CN114591214B - Isatin derivative and preparation method thereof - Google Patents
Isatin derivative and preparation method thereof Download PDFInfo
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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. From commercially available isatin, the synthesis of the isatin-magnolol splice CT5-1 and the synthesis of the isatin-and magnolol splice CT5-2 are realized by respectively introducing magnolol and a honokiol fragment through an N-alkylation reaction, from commercially available compound 3, the isatin-phthalimide splice CT5-3 can be efficiently obtained by introducing a phthalimide fragment through an S-alkylation reaction, and then the oxidation states of sulfur atoms are adjusted by utilizing m-chloroperoxybenzoic acid conditions to obtain CT5-4 and CT5-5 respectively. 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 framework, but also has obviously better in vitro activity against rheumatoid arthritis than the positive control drug methotrexate, and the in vivo activity is equivalent to that of the positive control drug methotrexate.
Description
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 (Rheumatoid arthritis, RA) is an autoimmune disease with high morbidity, complex etiology and high disability rate, and is characterized mainly by joint synovial thickening, inflammatory cell infiltration, pannus formation and destruction of articular cartilage and bone (Nat Rev Rheumatol,2020,16 (6): 316-333.). The etiology and pathogenesis of RA are complex, and in RA synovial tissue, synovial Fibroblasts (FLS) are main effector cells of synovial hyperplasia, and have biological characteristics of tumor-like 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, playing an important role in RA pathology (Nat Rev Rheumatol,2020,16 (6): 316-333.). More and more studies have shown that promoting apoptosis of FLS, or inhibiting proliferation, migration and invasion of FLS, is effective in preventing the occurrence and progression of RA (Immunol rev.2010,233 (1): 233-55.). Methotrexate (MTX) acts as a first-line drug in treating RA patients by inhibiting DNA biosynthesis and FLS proliferation (Br J Clin Pharmacol,2019,85 (10): 2228-2234.).
In recent years, new drug researches using natural products as lead compounds promote the development of modern medicines, and the specific gravity of drugs related to the natural products in various drugs marketed in batches is also increasing. Therefore, modification of natural products by means of pharmaceutical chemistry research is very important for new drug research.
Isatin (indoline-2, 3-dione, isatin) and its derivatives are an important class of azaheterocyclic compounds with a wide range of biological activities, including anti-inflammatory and anti-tumor activities, among others (Guangdong chemical 2015,14 (42): 106,113; pharmacol Rep.2013;65 (2): 313-35). Although research on isatin is increasing at home and abroad in recent years, isatin is also modified into derivatives by students to enhance the activity thereof, more isatin derivatives still need to be developed to meet basic research and clinical transformation requirements.
Disclosure of Invention
The invention provides isatin derivatives CT5-1, CT5-2, CT5-3, CT5-4 and CT5-5 and a preparation method thereof. From commercially available isatin, the synthesis of an isatin-magnolol splice CT5-1 and the synthesis of an isatin-and magnolol splice CT5-2 are realized by respectively introducing magnolol and a honokiol fragment through an N-alkylation reaction, from commercially available compound 3, an isatin-phthalimide fragment is introduced through an S-alkylation reaction, so that the isatin-phthalimide splice CT5-3 can be efficiently obtained, and then the oxidation states of sulfur atoms are adjusted by utilizing m-chloroperoxybenzoic acid conditions to obtain CT5-4 and CT5-5 respectively.
Wherein the isatin derivative CT5-1 has the following structure:
CT5-1 can be prepared by the following method:
dissolving isatoic acid in N, N-dimethylformamide solvent, carrying out N-alkylation reaction with a compound 1 under the condition of potassium carbonate, introducing magnolol fragments, and then removing phenolic hydroxyl protection under the condition of tetrabutylammonium fluoride/tetrahydrofuran to obtain CT5-1;
the reaction temperature is 60 ℃, the reaction time is 12 hours, the volume mole ratio of the N, N-dimethylformamide to the isatin is 10:1, and the mole ratio of the isatin, the compound 1 and the potassium carbonate is 1:2:2; the volume mole ratio of tetrahydrofuran to isatin is 10:1, the molar ratio of tetrabutylammonium fluoride to isatin is 1:1.
isatin derivative CT5-2 has the following structure:
CT5-2 can be prepared by the following method:
dissolving isatoic in N, N-dimethylformamide solvent, and carrying out N-alkylation reaction with a compound 2 under the condition of potassium carbonate to obtain CT5-2;
the reaction temperature is 60 ℃, the reaction time is 24 hours, the volume mole ratio of the N, N-dimethylformamide to the isatin is 10:1, and the mole ratio of the isatin, the compound 2 and the potassium carbonate is 4:1:2.
isatin derivative CT5-3 has the following structure:
CT5-3 can be prepared by the following method:
dissolving a bromoderivative compound 3 of isatin in tetrahydrofuran, and carrying out S-alkylation reaction with a compound 4 under the condition of sodium hydride to obtain CT5-3;
the reaction temperature is 25 ℃, the reaction time is 1 hour, the volume mole ratio of tetrahydrofuran to isatin is 10:1, and the mole ratio of sodium hydride, compound 4 to isatin is 2:2:1.
the isatin derivatives CT5-4 and CT5-5 have the following structures:
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, extracting, concentrating and separating to obtain CT5-4 and CT5-5;
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 framework, but also has obviously better in vitro activity against rheumatoid arthritis than the positive control drug methotrexate, and the in vivo activity is equivalent to that of the positive control drug methotrexate.
Description of the drawings:
FIG. 1 effect of CT5-2 on arthritis in CIA mice
FIG. 2 effect of CT5-2 on CIA mouse arthritis score
The specific embodiment 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 for 12 hours at 60 ℃, adding 50mL of water into the system, extracting with ethyl acetate (50 mL of 3), drying the combined organic phases with sodium sulfate, concentrating, separating the concentrated organic phases with a silica gel column, concentrating, adding 100mL of tetrahydrofuran, 10mmol of tetrabutylammonium fluoride, reacting for 15 minutes at 25 ℃, adding 50mL of water into the system, extracting with ethyl acetate (50 mL of 3), drying the combined organic phases with sodium sulfate, concentrating, separating the concentrated organic phases with a silica gel column to obtain CT5-1 (8 mmol,3.5 g) as a pale yellow liquid, and obtaining 80 percent yield;
1 H 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).
13 C NMR(126MHz,CDCl 3 )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 C 28 H 25 NNaO 4 [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 for 24 hours at 60 ℃, adding 50mL of water into the system, extracting with ethyl acetate (50 mL of 3), drying the combined organic phases with sodium sulfate, concentrating, and finally separating the concentrated organic phases with a silica gel column to obtain CT5-2 (9 mmol,5.4 g) as a pale yellow liquid with the yield of 88%;
1 H 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).
13 C NMR(101MHz,CDCl 3 )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 C 38 H 32 N 2 NaO 6 [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 ℃ to react for 1 hour, adding 50mL of water into the system, extracting with ethyl acetate (50 mL of 3), combining organic phases, drying the organic phases with sodium sulfate, concentrating the organic phases, and finally separating the concentrated organic phases with a silica gel column to obtain CT5-3 (8 mmol,3.3 g) as a pale yellow liquid with the yield of 82%;
1 H 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).
13 C 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 C 22 H 20 N 2 NaO 4 S[M+Na]+:431.1041,found:431.0907
HPLC purity 98.1%.
Example 4 preparation of CT5-4 and CT5-5
10mmol of CT5-3 was dissolved in 100mL of dichloromethane, and a solution of 10mmol of m-chloroperoxybenzoic acid in dichloromethane (10 mL) was slowly added to the system at 0deg.C; reacting at 0 ℃ for 20 minutes, adding 50mL of saturated sodium bicarbonate aqueous solution and 50mL of saturated sodium sulfite aqueous solution into the system, extracting with dichloromethane (50 mL is 3), combining organic phases, drying the organic phases with sodium sulfate, concentrating the organic phases, and finally separating the concentrated organic phases by a silica gel column to respectively obtain CT5-4 (6 mmol,2.5 g), light yellow liquid and yield of 60%; CT5-5 (4 mmol,1.8 g), pale yellow liquid, yield 40%;
CT5-4：
1 H 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).
13 C 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 C 22 H 20 N 2 NaO 5 S[M+Na]+:447.0991,found:447.0988
HPLC purity 99.1%.
CT5-5：
1 H 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).
13 C 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 C 22 H 20 N 2 NaO 6 S[M+Na]+:463.0940,found:463.0936
HPLC purity 99.3%.
Experimental example 1 influence of CT5-1, CT5-2, CT5-3, CT5-4, CT5-5 on proliferation of synovial fibroblasts (RA-FLS) from primary rheumatoid arthritis
The purpose of the experiment is as follows: investigation of the influence of CT5-1, CT5-2, CT5-3, CT5-4, CT5-5 on proliferation of primary RA-FLS
1.1. Experimental materials
1.1.1 cells: primary RA-FLS
1.1.2 reagents and instrumentation: DMEM medium (Gibco), fetal bovine serum (FBS, BI), 0.25% trypsin (Gibco), phosphate buffer (PBS, hyclone), DMSO (Sigma), CCK8 kit (APExBIO), multifunctional microplate reader (Synergy H1, bioTek), inverted microscope (Leica), ultra clean bench (singapore high tech), thermostatic water bath (Shanghai macrolaboratory equipment), low speed centrifuge (hunan henna instrument equipment), cell incubator (singapore high tech).
1.2. Experimental method
1.2.1 culturing RA-FLS cells to logarithmic growth phase, digesting the cells with 0.25% trypsin, centrifuging at 1000rpm for 5 minutes, resuspending the cell pellet with DMEM medium containing 10% FBS, seeding the cells in 96 well plates at a density of 5000 cells/well in a volume of 100. Mu.L, then placing in an incubator at 37℃and 5% CO 2 Culturing overnight under saturated humidity condition.
1.2.2 6 duplicate wells were set per group, each well was dosed as follows, and then incubation was continued for 24 hours.
Control group: i.e. no dosing group.
Positive control group: positive control groups were divided into 3 groups:
positive control group 1: methotrexate was added at a final concentration of 5 μm.
Positive control group 2: methotrexate was added at a final concentration of 10 μm.
Positive control group 3: methotrexate was added at a final concentration of 20 μm.
CT5-1 group: CT5-1 group was divided into 3 groups:
CT5-1 group 1: CT5-1 was added at 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 at a final concentration of 20. Mu.M.
CT5-2 group: CT5-2 groups were divided into 3 groups:
CT5-2 group 1: CT5-2 was added at 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 at a final concentration of 20. Mu.M.
CT5-3 group: CT5-3 groups were divided into 3 groups:
CT5-3 group 1: CT5-3 was added at a final concentration of 5. Mu.M.
CT5-3 group 2: CT5-3 was added at a final concentration of 10. Mu.M.
CT5-3 group 3: CT5-3 was added at a final concentration of 20. Mu.M.
CT5-4 group: CT5-4 groups were divided into 3 groups:
CT5-4 group 1: CT5-4 was added at a final concentration of 5. Mu.M.
CT5-4 group 2: CT5-4 was added at a final concentration of 10. Mu.M.
CT5-4 group 3: CT5-4 was added at a final concentration of 20. Mu.M.
CT5-5 group: CT5-5 groups were divided into 3 groups:
CT5-5 group 1: CT5-5 was added at a final concentration of 5. Mu.M.
CT5-5 group 2: CT5-5 was added at a final concentration of 10. Mu.M.
CT5-5 group 3: CT5-5 was added at a final concentration of 20. Mu.M.
After the cells were further cultured for 24 hours, the effect of the drug on proliferation of RA-FLS cells was examined using CCK8 kit. The specific operation is as follows: after adding 10. Mu.L of CCK8 to each well and further culturing for 2 hours, the absorbance (OD) value at a wavelength of 450nm was measured by a microplate reader, and the cell proliferation inhibition ratio of each group was calculated with respect to the control group as 0%.
1.3 experimental results
TABLE 1 Effect of novel compounds on primary RA-FLS proliferation
As shown in table 1, CT5-2 was able to significantly inhibit proliferation of primary RA-FLS, whose proliferation inhibiting effect was dose-dependent, increasing with increasing dose; and the effect of inhibiting proliferation is obviously better than that of positive control medicine methotrexate.
Experimental example 2 influence of CT5-1, CT5-2, CT5-3, CT5-4, CT5-5 on proliferation of rheumatoid arthritis synovial fibroblasts MH7A
The purpose of the experiment is as follows: study of the Effect of CT5-1, CT5-2, CT5-3, CT5-4, CT5-5 on proliferation of MH7A
2.1 Experimental materials
2.1.1 cells: MH7A
2.1.2 reagents and instrumentation: DMEM medium (Gibco), fetal bovine serum (FBS, BI), 0.25% trypsin (Gibco), phosphate buffer (PBS, hyclone), DMSO (Sigma), CCK8 kit (APExBIO), multifunctional microplate reader (Synergy H1, bioTek), inverted microscope (Leica), ultra clean bench (singapore high tech), thermostatic water bath (Shanghai macrolaboratory equipment), low speed centrifuge (hunan henna instrument equipment), cell incubator (singapore high tech).
2.2. Experimental method
2.2.1 culturing MH7A cells to log phase of growth, cells were digested with 0.25% trypsinCentrifugation at 1000rpm for 5 min, cell pellet was resuspended in DMEM medium containing 10% FBS, cells were seeded at a density of 5000 cells/well in a volume of 100. Mu.L in 96 well plates and then placed in an incubator at 37℃in 5% CO 2 Culturing overnight under saturated humidity condition.
2.2.2 6 duplicate wells were set per group, each well was dosed as follows, and then incubation was continued for 24 hours.
Control group: i.e. no dosing group.
Positive control group: positive control groups were divided into 3 groups:
positive control group 1: methotrexate was added at a final concentration of 5 μm.
Positive control group 2: methotrexate was added at a final concentration of 10 μm.
Positive control group 3: methotrexate was added at a final concentration of 20 μm.
CT5-1 group: CT5-1 group was divided into 3 groups:
CT5-1 group 1: CT5-1 was added at 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 at a final concentration of 20. Mu.M.
CT5-2 group: CT5-2 groups were divided into 3 groups:
CT5-2 group 1: CT5-2 was added at 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 at a final concentration of 20. Mu.M.
CT5-3 group: CT5-3 groups were divided into 3 groups:
CT5-3 group 1: CT5-3 was added at a final concentration of 5. Mu.M.
CT5-3 group 2: CT5-3 was added at a final concentration of 10. Mu.M.
CT5-3 group 3: CT5-3 was added at a final concentration of 20. Mu.M.
CT5-4 group: CT5-4 groups were divided into 3 groups:
CT5-4 group 1: CT5-4 was added at a final concentration of 5. Mu.M.
CT5-4 group 2: CT5-4 was added at a final concentration of 10. Mu.M.
CT5-4 group 3: CT5-4 was added at a final concentration of 20. Mu.M.
CT5-5 group: CT5-5 groups were divided into 3 groups:
CT5-5 group 1: CT5-5 was added at a final concentration of 5. Mu.M.
CT5-5 group 2: CT5-5 was added at a final concentration of 10. Mu.M.
CT5-5 group 3: CT5-5 was added at a final concentration of 20. Mu.M.
After the cells were further cultured for 24 hours, the effect of the drug on proliferation of MH7A cells was examined using the CCK8 kit. The specific operation is as follows: after adding 10. Mu.L of CCK8 to each well and further culturing for 2 hours, the absorbance (OD) value at a wavelength of 450nm was measured by a microplate reader, and the cell proliferation inhibition ratio of each group was calculated with respect to the control group as 0%.
2.3 experimental results
TABLE 2 Effect of novel compounds on MH7A proliferation
As shown in table 2, CT5-2 was able to significantly inhibit proliferation of MH7A, whose proliferation inhibiting effect was dose-dependent, increasing with increasing dose; and the effect of inhibiting proliferation is obviously better than that of positive control medicine methotrexate.
Experimental example 3 influence of CT5-2 on collagen-induced rheumatoid arthritis (collagen induced arthritis, CIA) mouse model
The purpose of the experiment is as follows: study of the therapeutic Effect of CT5-2 on CIA mouse model
3.1. Experimental animal
SPF-grade DBA/1 mice, males, 6-8 weeks of age.
3.2. Experimental method
The mice were randomly divided into 4 groups, each: control group, CIA model group, positive control group, CT5-2 group. CIA model establishment and drug administration: the DBA/1 mouse CIA model was induced using bovine type II Collagen (CII). The specific method comprises the following steps: (1) dissolving 2mg/mL bovine type II collagen in 0.1mol/L acetic acid solution, mixing with complete Freund's adjuvant containing 1mg/mL M.tubulosis according to volume of 1:1, and emulsifying and mixing uniformly on ice water bath by using a tissue homogenizer; (2) selecting male DBA/1 mice with age of 6-8 weeks, performing primary immunization on day 0, and subcutaneously injecting 0.1mL of the prepared emulsifier at a position 2 cm away from the root of the tail of the mice to sensitize the emulsifier; (3) boosting on day 21, mixing 2mg/mL bovine type II collagen solution with incomplete Freund's adjuvant at a volume of 1:1, emulsifying and mixing uniformly on ice-water bath with a tissue homogenizer, and subcutaneously injecting 0.1mL of emulsifier at a distance of 0.5 cm from the root of the tail of the mouse; (4) the disease condition of the joints of the four limbs of the mice is continuously observed. Starting from the day of secondary immunization (day 21), the mice of the CIA model group were given by intraperitoneal injection of the corresponding solvents, the mice of the positive control group were given by intraperitoneal injection of 20mg/kg of methotrexate, the CT5-2 groups were given by intraperitoneal injection of 20mg/kg of CT5-2, and the disease of the joints of the limbs of each group was observed. The extent of arthritis lesions in the extremities of mice was scored according to the following criteria, and the therapeutic effect of CT5-2 on arthritis in the CIA mouse model was evaluated: the scale of 0 is normal, 1 is mild red swelling, 2 is moderate red swelling, 3 is severe swelling, and 4 is joint deformation and rigidity. The composite score for each mouse was the sum of the 4 joint scores, with a top score of 16.
3.3. Experimental results
In vivo experimental results show that CT5-2 can obviously relieve arthritis of CIA mice (figure 1), reduce arthritis score (figure 2, compared with CIA model group, ** p < 0.01), and the therapeutic effect is equivalent to that of the first-line clinical medicine (positive control medicine) methotrexate.
According to the pharmacological and pharmacodynamic experiments, the in vitro activity of the CT5-2 anti-rheumatoid arthritis compound prepared by the invention is obviously superior to that of methotrexate, the in vivo activity is equivalent to that of the methotrexate, the skeleton is novel, the synthesis 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 medicaments.
Claims (13)
1. A process for the preparation of isatin derivatives, comprising the steps of:
dissolving isatin in N, N-dimethylformamide solvent, and carrying out N-alkylation reaction with another compound under the action of potassium carbonate to obtain one of isatin derivatives CT5-1 and CT5-2;
the other compound is one of a compound 1 and a compound 2, wherein the compound 1 has the following structure:
the compound 2 has the following structure:
the isatin derivative obtained by adding the compound 1 for N-alkylation is CT5-1, and the CT5-1 has the following structure:
the isatin derivative obtained by adding the compound 2 for N-alkylation is CT5-2, and the CT5-2 has the following structure:
2. the process for the preparation of isatin derivatives according to claim 1, characterized in that it further comprises a deprotection step when the other compound is compound 1.
3. The process for the preparation of isatin derivatives according to claim 2, characterized in that the deprotection step is carried out under tetrabutylammonium fluoride and tetrahydrofuran conditions.
4. The process for the preparation of isatin derivatives according to claim 1, characterized in that the molar volume ratio of isatin to N, N-dimethylformamide is 1:10.
5. the process for the preparation of isatin derivatives according to claim 1, characterized in that when the other compound is compound 1, the molar ratio of isatin, potassium carbonate to compound 1 is 1:2:2; when the other compound is compound 2, the mol ratio of the isatin to the potassium carbonate to compound 2 is 4:2:1.
6. the process for the preparation of isatin derivatives according to claim 1, characterized in that the reaction temperature of the N-alkylation reaction is 60 ℃.
7. A process for the preparation of isatin derivatives according to claim 3, characterized in that the molar volume ratio of isatin to tetrahydrofuran is 1:10, the mol ratio of isatin to tetrabutylammonium fluoride is 1:1.
8. an isatin derivative CT5-1 having the formula:
9. an isatin derivative CT5-2 having the formula:
10. a process for the preparation of isatin derivatives, comprising the steps of:
dissolving a bromoderivative 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 CT5-3;
the bromoderivative compound 3 of isatin has the following structure:
the compound 4 has the following structure:
the CT5-3 has the following structure:
11. the process for the preparation of isatin derivatives according to claim 10, characterized in that it further comprises the following steps:
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, extracting, concentrating and separating to obtain CT5-4 and CT5-5;
the CT5-4 has the following structure:
the CT5-5 has the following structure:
12. the process for the preparation of isatin derivatives according to claim 10, characterized in that the ratio of the molar volume of the brominated derivative compound of isatin 3 to tetrahydrofuran is 1:10; the mol ratio of the bromoderivative compound 3 of isatin to the sodium hydride to the compound 4 is 1:2:2.
13. the process for the preparation of isatin derivatives according to claim 11, characterized in that the volumetric molar ratio of dichloromethane solvent to isatin derivative CT5-3 is 10:1 and the molar ratio of meta-chloroperoxybenzoic acid to isatin derivative CT5-3 is 1:1.
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