CN114591214A - Isatin derivative and preparation method thereof - Google Patents

Isatin derivative and preparation method thereof Download PDF

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CN114591214A
CN114591214A CN202210306601.XA CN202210306601A CN114591214A CN 114591214 A CN114591214 A CN 114591214A CN 202210306601 A CN202210306601 A CN 202210306601A CN 114591214 A CN114591214 A CN 114591214A
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isatin
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王庆文
陈健
林献
陶成
李鑫
张楚楚
邓永星
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Peking University Shenzhen Hospital
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Abstract

The invention provides isatin derivatives CT5-1, CT5-2, CT5-3, CT5-4 and CT5-5 and a preparation method thereof. Starting from commercially available isatin, magnolol and honokiol fragments are respectively introduced through an N-alkylation reaction, synthesis of an isatin-magnolol splicer CT5-1 and synthesis of an isatin-honokiol splicer CT5-2 are achieved, starting from commercially available compound 3, a phthalimide fragment is introduced through an S-alkylation reaction, an isatin-phthalimide splicer CT5-3 can be efficiently obtained, and then the oxidation state of a sulfur atom is adjusted by using m-chloroperoxybenzoic acid conditions to respectively obtain CT5-4 and CT 5-5. The isatin derivatives CT5-1, CT5-2, CT5-3, CT5-4 and CT5-5 are not reported in the prior art; experiments prove that the compound CT5-2 not only has novel skeleton, but also has the in vitro activity of resisting the rheumatoid arthritis obviously superior to that of a positive control medicament methotrexate, and the in vivo activity is equivalent to that of the methotrexate.

Description

Isatin derivative and preparation method thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an isatin derivative and a preparation method thereof.
Background
Rheumatoid Arthritis (RA) is an autoimmune disease with high morbidity, complex etiology and high disability rate, and is characterized by joint synovial thickening, inflammatory cell infiltration, pannus formation and joint cartilage and bone destruction (Nat Rev Rheumatotol, 2020,16(6): 316-333.). The etiology and pathogenesis of RA are complex, and in RA synovial membrane tissue, synovial Fibroblast (FLS) are the main effector cells of synovial membrane hyperplasia, and have the biological properties of tumor cells such as abnormal proliferation, migration, adhesion, invasion and the like. FLS plays a key role in pannus formation by migrating and invading bone and cartilage, and plays an important role in the pathological process of RA (Nat Rev Rheumatotol, 2020,16(6): 316-. More and more studies have shown that promotion of apoptosis of FLS, or inhibition of proliferation, migration and invasion of FLS, is effective in preventing the occurrence and development of RA (Immunol rev.2010,233(1): 233-55.). Methotrexate (MTX) acts as a first-line drug for the treatment of RA patients by inhibiting DNA biosynthesis and FLS proliferation (Br J Clin Pharmacol,2019,85(10): 2228-2234.).
In recent years, the development of modern medicine is promoted by the research of new drugs using natural products as lead compounds, and the proportion of the drugs related to natural products among various approved drugs on the market is also increasing. Therefore, modification of natural products by means of pharmaceutical chemistry research means is very important for new drug research.
Isatin (indoline-2, 3-dione, Isatin) and its derivatives are an important class of nitrogen heterocyclic compounds with a wide range of biological activities, including anti-inflammatory and anti-tumor activities (Guangdong chemical industry 2015,14(42):106, 113.; Pharmacol Rep.2013; 65(2): 313-35). Although research on isatin has been increasing at home and abroad in recent years, isatin is also modified into derivatives by scholars to enhance its activity, there is still a need to develop more isatin derivatives to meet the needs of basic research and clinical transformation.
Disclosure of Invention
The invention provides isatin derivatives CT5-1, CT5-2, CT5-3, CT5-4 and CT5-5 and a preparation method thereof. Starting from commercially available isatin, magnolol and honokiol fragments are respectively introduced through an N-alkylation reaction, synthesis of an isatin-magnolol splicer CT5-1 and synthesis of an isatin-honokiol splicer CT5-2 are achieved, starting from commercially available compound 3, a phthalimide fragment is introduced through an S-alkylation reaction, an isatin-phthalimide splicer CT5-3 can be efficiently obtained, and then the oxidation state of a sulfur atom is adjusted by using m-chloroperoxybenzoic acid conditions to respectively obtain CT5-4 and CT 5-5.
Wherein the isatin derivative CT5-1 has the following structure:
Figure BDA0003565540370000021
CT5-1 can be prepared by the following method:
dissolving isatin in an N, N-dimethylformamide solvent, carrying out N-alkylation reaction with a compound 1 under the condition of potassium carbonate, introducing a magnolol fragment, and then removing phenolic hydroxyl protection under the condition of tetrabutylammonium fluoride/tetrahydrofuran to obtain CT 5-1;
Figure BDA0003565540370000022
the reaction temperature is 60 ℃, the reaction time is 12 hours, the volume molar ratio of the N, N-dimethylformamide to the isatin is 10:1, and the molar ratio of the isatin to the compound 1 to the potassium carbonate is 1: 2: 2; the volume molar ratio of the tetrahydrofuran to the isatin is 10:1, the molar ratio of tetrabutylammonium fluoride to isatin is 1: 1.
isatin derivative CT5-2 has the following structure:
Figure BDA0003565540370000023
CT5-2 can be prepared by the following method:
dissolving isatin in an N, N-dimethylformamide solvent, and carrying out N-alkylation reaction with a compound 2 under the condition of potassium carbonate to obtain CT 5-2;
Figure BDA0003565540370000031
the reaction temperature is 60 ℃, the reaction time is 24 hours, the volume molar ratio of the N, N-dimethylformamide to the isatin is 10:1, and the molar ratio of the isatin to the compound 2 to the potassium carbonate is 4: 1: 2.
isatin derivative CT5-3 has the following structure:
Figure BDA0003565540370000032
CT5-3 can be prepared by the following method:
dissolving the bromo-derivative compound 3 of isatin in tetrahydrofuran, and carrying out S-alkylation reaction with the compound 4 under the condition of sodium hydride to obtain CT 5-3;
Figure BDA0003565540370000041
the reaction temperature is 25 ℃, the reaction time is 1 hour, the volume molar ratio of tetrahydrofuran to isatin is 10:1, the molar ratio of sodium hydride to compound 4 to isatin is 2: 2: 1.
isatin derivatives CT5-4 and CT5-5 have the following structures:
Figure BDA0003565540370000042
CT5-4 and CT5-5 can be prepared by the following methods:
dissolving isatin derivative CT5-3 in dichloromethane solvent, controlling the temperature to be 0 ℃ to react with m-chloroperoxybenzoic acid, adding saturated sodium bicarbonate aqueous solution and saturated sodium sulfite aqueous solution after the reaction is finished, and extracting, concentrating and separating to obtain CT5-4 and CT 5-5;
Figure BDA0003565540370000051
the volume molar ratio of the dichloromethane solvent to the isatin derivative CT5-3 is 10:1, and the molar ratio of the m-chloroperoxybenzoic acid to the isatin derivative CT5-3 is 1: 1.
The invention has the following beneficial technical effects:
the isatin derivatives CT5-1, CT5-2, CT5-3, CT5-4 and CT5-5 are not reported in the prior art; experiments prove that the compound CT5-2 not only has novel skeleton, but also has the in vitro activity of resisting the rheumatoid arthritis obviously superior to that of a positive control medicament methotrexate, and the in vivo activity is equivalent to that of the methotrexate.
Description of the drawings:
FIG. 1 Effect of CT5-2 on arthritis in CIA mice
FIG. 2 Effect of CT5-2 on the arthritis score of CIA mice
The specific implementation mode is as follows:
example 1 preparation of CT5-1
Dissolving 10mmol of isatin in 100mLN, N-dimethylformamide, adding 20mmol of potassium carbonate and 20mmol of compound 1 into the system, reacting at 60 ℃ for 12 hours, adding 50mL of water into the system, extracting with ethyl acetate (50mL of 3), combining the organic phases, drying with sodium sulfate, concentrating, separating the concentrated organic phase with a silica gel column, concentrating, adding 100mL of tetrahydrofuran and 10mmol of tetrabutylammonium fluoride, reacting at 25 ℃ for 15 minutes, adding 50mL of water into the system, extracting with ethyl acetate (50mL of 3), combining the organic phases, drying with sodium sulfate, concentrating, separating the concentrated organic phase with a silica gel column to obtain CT5-1(8mmol, 3.5g), and obtaining a light yellow liquid with a yield of 80%;
1H NMR(500MHz,Chloroform-d)δ7.50(dd,J＝7.4,1.3Hz,1H),7.23–7.12(m,2H),7.11–7.05(m,2H),6.98(t,J＝7.5Hz,1H),6.92(dd,J＝5.4,3.1Hz,2H),6.82(d,J＝8.2Hz,1H),6.57(d,J＝8.0Hz,1H),6.05–5.80(m,2H),5.48(s,1H),5.15–4.91(m,4H),4.26(t,J＝4.9Hz,2H),4.02(t,J＝4.9Hz,2H),3.33(dd,J＝14.1,6.7Hz,4H).
13C NMR(126MHz,CDCl3)δ182.88,158.50,153.18,151.45,150.83,138.40,137.67,137.12,134.59,132.58,132.11,130.92,129.48,129.29,126.80,125.38,125.01,123.37,117.36,116.37,115.98,115.55,113.25,110.61,67.40,40.14,39.36,39.23,29.66
HRMS(ESI):m/z calcd for C28H25NNaO4[M+Na]+:462.1681,found:462.1675
HPLC purity 99.1%.
Example 2 preparation of CT5-2
Dissolving 40mmol of isatin in 100mLN, N-dimethylformamide, adding 20mmol of potassium carbonate and 10mmol of compound 2 into the system, reacting at 60 ℃ for 24 hours, adding 50mL of water into the system, extracting with ethyl acetate (50mL of 3), combining organic phases, drying with sodium sulfate, concentrating, and separating the concentrated organic phase with a silica gel column to obtain CT5-2(9mmol, 5.4g) as a light yellow liquid with a yield of 88%;
1H NMR(400MHz,Chloroform-d)δ7.65–7.53(m,2H),7.40(dd,J＝7.5,1.3Hz,1H),7.19–7.07(m,5H),7.04(dd,J＝8.3,2.3Hz,1H),6.98(d,J＝2.3Hz,1H),6.94–6.81(m,2H),6.72(d,J＝8.4Hz,1H),6.60(d,J＝8.0Hz,1H),5.98–5.84(m,1H),5.84–5.69(m,1H),5.10–4.96(m,2H),4.96–4.77(m,2H),4.31(t,J＝5.1Hz,2H),4.21(t,J＝5.1Hz,2H),4.13(t,J＝5.1Hz,2H),3.95(t,J＝5.1Hz,2H),3.30(d,J＝6.7Hz,2H),3.16(d,J＝6.3Hz,2H).
13C NMR(101MHz,CDCl3)δ183.05,182.91,158.34,158.19,154.72,153.28,151.13,150.81,138.23,137.96,137.31,136.40,133.40,131.29,131.01,128.24,128.08,127.73,125.13,124.73,123.67,123.24,117.39,117.16,115.57,115.24,113.49,110.81,110.42,66.56,65.25,40.05,39.92,39.16,34.09,29.52.
HRMS(ESI):m/z calcd for C38H32N2NaO6[M+Na]+:635.2158,found:635.2152
HPLC purity 98.5%.
Example 3 preparation of CT5-3
Dissolving 10mmol of compound 3 in 100mL of tetrahydrofuran, adding 20mmol of sodium hydride and 20mmol of compound 4 into the system at 0 ℃, reacting for 1 hour at 25 ℃, adding 50mL of water into the system, extracting with ethyl acetate (50mL of 3), combining organic phases, drying with sodium sulfate, concentrating, and finally separating the concentrated organic phase with a silica gel column to obtain CT5-3(8mmol, 3.3g) as a light yellow liquid with the yield of 82%;
1H NMR(400MHz,Chloroform-d)δ7.85(dd,J＝5.5,3.1Hz,2H),7.73(dd,J＝5.4,3.1Hz,2H),7.68–7.56(m,2H),7.24–7.02(m,1H),7.05–6.78(m,1H),4.01–3.88(m,2H),3.71(t,J＝7.0Hz,2H),2.83(t,J＝7.2Hz,2H),2.67(t,J＝7.2Hz,2H),1.81(ddd,J＝8.0,6.6,1.2Hz,2H),1.70–1.56(m,2H).
13C NMR(125MHz,Chloroform-d)δ185.76,168.22,161.27,147.44,134.94,132.95,131.85,128.92,123.56,123.34,122.00,113.89,42.78,38.32,31.87,31.75,27.00,26.54.
HRMS(ESI):m/z calcd for C22H20N2NaO4S[M+Na]+:431.1041,found:431.0907
HPLC purity 98.1%.
Example 4 preparation of CT5-4 and CT5-5
Dissolving 10mmol of CT5-3 in 100mL of dichloromethane, and slowly adding 10mmol of m-chloroperoxybenzoic acid in dichloromethane (10mL) at 0 ℃; reacting at 0 ℃ for 20 minutes, adding 50mL of saturated aqueous sodium bicarbonate solution and 50mL of saturated aqueous sodium sulfite solution into the system, extracting with dichloromethane (50mL × 3), combining the organic phases, drying with sodium sulfate, concentrating, and separating the concentrated organic phase with a silica gel column to obtain CT5-4(6mmol, 2.5g) and a light yellow liquid with a yield of 60%; CT5-5(4mmol, 1.8g), light yellow liquid, yield 40%;
CT5-4：
1H NMR(500MHz,Chloroform-d)δ7.81(dd,J＝5.7,3.2Hz,2H),7.74–7.65(m,3H),7.60(ddd,J＝7.9,6.9,1.2Hz,1H),7.48(dd,J＝7.9,1.2Hz,1H),7.19(ddd,J＝8.0,6.9,1.3Hz,1H),4.34(t,J＝5.8Hz,2H),3.78–3.62(m,2H),2.97(t,J＝5.9Hz,2H),2.63(t,J＝5.9Hz,2H),1.91–1.82(m,2H),1.78(pd,J＝6.3,1.0Hz,2H).
13C NMR(125MHz,Chloroform-d)δ185.77,168.22,161.44,148.71,134.83,132.95,131.83,128.92,123.60,123.20,121.98,113.84,53.12,52.63,38.55,38.24,26.46,19.75.
HRMS(ESI):m/z calcd for C22H20N2NaO5S[M+Na]+:447.0991,found:447.0988
HPLC purity 99.1%.
CT5-5：
1H NMR(500MHz,Chloroform-d)δ7.82(dd,J＝5.8,3.3Hz,2H),7.75–7.65(m,3H),7.57(ddd,J＝8.1,6.9,1.3Hz,1H),7.47(dd,J＝7.9,1.3Hz,1H),7.12(ddd,J＝8.0,6.9,1.3Hz,1H),4.43(t,J＝6.2Hz,2H),3.79–3.63(m,2H),3.40(t,J＝6.1Hz,2H),3.14(t,J＝6.1Hz,2H),1.87–1.78(m,2H),1.77–1.68(m,2H).
13C NMR(125MHz,Chloroform-d)δ185.79,168.23,160.79,147.68,134.66,132.95,131.83,128.94,123.64,123.18,121.98,113.87,55.06,53.79,38.46,38.21,26.21,21.64.
HRMS(ESI):m/z calcd for C22H20N2NaO6S[M+Na]+:463.0940,found:463.0936
HPLC purity 99.3%.
Experimental examples 1 Effect of CT5-1, CT5-2, CT5-3, CT5-4 and CT5-5 on proliferation of Primary Rheumatoid arthritis synovial fibroblasts (RA-FLS)
Purpose of the experiment: study on the effects of CT5-1, CT5-2, CT5-3, CT5-4 and CT5-5 on the proliferation of primary RA-FLS
1.1. Experimental Material
1.1.1 cells: primary RA-FLS
1.1.2 reagents and instruments: DMEM medium (Gibco), fetal bovine serum (FBS, BI), 0.25% trypsin (Gibco), phosphate buffered saline (PBS, Hyclone), DMSO (Sigma), CCK8 kit (APExBIO), multifunctional microplate reader (Synergy H1, BioTek), inverted microscope (Leica), ultra clean bench (singapore technologies ltd), thermostatted water bath (shanghai seminal macro experimental facilities ltd), low speed centrifuge (hunan chemometrics instruments ltd), cell culture chamber (singapore technologies ltd).
1.2. Experimental methods
1.2.1 culture of RA-FLS cells to logarithmic growth phase, digestion of cells with 0.25% trypsin, centrifugation at 1000rpm for 5 minutes, resuspension of the cell pellet in 10% FBS-containing DMEM, inoculation of cells in a 96-well plate at a density of 5000 cells/well in a volume of 100. mu.L, then placing in an incubator at 37 ℃ and 5% CO2And culturing overnight under saturated humidity conditions.
1.2.2 multiple wells were set for each group, and each well was dosed with the following conditions, followed by further incubation for 24 hours.
Control group: namely, no medicine group is added.
Positive control group: positive control groups were divided into 3 groups:
positive control group 1: methotrexate was added at a final concentration of 5 μ M.
Positive control group 2: methotrexate was added to a final concentration of 10. mu.M.
Positive control group 3: methotrexate was added to a final concentration of 20 μ M.
CT5-1 group: the CT5-1 components were divided into 3 groups:
CT5-1 group 1: CT5-1 was added to a final concentration of 5. mu.M.
CT5-1 group 2: CT5-1 was added at a final concentration of 10. mu.M.
CT5-1 group 3: CT5-1 was added to a final concentration of 20. mu.M.
CT5-2 group: the CT5-2 was divided into 3 groups:
CT5-2 group 1: CT5-2 was added to a final concentration of 5. mu.M.
CT5-2 group 2: CT5-2 was added to a final concentration of 10. mu.M.
CT5-2 group 3: CT5-2 was added to a final concentration of 20. mu.M.
CT5-3 group: the CT5-3 components were divided into 3 groups:
CT5-3 group 1: CT5-3 was added to a final concentration of 5. mu.M.
CT5-3 group 2: CT5-3 was added to a final concentration of 10. mu.M.
CT5-3 group 3: CT5-3 was added to a final concentration of 20. mu.M.
CT5-4 group: the CT5-4 components were divided into 3 groups:
CT5-4 group 1: CT5-4 was added to a final concentration of 5. mu.M.
CT5-4 group 2: CT5-4 was added to a final concentration of 10. mu.M.
CT5-4 group 3: CT5-4 was added to a final concentration of 20. mu.M.
CT5-5 group: the CT5-5 components were divided into 3 groups:
CT5-5 group 1: CT5-5 was added to a final concentration of 5. mu.M.
CT5-5 group 2: CT5-5 was added to a final concentration of 10. mu.M.
CT5-5 group 3: CT5-5 was added to a final concentration of 20. mu.M.
After the cells were cultured for 24 hours, the effect of the drug on the proliferation of RA-FLS cells was examined using CCK8 kit. The specific operation is as follows: 10. mu.L of CCK8 was added to each well, and after further culturing for 2 hours, the absorbance (OD) at a wavelength of 450nm was measured with a microplate reader, and the inhibition rate of cell proliferation of each group was calculated with the inhibition rate of cell proliferation of the control group being 0%.
1.3 results of the experiment
TABLE 1 Effect of various novel Compounds on proliferation of Primary RA-FLS
Figure BDA0003565540370000091
Figure BDA0003565540370000101
As shown in Table 1, CT5-2 can obviously inhibit the proliferation of primary RA-FLS, and the proliferation inhibition effect is dose-dependent and is increased with the increase of the dose; and the effect of inhibiting proliferation is obviously better than that of positive control drug methotrexate.
Experimental examples 2 Effect of CT5-1, CT5-2, CT5-3, CT5-4 and CT5-5 on proliferation of synovial fibroblasts MH7A in rheumatoid arthritis
Purpose of the experiment: study on the effects of CT5-1, CT5-2, CT5-3, CT5-4 and CT5-5 on MH7A proliferation
2.1 materials of the experiment
2.1.1 cells: MH7A
2.1.2 reagents and instruments: DMEM medium (Gibco), fetal bovine serum (FBS, BI), 0.25% trypsin (Gibco), phosphate buffered saline (PBS, Hyclone), DMSO (Sigma), CCK8 kit (APExBIO), multifunctional microplate reader (Synergy H1, BioTek), inverted microscope (Leica), ultra clean bench (singapore technologies ltd), thermostatted water bath (shanghai seminal macro laboratories ltd), low speed centrifuge (hunan chemostat instruments ltd), cell culture chamber (singapore technologies ltd).
2.2. Experimental methods
2.2.1 culture MH7A cells to logarithmic growth phase, digestion of the cells with 0.25% trypsin, centrifugation at 1000rpm for 5 minutes, resuspension of the cell pellet in DMEM medium containing 10% FBS, inoculation of the cells in a 96 well plate at a density of 5000 μ L/well and a volume of 100 μ L, followed by placement in an incubator at 37 ℃ with 5% CO2And culturing overnight under saturated humidity conditions.
2.2.2 multiple wells of 6 per group, each well was dosed as follows, and then culture was continued for 24 hours.
Control group: namely, no medicine group is added.
Positive control group: positive control groups were divided into 3 groups:
positive control group 1: methotrexate was added at a final concentration of 5. mu.M.
Positive control group 2: methotrexate was added to a final concentration of 10. mu.M.
Positive control group 3: methotrexate was added to a final concentration of 20 μ M.
CT5-1 group: the CT5-1 component was divided into 3 groups:
CT5-1 group 1: CT5-1 was added to a final concentration of 5. mu.M.
CT5-1 group 2: CT5-1 was added to a final concentration of 10. mu.M.
CT5-1 group 3: CT5-1 was added to a final concentration of 20. mu.M.
CT5-2 group: the CT5-2 component was divided into 3 groups:
CT5-2 group 1: CT5-2 was added to a final concentration of 5. mu.M.
CT5-2 group 2: CT5-2 was added at a final concentration of 10. mu.M.
CT5-2 group 3: CT5-2 was added to a final concentration of 20. mu.M.
CT5-3 group: the CT5-3 components were divided into 3 groups:
CT5-3 group 1: CT5-3 was added to a final concentration of 5. mu.M.
CT5-3 group 2: CT5-3 was added to a final concentration of 10. mu.M.
CT5-3 group 3: CT5-3 was added to a final concentration of 20. mu.M.
CT5-4 group: the CT5-4 components were divided into 3 groups:
CT5-4 group 1: CT5-4 was added to a final concentration of 5. mu.M.
CT5-4 group 2: CT5-4 was added to a final concentration of 10. mu.M.
CT5-4 group 3: CT5-4 was added to a final concentration of 20. mu.M.
CT5-5 group: the CT5-5 components were divided into 3 groups:
CT5-5 group 1: CT5-5 was added to a final concentration of 5. mu.M.
CT5-5 group 2: CT5-5 was added to a final concentration of 10. mu.M.
CT5-5 group 3: CT5-5 was added to a final concentration of 20. mu.M.
After the above cells were cultured for further 24 hours, the effect of the drug on the proliferation of MH7A cells was examined using the CCK8 kit. The specific operation is as follows: 10. mu.L of CCK8 was added to each well, and after further culturing for 2 hours, the absorbance (OD) at a wavelength of 450nm was measured with a microplate reader, and the inhibition rate of cell proliferation of each group was calculated with the inhibition rate of cell proliferation of the control group being 0%.
2.3 results of the experiment
Table 2 effect of each of the novel compounds on MH7A proliferation
Figure BDA0003565540370000111
Figure BDA0003565540370000121
As shown in table 2, CT5-2 can significantly inhibit the proliferation of MH7A, and the proliferation inhibition effect is dose-dependent and increases progressively with increasing dose; and the effect of inhibiting proliferation is obviously better than that of positive control drug methotrexate.
Experimental example 3 Effect of CT5-2 on collagen-induced rheumatoid arthritis (CIA) mouse model
Purpose of the experiment: study of the therapeutic Effect of CT5-2 on the CIA mouse model
3.1. Laboratory animal
SPF grade DBA/1 mice, male, 6-8 weeks old.
3.2. Experimental methods
Mice were randomly divided into 4 groups, which were: a control group, a CIA model group, a positive control group and a CT5-2 group. Establishing a CIA model and administering: the DBA/1 mouse CIA model was induced using bovine type II collagen (C II). The specific method comprises the following steps: dissolving 2mg/mL bovine type II collagen in 0.1mol/L acetic acid solution, mixing with 1mg/mL M.tuberculosis-containing complete Freund's adjuvant according to the volume of 1:1, and emulsifying and uniformly mixing on an ice-water bath by using a tissue homogenizer; selecting a male DBA/1 mouse with the age of 6-8 weeks, carrying out primary immunization on the 0 th day, and injecting 0.1mL of emulsifier subcutaneously at a position 2 cm away from the tail of the mouse for sensitization; thirdly, strengthening immunity on day 21, mixing 2mg/mL bovine II type collagen solution with incomplete Freund's adjuvant according to the volume of 1:1, emulsifying and uniformly mixing the mixture on an ice-water bath by using a tissue homogenizer, and injecting 0.1mL emulsifier subcutaneously at a position 0.5 cm away from the tail of a mouse; and fourthly, continuously observing the morbidity of the joints of the four limbs of the mouse. Starting from the day of secondary immunization (day 21), the mice in the group of the CIA model were intraperitoneally injected with the corresponding solvent, the mice in the positive control group were intraperitoneally injected with 20mg/kg of methotrexate, and the mice in the CT5-2 group were intraperitoneally injected with 20mg/kg of CT5-2, and the four limb joint morbidity of the mice in each group was observed. The degree of arthritis lesions of the four limbs of the mice was scored according to the following criteria, and the therapeutic effect of CT5-2 on the arthritis of the CIA mouse model was evaluated: normal score 0, mild redness and swelling score 1, moderate redness and swelling score 2, severe swelling score 3, and joint deformity and stiffness score 4. The composite score for each mouse was the sum of the scores of 4 joints, with the highest score of 16.
3.3. Results of the experiment
In vivo results show that CT5-2 can significantly reduce the arthritis in CIA mice (figure 1), reduce the arthritis score (figure 2, compared with the CIA model group,**p < 0.01) and the therapeutic effect is equivalent to that of methotrexate which is a first-line clinical drug (positive control drug).
According to the pharmacological and pharmacodynamic experiments, the in vitro activity of the prepared CT5-2 for resisting rheumatoid arthritis is obviously superior to that of methotrexate, the in vivo activity is equivalent to that of the methotrexate, the skeleton is novel, the synthetic method is innovative, and the compound is an anti-rheumatoid arthritis compound with development potential and can be directly used for treating related diseases and preparing related medicines.

Claims (19)

1. A method for preparing isatin derivative. The method is characterized by comprising the following steps:
the isatin is dissolved in N, N-dimethylformamide solvent, and undergoes N-alkylation reaction with another compound under the action of potassium carbonate to obtain one of isatin derivatives CT5-1 and CT 5-2.
2. The method for producing an isatin derivative according to claim 1, wherein the other compound is one of compound 1 and compound 2, and the compound 1 has the following structure:
Figure FDA0003565540360000011
the compound 2 has the following structure:
Figure FDA0003565540360000012
3. the method for producing an isatin derivative according to claim 2, wherein when the other compound is compound 1, the production method further includes a deprotection step.
4. The method for producing isatin derivative according to claim 3, wherein the deprotection step is performed under the conditions of tetrabutylammonium fluoride and tetrahydrofuran.
5. The method for preparing isatin derivatives according to claim 2, wherein the isatin derivative obtained by N-alkylation with the addition of compound 1 is CT5-1, and CT5-1 has the following structure:
Figure FDA0003565540360000013
6. the method for preparing isatin derivative according to claim 2, wherein the isatin derivative obtained by N-alkylation with the addition of compound 2 is CT5-2, CT5-2 has the following structure:
Figure FDA0003565540360000021
7. the process for preparing isatin derivative according to claim 1, wherein the molar volume ratio of isatin to N, N-dimethylformamide is 1: 10.
8. the method for preparing isatin derivative according to claim 2, wherein when the other compound is compound 1, the molar ratio of isatin, potassium carbonate and compound 1 is 1: 2: 2; when the other compound is compound 2, the molar ratio of isatin, potassium carbonate to compound 2 is 4: 2: 1.
9. the method for preparing isatin derivative according to claim 1, wherein the reaction temperature of the N-alkylation reaction is 60 ℃.
10. The method for preparing isatin derivative according to claim 4, wherein the molar volume ratio of isatin to tetrahydrofuran is 1: 10, the mol ratio of isatin to tetrabutylammonium fluoride is 1: 1.
11. an isatin derivative CT5-1 having the formula:
Figure FDA0003565540360000022
12. an isatin derivative CT5-2 having the formula:
Figure FDA0003565540360000031
13. a method for preparing isatin derivative. The method is characterized by comprising the following steps:
dissolving a bromo-derivative compound 3 of isatin in a tetrahydrofuran solvent, and carrying out S-alkylation reaction with a compound 4 under the action of sodium hydride to obtain an isatin derivative CT 5-3;
the bromo-derivative compound 3 of isatin has the following structure:
Figure FDA0003565540360000032
the compound 4 has the following structure:
Figure FDA0003565540360000033
14. the method for producing isatin derivative according to claim 13, further comprising the steps of:
dissolving isatin derivative CT5-3 in dichloromethane solvent, controlling the temperature to be 0 ℃ to react with m-chloroperoxybenzoic acid, adding saturated sodium bicarbonate aqueous solution and saturated sodium sulfite aqueous solution after the reaction is finished, and extracting, concentrating and separating to obtain CT5-4 and CT 5-5.
15. The method for preparing isatin derivative according to claim 13, wherein the molar volume ratio of the brominated derivative compound of isatin 3 to tetrahydrofuran is 1: 10; the mol ratio of the isatin bromo-derivative compound 3 to the sodium hydride to the compound 4 is 1: 2: 2.
16. the method for preparing isatin derivative according to claim 14, wherein the volume molar ratio of the dichloromethane solvent to the isatin derivative CT5-3 is 10:1, and the molar ratio of the m-chloroperoxybenzoic acid to the isatin derivative CT5-3 is 1: 1.
17. The method for preparing isatin derivative according to claim 13, wherein CT5-3 has the following structure:
Figure FDA0003565540360000041
18. the method for preparing isatin derivative according to claim 14, wherein CT5-4 has the following structure:
Figure FDA0003565540360000042
19. the method for preparing isatin derivative according to claim 14, wherein CT5-5 has the following structure:
Figure FDA0003565540360000043
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