Nitroreductase activation-based near-infrared fluorescence and photoacoustic dual-mode small molecule probe as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation, the small molecule probe can be activated by nitroreductase to be converted into a compound with fluorescence and photoacoustic signals, the general structural formula of the compound of the small molecule probe is # imgabs0 #, R can be substituted at 4, 5, 6 and 7 sites, and the substituent group is one of H, OCH3, F and Cl; the small-molecule probe compound has no fluorescence and photoacoustic, and under the catalysis of nitroreductase, nitryl is reduced into amino to be converted into a compound integrating optical imaging and photoacoustic imaging, so that when the small-molecule probe is applied to tumor hypoxia imaging, the tumor hypoxia imaging can be realized only through activation of the nitroreductase in tumor cells. Near-infrared fluorescence and photoacoustic dual modes can be started at the same time, and high-specificity, high-resolution, high-sensitivity and high-penetration-depth imaging of tumors can be achieved.

Description

Technical Field

[0001] The invention discloses a small molecule probe for tumor hypoxia imaging, and particularly relates to a near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation and a preparation method thereof. Background Art

[0002] Hypoxia is one of the specific markers of the tumor microenvironment. The hypoxic microenvironment is not only closely related to the development and metastasis of cancer, but also reduces the sensitivity of tumors to chemotherapy, radiotherapy and photodynamic therapy, seriously affecting the therapeutic effect of cancer. As a marker of tumor hypoxia, the expression of nitroreductase (NTR) is closely related to the degree of tumor hypoxia. Therefore, the detection of NTR can realize the hypoxia assessment of tumors. At present, in tumor hypoxia imaging, positron emission tomography, computed tomography, and magnetic resonance imaging technologies lack molecular sensitivity and specificity. Although the resolution and sensitivity of optical imaging alone are high, the penetration depth is usually limited (<1mm), and only superficial tumor hypoxia can be observed. The imaging depth of photoacoustic imaging in the body can be as high as several centimeters, but the resolution and sensitivity are not high.

[0003] Therefore, there is a need to combine optical imaging with photoacoustic imaging to overcome the shortcomings of using photoacoustic imaging or photoacoustic imaging alone. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation and a preparation method thereof, which activates nitroreductase in tumor cells, simultaneously turns on the near-infrared fluorescence and photoacoustic dual modes, and applies them to tumor hypoxia imaging, which can achieve high-specificity, high-resolution, high-sensitivity, and high-penetration depth imaging of tumors.

[0005] The present invention discloses a near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation. The small molecule probe can be activated by nitroreductase and converted into a compound having fluorescence and photoacoustic signals. The compound structural formula of the small molecule probe is:

[0006] Wherein R can be located at the 4, 5, 6, 7 position on the benzothiazole ring and is one of H, OCH3, F, and Cl;

[0007] Furthermore, under the catalysis of nitroreductase, the nitro group of the small molecule probe is reduced to an amino group, and the small molecule probe is converted into a compound having fluorescence and photoacoustic signals, and the general formula of the compound is:

[0008]

[0009] Furthermore, the structural formula of the compound of the small molecule probe is:

[0010]

[0011] Further, the catalytic reduction reaction of the small molecule probe is:

[0012]

[0013] The present invention also discloses a method for preparing a near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation, comprising the following steps:

[0014] S1, dissolving 2-methylbenzothiazole and 1,3-propane sultone in acetonitrile, reacting sufficiently, cooling to room temperature, and collecting a white solid product by filtration;

[0015] S2, dissolving 2-hydroxy-4-nitrobenzaldehyde in N,N-dimethylformamide, cooling to -15 to -10 degrees, adding cesium carbonate, and then adding 2-bromo-1-cyclohexene-1-carboxaldehyde solution under nitrogen protection, filtering to remove alkali after sufficient reaction, and then quenching the reaction with water and diluting the reaction with dichloride, washing to remove most of the N,N-dimethylformamide, and finally drying the fraction by spin drying and column chromatography; collecting the light yellow product;

[0016] S3, dissolving the white solid product in step S1 and the light yellow product in step S2 in Ac2O, then adding sodium acetate and stirring at a temperature of 60-80° C., and finally separating to obtain a small molecule probe compound;

[0017] Furthermore, the synthesis route of the small molecule probe is:

[0018]

[0019]

[0020] The invention discloses an application of the near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation in the preparation of in vivo optical and photoacoustic imaging contrast agents or tumor surgery navigation probes.

[0021] The beneficial effects of the present invention are as follows: the near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation and the preparation method thereof, the small molecule probe compound itself has no fluorescence and photoacoustic, under the catalysis of nitroreductase, the nitro group is reduced to the amino group and converted into a compound integrating optical imaging and photoacoustic imaging, therefore, the small molecule probe is applied to tumor hypoxia imaging, and the near-infrared fluorescence and photoacoustic dual-modes can be simultaneously turned on by activating the nitroreductase in the tumor cells, which can achieve high-specificity, high-resolution, high-sensitivity and high-penetration depth imaging of the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0023] Figure 1 The results of hydrogen spectrum and HPLC-MS / MS of small molecule probe 7-1 are shown;

[0024] Figure 2 This is the excitation emission spectrum of the product of the small molecule probe 7-1 reduced by nitroreductase;

[0025] Figure 3 The fluorescence spectra of the small molecule probe 7-1 after reacting with different concentrations of nitroreductase;

[0026] Figure 4 It is the linear graph after the small molecule probe 7-1 reacts with different concentrations of nitroreductase;

[0027] Figure 5 The UV-visible absorption spectra of probe 7-1 after reacting with different concentrations of nitroreductase;

[0028] Figure 6 is the UV-visible absorption linear graph after the reaction of probe 7-1 with different concentrations of nitroreductase;

[0029] Figure 7 The photoacoustic (PA) spectra of probe 7-1 after reacting with different concentrations of nitroreductase;

[0030] Figure 8 The photoacoustic (PA) linear graph after the reaction of probe 7-1 with different concentrations of nitroreductase;

[0031] Fig. 9 The fluorescence spectra of probe 7-1, probe 7-1+nitroreductase+NADH, and probe 7-1+nitroreductase+NADH+dicoumarol (DIC), an inhibitor of nitroreductase at different concentrations;

[0032] Fig.10 The fluorescence histogram of the small molecule probe 7-1 used to react with different substances (including anions, cations, amino acids, and other enzymes);

[0033] Fig.11 It is a bar graph of cell survival rate at different small molecule probe concentrations;

[0034] Fig.12 This is the fluorescence image after 10 μM small molecule probe 7-1 was incubated with Hala cells for 30 minutes;

[0035] Fig.13 This is the photoacoustic (PA) image after the small molecule probe 7-1 was incubated with Hala cells. DETAILED DESCRIPTION

[0036] Embodiment 1

[0037] The synthesis method and steps of the small molecule probe of this embodiment are as follows:

[0038] Step 1

[0039]

[0040] SM1-1 (R is H) (3 g, 1 eq), SM2 (1.6 g 1.5 eq) in CH3CN (30 ml), the reaction system was refluxed overnight.

[0041] Work up: The reaction system was cooled to room temperature, the solid was collected by filtration, the filter cake was rinsed with ether, and 2.3 g of off-white solid was collected.

[0042] Step 2:

[0043]

[0044] SM5 (1.06 g, 1.2 eq) in DMF (20 ml), cooled to -15 to -10 degrees, added cesium carbonate (5.2 g, 3 eq). Under nitrogen protection, SM4 (1 g, 1 eq) in DMF (2 ml) was slowly added dropwise to the reaction system. After reacting at low temperature for 2 hours, the temperature naturally rose to room temperature and the reaction was continued overnight.

[0045] Work up: Filter to remove alkali; add water to quench the reaction; dilute the reaction with dichloromethane; wash off most of the DMF with water; dry the fractions and perform column chromatography; collect 85 mg of light yellow product.

[0046] Step 3:

[0047]

[0048] SM6 (85 mg, 1 eq) and SM 3-1 (268 mg, 3 eq) were added to Ac2O (3 ml), and sodium acetate (260 mg, 3 eq) was added. The mixture was stirred at 70 degrees overnight.

[0049] Work up: spin off the solvent, column chromatography (DCM / MeOH), collect 45 mg of product.

[0050] Prepared small molecule probe (Compound 7-1): 3-(2-(2-(6-nitro-2,3-dihydro-1H-xanthen-4-yl)vinyl)benzo[d]thiazol-3-ium-3-yl)propane-1-sulfonate

[0051] 3-(2-(2-(6-nitro-2,3-dihydro-1H-xanthen-4-yl)vinyl)benzo[d]thiaz ol-3-ium-3-yl)propane-1-sulfonate

[0052] The results of hydrogen spectrum and HPLC mass spectrometry of compound 7-1 are as follows Figure 1 As shown:

[0053] Proton spectrum results of compound 7-1 1 H NMR(400MHz, DMSO-d6)δ8.43(d,J=15.0Hz,1H),8.31(dd,J=8.3,4.3Hz,2H),8. 19(s,1H),8.02(dd,J=8.3,2.4Hz,1H),7.81(t,J=8.0Hz,1H),7.72(t,J=7.7Hz, 1H),7.62(d,J=8.4Hz,1H),7.40(d,J=15.1Hz,1H),7.18(s,1H),5.00(t,J=7.9H z,2H),2.71(s,4H),2.62(t,J=6.4Hz,2H),2.15(s,2H),1.86(d,J=30.3Hz,2H).

[0054] Mass spectrometry result: m / z = 511.10

[0055] The small molecule probe is activated by nitroreductase, and the reaction is as follows:

[0056]

[0057] To the system of SM 7-1 (40 mg, 1 eq) and SnCl2 (150 mg, 10 eq), add dioxane hydrochloride solution (1.5 ml) and react at 60 degrees for 4 hours.

[0058] Work up: spin dry, column chromatography (DCM / MeOH) (very polar), collect 3 mg of product.

[0059] Compound 8-1: 3-(2-(2-(6-amino-2,3-dihydro-1H-xanthen-4-yl)vinyl)benzo[d]thiazol-3-ium-3-yl)propane-1-sulfonate

[0060] 3-(2-(2-(6-amino-2,3-dihydro-1H-xanthen-4-yl)vinyl)benzo[d]thiaz ol-3-ium-3-yl)propane-1-sulfonate

[0061] Proton spectrum results of compound 8-1 1 H NMR (400MHz, DMSO-d6) δ8.21(d,J=14.1Hz,1H),8.13(d,J=7.7Hz,1H),8.04(d,J =8.6Hz,1H),7.68(d,J=8.2Hz,1H),7.54(t,J=7.6Hz,1H),7.32(s,1H),7.25(d, J=8.6Hz,1H),6.92(d,J=14.0Hz,1H),6.59(d,J=7.1Hz,2H),6.43(s,2H),4.77( s,2H),3.17(d,J=5.5Hz,1H),2.63(d,J=23.7Hz,6H),2.09(s,2H),1.79(s,2H).

[0062] The mass spectrum result was m / z=481.15.

[0063] Compound 8-1 turns on fluorescence and photoacoustic signals under the action of intramolecular charge transfer. The fluorescence excitation wavelength is 680nm, the emission wavelength is 723nm, and the photoacoustic (PA) excitation wavelength is 710nm. Figure 2 As shown, the excitation-emission spectrum of the product of the probe reduced by nitroreductase.

[0064] The small molecule probe prepared by the present invention successfully realizes the high-sensitivity detection of the hypoxia degree of cancer cells. The small molecule probe is used to detect nitroreductase in vitro, which can realize the high-sensitivity detection of three modes: colorimetric, fluorescence, and photoacoustic (PA), and realizes the dual-mode detection of the hypoxia degree by fluorescence and photoacoustic.

[0065] The detection method is as follows:

[0066] Colorimetry

[0067] Linear range: 0.5-9ug

[0068] Limit of detection (LOD) = 88.0 ng / mL

[0069] Fluorescence method

[0070] Linear range: 0-2μg / mL

[0071] Limit of detection (LOD) = 7.8 ng / mL

[0072] Photoacoustic (PA) method

[0073] Linear range: 0-5μg / mL

[0074] Limit of detection (LOD) = 132.6 ng / mL

[0075] Cell experiments

[0076] The test results are as follows:

[0077] The small molecule probe prepared in the above example was used to react with nitroreductase at different concentrations. The fluorescence spectra after the reaction were as shown in FIG. Figure 3 As shown in the figure, the fluorescence intensity of nitroreductase increases with the increase of enzyme concentration in the range of 0-4μg / mL. The linear graph of the reaction between the small molecule probe and different concentrations of nitroreductase is shown in the figure. Figure 4 Linear range: 0-2 μg / mL, limit of detection (LOD) = 7.8 ng / mL. Figure 5 The UV-visible absorption spectra of the probe after reacting with different concentrations of nitroreductase. It can be seen from the figure that when the nitroreductase is in the range of 0-9 μg / mL, the absorption at 680 nm increases with the increase of enzyme concentration. Figure 6 Figure 2 shows the UV-visible absorption linearity graph of the probe after reacting with different concentrations of nitroreductase, using colorimetry: linear range: 0.5-9ug; detection limit (LOD) = 88.0ng / mL. The photoacoustic (PA) spectra of the probe after reacting with different concentrations of nitroreductase are shown in Figure 2. Figure 7 As shown, 710 nm PA increased with the increase of nitroreductase concentration in the range of 0-5 μg / mL. Figure 8 The photoacoustic (PA) linearity graph after the probe reacted with different concentrations of nitroreductase. The linear range of the photoacoustic (PA) method is 0-5 μg / mL, and the detection limit (LOD) is 132.6 ng / mL. Fig. 9 These are fluorescence spectra of the probe, probe + nitroreductase + NADH, and probe + nitroreductase + NADH + different concentrations of the nitroreductase inhibitor dicoumarol (DIC). This proves that the nitroreductase inhibitor dicoumarol can inhibit fluorescence, indicating that fluorescence is the result of the action of nitroreductase and the probe.

[0078] The small molecule probe is used to react with different substances (including anions, cations, amino acids, and other enzymes). The reaction results are as follows: Fig.10 As shown, it is proved that the probe can only turn on fluorescence when it reacts with nitroreductase, and the results prove the selectivity of the small molecule probe for nitroreductase.

[0079] The small molecule probe was used in the cytotoxicity experiment. The cell survival under different probe concentrations was as follows. Fig.11 As shown, it was shown that the probe had very low cytotoxicity within 20 μM.

[0080] 10 μM small molecule probe was incubated with Hala cells for 30 min. Fig.12As shown, the results show that the higher the degree of hypoxia, the stronger the fluorescence. And it can sensitively detect the degree of cell hypoxia at the cellular level. The nitroreductase inhibitor dicoumarol (DIC) can inhibit fluorescence, proving that the fluorescence is the result of the interaction between nitroreductase and the probe. Photoacoustic (PA) image after incubation of small molecule probe with Hala cells ( Fig.13 ) It can be seen that the photoacoustic (PA) signal can only be turned on at 710nm when the probe is incubated with cells, and the nitroreductase inhibitor dicoumarol (DIC) can inhibit the photoacoustic (PA) signal, proving that the photoacoustic is the result of the interaction between nitroreductase and the probe.

[0081] Embodiment 2

[0082] The structural formula of small molecule probe 7-2 is: Its synthetic route is:

[0083]

[0084]

[0085] Under the catalysis of nitroreductase, the nitro group is reduced to the amino group, and the small molecule probe is transformed into a compound with fluorescence and photoacoustic signals. The reaction formula is:

[0086]

[0087] The detection method of Example 1 was used for detection, and the detection results showed that the small molecule probe can simultaneously activate near-infrared fluorescence and photoacoustic dual modes, and can achieve high-specificity, high-resolution, high-sensitivity, and high-penetration depth imaging of tumors.

[0088] Embodiment 3

[0089] The small molecule probe 7-3 of this example is: Its synthetic route is:

[0090]

[0091] Under the catalysis of nitroreductase, the nitro group is reduced to the amino group, and the small molecule probe is transformed into a compound with fluorescence and photoacoustic signals. The reaction formula is:

[0092]

[0093] The detection method of Example 1 was used for detection, and the detection results showed that the small molecule probe can simultaneously activate near-infrared fluorescence and photoacoustic dual modes, and can achieve high-specificity, high-resolution, high-sensitivity, and high-penetration depth imaging of tumors.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation, characterized in that: The small molecule probe can be activated by nitroreductase to be converted into a compound having fluorescence and photoacoustic signals. The general structural formula of the compound of the small molecule probe is: Wherein R can be located at the 4, 5, 6, or 7 position on the benzothiazole ring and can be one of H, OCH3, F, and Cl.

2. The near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation according to claim 1, characterized in that: The nitro group of the small molecule probe is reduced to an amino group under the catalysis of nitroreductase, and the small molecule probe is converted into a compound having fluorescence and photoacoustic signals, and the compound is:

3. The near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation according to claim 1, characterized in that: The structural formula of the compound of the small molecule probe is:

4. The near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation according to claim 3, characterized in that: The catalytic reduction reaction of the small molecule probe is:

5. The method for preparing a nitroreductase-activated near-infrared fluorescence and photoacoustic dual-mode small molecule probe according to claim 1, characterized in that: The following steps are involved: S1, dissolving 2-methylbenzothiazole and 1,3-propane sultone in acetonitrile, reacting sufficiently, cooling to room temperature, and collecting a white solid product by filtration; S2, dissolving 2-hydroxy-4-nitrobenzaldehyde in N,N-dimethylformamide, cooling to -15 to -10 degrees, adding cesium carbonate, and then adding 2-bromo-1-cyclohexene-1-carboxaldehyde solution under nitrogen protection, filtering to remove alkali after sufficient reaction, and then quenching the reaction with water and diluting the reaction with dichloride, washing to remove most of the N,N-dimethylformamide, and finally drying the fraction by spin drying and column chromatography; collecting the light yellow product; S3, dissolving the white solid product in step S1 and the light yellow product in step S2 in Ac2O, then adding sodium acetate and stirring sufficiently at a temperature of 60-80° C., and finally separating to obtain a small molecule probe compound.

6. The method for preparing the near-infrared fluorescence and photoacoustic dual-mode small molecule probe based on nitroreductase activation according to claim 5, characterized in that: The synthetic route of the small molecule probe is:

7. Use of the nitroreductase-activated near-infrared fluorescence and photoacoustic dual-mode small molecule probe according to claim 1 in the preparation of in vivo optical and photoacoustic imaging contrast agents or tumor surgery navigation probes.

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