Synthetic method and application of pyrazolidine derivative

Abstract

The invention discloses a synthesis method and application of a pyrazolidine derivative, and belongs to the technical field of chemical medicine and organic synthesis. The invention provides a pyrazolidine compound as shown in a general formula III or IV, and the pyrazolidine compound comprises pharmaceutically acceptable salt and a deuterated substance of the pyrazolidine compound. The synthesis method of the compound comprises the following steps: taking a cyclohexadienone compound and azomethine as raw materials, and reacting under a catalyst-free condition to obtain the compound. The method has the advantages of simplicity and convenience in operation, environment friendliness, wide substrate application range, high yield and the like. The synthesized compound shows remarkable anti-inflammatory activity and has a wide application prospect in the field of neuroinflammation resistance. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of chemical medicine and organic synthesis, and specifically relates to a synthesis method and application of a pyrazolidine derivative. Background Art

[0002] As a core synthetic intermediate in heterocyclic chemistry, pyrazolidine is not only an important unit for constructing typical heterocyclic compounds such as pyrrole and pyrazine, but is also widely used as a key transformation unit in the synthesis of natural products and drugs. It is worth noting that the pyrazolidine skeleton is widely present in a variety of natural products and drug molecules with significant biological activity, showing diverse pharmacological effects. For example: (1) PTP1B inhibitors, which have shown significant efficacy in controlling obesity and type II diabetes; (2) Zanubrutinib, a highly effective and selective Bruton's tyrosine kinase (BTK) inhibitor, has shown excellent activity and good tolerability in patients with relapsed, refractory and newly diagnosed chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL); (3) Edaravone, which effectively reduces oxidative damage to brain cells, vascular endothelial cells and nerve cells by scavenging free radicals and inhibiting lipid peroxidation; (4) Compounds with anti-epileptic effects, which can be used to treat or prevent epileptic seizures; (5) Anti-Alzheimer's disease drugs, which are used to improve cognitive dysfunction, such as Alzheimer's disease; (6) Herbicides and pesticides, which can be used to treat human cognitive dysfunction and animal memory disorders and other related diseases. In summary, given the significant biological activity of pyrazolidine compounds and their broad application prospects in drug development, the development of a rapid and efficient method for the synthesis of pyrazolidine compounds has become an important research direction in the field of organic chemistry and medicinal chemistry.

[0003]

[0004] In view of the remarkable biological properties exhibited by pyrazolidine compounds, researchers have developed a variety of synthetic methods to construct pyrazolidine skeletons in the past few decades. However, existing methods usually rely on expensive metal catalysts and complex raw materials, and there are problems such as the synthesis process is not green enough and the product structure is single. In particular, for tetracyclic pyrazolidine derivatives with more diverse and complex spatial structures, the difficulty of synthesis increases significantly, and the currently reported synthetic strategies are extremely limited, which seriously restricts the development of drugs based on such skeletons.

[0005] In response to the above problems, the present invention has developed a new method for efficiently constructing a tetracyclic pyrazolidine skeleton, which not only enriches the library of pyrazolidine compounds, but also finds that such compounds have significant anti-neuroinflammatory activity. Summary of the invention

[0006] The present invention aims to solve the deficiencies in the prior art and provide a method for synthesizing a 6 / 5 / 6 / 6 tetra-fused ring pyrazolidine compound. The compound has a novel structure and four consecutive chiral centers. Its complexity and diversity significantly enrich the types of heterocyclic compounds and provide a new heterocyclic skeleton for drug molecular structure design research. In addition, the compound synthesized by the present invention exhibits excellent anti-neuroinflammatory activity and has potential drug development value.

[0007] The synthesis method of the invention is simple, the starting raw materials are easily available, it is green and environmentally friendly, the substrate has a wide range of application, the yield is high, no organic catalyst is used, and it has high atom economy.

[0008] Another object of the present invention is to provide a pyrazolidine compound and its application in the preparation of anti-neuroinflammatory drugs.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The present invention provides a pyrazolidine compound as shown in the general formula III or IV, or a pharmaceutically acceptable salt or deuterated product thereof;

[0011]

[0012] In the formula,

[0013] R is selected from: C1-C6 straight chain or branched alkyl, C3-C6 cycloalkyl, C1-C6 straight chain or branched alkoxy, -(CH 2 ) n -R a , substituted or unsubstituted aryl; the substitution on the aryl includes any one of mono- to tri-substitution, and the substituent is selected from halogen (F, Cl, Br, I), cyano, C1-C8 straight or branched alkyl, C1-C8 alkoxy; n is 1, 2, 3, R a It is a C2-C7 ester group, a 4-10 membered alicyclic group, or a siloxy group;

[0014] R' is NHPG or OR", wherein PG refers to Ts, Boc, Cbz, Bz or Ac, and R" is H or a C1-C6 straight or branched chain alkyl;

[0015] R 1 Selected from: H, halogen (F, Cl, Br, I), C1-C6 straight or branched alkyl, phenyl;

[0016] R 2 Selected from: H, halogen (F, Cl, Br, I), C1-C6 alkoxy;

[0017] R 3 Selected from: H, C1-C6 straight chain or branched alkyl;

[0018] R 4 Selected from: H, -C(O)R 5 ; R 5 Selected from C2-C8 alkenyl, halogen-containing C1-C6 straight or branched chain alkyl.

[0019] In one embodiment of the present invention, the aryl group is selected from: phenyl, naphthyl, thienyl.

[0020] In one embodiment of the present invention, the C2-C7 ester group is -COOR b , R b It is a C1-C6 straight chain or branched chain alkyl group.

[0021] In one embodiment of the present invention, the 4-10 membered alicyclic heterocyclic group contains 1-3 heteroatoms, and the heteroatoms include N, O, and S.

[0022] In one embodiment of the present invention, the silyl group is -O-SiR c R d R e , R c , R d , R e Each is independently selected from C1-C6 straight chain or branched alkyl.

[0023] In one embodiment of the present invention, R is further selected from:

[0024] In one embodiment of the present invention, R 1 Further selected from:

[0025] In one embodiment of the present invention, R 2 Further selected from:

[0026] In one embodiment of the present invention, R 3 Further selected from:

[0027] In one embodiment of the present invention, R 4 Further selected from:

[0028] In one embodiment of the present invention, the pharmaceutically acceptable salt includes an inorganic salt or an organic salt; wherein the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salt is selected from formates, acetates, trifluoroacetates, propionates, pyruvates, glycolates, oxalates, malonates, succinates, glutarates, fumarates, maleates, lactates, malates, citrates, tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, salicylates, p-toluenesulfonates, and ascorbates.

[0029] In one embodiment of the present invention, most preferably, the pyrazolidine compound is specifically selected from:

[0030]

[0031]

[0032] The present invention also provides a method for synthesizing the above-mentioned pyrazolidine derivatives, which comprises:

[0033] The cyclohexadienone compound shown in formula I and the azomethine imine shown in formula II are used as raw materials, and the tetracyclic pyrazolidine compound shown in formula III can be obtained by reaction:

[0034]

[0035] Among them, R, R', R 1 , R 2 , R 3 Same definition as above.

[0036] In one embodiment of the present invention, the molar ratio of the cyclohexadienone compound shown in Formula I to the azomethine imine shown in Formula II is 1.0:1.2.

[0037] In one embodiment of the present invention, the reaction is carried out in an organic solvent;

[0038] If R' is -OR", the organic solvent is selected from any one or more of the following: dichloromethane (DCM), dichloroethane (DCE), toluene, chloroform, N,N-dimethylformamide (DMF), acetonitrile (CH 3 CN), tetrahydrofuran (THF), acetone;

[0039] If R' is -NHPG, the organic solvent is selected from any one or more of the following: dichloromethane, dichloroethane, chloroform, acetonitrile, tetrahydrofuran, acetone, N,N-dimethylformamide.

[0040] Furthermore, the organic solvent is preferably dichloromethane.

[0041] In one embodiment of the present invention, the amount of the organic solvent added is 1 to 10 mL / mmol based on the amount of the cyclohexadienone compound represented by Formula I, preferably 5 mL / mmol.

[0042] In one embodiment of the present invention, no catalyst is added to the reaction.

[0043] In one embodiment of the present invention, a catalyst may be added during the reaction.

[0044] If R' is -OR", the catalyst is selected from any one or more of the following: DABCO, DIPEA, DBU, TsOH;

[0045] If R' is -NHPG, the catalyst is selected from any one or more of the following: NHTf 2 , K 2 CO 3 DABCO, Sc(OTf) 3 、DIPEA.

[0046] In one embodiment of the present invention, the amount of the catalyst added is 0-1 equivalent based on the amount of the cyclohexadienone compound shown in Formula I.

[0047] In one embodiment of the present invention, the reaction temperature is 20° C. to 80° C., and the reaction time is 12 to 160 hours.

[0048] Further, if R' is -OR", the reaction temperature is preferably 60°C to 80°C;

[0049] If R' is -NHPG, the reaction temperature is preferably 20°C to 40°C.

[0050] The present invention also provides a method for synthesizing a pyrazolidine derivative. The structure of the pyrazolidine derivative is shown in Formula IV. The reaction route of the synthetic method is:

[0051]

[0052] Among them, R 1 , R 2 , R 4 H, R, R', R 3 Same definition as above.

[0053] (1) using a cyclohexadienone compound represented by formula I and an azomethine imine represented by formula II as raw materials, and reacting to obtain a tetracyclic pyrazolidine compound represented by formula III;

[0054] (2) The obtained tetracyclic pyrazolidine compound is reacted in a sodium borohydride and methanol reaction system to obtain a pyrazolidine derivative represented by formula IV.

[0055] In one embodiment of the present invention, the process of step (1) is the same as above.

[0056] In one embodiment of the present invention, in step (2), a tetracyclic pyrazolidine compound is mixed with methanol, and the temperature is controlled at -5°C-10°C, sodium borohydride is slowly added under stirring, and then the mixture is heated to room temperature (20-30°C) and reacted for a period of time.

[0057] In one embodiment of the present invention, in step (2), the molar ratio of sodium borohydride to tetracyclic pyrazolidine compound is 1.0-1.5:1.

[0058] In one embodiment of the present invention, in step (2), the amount of methanol relative to the tetracyclic pyrazolidine compound is 10-20 mL / mmol, and specifically 12.5 mL / mmol.

[0059] In one embodiment of the present invention, in step (2), the reaction time is 20-40 min.

[0060] The present invention also provides a pharmaceutical composition for preventing neuroinflammation, which contains a pyrazolidine compound represented by general formula III or IV or a pharmaceutically acceptable salt or deuterated product thereof, and pharmaceutical excipients.

[0061] The present invention also provides the use of the pyrazolidine derivatives or pharmaceutically acceptable salts or deuterated derivatives thereof in the preparation of drugs for preventing or treating diseases associated with neuroinflammation.

[0062] The diseases associated with neuroinflammation generally include Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, stroke, brain trauma, epilepsy, depression, schizophrenia, chronic pain, viral encephalitis, bacterial meningitis, autoimmune encephalitis, Huntington's disease, HIV-related neurocognitive disorders, etc.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] In the prior art, the reported synthesis methods of pyrazolidine compounds have significant limitations: some methods rely on expensive metal catalysts or complex ligands, and are mainly limited to the construction of monocyclic pyrazolidine scaffolds, resulting in a single product structure and lack of diversity. In addition, the synthesis of complex polycyclic pyrazolidine compounds and their application in drug molecules have not been fully explored. In view of the above problems, the present invention provides a synthesis method with significant advantages: 6 / 5 / 6 / 6-fused tetracyclic pyrazolidine derivatives with four continuous stereo centers can be efficiently constructed. The method is simple to operate, the reaction conditions are mild and economical, with high yield and excellent stereoselectivity, a wide range of substrate applications, and no catalyst is required. The implementation of the present invention greatly enriches the structural diversity of pyrazolidine skeleton compounds, and provides important technical support for the drug development of such skeleton compounds.

[0065] The experimental results show that the pyrazolidine compounds synthesized by the present invention can significantly inhibit the neuroinflammatory response of BV2 microglia induced by lipopolysaccharide (LPS). Among them, compounds P5, P52 and P56 showed excellent inhibitory effects in reducing the level of cellular nitric oxide (NO) and the expression of inflammatory markers, and no obvious cytotoxicity was observed within the pharmacological concentration range. These characteristics show that the pyrazolidine compounds provided by the present invention have broad application prospects in the treatment of neuroinflammatory related diseases, and can be used as potential active lead compounds for further in-depth research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 The anti-neuroinflammatory activity of LPS-treated BV2 microglia after 24 hours of treatment was evaluated by high-throughput screening. A is a schematic diagram showing high-throughput screening of neuroinflammatory inhibitors by detecting cellular NO levels and cell viability. B, C, and D are the dose-dependent effect results of compounds 5, 52, and 56 in reducing cellular NO levels and cytotoxicity, respectively.

[0067] Figure 2 The expression level of inflammatory markers (E, F, G, and H correspond to the following markers: iNOS, Cox 2, Tnf-α, and Il-6, respectively) is shown in the figure. (N = 3 independent biological experiments. Data are expressed as "mean ± standard deviation". *p<0.05, **p<0.01, ***p<0.001, compared with the blank group cells. #p<0.05, ##p<0.01, ###p<0.001, compared with the LPS-treated control group cells.)

[0068] Figure 3 These are the X-ray single crystal diffraction structures of P1, P21, and P51. DETAILED DESCRIPTION

[0069] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0070] The present invention relates to the following chemical abbreviations: Me is methyl, Et is ethyl, n-Pr is n-propyl, i-Pr is isopropyl, n-Bu is n-butyl, Ph is phenyl, OMe is methoxy, n-Hexyl is n-hexyl, Boc is tert-butyloxycarbonyl, Ts is p-toluenesulfonyl, Ac is acetyl, and Bz is benzoyl.

[0071] The 4-aminocyclohexadienone shown in formula I (such as N1-N21 in the following embodiments) involved in the present invention can be prepared by itself according to existing literature, such as Org.Lett., 2021, 23, 7873-7877.

[0072] The 4-aminocyclohexadienone shown in formula I involved in the present invention (such as N22-N38 in the following embodiments) can be prepared by itself according to existing literature, such as J.Org.Chem., 2020, 85, 4515-4524.

[0073] The azomethine imine represented by formula II of the present invention (such as S1-S6 in the following examples) can be prepared according to existing literature, such as Tetrahedron, 2015, 71, 4473-4477.

[0074] Example 1

[0075] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0076]

[0077] The preparation method is as follows: Under air conditions, N1 (0.2 mmol, 55.4 mg) and S1 (0.24 mmol, 71.8 mg) are dissolved in CH 2 Cl 2 (1.0 mL), and the reaction was terminated after 36 hours at 70°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1), and the product P1 (101.4 mg, 88%) was obtained as a white solid.

[0078] Characterization data: 1H NMR(600MHz,Chloroform-d)δ7.89(d,J=1.3Hz,2H),7.88(d,J=1.3Hz,2H),7.43(dd,J=10.5,2.2Hz,1H),7 .38(d,J=8.1Hz,2H),7.34(d,J=8.0Hz,2H),7.29(td,J=7.6,1.5Hz,1H),7.20(td,J=7.5,1.3Hz,1H),7.11( dd,J=7.8,1.2Hz,1H),7.04(dd,J=7.7,1.4Hz,1H),6.82(s,1H),6.38(s,1H),5.88(d,J=10.4Hz,1H),4.97( d,J=6.9Hz,1H),4.62(dd,J=7.1,2.3Hz,1H),3.44(t,J=7.0Hz,1H),2.47(s,3H),2.45(s,3H),1.44(s,3H). 13 C NMR(151MHz,Chloroform-d)δ190.0,152.0,150.9,146.8,143.6,140.6,138.3,131.2,130.5,130.1,130.0,129 .7,128.8,127.8,127.0,126.81,126.78,120.4,64.9,63.0,57.3,56.9,29.4,22.0,21.7.HRMS(ESI)calculated for C 29 H 29 N 4 O 5 S 2 [M+H] + :577.1574, found 577.1567.

[0079] Example 2-16

[0080]

[0081] Referring to Example 1, different cyclohexadienone compound raw materials (N2-N16) were replaced to obtain corresponding products P2-P16.

[0082]

[0083]

[0084]

[0085]

[0086] Examples 17-19

[0087] Referring to Example 1, the corresponding cyclohexadienone compound raw materials (N17-N19) were replaced to obtain the corresponding products P17-P19.

[0088]

[0089]

[0090] Embodiment 20

[0091] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0092]

[0093] The preparation method is as follows: N20 (0.2 mmol, 30.8 mg) and S1 (0.24 mmol, 71.8 mg) are dissolved in CH 2 Cl 2 (1.0 mL), reacted at 70°C for 18 hours. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=1:1), to obtain product P20 (79.9 mg, 88%), which was a yellow solid.

[0094] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.88-7.85(m,2H),7.36(dd,J=8.6,0.8Hz,2H),7.23(dd,J=7 .6,1.6Hz,1H),7.16(td,J=7.5,1.4Hz,1H),7.08(dd,J=7.8,1.3Hz,1H),6.99-6.95(m,1H) ,6.63(dd,J=10.2,2.0Hz,1H),6.55(s,1H),5.96(d,J=10.2Hz,1H),4.92(d,J=7.5Hz,1H), 4.89(dd,J=7.3,2.0Hz,1H),3.60(s,3H),3.57(t,J=7.4Hz,1H),3.14(s,3H),2.45(s,3H). 13C NMR(101MHz,Chloroform-d)δ193.4,150.8,147.6,145.9,138.6,133.6,132.8,130.2,129.8,12 9.3,128.1,126.9,126.6,120.2,97.4,61.7,59.6,59.1,50.1,49.9,21.9.HRMS(ESI)calculated for C 23 H 24 N 3 O 5 S[M+H] + :454.1431, found 454.1429.

[0095] Embodiment 21

[0096] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0097]

[0098] The preparation method is as follows: N21 (0.2 mmol, 24.8 mg) and S1 (0.24 mmol, 71.8 mg) are dissolved in CH 2 Cl 2 (1.0 mL), and the reaction was stopped at 40°C for 40 hours. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1), to obtain product P21 (75.3 mg, 89%), which was a white solid.

[0099] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.88(d,J=8.4Hz,2H),7.38(d,J=8.0Hz,2H),7.30(td,J=7.7, 1.5Hz,1H),7.20(td,J=7.5,1.4Hz,1H),7.14(dd,J=7.8,1.3Hz,1H),7.01(dd,J=7.7,1.5Hz, 1H),6.88(dd,J=10.3,2.3Hz,1H),6.60(s,1H),5.90(d,J=10.3Hz,1H),4.66(d,J=6.5Hz,1H ),4.56(dd,J=6.9,2.4Hz,1H),4.03(s,1H),3.35(t,J=6.7Hz,1H),2.47(s,3H),1.51(s,3H). 13C NMR(101MHz,Chloroform-d)δ190.3,154.8,151.6,146.5,138.5,131.4,130.4,130.0,129.6, 128.4,127.5,126.9,126.8,120.8,69.9,65.5,63.0,56.4,31.2,22.0.HRMS(ESI)calculated for C 22 H 22 N 3 O 4 S[M+H] + :424.1326, found 424.1323.

[0100] Examples 22-34

[0101]

[0102] Referring to Example 21, the corresponding cyclohexadienone compound raw materials (N22-N34) were replaced to obtain the corresponding products P22-P34.

[0103]

[0104]

[0105]

[0106] Examples 35-37

[0107] Referring to Example 21, the corresponding cyclohexadienone compound raw materials (N35-N37) were replaced to obtain the corresponding products P35-P37.

[0108]

[0109] Embodiment 38

[0110] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0111]

[0112] The preparation method is as follows: N2 (0.2 mmol, 44.6 mg) and S2 (0.24 mmol, 76.1 mg) are dissolved in CH 2 Cl 2(1.0 mL), and the reaction was terminated after 45 hours at 70°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=3:1), to obtain the product P38 (100.3 mg, 93%), which was a white solid.

[0113] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.93-7.78(m,2H),7.46(d,J=10.5Hz,1H),7.38(d,J= 8.0Hz, 2H), 7.10 (dd, J=8.7, 5.2Hz, 1H), 6.96 (td, J=8.5, 2.9Hz, 1H), 6.70 (dd, J=8. 7,2.8Hz,1H),6.60(s,1H),6.54(s,1H),5.90(d,J=10.3Hz,1H),4.59(d,J=6.8Hz,1 H),4.54(d,J=7.1Hz,1H),3.49-3.39(m,1H),2.47(s,3H),1.63(s,3H),1.52(s,9H). 13 C NMR(101MHz,Chloroform-d)δ190.5,160.7(d,J CF =248.4Hz),154.8,152.5,150.7,146.5,134.9,131.6,130.4,129.5,128.4(d,J CF =8.7Hz),127.9,122.4(d,J CF =8.6Hz),116.8(d,J CF =22.4Hz),114.5(d,J CF =24.2Hz),80.1,64.9,62.3,56.7,54.5,29.7,28.7,21.9. 19 F NMR(376MHz,Chloroform-d)δ-113.8.HRMS(ESI)calculated forC 27 H 30 FN 4 O 5 S[M+H] + :541.1915, found 541.1910.

[0114] Embodiment 39

[0115] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0116]

[0117] The preparation method is as follows: N2 (0.2 mmol, 44.6 mg) and S3 (0.24 mmol, 79.0 mg) are dissolved in CH 2 Cl 2 (1.0 mL), and the reaction was terminated after 6 days at 70°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1), to obtain the product P39 (88.5 mg, 80%), which was a white solid.

[0118] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.85(d,J=7.9Hz,2H),7.44(d,J=10.4Hz,1H),7.37(d,J=8 .0Hz,2H),6.89(d,J=8.4Hz,1H),6.74(dd,J=8.6,2.6Hz,1H),6.67(d,J=2.6Hz,1H),6.6 4(s,1H),6.57(s,1H),5.89(d,J=10.4Hz,1H),4.62(d,J=7.0Hz,1H),4.53(d,J=7.1Hz,1 H),3.77(d,J=1.1Hz,3H),3.39(t,J=7.1Hz,1H),2.46(s,3H),1.63(s,3H),1.53(s,9H). 13 C NMR(101MHz,Chloroform-d)δ191.1,160.7,154.8,152.1,151.7,146.4,139.6,131.7,130.4,129.5,128. 5,128.1,113.4,112.4,111.2,80.0,64.9,62.5,56.9,55.4,54.5,29.6,28.7,21.9.HRMS(ESI)calculated for C 28 H 33 N 4 O 6 S[M+H] + :553.2115, found 553.2111.

[0119] Embodiment 40

[0120] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0121]

[0122] The preparation method is as follows: N2 (0.2 mmol, 44.6 mg) and S4 (0.24 mmol, 86.2 mg) are dissolved in CH 2 Cl 2 (1.0 mL), and the reaction was terminated after 5 days at 70°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=1:1), to obtain the product P40 (87.3 mg, 75%), which was a yellow solid.

[0123] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.85(d,J=8.4Hz,2H),7.43(d,J=10.4Hz,1H),7. 37(d,J=7.8Hz,2H),6.66(s,1H),6.55(s,1H),6.51(s,1H),6.45(s,1H),5.89( d,J=10.4Hz,1H),4.69(d,J=6.8Hz,1H),4.55(dd,J=7.0,2.1Hz,1H),3.87(s,3 H),3.82(s,3H),3.43(t,J=7.0Hz,1H),2.46(s,3H),1.63(s,3H),1.52(s,9H). 13 C NMR(101MHz,Chloroform-d)δ191.0,154.8,151.9,149.9,149.8,147.8,146.3,132.4,131.8,130.3,129.5,1 28.1,112.1,109.9,109.7,80.0,64.7,62.8,56.8,56.4,55.9,54.5,29.6,28.7,21.9.HRMS(ESI)calculated for C 29 H 35 N 4 O 7 S[M+H] + :583.2221, found 583.2217.

[0124] Embodiment 41

[0125] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0126]

[0127] The preparation method is as follows: N2 (0.2 mmol, 44.6 mg) and S5 (0.24 mmol, 68.4 mg) are dissolved in CH2 Cl 2 (1.0 mL), and the reaction was terminated after 5 days at 70°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1), to obtain the product P41 (75.8 mg, 75%), which was a white solid.

[0128] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ8.01-7.97(m,2H),7.74-7.70(m,1H),7.59(t,J=7.8Hz,2H),7 .44(d,J=10.4Hz,1H),7.28(td,J=7.6,1.5Hz,1H),7.18(td,J=7.5,1.4Hz,1H),7.13(dd,J= 7.7,1.3Hz,1H),6.98(d,J=7.5Hz,1H),6.64(s,1H),6.50(s,1H),5.90(d,J=10.4Hz,1H),4. 63(d,J=6.9Hz,1H),4.58(d,J=6.5Hz,1H),3.43(t,J=7.0Hz,1H),1.64(s,3H),1.53(s,9H). 13 C NMR(101MHz,Chloroform-d)δ190.7,154.8,152.2,151.3,138.4,135.0,134.8,130.0,129.7,129 .5,128.1,127.7,126.9,126.7,120.5,80.1,65.0,62.6,56.8,54.5,28.7.HRMS(ESI)calculated for C 26 H 29 N 4 O 5 S[M+H] + :509.1853, found 509.1851.

[0129] Embodiment 42

[0130] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0131]

[0132] The preparation method is as follows: N2 (0.2 mmol, 44.6 mg) and S6 (0.24 mmol, 75.6 mg) are dissolved in CH 2 Cl 2(1.0 mL), and the reaction was terminated after 57 hours at 70°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1), to obtain the product P42 (105.6 mg, 98%), which was a yellow solid.

[0133] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.92-7.87(m,2H),7.44(d,J=10.4Hz,1H),7.28(td,J=7.5 ,1.5Hz,1H),7.19(td,J=7.5,1.4Hz,1H),7.13(dd,J=7.8,1.3Hz,1H),7.03(d,J=2.0Hz,1 H),7.02-6.99(m,2H),6.61(d,J=13.1Hz,2H),5.90(d,J=10.4Hz,1H),4.71(d,J=6.9Hz, 1H), 4.54 (d, J = 6.2Hz, 1H), 3.89 (s, 3H), 3.45 (t, J = 7.0Hz, 1H), 1.64 (s, 3H), 1.53 (s, 9H). 13 C NMR(101MHz,Chloroform-d)δ190.9,164.8,154.9,152.2,151.4,138.5,131.8,129.9,128.0,127.8,12 6.8,126.7,125.7,120.6,115.0,80.0,64.9,62.6,57.0,56.0,54.5,29.6,28.7.HRMS(ESI)calculated forC 27 H 31 N 4 O 6 S[M+H] + :539.1959, found 539.1956.

[0134] Embodiment 43

[0135] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0136]

[0137] The preparation method is as follows: N2 (0.2 mmol, 44.6 mg) and S7 (0.24 mmol, 77.8 mg) are dissolved in CH 2 Cl 2(1.0 mL), reacted at 70°C for 47 hours. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1), to obtain product P43 (95.2 mg, 86%), which was a white solid.

[0138] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.57(dd,J=10.3,2.2Hz,1H),7.29(td,J=7.6,1.6Hz,1H),7 .23(td,J=7.5,1.4Hz,1H),7.16(dd,J=7.5,1.6Hz,1H),7.10(dd,J=7.7,1.3Hz,1H),6.99( d,J=6.8Hz,2H),6.21(s,1H),5.92(d,J=10.3Hz,1H),5.33(d,J=6.5Hz,1H),4.65(dd,J=6 .7,2.3Hz,1H),3.56(t,J=6.6Hz,1H),2.65(s,6H),2.30(s,3H),1.69(s,3H),1.49(s,9H). 13 C NMR(101MHz,Chloroform-d)δ191.0,154.5,151.6,150.8,145.3,138.3,132.6,130.0,128.5,128.3,1 27.8,127.0,126.7,120.6,80.0,63.9,62.7,57.0,54.6,29.2,28.6(3C),21.3.HRMS(ESI)calculated for C 29 H 35 N 4 O 5 S[M+H] + :551.2323, found 551.2322.

[0139] Embodiment 44

[0140] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0141]

[0142] The preparation method is as follows: N22 (0.2 mmol, 24.8 mg) and S2 (0.24 mmol, 76.1 mg) are dissolved in CH 2 Cl 2(1.0 mL), and the reaction was terminated after 44 hours at 40°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=1:1), to obtain product P44 (70.6 mg, 80%), which was a white solid.

[0143] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.91-7.84(m,2H),7.38(d,J=8.1Hz,2H),7.11(dd,J= 8.7,5.2Hz,1H),6.97(td,J=8.4,2.8Hz,1H),6.89(dd,J=10.3,2.3Hz,1H),6.72(dd, J=8.2,2.8Hz,1H),6.56(s,1H),5.89(d,J=10.3Hz,1H),4.57(d,J=6.4Hz,1H),4.54( dd,J=6.8,2.4Hz,1H),4.02(s,1H),3.34(t,J=6.6Hz,1H),2.47(s,3H),1.51(s,3H). 13 C NMR(101MHz,Chloroform-d)δ190.0,160.8(d,J CF =248.5Hz),155.0,150.9(d,J CF =2.4Hz),146.6,134.9(d,J CF =2.9Hz),131.3,130.4,129.6,128.4(d,J CF =8.5Hz),128.2,122.6(d,J CF =8.6Hz),116.8(d,J CF =22.4Hz),114.3(d,J CF =24.1Hz),69.8,65.5,62.7(d,J CF =2.0Hz),56.1,31.3,22.0. 19 F NMR(376MHz,Chloroform-d)δ-113.6(q,J=8.5Hz).HRMS(ESI)calculated for C 22 H 21 FN 3 O 4 S[M+H] + :442.1231, found 442.1233.

[0144] Embodiment 45

[0145] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0146]

[0147] The preparation method is as follows: N22 (0.2 mmol, 24.8 mg) and S3 (0.24 mmol, 79.0 mg) are dissolved in CH 2 Cl 2 (1.0 mL), reacted at 40°C for 26 hours. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=1:1), to obtain product P45 (68.0 mg, 75%), which was a yellow solid.

[0148] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.87(d,J=8.3Hz,2H),7.37(d,J=8.1Hz,2H),6.93-6.84(m,2H),6.75(dd,J=8.4,2.6Hz,1H),6.68(d,J=2.6Hz,1H),6.62(s, 1H),5.89(d,J=10.3Hz,1H),4.60(d,J=6.6Hz,1H),4.53(dd,J=6.9,2.4Hz,1H ),4.01(s,1H),3.77(s,3H),3.30(t,J=6.7Hz,1H),2.46(s,3H),1.50(s,3H). 13 C NMR(101MHz,Chloroform-d)δ190.5,160.7,154.8,151.9,146.5,139.5,131.3,130.4,129.6,12 8.4,128.3,113.5,112.6,111.2,69.9,65.6,62.8,56.4,55.4,31.2,21.9.HRMS(ESI)calculated for C 23 H 24 N 3 O 5 S[M+H] + :454.1431, found 454.1432.

[0149] Embodiment 46

[0150] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0151]

[0152] The preparation method is as follows: N22 (0.2 mmol, 24.8 mg) and S4 (0.24 mmol, 86.2 mg) are dissolved in CH 2 Cl 2 (1.0 mL), and the reaction was terminated after 3 days at 40°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:3) to obtain the product P46 (68.6 mg, 71%), which was a yellow solid.

[0153] Data Characterization: 1 H NMR(600MHz,Chloroform-d)δ7.87(dd,J=8.2,2.5Hz,2H),7.37(d,J=7.9Hz,2H),6.86(d,J=10.3Hz,1H),6.67(s,1H),6.48(d,J=15.5Hz,2H),5.88(d ,J=10.3Hz,1H),4.64(d,J=6.5Hz,1H),4.54(d,J=6.7Hz,1H),4.02(s,1H), 3.87(s,3H),3.83(s,3H),3.35(d,J=6.9Hz,1H),2.45(s,3H),1.50(s,3H). 13 C NMR(151MHz,Chloroform-d)δ190.5,154.6,150.1,149.8,147.8,146.4,132.4,131.5,130.3,129.6 ,128.4,112.3,109.7,109.7,69.8,65.4,63.1,56.3,56.2,55.9,31.2,21.9.HRMS(ESI)calculated forC 24 H 26 N 3 O 6 S[M+H] + :484.1537, found 484.1536.

[0154] Embodiment 47

[0155] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0156]

[0157] The preparation method is as follows: N22 (0.2 mmol, 24.8 mg) and S5 (0.24 mmol, 68.4 mg) are dissolved in CH 2Cl 2 (1.0 mL), and the reaction was terminated after 5 days at 40°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=1:1), to obtain the product P47 (59.7 mg, 73%), which was a white solid.

[0158] Data Characterization: 1 H NMR(600MHz,Chloroform-d)δ8.04-7.98(m,2H),7.72(t,J=7.5Hz,1H),7.59(t,J=7.7 Hz,2H),7.33-7.28(m,1H),7.22-7.16(m,1H),7.14(d,J=7.8Hz,1H),7.00(d,J=7.6Hz ,1H),6.88(dd,J=10.3,2.3Hz,1H),6.61(s,1H),5.90(d,J=10.3Hz,1H),4.60(d,J=6. 6Hz,1H),4.57(dd,J=6.9,2.4Hz,1H),3.97(s,1H),3.35(t,J=6.7Hz,1H),1.51(s,3H). 13 C NMR(151MHz,Chloroform-d)δ190.2,154.7,151.5,138.4,135.1,134.5,130.0,129.7,129 .5,128.4,127.5,127.0,126.8,120.7,69.9,65.6,63.0,56.3,31.2.HRMS(ESI)calculated for C 21 H 20 N 3 O 4 S[M+H] + :410.1169,found 410.1169.

[0159] Embodiment 48

[0160] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0161]

[0162] The preparation method is as follows: N22 (0.2 mmol, 24.8 mg) and S6 (0.24 mmol, 75.6 mg) are dissolved in CH 2 Cl 2(1.0 mL), and the reaction was terminated after 13 hours at 40°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=1:1), to obtain the product P48 (84.8 mg, 97%), which was a yellow solid.

[0163] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.94-7.90(m,2H),7.30(td,J=7.6,1.5Hz,1H),7.20( td,J=7.5,1.3Hz,1H),7.14(dd,J=7.8,1.3Hz,1H),7.04-7.00(m,3H),6.88(dd,J=1 0.3,2.3Hz,1H),6.61(s,1H),5.90(d,J=10.3Hz,1H),4.69(d,J=6.6Hz,1H),4.54(d d,J=6.8,2.4Hz,1H),4.06(s,1H),3.89(s,3H),3.35(t,J=6.7Hz,1H),1.51(s,3H). 13 C NMR(101MHz,Chloroform-d)δ190.4,164.9,154.9,151.6,138.5,131.8,130.0,128.3,127.6, 126.9,126.7,125.4,120.8,115.0,69.9,65.5,62.9,56.5,56.0,31.2.HRMS(ESI)calculated for C 22 H 22 N 3 O 5 S[M+H] + :440.1275,found 440.1273.

[0164] Embodiment 49

[0165] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0166]

[0167] The preparation method is as follows: N22 (0.2 mmol, 24.8 mg) and S6 (0.24 mmol, 77.8 mg) are dissolved in CH 2 Cl 2(1.0 mL), and the reaction was terminated after 37 hours at 40°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1), to obtain the product P49 (75.3 mg, 83%), which was a yellow solid.

[0168] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.30(td,J=7.6,1.6Hz,1H),7.23(td,J=7.4,1.4Hz,1H),7 .16(dd,J=7.6,1.6Hz,1H),7.11(dd,J=7.7,1.4Hz,1H),6.98(s,2H),6.91(dd,J=10.3,2. 4Hz,1H),6.25(s,1H),5.90(d,J=10.3Hz,1H),5.25(d,J=6.3Hz,1H),4.70(dd,J=6.6,2.5 Hz,1H),4.08(s,1H),3.45(t,J=6.5Hz,1H),2.82-2.47(m,6H),2.30(s,3H),1.54(s,3H). 13 C NMR(101MHz,Chloroform-d)δ190.5,154.8,151.0,145.2,138.4,132.6(2C),129.9,128.3,128 .2,127.6,127.0,126.7,120.8,70.1,64.3,63.0,56.4,31.5,23.4,21.3.HRMS(ESI)calculated for C 24 H 26 N 3 O 4 S[M+H] + :452.1639,found 452.1639.

[0169] Embodiment 50

[0170] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0171]

[0172] At room temperature, P2 (0.4mmol, 209.2mg) and methanol (5ml) were added to the reaction bottle. After cooling to 0°C, sodium borohydride (0.44mmol, 16.7mg) was slowly added under stirring, and then the reaction system was moved to room temperature and stirred for 20 minutes. After the reaction was completed, saturated NH4Cl solution (1.5mL) was added to quench, extracted with ethyl acetate, and the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was slurried with methanol to obtain a white solid product P50 (189.7mg, 90%).

[0173] Characterization data: 1 H NMR(400MHz,Chloroform-d)δ7.94-7.84(m,2H),7.34(d,J=8.0Hz,2H),7.05(ddd,J=8.4,6.3,2 .5Hz,1H),6.76(d,J=6.4Hz,2H),6.50(d,J=8.1Hz,1H),6.42(s,1H),4.67(dd,J=6.6,1.3Hz,1H) ,4.51-4.39(m,1H),4.35(s,2H),3.32(ddd,J=7.0,5.2,1.9Hz,1H),2.63(dd,J=14.2,8.6Hz,1H) ,2.54-2.48(m,1H),2.46(s,3H),2.39(d,J=14.0Hz,1H),1.61(s,1H),1.49(s,9H),1.40(s,3H). 13 C NMR(101MHz,Chloroform-d)δ209.8,155.0,145.1,138.5,134.4,130.0,129.5,128.8,127.8,120. 2,116.6,115.8,79.3,72.7,69.8,59.1,55.5,53.5,43.0,28.7,24.1,21.8.HRMS(ESI)calculated forC 27 H 33 N 4 O 5 S[M+H] + :525.2166, found 525.2159.

[0174] Embodiment 51

[0175] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0176]

[0177] At room temperature, P50 (0.3mmol, 158.1mg), sodium bicarbonate (0.36mmol, 30.2mg) and dichloromethane (5ml) were added to the reaction bottle. After cooling to 0°C, chloroacetyl chloride (0.36mmol, 28.8ul) was slowly added under stirring, and then the reaction system was moved to room temperature and stirred for 4 hours. After the reaction was completed, water was added to quench, ethyl acetate was extracted, and the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 4:1) to obtain a white solid product P51 (162.8mg, 90%).

[0178] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.90(d,J=8.3Hz,2H),7.48(d,J=8.1Hz,1H),7.41-7.35(m,2H),7.29(td,J=8.4,7.9,1.8 Hz,1H),7.22(td,J=7.5,1.3Hz,1H),7.00(dd,J=7.7,1.5Hz,1H),6.30(s,1H),5.21(s,1H),4.83(d,J=5.0Hz,1H),4.73 (dd,J=6.9,1.2Hz,1H),3.90(d,J=13.0Hz,1H),3.80(d,J=12.9Hz,1H),3.32(ddd,J=7.1,5.5,1.9Hz,1H),2.69(d,J=9. 5Hz,1H),2.52(d,J=9.2Hz,1H),2.49(s,3H),2.46(s,1H),1.66(s,1H),1.50(s,9H),1.40(s,3H),1.25(d,J=4.8Hz,1H). 13 C NMR(101MHz,Chloroform-d)δ207.1,165.6,154.9,145.6,134.2,131.9,130.0,129.1,128.8,127.4,127.3, 124.6,123.5,79.6,72.1,69.1,59.2,53.7,51.2,43.9,41.5,29.8,28.7,24.7,21.9.HRMS(ESI)calculated for C 29 H 32 C1N 4 O 6 S[MH]:599.1731, found 599.1738.

[0179] Embodiment 52

[0180] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0181]

[0182] At room temperature, P51 (0.1mmol, 60.3mg) and dichloromethane (1.5ml) were added to the reaction bottle. After cooling to 0°C, trifluoroacetic acid (3.0mmol, 223ul) was slowly added under stirring, and stirred at 0°C for 4 hours until the reaction was completed. After the reaction was completed, NaOH aqueous solution (3.0M) was added at 0°C to quench until the pH became alkaline, extracted with dichloromethane, and the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was slurried with methanol to obtain a white solid product P52 (48.3mg, 96%).

[0183] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.94-7.89(m,2H),7.47(d,J=8.5Hz,1H),7.38(d,J=8.3Hz,2H),7.2 9(dd,J=7.5,1.7Hz,1H),7.21(td,J=7.5,1.3Hz,1H),6.98(dd,J=7.7,1.8Hz,1H),5.26(s,1H),4. 85(d,J=5.5Hz,1H),4.50(d,J=6.9Hz,1H),3.93(d,J=12.9Hz,1H),3.83(d,J=13.0Hz,1H),3.27(d dd,J=7.1,5.5,1.9Hz,1H),2.48(s,3H),2.46(s,1H),2.20-2.15(m,2H),1.77(s,2H),1.20(s,3H). 13 C NMR(101MHz,Chloroform-d)δ207.7,165.7,145.5,134.2,132.0,130.0,129.0,128.9,127.4,127. 3,124.6,123.8,79.5,76.2,68.8,59.8,51.9,51.2,44.2,41.5,27.2,21.9.HRMS(ESI)calculated for C 24 H 26 C1N 4 O 4 S[M+H] + :501.1358,found501.1356.

[0184] Embodiment 53

[0185] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0186]

[0187] At room temperature, P2 (0.1mmol, 52.3mg) and dichloromethane (2ml) were added to the reaction flask. After cooling to 0°C, trifluoroacetic acid (3.0mmol, 223ul) was slowly added under stirring, and the mixture was stirred at 0°C for 40 minutes until the reaction was completed. After the reaction was completed, an aqueous NaOH solution (3.0M) was added at 0°C to quench the mixture until the pH became alkaline, and the mixture was extracted with dichloromethane. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain a white solid product P53 (42.4mg, quant).

[0188] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.84(d,J=8.2Hz,2H),7.36(d,J=8.1Hz,2H),7.27(t,J= 8.0Hz,1H),7.18(t,J=7.4Hz,1H),7.11(d,J=7.9Hz,1H),6.99(d,J=7.5Hz,1H),6.74(d d,J=10.3,2.1Hz,1H),6.54(s,1H),5.90(d,J=10.3Hz,1H),4.75(d,J=7.4Hz,1H),4.4 5(dd,J=7.2,2.1Hz,1H),3.39(t,J=7.1Hz,1H),2.46(s,3H),2.01(s,2H),1.39(s,3H). 13 C NMR(151MHz,Chloroform-d)δ191.3,156.1,151.4,146.2,138.5,132.0,130.3,129.9,129.8, 129.4,127.7,126.8,126.6,120.7,67.4,63.2,56.7,52.4,31.2,21.9.HRMS(ESI)calculated for C 22 H 25 N 4 O 3 S[M+H] + :423.1485, found 423.1488.

[0189] Embodiment 54

[0190] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0191]

[0192] At room temperature, P53 (0.09mmol, 37.4mg) and methanol (5ml) were added to the reaction bottle. After cooling to 0°C, sodium borohydride (0.1mmol, 3.7mg) was slowly added under stirring, and then the reaction system was moved to room temperature and stirred for 20 minutes. After the reaction was completed, saturated NH4Cl solution (1.0mL) was added to quench, extracted with ethyl acetate, and the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was slurried with methanol to obtain a white solid product P54 (33.8mg, 90%).

[0193] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.90(d,J=8.4Hz,2H),7.34(d,J=8.1Hz,2H),7.04(ddd,J=8.3,6.8,2.1H z,1H),6.77-6.67(m,2H),6.51(d,J=7.6Hz,1H),4.44(d,J=4.4Hz,1H),4.40(dd,J=6.6,1.3Hz,1H),4. 37(d,J=5.0Hz,1H),4.31(d,J=4.8Hz,1H),3.24(ddd,J=6.9,5.3,2.0Hz,1H),2.48(d,J=9.0Hz,1H),2. 45(s,3H),2.14(d,J=14.0Hz,1H),2.09-2.02(m,1H),1.75(s,3H),1.25(d,J=2.7Hz,1H),1.18(s,3H). 13 CNMR(101MHz,Chloroform-d)δ210.8,145.0,138.5,134.2,130.0,129.4,128.9,127.7,120. 1,116.9,115.9,79.5,69.3,59.7,55.5,51.5,43.2,29.8,26.9,21.8.HRMS(ESI)calculated for C 22 H 25 N 4 O 3 S[M+H] + :425.1642,found 425.1641.

[0194] Embodiment 55

[0195] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0196]

[0197] At room temperature, P22 (0.3mmol, 126.9mg) and methanol (6ml) were added to the reaction bottle. After cooling to 0°C, sodium borohydride (0.33mmol, 12.5mg) was slowly added under stirring, and then the reaction system was moved to room temperature and stirred for 20 minutes. After the reaction was completed, saturated NH4Cl solution (1.5mL) was added to quench, extracted with ethyl acetate, and the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was slurried with methanol to obtain a white solid product P55 (117.3mg, 92%).

[0198] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.94-7.90(m,2H),7.39-7.34(m,2H),7.04(ddd,J=8.1,7.0,1.8Hz,1 H),6.75-6.67(m,2H),6.52(dd,J=8.0,1.0Hz,1H),4.59(dt,J=5.1,1.4Hz,1H),4.48(dd,J=6.7,1.3 Hz,1H),4.42(d,J=5.1Hz,1H),4.14(d,J=5.1Hz,1H),3.57(s,1H),3.24(ddd,J=7.0,5.2,2.0Hz,1H ),2.52(d,J=9.1Hz,1H),2.46(s,4H),2.16(ddd,J=13.8,8.8,1.5Hz,1H),1.65(s,1H),1.26(s,3H). 13 C NMR(101MHz,Chloroform-d)δ209.8,145.3,138.5,133.7,130.2,129.6,128.8,127.6,120. 1,116.4,115.8,79.6,74.8,70.0,69.3,59.6,55.9,43.7,26.6,21.9.HRMS(ESI)calculated forC 22 H 24 N 3 O 4 S[M+H] + :426.1482,found426.1482.

[0199] Embodiment 56

[0200] The reaction formula for preparing the pyrazolidine compound in this embodiment is as follows:

[0201]

[0202] At room temperature, P55 (0.15mmol, 63.8mg), potassium carbonate (0.18mmol, 24.8mg) and dichloromethane (3ml) were added to the reaction bottle. After cooling to 0°C, acryloyl chloride (0.18mmol, 14.5ul) was slowly added under stirring, and then the reaction system was moved to room temperature and stirred for 4 hours. After the reaction was completed, water was added to quench, and dichloromethane was extracted. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The crude product was purified by flash column chromatography on silica gel (petroleum ether: ethyl acetate = 3:2) to obtain a white solid product P56 (57.5mg, 80%).

[0203] Data Characterization: 1 H NMR(400MHz,Chloroform-d)δ7.95-7.89(m,2H),7.35(d,J=8.1Hz,2H),7.22(td,J=7.7,1.6Hz,1H),7.16(td,J= 7.5,1.3Hz,1H),7.12(d,J=8.1Hz,1H),6.97(dd,J=7.7,1.6Hz,1H),6.45-6.31(m,2H),5.74(dd,J=9.3,2.7Hz,1H ),5.71(s,1H),4.70(d,J=5.3Hz,1H),4.63(dd,J=6.8,1.3Hz,1H),3.26(d,J=5.0Hz,1H),3.23(td,J=5.3,2.7Hz, 1H), 2.57 (d, J = 14.0Hz, 1H), 2.48 (d, J = 1.9Hz, 1H), 2.46 (s, 3H), 2.23 (ddd, J = 14.0, 8.7, 1.4Hz, 1H), 1.27 (s, 3H). 13 C NMR(101MHz,Chloroform-d)δ207.9,165.1,145.4,133.9,132.5,130.1,129.9,129.2,128.8,128.7,1 27.5,126.3,123.9,122.9,77.9,74.2,70.1,69.4,59.9,51.8,44.3,27.1,21.9.HRMS(ESI)calculated for C 25 H 26 N 3 O 5 S[M+H] + :480.1588,found 480.1586.

[0204] Example 57: Amplification reaction

[0205] Referring to Example 2, the preparation method is as follows: under air conditions, N2 (3 mmol, 669.0 mg) and S1 (3.6 mmol, 1076.4 mg) are dissolved in CH 2 Cl 2 (15.0 mL), and the reaction was terminated after 56 hours at 70°C. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1), and the product P2 (1.41 g, 90%) was obtained as a white solid.

[0206] Referring to Example 22, the preparation method is as follows: under air conditions, N21 (3 mmol, 372.4 mg) and S1 (3.6 mmol, 1076.4 mg) are dissolved in CH 2 Cl 2 (15.0 mL), reacted at 40°C for 24 hours. The reaction solution was directly concentrated under reduced pressure, and the crude product was separated by silica gel column chromatography, eluted with petroleum ether and ethyl acetate (petroleum ether:ethyl acetate=2:1), to obtain product P21 (1.1 g, 87%), which was a white solid.

[0207] Stereo configuration characterization of the above products:

[0208] Figure 1 The single crystal structures of products P1 and P21 and the corresponding X-ray single crystal diffraction data are shown below:

[0209]

[0210]

[0211] Correspondingly, other products P1~P49 and P53 also have the same configuration.

[0212] Figure 2 The single crystal structure diagram of the product P51 and the corresponding X-ray single crystal diffraction data are shown below:

[0213]

[0214]

[0215]

[0216] Correspondingly, other products P52 and P54-P56 also have consistent configurations.

[0217] Example 58: Comparison of solvents

[0218] Referring to Example 1, the reaction solvent was replaced with other solvents shown in Table 1, the reaction temperature was changed to 60° C., and the other conditions remained unchanged. The results of the corresponding reactions are shown in Table 1.

[0219] Table 1 Effect of solvent on the preparation of pyrazolidine

[0220] Solvents P1 yield (%) DCM 79 Chloroform 55 <![CDATA[CH 3 OH]]> trace <![CDATA[CH 3 CN]]> 30 THF 20 Toluene 50 Chlorobenzene 50 Acetone 20 DMF 40 DCE 70

[0221] Correspondingly, the other products P1-P20 and P38-P43 also have the same conditions.

[0222] Referring to Example 21, only the reaction solvent was replaced with other solvents shown in Table 2, and the other conditions remained unchanged. The results of the corresponding reactions are shown in Table 2.

[0223] Table 2 Effect of solvent on the preparation of pyrazolidine

[0224]

[0225]

[0226] Correspondingly, the other products P21-P37 and P44-P49 also have the same conditions.

[0227] Example 59: Catalyst Exploration and Comparison

[0228] Referring to Example 1, the catalyst shown in Table 3 was added, the reaction temperature was changed to 60° C., and the other conditions remained unchanged. The results of the corresponding reactions are shown in Table 3.

[0229] Table 3 Effect of catalyst on the preparation of pyrazolidine

[0230] catalyst P1 yield (%) <![CDATA[K 2 WHAT 3 (1.2eq)]]> n.r. <![CDATA[Cs 2 WHAT 3 (1.2eq)]]> n.r. DABCO(0.2eq) 64 DIPEA (0.2eq) 65 DBU(0.2eq) 64 TsOH (0.2eq) 67 / 79

[0231] Correspondingly, the other products P1-P20 and P38-P43 also have the same conditions.

[0232] Referring to Example 21, the catalyst shown in Table 4 was added, and the other conditions remained unchanged. The results of the corresponding reactions are shown in Table 4.

[0233] Table 4 Effect of catalyst on the preparation of pyrazolidine

[0234] catalyst P21 yield (%) <![CDATA[K 2 WHAT 3 (1.0eq)]]> 75 <![CDATA[Cs 2 WHAT 3 (1.0eq)]]> messy DABCO(0.2eq) 40 DIPEA (0.2eq) 35 DBU(0.2eq) messy TsOH (0.2eq) messy <![CDATA[(PhO) 2 PO 2 H(0.2eq)]]> messy <![CDATA[Sc(OTf) 3 (0.2eq)]]> 40 <![CDATA[In(OTf) 3 (0.2eq)]]> messy <![CDATA[NHTf 2 (0.2eq)]]> 70 / 89

[0235] Correspondingly, the other products P21-P37 and P44-P49 also have the same conditions.

[0236] Example 60: Temperature Exploration and Comparison

[0237] Referring to Example 1, only the temperature was replaced with other temperatures shown in Table 5, and the other conditions remained unchanged. The results of the corresponding reactions are shown in Table 5.

[0238] Table 5 Effect of temperature on the preparation of pyrazolidine

[0239] temperature P1 yield (%) 60℃ 79 70°C (Example 1) 88 80℃ 89

[0240] Correspondingly, the other products P1-P20 and P38-P43 also have the same conditions.

[0241] Referring to Example 21, only the temperature was replaced with other temperatures shown in Table 6, and the other conditions remained unchanged. The results of the corresponding reactions are shown in Table 6.

[0242] Table 6 Effect of temperature on the preparation of pyrazolidine

[0243]

[0244]

[0245] Correspondingly, the other products P21-P37 and P44-P49 also have the same conditions.

[0246] Example 61: Anti-inflammatory activity test of the compounds of the present invention

[0247] BV2 cells were induced with 1 μg / mL LPS to construct a neuroinflammatory cell model. BV2 cells were treated with compounds at a concentration of 20 μM for 24 hours. Nitric oxide (NO) was used as an important signaling molecule for evaluating neuroinflammation. Its inhibition rate could be used to evaluate the inhibitory activity of neuroinflammation. The Griess kit method was used to detect the NO secretion level in the supernatant of BV2 cells after 24 hours of treatment. Finally, different concentrations of sodium nitrite were diluted to detect the NO content released. The standard curve was fitted, and the NO inhibition percentage of the test compound was calculated with the LPS model group as 100%.

[0248] When testing the EC50 of the compound, the compound to be tested was diluted with different concentrations, and the same treatment was performed according to the above method. The NO inhibition rate at different compound concentrations was calculated, and the compound EC50 was fitted using Grapad mapping software.

[0249] The cells were seeded in a 96-well culture plate at a density of 10,000 cells per well, treated with 20 μM compounds, and incubated for 30 min in a cell culture incubator after 24 h with CCK8 reagent. Finally, the absorption rate (OD) at 450 nm was measured using an ELISA reader. The preferred compounds were diluted to different concentrations and treated according to the above method. Finally, the cell viability of the compounds at different concentrations was calculated, and the compound IC50 was calculated using the fitting curve. The cell viability calculation formula for BV-2 cells is as follows: Cell viability (%) = (OD sample-OD blank) / (OD blank-OD control) X 100%.

[0250] Extract RNA using Trizol method: a. Preparation of operating consumables: Prepare uncontaminated DEPC water and unopened gun tips and other consumables in advance. b. Trizol digestion: discard the culture medium, add pre-cooled PBS to wash once, and add 1mL of Trizol to each well of the 6-well plate to digest the cells for 5 minutes. c. Add chloroform: transfer the cells and Trizol solution to a centrifuge tube, and add 300μL of chloroform, shake violently, and let it stand for 4 minutes after emulsification. d. Centrifugation: Put the centrifuge tube in a refrigerated centrifuge for centrifugation under the conditions of: 4℃, 12000rpm', 15min. After centrifugation, the vertical centrifuge tube can be seen to have three layers of liquid, namely, colorless aqueous phase layer, protein layer, and phenol-chloroform layer. Aspirate the top colorless aqueous phase layer. If pink substance is aspirated, it can be centrifuged again and taken again. Generally, 300uL is enough for subsequent experiments. e. Mixing with isopropanol: According to the above steps, take an equal volume of supernatant and add analytical grade isopropanol, use a gun to blow and hit the liquid in the centrifuge tube to mix it, and place the centrifuge tube on a normal temperature tube rack for 15 minutes. f. Centrifugation: After the standing is over, centrifuge the liquid in the centrifuge tube at 4°C, 12000rpm, 15min. After the centrifugation is over, use the tip of the gun to absorb the supernatant, and finally a trace of white precipitate can be seen under the tube wall. g. Wash the precipitate with ethanol: Use DEPC water to prepare 75% ethanol, add 1mL of anhydrous ethanol to each centrifuge tube, and finally use the tip of the gun to gently mix the white precipitate. Generally, it can be centrifuged after washing 5 times. The centrifugation conditions are: 4°C, 8000rpm centrifugation for 5min, and the supernatant is discarded after the centrifugation is over. h. Dissolving the precipitate: Open the cap of the centrifuge tube, place the centrifuge tube horizontally under the fume hood, wait for the anhydrous ethanol in the centrifuge tube to evaporate, and the white precipitate to melt at the same time. Generally, this process can be completed in 20 minutes. When the white precipitate turns colorless, add different volumes of DEPC water to each tube according to the amount of precipitate. Generally, add a small amount of DEPC water first, and then add more if the subsequent concentration is high. After adding DEPC, tap the bottom of the EP tube, separate it at 4℃, and place it on ice.

[0251] RNA concentration determination: According to the instruction manual of the protein nucleic acid quantification instrument, the RNA concentration was determined. The probe was first washed with DEPC water, and DEPC water was used as a blank control. 1 μL of sample was taken on the probe of the protein nucleic acid quantification instrument for RNA content and purity detection. If the A260 / 280 value on the left side of the instrument is between 1.8-2.0, it indicates that the RNA purity is high. If A260 / 280 is less than 1.8, it indicates that the extracted RNA contains impurities such as protein. If A260 / 280 is greater than 2.0, it indicates that RNA degradation exists. If the RNA concentration is too high, DEPC water can be added for dilution. The general measured concentration is about 200 ng / μL. In the next step, the reverse transcription template RNA should be loaded as much as possible, and less DEPC water should be added to ensure the accuracy of loading.

[0252] Reverse transcription process: a. Genome removal: According to the total system of 16μL, the amount of RNA is 1μg, add the required volume of each sample, add 4μL of genome reagent, and add DEPC water to the total system to 20μL, centrifuge, and finally perform genome removal on the PCR instrument at 42℃, 2min. b. Reverse transcription sample: Take 16μL of genome removal sample and add 4μL of 5xMix to the PCR tube, and reverse transcribe the sample into cDNA according to the procedure.

[0253] Fluorescence quantitative PCR experiment:

[0254] a. Template loading: Add 180 μL of DEPC water to the reverse transcription sample to dilute the sample 10 times, and add various reaction components into the 96-well plate according to the SYBR qPCRMix instructions. The specific system is as follows:

[0255]

[0256]

[0257] b. After the sample is loaded, centrifuge it and use ABI's Quantstudio TM 7Pro Real-Time PCR System was used for detection. The running program settings are shown in the following table.

[0258]

[0259] c. Data analysis: After the run is completed, check whether the amplification curve is complete and whether the melting curve is a single peak on the instrument, export the CT value, process the data in Excel, and use GraphPrism to draw graphs.

[0260] A total of nine compounds, including compounds P5, P21, and P52-P56, were preliminarily tested for their anti-inflammatory activity. The NO level and cell survival rate were tested respectively. By comparing compounds P5, P52, and P56, it was found that these three compounds had little effect on the survival rate of BV2 cells, and the NO level was low, indicating that these four compounds have good anti-neuroinflammatory activity. Therefore, different concentrations of these four compounds were set to test the NO level, and the specific EC was calculated using Granpad fitting. 50 value.

[0261] Table 7 Preliminary measurement of NO levels and cell viability of 9 compounds

[0262] Group NO level (n=4) average Cell survival rate (%) LPS 100 100 DEX 31.4 83.4 P5 10.9 110.2 P21 7.3 23.6 P30 5.5 23.9 P32 5.5 24.6 P52 6.1 89.7 P53 6.7 27.4 P54 48.8 90.0 P55 54.3 90.4 P56 8.5 102.4

[0263] Note: For the initial test of NO, LPS was taken as 100% and the initial test concentration of the compound was 20 μM. For the effect of the compound concentration of 20 μM on the survival rate of BV2 cells, no addition of the compound was taken as 100%.

[0264] As shown in Table 1, the compounds of the present invention can reduce the cellular NO level. Among them, compounds P5, P52 and P56 significantly reduced the cellular NO level in LPS-treated microglia in a dose-dependent manner. 50 The values ​​were 2.4, 0.27 and 3.2 μM, respectively, indicating significant anti-inflammatory activity. Compounds P5, P52, P54, P55 and P56 all showed good cell tolerance. Figure 1 BD)

[0265] In addition, by detecting the expression levels of inflammatory markers, it was found that compound P52 could significantly reverse the upregulation of iNOS, Cox 2, Tnf-α and Il-6 induced by LPS ( Figure 2 EH).

[0266] In summary, the compounds provided by the present invention show broad application prospects in the development of anti-neuroinflammatory drugs.

[0267] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A pyrazolidine compound as shown in the general formula III or IV, or a pharmaceutically acceptable salt or deuterated product thereof; In the formula, R is selected from: C1-C6 straight chain or branched alkyl, C3-C6 cycloalkyl, C1-C6 straight chain or branched alkoxy, -(CH2) n -R a , substituted or unsubstituted aryl; the substitution on the aryl includes any one of mono- to tri-substitution, and the substituent is selected from halogen, cyano, C1-C8 straight or branched alkyl, C1-C8 alkoxy; n is 1, 2, 3, R a It is a C2-C7 ester group, a 4-10 membered alicyclic group, or a siloxy group; R' is NHPG or OR", where PG means Ts, Boc, Cbz, Bz or Ac, R" is H or C1-C6 straight or branched chain alkyl; R 1 Selected from: H, halogen, C1-C6 straight or branched alkyl, phenyl; R 2 Selected from: H, halogen, C1-C6 alkoxy; R 3 Selected from: H, C1-C6 straight chain or branched alkyl; R 4 Selected from: H, -C(O)R5; R5 is selected from C2-C8 alkenyl, halogen-containing C1-C6 straight or branched alkyl.

2. The pyrazolidine compound according to claim 1, or a pharmaceutically acceptable salt or deuterated product thereof, characterized in that: Aryl is selected from: phenyl, naphthyl, thienyl; C2-C7 ester group is -COOR b , R b is a C1-C6 straight chain or branched chain alkyl; The 4-10 membered aliphatic heterocyclic group contains 1-3 heteroatoms, including N, O, and S; Siloxy is -O-SiR c R d R e , R c , R d , R e Each is independently selected from C1-C6 straight chain or branched alkyl.

3. The pyrazolidine compound according to claim 1, or a pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: R is specifically selected from: R 1 Specific selection: R 2 Specific selection: R 3 Specific selection: R 4 Specific selection:

4. The pyrazolidine compound according to claim 1, or a pharmaceutically acceptable salt or deuterated substance thereof, characterized in that: The pharmaceutically acceptable salt is an inorganic salt or an organic salt; wherein the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; and the organic salt is selected from formates, acetates, trifluoroacetates, propionates, pyruvates, glycolates, oxalates, malonates, succinates, glutarates, fumarates, maleates, lactates, malates, citrates, tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, salicylates, p-toluenesulfonates, and ascorbates.

5. The pyrazolidine compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt or deuterated product thereof, characterized in that: The pyrazolidine compound is specifically selected from:

6. A method for synthesizing the pyrazolidine derivative represented by formula III as claimed in claim 1, characterized in that: The method is: The cyclohexadienone compound shown in formula I and the azomethine imine shown in formula II are used as raw materials, and the tetracyclic pyrazolidine compound shown in formula III can be obtained by reaction: Among them, R, R', R 1 , R 2 , R 3 The definition is the same as that of claim 1.

7. A method for synthesizing a pyrazolidine derivative represented by formula IV, characterized in that: The reaction route of the synthetic method is: Among them, R 1 , R 2 , R 4 H, R, R', R 3 Same as the definition in claim 1, (1) using a cyclohexadienone compound represented by formula I and an azomethine imine represented by formula II as raw materials, and reacting to obtain a tetracyclic pyrazolidine compound represented by formula III; (2) The obtained tetracyclic pyrazolidine compound is reacted in a sodium borohydride and methanol reaction system to obtain a pyrazolidine derivative represented by formula IV.

8. A pharmaceutical composition for anti-neuroinflammation, characterized in that: It contains the pyrazolidine compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or deuterated product thereof, and pharmaceutical excipients.

9. Use of the pyrazolidine derivative according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or deuterated product thereof in the preparation of a medicament for preventing or treating diseases associated with neuroinflammation.

10. The use according to claim 9, characterized in that: The diseases associated with neuroinflammation generally include Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, stroke, brain trauma, epilepsy, depression, schizophrenia, chronic pain, viral encephalitis, bacterial meningitis, autoimmune encephalitis, Huntington's disease, and HIV-related neurocognitive disorders.

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