provides a PET imaging agent for diagnosing hypoxia-related diseases, which comprises the nitroimidazole derivatives.

It is another object of the present invention to provide the use of the hypoxic imaging agent in the diagnosis of tumors, stroke and atherosclerosis.

The technical scheme of the invention has the following beneficial effects:

the nitroimidazole derivative containing the amino acid characteristic group provided by the invention has the amino acid characteristic group, so that the nitroimidazole derivative can enter cells by means of a transport carrier of amino acid, and has stronger targeting property. The nitroimidazole derivative provided by the invention has high uptake in pathological parts such as tumors and the like, effectively improves the signal-to-noise ratio in the image diagnosis process, and animal experiments show that the nitroimidazole derivative provided by the invention obviously improves the pharmacokinetic performance of a hypoxia imaging agent.

Drawings

FIG. 1 is a PET image of the tumor of example 4, with the encircled areas representing the tumor.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

The preparation method of the compound shown in the formula II is shown in the reaction formula I:

using a compound containing a labeled precursor of formula V and containing an activating group, carrying out fluorination to obtain an intermediate compound, and carrying out hydrolysis to obtain the compound of formula I.

The preparation method of the compound shown in the formula III is shown in a reaction formula II:

using a compound containing an activating group and a labeled precursor shown as a formula VI, fluorinating to obtain an intermediate compound, and hydrolyzing to obtain a compound shown as a formula III.

The preparation method of the compound shown in the formula IV is shown as a reaction formula III:

using a labeled precursor compound containing an activating group, fluorinating to obtain an intermediate compound, and hydrolyzing to obtain a compound of a formula IV.

In the above reaction formulae I, II, III,

r is C1-8 alkyl, saturated alkoxy or amido, and the substituent is selected from- (CH)₂)n、-(OCH₂CH₂) n- (n is an integer of 1 to 4);

f is¹⁹F or¹⁸F；

Wherein R is₆And R₇The amino protecting groups are respectively selected independently and are the same or different, and the amino protecting group is preferably any one of 2,4, 6-trimethoxybenzyl, 4-methoxybenzyl or tert-butylcarbonyl;

R₈is any one of tert-butyloxycarbonyl (Boc), Trityl (Trityl), 2,4, 6-trimethoxybenzyl, 2, 4-dimethoxybenzyl or 4-methoxybenzyl;

LG is a leaving group, preferably a sulfonate leaving group or a halogen.

Example 1(S) -2-amino-3- (4- (2- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) ethoxy) phenyl) propanoic acid

The structural formula is as follows:

the synthetic route is as follows:

1) tert-butyl (2S) -3- (4- (2- (benzyl (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) ethoxy) phenyl) -2- ((tert-butoxycarbonyl) amino) propionate

Tert-butyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (tolyloxy) ethoxy) phenyl) propanoate (2g, 3.74mmol) was dissolved in 40mL acetonitrile and N-benzyl-3- ((tetrahydro-2H-pyran-2-yl) oxy) propan-1-amine (1.02g, 4.11mmol), sodium iodide (500mg, 2.24mmol) and potassium carbonate (1.03g, 7.48mmol) were added in that order and refluxed at 100 ℃ for 28H. Then filtered, the filtrate was spin-dried and taken up, by flash chromatography (petroleum ether: ethyl acetate ═ 80:20), to give tert-butyl (2S) -3- (4- (2- (benzyl (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) ethoxy) phenyl) -2- ((tert-butoxycarbonyl) amino) propionate (1.4g, 70.0%).¹H NMR(300MHz,CDCl₃)δ7.40–7.28(m,5H),7.06(d,J＝8.4Hz,2H),6.78(d,J＝8.3Hz,2H),4.97(d,J＝8.0Hz,1H),4.54(s,1H),4.41(d,J＝4.8Hz,1H),4.00(t,J＝6.0Hz,2H),3.92–3.63(m,4H),3.57–3.36(m,2H),3.01-2.98(d,J＝5.5Hz,1H),2.89(t,J＝5.5Hz,1H),2.78–2.53(m,1H),1.91–1.61(m,4H),1.46-1.41(m,18H).HRMS calcd for C35H52N2O7 612.3775；found,613.1832[M+H]⁺。

2) Tert-butyl (2S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- ((3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) ethoxy) phenyl) propionate

Tert-butyl (2S) -3- (4- (2- (benzyl (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) ethoxy) phenyl) -2- ((tert-butoxycarbonyl) amino) propionate (1).4g, 2.28mmol) was dissolved in ethanol, palladium on carbon (800mg, 7.54mmol) was added, vacuum was applied, and H was charged₂The reaction was allowed to proceed at room temperature for 2H, then filtered through celite, the filtrate was spun dry and taken up by flash chromatography (dichloromethane: methanol ═ 95:5) to give tert-butyl (2S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- ((3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) ethoxy) phenyl) propionate (1.09g, 92.1%).¹H NMR(300MHz,CDCl₃)δ7.08(d,J＝8.4Hz,2H),6.83(d,J＝8.5Hz,2H),4.98(d,J＝7.7Hz,1H),4.60(s,1H),4.41(d,J＝6.9Hz,1H),4.09(t,J＝4.9Hz,2H),3.87-3.84(m,2H),3.54-3.49(m,2H),3.04-2.98(m,4H),2.85(t,J＝6.8Hz,1H),1.89-1.87(m,4H),1.44(s,9H),1.42(s,9H).HRMS calcd for C28H46N2O7522.3305；found,523.1219[M+H]⁺.

3) Tert-butyl (2S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) acetamido) ethoxy) phenyl) propionate

2- (2-Nitroimidazol-1-yl) acetic acid (253mg, 1.48mmol) was dissolved in 20mL of DMF, and 2mL of triethylamine and tert-butyl (2S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- ((3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) ethoxy) phenyl) propionate (700mg, 1.34mmol) were added, and HBTU (1.27g, 3.35mmol) was added and reacted at room temperature for 2H. Then extracting with ethyl acetate, washing with saturated saline solution for three times, collecting organic phase layer, and adding Na₂SO₄Dried and purified by flash purification chromatography (petroleum ether: ethyl acetate 25:75) to give tert-butyl (2S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) acetamido) ethoxy) phenyl) propionate (600mg, 66.3%).¹H NMR(300MHz,CDCl₃)δ7.24–7.03(m,4H),6.87-6.78(m,2H),5.48-5.41(m,2H),4.98(d,J＝7.1Hz,1H),4.55(t,J＝7.1Hz,1H),4.40(s,1H),4.17-4.10(m,1H),3.98–3.43(m,8H),2.07–1.71(m,4H),1.44(s,18H).HRMS calcd for C33H49N5O10 675.3479；found,676.2810[M+H]⁺.

4) Tert-butyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (N- (3-hydroxypropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) benzene) propionate

Tert-butyl (2S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) acetamido) ethoxy) phenyl) propionate (1.5g, 2.22mmol) was dissolved in a mixed solution of 20mL of ethanol and 20mL of tetrahydrofuran, pyridine p-toluenesulfonate (335mg, 1.33mmol) was added, reacted at 50 ℃ for 2H, followed by spin-drying and sample-stirring, and subjected to flash purification chromatography (dichloromethane: methanol ═ 95:5) to give tert-butyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (N- (3-hydroxypropyl) -2- (2-nitro-1H-yl) amino) -3- (4-nitro-1H-methyl ether) -imidazol-1-yl) acetamido) benzene) propionate (600mg, 42.7%).¹H NMR(300MHz,CDCl₃)δ7.26–7.01(m,4H),6.87-6.78(d,J＝8.4Hz,2H),5.48-5.41(m,2H),4.99(s,1H),4.51–4.35(m,1H),4.24–4.06(m,2H),3.84–3.52(m,6H),3.15–2.86(m,2H),2.05–1.91(m,1H),1.85–1.69(m,1H),1.44(s,18H).HRMS calcd for C28H41N5O9591.2904；found,592.2012[M+H]⁺.

5) Tert-butyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- (p-tolyloxy) propyl) acetamido) benzene) propionate

Tert-butyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (N- (3-hydroxypropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) benzene) propionate (600mg, 0.95mmol) was dissolved in 25mL of dichloromethane, TsCl (760mg, 4.0mmol), 0.6mL of triethylamine and 10mg of DMAP were added in this order under ice bath, reacted at room temperature for 12 hours, washed with water three times, and the organic phase layer was washed with Na₂SO₄Dried and purified by flash chromatography (ethyl acetate) to give tert-butyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- (p-tolyloxy) propyl) acetamido) benzene) propionate (283mg, 40.0%).¹H NMR(300MHz,CDCl₃)δ7.83–7.74(m,2H),7.36(d,J＝8.0Hz,2H),7.21(s,1H),7.13-7.03(m,3H),6.86(d,J＝8.4Hz,1H),6.76(d,J＝8.4Hz,1H),5.43(s,1H),5.29(s,1H),4.99(s,1H),4.42(s,1H),4.13-4.03(m,5H),3.80(s,1H),3.66-3.60(m,1H),3.50(s,2H),3.02(d,J＝5.7Hz,2H),2.46(s,3H),2.19(s,1H),2.06(s,2H),2.04–1.93(m,1H),1.44(s,18H).HRMS calcd for C35H47N5O11S 745.2993；found,746.1320[M+H]⁺.

6) (S) -tert-butyl 2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) phenyl) propionate

Tert-butyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- (p-tolyloxy) propyl) acetamido) benzene) propionate (100mg, 0.13mmol) was dissolved in 20mL tetrahydrofuran, 0.5mL tetrabutylammonium fluoride solution (1M) was added, reacted at 50 ℃ for 12H, then the tetrahydrofuran was spin-dried, extracted with ethyl acetate, washed with water three times, and the organic phase layer was Na-washed₂SO₄Dried and then purified by flash chromatography (petroleum ether: ethyl acetate 50:50) to give tert-butyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) phenyl) propionate (26mg, 32.5%).¹H NMR(300MHz,CDCl₃)δ7.22–7.00(m,4H),6.88–6.68(m,2H),5.43(s,1H),5.35–5.15(m,1H),5.03(s,1H),4.73–4.47(m,1H),4.38(s,1H),4.14-4.10(m,1H),3.79-3.51(m,2H),3.00(s,2H),2.28–2.00(m,2H),1.42(s,18H).13C NMR(75MHz,CDCl₃)δ170.93,165.60,165.18,165.01,157.22,156.86,154.98,145.27,130.83,130.64,129.77,128.99,128.12,127.11,114.30,114.16,82.68,82.03,81.65,80.51,79.64,79.53,65.89,65.33,60.37,54.95,51.29,51.09,49.74,47.55,46.74,44.74,37.49,28.30,27.91,14.37.HRMS calcd for C28H40FN5O8 593.2861；found,594.1963[M+H]⁺.

7) (S) -2-amino-3- (4- (2- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) ethoxy) phenyl) propanoic acid

Tert-butyl (S) -2- ((tert-butoxycarbonyl) amino) -3- (4- (2- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) phenyl) propanoate (30mg, 0.05mmol) was dissolved in 2mL trifluoroacetic acid, reacted at room temperature, monitored by TLC, and then trifluoroacetic acid was removed to give (S) -2-amino-3- (4- (2- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) ethoxy) phenyl) propanoate (21.4mg, 98%).¹H NMR(300MHz,DMSO)δ8.24(s,2H),7.60(s,1H),7.20(d,J＝6.7Hz,2H),7.02-6.89(m,2H),5.58(s,1H),5.58-5.46(m,2H),4.52-4.33(m,2H),4.18-4.04(m,4H),3.80(s,1H),3.69–3.52(m,2H),3.03(s,2H),2.15-2.05(m,1H),1.99–1.75(m,1H).HRMS calcd for C19H24FN5O6437.1711；found,438.2641[M+H]⁺.

Example 2(2S, 4R) -2-amino-4- (3- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) propyl) glutaric acid

The structural formula is as follows:

the synthetic route is as follows:

1) di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (p-tolyloxy) propyl) glutarate

Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3-hydroxypropyl) glutarate (900mg, 2.16mmol) was dissolved in 25mL of dichloromethane, p-toluenesulfonyl chloride (1.64g, 8.63mmol), triethylamine (6.67mL) and 4-dimethylaminopyridine (10mg, 0.08mmol) were added under ice-bath conditions, reacted at room temperature for 12h, and then washed three times with water, Na₂SO₄After drying, by flash chromatography (petroleum ether: ethyl acetate 80:20), di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (p-tolyloxy) propyl) glutarate (790mg, 64.1%) was obtained.¹H NMR(300MHz,CDCl₃)δ7.80(d,J＝8.2Hz,2H),7.36(d,J＝8.0Hz,2H),4.86(d,J＝8.6Hz,1H),4.04(dd,J＝16.9,11.2Hz,3H),2.47(s,3H),2.30(s,1H),1.90-1.56(m,2H),1.42(s,27H).HRMS calcd for C28H45NO9S 571.2815；found,572.3193[M+H]⁺。

2) Di-tert-butyl (2R, 4S) -2- (3- (benzyl (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) propyl) -4- ((tert-butoxycarbonyl) amino) glutarate

Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (p-tolyloxy) propyl) glutarate (700mg, 1.23mmol) was dissolved in 40mL acetonitrile and N-benzyl-3- ((tetrahydro-2H-pyran-2-yl) oxy) propan-1-amine (336mg, 1.35mmol) was addedSodium iodide (198mg, 1.23mmol) and potassium carbonate (339mg, 2.46mmol) were refluxed at 100 ℃ for 48H and then filtered, the filtrate was taken up with rotary drying and chromatography by flash purification (petroleum ether: ethyl acetate ═ 70:30) to give di-tert-butyl (2R, 4S) -2- (3- (benzyl (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) propyl) -4- ((tert-butoxycarbonyl) amino) glutarate (545mg, 68.3%).¹H NMR(300MHz,CDCl₃)δ7.37–7.28(m,5H),4.87(d,J＝8.2Hz,1H),4.52(s,2H),4.11(d,J＝8.6Hz,1H),3.90–3.66(m,2H),3.64–3.34(m,5H),2.62–2.36(m,4H),2.29(d,J＝6.3Hz,1H),1.81-1.75(m,6H),1.65(d,J＝9.3Hz,3H),1.46(d,J＝8.5Hz,31H).¹³C NMR(75MHz,CDCl₃)δ175.07,171.84,155.39,139.94,128.80,128.10,126.70,99.66,98.84,81.73,65.77,62.25,58.53,53.49,50.51,43.50,31.58,30.70,28.30,28.09,27.98,27.30,25.47,24.33,22.65,19.60.HRMS calcd for C36H60N2O8 648.4350；found,649.3182[M+H]⁺.

3) Glutaric acid di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- ((3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) propyl ester

Di-tert-butyl (2R, 4S) -2- (3- (benzyl (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) propyl) -4- ((tert-butoxycarbonyl) amino) glutarate (500mg, 0.77mmol) was dissolved in ethanol, palladium on carbon (50mg, 0.47mmol) was added, vacuum was applied, and H-charged₂Reacted at room temperature for 12H, then filtered through celite, spin dried, and taken up, by flash chromatography (petroleum ether: ethyl acetate: 50) to give di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- ((3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) amino) glutarate (391mg, 91.0%).¹H NMR(300MHz,CDCl₃)δ5.08(d,J＝6.7Hz,1H),4.53(s,1H),4.12(s,1H),3.90–3.72(m,2H),3.57–3.40(m,2H),3.14(s,4H),2.48–2.26(m,1H),2.12(s,2H),1.97–1.60(m,7H),1.47-1.37(m,32H).¹³C NMR(75MHz,CDCl₃)δ174.39,98.93,81.07,77.44,77.02,76.59,64.42,62.51,45.57,43.00,30.61,28.31,28.07,27.99,24.47,20.00,8.61.HRMS calcd for C29H54N2O8 558.3880；found,559.0124[M+H]⁺.

4) Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) acetamido) propyl) glutarate

2- (2-Nitroimidazol-1-yl) acetic acid (87mg, 0.51mmol) was dissolved in 10mL of DMF, 2mL of triethylamine and di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) glutarate (300mg, 0.54mmol) were added, HBTU (439mg, 1.16mmol) was finally added, the reaction was carried out at room temperature for 2H, extraction was carried out with ethyl acetate, the mixture was washed with saturated brine three times, and the organic phase layer was washed with Na₂SO₄Dried and then purified by flash chromatography (petroleum ether: ethyl acetate 80:20) to give di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) acetamido) propyl) glutarate (220mg, 57.3%).¹H NMR(300MHz,CDCl₃)δ7.20–7.02(m,2H),5.36(s,1H),5.23(s,1H),5.03–4.78(m,1H),4.53(s,1H),4.11(s,1H),3.84-3.75(m,2H),3.54–3.34(m,5H),2.31(s,1H),1.96–1.59(m,9H),1.87-1.42(m,30H).HRMS calcd for C34H57N5O11 711.4055；found,712.0483[M+H]⁺.

5) Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (N- (3-hydroxypropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) propyl glutarate

Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) acetamido) propyl) glutarate (500mg, 0.70mmol) was dissolved in a mixed solution of 20mL of ethanol and 20mL of tetrahydrofuran, pyridine p-toluenesulfonate (106mg, 0.42mmol) was added, reacted at 50 ℃ for 2H, and then spin-dried and sample-stirred, and by rapid purification chromatography (ethyl acetate), di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (N- (3-hydroxypropyl) -2- (2-nitro-1H-imidazol-1-yl) glutarate was obtained -yl) acetamido) propyl ester (300mg, 68.3%). HRMS calcd for C29H49N5O10627.3479; found,628.0521[ M + H [ ]]⁺.

6) Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- (p-tolyloxy) propyl) acetamido) propyl) glutarate

Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (N- (3-hydroxypropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) propyl glutarate (500mg, 0.79mmol) was dissolved in 25mL of dichloromethane, TsCl (59.2mg, 3.12mmol), 0.5mL of triethylamine and 10mg of DMAP were sequentially added under ice bath, reacted at room temperature for 12 hours, washed with water three times, and the organic phase layer was Na₂SO₄Dried and then purified by flash chromatography (dichloromethane: methanol ═ 95:5) to give di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- (p-tolyloxy) propyl) acetamido) propyl) glutarate (265mg, 43.0%). HRMS calcd for C36H55N5O12S 781.3568; found,782.1045[ M + H [ ]]⁺.

7) Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) propyl) glutarate

Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (2- (2-nitro-1H-imidazol-1-yl) -N- (3- (p-tolyloxy) propyl) acetamido) propyl) glutarate (100mg, 0.12mmol) was dissolved in 20mL of tetrahydrofuran, 0.5mL of tetrabutylammonium fluoride solution (1M) was added, the reaction was carried out at 50 ℃ for 12H, then the tetrahydrofuran was removed, extraction was carried out with ethyl acetate, washing was carried out three times, and the organic phase layer was washed with Na₂SO₄Dried and then purified by flash chromatography (dichloromethane: methanol ═ 95:5) to give di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) propyl) glutarate (23mg, 30.2%).¹H NMR(300MHz,CDCl₃)δ7.12(d,J＝8.7Hz,2H),5.36–5.16(m,2H),4.99-4.88(m,1H),4.65-4.35(m,1H),4.17-4.09(m,1H),3.56–3.25(m,4H),2.37-2.29(m,1H),2.13-2.02(m,2H),1.83–1.66(m,5H),1.45-1.41(m,27H).¹³C NMR(75MHz,CDCl₃)δ174.59,174.30,171.55,164.74,164.64,155.64,145.19,128.04,127.18,82.24,82.00,81.22,80.91,79.85,52.59,51.15,47.56,45.66,43.05,42.73,42.42,35.02,34.33,30.32,29.16,28.64,28.24,28.02,27.93,26.15.HRMS calcd for C29H48FN5O9 629.3436；found,630.0230[M+H]⁺.

8) (2S, 4R) -2-amino-4- (3- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) propyl) glutaric acid

Di-tert-butyl (2S, 4R) -2- ((tert-butoxycarbonyl) amino) -4- (3- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) propyl) glutarate (10mg, 0.016mmol) was dissolved in 1mL of trifluoroacetic acid and reacted at room temperature for 12H, then the trifluoroacetic acid was removed, an appropriate amount of ether was added, and the cake was filtered to give (2S, 4R) -2-amino-4- (3- (N- (3-fluoropropyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) propyl) glutarate (7mg, 90%). HRMS calcd for C16H24FN5O7417.1660; found,418.2019[ M + H [ ]]⁺.

Example 32-amino-7-fluoro-4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) heptanoic acid

The structural formula is as follows:

the synthetic route is as follows:

1)2- ((tert-Butoxycarbonyl) amino) -4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) -7- ((tetrahydro-2H-pyran-2-yl) oxy) heptanoic acid tert-butyl ester

2- (2-Nitro-1H-imidazol-1-yl) ethan-1-amine (283mg, 1.81mmol) was dissolved in DCM, 15mL of triethylamine was added and stirred at room temperature for 1H, and 5- (tert-butoxy) -4- ((tert-butoxycarbonyl) amino) -5-oxo-2- (3- ((tetrahydro-2H-pyran-2-yl) oxy) propyl) pentanoic acid (807mg, 1.81mmol), HOBT (80mg, 0.52mmol), EDCI (1g, 5.23mmol) were added in that order and reacted at room temperature for 1 day, followed by washing with water three times, and the organic phase layer was washed with Na₂SO₄Dried and then purified by flash chromatography (ethyl acetate) to give tert-butyl 2- ((tert-butoxycarbonyl) amino) -4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) -7- ((tetrahydro-2H-pyran-2-yl) oxy) heptanoate (400mg, 37.9%). HRMS calcd for C27H45N5O9583.3217; found,584.2510[ M + H [ ]]⁺.

2) Heptoic acid tert-butyl ester 2- ((tert-butoxycarbonyl) amino) -7-hydroxy-4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl)

Tert-butyl 2- ((tert-butoxycarbonyl) amino) -4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) -7- ((tetrahydro-2H-pyran-2-yl) oxy) heptanoate (500mg, 0.85mmol) and pyridine p-toluenesulfonate (65mg, 0.25mmol) were dissolved in ethanol, reacted at 50 ℃ for 12H, then spin-dried and sample-mixed to give tert-butyl heptanoate 2- ((tert-butoxycarbonyl) amino) -7-hydroxy-4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) (400mg, 94.3%) by flash purification chromatography (dichloromethane: methanol ═ 10: 1). 1H NMR (300MHz, CDCl3) δ 7.21(s,1H),7.08(s,2H),5.17(s,1H),4.60(s,2H),4.06(s,1H), 3.79-3.60 (m,4H),2.42(s,1H),2.17(s,1H), 1.95-1.83 (m,2H), 1.75-1.61 (m,2H),1.45(d, J ═ 16.0Hz,18H), 13C NMR (75MHz, CDCl3) δ 176.05,171.48,155.78,144.56,128.19,127.43,82.38,80.20,77.41,77.19,76.99,76.57,62.16,58.40,53.18,49.54,43.10, 39.40.45, 29.70,29.15,28.29, ms 27.96,18.39, hrcalc 25; found,500.1402[ M + H [ ]]⁺.

3)2- ((tert-Butoxycarbonyl) amino) -4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) -7- (tolyloxy) heptanoic acid tert-butyl ester

Tert-butyl heptanoate 2- ((tert-butoxycarbonyl) amino) -7-hydroxy-4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) (220mg, 0.44mmol) was dissolved in dichloromethane, and TsCl (430mg, 2.26mmol) and triethylamine (5mL) were added in this order under ice-cooling, followed by reaction at room temperature for 12 hours, washing with water three times, and the organic phase layer was Na₂SO₄Dried and then purified by flash chromatography (petroleum ether: ethyl acetate ═ 30:70) to give tert-butyl 2- ((tert-butoxycarbonyl) amino) -4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) -7- (tolyloxy) heptanoate (200mg, 69.6%). 1H NMR (300MHz, CDCl3) δ 7.71(d, J ═ 8.2Hz,2H), 7.37-7.25 (m,2H),7.19(s,1H),6.96(s,1H),5.16(s,1H),4.52(d, J ═ 4.7Hz,2H),3.95(s,3H),3.70(s,2H),2.41(s,3H),1.91(s,1H),1.80(s,1H),1.62(s,3H),1.38(d, J ═ 19.4Hz,20H), 13C NMR (75MHz, CDCl3) δ 175.37,171.48,155.51,144.88,144.16,132.77,129.88,128.23,127.80,82.06,79.78,77.53,77.11,76.68,70.22,60.32,53.29,50.21,42.88,38.95,34.44,28.71,28.24,27.90,26.47,21.57,20.98,14.13.HRMS calcd for C29H43N5O10S 653.2731；found,654.1132[M+H]⁺.

4) tert-butyl 2- ((tert-butoxycarbonyl) amino) -7-fluoro-4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) heptanoate

Tert-butyl 2- ((tert-butoxycarbonyl) amino) -4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) -7- (tolyloxy) heptanoate (100mg, 0.15mmol) was dissolved in a small amount of THF, 0.2mL of tetrabutylammonium fluoride solution (1M) was added under nitrogen protection, reaction was carried out at 50 ℃ for 2 days, THF was then removed, extraction was carried out with ethyl acetate, washing was carried out three times, and the organic phase layer was washed with Na₂SO₄After drying, by flash purification chromatography (ethyl acetate), tert-butyl 2- ((tert-butoxycarbonyl) amino) -7-fluoro-4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) heptanoate (10mg, 13.3%) was obtained. HRMS calcd for C22H36FN5O7501.2599; found,502.2056[ M + H [ ]]⁺.

5) 2-amino-7-fluoro-4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) heptanoic acid

Tert-butyl 2- ((tert-butoxycarbonyl) amino) -7-fluoro-4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) heptanoate (10mg, 0.02mmol) was dissolved in 1mL trifluoroacetic acid, stirred at room temperature for 2-3H, and then the trifluoroacetic acid was removed to give 2-amino-7-fluoro-4- ((2- (2-nitro-1H-imidazol-1-yl) ethyl) carbamoyl) heptanoic acid (6.8mg, 97%). HRMS calcd for C13H20FN5O5345.1448; found,346.3189[ M + H [ ]]⁺.

Example 4(S) -2-amino-3- (4- (2- (N- (3- (fluoro-))¹⁸F) Propyl) -2- (2-nitro-1H-imidazol-1-yl) acetamido) ethoxy) phenylpropionic acid

The structural formula is as follows:

the labeling route is as follows:

the experimental procedure was as follows:

a) will accelerator produced¹⁸F]F^-The solution is passed through QMA column¹⁸F]F^-Adsorbed on the column; (QMA column activation method: 10mL of 0.5M sodium bicarbonate solution was used to elute QMA column, 10mL was air-dried, 10mL was used to elute high purity water, and 10mL was air-dried.)

b) 1mL of 18-crown-6/KHCO was used₃The leacheate will¹⁸F]F^-Eluting from the QMA column into a reaction tube; (solution preparation method: 480mg of 18-crown-6 is dissolved in 18.6mL of acetonitrile, 90mg of potassium bicarbonate is dissolved in 3.4mL of water, and the two liquids are mixed uniformly.)

c) The eluent is put in an oil bath at 100 ℃ and is added with N₂Blowing and evaporating to dryness;

d) adding 1mL of anhydrous acetonitrile into a reaction flask to conduct azeotropic dehydration, and repeating the process three times to obtain dried [ alpha ], [ alpha ] form¹⁸F]F^-18-crown-6/KHCO₃A complex compound;

e) to the above-mentioned dried [ 2 ]¹⁸F]F^-18-crown-6/KHCO₃To the complex was added 1mL of a solution of 5mg of the labeled precursor in t-amyl alcohol/acetonitrile (V/V. 9/1);

f) placing the reaction solution in an oil bath at 100 ℃ for reaction for 10 min;

g) after the reaction is finished, cooling the reaction solution in an ice bath, adding 9mL of water into the reaction tube, and passing through an Oasis HLB solid-phase extraction column; (Oasis column activation method: the Oasis column was rinsed with 10mL of ethanol, the column was dried under 10mL of air, the column was rinsed with 10mL of high purity water, and finally the column was dried under 10mL of air.)

h) Pumping 10mL of water to wash the Oasis HLB solid phase extraction column, pushing air to dry the column, leaching the intermediate adsorbed on the column into a clean penicillin bottle by using 1mL of ethanol, and taking a proper amount of intermediate sample for Radio-HPLC analysis;

i) adding ethanol solution containing intermediate in N₂Drying, adding 1mL of trifluoroacetic acid, uniformly mixing in a vortex mode, placing in an oil bath at 50 ℃ for reacting for 5min, drying the solvent after the reaction is finished, and adjusting the pH value to be neutral;

j) adding 1mL of physiological saline, uniformly mixing by vortex, filtering by using a sterile filter membrane of 0.22 mu m, taking a proper amount of sample for Radio-HPLC analysis, and calculating the yield and the radiochemical purity (RCP) of the labeling reaction; the product is identified by HPLC (Phenomenex Gemini-Nx C18110A (250X 4.6mm), 0-30min, 0.1% formic acid water, 10% -90% acetonitrile, 11.5min of peak time and more than 95% purity;

k) the labeled compounds were placed in PBS and mouse serum, incubated for 2 hours at 37 deg.C, removed at 5, 30, 60 and 120min and assayed for RCP by Radio-HPLC. From the experimental results, it was found that the compound provided in example 4 has good stability.

Example 5A marker compound and [ 2 ]¹⁸F]EMT-6 cell uptake assay in vitro by FMISO

The experimental procedure was as follows:

1) human breast cancer EMT-6 cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Digesting the cells from the culture dish into a single cell suspension, uniformly blowing, and taking 10mL (the concentration is 2 multiplied by 10)⁶cells/mL), transferred into two sterilized and disinfected 25mL three-necked bottles, and the water bath temperature is constant at 37 ℃ and the rotation speed is 200-300 rpm. Introducing 95% nitrogen-5% carbon dioxide saturated with water into the oxygen-lacking system, introducing 95% oxygen-5% carbon dioxide saturated with water into the oxygen-containing body, and stirring and pre-balancing for 1 hour;

2) taking 100. mu.L of the labeled compound and [ [ solution ] ]¹⁸F]FMISO (1 mCi/mL) is added into two reaction bottles respectively, the mixture is shaken up fully, and timing is started;

3) taking 200 μ L cell suspension sample in a centrifuge tube at 5, 30, 60, 120, 180 and 240min, and taking 4 times in parallel;

4) centrifuging the sample at 2000rpm for 5min to separate cells and culture solution;

5) a radioactive count of 90. mu.L of the supernatant was taken and measured, along with the radioactive count of the remaining 110. mu.L of EMT-6-containing cell line solution. The uptake of the compound in the cell is expressed as Cin/Cout;

6) after the experiment is finished, cell activity is determined by using a trypan blue staining experiment, and the cell activity is more than 90% in the whole experiment process;

table 1: cellular uptake assay of Compounds

As can be seen from Table 1, the nitroimidazole derivatives provided by the invention have high tumor cell uptake and are very suitable for hypoxia imaging.

EXAMPLE 6 biodistribution of labeled Compounds in EMT-6 tumor mice

Female EMT-6 breast cancer model mice (BALB/c mice) were selected for 60min treatment, with 5 mice per group. The labeled compound diluted in 10% ethanol-physiological saline was injected tail vein (0.1mL) into mice and timed. At the time point, sacrifice, dissect each organ, weigh, measure the radioactivity count of each tissue, calculate uptake and target to non-target ratios (including tumor to blood ratio, tumor to muscle ratio, tumor to liver ratio) for each organ. As can also be seen from table 2, the nitroimidazole derivatives provided by the present invention have significantly higher tumor uptake, and are very suitable for hypoxia imaging.

Table 2: tumor to blood ratio and tumor to meat ratio of the compound

Compound (I)	Tumor to blood ratio	Tumor meat ratio
			F-MISO	0.95±0.08	1.06±0.11
Example 4	3.26±0.14	2.59±0.17
			18F-example 2	3.11±0.09	3.11±0.11
18F-example 3	2.72±0.21	2.83±0.09

Example 7 Micro-PET imaging in EMT-6 tumor mice

EMT-6 breast cancer model mice (BALB/c mice) are prepared before the experiment, Micro-PET imaging is carried out when tumors grow to be about 0.5cm, and fasting is carried out for 8 hours before imaging. On the day of the experiment, the labeled compound was diluted with saline, and the drug (-4 MBq, 0.2mL) was aspirated through an insulin needle and injected into the mice via the tail vein. Mice were anesthetized with 2% isoflurane gas and then fixed on a PET examination couch, then CT scout scan was performed, and PET scan was performed 60min after injection, and scan images were analyzed using AMIDE software. As can be seen from FIG. 1, (S) -2-amino-3- (4- (2- (N- (3- (fluoro-)) prepared according to example 4 of the present invention¹⁸F) The propyl) -2- (2-nitro-1H-imidazole-1-yl) acetamido) ethoxy) phenylpropionic acid can realize good imaging effect on tumors.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.