TWI704127B - Fullerene derivatives and the uses thereof - Google Patents

Abstract

The present invention provides a use of fullerene derivatives for the manufacture of a medicament for regulating central nervous system (CNS), wherein the fullerene derivatives includes five hydrophilic groups attached to one semisphere of the fullerene cage, and the other one remains nonfunctionalized. The fullerene derivatives of the present invention can repair CNS deficit or killing brain tumor.

Description

Fullerene derivatives and their uses

The present invention relates to a fullerene derivative and its use, particularly to the fullerene derivative and its use for regulating the central nervous system.

In addition to suffering from schizophrenia, depression, addiction, and other mental illnesses, the economic loss caused by the loss of productivity can reach billions of dollars a year. According to estimates from the World Health Organization (WHO), if we calculate the number of years of life lost due to premature death and loss of healthy quality of life in the world, neurological and mental diseases account for 13%.

Although we urgently need newer and better drugs to treat various psychiatric and neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease, for large pharmaceutical companies, the development of such drugs is not only complicated but also expensive It has spent millions of dollars to develop new drugs, but in the end it may lose money and the risks are too great. This is why many large pharmaceutical companies have retreated to the development of neurological disorders and other central nervous system (CNS) drugs.

It is far more difficult to develop central nervous system drugs than most other drugs. In addition to the same time and cost as other drugs, there is another difficulty in developing central nervous system drugs-it is best to be able to cross the blood brain barrier. barrier; referred to as BBB). The blood-brain barrier refers to a "barrier" between the blood vessels and the brain that selectively prevents certain substances from entering the brain from the blood. The blood-brain barrier prevents almost all substances from passing through. Except for oxygen, carbon dioxide and blood sugar, most drugs and proteins generally cannot pass through due to their molecular structure. Compared with other tissues, such as the capillary endothelial cells of muscle tissue, the pinocytosis of brain capillary endothelial cells is very weak. Therefore, for brain capillary endothelial cells, the ability to transport substances (macromolecules and electrolytes) through pinocytosis is very limited, which further strengthens the barrier function of brain capillary walls. blood The function of the brain barrier is to prevent the brain from being affected by chemical transmitters. Since many functions of the body are controlled by the secretion of hormones by the brain, if chemical conductive substances are allowed to flow freely in the brain, it may cause feedback phenomena. Therefore, for the normal operation of the brain, the existence of the blood-brain barrier is necessary. On the other hand, the existence of the blood-brain barrier prevents the brain from being infected by germs.

At present, the market for central nervous system drugs is under development. Compared with the development of relatively mature cardiovascular drugs, central nervous system drugs have grown by more than 500% in recent years. The main reason for the insufficient development of central nervous system drugs is that most drugs cannot pass through the blood-brain barrier of humans. In terms of molecular weight, all macromolecular drugs cannot pass the blood-brain barrier, and only a small portion (~2%) of small-molecule high-fat-soluble neuropharmacological drugs can pass the blood-brain barrier. Of these small molecule drugs that account for about 2% of the blood-brain barrier, only a few brain diseases respond to these small molecule drugs. Many neurological disorders (ie, brain tumors, Alzheimer's disease, and other central nervous system disorders) still do not respond well to the traditional treatments of these small molecule drugs. For people suffering from neurological diseases, although the development of central nervous system drugs faces many difficulties and challenges, they are still eagerly looking forward to better new drugs or new treatments.

In view of the above problems, the present invention provides a use of a fullerene derivative for preparing a pharmaceutical composition for regulating the central nervous system, wherein the fullerene derivative contains 5 hydrophilic groups in half of the fullerene framework ( Fullerene cage), the other half of the fullerene frame does not have any functional groups. Among them, regulating the central nervous system may include the effects of promoting the proliferation of central nerve cells and inhibiting the growth of brain tumor cells.

其中R具有下列之一的結構：

，或

；且R’為H或Cl。 In an embodiment of the present invention, the half of the fullerene frame without functional groups is hydrophobic and has a structural formula as Formula I:

Where R has one of the following structures:

,or

; And R'is H or Cl.

該富勒烯衍生物有促進神經幹細胞增殖的效果；當R具有下列之一的結構時：

該富勒烯衍生物有促進神經幹細胞之神經元分化的效果；當R具有下列之一的結構時：

該富勒烯衍生物有保持神經前驅細胞之自我更新能力的效果；當R具有下列之一的結構時：

該富勒烯衍生物有抑制腦神經膠質瘤細胞增殖的效果；當R為具有下列之一的結構時：

該富勒烯衍生物有抗腦腫瘤的效果；當R為具有下列之一的結構時：

該富勒烯衍生物有恢復受損之中樞神經系統的效果。 In an embodiment of the present invention, when R has one of the following structures:

The fullerene derivative has the effect of promoting the proliferation of neural stem cells; when R has one of the following structures:

The fullerene derivative has the effect of promoting neuron differentiation of neural stem cells; when R has one of the following structures:

The fullerene derivative has the effect of maintaining the self-renewal ability of neural precursor cells; when R has one of the following structures:

The fullerene derivative has the effect of inhibiting the proliferation of brain glioma cells; when R has one of the following structures:

The fullerene derivative has an anti-brain tumor effect; when R has one of the following structures:

The fullerene derivative has the effect of restoring damaged central nervous system.

其中R具有下列之一的結構：

The present invention also provides a novel fullerene derivative, which has a structural formula as Formula I:

Where R has one of the following structures:

，或

；R’係為H或Cl。

,or

; R'is H or Cl.

Through the technical features of the present invention, the fullerene derivatives provided by the present invention solve the problem that most traditional drugs used to treat the central nervous system are difficult to cross the blood-brain barrier. It can promote the growth, proliferation and anti-tumor of neural stem cells, and it has been proved in animal experiments that it can repair the damage of the central nervous system or resist brain tumors. The present invention provides a novel treatment method for brain tumors and nerve injury diseases.

Figure 1 is an example of the water-soluble C ₆₀ fullerene derivatives of the present invention, which are compounds 1-11.

Figure 2 is a dynamic light scattering (DLS) spectrum of the aqueous solutions of compounds 1-9 of the present invention.

Figure 3 is a statistical graph showing the survival rate (%) of neural stem cells and C6 cells (Figure 3B) and C6 cells (Figure 3C) relative to the control group after treating neural stem cells and C6 cells with compounds 1-11 of the present invention (Figure 3A); * Indicates p<0.05.

Figure 4 is a statistical graph showing the relative gene expression rates of neural stem cells' nestin, β-tubulin and MAP2 genes based on the control group after treating neural stem cells with compounds 1 to 6 of the present invention (Figure 4A) (Figure 4B); * indicates p<0.05.

Figure 5 shows the HIF1α, Nrf-2, and AP1 genes of the two cells after treating neural stem cells with compounds 3~5, 7~8 of the present invention and C6 cells with compounds 3~4, 7~8 as the control group The benchmark relative gene expression rate statistics chart; * means p<0.05.

Figure 6 is a graph showing the oxygen consumption rate (OCR) statistics of the two cells in the basal, ATP production, and non-mitochondrial respiration after treating neural stem cells with compounds 3~4 of the present invention and C6 cells with compounds 7~8 ; * Means p<0.05.

Figure 7 shows the contraction and curling behavior of each embryo (Figure 6B), the frequency of tail flicking (Figure 6C), and hatching after treatment of zebrafish embryos with central nervous system defects with the compounds 3~5 and 7~8 of the present invention (Figure 6A) Rate (Figure 6D) statistical graph; * means p<0.05.

Figure 8 is a statistical chart of the swimming speed recovery rate (%) of each zebrafish after treating adult zebrafish with damaged cerebellum with compounds 3 to 4 of the present invention (Figure 7A) (Figure 7B).

Figure 9 shows the fluorescence micrographs of hatched larvae (Figure 8B) and tumor growth rate (%) after treatment of zebrafish embryos injected with C6 cells with compounds 3~4 and 7~8 of the present invention (Figure 8A) Statistics chart (Figure 8C); * means p<0.05.

definition

Unless otherwise specified, the "compounds" 1-11 referred to in the specification of the present invention are synonymous with "fullerene derivatives" 1-11, and both refer to the compounds of the structural formulas 1-11 shown in FIG.

"About", "about" or "approximately" generally refers to 20%, preferably 10%, and most preferably 5%. The numerical values in this article are approximate values, and the meanings of "approximately", "approximately" or "approximately" can be implied without a clear definition.

In the present invention, we synthesized 11 different water-soluble C ₆₀ fullerene derivatives and evaluated their effects in repairing the central nervous system or killing brain tumors. Neural stem cells (NSCs) and glioma cells (glioma cells) are used for in vitro experiments to screen candidate compounds. Use zebrafish model organisms to evaluate in vivo effects. Our experimental results indicate that functionalized C ₆₀ may have the unique property of regulating the oxygen metabolism of target cells, and its neuroprotective or apoptotic effects can be used in the treatment of central nervous system diseases.

The molecular structure of the water-soluble fullerene derivative of the present invention is shown in Figure 1. Compounds 1 to 3 have groups of phenylacetic acid, phenylpropionic acid and phenylbutyric acid, respectively, and were synthesized according to the method previously developed by our team, see Troshina et al. ¹ . Sulfur-containing fullerene derivatives 4 and 5 were synthesized in the manner previously reported, see Khakina et al. ² . Compound 4 contains 5 groups of the commercial drug captopril in its molecular structure. The spectral characteristics of compounds 4 to 5 are shown below. The synthesis method and characteristics of the fullerene derivative 6 have been disclosed in the previous report of our team, see Yurkova et al. ³ . Fullerene derivatives 7-9 have amino acid groups, and their amino acid groups are phenyl alanine, serine and β-alanine, based on For the synthesis method previously reported, see Kornev et al. ⁴ . In addition, compounds 10-11 are synthesized in the same way as compounds 7-9. The spectral characteristics of the novel compound 9 (protected tert-butyl ester form) are also shown below.

Spectral characteristics of compound 4:

^{_{1 HNMR ((CD 3) 2}} CO: CS 2 (1: 1), 600MHz, δ, ppm.): 1.15-1.26 (m, 15H, C H 3), 1.76-2.34 (m, 20H, C H 2 ), 2.53-2.97 (m, 10H, C H 2 -S, C H -C (O)), 3.1-3.23 (m, 5H, C H 2 -S), 3.32-3.78 (m, 10H, C H ₂ -N), 4.15-4.33 (m, 5H, C H -COOH), 4.95 (s, 1H, C ₆₀ - H ).

¹³ C NMR ((CD ₃ ) ₂ CO: CS ₂ (1:1), 150MHz, δ, ppm.): 16.54( C H ₃ ), 16.65( C H ₃ ), 24.37( C H ₂ ), 24.42( C H ₂ ), 27.08( C H ₂ ), 28.72( C H ₂ ), 41.14( C H-C(O)), 41.33( C H-C(O)), 46.44(S- C H ₂ ), 46.50(S- C H ₂ ), 46.55 (S- C H ₂ ), 54.00 (cage sp ³ C ), 55.92 (cage sp ³ C ), 56.60 (cage sp ³ C ), 58.26 ( C H-COOH), 58.30 ( C H -COOH),58.34( C H-COOH),59.06(cage sp ³ C -H),140.06,141.95,142.23,142.48,142.58,142.61,142.72,143.27,143.65,143.76,144.07,145.68,146.12,146.16, 146.22,146.27,146.31,146.35,147.45,147.51,147.53,147.65,147.73,147.81,147.87,147.99,148.17,148.20,172.72 (N- C = O), 173.25 (N- C = O), 173.30 (C OOH ), 173.34( C OOH), 173.45( C OOH).

ESI MS : m/z=1800([MH] ^- ),900([MH] ^2- ).

Spectral characteristics of compound 5:

¹ H NMR (D ₂ O, 500MHz, δ, ppm): 2.00-2.19 (M, 10H), 2.87-2.91 (M, 6H), 2.93-3.00 (M, 4H), 3.08-3.12 (M, 6H) , 3.20-3.23 (M, 4H), 3.45 (c, 1H).

¹³ C NMR (D ₂ O, 125MHz, δ, ppm): 23.02 ( C H ₂ ), 24.50 ( C H ₂ ), 30.39 ( C H ₂ ), 30.64 ( C H ₂ ), 31.13 ( C H ₂ ), 48.27 ( C H ₂ ), 48.84 ( C H ₂ ), 52.50 (cage sp ³ C ), 54.44 (cage sp ³ C ), 55.03 (cage sp ³ C ), 59.38 (cage sp ³ C ), 141.67, 141.72, 141.78,141.88,142.41,142.53,142.58,142.79,143.16,143.58,143.71,144.99,145.20,145.27,145.42,145.99,146.36,146.52,146.71,146.92,147.02,147.16,148.96, 149.70,152.00,152.15, 152.88.

FTIR (KBr pellet, ν ,cm ^-1 ): 454.00(W), 498.00(W), 528.00(M), 542.00(M), 558.00(W), 606.00(M), 668.00(W), 738.00(W) ),808.00(VW),862.00(VW),944.00(W),1052.00(VS),1114.00(M),1190.00(VS),1350.00(W),1418.00(W),1634.00(M),1700.00(W ).

Spectral characteristics of compound 9 (protected tert-butyl ester form):

_{^{1 H NMR (CDCl 3, 600MHz}} , δ, ppm.): 1.50 (m, 45H, C (C H 3) 3), 2.67-2.75 (m, 10H, C H 2 -N), 3.50-3.62 (m ,10H,C H ₂ -C(O)).

¹³ C NMR (CDCl ₃ , 150MHz, δ, ppm.): 28.20 ( C (CH ₃ ) ₃ ), 28.23 ( C (CH ₃ ) ₃ ), 28.25 (C ( C H ₃ ) ₃ ), 36.07 ( C H ₂ -CH ₂ -N), 36.18 ( C H ₂ -CH ₂ -N), 36.93 ( C H ₂ -CH ₂ -N), 42.54 ( C H ₂ -N), 42.86 ( C H ₂ -N), 44.61( C H ₂ -N), 65.32(cage sp ³ C ), 67.73(cage sp ³ C ), 65.59(cage sp ³ C ), 80.54(O- C ), 80.68(O- C ), 80.70(O -C ),140.30,142.57,143.19,143.58,143.89,144.06,144.19,144.23,144.41,144.49,144.85,145.15,145.48,147.21,147.24,147.31,147.90,148.35,148.40,148.45,148.71,148.88,149.71, ^{150.87,154.04,154.14,171.93 (C OOBu t), 172.19} (C OOBu t), 172.21 (C OOBu t).

ESI MS : m/z=1442 ([M-Cl] ^- ).

Compounds 1-11 contain 5 hydrophilic groups on half of the fullerene cage, while the other half of the fullerene cage does not have any functional groups and is highly hydrophobic. On the other hand, the unique molecular structures of compounds 1-11 are quite different from other known water-soluble fullerene derivatives. On the other hand, the arrangement of the organic groups of the aforementioned compounds 1-11 on the fullerene carbon framework makes it hydrophilic and has a strong tendency to self-assemble in polar solvents, especially in water. Therefore, compounds 1-11 will have a larger nanoparticle size in the aqueous solution than the dissolved molecules alone.

Figure 2 shows the particle size distribution spectrum of the aqueous solutions of compounds 1-9 (concentration 2.75×10 ^-3 M) detected by dynamic light scattering (DLS). Taking compound 3 as an example, it can be found in the spectrum that the smallest particle has an average hydrodynamic radius (<R _h >) close to 1 nm, indicating that compound 3 can be dissolved in the solvent alone. The middle-sized particles have an average hydrodynamic radius of about 6 nm, because small fullerene clusters (clusters) become like micelles. The largest clusters have an average hydrodynamic radius close to 50 nm, and most have the structure of double-layer fullerene vesicles. Similar vesicle structures are also found in other hydrophilic fullerene derivatives. Vesicles composed of fullerenes can self-assemble to form larger supramolecular architectures close to 1-100μm. Similar characteristics also appear in compounds 2 and 5.

Figure 2 shows the aggregation characteristics of compounds 1-9, and also shows that all the water-soluble fullerene derivatives of the present invention present nano-particles composed of a large number of individual molecules. These supramolecular structures are bonded by relatively weak Van der Waals forces, so they should be unstable structures and will coexist with some solitary molecules under dynamic equilibrium. Therefore, both nanoparticles and individual molecules are used for different biological effects in cells and animals.

In vivo toxicity test in animals

Some time ago, researchers disputed whether fullerenes are toxic. Recent studies have shown that unmodified C ₆₀ has no significant toxicity to animals. This means that the toxicity of fullerene derivatives depends on the chemical structure of the organic groups on it and the solubility of the compound in an aqueous medium. From Table 1, we found that although compounds 1 to 6 and 8 have acute toxicity in mice (via intraperitoneal injection), these fullerene derivatives with soluble carboxyl groups have very low toxicity, and their LD ₅₀ value exceeds 300 mg. /kg. Sulfonic acid group (compound 5) and phosphoric acid group (compound 6) are more toxic, with an LD ₅₀ value of 150-200 mg/kg. The least toxic compounds 4 and 8 have excellent LD ₅₀ values of 730 and 930 mg/kg, respectively. Therefore, the fullerene derivatives of the present invention have moderate to low toxicity and are quite safe to use in different animal studies.

In vitro ( in vitro ) experiment

We conducted in vitro experiments with neural stem cells (NSCs) and glioma cells (brain glioma cells; C6 cells) to test the effects of water-soluble C ₆₀ fullerene derivatives on the survival and growth of these cells.

The neural stem cell lines in the following experiments are murine neural stem cells (murine NSCs), which are isolated from the brains of adult mice, and the cells are transfected with the promoter F1B-green fluorescent protein (F1B-GFP). Neural stem cells were cultured in DMEM/F12 medium (Dulbecco's Modified Eagie's Medium and Ham's F-12, Gibco, USA), which contained 10% fetal bovine serum (FBS, Gibco, USA), 400μg/ml G418 (Invitrogen), and 100U/ ml penicillin-streptomycin (Caisson Labs, USA), and stored in a 37°C, 5% carbon dioxide incubator. The medium is changed every 2 days.

The glioma cell lines in the following experiments were rat C6 glioma cell lines (ATCC, Rockville, Maryland, USA), which were grown in DMEM medium (Invitrogen, Burlington, Ontario, Canada). A monolayer culture that contains heat-deactivated 10% fetal bovine serum (Invitrogen, Burlington, Ontario), glutamax-1 glutamate supplement (Invitrogen, Burlington, Ontario), and Penicillin-Streptomycin (Invitrogen, Burlington, Ontario). The cell lines are kept at 37°C and 5% carbon dioxide. The medium is changed every 2 days.

We evaluated the effects of water-soluble C ₆₀ fullerene derivatives 1-11 on the survival and growth of neural stem cells and glioma cells (Figure 3A). The evaluation of cell survival was calculated using MTT analysis. The MTT analysis system is to wash the cells with phosphate buffered saline (PBS) after 24 hours in contact with the compound, and add 100μl 0.5mg/ml tetrazolium dye (Sigma-Aldrich) to incubate at 37°C for 1 hour, at the end of the incubation Remove the tetrazolium dye. Dissolve the formed purple crystals with DMSO, and read the absorbance at 570nm with a microdisk analyzer (SpectraMax® M5, USA). Each test was repeated three independent experiments. In each independent experiment, each group has 4 samples.

The survival rate of neural stem cells was significantly improved after being treated with 100nM compound 4, 5~6, and 1~3 for 24 hours (approximately 130%, 120%, and 120%, respectively), and the survival rate was significant after treatment with compound 8 and 9 reduce. There was no significant difference in survival rate of neural stem cells treated with compound 7 (Figure 3B). These results show that the fullerene derivatives of compounds 1 to 6 can increase the survival rate and proliferation of brain neural progenitors. In addition, in the results of compounds 10 and 11, both compounds 10 and 11 can increase the survival rate of neural stem cells by about 2 times that of the control group. It is obvious that compounds 10 and 11 can also increase the survival of brain neural progenitors. Rate and proliferation.

C6 cells were treated with compounds 4 to 6 containing phosphorus and sulfur, and their survival rates increased significantly (approximately 130%, 110%, and 115%, respectively). In contrast, compounds 7, 8, and 9 significantly reduced the survival rate of C6 cells. At the same time, compounds 1, 2, and 3 had no significant effect on the survival rate of glioma cells (Figure 3C). These results show that the fullerene derivatives 7-9 with amino acid groups have the effect of inhibiting the proliferation of brain glioma cells. In addition, among the results of compounds 10 and 11, compound 11 also has the effect of inhibiting the proliferation of brain glioma cells.

Interestingly, compounds 1 to 3 with phenylacetic acid, phenylpropionic acid and phenylbutyric acid groups have the effect of promoting the proliferation of neural stem cells, but have no effect on C6 cells. In contrast, compound 7 with a phenylalanine group inhibits C6 cells, but has no effect on the survival rate of neural stem cells.

Then the cells were treated with 100nM compound 1~6, and the gene expression level was analyzed by reverse transcription polymerase chain reaction to evaluate the cell status. The effect of water-soluble fullerene derivatives on neural stem cell differentiation is shown in Figure 4. Compared with the control group, the expression of nestin gene (neural stem cell target) was significantly improved (about 3.5 and 5 times) after the neural stem cells were treated with compounds 4 and 3 for 7 days, but the group treated with compounds 2 and 1 Significantly reduced. Compared with the control group, neural stem cells treated with compounds 1 to 6 showed more β-tubulin (β-tubulin; early neural marker) and MAP2 (mature neural marker) on the 7th day. Among all C ₆₀ fullerene derivatives of the present invention, compound 4 induces β-tubulin and MAP2 to express the most. The above results show that fullerene derivatives 1~6 can promote neuronal differentiation of neural stem cells. In addition, compounds 3~6 can maintain the self-renewal ability of some neural precursor cells.

The effect of compound 3~5, 7, 8 on mitochondria

Because the mitochondria is a key regulator of cell death, proliferation and differentiation, and plays an important role in the formation of neurodegenerative diseases and brain tumors. Therefore, we tested whether these water-soluble fullerene derivatives have anti-oxidant or anti-tumor effects related to mitochondrial cell protection. We treated the cells with 100nM compounds 3~5, 7, and 8, and used quantitative reverse transcription-polymerase chain reaction to detect the relative markers of mitochondria HIF1α, Nrf-2, and AP1. The relative gene expression rate of these genes in the control group. The results are shown in Figure 5. Neural stem cells were treated with 100 nM compound 3~5, 7~8 for 2 days, and they showed more mitochondrial related genes (HIF1α, Nrf-2, and AP1) than the control group. Among all C ₆₀ fullerene derivatives, neural stem cells treated with compound 4 have the highest expression of mitochondrial-related genes. On the other hand, after C6 cells were treated with the amino acid-containing fullerene derivatives of compounds 7 and 8, the expression of mitochondrial-related genes was reduced. At the same time, the expression of mitochondrial-related genes in C6 cells slightly increased after treatment with compound 3, indicating that compound 3 did not induce tumor growth. These gene expression results show that functionalized fullerenes can regulate mitochondrial activity in different types of cells and have cell protection (antioxidant) or anti-tumor effects.

In order to directly evaluate the effects of functionalized fullerene derivatives 3, 4, 7, and 8 on neural stem cells and C6 cell intramitochondrial function, a bioenergetic assay was used to evaluate the oxygen consumption rate of cellular energy. , OCR for short). We used XF-96 extracellular analyzer (Seahorse Bioscience) to analyze the cell energy and oxygen consumption rate at 37°C. Neural stem cells or C6 cells were implanted into an 8-well culture dish at a density of 20,000 cells/well, and cultured in an incubator for 24 hours. Then, 100 nM of compound 3, 4 or 0.5 mM of compound 7, 8 were added, respectively, and cultured for 24 hours. Add the culture medium and replace it with unbuffered DMEM (DMEM with 25mM glucose, 1mM sodium pyruvate, 31mM NaCl, 2mM GlutaMax ^TM medium, pH 7.4), the hydrogel (hydrogel) at 37 ℃ without adding 5% Incubate for 1 hour in a CO ₂ environment. For all reagents to be analyzed, adjust the pH to 7.4 on the day of analysis. The Seahorse XF~96 software automatically calculates the oxygen consumption rate of the cells. The data is the average of the detection results of 3 to 6 wells in the culture plate.

Oxygen consumption is an important indicator of normal cell function, and it is also a parameter used to study mitochondrial function. In cancer cells, the rate of oxygen consumption is seen as a marker for the transition from healthy oxidative phosphorylation to aerobic glycolysis. The results of bioenergy evaluation are shown in Figure 6. Neural stem cells treated with 100 nM compound 3 or 4 had a significant increase in their basal oxygen consumption rate and adenosine triphosphate (ATP) production. The changes in non-mitochondrial respiratory oxygen consumption of neural stem cells treated with compounds 3 and 4 were not obvious. C6 cells were treated with amino acid-containing fullerene derivatives 7~8 (concentration 0.5mM) for 24 hours, and the basic oxygen consumption rate, ATP production (calculated from the measured oxygen consumption rate) and non- The mitochondrial respiratory oxygen consumption decreases. Taken together, these results show that fullerene derivatives 3~4 have the best effect in increasing mitochondrial function to induce the proliferation and differentiation of neural stem cells. At the same time, compounds 7~8 can destroy the mitochondrial function and non-mitochondrial respiration, and have anti-tumor effects.

Use model animals with central nervous system defects to test the effects of compounds 3~5, 7, 8

Zebrafish ( Danio rerio ) is an important vertebrate model in neuroscience. As shown in the experimental procedure of Figure 7A, the zebrafish embryos were directly immersed in 2% ethanol (EtOH) for 4 hours, and then replaced with a normal medium containing 100 nM fullerene derivatives 3~5, 7, and 8. Soaking in 2% ethanol for 4 hours can cause damage to the central nervous system of zebrafish. The zebrafish embryo is tested at the 18 somite stage (somite stage), and its early motor behavior has spontaneous coiling contraction. As shown in Figure 7B, wild-type embryos that were not exposed to ethanol (WT, blank control group) showed a complete side-to-side contraction of approximately 85% and a slight reaction (very slight) of approximately 15%. The curling or single-side contraction curling); abnormal embryos (EtOH, damaged by ethanol) without any treatment have about 76% no response (no contraction) and about 24% mild response. After treatment with compound 7, abnormal embryos had about 28.6% of complete contraction, about 43% of mild reactions, and 28.4% of non-responses. About 10% of embryos treated with compound 8 had mild reactions and about 90% had no reactions. It is worth mentioning that the embryos treated with compound 3 and sulfur-containing fullerene derivatives 4~5 showed about 70%, 55%, and 65% complete shrinkage, respectively.

The frequency of zebrafish tail flicks was measured 24 hours after fertilization, as shown in Figure 7C. The tail flick frequency of wild-type zebrafish embryos was about 0.075 Hz, while that of the untreated neurologically damaged group was significantly lower, about 0.03 Hz. In the group treated with compound 7, the tail flick frequency slightly increased to about 0.05 Hz. Surprisingly, the tail flick frequency of the group treated with compound 8 was reduced to 0.01 Hz. For groups of embryos treated with Compounds 3, 4, and 5, the tail flick frequency was significantly increased to approximately 0.065, 0.10, and 0.15 Hz, respectively.

Hatchability is the most important parameter for evaluating functional recovery after ethanol-induced central nervous system damage. As shown in Figure 7D, wild-type embryos have a 95% hatching rate. After ethanol-induced central nervous system injury, the untreated group had a 17% hatching rate. The group treated with compound 7 had a hatching rate of 25%. On the other hand, the group treated with compound 8 did not hatch any embryos from the eggs. The embryos treated with captopril-based fullerene derivative 4 had the highest hatching rate of about 65%, while the embryos treated with compounds 3 and 5 had a hatching rate of about 55%. The central nervous system damage model shows that fullerene derivatives 3~5 can significantly improve the damaged nervous system function during the embryonic period.

We also evaluated the effects of functionalized fullerenes 3 and 4 in repairing the central nervous system of adult zebrafish by comparing adult zebrafish without brain damage (blank control group) and using a syringe (27G, Thermo Scientific , U.S.) An adult zebra with damage to the cerebellum Fish (control group) and adult zebrafish injected with 10 μl of 100 nM fullerene derivatives 3 or 4 immediately after cerebellar injury. The rescue rate is because adult zebrafish with traumatic cerebellar injury will Unable to move or imbalance while swimming. Figure 8A illustrates that adult zebrafish with cerebellar damage were injected with fullerene derivatives and their swimming speed was recorded by a camera. There were 11 zebrafish tested in each group, and the experiment was repeated twice. The zebrafish for swimming speed measurement are all trained to swim in a 260mm×90mm×50mm water tank. The swimming speed is analyzed using the software ImageJ (National Institutes of Health, USA); with no injection or fuller injection The speed recovery rate (%) of the zebrafish swimming speed of the ene derivative to the cerebellar injury was calculated relative to the zebrafish swimming speed of the blank control group. As shown in Figure 8B, the zebrafish with cerebellar damage were injected with a captopril-based fullerene derivative 4, and the recovery rate of zebrafish movement was 34 after the damage and at 2, 4, and 6 days after injection. %, 57%, and 72%. In addition, the fullerene derivative 4 also has a good brain damage recovery effect. In contrast, the control group did not have any functional recovery after 4 days.

To evaluate the effect of C ₆₀ fullerene derivatives on brain tumors in vivo , we used a tumor xenograft model to evaluate the apoptotic effect of C6 cells. As shown in Figure 9A, C6 cells (2×10 ² cells/embryo) were injected into the mid-posterior brain region of zebrafish embryos 24 hours after fertilization, and 100 nM fullerene was derived after tumor transplantation (48 hours after fertilization) Compounds 3, 4 or 0.5 mM fullerene derivatives 7, 8 were injected into the tumor transplantation site.

In terms of cell labeling, C6 cells were labeled with red fluorescent dye (PKH26 red fluorescent cell connection kit, Sigma) in vitro. The cells were mixed with 2×10 ^-6 M PKH26 at a density of 1×10 ⁷ cells/ml for labeling. The long fat tail of PKH26 can stably bind to the cell membrane. The labeling process is terminated with complete medium, and the labeled cells are washed for use.

In this example, Tg ( flila : EGFP) zebrafish were used, and embryos were purchased from the Zebrafish International Resource Center (ZIRC, Oregon, USA), and were cultured and mated under standard conditions. The green fluorescent protein (EGFP) driven by the fli1a promoter is mainly expressed in vascular endothelial cells and endocardial cells, which can monitor vascular proliferation immediately. At 28 ℃, E3 embryo culture dishes containing solution (5mM NaCl, 0.17mM KCl, 0.33mM CaCl 2, and 0.33mM MgSO ₄₎ culturing the zebrafish. The behavior of embryos and larvae was observed with a Leica (Z16 APO) stereo microscope, and images were taken with an Olympus camera (C-7070 CCD). Calculate the tumor volume with image J software, and divide the tumor volume 3 days after the tumor cell injection by the tumor volume 1 day after the tumor cell injection to convert it to the tumor growth rate.

The results are shown in Figures 9B and 9C, showing that compounds 7-8 significantly reduced tumor size. On the contrary, compared with the control group, compound 4 even promoted the degree of tumor expansion. At the same time, compound 3, which has a slight effect in in vitro experiments, has no significant effect on tumor size in zebrafish experiments.

From the foregoing, we present several examples of water-soluble fullerene derivatives of different chemical species. First, we study the different types of linkages between the fullerene cage and soluble groups: CC bonds (compounds 1~3), CS bonds (compounds 4~5), CP bonds (compound 6), and CN Bond (compounds 7-9). Second, we make soluble groups with different properties: most of the compounds (1~4 and 7~9) have carboxyl groups (-COO ^- K ⁺ ) in the form of potassium salt, and compound 5 has sulfonic acid in the form of sodium salt. Group (-SO ₃ ^- K ⁺ ), compound 6 has a phosphate group (-PO(O ^- K ⁺ ) ₂ ) in the form of a potassium salt. Third, the spacer structure between the carbon ball and the terminal soluble group is also replaceable. For example, we can increase the amount of CH ₂ units in the aliphatic chain of the CC bond system of compounds 1 to 3, or we can use different ones in CN compounds. Amino acid groups: phenylalanine (compound 7), serine (compound 8) and β-alanine (compound 9). Experimental results show that the type of link and the nature of the soluble group will affect the toxicity of the water-soluble fullerene derivative. For example, compound 5 and 6 each having a sulfonic acid group and phosphoric acid group, rendering a lower half lethal dose (median lethal dose) LD ₅₀ than other fullerene derivative having a carboxyl group.

All other biological effects of these fullerene derivatives mainly depend on the type of bonding between the fullerene cage and the soluble group. Therefore, compared with compounds 1~3 with CC bonds, compounds 4~5 with CS bonds and compound 6 with CP bonds, compounds 7~9 with CN bonds did not promote brain nerve precursor cells. The survival rate and proliferation effect. The three amino acid-containing fullerene derivatives 7-9 all have the same effect of inhibiting the growth of glioma cells in vivo and in vitro, while the other compounds have no anti-tumor activity. Interestingly, only compounds 4~5 with C-S bonding significantly induce basal oxygen consumption rate and ATP production in neural stem cells. In addition, the sulfur-containing compound 4 has been shown in in vivo experiments in zebrafish embryo and adult animal models. Show the strongest central nervous system protection.

It can be seen from the above-mentioned examples that the present invention proposes a fullerene derivative that can promote the growth, proliferation and anti-tumor of neural stem cells, and has been proven in animal experiments to repair damage to the central nervous system or resist brain tumors, providing a novel The treatment of brain tumors and nerve damage diseases. It is worth noting that the fullerene derivatives proposed in the present invention can cross the blood-brain barrier and reach the brain to achieve therapeutic effects. It does not need to be delivered directly to the brain to have therapeutic effects like many existing central nervous system drugs.

【references】

1. OA Troshina, PA Troshin, AS Peregudov, VI Kozlovskiy, J. Balzarini, RN Lyubovskaya, Chlorofullerene C ₆₀ Cl ₆ : a precursor for straightforward preparation of highly water-soluble polycarboxylic fullerene derivatives active against HIV. Org. Biomol. Chem. , 2007 , 5 , 2783-2791

2. EA Khakina, AA Yurkova, AA Peregudov, SI Troyanov, V. Trush, AI Vovk, AV Mumyatov, VM Martynenko, J. Balzarini, PA Troshin, Highly selective reactions of C ₆₀ Cl ₆ with thiols for synthesis of functionalized [60 ]fullerene derivatives. Chem. Commun. , 2012 , 48, 7158-7160

3. AA Yurkova, EA Khakina, SI Troyanov, A. Chernyak, L. Shmygleva, AA Peregudov, VM Martynenko, YA Dobrovolskiy, PA Troshin, Arbuzov chemistry with chlorofullerene C ₆₀ Cl ₆ : a powerful method for selective synthesis of highly functionalized [ 60]fullerene derivatives. Chem. Commun. , 2012 , 48, 8916-8918

4. AB Kornev, EA Khakina, SI Troyanov, AA Kushch, DG Deryabin, AS Peregudov, A. Vasilchenko, VM Martynenko, PA Troshin, Facile preparation of amine and amino acid adducts of [60]fullerene using chlorofullerene C ₆₀ Cl ₆ as a precursor. Chem. Commun. , 2012 , 48, 5461-5463

Claims (11)

Use of a fullerene derivative for preparing a pharmaceutical composition for promoting the proliferation of central nerve cells, wherein the fullerene derivative contains 5 hydrophilic groups on half of the fullerene cage, and the other half There is no functional group on the fullerene framework; and the fullerene derivative has a structural formula as Formula I:

Wherein, R is the hydrophilic group and has one of the following structures:

,or

; And R'is H or Cl. The use as described in item 1 of the scope of patent application, wherein the half of the fullerene frame without functional groups is hydrophobic. The use described in item 1 or item 2 of the scope of patent application, wherein the fullerene derivative has the effect of promoting the proliferation of neural stem cells. For the purposes described in item 1 or item 2 of the scope of patent application, when R has one of the following structures:

The fullerene derivative has the effect of promoting neuronal differentiation of neural stem cells. For the purposes described in item 1 or item 2 of the scope of patent application, when R has one of the following structures:

The fullerene derivative has the effect of maintaining the self-renewal ability of neural precursor cells. For the purposes described in item 1 or item 2 of the scope of patent application, when R has one of the following structures:

The fullerene derivative has the effect of restoring damaged central nervous system. A fullerene derivative is used to prepare a pharmaceutical composition for inhibiting the growth of brain tumor cells, wherein the fullerene derivative contains 5 hydrophilic groups on half of the fullerene cage, and the other half There is no functional group on the fullerene framework; and the fullerene derivative has a structural formula as Formula I:

Wherein, R is the hydrophilic group and has one of the following structures:

,

,

,or

; And R'is H or Cl. For the application described in item 7 of the scope of patent application, the half of the fullerene frame without functional groups is hydrophobic. The use according to item 7 or 8 of the scope of patent application, wherein the fullerene derivative has the effect of inhibiting the proliferation of brain glioma cells. The use described in item 7 or 8 of the scope of patent application, wherein the fullerene derivative has anti-brain tumor effect. A fullerene derivative, which has the structural formula of Formula I:

Where R has one of the following structures:

4, 5, 9,

R'is H or Cl.

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