A nitroaromatic ring-substituted imidazopyrazine compound, its preparation method and application

Abstract

This invention provides a nitro-aryl ring-substituted imidazopyrazine compound that can be used as a bioluminescent probe for detecting nitroreductases. In the NanoLuc luciferase bioluminescent system, this probe exhibits good responsiveness in in vitro experiments and can be selectively reduced by NTRs. Viable bacterial experiments and imaging experiments in a mouse model of bacterial infection both demonstrate that this probe is a highly selective and sensitive luminescent probe for detecting NTR activity, enabling the detection and imaging of NTR activity in vivo.

Description

Technical Field

[0001] This invention relates to the field of bioluminescence imaging technology, specifically to a nitroaromatic ring-substituted imidazopyrazine compound, its preparation method, and its application. Background Technology

[0002] Various endogenous species (such as bacteria in the in vivo microenvironment) play important roles in living organisms, and the detection of bioactive substances requires excellent imaging tools for visualization. Bioluminescence (BL) imaging, as a novel imaging technique, has become an indispensable technology in biomedical research and is currently a major strategy for studying and exploring life activities in living cells and organisms [1-2]. Compared with traditional fluorescence imaging techniques, bioluminescence imaging relies on the enzymatic reaction of luciferase oxidizing its substrate to emit light, avoiding interference and phototoxicity problems caused by exogenous excitation light, and can provide a very high signal-to-noise ratio in imaging [3-4]. As a non-invasive, longitudinal tracking technique, bioluminescence imaging is widely used at the forefront of exploring life mechanisms, from sensitive cell analysis to bioluminescence-based molecular imaging [5-6].

[0003] Since DeLuca’s lab first reported cloning firefly luciferase (FLuc, derived from Photinus pyralis) in E. coli[7], the widespread application of bioluminescence has spurred research on luciferase. As a result, researchers have successively discovered several luciferases and their corresponding substrates from Oplophorus gracilirostris (OLuc) and Gaussia princeps (GLuc)[8-10]. For example, the cDNA clone of OLuc consists of two 35 kDa subunits and two 19 kDa subunits, and its bioluminescent activity is associated only with the smaller subunit (OLuc-19)[11-12]. However, the low expression of this subunit protein and stability issues have limited its application. Mutagenesis optimization of this subunit has enabled Promega scientists to obtain stable and high bioluminescence pairs for NanoLuc (NLuc) and its corresponding substrate furimazine

[13] . As a novel bioluminescence imaging platform, NLuc-furimazine can generate high-intensity, glow-type luminescence signals (signal half-life > 2h), and its bioluminescence intensity is more than 150 times stronger than that of traditional firefly luciferase and coelenterin systems

[13] . Furthermore, NonaLuc enzymes have advantages such as small size, high expression rate, and good physical stability, making them suitable for various fields such as medicine

[14] , biology

[15] , physics

[16] , and multidisciplinary sciences [17-18].

[0004] Although some studies have been reported on the modification of corresponding substrates using the deep-sea bioluminescent enzyme NanoLuc [19-25], the development of small molecule probes based on the NLuc-furimazine bioluminescence system remains limited. Furthermore, to date, no studies have utilized the NLuc-furimazine luminescence system to develop nitroreductase probes. Nitroreductases (NTRs), as an important class of oxidoreductases in organisms, catalyze the formation of corresponding amino compounds from nitroaromatic compounds in the presence of NADH or nicotinamide adenine dinucleotide phosphate (NADPH). Abnormal NTR expression is closely related to various disease processes; therefore, accurate detection of NTR expression levels is of great significance for the early diagnosis, disease monitoring, and development of specialized diagnostic and therapeutic technologies for various diseases [26-29]. To this end, this invention develops a series of NTR bioluminescent probes based on the NLuc-furimazine system, aiming to further advance the development of diagnostic and therapeutic methods for flavoprotein-related diseases by broadening the applications of NanoLuc bioluminescence technology. Summary of the Invention

[0005] The purpose of this invention is to provide a nitroaromatic ring-substituted imidazopyrazine compound, its preparation method and application, which can be used as a probe for detecting nitroreductase, thus broadening the application of NanoLuc bioluminescence technology.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a nitroaromatic ring-substituted imidazopyrazine compound having the general formula (1):

[0008]

[0009] Among them, R 1 For H, OH, alkoxy groups, R 2 It is a nitro-substituted arylmethyl group.

[0010] In the above compounds, R 2 The aromatic ring in it is a benzene ring or a heteroaromatic ring.

[0011] Preferably, the heteroaromatic ring includes, but is not limited to, furan rings, thiophene rings, pyrrole rings, imidazole rings, etc.

[0012] Optimal choice, R 2 It is any one of the following groups:

[0013]

[0014] As a preferred embodiment, the compound is selected from:

[0015] Compound 1: 8-benzyl-2-(furan-2-ylmethyl)-3-((5-nitrofuran-2-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine;

[0016] Compound 2: 8-benzyl-2-(furan-2-ylmethyl)-3-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine;

[0017] Compound 3: 8-benzyl-2-(furan-2-ylmethyl)-3-((1-methyl-5-nitro-1H-imidazol-2-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine;

[0018] Compound 4: 8-benzyl-2-(furan-2-ylmethyl)-3-((5-nitrothiophen-2-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine;

[0019] Compound 5: 8-benzyl-2-(furan-2-ylmethyl)-3-((2-nitrobenzyl)oxy)-6-phenylimidazo[1,2-a]pyrazine;

[0020] Compound 6: 8-benzyl-2-(furan-2-ylmethyl)-3-((4-nitrobenzyl)oxy)-6-phenylimidazo[1,2-a]pyrazine;

[0021] Compound 7: 3-(8-benzyl-2-(furan-2-ylmethyl)-3-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)imidazo[1,2-a]pyrazin-6-yl)phenol.

[0022] In a second aspect, the present invention provides a method for preparing the compound as described above, the method comprising:

[0023] Under inert gas protection, 8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazine-3(7H)-one, whether protected or unprotected, was reacted with a methyl bromide derivative in the presence of potassium carbonate to obtain the compound shown in formula (1).

[0024] In a third aspect, the present invention provides the use of the compounds described above as bioluminescent probes for detecting nitroreductases.

[0025] Preferably, the probe is used as a substrate for the NanoLuc bioluminescence system, for bioluminescence imaging studies, or for detecting drug effects.

[0026] In a fourth aspect, the present invention provides a kit for detecting nitroreductase, comprising the compound as described in any one of claims 1 to 5.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) The compound with the structure of formula (1) provided by the present invention can be used as a nitro reductase probe. It exhibits good responsiveness in in vitro experiments and can be selectively reduced by NTR. It can detect and image the in vivo NTR activity at the cellular and animal levels.

[0029] (2) The nitroreductase probes with the structure of formula (1) provided by the present invention can all be used as corresponding substrates of the NanoLuc bioluminescence system, thus broadening the application of NanoLuc bioluminescence technology. Attached Figure Description

[0030] Figure 1 The figure shows the experimental results of the chemical stability test of the compound of this invention;

[0031] Figure 2 Figure 1 shows the experimental results verifying the mechanism of action of the compound of the present invention as a nitroreductase probe.

[0032] Figure 3 The figure shows the bioluminescence signal results of the compound of the present invention as a nitroreductase probe after incubation in vitro for different times.

[0033] Figure 4 The figure shows the concentration-dependent experimental results of the compound of this invention as a nitroreductase probe;

[0034] Figure 5 The figure shows the selective experimental results of the compound of the present invention as a nitroreductase probe;

[0035] Figure 6 The image shows the cytotoxicity test results of the compounds of this invention.

[0036] Figure 7 This is a bioluminescence imaging experiment showing the results of the compound of the present invention as a nitroreductase probe in bacterial culture.

[0037] Figure 8 This is a bioluminescence imaging experiment result of the compound of the present invention as a nitroreductase probe in an animal model;

[0038] Figure 9 The figure shows the experimental results of detecting the NTR activity of mouse intestinal bacteria using the compound of this invention as a nitroreductase probe. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," "outer," "front end," "rear end," "both ends," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0041] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0042] In order to clearly understand the technical content of the present invention, the specific implementation method of the present invention will be further described below in conjunction with specific embodiments.

[0043] Synthesis of nitroreductase probes

[0044] Example 1: Preparation of intermediate 1c

[0045]

[0046] Under ice-water bath conditions, NaBH4 (378 mg, 10 mmol) was added to a methanol (30 mL) solution of 5-nitrofuran-2-carboxaldehyde (705 mg, 5 mmol), and the resulting mixture was stirred at 0 °C for 0.5 h. After the initial reaction 1a was consumed, the mixture was quenched with water, and the aqueous layer was extracted with EA. The combined organic layers were dried over anhydrous Na2SO4, concentrated under reduced pressure, and subjected to silica gel column chromatography (PE / EA = 4 / 1) to give compound 1b at 465 mg (65%). TLC (PE / EA = 2 / 1): R f =0.23; 1H NMR (500MHz, CDCl3) δ7.28 (d, J = 3.7Hz, 1H), 6.55 (d, J = 3.7Hz, 1H), 4.70 (s, 2H), 2.56 (s, 1H).

[0047] Under ice-water bath conditions, hydroxymethyl derivative (1b) (3.5 mmol) was dissolved in dichloromethane (4 mL). A solution of phosphorus tribromide (4.47 mmol) in dichloromethane (3 mL) was added dropwise to the above solution, and the mixture was stirred for 30–60 min. The solution was quenched with water, and the aqueous layer was extracted with DCM. The combined organic layers were dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and subjected to silica gel column chromatography (PE / EA = 5 / 1) to give 2-(bromomethyl)-5-nitrofuran 1c at 339 mg (73%). TLC (PE / EA = 5 / 1): R f =0.33; 1 H NMR (500MHz, CDCl3) δ7.27 (d, J = 3.7Hz, 1H), 6.62 (d, J = 3.7Hz, 1H), 4.46 (s, 2H); 13 C NMR (126MHz, CDCl3) δ153.47, 151.77, 112.52, 112.43, 20.74.

[0048] Example 2: Preparation of intermediate 4c

[0049] (5-Nitrofuran-2-yl)methanol 4b (PE / EA = 4 / 1, 556 mg, 0.35 mmol, 70%) was prepared from 5-nitrothiophene-2-carboxaldehyde (785 mg, 5 mmol) and NaBH4 (378 mg, 10 mmol) according to the method described in 1c. TLC (PE / EA = 2 / 1): R f =0.15; 1 ¹H NMR (500 MHz, CDCl₃) δ 7.79 (d, J = 4.1 Hz, 1H), 6.91 (dd, J = 4.0, 1.0 Hz, 1H), 4.85 (s, 2H). Then, 2-(bromomethyl)-5-nitrofuran 4c (PE / EA = 5 / 1, 633 mg, 85%) was prepared by reacting 4b with PBr₃ (0.4 mL, 4.2 mmol) in DCM solution. TLC (PE / EA = 5 / 1): R f =0.53; 1 H NMR (500MHz, CDCl3) δ7.76 (d, J = 4.1Hz, 1H), 7.06 (d, J = 4.1Hz, 1H), 4.62 (s, 2H). 13C NMR (126MHz, CDCl3) δ151.77, 148.38, 128.46, 127.14, 24.65.

[0050] Example 3: Preparation of intermediate 2b

[0051]

[0052] Hydroxymethyl derivative 2a (550 mg, 3.5 mmol) was dissolved in dichloromethane (20 mL), and the reaction was placed in an ice bath. Then, pyridine (0.83 mL, 10.5 mmol) and SOCl2 (0.77 mL, 10.5 mmol) in dichloromethane (10 mL) were added dropwise. After the addition was complete, the mixture was stirred at 0 °C for 60 min, then heated to room temperature and reacted overnight. The reaction was quenched with water, and the aqueous layer was extracted with DCM. The combined organic layers were dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA = 1 / 1) to give 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazolium 2b (460 mg, 75%). TLC (PE / EA = 1 / 1): R f =0.38; 1 H NMR (500MHz, CDCl3) δ7.19(s,1H),4.63(s,2H),4.07(s,3H); 13 C NMR (126MHz, CDCl3) δ146.46, 132.83, 128.57, 34.26, 33.93.

[0053] Example 4: Preparation of intermediate 3b

[0054]

[0055] Following the synthesis of compound 2b, 2-(chloromethyl)-1-methyl-5-nitro-1H-imidazolium 3b was prepared in 80% yield. TLC (PE / EA = 1 / 1): R f =0.45; 1 H NMR (500MHz, CDCl3) δ7.94(s,1H),4.67(s,2H),4.04(s,3H); 13 C NMR (126MHz, CDCl3) δ147.18, 139.76, 131.82, 36.15, 33.83.

[0056] Example 5: Preparation of probes 1-6

[0057]

[0058] General procedure for synthesizing probes (1-6): Under a nitrogen atmosphere, methyl bromide (chloride) derivative (1.05 mmol) and K₂CO₃ (1.05 mmol) were added to a DMF (3.5 mL) solution of Fz (133 mg, 0.35 mmol). The mixture was stirred at room temperature for 8-10 hours. After the reaction was complete, water (30 mL) was added to quench the reaction and the mixture was extracted with EA (30 mL × 2). The combined organic layers were washed with brine, dried over anhydrous Na₂SO₄, and concentrated. Probes 1-6 were purified using a silica gel column (PE / EA) or by preparative high-performance liquid chromatography (CH₃CN / H₂O, flow rate 8 mL / min).

[0059] 8-Benzyl-2-(furan-2-ylmethyl)-3-((5-nitrofuran-2-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine: prepared from F2 and 2-(bromomethyl)-5-nitrofuran, and purified by preparative high performance liquid chromatography (65% CH3CN / H2O) to obtain probe 1 (103 mg, 58%), a yellow solid. 1 H NMR (500MHz, CDCl3) δ7.78–7.74(m,2H),7.43–7.39(m,3H),7.38–7.33(m,3H),7.30–7.27(m,2H),7.25–7.22(m,1H),7.14(d,J=1.9Hz,1H),7.01( d,J=3.6Hz,1H),6.14(dd,J=3.2,1.9Hz,1H),6.09(d,J=3.7Hz,1H),5.96 (d,J=3.2Hz,1H),4.18–4.11(m,2H),3.35(q,J=15.1Hz,2H),3.29(s,2H); 13 C NMR (126MHz, CDCl3) δ180.85,158.32,153.48,151.32,149.70,148.24,142.06,135.76,134.78,133.16,129.52(×2),128.81(×2),,12 8.58,128.43(×2),126.87,125.19(×2),112.37,111.73,110.39,108.83,108.66,74.47,39.51,35.30,34.62; HRMS(ESI)m / zcalc.for C 29 H 23 N4O5[M+H] + 507.1668, found 507.1671. HPLC purity 97.0%.

[0060] 8-Benzyl-2-(furan-2-ylmethyl)-3-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine: prepared from Fz and 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazolium, and purified by preparative high performance liquid chromatography (58% CH3CN / H2O) to obtain probe 2 (122 mg, 67%), an off-white solid. 1 HNMR (500MHz, CDCl3) δ7.87–7.83(m,3H),7.61–7.58(m,2H),7.46(td,J=7.2,6.3,1.3Hz,2H),7.41–7.37(m,1H),7.34(d,J=1.8Hz,1H),7.29(t,J= 7.7Hz,2H),7.21(d,J=7.3Hz,1H),7.11(s,1H),6.34(dd,J=3.3,1.9Hz,1H ),6.18–6.12(m,1H),5.05(s,2H),4.59(s,2H),4.16(s,2H),4.03(s,3H); 13 C NMR (126MHz, CDCl3) δ153.34,151.80,146.62,141.63,139.30,137.72,136.48,136.24,132.46,131.77,130.62,129.71(×2),12 9.66,128.95(×2),128.80,128.32(×2),126.55,126.20(×2),110.81,108.47,106.90,65.45,39.37,34.41,26.97; HRMS(ESI)m / z calc.for C 29 H 25 N6O4[M+H] + 521.1937, found. HPLC purity 98.0%.

[0061] 8-Benzyl-2-(furan-2-ylmethyl)-3-((1-methyl-5-nitro-1H-imidazol-2-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine: prepared from Fz and 2-(chloromethyl)-1-methyl-5-nitro-1H-imidazolium, purified by silica gel column chromatography (PE / EA=2 / 1) to obtain probe 3 (136 mg, 75%), an off-white solid. 1HNMR(500MHz, CDCl3)δ8.03(s,1H),7.97(s,1H),7.92–7.88(m,2H),7.61–7.57(m, 2H),7.46(dd,J=8.3,6.7Hz,2H),7.41–7.37(m,1H),7.34(dd,J=1.9,0.8Hz,1H),7 .29(dd,J=8.3,6.9Hz,2H),7.23–7.19(m,1H),6.35(dd,J=3.3,1.9Hz,1H),6.16(d d,J=3.2,1.0Hz,1H),5.12(s,2H),4.59(s,2H),4.20(d,J=0.9Hz,2H),3.99(s,3H); 13 C NMR(126MHz, CDCl3)δ153.14,151.94,146.29,141.57,139.16,137.77,136.57,136.29,132.48,131.94,130.59,129.70(×2),12 8.86(×2),128.68,128.29(×2),126.50,126.23(×2),110.78,108.95,106.87,99.99,67.98,39.35,33.73,26.92; HRMS(ESI)m / z calc.for C 29 H 25 N6O4[M+H] + 521.1937, found 521.1939. HPLC purity 98.0%.

[0062] 8-Benzyl-2-(furan-2-ylmethyl)-3-((5-nitrothiophene-2-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine: prepared from Fz and 2-(bromomethyl)-5-nitrothiophene, and purified by preparative high performance liquid chromatography (65% CH3CN / H2O) to obtain probe 4 (111 mg, 61%), a yellow solid. 1H NMR (500MHz, CDCl3) δ7.75–7.72(m,2H),7.55(d,J=4.1Hz,1H),7.44–7.37(m,5H),7.34(ddd,J=7.7,5.4,2.5Hz,3H),7.30–7.28(m,2H),7. 12(d,J=1.7Hz,1H),6.61(d,J=4.1Hz,1H),6.12(dd,J=3.2,1.9Hz,1H),5.92(d,J=3.2Hz,1H),4.21(s,2H),3.47–3.32(m,2H),3.27(s,2H); 13 C NMR (126MHz, CDCl3) δ180.69,158.39,151.15,149.84,148.28,144.56,142.01,135.60,134.60,133.18,129.65(×2),128.80(×2 ),128.64,128.54(×2),128.16,127.15,127.01,125.12(×2),110.36,108.72,108.37,75.71,39.64,36.76,35.41; HRMS(ESI)m / z calc.for C 29 H 23 N4O4S[M+H] + 523.1440, found 523.1439. HPLC purity 96.0%.

[0063] 8-Benzyl-2-(furan-2-ylmethyl)-3-((2-nitrobenzyl)oxy)-6-phenylimidazo[1,2-a]pyrazine: prepared from Fz and 1-(bromomethyl)-2-nitrobenzene, purified by silica gel column chromatography (PE / EA=3 / 1) to obtain probe 5 (143 mg, 79%), an off-white solid. 1H NMR(500MHz, CDCl3)δ8.17(dd,J=8.2,1.3Hz,1H),8.08(s,1H),7.93–7.90(m,2H),7.85(dd, J=7.8,1.3Hz,1H),7.74(dd,J=7.5,1.3Hz,1H),7.63–7.60(m,2H),7.57(td,J=7.9,1.4Hz,1 H),7.45(dd,J=8.4,6.9Hz,2H),7.38(d,J=7.3Hz,1H),7.32–7.27(m,3H),7.23–7.19(m,1H) ,6.27(dd,J=3.2,1.9Hz,1H),6.13(d,J=3.1Hz,1H),5.45(s,2H),4.61(s,2H),4.22(s,2H); 13 C NMR (126MHz, CDCl3) δ152.98,152.04,147.23,141.55,138.86,137.95,136.98,136.79,134.09,132.40,131.90,130.37,129.74(×2),129 .39,129.38,128.81(×2),128.52,128.29(×2),126.47,126.28(×2),125.13,110.55,109.08,106.77,73.60,39.34,26.90; HRMS(ESI)m / z calc.for C 31 H 25 N4O4[M+H] + 517.1876, found 517.1875. HPLC purity 97.0%.

[0064] 8-Benzyl-2-(furan-2-ylmethyl)-3-((4-nitrobenzyl)oxy)-6-phenylimidazo[1,2-a]pyrazine: prepared from Fz and 1-(bromomethyl)-4-nitrobenzene, purified by silica gel column chromatography (PE / EA=3 / 1) to obtain probe 6 (148 mg, 82%), an off-white solid. 1H NMR (500MHz, CDCl3) δ8.27–8.23(m,2H),7.88(s,1H),7.84–7.81(m,2H),7.62–7.59(m,2H),7.57–7.54(m,2H),7.46–7.42(m,2H),7.40–7.35(m, 1H),7.33–7.27(m,3H),7.24–7.19(m,1H),6.32(dd,J=3.2,1.9Hz,1H),6 .13(dd,J=3.2,1.0Hz,1H),5.13(s,2H),4.59(s,2H),4.19–4.17(m,2H); 13 C NMR (126MHz, CDCl3) δ153.13,152.07,148.15,142.66,141.51,138.92,137.86,136.94,136.69,132.40,130.36,129.74(×2),128.85( ×2),128.62,128.61(×2),128.27(×2),126.48,126.19(×2),124.00(×2),110.63,108.77,106.79,75.60,39.35,26.88; HRMS(ESI)m / z calc.for C 31 H 25 N4O4[M+H] + 517.1876, found 517.1877. HPLC purity 98.0%.

[0065] Example 6: Preparation of probe 7

[0066]

[0067] 8-Benzyl-6-(3-((tert-butyldimethylsilyl)oxy)phenyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one (7a): Compound 7a was obtained according to previously reported methods

[21] . 1H NMR(500MHz, CDCl3)δ7.78(d,J=3.8Hz,1H),7.74(d,J=1.6Hz,1H),7.71(s, 1H),7.57–7.54(m,2H),7.45(s,1H),7.40(dd,J=7.9,1.4Hz,1H),7.35–7.3 1(m,3H),7.29(d,J=7.9Hz,1H),7.24(d,J=7.5Hz,1H),6.83(dd,J=8.0,2.4 Hz, 1H), 6.74 (dd, J = 3.8, 1.6Hz, 1H), 4.38 (s, 2H), 1.01 (s, 9H), 0.22 (s, 6H).

[0068] 3-(8-benzyl-2-(furan-2-ylmethyl)-3-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)imidazo[1,2-a]pyrazin-6-yl)phenol: Under nitrogen protection, 7a (179 mg, 0.35 mmol), 5-(chloromethyl)-1-methyl-2-nitro-1H-imidazol (2b) (184 mg, 1.05 mmol), and K₂CO₃ (145 mg, 1.05 mmol) were dissolved in 5 mL of DMF and reacted overnight at room temperature. After the reaction was complete, the mixture was quenched with 30 mL of water, extracted with ethyl acetate (2 × 30 mL), and the organic phases were combined, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to give a crude reddish-brown solid. The crude product was dissolved in 10 mL of methanol solution, 0.5 mL of HCl (6 M) solution was added, and the reaction was stirred at room temperature for 1 hour. After the reaction was complete, 30 mL of water was added and the mixture was poured into a separatory funnel. The aqueous layer was extracted with dichloromethane, the organic layers were combined, dried over anhydrous Na2SO4, and concentrated on a rotary evaporator. The crude product was purified by preparative high-performance liquid chromatography (55% CH3CN / H2O, flow rate 8 mL / min) to obtain probe 7 (118 mg, 63%) as a yellow solid. 1 HNMR(500MHz, CDCl3)δ7.81(s,1H),7.53–7.48(m,2H),7.38–7.33(m,2H),7.29( d,J=1.8Hz,1H),7.24(t,J=7.9Hz,1H),7.18(t,J=7.5Hz,2H),7.13–7.09(m,1H) ,7.06(s,1H),6.84(dd,J=8.0,2.4Hz,1H),6.29(dd,J=3.2,1.9Hz,1H),6.11(d, J=3.2Hz,1H),5.71(s,1H),5.00(s,2H),4.53(s,2H),4.14(s,2H),3.94(s,3H); 13C NMR (126MHz, CDCl3) δ157.00,152.96,151.63,146.40,141.63,139.02,137.74,137.56,136.35,132.41,132.03,130.64,130.15,12 9.58(×2),129.46,128.29(×2),126.51,118.06,116.16,113.17,110.81,108.77,106.99,65.43,39.24,34.39,26.77; HRMS(ESI)m / z calc.forC 29 H 29 N6O5[M+H] + 537.1886, found 537.1883. HPLC purity 97.0%.

[0069] Activity evaluation of nitroreductase probes

[0070] Reagents and materials

[0071] 50mM Tris-HCl (pH=7.40) buffer solution a: purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0072] Preparation of 50mM Tris-HCl (containing 0.5mM NADH, pH=7.40) buffer solution b: Dissolve 70.94mg of disodium NADH in 200mL of MiliQ ultrapure water and use immediately.

[0073] Preparation of nitroreductase probe solution: Weigh a certain amount of nitroreductase probe, dissolve it in DMSO to prepare a 25mM concentrated stock solution, and dilute it with buffer solution to the required concentration. Prepare fresh solution before use.

[0074] Preparation of nitroreductase solution: Dissolve 1 mg of nitroreductase in 1 mL of PBS on ice, and dilute with buffer solution to the required concentration.

[0075] The Escherichia coli strain BL21(DE3) was derived from Sigma-Aldrich (St. Louis, MO).

[0076] NanoLuc luciferase was prepared in the laboratory: NanoLuc was constructed into the pMKH vector, the pMKH-NanoLuc plasmid was transformed into E. coli BL21(DE3) strain, the strain was cultured and protein expression was induced, and then the target protein was obtained by purification.

[0077] Example 7: Chemical stability test of the probe

[0078] To test the chemical stability of the probe, a 500 μM NTR probe or F2 ethanol solution was first prepared and stored in the dark at 25 °C for a certain period of time (0-48 hours). The change in content was monitored by HPLC and the chromatogram was recorded at an absorbance of 254 nm. Figure 1 The changes in the content of the probe in an ethanol solution (500 μM) after storage at 25 °C for a certain period of time (0-48 h) are shown.

[0079] Example 8: Verification of the probe's mechanism of action

[0080] Add 100 μL of probe 7 solution (5 mM) and 900 μL of nitroreductase (10 μg / mL) in Tris-HCl buffer to an EP tube, mix thoroughly, and incubate at 37°C for 5 hours. Simultaneously, add 1000 μL of probe 7 solution (500 μM) or luciferase solution (250 μM) to the EP tube as a standard sample.

[0081] After filtration through Millipore, the sample was transferred to a high-performance liquid chromatography (HPLC) vial and analyzed using a Waters-2695 HPLC system. The HPLC analysis results show... Figure 2 In the presence of NADH, probe 7 can be reduced by nitroreductase, and the retention time of the reduction product in the spectral peak matches that of fluorescein. Therefore, the reduction mechanism of the probe is confirmed, and more in-depth activity testing can be performed.

[0082] Among them Figure 2 In the curve, S1 is probe 7 (500 μM) + NTR (5 μg / mL) + NADH (500 μM), S2 is fluorescein (250 μM), and S3 is probe 7 (500 μM).

[0083] Example 9: Time-dependent experiment on probe bioluminescence intensity

[0084] Time-dependent measurements were performed using Tris-HCl buffer. The probe stock solution (25 mM) was diluted to 25 μM with Tris-HCl buffer. 50 μL of the diluted probe solution and 50 μL of 5 μg / mL nitroreductase solution (containing 0.5 mM NADH) were added to each well of a 96-well plate (WHB, black), and incubated at 37°C for 30, 40, 60, or 90 min (three replicates per group). After incubation, 50 μL of NanoLuc luciferase (10 μg / mL) in Tris-HCl buffer was added to each well, and the bioluminescence intensity of each well was immediately measured using an AniView 100 multi-mode animal in vivo imaging system for 5 s. Figure 3 The bioluminescent signal response diagrams of the probe and nitroreductase after incubation for different times in vitro are shown, such as... Figure 3 As shown, the bioluminescence signal gradually increases with increasing incubation time, reaching a maximum at 30 min and decreasing slightly at 90 min.

[0085] Example 10: Experiment on the dependence of probe bioluminescence intensity on nitroreductase concentration

[0086] The concentration-dependent experiments of the nitroreductase probe were conducted in a Tris-HCl buffer system.

[0087] First, the prepared nitroreductase was diluted with buffer solution b to final concentrations of 0 μg / mL, 0.039 μg / mL, 0.078 μg / mL, 0.156 μg / mL, 0.312 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL. Then, 50 μL of probe solution (25 μM) and 50 μL of nitroreductase solutions of different concentrations were added sequentially to each of the 96-well black plates, mixed thoroughly, and incubated at 37°C on a shaker for 30 min (three replicates per group). Subsequently, 50 μL of NanoLuc luciferase (10 μg / mL) solution was added to each well, and the bioluminescence intensity of each well was immediately measured using an AniView100 multi-mode animal in vivo imaging system for 5 s exposure time. Relative luminescence intensity = number of photons emitted by the experimental group [p / s / cm2 / sr] / number of photons emitted by the control group [p / s / cm2 / sr]. Figure 4 The results of the probe sensitivity experiment are shown, in which... Figure 4 A shows bioluminescent imaging of the probe after incubation with different concentrations of NTR; Figure 4 B shows the relative bioluminescence intensities of probes 1-7 after incubation with different concentrations of NTR compared to the control group; Figure 4 C shows the relationship between NTR concentration and relative bioluminescence intensity of probe 7 in the range of 0-5 μg / mL; Figure 4 D shows the linear relationship (mean ± SD, n = 3) between NTR concentration and the relative bioluminescence intensity of probe 7 in the range of 0–0.625 μg / mL. From the experimental results ( Figure 4 As the NTR reductase concentration increased, the bioluminescent signal of all probes gradually increased. When CNTR = 5 μg / mL, the bioluminescent intensity of probes 1-7 increased by approximately 20-fold, 770-fold, 320-fold, 40-fold, 20-fold, 280-fold, and 560-fold compared to the blank. Figure 4 (B) Within the range of 0-0.625 μg / mL, the relative bioluminescence intensity of probe 7 is linearly related to the concentration of NTR, with a detection limit of 0.016 μg / mL, and can be used for the quantitative detection of NTR content in aqueous solution.

[0088] Example 11: Probe Selectivity Test for Nitro Reductase

[0089] Similarly, the selectivity experiment of the nitroreductase probe was performed in a Tris-HCl buffer system.

[0090] First, add 50 μL of diluted probe solution (25 μM) to each well of a 96-well plate. Then, add 50 μL of different reducing agents to the probe solution, including nitroreductase (10 μg / mL) + NADPH (0.5 mM), nitroreductase (10 μg / mL), NADPH (0.5 mM), reduced glutathione (GSH, 1000 μM), cysteine ​​(Cys, 1000 μM), glutamate (Glu, 1000 μM), sodium ascorbate (VcNa, 1000 μM), and sodium hydrosulfide (NaSH, 1000 μM) in Tris-HCl solution (three replicates per group). After incubation at 37°C for 30 min, 50 μL of NanoLuc luciferase (10 μg / mL) solution was added to each well, and the bioluminescence intensity of each well was immediately measured using an AniView100 multi-mode animal in vivo imaging system for 5 s exposure time. The control group contained only the probe and buffer solution. Figure 5 The experimental results on the selectivity of the probe are shown, in which Figure 5 A shows bioluminescent imaging of the probe and related substances; Figure 5 B shows the relative bioluminescence intensities (mean ± SD, n = 3, *p < 0.0001) of probes 1-7 and related substances compared to the control group. From the experimental results ( Figure 5 The results showed that, compared to the control group, the bioluminescence intensity was significantly enhanced only when the probe was co-incubated with NTR-NADH; under other reducing conditions, the bioluminescence intensity remained almost unchanged. This indicates that the probe is more sensitive to the NTR-NADH system and is easily reduced to luciferin.

[0091] Example 12: Probe Cytotoxicity Test

[0092] LX2 cells in logarithmic growth phase were digested and seeded into 96-well plates (5*10⁶ cells / wells). 4 ( / well), cultured until adherent, discard the old medium, add complete medium containing the test compound (DEME + 10% FBS + 0.1% antibiotics), culture at 37℃ and 5% CO2 for 24h, add 10uL of CCK-8 solution (Beyotime) to each well, continue incubation for 1h, and measure the UV absorbance at 450nm. Figure 6 The effect of different concentrations of probes 2 and 7 (10 μM, 20 μM) on cell viability after incubation with LX2 cells for 24 h was shown by the CCK8 method (mean ± SD, n = 3).

[0093] Example 13: Application of probes at the cellular level

[0094] E. coli BL21 stored at -80℃ was streaked on plates for 12 h, and single colonies were picked and inoculated into 50 mL of liquid LB medium (C Kan =100μg / mL), cultured at 37℃ and 180rpm until the OD value of the bacterial culture reached 0.6, set the shaking temperature to 16℃, and continue to culture for 2h. Then add IPTG to the final concentration of 0.2mM, and induce protein expression at 16℃ and 180rpm for 12-16h.

[0095] Add 50 μL of probe solution (25 μM) and 50 μL of E. coli solution suspended in LB to a 96-well plate (0-10 μL). 8 Bacteria / mL were collected and incubated at 37°C. The bioluminescence intensity of each well was then measured using an Aniview 100 IVIS imaging system with an exposure time of 5 s. For the NTR inhibitor assay, LB suspensions of *E. coli* were pretreated with or without dicumarol (0.1 mM) for 12 h. The suspensions were then treated with 25 μM probe 2 or 7 for 30 min. Bioluminescence measurements of the bacterial solutions were performed using an EnVision Multimode Microplate Reader (PerkinElmer, USA). Figure 7 Experimental results showing the bioluminescence imaging of the probe in bacterial culture are presented, in which... Figure 7 A shows the curve of bioluminescence intensity changing over time when the probe is co-incubated with bacterial culture; Figure 7 B shows the probes interacting with E. coliN Luc. + bacteria, E. coli N Luc - Bioluminescent signals during bacterial incubation; Figure 7 C shows the relative bioluminescence intensity with and without inhibitor pretreatment before the probe is incubated with the bacterial culture; Figure 7 D shows the bioluminescence intensity (mean ± SD, n = 3, ***p < 0.001) after the probe was incubated with different amounts of bacterial culture. Figure 7 As shown in Figure A, the bioluminescence signal value was highest after 30 minutes of co-incubation of the probe with bacterial culture, and the bioluminescence intensity gradually weakened with increasing time. The probe was compared among different *E. coli* strains, such as... Figure 7 As shown in B, compared with Escherichia coli Luc-, from Escherichia coli NLuc + Significant bioluminescent signals were detected. Furthermore, the signal intensities of bacterial cultures treated with probes 2 and 7 were relatively high. Compared to Fz, the bioluminescent signals of probes 2 and 7 were comparable after 30 min of incubation. Figure 7C), and the bioluminescence intensity decreased with the decrease in bacterial count. The anticoagulant dihydroxycoumarin is an effective inhibitor of many flavinases. With the bioluminescence intensity of the group without coumarin set as 1, the bioluminescence intensity of the bacterial culture significantly decreased after coumarin pretreatment. Figure 7 D). The results showed that the probe had good bioluminescent activity at the cellular level, demonstrating its potential for application in cellular-level bioluminescence imaging.

[0096] Example 14: Application of probes at the animal level

[0097] The bacteria were incubated at 37°C until OD values ​​in LB medium containing 100 μg / mL Kan were reached. 600 The culture was harvested by centrifugation (4000×g, 10 min) after reaching a concentration of 0.6, and washed three times in PBS. After washing, the bacteria were resuspended in PBS. To obtain a relatively accurate luminescent signal, hair was removed from the hind legs of BALB / c mice before modeling, and then 50 μL of bacteria suspended in PBS was injected directly into the quadriceps muscle to a depth of approximately 5 mm. (The concentration range of the bacterial suspension used for injection was 10...) 6 Up to 10 8 (bacteria / mL). One hour after bacterial inoculation, 200 μL of 10 mM probe or 200 μL of 10 mM MF2 was injected intraperitoneally (IP). For in vivo experiments involving different probe concentrations, the injected NCL probe concentration was 200 μL of 1 mM or 10 mM solution. Bioluminescence imaging of mice was performed periodically after probe injection. Figure 8 Experimental results of bioluminescence imaging of the probe in animal models are shown, in which... Figure 8 A represents probes 2 (1 mg / mouse) and 7 (1 mg / mouse) against infecting bacteria (10... 8 The results of bioluminescence imaging of model mice (bacteria / mL); Figure 8 B represents the bioluminescence imaging results obtained by injecting probe 7 (1 mg / mouse) or Fz (0.76 mg / mouse) into a mouse model of bacterial infection; Figure 7 C represents the injection probe 7 (1 mg / mouse) for different quantities of bacterial suspension (10 6 -10 8 The results of bioluminescence intensity measurement in a model mouse (bacteria / mL); Figure 7 D represents the injection of probe 7 at different concentrations (0.1 mg / mouse or 1 mg / mouse) against infecting bacteria (10 8 Results of bioluminescence imaging in model mice (n=4) with bacteria / mL. Figure 8As shown in Figure A, the bioluminescent signal of probe 7 was significantly higher than that of probe 2, indicating that probe 7 is more suitable for in vivo imaging in animals. A significant bioluminescent signal was detected 30 minutes after probe injection, then gradually increased until reaching a plateau at 90 minutes. The signal intensity did not show significant decay up to 4 hours and was correlated with the injected probe dosage and the amount of infected bacteria. Figure 8 A, D). Compared to Fz, the bioluminescence intensity in animals injected with probe 7 was slightly lower, but the signal decay in the Fz group was faster, and at 2 hours it was consistent with that of the probe 7 group. Figure 8 B). The above results indicate that probe 7 provides good imaging performance in animals and can be used to detect bacterial infection-related models.

[0098] Example 15: Probe for the detection of NTR activity in intestinal bacteria

[0099] To measure the nitroreductase activity of the mouse gut microbiota, fresh mouse feces were collected, weighed, and then resuspended in PBS at a ratio of 1:5 (g / ml). After mixing, magnetic beads were added and the mixture was vortexed, followed by centrifugation at 4°C (200×g, 10 min) to obtain a bacterial suspension. The bacterial strain was then washed twice in PBS, lysed, and centrifuged (8000×g, 5 min, 4°C) to remove the supernatant. The bacterial suspension was washed once with Tris buffer (pH 7.4), centrifuged at 8000×g, 4°C for 5 min, and the supernatant was removed. The bacterial pellet was resuspended in Tris buffer, homogenized, and the supernatant was collected by centrifugation (15000×g, 15 min, 4°C). The supernatant was treated with 12.5 μM probe 7 for 30 min (6 samples per group), followed by the addition of 50 μL of NanoLuc luciferase (10 μg / mL) solution. The bioluminescence intensity of the reaction solution was immediately measured using an EnVision multimode microplate reader (PerkinElmer). The standard curve was obtained by co-incubating probe 7 with nitroreductase at different concentrations (0 μg / mL, 0.039 μg / mL, 0.078 μg / mL, 0.156 μg / mL, 0.312 μg / mL, 0.625 μg / mL). Figure 9 The results of the detection of NTR activity of probe 7 against mouse intestinal bacteria are shown, among which Figure 9 A shows a standard curve of the probe's luminescence intensity as a function of NTR concentration; Figure 9 B shows the results of the mouse intestinal bacterial NTR activity assay (HFD vs ND, ***P<0.001, n=6). For example... Figure 9 As shown, a high-fat diet alters the gut microbiota structure. Compared with the ND group, the HFD group showed significantly higher NTR activity in gut microbiota, with a statistically significant difference (***p<0.001). Therefore, a high-fat diet can alter the gut microbiota and increase NTR activity.

[0100] Any aspects of this invention not described in detail are well-known to those skilled in the art.

[0101] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0102] References

[0103] [1]ZMKaskova,ASTsarkova,IVYampolsky,1001lights:Luciferins,luciferases,their mechanisms of action and applications in chemical analysis,biology and medicine,Chem.Soc.Rev.45(2016)6048-6077.

[0104] [2] LFGreer 3rd, AASzalay, Imaging of light emission from the expression of luciferases in living cells and organisms: a review, Luminescence. 17 (2002) 43-74.

[0105] [3] JAPrescher, CHContag, Guided by the light: visualizing biomolecular processes in living animals with bioluminescence, Curr. Opin. Chem. Biol. 14 (2010) 80-89.

[0106] [4]DKWelsh,SAKay,Bioluminescence imaging in living organisms,Curr.Opin.Biotechnol.16(2005)73-78.

[0107] [5]C.G.England,E.B.Ehlerding,W.Cai,NanoLuc:A Small Luciferase IsBrightening Up the Field ofBioluminescence,Bioconjug.Chem.27(2016)1175-1187.

[0108] [6]S.Iwano,M.Sugiyama,H.Hama,A.Watakabe,N.Hasegawa,T.Kuchimaru,K.Z.Tanaka,M.Takahashi,Y.Ishida,J.Hata,S.Shimozono,K.Namiki,T.Fukano,M.Kiyama,H.Okano,S.Kizaka-Kondoh,T.J.McHugh,T.Yamamori,H.Hioki,S.Maki,A.Miyawaki,Single-cell bioluminescence imaging of deep tissue in freelymoving animals,Science.359(2018)935-939.

[0109] [7]L.J.Kricka,F.R.Leach,In memoriam Dr Marlene DeLuca.1987O.M.SmithLecture.Firefly luciferase:mechanism ofaction,cloning and expression oftheactive enzyme,J.Biolumin.Chemilumin.3(1989)1-5.

[0110] [8]I.Yamaguchi,Oplophorus oxyluciferin and a model luciferin compoundbiologically active with Oplophorus luciferase,Biochem.J.151(1975)9-15.

[0111] [9]O.Shimomura,T.Masugi,F.H.Johnson,Y.Haneda,Properties and reactionmechanism ofthe bioluminescence system ofthe deep-sea shrimp Oplophorusgracilorostris,Biochemistry.17(1978)994-998.

[0112]

[10] B.A.Tannous,D.E.Kim,J.L.Fernandez,R.Weissleder,X.O.Breakefield,Codon-optimized Gaussia luciferase cDNA for mammalian gene expression inculture and in vivo,Mol.Ther.11(2005)435-443.

[0113]

[11] S.Inouye,K.Watanabe,H.Nakamura,O.Shimomura,Secretional luciferaseofthe luminous shrimp Oplophorus gracilirostris:cDNA cloning of a novelimidazopyrazinone luciferase(1),FEBS Lett.481(2000)19-25.

[0114]

[12] O.Shimomura,T.Masugi,F.H.Johnson,Y.Haneda,Properties and reactionmechanism ofthe bioluminescence system ofthe deep-sea shrimp Oplophorusgracilorostris,Biochemistry.17(1978)994-998.

[0115]

[13] M.P.Hall,J.Unch,B.F.Binkowski,M.P.Valley,B.L.Butler,M.G.Wood,P.Otto,K.Zimmerman,G.Vidugiris,T.Machleidt,M.B.Robers,H.A.Benink,C.T.Eggers,M.R.Slater,P.L.Meisenheimer,D.H.Klaubert,F.Fan,L.P.Encell,K.V.Wood,Engineeredluciferase reporter from a deep sea shrimp utilizing a novelimidazopyrazinone substrate,ACS.Chem.Biol.7(2012)1848-1857.

[0116]

[14] I.Pavlinov,M.Salkovski,L.N.Aldrich,Beclin 1-ATG14L Protein-Protein Interaction Inhibitor Selectively Inhibits Autophagy throughDisruption ofVPS34 Complex I,J.Am.Chem.Soc.142(2020)8174-8182.

[0117]

[15] A.S.Dixon,M.K.Schwinn,M.P.Hall,K.Zimmerman,P.Otto,T.H.Lubben,B.L.Butler,B.F.Binkowski,T.Machleidt,T.A.Kirkland,M.G.Wood,C.T.Eggers,L.P.Encell,K.V.Wood,NanoLuc Complementation Reporter Optimized for AccurateMeasurement of Protein Interactions in Cells,ACS.Chem.Biol.11(2016)400-408.

[0118]

[16] N.C.Dale,E.K.M.Johnstone,C.W.White,K.D.G.Pfleger,NanoBRET:TheBright Future ofProximity-BasedAssays,Front.Bioeng.Biotechnol.7(2019)56.

[0119]

[17] H.Shigeto,T.Ikeda,A.Kuroda,H.Funabashi,ABRET-based homogeneousinsulin assay using interacting domains in the primary binding site of theinsulin receptor,Anal,Chem.87(2015)2764-2770.

[0120]

[18] K.Krawczyk,S.Xue,P.Buchmann,G.Charpin-El-Hamri,P.Saxena,M.D.Hussherr,J.Shao,H.Ye,M.Xie,M.Fussenegger,Electrogenetic cellular insulinrelease for real-time glycemic control in type 1 diabetic mice,Science.368(2020)993-1001.

[0121]

[19] C.Yan,L.Du,M.Li,Novel NanoLuc-type substrates with various C-6substitutions,Bioorg.Med.Chem.Lett.30(2020)127085.

[0122]

[20] J.Li,X.Wang,G.Dong,C.Yan,Y.Cui,Z.Zhang,L.Du,M.Li,Novel furimazinederivatives for nanoluciferase bioluminescence with various C-6 and C-8substituents,Org,Biomol.Chem.19(2021)7930-7936.

[0123]

[21] Y.Su,J.R.Walker,T.P.Smith,L.X.Liu,M.P.Hall,L Labanieh,R Hurst,D.C.Wang,L.P.Encell,N Kim,F Zhang,M.A.Kay,K.M.Casey,R.G.Majzner,J.R.Cochran,C.L.Mackall,T.A.Kirkland,M.Z.Lin,Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals,Nat.Methods.17(2020)852-860.

[0124]

[22] Y.Su,J.R.Walker,M.P.Hall,M.A.Klein,X.Wu,L.P.Encell,K.M.Casey,L.X.Liu,G.Hong,M.Z.Lin,T.A.Kirkland,An optimized bioluminescent substrate fornon-invasive imaging in the brain,Nat.Chem.Biol.(2023).DOI:10.1038 / s41589-023-01265-x.

[0125]

[23] M.Hattori,G.Kawamura,R.Kojima,M.Kamiya,Y.Urano,T.Ozawa,ConfocalBioluminescence Imaging for Living Tissues with a Caged Substrate ofLuciferin,Anal.Chem.88(2016):6231-6238.

[0126]

[24] Y.Mizui,M.Eguchi,M.Tanaka,Y.Ikeda,H.Yoshimura,T.Ozawa,D.Citterio,Y.Hiruta,Long-term single cell bioluminescence imaging with C-3 positionprotected coelenterazine analogues,Org.Biomol.Chem.19(2021)579-586.

[0127]

[25] M.Orioka,M.Eguchi,Y.Mizui,Y.Ikeda,A.Sakama,Q.Li,H.Yoshimura,T.Ozawa,D.Citterio,Y.Hiruta,A Series of Furimazine Derivatives for SustainedLive-Cell Bioluminescence Imaging and Application to the Monitoring ofMyogenesis at the Single-Cell Level,Bioconjug.Chem.33(2022)496-504.

[0128]

[26] D.W.Bryant,D.R.McCalla,M.Leeksma,P.Laneuville,Type Initroreductases of Escherichia coli,Can.J.Microbiol.(1981)81-86.

[0129]

[27] S.Kitamura,N.Narai,K.Tatsumi,Studies on bacterialnitroreductases.Enzymes involved in reduction ofaromatic nitro compounds inEscherichia coli,J.Pharmacobiodyn.6(1983)18-24.

[0130]

[28] M.D.Roldán,E.Pérez-Reinado,F.Castillo,C.Moreno-Vivián,Reductionof polynitroaromatic compounds:the bacterial nitroreductases,FEMS.Microbiol.Rev.32(2008)474-500.

[0131]

[29] E.Akiva,J.N.Copp,N.Tokuriki,P.C.Babbitt,Evolutionary andmolecular foundations ofmultiple contemporary functions ofthe nitroreductasesuperfamily,Proc.Natl.Acad.Sci.U S A.114(2017)E9549-E9558。

Claims

1. A nitroaromatic ring-substituted imidazopyrazine compound, characterized in that, The compound has the general formula (1): ; Among them, R 1 For H, OH, R 2 It is any one of the following groups: 。 2. The nitroaromatic-substituted imidazopyrazine compound according to claim 1, characterized in that, The compound is selected from: Compound 1: 8-benzyl-2-(furan-2-ylmethyl)-3-((5-nitrofuran-2-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine; Compound 2: 8-benzyl-2-(furan-2-ylmethyl)-3-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine; Compound 3: 8-benzyl-2-(furan-2-ylmethyl)-3-((1-methyl-5-nitro-1H-imidazol-2-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine; Compound 4: 8-benzyl-2-(furan-2-ylmethyl)-3-((5-nitrothiophen-2-yl)methoxy)-6-phenylimidazo[1,2-a]pyrazine; Compound 5: 8-benzyl-2-(furan-2-ylmethyl)-3-((2-nitrobenzyl)oxy)-6-phenylimidazo[1,2-a]pyrazine; Compound 6: 8-benzyl-2-(furan-2-ylmethyl)-3-((4-nitrobenzyl)oxy)-6-phenylimidazo[1,2-a]pyrazine; Compound 7: 3-(8-benzyl-2-(furan-2-ylmethyl)-3-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)imidazo[1,2-a]pyrazin-6-yl)phenol.

3. The method for preparing the compound according to any one of claims 1 to 2, characterized in that, The method includes: Under inert gas protection, 8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazine-3(7H)-one, whether protected or unprotected, was reacted with a methyl bromide derivative in the presence of potassium carbonate to obtain the compound shown in formula (1).

4. Use of the compound according to any one of claims 1 to 2, characterized in that, Used to prepare bioluminescent probes for detecting nitroreductase.

5. The use as described in claim 4, characterized in that, The probe is used as a substrate for the NanoLuc bioluminescence system, for bioluminescence imaging studies, or for detecting drug effects.

6. A kit for detecting nitroreductase, characterized in that, It includes the compound as described in any one of claims 1 to 2, NanoLuc luciferase, NADH, and a 50 mM Tris-HCl buffer solution at pH 7.40.

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