CN102702113A - 5-Fluorouracil derivative and preparation method and uses - Google Patents

Abstract

式1其中R为-COCH₂C₆H₄NO₂-o，或-COCH₂CH₂C₆H₄NO₂-o或-COCH₂CH₂OC₆H₄NO₂-o，或-COCH₂OC₆H₄NO₂-o，或-COC₆H₄NO₂-o。The present invention relates to a class of new organic compounds, uses and preparation methods of such compounds. More precisely, it is a 5-fluorouracil derivative, and the use and preparation method of this compound. The compound 5-fluorouracil derivative of the present invention is shown in the following formula,

Formula 1 wherein R is -COCH ₂ C ₆ H ₄ NO ₂ -o, or -COCH ₂ CH ₂ C ₆ H ₄ NO ₂ -o or -COCH ₂ CH ₂ OC ₆ H ₄ NO ₂ -o, or -COCH _{2 OC6H4NO2 -} o _, or _{-COC6H4NO2 -} o _.

Description

5-Fluorouracil derivatives and their preparation and use

technical field

The present invention relates to a class of new organic compounds, uses and preparation methods of such compounds. More precisely, it is a 5-fluorouracil derivative, and the use and preparation method of this compound. the

Background technique

In 1957, 5-fluorouracil (hereinafter referred to as 5-Fu) was synthesized by Duschinsky et al., and it was first applied clinically by Curreri and Ausfield in the same year. Proliferating cells have a lethal effect. It has played an irreplaceable important role in clinical practice. It is based on the concept of electron isosteres. After replacing the hydrogen atoms in uracil with fluorine atoms, since the atomic radius of fluorine is similar to that of hydrogen, the volume of fluoride is almost equal to that of the original compound, and the C-F bond is particularly stable. It is not easy to decompose during the process, and replaces normal metabolites at the molecular level, so it is a thymine synthase (TS) inhibitor. 5-Fu and its derivatives are first converted into fluorouracil deoxyribonucleotides (FUDRP) in the body, combined with TS, and then interacted with the coenzyme 5,10-methinetetrahydrofolate. Due to the stability of the C-F bond, it cannot be synthesized effectively Thymidine deoxynucleotide (TDRP) inactivates the enzyme (TS), thereby inhibiting DNA synthesis, and finally tumor cell death. the

Although 5-Fu has a good curative effect, its main disadvantages are difficulty in oral absorption, high toxicity and side effects, and low fat solubility, which can cause severe gastrointestinal reactions and bone marrow suppression and other side effects. In order to reduce toxicity and improve curative effect, people have carried out a lot of chemical modification work on 5-Fu and developed a large number of derivatives over the years. According to the structural characteristics of 5-Fu, N in its molecule is the main modification site. There are currently several drugs on the market: ① Tegafur and Difuradin; ② Carmofur; ③ Doxifluridine (Doxifuridine, Doxifluridine, 5'-DUFR). the

5-Fluorouracil is a kind of anti-tumor anti-metabolism drug that is widely used. It is the product of replacing the hydrogen atom in uracil with fluorine atoms, and inhibits the proliferation of cancer cells by interfering with the synthesis of nucleic acids. 5-fluorouracil also has a certain inhibitory effect on normal tissue cell proliferation, so it has many toxic and side effects and its selectivity is low. The strength of the chemical bond between 5-fluorouracil and the carrier is an important factor affecting its anti-tumor activity and toxicity. The weaker the chemical bond is, the higher the anti-tumor activity is, but the corresponding toxicity is also greater. For prior art, please refer to: Duschinsky R, Pleven E, Heidelberger C. The synthesis of 5-fluoropyrimidines. J Am Chem Sci, 1957, 79 (16): 4559-4560. Wang Yanguang, Li Jingxin, etc., fluorouracil spin-labeled derivatives Synthesis and antitumor activity, Chemical Journal of Chinese Universities, 1993, 14:1399. L Caram, M Doeleman, J H Perrin, Synthesis of 1-and 3-Arylcarbonyl Derivatives of 5-Fluorouracil[J], J Heterocyclic Chem 1999, 36, 397-401.Tai-Shun Lin, Lin Wang, (o-and p -Nitrobenzyloxycarbonyl)-5-Fluorouracil Derivatives as Potential Conjugated Bioreductive Alkylating Agents[J], J Med Chem, 1986, 29, 84-9.K Kigasaws, M Hiiragi, K Wakisaka, et al, Studies on the Synthesis of the Synthesis of Chemoics: and Antitumor Activity of N-Acyl-and N-(Alkoxycarbonyl)-5-Fluorouracil Derivatives[J], J Med Chem, 1980, 23, 1324-9.

Contents of the invention

The invention provides a new class of 5-fluorouracil derivatives, which can overcome the shortcomings of the prior art, can improve the selectivity and antitumor activity of 5-fluorouracil, and can better meet the needs of clinical medicine. At the same time, the present invention also provides the use of this compound and its preparation method. the

Compound 5-fluorouracil derivatives of the present invention are shown in formula 1,

Formula 1

wherein R is -COCH ₂ C ₆ H ₄ NO ₂ -o, or -COCH ₂ CH ₂ C ₆ H ₄ NO ₂ -o or -COCH ₂ CH ₂ OC ₆ H ₄ NO ₂ -o, or -COCH ₂ OC ₆ H ₄ NO ₂ -o, or -COC ₆ H ₄ NO ₂ -o.

The preferred preparation method of the 5-fluorouracil derivative of the present invention is to add 10mmol 5-fluorouracil to 10mmol triethylamine and 15ml DMF, slowly add 11mmol of the corresponding aroyl chloride dropwise to the above mixture under stirring and ice-bath conditions, and mix well Finally, remove the ice bath, remove DMF after fully reacting at room temperature, add 30ml ether to the residue and stir until a solid appears, pour off the ether layer, add 30ml ether, stir, filter, wash with water, and dry to obtain the target compound. the

The 5-fluorouracil derivative of the invention can be used in the preparation of antitumor drugs. the

The present invention condenses o-nitrophenylacetic acid and its analogs with the 1-position of 5-FU to passivate 5-FU, so that FUDRP cannot be formed, cannot be incorporated into DNA synthesis pathways, and cannot lead to lethal synthesis. However, the activity of amidohydrolase and reductase in tumor cells is higher than that of normal cells, and the pH value is low, so that the nitro group is preferentially reduced in tumor cells, and amides are highly active amidohydrolase, low pH and the amino group obtained by nitro reduction. Under the neighbor-helping effect, o-nitrophenylacetic acid and its analogs rapidly cyclize into stable five-membered rings or six-membered rings, releasing a sufficient amount of activated 5-FU to produce cytotoxicity in tumor cells, increasing the 5-FU Selectivity to tumor cells; at the same time, the nitro group is not reduced and the amide is directly hydrolyzed to release and activate 5-FU and o-nitrophenylacetic acid and its analogues. The two have synergistic anti-tumor effects, and the activation pathway of 5-FU is also on Tumor cells are selective. the

According to relevant experiments, it can be seen that the antitumor activity of the 5-fluorouracil derivatives of the present invention is obviously better than that of the 5-fluorouracil currently used clinically, and is a very promising antitumor drug; the preparation method reaction conditions provided by the present invention It is mild, the reagent is cheap and has low toxicity, the reactant is easy to handle, and the pure target compound can be obtained without column chromatography, and the product yield is high. the

Description of drawings

Figures 1 to 7 are photos of the signs of the mice tested, wherein: Figures 1 to 5 are pictures of the signs of the mice taking compounds 1 to 5 of the present invention, and Figure 6 is a photo of the signs of the mice taking 5-fluorouracil, Figure 7 is a photo of the signs of the mice in the model group. the

Figure 8 to Figure 14 are the pathological slices of the tumor body, wherein: Figure 8 is the pathological slice of the tumor in the model group, Figure 9 is the pathological slice of the tumor in the 5-Fu group, and Figure 10 is the pathological slice of the tumor in the compound 1 medium dose group Figure 11 is a pathological section of the tumor in the compound 2 medium-dose group, Figure 12 is a pathological section of the tumor in the compound 3 medium-dose group, Figure 13 is a pathological section of the tumor in the compound 4 medium-dose group, and Figure 14 is a pathological section of the tumor in the compound 5 Pathological section diagram of tumor body in dose group. the

Detailed ways

Examples of the invention are provided below. the

Compound preparation method of the present invention is:

Put 5-Fu (10mmol) in a dry reaction flask, add triethylamine (10mmol), DMF15ml and stir, slowly add aroyl chloride (11mmol) to the above mixture in an ice bath, remove the ice bath after 0.5h, and react at room temperature 24h to stop the reaction. Evaporate DMF under reduced pressure at 30°C, add 30ml of diethyl ether to the residue and stir until a solid appears, pour off the diethyl ether layer, add 30ml of diethyl ether, stir, filter, wash with water, and dry to obtain the target compound. The reaction route is shown in formula 2. the

X=--, CH ₂ , CH ₂ CH ₂ , OCH ₂ CH ₂ , OCH ₂

Formula 2

The following is the specific preparation process of the compound of the present invention:

1. Drug preparation

(1) First prepare aromatic acids: o-nitrophenylpropionic acid (C ₉ H ₉ NO ₄ ), o-nitrophenoxyacetic acid (C ₈ H ₇ NO ₅ ) and o-nitrophenoxypropionic acid (C ₉ H ₉ NO ₅ ), or prepared separately as follows:

1. Preparation of o-nitrophenylpropionic acid

Take 39g of malonic acid and 45mL of acetic anhydride in a 150mL Erlenmeyer flask, slowly add 45 drops of H ₂ SO ₄ (concentrated) dropwise under ice bath stirring, add 33mL of acetone after 0.5h, stir until solid appears, and place in the refrigerator for 12h , filtered, and washed with water until the yellow color faded to obtain 43.7 g of white prismatic crystals of isopropylidene malonate, with a yield of 94.9%. (Consistent with the literature: a: Tao Ma, Li Liu, Hai Xue, Li Li, Chun-Yan Han, Lin Wang, Zhi-Wei Chen, Gang Liu, Chemical Library and Structure-Activity Relationships of 11-Demethyl-12-oxo Calanolide A Analogues as Anti-HIV-1 Agents, Journal of Medicinal Chemistry. 51(2008) 1432-1446.

b: Jorge E.Gomez-Galeno, Qun Dang, Thanh H.Nguyen, Serge H.Boyer, Matthew P.Grote, Zhili Sun, Mingwei Chen, A potent and selective AMPK activator that inhibits de novo lipogenesis, ACS Medicinal Chemistry Letters. 1(2010)478-482.)

Put 37g of o-nitrobenzaldehyde, 42g of isopropylidene malonate, 3mL of piperidine, 7.6mL of acetic acid, 400mL of benzene in a 500mL single-necked bottle, install a water separator, heat and stir under reflux for 4 hours, evaporate the benzene, add 100mL of ethanol after cooling , stirred, and filtered to obtain 58 g of isopropyl o-nitrophenylacrylate as a yellow powder solid, with a yield of 87%. mp119-120℃ (consistent with literature: Y.Hu, P.Wei, H.Huang, Z.-G.Le, Z.-C.Chen, Organic reactions in ionic liquids: ionic liquid ethylammonium nitrate-promoted knoevenagel condensation of meldrum's acid with aromatic aldehydes, Synth. Commun. 35 (2005) 23, 2955-2960.). the

58g of isopropyl o-nitrophenyl acrylate was placed in a 500mL single-necked bottle, 270mL of methanol was added, 8.3g of _NaBH4 was added in batches at room temperature, and dilute hydrochloric acid (1:1) was added to the mixture after stirring for 0.5h. Stir until a solid appears, add an appropriate amount of water to the mixture, filter the mixture, and wash with water to obtain 58 g of isopropyl o-nitrophenylmalonate as a yellow powder, with a yield of 99%. mp 118-119°C (consistent with literature Dhevalapally B. Ramachary, Mamillapalli Kishor, Y. Vijayendar Reddy, Development of Pharmaceutical Drugs, Drug Intermediates and Ingredients by Using Direct Organo-Click Reactions, Eur.J.Org.Chem.2008, 975 -993.).

Put 58g of isopropyl o-nitrophenylmalonate in a 500mL single-necked bottle, add 250mL of dilute acetic acid (4:1), reflux for 4 hours, add 30mL of concentrated hydrochloric acid, continue to reflux for 4 hours, cool, add an appropriate amount of water to stir, filter, 34 g of o-nitrophenylpropionic acid was obtained as a pale yellow solid. mp 113-114℃ (consistent with literature Abdol Reza Hajipour, Arnold E. Ruoho, Nitric acid in the presence of P2O5 supported on silica gel-a useful reagent for nitration of aromatic compounds under solvent-free conditions, Tetrahedron 5 Letters (200.46 Letters )8307-8310.). the

2. Preparation of o-nitrophenoxyacetic acid

Weigh 2.28g of NaOH to prepare a 25% solution, put 6.05g of 2-chloroacetic acid in a small beaker, add a small amount of water to dissolve, add the above NaOH solution drop by drop under ice bath, and measure the pH value between 9 and 10 after dropping , to obtain sodium chloroacetate solution for later use. the

Add 5.56g o-nitrophenol to the three-necked flask, add 25% solution containing 1.76g NaOH, KI with 10% chloroacetic acid at the same time, heat the mixture to dissolve, slowly add the above sodium chloroacetate solution dropwise at about 60°C, and reflux for 6h During reflux, check the pH value every 0.5h, adjust the pH value by adding NaOH solution and keep it between 9 and 10, after cooling, acidify with concentrated HCl to pH<1, and a large amount of solids appear, after recrystallization 3.0 g of light yellow needle-like crystals were obtained, with a yield of 38%. mp 156-158°C; ¹ HNMR (TMS, Me ₂ SO-d ₆ , 400MHz); δ: 13.25 (s, 1H, OH), 7.88 (d, 1H, J=8.0Hz, H-3), 7.62 ( t, 1H, J=8.0Hz, H-5), 7.27(d, 1H, J=8.4Hz, H-6), 7.14(t, 1H, J=8.2Hz, H-4), 4.93(s, 2H, -CH ₂ ). ¹³ CNMR δ: 169.37 (CO), 150.44 (C ¹ ), 139.73 (C ⁵ ), 134.12 (C ² ), 124.94 (C ³ ), 121.08 (C ⁴ ), 115.06 (C ⁶ ), 65.28 (-CH ₂ ); IR (KBr) v: 3116 (OH), 3050 (Ph-H), 2989 (CH ₂ ), 1734 (C=O), 1607-1490 (PhC=C), 1253 (Ph—O), 1098 (O—CH ₂ ) cm ⁻¹ .

3. Preparation of o-nitrophenoxypropionic acid

According to the preparation method of o-nitrophenoxyacetic acid, light yellow needle-like crystal o-nitrophenoxypropionic acid was obtained from o-nitrophenol and 3-chloropropionic acid with a yield of 32%. mp 158-159°C; ¹ HNMR (TMS, DMSO-d ₆ , 400MHz) δ: 13.26 (s, 1H, OH), 7.88 (d, 1H, J=6.8Hz, H-3), 7.63 (t, 1H , J=7.2Hz, H-5), 7.27(d, 1H, J=8.0Hz, H-4), 7.14(t, 1H, J=7.6Hz, H-6), 4.93(s, 2H, - CH ₂ CO), 2.09 (s, 2H, OCH ₂ -). ¹³ CNMR δ: 176.24 (CO), 134.08 (C ^2,5 ), 125.60 (C ¹ ), 121.01 (C ^3,4 ), 115.11 (C ⁶ ), 64.97 (Ph OCH ₂ ), 33.97 (PhOCH ₂ CH ₂ ). IR (KBr) v: 3036 (Ph-H), 2933 (PhOCH ₂ -), 2784 (-CH ₂ CO), 1703 (C= O), 1610-1488 (PhC=C), 1255 (Ph-O), 1030 (O-CH ₂ ) cm ^-1 .

(2) Preparation of aroyl chlorides

Add 10 mmol of the aromatic acid obtained as described above into 15 mmol of thionyl chloride, reflux at 50-60°C for 2-3 hours, and then remove excess thionyl chloride under reduced pressure to obtain the corresponding oily aroyl chloride, namely: The yields of o-nitrophenylacetyl chloride, o-nitrophenylpropionyl chloride, o-nitrophenoxypropionyl chloride, o-nitrophenoxyacetyl chloride, and o-nitrobenzoyl chloride are all above 90%. the

(3) Preparation of the target compound

Put 5-fluorouracil (hereinafter referred to as 5-Fu) (10mmol) in a dry reaction bottle, add triethylamine (10mmol), DMF15ml and stir, slowly add 11mmol of the corresponding aroyl chloride to the above mixture in an ice bath, 0.5h Afterwards, the ice bath was removed, and the reaction was stopped at room temperature for 24 hours. Evaporate DMF under reduced pressure at 30°C, add 30ml of diethyl ether to the residue and stir until a solid appears, pour off the diethyl ether layer, add 30ml of diethyl ether and stir, filter, wash with water, and dry to obtain the target compound. The following are the obtained compounds and characterization test data:

The structural formula of compound 1-N ₁ -o-nitrophenylacetyl-5-fluorouracil is as formula 3,

Formula 3

It is light yellow crystal, the yield is 54%, mp (begins to turn black at 160°C, and finally does not melt); R _f =0.41 (CHCl ₃ : EtOAc=4:1), ¹ HNMR (TMS, Me ₂ SO-d ₆ , 400MHz) δ: 12.33(s, 1H, 5-Fu-N ₃ -H), 8.38(d, 1H, J=7.2Hz, 3′-HC ₆ H ₄ NO ₂ -o), 8.16(d, 1H, _JHF =8.0Hz, 5-Fu- _C6 -H), 7.77 ₍ t, 1H, J= _7.4Hz , 5'-HC6H4NO2 _- o), 7.64(d, 1H, J=7.6Hz , 6′-HC ₆ H ₄ NO ₂ -o), 7.59 (t, 1H, J=7.6Hz, 4′-HC ₆ H ₄ NO ₂ -o), 4.8 (s, 2H, -CH ₂ C ₆ H ₄ NO ₂ -o); ¹³ CNMR δ: 169.89 (PhCH ₂ CO), 157.08 (d, J=17.6Hz, 5-Fu-C ⁴ ), 148.36 (5-Fu-C ² ), 142.64 (C ² ′ ), 140.28(5-Fu-C ⁵ ), 134.11(C ⁵ ′), 133.71(C ¹ ′), 129.72(C ⁶ ′), 129.05(C ⁴ ′), 124.89(C ³ ′), 121.44(d , J=37.0Hz, 5-Fu-C ⁶ ), 43.51 (PhCH ₂ ); m/z (%): 293 (0.21), 256 (0.36), 206 (0.37), 171 (0.16), 164 (100 ), 136(50.57), 130(79.73), 92(75.78), 78(48.54); HRMS(ESI): (M+NH ₄ ⁺ )311.0781(calculated 311.0786), error=1.6ppm; IR(KBr)v : 3196(NH), 3149(NH), 3100(CH=CF), 1741-1683(C=O), 1610-1460(PhC=C)cm ^-1 .

The structural formula of compound 2-N ₁ -o-nitrophenylpropionyl-5-fluorouracil is as formula 4,

Formula 4

As white crystals, yield 64%, mp 188-190°C; R _f =0.53 (CHCl _{3 :} EtOAc=4:1); ¹ HNMR (TMS, Me ₂ SO-d ₆ , 400MHz) δ: 12.16(s, 1H, 5-Fu-N ₃ -H), 8.39 (d, 1H, J = 7.6Hz, 3'-HC ₆ H ₄ NO ₂ -o), 7.96 (d, 1H, _JHF = 8.4Hz, 5- Fu-C ₆ -H), 7.69 (t, 1H, J=7.6Hz, 5′-HC ₆ H ₄ NO ₂ -o), 7.59 (d, 1H, J=7.6Hz, 6′-HC ₆ H ₄ NO ₂ -o), 7.51 (t, 1H, J = 7.6 Hz, 4'-HC ₆ H ₄ NO ₂ -o), 3.43 (t, 2H, J = 7.4 Hz, -CH ₂ C ₆ H ₄ NO ₂ -o), 3.16 (t, 2H, J = 7.2 Hz, -CH ₂ CH ₂ C ₆ H ₄ NO ₂ -o); ¹³ CNMRδ: 171.47 (PhCH ₂ CH ₂ CO), 157.18 (d, J = 27.6 Hz , 5-Fu-C ⁴ ), 149.25 (5-Fu-C ² ), 148.33 (C ² ′), 141.23 (d, J=235.5Hz, 5-Fu-C ⁵ ), 134.87 (C ¹ ′), 133.43(d, J=11.0Hz, C ⁵ '), 131.80(C ⁶ '), 127.82(d, J=14.2Hz, C ⁴ '), 124.43(d, J=8.6Hz, C ³ '), 121.60 (d, J=36.2Hz, 5-Fu-C ⁶ ), 34.24 (PhCH ₂ CH ₂ ), 26.97 (d, J=29.9Hz, PhCH ₂ ), m/z (%): 307 (0.09), 256 (0.26), 218(0.55), 185(0.13), 178(100), 150(56.26), 130(25.79), 77(34.79); HRMS(ESI): (M+NH ₄ ⁺ )325.0940(calculated 325.0943 ), error=0.9ppm; IR(KBr)v: 3240(NH), 3095(CH=CF), 1740-1674, (C=O), 1608-1443(PhC=C) cm ^-1 .

The structural formula of compound 3-N ₁ -o-nitrophenoxypropionyl-5-fluorouracil is as formula 6,

Formula 6

It is white powder, yield 73.33%, mp 150-153°C; R _f =0.38 (CHCl _{3 :} EtOAc=4:1), ¹ HNMR (TMS, Me ₂ SO-d ₆ , 400MHz) δ: 11.84(s, 1H, 5-Fu-N ₃ -H), 8.13 (d, 1H, J = 8.0Hz, 3'-HC ₆ H ₄ NO ₂ -o), 8.08 (d, 1H, J _HF = 6.4Hz, 5- Fu-C ₆ -H), 7.82(t, 1H, J=7.8Hz, 5′-HC ₆ H ₄ NO ₂ -o), 7.54(t, 1H, J=7.6Hz, 4′-HC ₆ H ₄ NO ₂ -o), 7.47 (d, 1H, J=8.4Hz, 6′-HC ₆ H ₄ NO ₂ -o), 3.96 (t, 2H, -CH ₂ CH ₂ OC ₆ H ₄ NO ₂ -o) , 3.08 (t, 2H, -CH ₂ OC ₆ H ₄ NO ₂ -o); ¹³ CNMR δ: 169.03 (N ¹ CO), 157.49 (d, J=26.0Hz, 5-Fu-C ⁴ ), 149.59 ( 5-Fu-C ² ), 141.66 (d, J=230.8Hz, 5-Fu-C ⁵ ), 141.42 (C ⁵ ′), 138.22 (C ² ′), 135.59 (C ³ ′), 130.72 (C ⁴ ′), 130.39 (C ⁶ ′), 127.45 (C ¹ ′), 125.43 (d, J=48.1Hz, 5-Fu-C ⁶ ), 43.83 (PhOCH ₂ ), 32.24 (PhOCH ₂ CH ₂ ); HRMS ( ESI): (M+NH ₄ ⁺ ) 341.0896 (calculated 341.0892), error=1.2ppm; IR (KBr) v: 3190 (NH), 3072 (CH=CF), 1768-1660 (C=O), 1603- 1450 (PhC=C) cm ^-1 .

The structural formula of compound 4-N ₁ -o-nitrophenoxyacetyl-5-fluorouracil is as formula 5,

Formula 5

It is white powder, yield 84%, mp 136-140°C; R _f =0.34 (CHCl _{3 :} EtOAc=4:1), ¹ HNMR (TMS, Me ₂ SO-d ₆ , 400MHz) δ: 12.39(s, 1H, 5-Fu-N ₃ -H), 8.41 (d, 1H, J = 5.2Hz, 3'-HC ₆ H ₄ NO ₂ -o), 7.9 (d, 1H, _JHF = 7.6Hz, 5- Fu-C ₆ -H), 7.59 (t, 1H, J=7.8Hz, 5′-HC ₆ H ₄ NO ₂ -o), 7.4 (d, 1H, J=8.4Hz, 6′-HC ₆ H ₄ NO ₂ -o), 7.15(t, 1H, J=7.6Hz, 4′-HC ₆ H ₄ NO ₂ -o), 5.54(s, 2H, -CH ₂ OC ₆ H ₄ NOX-o), ¹³ CNMR δ: 167.81 (PhOCH ₂ CO), 157.03 (d, J=28.4Hz, 5-Fu-C ⁴ ), 150.31 (5-Fu-C ² ), 148.25 (C ¹ ′), 141.47 (d, J=238.7 Hz, 5-Fu-C ⁵ ), 139.70 (C ⁵ ′), 133.99 (C ² ′), 124.88 (C ³ ′), 121.30 (C ⁴ ′), 120.95 (C ⁶ ′), 115.50 (5-Fu -C ⁶ ), 69.91 (PhOCH ₂ ); m/z (%): 309 (0.09), 220 (0.25), 197 (0.23), 171 (53.38), 152 (58.24), 130 (29.07), 122 ( 100), 78 (65.31); HRMS (ESI): (M+NH ₄ ⁺ ) 327.0731 (calculated 327.0735), error=1.2ppm, IR (KBr) v: 3189 (NH), 3147 (NH), 3070 ( CH=CF), 1741-1682 (C=O), 1605-1486 (PhC=C) cm ^-1 .

The structural formula of compound 5-N ₁ -o-nitrobenzoyl-5-fluorouracil is as formula 7,

Formula 7

It is white powder, yield 84%, mp 209-212°C; R _f =0.52 (CHCl _{3 :} EtOAc=4:1), ¹ HNMR (TMS, Me ₂ SO-d ₆ , 400MHz) δ: 12.25(s, 1H, 5-Fu-N ₃ -H), 8.67 (d, 1H, J=6.8Hz, 3′-HC ₆ H ₄ NO ₂ -o), 8.32 (d, 1H, J=8.0Hz, 6′- HC ₆ H ₄ NO ₂ -o), 7.93(t, 1H, J=7.6Hz, 5'-HC ₆ H ₄ NO ₂ -o), 7.78(t, 1H, J=8.0Hz, 4'-HC ₆ H ₄ NO ₂ -o), 7.71 (d, 1H, _JHF = 7.6Hz, 5-Fu-C ₆ -H); ¹³ CNMR δ: 164.90 (PhCO), 157.06 (d, J = 28.3Hz, 5- Fu-C ⁴ ), 147.49 (5-Fu-C ² ), 144.17 (C ² ′), 141.89 (d, J=239.4Hz, 5-Fu-C ⁵ ), 135.18 (C ⁵ ′), 132.45 (C ⁴ ′), 130.81 (C ¹ ′), 127.35 (C ⁶ ′), 24.01 (C ³ ′), 121.18 (d, J=38.6Hz, 5-Fu-C ⁶ ); m/z (%): 279 (0.13), 233 (0.17), 167 (0.49), 150 (100), 76 (66.33); HRMS (ESI): (M+NH ₄ ⁺ ) 297.0629 (calculated 297.0630), error=0.3ppm; IR ( KBr)v: 3197(NH), 3151(NH), 3064(CH=CF), 1729-1688(C=O), 1576-1458(PhC=C)cm ^-1 .

2. Antitumor activity evaluation of 5-fluorouracil derivatives of the present invention

1 In vitro cytotoxicity test

1.1 Test method

The five 5-fluorouracil derivatives synthesized were entrusted to the School of Life Sciences of Lanzhou University to conduct in vitro screening tests for biological activity. The tetrazolium salt reduction method (MTT) was used to treat K562 (human erythrocytic leukemia cell line), MGC-830 (gastric adenocarcinoma cell line) respectively. cell line), A549 (human lung adenocarcinoma cell line) and primary cultured normal human fibroblasts (Fibroblast) for cell viability tests. the

1.2 Drugs, reagents and instruments

Tetramethylazolium salt (MTT) was produced by Sigma; DMEM and trypsin were produced by Gibco; newborn bovine serum (NBS) was produced by the Scientific Research Center of the _Fourth Affiliated Hospital of Hebei Medical University; type; the automatic microplate reader is the Finnish MultiskanMK3 type.

1.3 Cell lines

The selected cells were cultured in DMEM medium containing 10% NBS (containing penicillin 1×10 ⁵ IU.L ^-1 , streptomycin 100 mg.L ^-1 ) in a 37°C incubator containing 5% CO ₂ . 0.25% trypsin digestion and passage. Primary culture of human fibroblasts.

1.4 Cell proliferation inhibition test (MTT method)

The tetrazolium blue (MTT) method is as follows: K562 cells in the logarithmic growth phase were taken and added to a 96-well culture plate, 90 μL/well. The negative control was normal saline, and the positive control was 5-fluorouracil. 10 μL of samples with different concentrations were added to each well, and 4 replicate wells were set up in each concentration adding sample group and control group, and each plate was set up with 12 blank control wells. The final concentration of each compound and positive control 5-fluorouracil was 10 ^-3 mol.L ^-1 , 10 ^-4 mol.L ^-1 , 10 ^-5 mol.L ^-1 , 10 ^-6 mol.L ^-1 , and the cells were kept in CO ₂ After incubating in the incubator for 72 hours, 10 μL/well of MTT was added. Continue culturing for 4 hours, add 100 μL/well of triple solution [10% SDS-5% isobutanol-0.012mol/L HCl (w/v/v)], leave it overnight, and use a microplate reader to measure at dual wavelengths of 570 and 630 nm respectively The OD (absorbance) value of each well was subtracted from the absorbance value of the blank well. The inhibition rate (IR%) of the drug on the in vitro proliferation of each cell line was calculated according to the following formula: IR%=(1-OD _sample /OD _control )×100%. The half-maximum inhibitory concentration (IC ₅₀ ) of the target compound was calculated with SPSS17.0 statistical software package, and the data of three measurements were processed with this statistical software package. The experimental results are shown in Table 1:

Table 1 5-Fu and the half maximal inhibitory concentration (IC ₅₀ ) of each compound

It can be seen from the table that the tested 5-fluorouracil derivatives have very good inhibitory effects on the three tumor cell lines, especially the inhibitory effect on K562 cells is significantly better than that of 5-Fu. The cytotoxic activity of these 5 tested compounds on normal fibroblasts is low, reflecting the difference between the cytotoxic effect on tumor cells and normal cells, but this phenomenon can not be used as a selective indicator of the tested compounds. Judgment basis, because human fibroblasts are only one type of normal human tissue cells. The test of the selectivity of the test compound needs to be further explained by the following acute toxicity test and in vivo anti-tumor activity test. the

2 Acute toxicity test (determination of LD ₅₀ )

2.1 Experimental materials

Kunming mice, weighing 20-25 grams, can be used for both sexes, and were purchased from the Animal Experiment Center of Lanzhou University. The animals were kept in an environment of 20-25° C. with a relative humidity of 35-45%. The animals were fasted 12 hours before the experiment but had free access to water. 1mL syringe, electronic scale, all test compounds were dissolved in physiological saline containing 0.5% sodium carboxymethylcellulose, and administered by intraperitoneal injection. the

2.2 Experimental methods and results

Through the preliminary activity screening, the compounds with effective anti-tumor effects were further tested for activity, and the LD ₅₀ of each compound was determined by the modified Karber method, and the acute toxicity of the synthesized compounds was quantitatively evaluated. The results are shown in Table 2.

Table 2 LD ₅₀ and confidence limits

3 In vivo anti-tumor activity experiment

3.1 Experimental materials

Kunming mice, weighing 20-25 grams, can be used for both sexes, and were purchased from the Animal Experiment Center of Lanzhou University. One week before the experiment, the animals were raised in an environment of 20-25°C, with a relative humidity of 35-45%. Animals were free to forage and drink water except during the experiment. 1mL syringe, electronic scale, containing 5‰ CMC-Na saline, S ₁₈₀ cell line.

3.2 Experimental method

3.2.1 Transplanted tumor strains in mice

Passage of S ₁₈₀ cell line: the resuscitated S ₁₈₀ tumor line was injected into the abdominal cavity of Kunming mice. After 5-7 days, the abdominal circumference of the mice increased to the maximum. 3 times, spare.

3.2.2 Tumor model establishment and group administration

In the ultra-clean workbench, first take 0.1mL of S ₁₈₀ sarcoma liquid and dilute it 20 times with normal saline. Calculate the number of living tumor cells in 1mL of tumor fluid under an optical microscope, and calculate the dilution factor of the tumor fluid, dilute it with normal saline to 2*10 ⁶ cells/0.2ml, store it in ice water, and sterilize the mouse right axillary skin with 75% alcohol , 0.2 mL per mouse was inoculated subcutaneously in the right armpit of the mouse.

24 hours after inoculation, the tested mice were randomly divided into the following 18 groups, with 8 mice in each group (half male and half male), in which: the first group was the blank control group, the second group was the pathological model control group, and the third group It is a 5-FU dose group, and groups 4-18 are respectively high, middle and low dose test groups of compounds 1-5. The doses in Table 2.1 were administered by intraperitoneal injection, wherein: Group 1 (high, medium and low doses) used the compound 1-N ₁ -o-nitrophenylacetyl-5-fluorouracil, Group 2 (high, medium and low doses) ) group used the compound 2-N ₁ -o-nitrophenylpropionyl-5-fluorouracil, and group 3 (high, medium and low doses) used the compound 3-N ₁ -o-nitrophenoxypropionyl-5 -Fluorouracil, group 4 (high, medium and low dose) uses compound 4-N ₁ -o-nitrophenoxyacetyl-5-fluorouracil, group 5 (high, medium and low dose) uses compound 5- N ₁ -o-nitrobenzoyl-5-fluorouracil. The tumor formation rate of the mice inoculated was 100%, and the average tumor weight was 0.34±0.08g. In the experiment, the mice in the administration group were administered once a day for 10 consecutive days, and the mice in the blank control group and the pathological model control group were given 0.6ml of normal saline every day. See Table 1 for the dosage of mice in the test group.

Table 3 compound dosage (mg/Kg/d)

3.2.3 Observation method

After modeling, the local tumor tissue of the mice in each group, the activity, eating, and mental state of the mice were observed. After modeling, mice in each group had local foot tissue swelling, deformed left foot, and had difficulty moving. In severe cases, the left lower limb was swollen and deformed. After administration, the activity of the mice in each group decreased to varying degrees, and the coat color became darker. During the experiment, the mice died due to various reasons, and their biological characteristics are shown in Figures 1 to 7. In the test, it was found that the signs of the test mice taking each compound of the present invention were better than those taking 5-fluorouracil, and the fur state and spirit of the test mice in each group were better than those of the model group and those taking 5-fluorouracil. the

3.2.4 Material collection and pathological film production

On the next day after drug withdrawal, the mice in each group were weighed, blood was collected by eyeball removal, and the thymus and spleen were collected and weighed after death by dislocation of the cervical vertebrae. Strictly peel off the subcutaneous tumor tissue of the right paw pad of the mice in each group, avoid necrosis and hemorrhage, use an electronic scale to weigh the tumor weight (accurate to 0.01g), put it in formalin solution immediately after weighing, and fix it for 12 hours Afterwards, they were routinely dehydrated, transparentized, and embedded in paraffin. Slice with an ultra-thin microtome, 5 μm thick for HE staining and 4 μm thick for immunohistochemical staining, dry naturally, and observe under a microscope, see Figures 8 to 14. the

Observation of pathological sections showed that the sarcoma cells in the model group were closely arranged, with large and deeply stained nuclei, and frequent mitotic phases, indicating that the tumor tissue grew vigorously. The cells in the 5-fluorouracil group were loosely arranged, but there were fewer necrotic cells. In the middle dose group of each drug tested, the cells were sparsely arranged, large areas of necrosis were seen, the cell structure was severely damaged, nuclear pyknosis, fragmentation, and dissolution, vacuoles were seen in the cytoplasm, and the cytoplasm boundary was not obvious. The results showed that, compared with the 5-fluorouracil group, the anti-tumor effect of the middle-dose group of each drug tested was more prominent. the

3.2.5 Determination of immune organ index and tumor inhibition rate

表示，采用t检验；等级资料比较采用秩和检验。  Spleen index and thymus index (mg/10g) were calculated according to the following formulas, respectively. Spleen index=spleen mass (mg)/body mass (g), thymus index=thymus mass (mg)/body mass (g), tumor inhibition rate=(average tumor weight of control group-average tumor weight of drug-administered group)/control The average tumor weight of each group × 100%, the relevant immune organ index and tumor inhibition rate of S ₁₈₀ tumor mice in each group are shown in Table 4. All data were statistically analyzed using SPSS 17.0 statistical software package, and measurement data were expressed as mean ± standard deviation

Indicates that the t test is used; the rank sum test is used for the comparison of rank data.

Table 4 The relevant immune organ index mg/g of each group and tumor inhibition rate

Compared with the test drug group and the pathological model control group, *P<0.05, **P<0.01, ***P<0.001

Experimental results show that the tumor weight of mice in the compound group of the present invention is significantly smaller than that in the blank group, indicating that these compounds have a positive effect on inhibiting tumor size, and it will be beneficial to treat tumors clinically. It can be seen from the table that the high, medium and low dose groups of each compound can improve the thymus index, spleen index and white blood cell count of _S180 tumor mice. Compared with the pathological model control group, the difference is statistically significant (*P<0.05 , **P<0.01, ***P<0.001). In addition, the tumor inhibition rates of the tested compounds in each group were higher than those of the 5-FU group. In particular, compounds 3 and 4 also showed better antitumor activity than 5-Fu at lower doses than 5-Fu (the dose of 5-Fu was equivalent to 1/7 LD ₅₀ , and the doses of compounds 3 and 4 were equivalent In 1/9 or 1/12LD ₅₀ ), it shows that the compounds of the present invention have better tumor inhibitory effect than 5-FU and their selectivity is greatly improved than 5-Fu, that is, their safety index is higher than 5-FU Fu.

5 Determination of fat-water partition coefficient

Using n-octanol-water as a simulated system, the lipid-water partition coefficient of each compound was determined by shake flask method-UV spectrophotometry. The formula is expressed as follows: LgP=Lg(Co/Cw). where Co and Cw denote the equilibrium concentration of the compound in the non-aqueous phase and its neutral form in the aqueous phase, respectively. The results are shown in Table 4. the

Table 5 Compound lipid-water partition coefficient LgP (n-octanol-water)

It can be seen from the table that the fat solubility of compounds 3 and 5 of the present invention has been greatly improved compared with 5-Fu. If they can be used as clinical medicine, their physical and chemical properties can meet the requirements of clinical needs. the

In summary: the anti-tumor effect of the new compound is better than that of 5-fluorouracil and its selectivity is higher than that of 5-fluorouracil; the new compound can significantly improve the thymus index, spleen index and white blood cell count of the S180 transplanted mouse model, and its effect is better than that of 5-fluorouracil. 5-fluorouracil. In addition, the lipid solubility of compounds 3 and 5 has been greatly improved compared with 5-Fu. Therefore, the new compound can better meet the needs of clinical medicine. the

Claims (4)

1. 5-fluorouracil derivatives as shown in formula 1,

Formula 1 wherein R is -COCH ₂ C ₆ H ₄ NO ₂ -o, or -COCH ₂ CH ₂ C ₆ H ₄ NO ₂ -o, or -COCH ₂ CH ₂ OC ₆ H ₄ NO ₂ -o, or -COCH ₂ OC ₆ H ₄ NO ₂ -o, or -COC ₆ H ₄ NO ₂ -o. 2. the preparation method of the described 5-fluorouracil derivative of claim 1 is characterized in that 5-fluorouracil is added in the mixed solution of triethylamine and DMF, stirs, and under ice-bath condition, corresponding aroyl chloride is slowly added dropwise to In the mixture, stir well and react fully at room temperature, remove DMF, add diethyl ether to the residue and stir until a solid appears, remove the diethyl ether layer, add diethyl ether again and stir, then filter, wash with water, and dry to obtain the target compound. 3. the preparation method of the described 5-fluorouracil derivative of claim 2 is characterized in that 10mmol 5-fluorouracil is added in 10mmol triethylamine and 15ml DMF, under stirring and ice bath condition, corresponding aroyl chloride 11mmol is slowly added dropwise to above-mentioned In the mixture, remove the ice bath after mixing evenly, remove DMF after fully reacting at room temperature, add 30ml ether to the residue and stir until a solid appears, pour off the ether layer, add 30ml ether and stir, filter, wash with water, and dry to obtain the target compound . 4. The application of the 5-fluorouracil derivative as claimed in claim 1 in the preparation of antitumor drugs.

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