CN103467549A - Sulfur-bearing uridine anticancer drug, intermediate and synthesis method - Google Patents

Abstract

The present invention uses uridine as the main raw material, which is treated with iodine and dilute nitric acid to obtain 5-iodouridine, and then treated with acetic anhydride to obtain 2', 3', 5'-O-trioxyacetyl-5-iodo Uridine, and then react with 2-(tributylstannyl)furan or thiophene under the action of bistriphenylphosphopalladium chloride to obtain an intermediate, which is then treated with phosphorus pentasulfide, and then deprotected under stirring in a saturated ammonia methanol solution at room temperature , to give 5-(2-furyl or thienyl)-4-thiouridine. The compound has absorption at 363nm, is highly sensitive to UVA light and can enter the inner layer of the tissue, and can selectively act on cancer cells, overcoming the fact that the photosensitizing reagents used in phototherapy do not gather in the nucleus and cannot act selectively The disadvantages of cancer cells; the compound of the present invention is a new type of base sulfur-containing nucleoside compound, which absorbs light in the long-wave region and can selectively act on cancer cells in the inner layer of the tissue. It has potential medicinal value and is easy to enter tissue lining cells.

Description

Thiuridine-containing anticancer drug, intermediate and synthesis method

technical field

The present invention relates to a new compound for treating cancer and its preparation method, in particular, it relates to 5-(2-furyl or thienyl)-4-thiouridine and its analogs, intermediates and synthesis methods, and the synthesized products The ultraviolet spectrum has absorption at 363nm, is sensitive to UVA light, and is a drug suitable for treating cancer. the

Background technique

Malignant tumors are currently the No. 1 killer threatening human life. Although chemotherapy can reduce cancer mortality, existing anticancer drugs have relatively large side effects on normal cells while killing cancer cells. Radiation therapy focuses rays precisely on target tissue, but high-energy rays can also damage normal cells while killing cancer cells. Surgical treatment is still the most effective treatment for some tumors, but it is not suitable for all malignant tumors. Surgical treatment is basically powerless for advanced cancer. Obviously, chemotherapy, radiotherapy and surgery all have disadvantages for patients, so we urgently need a better way to treat cancer, which combines the advantages of chemotherapy and radiotherapy: the effect of chemotherapy drugs on cancer cells and the target radiation of radiotherapy effect. Therefore, it has become an important frontier topic to find anticancer drugs with precise targets and no or less damage to normal cells. The popular photodynamic therapy ( P hoto dynamic therapy , PDT) and the near-ultraviolet light (UVA) assisted anticancer drug therapy under development are two hot spots.

Since 2001, Dr. Peter Karran and Dr. Xu Yaozhong from the United Kingdom have conducted a lot of research and found that the synergistic effect of 4-sulfur thymidine (referred to as: 4-thiothymidine) and near ultraviolet light (UVA) can selectively To destroy the DNA of cancer cells, and kill cancer cells and their tissues, a novel cancer treatment method—near-ultraviolet light-assisted thiothymidine (UVA/4-thiothymidine) therapy is proposed, which uses near-ultraviolet light and 4-Thiothymidine acts synergistically to damage DNA and its cells. Due to the need to continuously replicate DNA during the rapid growth of cancer cells, resulting in the enrichment of 4-thiothymidine, it is more susceptible to near-ultraviolet radiation than normal tissue cells. Studies have shown that low-dose near-ultraviolet light that is harmless to normal tissue cells can easily kill cancer cells containing 4-thiothymidine DNA. This near-ultraviolet light/thiothymidine therapy is also a kind of photochemotherapy. Its principle is: a small molecule photosensitizer (such as 4-thiothymidine) enters the DNA of the target cell, and passes through a specific wavelength (near-ultraviolet light). , visible, or near-infrared) to kill cancer cells by sensitizing them briefly to light at a wavelength that is well matched to the optical properties of the photosensitizer. Using the synergistic effect of near ultraviolet light and 4-thiothymidine to damage the DNA in proliferating cells, as shown in Figure 1. the

Studies have shown that low-dose near-ultraviolet light that is harmless to normal cells can easily kill proliferating cells containing 4-thiothymidine DNA. It can be seen that the therapy of 4-thymidine combined with near-ultraviolet light has the potential of low toxicity and high selectivity to treat cancer. It has a killing effect on skin cancer and bladder cancer cells. It is a potential anticancer drug, especially for The treatment of skin cancer and some other light-sensitive tumors has received attention. Although 4-thiothymidine can be used as a new type of nucleoside anticancer drug, the near-ultraviolet wavelength of 4-thiothymidine is at 335nm, which has poor tissue penetration ability, and the ability of light to penetrate tissue is similar to that of photosensitizers. It is related to the excitation wavelength of the light received. The longer the wavelength of the light, the stronger the ability to penetrate the tissue, and it is easier to enter the cells in the inner layer of the tissue. Therefore, the development of photosensitizers for new anticancer drugs that absorb light in the long-wave region has attracted people's attention. the

"Photosensitive drugs" are needed in both photodynamic therapy and the near-ultraviolet light-assisted anticancer drug therapy approach that is being developed. Therefore, the development of new targeted and highly selective anti-tumor "photosensitive drugs" is an urgent need to effectively control malignant tumors. the

Contents of the invention

The purpose of the present invention is to provide a compound that absorbs ultraviolet spectrum at 363nm and has strong sensitivity to UVA light to treat cancer, that is, an anticancer drug containing thiouridine, an intermediate of the drug (compound) and a synthetic method . the

In order to achieve the above-mentioned purpose of the present invention, the thiouridine-containing anticancer drug provided by the present invention is 5-(2-furyl or thienyl)-4-thiouridine or 5-(2-furyl or thienyl) -4-thio-2 ^, -deoxyuridine drug, characterized in that 5-(2-furyl or thienyl)-4-thiouridine or 5-(2-furyl or thienyl)-4-thio -2 ^, the general chemical formula I of -deoxyuridine is:

in:

R ₁ represents H;

R ₂ represents H or OH.

The thiouridine-containing anticancer drug intermediate of the present invention is 5-(2-furyl or thienyl)-4-thiouridine or 5-(2-furyl or thienyl)-4-thio-2 ^, -The intermediate of deoxyuridine medicine, it is characterized in that, the chemical general formula I of intermediate is:

in:

R ₁ represents an acetyl group;

R ₂ represents OR ₁ or H.

The synthesis method of thiouridine-containing anticancer drug of the present invention, that is, the synthesis reaction route of the compound of general formula I is as follows:

The synthetic method of compound described in general formula ( I ), concrete synthesis comprises the steps:

The first step is to prepare 5-iodouridine: under the condition of 110°C, uridine and elemental iodine are in 3 mol/L nitric acid solution, and the reaction is monitored by thin-layer chromatography (TLC) (developing agent is the volume ratio of petroleum Ether: ethyl acetate = 1:1), the product was extracted with petroleum ether until the lower layer solution was colorless, and the lower layer solution was left to stand and recrystallized to obtain the pure product

The second step, acetylation: under the condition of 0°C, 5-iodo-2 ^, -deoxyuridine or 5-iodouridine reacts with acetic anhydride, the protection reagent of hydroxyl, in a pyridine solvent, and the product is prepared with ethyl acetate and Petroleum ether recrystallization affords the product protected via the _R

The third step, upper thiophene ring or furan ring: under the protection of argon at 90°C, the product obtained in the second step is mixed with 2-(tributylstannyl)furan or thiophene, bistriphenyl Phosphorus dichloride palladium reaction, thin layer chromatography (TLC) to monitor the reaction (developing agent is the volume ratio of petroleum ether: ethyl acetate = 1:1), the product is added to n-hexane for oscillation, and the obtained solid is mixed with dichloromethane: methanol =1:1 recrystallization, pure product

The fourth step, sulfur replaces oxygen: the product obtained in the third step is reacted with phosphorus pentasulfide in a dioxane solvent to obtain 4-position sulfur-substituted uridine and its analogues; wherein, the amount of phosphorus pentasulfide is 1% of the product of the third step ~1.5 times equivalent;

The fifth step, deacetylation: Inflate ammonia gas into the methanol solution to prepare an ammonia-saturated methanol solution, and then add the product obtained in the fourth step to the ammonia-saturated methanol solution for reaction to obtain 5-(2-furyl or Thienyl)-4-thiouridine and its analogues.

The preferred synthetic method of 5-(2-furyl or thienyl)-4-thiouridine comprises the steps:

The first step is to prepare 5-iodouridine: under the condition of 110°C, uridine and elemental iodine are in 3 mol/L nitric acid solution, and the reaction is monitored by thin-layer chromatography (TLC) (developing agent is the volume ratio of petroleum Ether: ethyl acetate = 1:1), the product was extracted with petroleum ether until the lower layer solution was colorless, and the lower layer solution was left to stand and recrystallized to obtain the pure product

The second step, acetylation: under the condition of 0°C, 5-iodo-2 ^, -deoxyuridine or 5-iodouridine reacts with acetic anhydride, the protection reagent of hydroxyl, in a pyridine solvent, and the product is prepared with ethyl acetate and Petroleum ether recrystallization affords the product protected via the _R

The third step, upper thiophene ring or furan ring: under the protection of argon at 90°C, the product obtained in the second step is mixed with 2-(tributylstannyl)furan or thiophene, bistriphenyl Phosphorus dichloride palladium reaction, thin layer chromatography (TLC) to monitor the reaction (developing agent is the volume ratio of petroleum ether: ethyl acetate = 1:1), the obtained oily product was added to n-hexane for oscillation, and the obtained solid was washed with dichloromethane : Methanol=1:1 recrystallization, get pure product

The fourth step, sulfur replaces oxygen: the product obtained in the third step is reacted with phosphorus pentasulfide in a dioxane solvent to obtain the 4-position sulfur-substituted uridine and its analogues; wherein, the amount of phosphorus pentasulfide is 1% of the product obtained in the third step 1~1.5 times equivalent

The fifth step, deacetylation: Inflate ammonia gas into the methanol solution to prepare an ammonia-saturated methanol solution, and then add the product obtained in the fourth step to the ammonia-saturated methanol solution for reaction to obtain 5-(2-furyl or Thienyl)-4-thiouridine and its analogues.

Wherein, the method for removing impurities in the fourth step is: after the reaction, the solvent is firstly distilled off under reduced pressure, and the crude product is purified with a silica gel chromatography column, and the eluent is dichloromethane:methanol with a volume ratio of 200:1. The solvent was distilled off to obtain yellow needle crystals. the

The method for the fifth step to purify the product is to remove the solvent under reduced pressure distillation, and the crude product is purified with a silica gel chromatography column, and the eluent is successively a, dichloromethane b, dichloromethane:methanol=200:1 c, dichloromethane Chloromethane:methanol=50:1, then the solvent was removed under vacuum to give yellow needle crystals. the

The ultraviolet spectrum of the compound of the present invention and its derivatives has absorption at 363nm, and is more sensitive to UVA light. Therefore, the compound of the present invention belongs to a novel nucleoside compound containing a sulfur base, which can selectively act on cancer cells, and overcomes the problem that the photosensitizing drugs used in existing phototherapy cannot selectively act on cancer cells. The compound not only has anticancer activity but also can selectively act on cancer cells under ultraviolet light irradiation, and has less toxic and side effects. It will be a new type of derivative for near ultraviolet light (UVA) assisted anticancer drug therapy, and further development and research And the clinical application value is high, and the application prospect is extensive, especially in the treatment of skin cancer. the

In addition, the synthesis method has the advantages of simple reaction conditions, readily available raw materials, low cost, high product yield and high purity. In particular, the use of acetic anhydride as the acetylation reagent in the present invention not only has cheap raw material prices, high product conversion, high catalyst activity, small dosage, mild reaction conditions (compared to acetyl chloride), and is environmentally friendly, but also simple and easy to implement after-treatment. Thereby greatly improving the application prospect of its industrialization. the

Description of drawings

Figure 1 is a schematic diagram of photochemotherapy (near ultraviolet light/4-thymidine therapy); Figure 2 is a graph of 4S5ThioU concentration and cell survival; Figure 3 is a graph of 4S5ThiodU concentration and cell survival; Figure 4 is a graph of 4SFurU concentration and cell survival Rate map; Figure 5 is a graph of 4SFurdU concentration and cell viability. the

Detailed ways

The embodiments of the present invention are described in detail below: the present embodiment is implemented under the premise of the technical solution of the present invention, and detailed implementation and specific operation process are provided, but the protection scope of the present invention is not limited to the following implementation example. the

The present invention relates to the synthesis method of general formula 4-thio-5-(2-furyl or thienyl)uridine and its derivatives. the

R ₁ represents a protecting group of O or H;

R ₂ represents OR ₁ or OH.

Preferred specific O protecting groups include alkanoyl and aroyl groups (e.g. acyl, especially benzoyl), trisubstituted arylmethyl groups (trityldimethoxy) and silyl groups (e.g. trialkylsilanes, especially trimethylsilane). the

Furthermore, compound (I) may exhibit tautomerism. Discoveries can now include all tautomeric forms and mixtures. The series of compounds (I) belong to sulfur-containing nucleoside drugs for treating cancer, and can be used for treating cancer-related diseases. the

The specific synthetic steps of general formula (I) compound are:

A. Iodination reaction The 5-position iodination reaction of uridine or deoxyuridine under acidic conditions

B. Protect the hydroxyl group on the sugar ring Protect 5-iododeoxyuridine (thymidine) or 5-iodouridine through the protecting group shown in general formula (I)

C. Heterocyclic substituted iodine React the compound protected by the protecting group represented by the general formula (I) with tributylstannyl furan or thiophene to obtain protected 5-(2-furyl or thienyl)uridine and derivatives thing

D. Sulfur-substituted oxygen React the protected 5-(2-furyl or thienyl) uridine and its derivatives with phosphorus pentasulfide to obtain 4-position sulfur-substituted 5-(2-furyl or thienyl) uridine and its analog

E. Remove the protecting group of O as shown in general formula (I) Fill ammonia gas into methanol solution to prepare ammonia-saturated methanol solution, then add the product obtained in step D to reaction in ammonia-saturated methanol solution, then Get 4-thio-5-(2-furyl or thienyl)uridine and its derivatives.

The described A step is specifically: under the condition of 110°C, uridine and elemental iodine are in a 3 mol/L nitric acid solution, and the reaction is monitored by thin layer chromatography (TLC) (developing agent is petroleum ether in volume ratio: Ethyl acetate = 1:1), the product was extracted with petroleum ether until the lower layer solution was colorless, and the lower layer solution was left to stand and recrystallized to obtain the pure product. the

The B step is specifically: under the condition of 0°C, 5-iodo-2 ^, -deoxyuridine or 5-iodouridine and a hydroxyl protection reagent such as acetic anhydride, benzyl chloride, trimethylchlorosilane etc., react in pyridine or tetrahydrofuran solvent, and the product is recrystallized from ethyl acetate and petroleum ether to obtain the product protected by R ₁ .

The C step is specifically: under the condition of argon protection at 90°C, the product obtained in the B step is mixed with 2-(tributylstannyl)furan or thiophene, bistriphenylphosphine dichloride in a dioxane solvent Palladium reaction, thin layer chromatography (TLC) to monitor the reaction (petroleum ether: ethyl acetate = 1:1 in volume ratio as the developer), the product was added to n-hexane for oscillation, and the obtained solid was dichloromethane: methanol = 1: 1. Recrystallize to obtain pure product. the

The D step is specifically: reacting the product obtained in the C step with phosphorus pentasulfide in a dioxane solvent to obtain 4-sulfur-substituted 5-(2-furyl or thienyl)uridine and its derivatives; wherein , the consumption of phosphorus pentasulfide is 1～1.5 times equivalent of the product obtained in step C. the

The E step is specifically: filling ammonia gas into the methanol solution to prepare an ammonia-saturated methanol solution, and then adding the product obtained in step D to the ammonia-saturated methanol solution for reaction to obtain 5-(2-furyl or thiophene base)-4-thiouridine and its analogues. the

Embodiment 1: a kind of 4-thio-5-(2-furyl) uridine and its intermediate and synthetic method comprise the following steps:

(1) Synthesis of 5-iodouridine

       

 

将尿苷 (0.55 g, 2 mmol)溶于10 mL 0.3mol/L的稀硝酸中，加入碘单质(0.504 g, 1.98 mmol)，回流加热100 min直到原料完全反应(薄层色谱法跟踪监测)。停止反应后热过滤，除去过量的碘单质，滤液用石油醚萃取至溶液澄清，置于4℃下得到大量无色的针状晶体5-碘尿苷，干燥后称重得0.63 g，收率75%。m. p. 205-207℃，(文献值m. p. 208-210℃)；

Dissolve uridine (0.55 g, 2 mmol) in 10 mL of 0.3 mol/L dilute nitric acid, add simple iodine (0.504 g, 1.98 mmol), and heat under reflux for 100 min until the raw material is completely reacted (monitored by thin-layer chromatography). . After stopping the reaction, heat filtration to remove excess elemental iodine, the filtrate was extracted with petroleum ether until the solution was clear, and placed at 4°C to obtain a large amount of colorless needle-like crystals of 5-iodouridine, which were weighed to obtain 0.63 g after drying. 75%. m.p. 205-207℃, (literature value m.p. 208-210℃);

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 11.71(br s,1H, NH), 8.50(s, 1H, 6-H), 5.74(d, 1H, J = 4.0 Hz, 1' -H), 5.10, 5.29, 5.44(d, t, d, 1H, 1H, and 1H, OH), 4.05 (dd, 1H, J = 4.0 Hz, 8.0Hz, 2'-H), 4.00 (dd, 1H, J = 4.0 Hz, 8.0 Hz, 3'-H), 3.89 (d, 1 H, J =4.0 Hz, 4'-H), 3.54-3.73 (m, 2H, 5'-H); UV- Vis (in CH ₃ CN), λmax/nm: 280.0, λmax/nm: 243.0.

(2) Synthesis of 2’,3’,5’-O-triacetyl-5-iodouridine

5-iodouridine (1.00 g, 2.70 mmol) was dissolved in 15 mL of anhydrous pyridine, and after it was fully dissolved, acetic anhydride (3.0 mL, 32 mmol) was added, and the reaction was carried out in an ice bath at 0°C for 5 h ( Thin-layer chromatography followed and monitored the reaction). After the reaction was stopped, the solvent was distilled off under reduced pressure. The crude product was recrystallized from ethanol and dried to obtain 1.23 g of white solid 2’,3’,5’-O-triacetyl-5-iodouridine with a yield of 92%. m.p. 175-176℃, (literature value m.p. 177-178℃);

¹ H NMR(400 MHz, DMSO-d ₆ ) (ppm): 11.83 (br s, 1H, NH), 8.18 (s, 1H,6-H), 5.88(d, J = 4.0 Hz, 1H, 1 ^, -H), 5.47 (dd, J=8.0 Hz and J = 8.0 Hz, 1H, 2'-H), 5.33~5.35 (m, 1H,3'-H), 4.31~4.36 (m, 1H, 4' -H), 4.21~4.27 (m, 2H, 5'-H), 2.06, 2.07, 2.08(3s, 3H, 3H, 3H, OAc); UV (in CH ₃ CN): λmin=257.0nm, λmax= 226.5nm.

(3) Synthesis of 2’,3’,5’-O-triacetyl-5-(2-furyl)uridine

Dissolve 2',3',5'-O-triacetyl-5-iodouridine (2 g, 4.02 mmol) in 94ml of anhydrous tetradioxane, protect with argon, and add tributylstannane Furan (8.70 g, 24.29 mmol) and bistriphenylphosphine palladium dichloride (0.0564 g, 0.08 mmol) were reacted at 90°C for 1 hour (reaction monitored by TLC). After stopping the reaction, cool, filter with diatomaceous earth, add 16ml of n-hexane to shake the yellow oil obtained after removing the solvent, filter, and dissolve the filter residue with a minimum amount of dichloromethane:methanol=1:1, and place it at 4°C to obtain A large amount of powdery solid weighed 1.18 g after drying, and the yield was 67.31%. m.p. 193-195°C. ¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 11.83(br s,1H, NH), 8.06(s, 1H, 6-H), 7.70(s,1H,5 ^,, -H), 6.91(s,1H,3 ^,, -H),6.56(s,1H,4 ^,, H)6.04-6.05(d, 1H, J = 4.0 Hz, 1'-H), 5.48-5.49(d, 1H , J = 4.0 Hz, 2'-H),5.37 (m, 1H, 3'-H),4.31-4.34 (d, 3H, J = 12.0 Hz, 4'-H, 5'-H), 2.10( br s, 9 H, CH ₃ ); UV-Vis (in CH ₃ CN): λmax/nm: 245.0, λmax/nm: 313.5.

(4) Synthesis of 4-thio-2',3',5'-O-triacetyl-5-(2-furyl)uridine

Dissolve 2',3',5'-O-triacetyl-5-(2-furyl)uridine (1.00 g, 2.32 mmol) in 50 ml of anhydrous tetradioxane, add phosphorus pentasulfide (1.00 g , 4.50 mmol), reacted at 105°C for 4 hours (trace and monitor the reaction by thin-layer chromatography). After stopping the reaction, cooling, filtering, solvent removal, column separation and purification to obtain 0.675g of yellow solid, yield 64.29%. m.p. 189-191°C.

¹ H NMR (400 MHz, DMSO-d ₆ )(ppm):13.07(brs,1H,NH),8.15(s,1H,6-H),7.71(s,1H,5 ^,, -H),7.34 -7.35(d,1H,J=4Hz,3 ^,, -H),6.57(s,1H,4 ^,, H)6.01-6.02(d, 1H, J = 4.0 Hz, 1'-H), 5.53- 5.56(t, 1H, J = 4.0 Hz, 2'-H),5.38-5.40 (t, 1H, J=4Hz,3'-H),4.36-4.37 (m, 3H, 4'-H, 5' -H), 2.04-2.08(t,9 H, CH ₃ ); UV-Vis (in CH ₃ CN): λmax/nm: 234.5, λmax/nm: 285, λmax/nm: 358.5.

(5) Synthesis of 4-thio-5-(2-furyl)uridine

Dissolve 4-thio-2',3',5'-O-triacetyl-5-(2-furyl)uridine (0.36 g, 0.91 mmol) in 96 ml ammonia methanol saturated solution, stir at room temperature for 5 Hours (reaction monitored by TLC follow-up). Column separation and purification yielded 0.1987 g of a yellow solid, with a yield of 70.96%. m.p. 211-213°C.

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 12.91(brs,1H, NH), 8.61(s,1H, 6-H), 7.61(s,1H,5 ^,, -H),7.29 -7.30(d,1H,J=4Hz,3 ^, -H),6.52(s,1H,4 ^,, -H),5.81 -5.82(d,1H, J = 4.0 Hz, 1'-H) , 4.13-4.15(t, 1H, J = 4.0 Hz, 2'-H),4.02-4.04 (t, 1H, J=4Hz,3'-H),3.94 (s, 1H, 4'-H), 3.71-3.74(d,1 H, J = 12.0 Hz 5'-H) 3.59-3.62(d,1 H, J = 12.0 Hz ,5'-H); UV-Vis (in CH ₃ CN): λmax/ nm: 234.5, λmax/nm: 286.5, λmax/nm: 362.5.

Embodiment 2: a kind of 4-thio-5-(2-thienyl) uridine and its intermediate and synthetic method comprise the following steps:

(1) Synthesis of 5-iodouridine

       

 

将尿苷 (0.55 g, 2 mmol)溶于10 mL 0.3mol/L的稀硝酸中，加入碘单质(0.504 g, 1.98 mmol)，回流加热100 min直到原料完全反应(薄层色谱法跟踪监测)。停止反应后热过滤，除去过量的碘单质，滤液用石油醚萃取至溶液澄清，置于4℃下得到大量无色的针状晶体5-碘尿苷，干燥后称重得0.63 g，收率75%。m. p. 205-207℃，(文献值m. p. 208-210℃)；

Dissolve uridine (0.55 g, 2 mmol) in 10 mL of 0.3 mol/L dilute nitric acid, add simple iodine (0.504 g, 1.98 mmol), and heat under reflux for 100 min until the raw material is completely reacted (monitored by thin-layer chromatography). . After stopping the reaction, heat filtration to remove excess elemental iodine, the filtrate was extracted with petroleum ether until the solution was clear, and placed at 4°C to obtain a large amount of colorless needle-like crystals of 5-iodouridine, which were weighed to obtain 0.63 g after drying. 75%. m.p. 205-207℃, (literature value m.p. 208-210℃);

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 11.71(br s,1H, NH), 8.50(s, 1H, 6-H), 5.74(d, 1H, J = 4.0 Hz, 1' -H), 5.10, 5.29, 5.44(d, t, d, 1H, 1H, and 1H, OH), 4.05 (dd, 1H, J = 4.0 Hz, 8.0 Hz, 2'-H), 4.00 (dd, 1H, J = 4.0 Hz, 8.0 Hz, 3'-H), 3.89 (d, 1 H, J =4.0 Hz, 4'-H), 3.54-3.73 (m, 2H, 5'-H); UV- Vis (in CH ₃ CN), λmax/nm: 280.0, λmax/nm: 243.0.

(2) Synthesis of 2’,3’,5’-O-triacetyl-5-iodouridine

5-iodouridine (1.00 g, 2.70 mmol) was dissolved in 15 mL of anhydrous pyridine, and after it was fully dissolved, acetic anhydride (3.0 mL, 32 mmol) was added, and the reaction was carried out in an ice bath at 0°C for 5 h ( Thin-layer chromatography followed and monitored the reaction). After the reaction was stopped, the solvent was distilled off under reduced pressure. The crude product was recrystallized from ethanol and dried to obtain 1.23 g of white solid 2’,3’,5’-O-triacetyl-5-iodouridine with a yield of 92%. m.p. 175-176℃, (literature value m.p. 177-178℃);

¹ H NMR(400 MHz, DMSO-d ₆ ) (ppm): 11.83 (br s, 1H, NH), 8.18 (s, 1H, 6-H), 5.88(d, J = 4.0 Hz, 1H, 1' -H), 5.47 (dd, J=8.0 Hz and J = 8.0 Hz, 1H, 2'-H), 5.33~5.35 (m, 1H, 3'-H), 4.31~4.36 (m, 1H, 4' -H), 4.21~4.27 (m, 2H, 5'-H), 2.06, 2.07, 2.08(3s, 3H, 3H, 3H, OAc); UV (in CH ₃ CN): λmax=257.0nm, λmax= 226.5nm.

(3) Synthesis of 2’,3’,5’-O-triacetyl-5-(2-thienyl)uridine

     

Dissolve 2',3',5'-O-triacetyl-5-iodouridine (0.60 g, 1.21 mmol) in 60 ml of anhydrous 14-dioxane, under argon protection, add tributyltin Alkylthiophene (1.03 g , 2.76 mmol) and bistriphenylphosphine palladium dichloride (0.02 g , 0.029 mmol) were reacted at 90°C for 3 hours (reaction monitored by TLC). After stopping the reaction, cool, filter with diatomaceous earth, remove the yellow oil obtained from the solvent, weigh 0.51 g after column separation, and the yield is 92.73%. m.p. 178-181°C.

¹ H NMR(400 MHz,DMSO-d ₆ )(ppm): 11.83(brs,1H,NH),8.17(s,1H,6-H),7.46-7.50(m ,2H,5 ^,, -H, 3 ^,, -H),7.07(m,1H,4 ^,, -H), 5.94-5.95(d, 1H, J = 4.0 Hz, 1 ^, -H), 5.57-5.60(t, 1H, J = 12.0 Hz, 3'-H),5.34-5.39 (m, 1H, 2'-H),4.25-4.36 (m, 3H, 4'-H, 5'-H), 2.04-2.06(m,9H, CH ₃ ); UV-Vis (in CH ₃ CN): λmax/nm: 262.0, λmax/nm: 304.0.

(4) Synthesis of 4-thio-2',3',5'-O-triacetyl-5-(2-thienyl)uridine

     

Dissolve 2',3',5'-O-triacetyl-5-(2-thienyl)uridine (0.40 g, 0.88 mmol) in 40 ml of anhydrous tetradioxane, add phosphorus pentasulfide (0.41 g , 1.83 mmol), and reacted at 105°C for 4 hours (reaction monitored by TLC). After the reaction was stopped, it was cooled, filtered, solvent removed, and column separation and purification gave 0.23 g of a yellow solid, with a yield of 56.1%. m.p.171-173°C.

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm):13.07(brs,1H,NH),8.05(s,1H,6-H),7.57-7.58(d,1H,J=4.0Hz,5 ^,, -H),7.29-7.30(d,1H,J=4Hz,3 ^,,- H),7.06-7.08(t,1H,J=4Hz,4 ^,, -H),5.93-5.94(d, 1H, J = 4.0 Hz, 1'-H), 5.60-5.63(t, 1H, J = 4.0 Hz,, 8.0Hz, 3'-H), 5.35-5.41 (m, 1H, 2'-H), 4.24-4.36 (m, 3H, 4'-H, 5'-H), 2.07(m,9 H, CH ₃ ); UV-Vis (in CH ₃ CN): λmax/nm: 239, λmax/nm: 285, λmax/nm: 348.

(5) Synthesis of 4-thio-5-(2-thienyl)uridine

     

Dissolve 4-thio-2',3',5'-O-triacetyl-5-(2-thienyl)uridine (1.00 g, 2.14 mmol) in 60 ml ammonia methanol saturated solution, add 10 ml ammonia water , 80ml of methanol, stirred at room temperature for 5 hours (thin-layer chromatography tracking and monitoring the reaction). Column separation and purification yielded 0.33 g of a yellow solid, with a yield of 45.21%. m.p.185-187°C.

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 12.89(brs,1H, NH), 8.51(s,1H, 6-H), 7.47-7.48(d,1H,J=4.0Hz,5 ^,, H),7.27-7.28(m,1H,3 ^,, -H),6.98-7.00(m,1H,4 ^,, -H),5.75 -5.76(d, 1H, J = 4.0 Hz, 1' -H), 5.50-5.51(d, 1H, J = 4.0 Hz, OH), 5.30-5.32 (t, 1H, J=4Hz, OH), 5.06-5.07 (d, 1H, J=4.0Hz 5'- OH), 4.10-4.14(m,1H,3 ^, -H) 4.00-4.04(m,1H,2 ^, -H),3.89-3.90(m,1H,4 ^, -H)3.56-3.72(m ,2H,5 ^, -H); UV-Vis (in CH ₃ CN): λmax/nm: 239, λmax/nm: 287, λmax/nm: 356.

Embodiment 3: A kind of 4-thio-5-(2-furyl)-2'-deoxyuridine and its intermediate and synthesis method, comprising the following steps:

(1) Synthesis of 3',5'-O-dioxoacetyl-5-iodo-2'-deoxyuridine

In a 100 mL three-necked flask, add 5-iodo-2'-deoxyuridine (1.0 g, 2.82 mmol), anhydrous pyridine (15 mL, 183 mmol), and add purified acetic anhydride after it is fully dissolved (3.0 mL, 32 mmol), reacted under ice bath conditions for 16 h, removed the solvent under reduced pressure, then added 10 mL each of dichloromethane and benzene, removed the solvent again under reduced pressure, and added dichloromethane (20 mL), the solvent was removed under reduced pressure. The crude product was dissolved in dichloromethane (250 mL), and saturated NaHCO ₃ (80 mL) was added, and extracted three times with dichloromethane. The organic phase was dried over anhydrous NaSO ₄ , filtered, and the filtrate was freed of solvent under reduced pressure. Add 1.5 mL of 95% ethanol, and then place the mixture in the freezer for 24 h to precipitate a white solid 3',5'-O-dioxoacetyl-5-iodo-2'-deoxyuridine (1.13 g , 2.58 mmol), the yield is 91%, m. p. 156-157 ℃.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 11.78 (br s, 1H, NH), 8.05 (s, 1H, 6-H), 6.11 (t, J=4.0 Hz, 8.0 Hz, 1H, 1 '-H), 5.17-5.19 (m, 1H, 3'-H), 4.26 (d, J=4.0 Hz, 2H, 5'-H), 4.20 (s, 1H, 4'-H), 2.28- 2.33 (m, 2H, 2'-H), 2.06 (s, 3H, -OC=OCH ₃ ), 2.11 (s, 3H, -OC=OCH ₃ ); HRMS (ES-TOF): 460.9842 [M+Na ⁺ ], Calcd for C ₁₃ H ₁₅ N ₂ O ₇ NaI: 460.9822; IR (film)/cm ^-1 1740.2, 1708.7(C=O), 1689.4 (amide C=O) , 1611.6(C=C).

(2) Synthesis of 3',5'-O-dioxoacetyl-5-(2-furyl)-2'-deoxyuridine

     

Dissolve 3',5'-O-dioxoacetyl-5-iodo-2'-deoxyuridine (0.50 g, 1.14 mmol) in 30 ml of anhydrous tetradioxane, under argon protection, Add tributylstannylfuran (1.22 g , 3.42 mmol) and bistriphenylphosphine palladium dichloride (0.016 g , 0.023 mmol) and react at 90°C for 3 hours (reaction monitored by TLC). After the reaction was stopped, it was cooled, filtered with celite, and the yellow oil obtained by removing the solvent was weighed to obtain 0.33 g of a white solid after column separation, with a yield of 75.84%. m.p.133-135°C.

¹ H NMR (500 MHz, DMSO-d ₆ ) (ppm): 11.72 (brs, 1H, NH), 7.98 (s, 1H, 6-H), 7.66 (m , 1H, 5 ^,, -H), 6.89 -6.90(m,1H,3 ^,, -H),6.54-6.55(dd,1H,J=4.0Hz,4 ^,,- H),6.22- 6.25(m,1H,1 ^, -H) 5.22- 5.24 (d, 1H, J = 8.0 Hz,3 ^, -H),4.27-4.29 (m, 3H, 4'-H,5'-H),2.35-2.46(m,2H,2 ^, -H), 2.08 -2.10(m,6 H, CH3); UV-Vis (in CH ₃ CN): λmax/nm:246.5, λmax/nm:313.5.

(3) Synthesis of 4-thio-3',5'-O-triacetyl-5-(2-furyl)-2'-deoxyuridine

     

Dissolve 3',5'-O-dioxoacetyl-5-(2-furyl)-2'-deoxyuridine (0.5876 g, 1.55 mmol) in 40 ml of anhydrous tetradioxane , add phosphorus pentasulfide (0.6985 g, 3,15 mmol), and react at 105°C for 3 hours (trace and monitor the reaction by thin-layer chromatography). After stopping the reaction, cooling, filtering, solvent removal, column separation and purification to obtain 0.30 g of yellow solid, yield 49.18%. m.p. 156-159°C.

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 13.00(br s,1H, NH), 8.08(s,1H, 6-H), 7.67(s,1H,5 ^,, -H), 7.37(s,1H, 3 ^,, -H),6.55(s,1H,4 ^,, H)6.14-6.17(t, 1H, J =8.0Hz, 4.0 Hz,1'-H),5.22(s, 1H,3'-H),4.27-4.33 (m,3H, 4'-H, 5'-H),3.57-3.64 (m, 3H,2 ^, -H), 2.03-2.08(m,9 H, CH ₃ ); UV-Vis (in CH ₃ CN): λmax/nm: 231.5, λmax/nm: 286, λmax/nm: 362.5.

(4) Synthesis of 4-thio-5-(2-furyl)-2'-deoxyuridine

Dissolve 4-thio-3',5'-O-triacetyl-5-(2-furyl)-2'-deoxyuridine (0.28 g, 0.71 mmol) in 45 ml ammonia methanol saturated solution, room temperature Stir for 5 hours (reaction followed by TLC). Column separation and purification yielded 0.10 g of a yellow solid with a yield of 45.45%. m.p.196-199°C.

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 12.83 (brs, 1H, NH), 8.43 (s, 1H, 6-H), 7.56 (s, 1H, 5 ^,, -H), 7.26 (m,1H, 3 ^,, -H),6.46-6.47(m,1H,4 ^,, H),,6.06-6.10(t, 1H, J = 8.0 Hz, 1'-H), 4.21-4.22( d, 1H, J = 4.0 Hz, 3'-H), ,3.81 (s, 1H, 4'-H), 3.51-3.60(dd,2H, J = 12.0 Hz 5'-H) 2.17-2.19( t,2 H, J = 4.0 Hz ,2'-H); UV-Vis (in CH ₃ CN): λmax/nm: 234.0, λmax/nm: 286.0, λmax/nm: 363.0.

Embodiment 4: A kind of 4-thio-5-(2-thienyl)-2'-deoxyuridine and its intermediate and synthesis method, comprising the following steps:

(1) Synthesis of 3',5'-O-dioxoacetyl-5-iodo-2'-deoxyuridine

In a 100 mL three-necked flask, add 5-iodo-2'-deoxyuridine (1.0 g, 2.82 mmol), anhydrous pyridine (15 mL, 183 mmol), and add purified acetic anhydride after it is fully dissolved (3.0 mL, 32 mmol), reacted under ice bath conditions for 16 h, removed the solvent under reduced pressure, then added 10 mL each of dichloromethane and benzene, removed the solvent again under reduced pressure, and added dichloromethane (20 mL), the solvent was removed under reduced pressure. The crude product was dissolved in dichloromethane (250 mL), then added saturated sodium bicarbonate (80 mL), and extracted three times with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was freed of solvent under reduced pressure. Add 1.5 mL of 95% ethanol, then place the mixture in the freezer for 24 h, and a white solid 3',5'-O-dioxoacetyl-5-iodo-2'-deoxyuridine (1.13 g , 2.58 mmol), the yield was 91%, m. p. 156-157 ° C.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ: 11.78 (br s, 1H, NH), 8.05 (s, 1H, 6-H), 6.11 (t, J=4.0 Hz, 8.0 Hz, 1H, 1 '-H), 5.17-5.19 (m, 1H, 3'-H), 4.26 (d, J=4.0 Hz, 2H, 5'-H), 4.20 (s, 1H, 4'-H), 2.28- 2.33 (m, 2H, 2'-H), 2.06 (s, 3H, -OC=OCH ₃ ), 2.11 (s, 3H, -OC=OCH ₃ ); HRMS (ES-TOF): 460.9842 [M+Na ⁺ ], Calcd for C ₁₃ H ₁₅ N ₂ O ₇ NaI: 460.9822; IR (film)/cm ^-1 1740.2, 1708.7(C=O), 1689.4 (amide C=O) , 1611.6(C=C).

(2) Synthesis of 3',5'-O-dioxoacetyl-5-(2-thienyl)-2'-deoxyuridine

     

Dissolve 3',5'-O-dioxoacetyl-5-iodo-2'-deoxyuridine (0.50 g, 1.14 mmol) in 40 ml of anhydrous tetradioxane, under argon protection, Tributylstannylthiophene (1.28 g, 3.42 mmol) and bistriphenylphosphine palladium dichloride (0.02 g, 0.0284 mmol) were added, and reacted at 95°C for 3 hours (the reaction was tracked and monitored by thin-layer chromatography). After stopping the reaction, cool, filter with diatomaceous earth, remove the yellow oil obtained by solvent removal, and weigh 0.40 g of white solid after column separation, with a yield of 88.89%. m.p.98-100℃.

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 11.78 (brs, 1H, NH), 8.03 (s, 1H, 6-H), 7.47-7.50 (m, 2H, 5 ^,, -H, 3 ^,, -H),7.07-7.09(t,1H,J=4.0Hz,4 ^,, -H), 6.18-6.21(t,1H,J=8.0Hz,4.0Hz,1 ^, -H)5.21- 5.22(d,1H,J=8.0Hz,3 ^, -H),4.30(m,2H,5 ^, -H)4.23(m,2H,4 ^, -H),2.58-2.65(m,1H,2 ^, -H),2.32-2.38(m,1H,2 ^, -H),2.01-2.07(m, 6H,CH3); UV-Vis(in CH ₃ CN): λmax/nm:258, λmax/nm:315

(3) Synthesis of 4-thio-3',5'-O-triacetyl-5-(2-thienyl)-2'-deoxyuridine

     

Dissolve 3',5'-O-dioxoacetyl-5-(2-thienyl)-2'-deoxyuridine (0.45 g, 1.14 mmol) in 25 ml of anhydrous tetradioxane , add phosphorus pentasulfide (0.45 g , 2.03 mmol), and react at 105°C for 3 hours (trace and monitor the reaction by thin-layer chromatography). After stopping the reaction, cooling, filtering, solvent removal, column separation and purification to obtain 0.27g of yellow solid, yield 57.45%. m.p.80-83°C.

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 13.01(br s,1H, NH), 7.90(s,1H, 6-H), 7.56-7.57(d,1H,J=4.0Hz, 5 ^,, -H),7.28-7.29(m,1H,3 ^,, -H),7.05-7.07(m,1H,4 ^,, H)6.11-6.15(m,1H,1'-H),5.20 -5.22(d,1H,J=8.0Hz,3'-H),4.27 (m,3H, 4'-H, 5'-H),2.62-2.67(m,1H,2 ^, -H),2.33 -2.40(m,1H,2 ^, -H),2.07(m,3H,CH ₃ ),1.91(m,3H,CH ₃ );UV-Vis (in CH ₃ CN): λmax/nm: 239.0, λmax /nm:286, λmax/nm:352.0.

(4) Synthesis of 4-thio-5-(2-thienyl)-2'-deoxyuridine

Dissolve 4-thio-3',5'-O-triacetyl-5-(2-thienyl)-2'-deoxyuridine (0.27 g, 0.66 mmol) in 43 ml ammonia methanol saturated solution, room temperature Stir for 5 hours (reaction followed by TLC). Column separation and purification yielded 0.10 g of a yellow solid, with a yield of 47.62%. m.p.181-183°C.

¹ H NMR (400 MHz, DMSO-d ₆ ) (ppm): 12.91 (brs, 1H, NH), 8.44 (s, 1H, 6-H), 7.50-

7.51(d,1H,J=4.0Hz,5 ^,, -H),7.31-7.32(d,1H,J=4.0Hz,3 ^,,- H),7.02-7.04(m,1H,4 ^,, - H),6.12-6.15(t,1H,J=8.0 Hz,4.0 Hz,1 ^, -H),5.28-5.29(d,1H,J=4.0Hz,5 ^, -OH),5.21-5.24(t, 1H,J = 4.0 Hz,8.0Hz,3'-OH),4.28- 4.30(m,1H,3 ^, -H),3.85-3.86(d,1H,J=4.0Hz,4'-H),3.58 -3.69(m,2 H,5'-H), 2.23-2.32(m,2 H, 2'-H); UV-Vis (in CH ₃ CN): λmax/nm: 239.0,λmax/nm: 287.0 ,λmax/nm: 356.0.

Embodiment 5: Test in vitro anti-mouse melanoma B16-F10 cell activity

1. Inhibitory effect on mouse melanoma B16-F10

Take the B16-F10 cells in the logarithmic growth phase, adjust the cell concentration to 10×10 ⁵ /ml with DMEM medium containing 10% fetal bovine serum and antibiotics, add 100ul per well (about 10 ⁴ cells) in a 37°C, 5% carbon dioxide incubator overnight. The next day, add the culture solution containing different drugs into the 96-well plate, 100ul per well, set three parallel wells for each drug combination, and the final concentration of the drug is 200um/L, 400um/L, 600um/L, 800um/L , 1000um/L, the control group only added culture medium. Place them in a 37°C, 5% carbon dioxide incubator for 48 hours.

2, MTT experiment (thiazolium blue colorimetric method)

After the incubation, add 20ul of MTT solution (5mg/ml) to each well and continue to incubate for 3 hours, take out the 96-well plate, discard all the supernatant in the well, and gently dry the residual liquid in the 96-well plate with a clean paper towel. Add 100ulDMSO to each well, dissolve for 30 minutes, and then use a microplate reader to take the absorbance at a wavelength of 570nm. Record the experimental results and process the data. The results are shown in Figure 2, Figure 3, Figure 4, and Figure 5. the

Table LD ₅₀ concentration of four substances substance LD ₅₀ Concentration/um/L 4S5ThioU 296 4S5ThiodU 415 4S5FurU 380 4S5FurdU 400

It can be seen from Figure 2, Figure 3, Figure 4, and Figure 5 that the four compounds have good inhibitory effects on mouse melanoma B16-F10 cells. It can be seen from the table that the four substances have the inhibitory effect on the mouse melanoma B16-F10 cells, and the inhibitory effect of 4-thio-5-(2-thienyl)uridine is relatively the best. the

Claims (5)

1. A thiouridine-containing anticancer drug, which is 5-(2-furyl or thienyl)-4-thiouridine or 5-(2-furyl or thienyl)-4-thio-2 ^, - Deoxyuridine drug, characterized in that 5-(2-furyl or thienyl)-4-thiouridine or 5-(2-furyl or thienyl)-4-thio-2 ^, -deoxyuridine General chemical formula I is: Among them: R ₁ represents H; R ₂ represents H or OH. 2. A thiouridine-containing anticancer drug intermediate, which is 5-(2-furyl or thienyl)-4-thiouridine or 5-(2-furyl or thienyl)-4-thio- ^2. An intermediate of a deoxyuridine drug, characterized in that the general chemical formula I of the intermediate is:

in: R ₁ represents an acetyl group; R ₂ represents OR ₁ or H. 3. A synthetic method for preparing thiouridine-containing anticancer drugs, comprising the steps of: The first step is to prepare 5-iodouridine: under the condition of 110°C, uridine and elemental iodine are in 3 mol/L nitric acid solution, and the reaction is monitored by thin-layer chromatography (TLC) (developing agent is the volume ratio of petroleum Ether: ethyl acetate = 1:1), the reaction product was extracted with petroleum ether until the lower layer solution was colorless, and the lower layer solution was left to stand for recrystallization to obtain pure 5-iodouridine; The second step, acetylation: under the condition of 0°C, 5-iodo-2 ^, -deoxyuridine or 5-iodouridine reacts with acetic anhydride, the protection reagent of hydroxyl, in a pyridine solvent, and the product is prepared with ethyl acetate and Petroleum ether recrystallization obtains the product of purification; The third step, upper thiophene ring or furan ring: under the protection of argon at 90°C, the product obtained in the second step is mixed with 2-(tributylstannyl)furan or thiophene, bistriphenyl Phosphorus dichloride palladium reaction, thin layer chromatography (TLC) to monitor the reaction (developing agent is the volume ratio of petroleum ether: ethyl acetate = 1:1), the obtained oily product was added to n-hexane for oscillation, and the obtained solid was washed with dichloro Methane: Methanol = 1:1 recrystallization to obtain pure product; The fourth step, sulfur replaces oxygen: the product obtained in the third step is reacted with phosphorus pentasulfide in a dioxane solvent to obtain a product in which 4-position sulfur is substituted for uridine; wherein, the amount of phosphorus pentasulfide is 1~5% of the product obtained in the third step 1.5 times equivalent; The fifth step, deacetylation: inject ammonia gas into the methanol solution to prepare a saturated methanol solution of ammonia, and then add the product obtained in the fourth step into a saturated methanol solution of ammonia for reaction to obtain 5-(2-furyl or thienyl)-4-thiouridine. 4. according to the synthetic method of the described 5-(2-furyl or thienyl)-4-thiouridine and its analogs of claim 3, it is characterized in that, in the third step, 2-(tributylstannane Base) the consumption of furan or thiophene is 4-6 times equivalent of the second step product. 5. according to the synthetic method of the described 5-(2-furyl or thienyl)-4-thiouridine and its analogs of claim 4, it is characterized in that, the method for described the 5th step purification product is, in reducing The solvent was removed under pressure distillation, and the crude product was purified by silica gel chromatography. The eluents were a, dichloromethane b, dichloromethane:methanol=200:1 c, dichloromethane:methanol=50:1, and then in vacuum to remove the solvent.

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