CN115806566A - Preparation method and application of a nitroreductase-activated multifunctional molecular prodrug for overcoming tumor oxygen heterogeneity distribution - Google Patents

Abstract

The invention discloses a preparation method and application of a multifunctional molecular prodrug activated by nitroreductase for overcoming the heterogeneous distribution of tumor oxygen, which utilizes the cytotoxic quinoline alkaloid chemotherapeutic drug camptothecin , introducing a nitrobenzene-modified fluoroboridipyrrole photosensitizer in response to tumor hypoxia at its active site to obtain a fluoroboridipyrrole-nitrobenzene-camptothecin conjugate, which combines photodynamic and hypoxia Oxygen-activated chemotherapy can overcome the problem of heterogeneity of tumor oxygen distribution and realize whole-tumor therapy. The present invention prepares a fluoroborate dipyrrole-nitrobenzene-camptothecin conjugate, which is used to overcome the heterogeneity of tumor oxygen distribution and achieve the goal of whole tumor treatment. The synthesis method of the invention is simple, the raw materials are easy to obtain, the cost is low, the side reaction is few, the yield is high, the purification is easy, and it is beneficial to industrialized production.

Description

A multifunctional nitroreductase-activated nitroreductase for overcoming the heterogeneous distribution of tumor oxygen Preparation method and application of molecular prodrug

technical field

The invention belongs to the field of anticancer drug design and synthesis, and in particular relates to a preparation method and application of a multifunctional molecular prodrug activated by nitroreductase for overcoming tumor oxygen heterogeneity distribution.

Background technique

Tumor hypoxia is caused by the uncontrolled proliferation of tumor cells and is one of the important features shared by the tumor microenvironment. Its manifestation is that the oxygen content level in intratumoral tissue cells is significantly lower than that in normal tissue cells. In response to this phenomenon, scientists use hypoxia-responsive functional groups (such as nitroaromatic derivatives, azobenzene, quinone derivatives) to chemically modify active drugs (such as paclitaxel, camptothecin, doxorubicin, etc.) , developed into a hypoxia-responsive prodrug. The prodrug protects and functionalizes the active site of the active drug, thereby improving the specificity of the drug in treating tumor tissue and reducing the systemic toxic and side effects of the drug. However, the oxygen distribution in solid tumors is characterized by heterogeneity. The tissue cells closer to the blood vessels are still in a normal oxygen state, and the tumor area far away from the blood vessels is in a hypoxic state, resulting in significant differences in the oxygen content of the entire tumor tissue. Therefore, hypoxia-responsive prodrugs have great application limitations, which can only treat tumor hypoxic regions, but residual tumor tissue cells can still lead to cancer recurrence.

Recently, the combined treatment mode of photodynamic therapy and chemotherapy has been extensively studied. This combined treatment method can significantly improve the efficiency of cancer treatment, reduce the dosage of drugs, and reduce the drug resistance of lesion tissues. Photodynamic therapy (PDT) is an early developed minimally invasive light therapy, and it has been gradually applied to the diagnosis and treatment of clinical cancer in recent years. Compared with traditional treatment methods such as surgery, radiotherapy, and chemotherapy, PDT has the advantages of minimal invasiveness, high selectivity, low side effects, no drug resistance, and a broad spectrum of tumor treatment. In the process of PDT, the light of a specific wavelength locally irradiates the tumor site, excites the photosensitizer enriched in the tumor tissue, and the excited state photosensitizer and the molecular oxygen in the tumor tissue undergo energy transfer to generate singlet oxygen (Singlet oxygen, ¹ O ₂ ). ¹ O ₂ has strong oxidative properties and can induce apoptosis or necrosis. In addition, PDT, as a precise light-controlled therapy, can rapidly consume oxygen in tumor tissue and cause severe hypoxia while generating ¹ O ₂ . Therefore, the integration of photosensitizers and hypoxia-responsive prodrugs can exert the maximum effect of photodynamic therapy and hypoxia-responsive chemotherapy, achieve the purpose of synergistic treatment, and overcome the treatment difficulties caused by the heterogeneity of oxygen distribution in tumor tissues, and achieve full tumor treatment.

Contents of the invention

The purpose of the present invention is to provide a preparation method and application of a nitroreductase-activated multifunctional molecular prodrug for overcoming the heterogeneous distribution of tumor oxygen. The present invention utilizes the hypoxia-responsive nitrobenzene connecting arm to covalently link the chemotherapeutic drug Camptothecin (CPT) and the fluorobodipyrrole photosensitizer to obtain a single-molecule precursor that integrates photodynamic therapy and chemotherapy. medicine. Since the hydroxyl active site of CPT is modified, resulting in the inhibition of the cytotoxicity of CPT, the whole molecular prodrug has extremely low dark toxicity. The prodrug synthesized by the invention has the advantages of simple synthesis method, easy purification and separation, less side reactions, high yield, readily available raw materials and low cost, and is favorable for industrialized production. The prodrug was enriched to the tumor site by tail vein injection. When the tumor tissue is locally irradiated, the drug can effectively perform photodynamic therapy in the normal oxygen area of the tumor, and at the same time consume oxygen to accelerate tumor hypoxia, promote the overexpression of nitrobenzene reductase in tumor cells, and nitrobenzene in the prodrug The group is reduced to aniline, and a further intramolecular transformation reaction occurs to release the chemotherapeutic drug CPT; while the drug enriched in the hypoxic region of the tumor can be reduced by the overexpressed nitroreductase, and the chemotherapeutic drug CPT is released, making up for photodynamic therapy. Disadvantages of undertreatment in hypoxic areas of the tumor. Therefore, the prodrug is expected to overcome the treatment difficulties caused by the heterogeneity of tumor oxygen distribution and achieve the goal of whole-tumor therapy.

To achieve the above object, the present invention adopts the following technical solutions:

A nitroreductase-activated multifunctional molecular prodrug for overcoming the heterogeneous distribution of tumor oxygen, which is a fluorobodipyrrole-nitrobenzene-camptothecin conjugate, and its chemical structure is:

为起始原料，合成氟硼二吡咯-硝基苯-喜树碱轭合物

(BDP-Nitro-CPT)。The preparation method of above-mentioned compound comprises the following steps: with compound

Synthesis of fluoroborate dipyrrole-nitrobenzene-camptothecin conjugate as starting material

(BDP-Nitro-CPT).

按照摩尔比1:2.2溶解于二氯甲烷中，然后加入2-3当量的4-二甲氨基吡啶(DMAP)、3滴N,N-二异丙基乙胺(DIPEA)以及三光气，室温条件下剧烈搅拌反应液18-36h；反应结束后，用二氯甲烷和水萃取，有机相经无水Na₂SO₄干燥后减压移除溶剂；最后以二氯甲烷-甲醇(体积比为50:1)为洗脱剂，经硅胶柱层析分离得到氟硼二吡咯-硝基苯-喜树碱轭合物

(BDP-Nitro-CPT)；Specifically: the compound

Dissolve in dichloromethane at a molar ratio of 1:2.2, then add 2-3 equivalents of 4-dimethylaminopyridine (DMAP), 3 drops of N,N-diisopropylethylamine (DIPEA) and triphosgene, room temperature The reaction solution was vigorously stirred under the conditions for 18-36h; after the reaction, extracted with dichloromethane and water, the organic phase was dried over anhydrous Na ₂ SO ₄ and the solvent was removed under reduced pressure; finally dichloromethane-methanol (volume ratio: 50:1) as eluent, separated by silica gel column chromatography to obtain fluorobodipyrrole-nitrobenzene-camptothecin conjugate

(BDP-Nitro-CPT);

的摩尔量计。The 2-3 equivalents of DMAP to

molarity meter.

The preparation method of the compound 2a comprises the following steps:

三溴化磷为起始原料，合成

(1) with

Phosphorus tribromide is used as starting material, synthesized

为起始原料，合成化合物

(2) With the compound synthesized in step (1)

As starting materials, synthetic compounds

Specifically:

三溴化磷按摩尔比2：1混合，并溶于乙腈中，然后在冰浴条件下反应2h；反应结束后，除去溶剂，以二氯甲烷为洗脱剂，经硅胶柱层析分离，得到黄色固体

(1) will

Phosphorus tribromide was mixed at a molar ratio of 2:1, dissolved in acetonitrile, and then reacted in an ice bath for 2 hours; after the reaction was completed, the solvent was removed, and dichloromethane was used as an eluent to separate by silica gel column chromatography. yielded a yellow solid

按摩尔比1：2溶于丙酮中，然后加入10当量的碳酸钾，反应在60℃油浴条件下回流1h，反应结束后，将碳酸钾过滤，并除去溶剂，随后用二氯甲烷和水萃取，有机相经无水Na₂SO₄干燥后减压移除溶剂，然后以二氯甲烷-甲醇(体积比为50：1)为洗脱剂，经硅胶柱层析分离得到绿色固体(2) Will

Dissolve in acetone at a molar ratio of 1:2, then add 10 equivalents of potassium carbonate, and reflux at 60°C for 1 hour in an oil bath. After the reaction, filter the potassium carbonate and remove the solvent, then use dichloromethane and water Extraction, the organic phase was dried over anhydrous Na ₂ SO ₄ , the solvent was removed under reduced pressure, and then dichloromethane-methanol (volume ratio 50:1) was used as the eluent, and a green solid was obtained by silica gel column chromatography

的摩尔量计。应用：所述的用于克服肿瘤氧异质性分布的硝基还原酶激活的多功能分子前药在制备抗癌药物中的应用。The 10 equivalents of potassium carbonate, to

molarity meter. Application: the application of the multifunctional molecular prodrug activated by nitroreductase for overcoming tumor oxygen heterogeneity distribution in the preparation of anticancer drugs.

The boron dipyrrole-nitrobenzene-camptothecin (BDP-Nitro-CPT) prodrug is used for synergistic treatment to overcome the heterogeneity of tumor oxygen distribution.

Alkaloids are low molecular weight biomolecules with a nitrogen content of 20%. Some alkaloids have strong biological activity and are often used for disease treatment. Currently clinically used alkaloid anticancer drugs include camptothecin, which is a type I DNA topoisomerase (Top I) inhibitor, and has certain curative effects on esophageal cancer, colon cancer, liver cancer and lung cancer. However, camptothecin also has some disadvantages: such as poor water solubility, unstable structure, and large systemic side effects. Photosensitizer is the core element of photodynamic therapy. Fluoroboron dipyrrole derivatives are one of the most widely studied fluorescent dyes at present, which meet a series of advantages of ideal photosensitizers: such as clear chemical structure, strong light absorption, and high photostability. , low dark toxicity, high singlet oxygen quantum yield after heavy atom modification, etc.

Based on this, the present invention firstly connects the hypoxic responsive nitrophenyl group to the boron dipyrrole photosensitizer, and then covalently connects it to the chemotherapeutic drug camptothecin to form a multifunctional molecular prodrug BDP- Nitro-CPT. When the tumor site is irradiated with red light, the drug enriched in the normal oxygen area of the tumor is excited by the light to generate photodynamic activity, which in turn causes hypoxia in the tumor site and induces the activation of the chemotherapeutic drug camptothecin, which plays the role of photodynamic-chemotherapy synergistic therapy. effect. Molecular prodrugs in the hypoxic region of tumors can directly activate the chemotherapeutic drug camptothecin, making up for the shortcomings of photodynamic therapy in the hypoxic region of tumors. Therefore, BDP-Nitro-CPT prodrugs are expected to overcome the heterogeneity of tumor oxygen distribution Treatment difficulties caused by sex, to achieve the purpose of whole tumor treatment.

Significant advantage of the present invention is:

(1) Camptothecin is an alkaloid that can directly damage the DNA structure. When the nitrobenzene linker is covalently linked to the fluorobodipyrrole photosensitizer, the stability of camptothecin in the blood circulation process can be improved. , and effectively reduce the systemic toxicity of the drug;

(2) The BDP-Nitro-CPT prodrug can be directly or indirectly reduced to aniline under hypoxic conditions or in the process of photodynamic therapy, and further undergoes an intramolecular conversion reaction to release the chemotherapeutic drug camptothecin, which acts as a Photodynamic therapy-chemotherapy synergistic effect;

(3) The absorption and emission spectra of the prodrug are both located in the red light region, and the red light has strong tissue penetration ability, and it is not easy to cause skin phototoxicity and low dark toxicity during photodynamic therapy, so it is an ideal prodrug;

(4) The structure of the target prodrug is single, the synthesis is simple, and the product is easy to purify;

(5) From the cytotoxicity experiments of cancer cells 4T1 (mouse breast cancer cells), HepG2 (human liver cancer cells), HeLa ((human cervical cancer cells), it can be seen that hypoxic cancer cells can induce BDP-Nitro-CPT The nitrophenyl group in BDP-Nitro-CPT is reduced, and then the chemotherapeutic drug camptothecin is released. Under light conditions, the photodynamic effect of BDP-Nitro-CPT can induce hypoxia in cancer cells and release camptothecin.

Description of drawings

Fig. 1 Cytotoxicity of BDP-Nitro-CPT and 2a to 4T1, HepG2 and HeLa cells cultured under different oxygen concentrations under light (660nm, 20mW/cm ² , 5min) and no light conditions;

Figure 2 Antitumor activity of different compounds with and without light; (a) tumor volume changes in different groups of mice during treatment; (b) tumor mass and (c) morphology of mice after 15 days; (d) 15 Changes in body weight of mice within days; (e) Morphological analysis of tumor tissues after H&E staining of mice in different groups after treatment for 15 days;

Fig. 3 Morphology of tissue sections after H&E staining of various organs of different groups of mice;

Fig. 4 is the mechanism of action of boron dipyrrole-nitrophenylcamptothecin (BDP-Nitro-CPT) prodrug;

Fig. 5 is the ¹ H NMR spectrogram of compound 1a in CDCl ₃ ;

Fig. 6 is the ¹³ C NMR spectrogram of compound 1a in CDCl ₃ ;

Fig. 7 is the high resolution mass spectrometry (HRMS) figure of compound 1a;

Fig. 8 is the ¹ H NMR spectrogram of compound 2a in CDCl ₃ ;

Fig. 9 is the ¹³ C NMR spectrogram of compound 2a in CDCl ₃ ;

Figure 10 is the HRMS spectrogram of compound 2a;

Fig. 11 is the ¹ H NMR spectrum of compound BDP-Nitro-CPT in CDCl ₃ ;

Figure 12 is the ¹³ C NMR spectrum of compound BDP-Nitro-CPT in _CDCl3 ;

Figure 13 is the HRMS spectrum of the compound BDP-Nitro-CPT.

Detailed ways

In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

The specific preparation process of the nitroreductase-activated multifunctional molecular prodrug fluorobodipyrrole-nitrobenzene-camptothecin conjugate that can be used to overcome the heterogeneous distribution of tumor oxygen includes:

加入到100mL的圆底烧瓶中，用乙腈溶解，称取0.5当量的三溴化磷溶于乙腈，再用恒压滴液漏斗在冰浴搅拌下将三溴化磷滴加到反应瓶，反应2h；反应结束后，除去溶剂乙腈，剩余物用二氯甲烷和水萃取三次，收集有机相，旋蒸除去二氯甲烷。以二氯甲烷为洗脱剂，经硅胶柱色谱分离再旋蒸得到黄色固体化合物

产率为35％-40％；(1) will

Add it into a 100mL round bottom flask, dissolve it with acetonitrile, weigh 0.5 equivalent of phosphorus tribromide and dissolve it in acetonitrile, then use a constant pressure dropping funnel to add phosphorus tribromide dropwise to the reaction flask under stirring in an ice bath, and react 2h; after the reaction, the solvent acetonitrile was removed, the residue was extracted three times with dichloromethane and water, the organic phase was collected, and the dichloromethane was removed by rotary evaporation. Using dichloromethane as the eluent, separated by silica gel column chromatography and rotary evaporation to obtain a yellow solid compound

The yield is 35%-40%;

按摩尔比1：2溶于丙酮中，然后加入10当量的碳酸钾，反应液在60℃油浴条件下回流1h，反应结束后，将碳酸钾过滤，并除去溶剂，随后用二氯甲烷和水萃取，有机相经无水Na₂SO₄干燥后减压移除溶剂，然后以二氯甲烷-甲醇(体积比为50:1)为洗脱剂，经硅胶柱层析分离得到绿色固体

产率为52％-58％。(2) Will

Dissolve in acetone at a molar ratio of 1:2, then add 10 equivalents of potassium carbonate, and reflux the reaction solution at 60°C for 1 hour in an oil bath. After the reaction, filter the potassium carbonate and remove the solvent, then use dichloromethane and Water extraction, the organic phase was dried over anhydrous Na ₂ SO ₄ and the solvent was removed under reduced pressure, and then dichloromethane-methanol (volume ratio 50:1) was used as the eluent and separated by silica gel column chromatography to obtain a green solid

The yield was 52%-58%.

按照摩尔比1：2.2溶解于二氯甲烷中，然后加入2-3当量的4-二甲氨基吡啶(DMAP)、3滴N,N-二异丙基乙胺(DIPEA)以及三光气，室温条件下剧烈搅拌反应液18-36h；反应结束后，用二氯甲烷和水萃取，有机相经无水Na₂SO₄干燥后减压移除溶剂；最后以二氯甲烷-甲醇(体积比为50:1)为洗脱剂，经硅胶柱层析分离得到氟硼二吡咯-硝基苯-喜树碱轭合物

(BDP-Nitro-CPT)，产率为69％-74％。(3) Will

Dissolve in dichloromethane at a molar ratio of 1:2.2, then add 2-3 equivalents of 4-dimethylaminopyridine (DMAP), 3 drops of N,N-diisopropylethylamine (DIPEA) and triphosgene at room temperature The reaction solution was vigorously stirred under the conditions for 18-36h; after the reaction, extracted with dichloromethane and water, the organic phase was dried over anhydrous Na ₂ SO ₄ and the solvent was removed under reduced pressure; finally dichloromethane-methanol (volume ratio: 50:1) as eluent, separated by silica gel column chromatography to obtain fluorobodipyrrole-nitrobenzene-camptothecin conjugate

(BDP-Nitro-CPT), the yield was 69%-74%.

Example 1

(0.30g,1.00mmol)加入到100mL的圆底烧瓶中，用15mL乙腈溶解；然后称取三溴化磷(0.14g,0.50mmol)溶于10mL乙腈，再用恒压滴液漏斗在冰浴搅拌下将三溴化磷滴加到反应瓶，反应2h；反应结束后，除去溶剂乙腈，剩余物用二氯甲烷和水萃取三次，收集有机相，旋蒸除去二氯甲烷。以二氯甲烷为洗脱剂，经过硅胶柱色谱分离再旋蒸得到黄色固体化合物

(0.13g,37％)。¹H NMR(500MHz,CDCl₃):δ(ppm)＝8.28(d,2H,J＝9.0Hz,ArH),7.69(d,2H,J＝9.0Hz,ArH),7.21(s,2H,ArH),5.20(s,2H,OCH₂),4.69(s,2H,OCH₂),4.53(s,2H,CH₂Br),2.34(s,3H,CH₃).¹³C NMR(150MHz,CDCl₃):δ(ppm)＝152.65,147.68,144.41,135.19,134.08,131.84,131.21,127.84,123.88,74.85,60.79,27.96,20.76.HRMS(ESI):理论计算值(m/z[M+Na]⁺)为:388.0160；实际测试值为388.0145。(1) will

(0.30g, 1.00mmol) was added to a 100mL round bottom flask, dissolved with 15mL acetonitrile; then weighed phosphorus tribromide (0.14g, 0.50mmol) dissolved in 10mL acetonitrile, and then used a constant pressure dropping funnel in an ice bath Phosphorus tribromide was added dropwise to the reaction flask under stirring, and reacted for 2 hours; after the reaction, the solvent acetonitrile was removed, the residue was extracted three times with dichloromethane and water, the organic phase was collected, and the dichloromethane was removed by rotary evaporation. Using dichloromethane as the eluent, separated by silica gel column chromatography and rotary evaporation to obtain a yellow solid compound

(0.13 g, 37%). ¹ H NMR (500MHz, CDCl ₃ ): δ (ppm) = 8.28 (d, 2H, J = 9.0Hz, ArH), 7.69 (d, 2H, J = 9.0Hz, ArH), 7.21 (s, 2H, ArH ), 5.20 (s, 2H, OCH ₂ ), 4.69 (s, 2H, OCH ₂ ), 4.53 (s, 2H, CH ₂ Br), 2.34 (s, 3H, CH ₃ ). ¹³ C NMR (150MHz, CDCl ₃ ): δ (ppm) = 152.65, 147.68, 144.41, 135.19, 134.08, 131.84, 131.21, 127.84, 123.88, 74.85, 60.79, 27.96, 20.76. HRMS (ESI): theoretically calculated value (m/z [M+Na ] ⁺ ) is: 388.0160; the actual test value is 388.0145.

(0.10g,0.10mmol)、

(0.074g,0.20mmol)溶于丙酮中，然后加入碳酸钾(0.14g，1.0mmol)，反应液在60℃油浴条件下回流1h，反应结束后，将碳酸钾过滤，并除去溶剂，随后用二氯甲烷和水萃取，有机相经无水Na₂SO₄干燥后减压移除溶剂，然后以二氯甲烷-甲醇(体积比为50:1)为洗脱剂，经硅胶柱层析分离得到绿色固体

(0.071g,53％)。¹H NMR(400MHz,CDCl₃):δ(ppm)＝8.23(d,J＝8.4Hz,2H,ArH),8.10(d,J＝16.4Hz,2H,CH＝CH),7.59-7.63(m,8H,ArH andCH＝CH),7.31(s,1H,ArH),7.30(s,1H,ArH),7.18(d,J＝8.4Hz,2H,ArH),7.08(d,J＝8.8Hz,2H,ArH),6.97(d,J＝8.8Hz,4H,ArH),5.15(s,2H,OCH₂),5.13(s,2H,OCH₂),4.76(s,2H,OCH₂),4.20(t,J＝4.8Hz,4H,OCH₂),3.90(t,J＝4.8Hz,4H,OCH₂),3.78-3.75(m,4H,OCH₂),3.72-3.66(m,8H,OCH₂),3.58-3.55(m,4H,OCH₂),3.39(s,6H,OCH₃),2.39(s,3H,CH₃),1.42(s,6H,CH₃)；¹³C NMR(150MHz,CDCl₃):δ(ppm)＝160.01,159.38,153.10,148.41,147.64,144.45,140.78,138.85,134.98,133.97,132.33,131.04,130.94,129.87,129.84,129.27,127.78,127.46,123.83,116.10,115.43,114.97,110.17,75.79,71.94,70.89,70.68,70.59,69.69,67.55,65.75,60.85,59.07,20.89,13.82.HRMS(ESI):C₆₃H₆₈BBr₂F₂N₃O₁₃的理论计算值(m/z[M+H]⁺)为1306.3052；实际测试值为1306.3081。(2) Will

(0.10g, 0.10mmol),

(0.074g, 0.20mmol) was dissolved in acetone, then potassium carbonate (0.14g, 1.0mmol) was added, and the reaction solution was refluxed at 60°C for 1h in an oil bath. After the reaction, potassium carbonate was filtered, and the solvent was removed, followed by Extract with dichloromethane and water, dry the organic phase over anhydrous Na ₂ SO ₄ and remove the solvent under reduced pressure, then use dichloromethane-methanol (volume ratio 50:1) as the eluent, and perform silica gel column chromatography A green solid was isolated

(0.071 g, 53%). ¹ H NMR (400MHz, CDCl ₃ ): δ (ppm) = 8.23 (d, J = 8.4Hz, 2H, ArH), 8.10 (d, J = 16.4Hz, 2H, CH = CH), 7.59-7.63 (m ,8H,ArH andCH=CH),7.31(s,1H,ArH),7.30(s,1H,ArH),7.18(d,J=8.4Hz,2H,ArH),7.08(d,J=8.8Hz, 2H, ArH), 6.97 (d, J=8.8Hz, 4H, ArH), 5.15 (s, 2H, OCH ₂ ), 5.13 (s, 2H, OCH ₂ ), 4.76 (s, 2H, OCH ₂ ), 4.20 (t, J = 4.8Hz, 4H, OCH ₂ ), 3.90 (t, J = 4.8Hz, 4H, OCH ₂ ), 3.78-3.75 (m, 4H, OCH ₂ ), 3.72-3.66 (m, 8H, OCH 2 ₂ ),3.58-3.55(m,4H,OCH ₂ ),3.39(s,6H,OCH ₃ ),2.39(s,3H,CH ₃ ),1.42(s,6H,CH ₃ ); ¹³ C NMR (150MHz ,CDCl ₃ ):δ(ppm)＝160.01,159.38,153.10,148.41,147.64,144.45,140.78,138.85,134.98,133.97,132.33,131.04,130.94,129.87,129.84,129.27,127.78,127.46,123.83,116.10, 115.43, 114.97, 110.17, 75.79, 71.94, 70.89, 70.68, 70.59, 69.69, 67.55, 65.75, 60.85, 59.07, 20.89, 13.82. Theoretical calculation of HRMS(ESI): C ₆₃ H ₆₈ BBr ₂ F ₂ N ₃ O ₁₃ The value (m/z[M+H] ⁺ ) is 1306.3052; the actual test value is 1306.3081.

(0.03g,0.086mmol)溶解于二氯甲烷中，然后加入4-二甲氨基吡啶(DMAP)(0.03g,0.246mmol)和3滴N,N-二异丙基乙胺(DIPEA)室温搅拌反应10分钟，然后滴加三光气的二氯甲烷溶液至反应液澄清时加入2a

(0.05g,0.039mmol),室温条件下剧烈搅拌反应液18h；反应结束后，用二氯甲烷和水萃取，有机相经无水Na₂SO₄干燥后减压移除溶剂；最后以二氯甲烷-甲醇(体积比为50:1)为洗脱剂，经硅胶柱层析分离得到氟硼二吡咯-硝基苯-喜树碱(BDP-Nitro-CPT)轭合物

(0.022g,70％)。¹HNMR(500MHz,CDCl₃):δ(ppm)＝8.38(s,1H,ArH),8.14(d,J＝8.5Hz,1H,ArH),8.12(d,J＝16.5Hz,2H,CH＝CH),7.96-7.94(m,3H,ArH),7.83(t,J＝7.5Hz,1H,ArH),7.67(t,J＝7.5Hz,ArH),7.63-7.60(m,6H,ArH and CH＝CH),7.42(d,J＝7.5Hz,2H,ArH),7.32(s,1H,ArH),7.31(s,1H,ArH),7.29(s,1H,ArH),7.15(d,J＝8.5Hz,2H,ArH),7.04(d,J＝8.5Hz,2H,ArH),6.96(d,J＝8.5Hz,4H,ArH),5.72(d,J＝17.0Hz,1H,OCH₂),5.41(d,J＝17.0Hz,1H,OCH₂),5.30-5.20(m,4H,OCH₂),5.07-5.03(m,4H,NCH₂ and OCH₂),4.20(t,J＝4.0Hz,4H,OCH₂),3.90(t,J＝4.5Hz,4H,OCH₂),3.77(t,J＝4.0Hz,4H,OCH₂),3.72-3.67(m,8H,OCH₂),3.57(m,4H,OCH₂),3.39(s,6H,OCH₃),2.33(s,3H,CH₃),2.30-2.13(m,2H,CH₂),1.39(s,6H,CH₃),0.88(t,J＝6.5Hz,3H,CH₃)；¹³C NMR(150MHz,CDCl₃):δ(ppm)＝160.01,160.09，159.33,157.29，153.78，153.67，152.22，148.84，148.47，147.55，146.51，145.60，143.89，140.79，138.94，138.43，135.18，131.59，132.50，132.36，131.28，130.86，129.93，129.50，129.34，128.48，128.43，128.29，128.26，127.94，127.56，123.54，120.42，116.14，115.46，115.05，110.23，95.78，78.13，76.37，72.01，70.95，70.74，70.66，69.75，67.62，67.22，65.83，50.05，32.09，20.85，13.89，7.69.HRMS(ESI):C₈₄H₈₂BBr₂F₂N₅O₁₈的理论计算值(m/z[M+H]+)为1680.3954；实际测量值为1680.4090。(3) Will

(0.03g, 0.086mmol) was dissolved in dichloromethane, then added 4-dimethylaminopyridine (DMAP) (0.03g, 0.246mmol) and 3 drops of N,N-diisopropylethylamine (DIPEA) and stirred at room temperature React for 10 minutes, then add triphosgene solution in dichloromethane dropwise until the reaction solution is clear, add 2a

(0.05g, 0.039mmol), the reaction solution was vigorously stirred at room temperature for 18h; after the reaction, extracted with dichloromethane and water, the organic phase was dried over anhydrous Na ₂ SO ₄ and the solvent was removed under reduced pressure; Methane-methanol (volume ratio: 50:1) was used as the eluent, and the fluorobodipyrrole-nitrobenzene-camptothecin (BDP-Nitro-CPT) conjugate was obtained by silica gel column chromatography

(0.022g, 70%). ¹ HNMR (500MHz, CDCl ₃ ): δ (ppm) = 8.38 (s, 1H, ArH), 8.14 (d, J = 8.5Hz, 1H, ArH), 8.12 (d, J = 16.5Hz, 2H, CH = CH),7.96-7.94(m,3H,ArH),7.83(t,J=7.5Hz,1H,ArH),7.67(t,J=7.5Hz,ArH),7.63-7.60(m,6H,ArH and CH=CH), 7.42(d, J=7.5Hz, 2H, ArH), 7.32(s, 1H, ArH), 7.31(s, 1H, ArH), 7.29(s, 1H, ArH), 7.15(d, J=8.5Hz, 2H, ArH), 7.04(d, J=8.5Hz, 2H, ArH), 6.96(d, J=8.5Hz, 4H, ArH), 5.72(d, J=17.0Hz, 1H, OCH ₂ ),5.41(d,J＝17.0Hz,1H,OCH ₂ ),5.30-5.20(m,4H,OCH ₂ ),5.07-5.03(m,4H,NCH ₂ and OCH ₂ ),4.20(t,J =4.0Hz, 4H, OCH ₂ ), 3.90 (t, J = 4.5Hz, 4H, OCH ₂ ), 3.77 (t, J = 4.0Hz, 4H, OCH ₂ ), 3.72-3.67 (m, 8H, OCH ₂ ),3.57(m,4H,OCH ₂ ),3.39(s,6H,OCH ₃ ),2.33(s,3H,CH ₃ ),2.30-2.13(m,2H,CH ₂ ),1.39(s,6H, CH ₃ ), 0.88 (t, J=6.5Hz, 3H, CH ₃ ); ¹³ C NMR (150MHz, CDCl ₃ ): δ(ppm)=160.01, 160.09, 159.33, 157.29, 153.78, 153.67, 152.22, 148.84, 148.47，147.55，146.51，145.60，143.89，140.79，138.94，138.43，135.18，131.59，132.50，132.36，131.28，130.86，129.93，129.50，129.34，128.48，128.43，128.29，128.26，127.94，127.56，123.54，120.42， 116.14, 115.46, 115.05, 110.23, 95.78, 78.13, 76.37, 72.01 _{, 70.95, 70.74, 70.66, 69.75, 67.62, 67.22,} 65.83, 50.05, 32.09, 20.85, _13.89 , 7.49 Theoretical calculated for ₂ F ₂ N ₅ O ₁₈ (m/z [M+H]+) 1680.3954; found 1680.4090.

Application example 1

The in vitro anticancer activity study of the molecular prodrug BDP-Nitro-CPT can verify the hypoxia response mechanism and synergistic therapeutic mechanism of the drug, and provide an experimental basis for in vivo experiments. Cell viability was determined by MTT method. MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) is a yellow solid whose aqueous solution can effectively identify living cells during cancer cell inhibition . The detection principle is that the succinate dehydrogenase in the mitochondria of living cells reduces the added MTT to water-insoluble purple crystalline formazan and deposits in the cells, while the dead cells cannot restore the added MTT due to their damaged cell structure. The cells were treated with MTT solution, and after the living cells produced formazan, dimethyl sulfoxide (DMSO) was added to the cells to dissolve the formazan, and the absorbance value (OD value) at 570 nm was detected with a microplate reader. The cell viability was plotted against the logarithmic concentration of the drug, and the half inhibitory concentration value (IC ₅₀ value) was calculated. Cell viability calculation formula: Cellviability%=(AA ₀ )/(A ₁ -A ₀ )×100%; where A is the OD value of the experimental group, A ₀ is the OD value of the blank group, and A ₁ is the OD of the cell control group value.

MTT experiment: select normally growing 4T1 cells (mouse breast cancer cells), HepG2 cells (human liver cancer cells), and HeLa cells (human cervical cancer cells) as cell lines for in vitro activity research. Spread approximately 7×10 ³ cancer cells evenly in each well of a 96-well plate, and incubate in a cell culture incubator at a constant temperature of 37°C for 24 hours. After the incubation, the old medium was replaced with fresh DMEM medium containing different concentrations of BDP-Nitro-CPT (or 2a). Continue to incubate for 8 h under oxygen (0.1% oxygen concentration) condition. After incubation, the cells were washed three times with PBS, and then fresh DMEM medium was added. The cells were treated with/without light (660nm, 20mW/cm ² , 5min) under normal oxygen, mild hypoxia and hypoxia conditions. Cells were incubated for 24 h after the end of light exposure. Then, 10 μL of MTT (5 mg/mL) solution was added to the 96-well plate, and continued to incubate for 4 h. After the incubation, use a pipette gun to remove the medium in the 96-well plate, and add 100 μL of DMSO to fully dissolve the formazan in the cells. The OD value at 490nm was detected with a microplate reader.

We used the MTT method to measure the molecular prodrug BDP-Nitro-CPT prepared in the example and the control drug 2a in normal oxygen (21% O ₂ ), mild hypoxia (6% O ₂ ) and hypoxia (0.1% O ₂ ). Cytotoxicity to 4T1 cells, HepG2 cells, and HeLa cells under certain conditions. It can be seen from Figure 1 that: under normal oxygen conditions (c, f, i in Figure 1), in the range of drug concentration of 0.01-10 μM, BDP-Nitro-CPT and 2a have no obvious killing effect on the three cancer cells, It shows that the drug has good biocompatibility. When the cells were under hypoxic conditions (a, d, g in Figure 1), BDP-Nitro-CPT had a certain killing effect on 4T1, HepG2, HeLa cells, while 2a had a certain killing effect on the three cancer cells treated under hypoxic conditions No obvious cytotoxicity. This result indicates that under hypoxic conditions, the nitrophenyl group in the BDP-Nitro-CPT prodrug can be reduced by the overexpressed nitroreductase in hypoxic cells, and then release the chemotherapeutic drug camptothecin, which induces cancer. cell death. Under light conditions (660nm, 20mW/cm ² , 5min), BDP-Nitro-CPT has high cytotoxicity to three kinds of cancer cells cultured under normoxic and mild hypoxic conditions, and it has high cytotoxicity to normal oxygen cultured The IC ₅₀ values of 4T1, HepG2, and HeLa cells were 0.66 μM, 0.67 μM, and 0.73 μM, and the IC ₅₀ values of 4T1, HepG2, and HeLa cells cultured under mild and hypoxic conditions were 0.73 μM, 0.66 μM, and 0.77 μM. The cytotoxicity was higher than that of the control group 2a (Figure 1, Table 1). It shows that BDP-Nitro-CPT can consume oxygen and promote the release of camptothecin in the process of photodynamic therapy, realizing the synergistic effect of photodynamic therapy-chemotherapy. In addition, the IC ₅₀ value of BDP-Nitro-CPT to hypoxic cells is higher than that of hypoxic cells treated by 2a, indicating that the camptothecin released _by BDP-Nitro-CPT prodrug under hypoxic conditions can compensate for the loss of light. Insufficient dynamic therapy.

Table 1. IC ₅₀ values of BDP-Nitro-CPT and 2a on 4T1, HepG2, HeLa cells

The in vivo anticancer activity of the molecular prodrug BDP-Nitro-CPT and the control photosensitizer 2a was studied. This experiment can further verify the hypoxia response mechanism and synergistic therapeutic mechanism of the BDP-Nitro-CPT prodrug. In this experiment, Balb/c mice were used to detect the anti-tumor effect for a period of 15 days. By observing the changes in tumor size during treatment, the tumor mass of mice in each group after treatment, and H&E staining of tumor tissue sections, the tumor inhibitory effect was evaluated, and the safety of the drug was evaluated.

Experimental method and specific steps:

(1) Establishment of the Balb/c mouse model: 30 5-week-old female Balb/c mice weighing about 20 g were selected, and 1×10 ⁶ 4T1 mouse breast cancer cells were subcutaneously implanted on the back of each mouse.

(2) Grouping and administration: After the average tumor size of the mice grew to 60mm ³ , the mice were randomly divided into six groups (BDP-Nitro-CPT+Light group, 2a+Light group, BDP-Nitro-CPT group, 2a group, Saline+Light group, Saline group), five mice in each group in parallel, each group of mice were given the corresponding drug (100 μL, 100 μM) by tail vein injection.

(3) Illumination treatment: the first administration was on the 0th day, and the illumination group received light treatment 24 hours after the administration, using a 660nm laser to irradiate the mouse tumor site for 10 minutes (power density: 300mW/cm ² ).

(4) Data measurement and recording: After the light treatment, the mice were weighed regularly every day and the tumor volume was measured, repeated for 15 days.

(5) H&E staining: After the treatment, one mouse was randomly selected in each group, and killed by cervical dislocation, and the tumor, heart, liver, spleen, lung, and kidney were taken out and soaked in 4% paraformaldehyde solution After being fixed in medium for 24 hours, the sections were wrapped in paraffin for H&E staining. The histological morphology of each tissue section was observed and photographed under an optical microscope.

The anti-tumor results of the experimental group and the control group are summarized in Figure 2. It can be seen from Figure 2 that compared with the Saline control group, the mice in the Saline+Light group and the 2a injection group without light had no obvious tumor inhibitory effect, indicating that neither injection of BDP photosensitizer 2a nor light administration alone had anti-tumor effects . However, the 2a+light and BDP-Nitro-CPT no-light groups had significant tumor-inhibitory effects, which were due to photodynamic therapy and tumor hypoxia-activated chemotherapy, respectively. In contrast, mice injected with BDP-Nitro-CPT into the tail vein and treated with light had the most significant anti-tumor effect, and the size and quality of the dissected mouse tumors were relatively small (c and b in Figure 2). There were even four mice whose tumors were completely eliminated without recurrence (Fig. 2c). In addition, H&E staining of tumor histology showed that the BDP-Nitro-CPT+Light group could induce massive necrosis and apoptosis of tumor cells (Fig. 2 e). The body weight of the mice fluctuated negligibly throughout the treatment period (Figure 2, d). In addition, it can be seen from the tissue sections of various organs that 2a and BDP-Nitro-CPT will not cause damage to normal tissues even under light conditions (Fig. 3). The experimental results showed that BDP-Nitro-CPT, which was enriched in the tumor area by light, had a photodynamic-chemotherapy synergistic effect, and while enhancing tumor suppression, BDP-Nitro-CPT showed good biological safety.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (8)

1. A multifunctional molecular prodrug activated by nitroreductase for overcoming the distribution of tumor oxygen heterogeneity, characterized in that: the prodrug is fluorobodipyrrole-nitrobenzene-camptothecin conjugated substance, its chemical structure is:

. 2. A method for preparing a multifunctional molecular prodrug activated by nitroreductase for overcoming tumor oxygen heterogeneity distribution as claimed in claim 1, characterized in that: the preparation method comprises the following steps:

,

As the starting material, fluoroboron dipyrrole-nitrobenzene-camptothecin conjugate was synthesized. 3. preparation method according to claim 2, is characterized in that, described preparation method specifically comprises the following steps:

,

Dissolve in dichloromethane at a molar ratio of 1:2.2, then add 2-3 equivalents of 4-dimethylaminopyridine DMAP, 3 drops of N,N-diisopropylethylamine DIPEA and triphosgene, and stir vigorously at room temperature The reaction solution was 18-36 h; after the reaction, extracted with dichloromethane and water, the organic phase was dried over anhydrous Na ₂ SO ₄ and the solvent was removed under reduced pressure; finally, dichloromethane-methanol with a volume ratio of 50:1 As the eluent, the fluorobodipyrrole-nitrobenzene-camptothecin conjugate was obtained by silica gel column chromatography. 4. preparation method according to claim 3, is characterized in that, the DMAP of described 2-3 equivalent, with

molarity meter. 5. according to the preparation method described in claim 2 or 3, it is characterized in that, described

The preparation method comprises the following steps: (1) to

, phosphorus tribromide as the starting material, synthesized

; (2) Synthesized in step (1)

,

as starting material, synthesized

. 6. preparation method according to claim 5, is characterized in that, described

The preparation method specifically comprises the following steps: (1) Will

and phosphorus tribromide were mixed at a molar ratio of 2:1, dissolved in acetonitrile, and then reacted under ice bath conditions for 2 h; Separated to give a yellow solid

; (2) Will

,

Dissolve in acetone at a molar ratio of 1:2, then add 10 equivalents of potassium carbonate, and reflux at 60°C for 1 h in an oil bath. After the reaction, filter the potassium carbonate and remove the solvent, then use dichloromethane and Extract with water, dry the organic phase over anhydrous Na ₂ SO ₄ and remove the solvent under reduced pressure, then use dichloromethane-methanol with a volume ratio of 50:1 as the eluent, and separate by silica gel column chromatography to obtain a green solid

. 7. preparation method according to claim 6 is characterized in that, the potassium carbonate of described 10 equivalents, with

Calculation of the molar mass. 8. The application of a multifunctional molecular prodrug activated by nitroreductase for overcoming tumor oxygen heterogeneity distribution as claimed in claim 1 in the preparation of anticancer drugs.

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