CN119241615A - A glycosylated JAK inhibitor prodrug and its preparation method and application - Google Patents

Abstract

The present invention discloses a glycosylated JAK inhibitor prodrug and its preparation method and application. The glycosylated JAK inhibitor prodrug has a general structural formula as shown in Formula 1, where JAKi represents a small molecule JAK inhibitor; Represents various sugar groups. The series of glycosylated JAK inhibitors prepared by the present invention have obvious colon-targeted drug release. After oral administration, the original drug components are almost undetectable in the small intestine and plasma. After reaching the colon, a large amount of active ingredients will be released to exert anti-inflammatory effects, which not only exerts a targeted drug release effect, but also greatly reduces the systemic toxicity and side effects of the drug. This shows that this type of glycosylated JAK inhibitor is an orally available IBD targeted anti-inflammatory drug. Compared with the original small molecule JAK inhibitor, it has higher safety and is expected to have more significant anti-inflammatory effects. It is a new type of prodrug that releases drugs locally and exerts its efficacy.

Description

Glycosylated JAK inhibitor prodrug as well as preparation method and application thereof

Technical Field

The invention belongs to the field of chemical medicaments, and in particular relates to a glycosylated JAK inhibitor, a pharmaceutical application thereof, a medicament or prodrug thereof and a preparation method thereof.

Background

Janus kinase (hereinafter JAK) is a non-transmembrane tyrosine kinase and plays an important role in the signaling pathway of many cytokines. The JAK family consists of JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK 2), which are key components in JAK signal transducers and activators of the transcriptional pathway that phosphorylate cytokine receptors and signaling molecules containing src homology domains, playing a key role in cytokine receptor signaling pathways. JAK is a key node for signal transduction, plays a key role in the occurrence and development of diseases such as inflammation, autoimmune diseases, malignant tumors and the like, and plays an important role in regulating and controlling the growth and development of hematopoietic cells. JAK inhibitors have been widely used in the treatment of a variety of diseases. For example, JAK1 and JAK3 inhibitors are used to treat arthritis and JAK2 inhibitors are used to treat breast cancer and blood cancer. These diseases are all associated with activation of cytokine signaling pathways, and JAK inhibitors can slow down disease progression by inhibiting activation of these signaling pathways. Currently, more than ten JAK inhibitors have been approved for the treatment of various diseases driven by immune mechanisms.

Inflammatory bowel disease (inflammatoryboweldisease, IBD) is a chronic, non-specific inflammatory disease of the intestinal tract, including Crohn's Disease (CD) and ulcerative colitis (ulcerativecolitis, UC), which has the characteristics of long course, difficult cure, easy recurrence, etc. Traditional therapeutic agents for IBD include 5-aminosalicylic acid, corticosteroids, and immunosuppressants. In recent years, more and more biological agents targeting cytokines, cytokine receptors, inflammatory signal pathways and the like and novel oral small molecule drugs are gradually coming into the public view. Wherein the novel small molecule drug JAK inhibitor inhibits the combination of JAK kinase and cytokine by blocking the JAK/STAT pathway, reduces the expression of inflammatory factors, and effectively inhibits the occurrence and development of inflammation in IBD. However, JAK inhibitors often accompany adverse effects such as dizziness, nausea, upper respiratory tract infections, and urinary tract infections, as JAK is widely present in the signaling pathway of many cytokines. Therefore, developing JAK inhibitors with intestinal targeting, low toxicity and mild side effects has important academic value and economic significance for the treatment of IBD.

Disclosure of Invention

The invention provides a small molecule JAK inhibitor glycosylation prodrug based on colorectal part drug release strategy, which can effectively reduce systemic toxic and side effects of the drug. The focal site of IBD, colorectal, has a rich diversity of intestinal flora that secrete large amounts of biological enzymes, a major class of which are glycosidases. The invention provides a glycosylation method of a JAK inhibitor and provides a series of glycosylated JAK inhibitor prodrugs, wherein the prodrugs can be orally taken, stably exist in the stomach and the small intestine, are safe and nontoxic, and cut off sugar chains under the action of intestinal flora enzyme-glycosidase after reaching the large intestine to release JAK inhibitor active molecules so as to achieve the anti-inflammatory effect of colorectal directional drug release.

The structural general formula of the glycosylation JAK inhibitor prodrug provided by the invention is shown as formula 1:

in formula 1 JAKi represents a small molecule JAK inhibitor,

Further, the small molecule JAK inhibitor is a JAK inhibitor with an anti-colitis effect;

Still further, the small molecule JAK inhibitors include, but are not limited to, JAK inhibitors represented by formulas I-VII;

In the formula 1, the components are mixed, Represents various glycosyl groups obtainable from various saccharide molecules (such as monosaccharides, disaccharides, trisaccharides and polysaccharides) including, but not limited to, saccharide molecules represented by formulae a-j;

further, the glycosylated JAK inhibitor prodrug may be selected from compounds of the following structure:

the action mechanism of the glycosylated JAK inhibitor prodrug provided by the invention is as follows:

the invention also provides a preparation method of the glycosylation JAK inhibitor prodrug shown in the formula 1.

The synthetic route for the glycosylated JAK inhibitor prodrug of formula 1 is shown below:

the preparation method of the glycosylation JAK inhibitor prodrug shown in the formula 1 specifically comprises the following steps:

1) Reacting a small molecule JAK inhibitor (JAKi) represented by formula 2 with formaldehyde to obtain a methylolated JAK inhibitor represented by formula 3;

2) Carrying out glycosylation coupling reaction on a methylolated JAK inhibitor shown in a formula 3 and a compound shown in a formula 5 under the catalysis of trimethyl silicone triflate (TMSOTF) to obtain a compound shown in a formula 6;

3) Deprotection of the protecting group R in the compound of formula 6 affords the glycosylated JAK inhibitor prodrug of formula 1.

In the step 1), the small molecule JAK inhibitor shown in the formula 2 includes, but is not limited to, JAK inhibitors shown in the formulas I to VII.

In the step 1), water is also added in the reaction, wherein the water solution JAKi is formaldehyde water solution (mass volume concentration is 37 percent), and the feeding ratio of the water is 1-1.2 mmol to 1.2-1.5 mL to 1-1.5 mL in sequence;

In the step 1), the reaction condition of the reaction is 45-55 ℃ for 1-3 hours.

In the above step 2), in the formula 5, r=ac (acetyl) or Bz (benzoyl), or a protecting group of other saccharide molecules.

In the step 2), the preparation method of the compound shown in the formula 5 comprises the following steps of taking a saccharide molecule shown in the formula 4 as a raw material, and sequentially carrying out conventional reactions in three-step saccharide chemistry of acylation, hydrolysis and trichloroacetimidation to obtain the compound shown in the formula 5.

The saccharide molecules represented by formula 4 include, but are not limited to, saccharide molecules represented by formulas a-j above.

In the step 2), the molar ratio of the compound shown in the formula 5, the methylolated JAK inhibitor shown in the formula 3 and the trimethylsilyl triflate is 1 (1.0-2.0) to 0.1-0.3.

In the step 2), the reaction condition of the reaction is 0-25 ℃ for 0.5-2 hours.

The invention also protects the use of the glycosylated JAK inhibitor prodrugs described above.

The application is the application of glycosylation JAK inhibitor prodrug in preparing anti-inflammatory drugs.

Further, the anti-inflammatory drug is a drug for preventing and/or treating inflammatory bowel disease (inflammatoryboweldisease, IBD).

Still further, the inflammatory bowel disease (inflammatoryboweldisease, IBD) includes Crohn's Disease (CD) and ulcerative colitis (ulcerativecolitis, UC).

Further, the anti-inflammatory drug has colon-targeted drug release effect.

The invention also protects an anti-inflammatory drug for colon targeted drug release.

The active ingredients of the medicine provided by the invention comprise glycosylated JAK inhibitor prodrugs shown in the formula 1.

Further, the anti-inflammatory drug has the efficacy of preventing and/or treating inflammatory bowel disease (inflammatoryboweldisease, IBD).

The invention also provides a method for preventing and/or treating inflammatory bowel disease (inflammatoryboweldisease, IBD).

The method comprises the step of administering to a recipient animal or human a glycosylated JAK inhibitor prodrug of formula 1 of the present invention.

Compared with the prior art, the invention has the following beneficial effects:

The series of glycosylated JAK inhibitors prepared by the invention have obvious colon targeted drug release effect, hardly detect the original drug ingredients in small intestine and blood plasma after oral administration, release a large amount of active ingredients to play an anti-inflammatory role after reaching colon, play a role in targeted drug release, and greatly reduce systemic toxic and side effects of the drug, so that the glycosylated JAK inhibitors are IBD targeted anti-inflammatory drugs which can be taken orally, have higher safety and are expected to have more obvious anti-inflammatory effects compared with the original drug small molecule JAK inhibitors, and are novel prodrugs which can be locally released and exert drug effects.

Drawings

FIG. 1 shows the histopathological score of mice in the colitis model after drug treatment, and the administration by gavage was performed 1 time a day with 5. Mu. Mol/mg/day for 7 consecutive days.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 Synthesis of glycosylated JAK inhibitor prodrugs

The synthesis process comprises the following steps:

1) Under the protection of nitrogen, the compound 2 (2-I-2-VII) (1.0 mmol,1.0 eq) is added into formaldehyde aqueous solution (37%, 1.2 mL) and water (1 mL) to react for 1 hour at 45 ℃. And (3) performing reduced pressure distillation and freeze drying by a freeze dryer to obtain a mixture white solid powder compound 3 (3-I-3-VII) which is directly used for the next reaction. (this step of reaction means step 1)

2) The peracetyl or perbenzoyl trichloroacetimidate compound 5 (5-a-5-j) (1.0-2.0 eq) can be synthesized by taking saccharide molecules 4 (4-a-4-j) as raw materials according to a conventional method, and the compound 3 (3-I-3-VII) (1.0 mmol,1.0 eq) is dissolved in dry acetonitrile (or dichloromethane, acetone), trimethyl silicone triflate (0.1-0.3 eq) is added, the mixture is reacted for 0.5 hours at 25 ℃, triethylamine is added for neutralization, and the liquid is concentrated to obtain a crude product (the reaction in the step is referred to as step 2).

3) Dissolving the crude product in a methanol-dichloromethane (1/1-1/4) mixed solution, dropwise adding 1M sodium methoxide to the pH of a system to 9-11, reacting for 2 hours at room temperature, adding acidic Resin IR120, stirring to the pH of the system to 7.0, filtering out the Resin, concentrating the filtrate, and passing through a silica gel column, wherein the mobile phase ethyl acetate is methanol: water=7:2:0-7:2:1, and obtaining a white powdery product 1 (1-a-I-1-j-VII). (this step of reaction means step 3)

Compound yield and structural characterization:

Yield rate 65.4％.¹H NMR(500MHz,D₂O):δ8.52(s,1H),7.48(s,1H),7.42(t,J＝2.9Hz,1H),6.99–7.02(m,1H),6.94(t,J＝6.0Hz,1H),5.33(d,J＝10.8Hz,1H),5.12(d,J＝10.8Hz,1H),4.37(d,J＝7.9Hz,1H),4.30–4.35(m,1H),4.09(d,J＝4.2Hz,1H),3.74–3.90(m,4H),3.68(dd,J＝10.3,6.9Hz,1H),3.62(dd,J＝11.6,4.0Hz,1H),3.40–3.47(m,1H),3.26(dd,J＝10.2,6.0Hz,1H),3.05–3.13(m,2H),2.95(td,J＝8.2,4.8Hz,1H),2.54(dt,J＝10.8,5.4Hz,1H),1.05–1.14(m,1H),0.76–0.85(m,1H),0.61–0.66(m,3H).ESI-HRMS:m/z calculated for C₂₄H₃₂F₃N₆O₇[M-H]^-:560.2206,found:560.2209.

Yield rate 63.5％.¹H NMR(500MHz,D₂O):δ8.51(s,1H),7.47(s,1H),7.40(t,J＝3.0Hz,1H),6.99–7.01(m,1H),6.92(t,J＝6.0Hz,1H),5.50(d,J＝11.0Hz,1H),5.31(d,J＝11.0Hz,1H),4.56(d,J＝7.7Hz,1H),4.31–4.36(m,1H),3.90(d,J＝3.5Hz,1H),3.60–3.91(m,7H),3.51(dd,J＝9.8,7.4Hz,1H),3.24(dd,J＝10.0,5.8Hz,1H),2.52(dt,J＝10.6,5.2Hz,1H),1.09–1.14(m,1H),0.77–0.84(m,1H),0.62–0.66(m,3H).ESI-HRMS:m/z calculated for C₂₄H₃₂F₃N₆O₇[M-H]^-:560.2206,found:560.2208.

Yield rate 62.8％.¹H NMR(500MHz,D₂O):δ8.52(s,1H),7.48(s,1H),7.42(t,J＝2.9Hz,1H),6.99–7.02(m,1H),6.94(t,J＝6.0Hz,1H),5.37(d,J＝11.1Hz,1H),5.27(d,J＝11.1Hz,1H),5.10(d,J＝1.6Hz,1H),4.30–4.35(m,1H),3.95(dd,J＝3.3,1.8Hz,1H),3.77–3.92(m,6H),3.71(dd,J＝12.4,6.4Hz,1H),3.68(dd,J＝10.3,6.9Hz,1H),3.56–3.66(m,2H),3.26(dd,J＝10.0,6.1Hz,1H),2.55(dt,J＝10.7,5.2Hz,1H),1.07–1.14(m,1H),0.75–0.82(m,1H),0.60–0.65(m,3H).ESI-HRMS:m/z calculated for C₂₄H₃₂F₃N₆O₇[M-H]^-:560.2206,found:560.2209.

Yield rate 65.1％.¹H NMR(500MHz,D₂O):δ8.68(s,1H),7.55(s,1H),7.32(t,J＝2.8Hz,1H),6.90–6.94(m,1H),6.86(t,J＝6.0Hz,1H),5.41(d,J＝11.3Hz,1H),5.30(d,J＝11.3Hz,1H),4.51(d,J＝7.8Hz,1H),4.35–4.38(m,1H),3.70–3.90(m,7H),3.61(dd,J＝9.9,3.4Hz,1H),3.49(dd,J＝9.9,7.8Hz,1H),3.26(dd,J＝10.0,6.1Hz,1H),2.51–2.59(m,1H),1.22(d,J＝6.2Hz,3H),1.09–1.17(m,1H),0.74–0.81(m,1H),0.62–0.67(m,3H).ESI-HRMS:m/z calculated for C₂₄H₃₀F₃N₆O₆[M-H]^-:555.2179,found:555.2171.

Yield rate 66.3％.¹H NMR(500MHz,D₂O):δ8.77(s,1H),7.68(s,1H),7.49(t,J＝2.4Hz,1H),7.04–7.12(m,1H),6.99(t,J＝6.0Hz,1H),5.59(d,J＝11.3Hz,1H),5.40(d,J＝11.3Hz,1H),4.56(d,J＝7.7Hz,1H),4.31–4.37(m,1H),3.92(dd,J＝11.4,5.2Hz,1H),3.74–3.90(m,4H),3.56–3.65(m,2H),3.41(t,J＝9.0Hz,1H),3.29–3.37(m,3H),2.57(dt,J＝10.4,5.0Hz,1H),1.02–1.12(m,1H),0.78–0.86(m,1H),0.60–0.64(m,3H).ESI-HRMS:m/z calculated for C₂₃H₂₈F₃N₆O₆[M-H]^-:541.2022,found:541.2012.

Yield rate 64.7％.¹H NMR(500MHz,D₂O):δ8.50(s,1H),7.44(s,1H),7.40(t,J＝3.2Hz,1H),6.94–7.00(m,1H),6.90(t,J＝6.0Hz,1H),5.32(d,J＝10.7Hz,1H),5.10(d,J＝10.8Hz,1H),4.44(d,J＝7.9Hz,1H),4.30–4.34(m,1H),4.25(d,J＝7.3Hz,1H),3.70–3.84(m,4H),3.57–3.63(m,3H),3.22(dd,J＝10.0,6.0Hz,1H),3.02(td,J＝8.2,5.3Hz,1H),2.57(dt,J＝10.8,5.4Hz,1H),1.09–1.15(m,1H),0.74–0.80(m,1H),0.65–0.69(m,3H).ESI-HRMS:m/z calculated for C₃₀H₄₀F₃N₆O₁₂[M-H]^-:733.2656,found:733.2650.

Yield rate 65.9％.¹H NMR(500MHz,D₂O):δ8.66(s,1H),7.71(s,1H),7.55(s,1H),6.91–7.02(m,2H),5.50(d,J＝11.3Hz,1H),5.40(d,J＝3.9Hz,1H),5.36(d,J＝11.3Hz,1H),4.68(d,J＝8.0Hz,1H),4.31–4.37(m,1H),3.70–3.91(m,8H),3.54–3.67(m,4H),3.48(dt,J＝6.7,3.1Hz,2H),3.32(t,J＝9.5Hz,1H),3.27–3.22(m,2H),2.50(dt,J＝10.7,5.2Hz,1H),1.00–1.11(m,1H),0.76–0.85(m,1H),0.61–0.66(m,3H).ESI-HRMS:m/zcalculated for C₃₀H₄₀F₃N₆O₁₂[M-H]^-:733.2656,found:733.2649.

Yield rate 55.2％.¹H NMR(500MHz,D₂O):δ8.57(s,1H),7.48(s,1H),7.40(t,J＝2.9Hz,1H),6.99–7.03(m,1H),6.91(t,J＝6.1Hz,1H),5.50(d,J＝11.3Hz,1H),5.36(d,J＝11.3Hz,1H),4.68(d,J＝8.0Hz,1H),4.58(d,J＝7.8Hz,1H),4.31–4.35(m,1H),3.59–3.91(m,10H),3.48(dt,J＝6.7,3.1Hz,2H),3.32(t,J＝9.5Hz,1H),3.22–3.27(m,2H),2.54(dt,J＝10.8,5.4Hz,1H),1.05–1.14(m,1H),0.79–0.88(m,1H),0.62–0.67(m,3H).ESI-HRMS:m/z calculated for C₃₀H₄₀F₃N₆O₁₂[M-H]^-:733.2656,found:733.2651.

Yield rate 50.1％.¹H NMR(500MHz,D₂O):δ8.52(s,1H),7.48(s,1H),7.42(t,J＝2.9Hz,1H),6.99–7.02(m,1H),6.94(t,J＝6.0Hz,1H),4.69–4.73(m,2H),4.65(d,J＝7.8Hz,1H),4.30–4.35(m,1H),3.42–3.90(m,21H),3.22(dd,J＝10.0,5.9Hz,1H),2.50(dt,J＝10.6,5.0Hz,1H),1.00–1.10(m,1H),0.72–0.80(m,1H),0.60–0.65(m,3H).ESI-HRMS:m/z calculated for C₃₆H₅₀F₃N₆O₁₇[M-H]^-:895.3185,found:895.3172.

Yield rate 52.9％.¹H NMR(500MHz,D₂O):δ8.44(s,1H),7.40(s,1H),7.33(t,J＝2.8Hz,1H),6.99–7.01(m,1H),6.90(t,J＝6.2Hz,1H),5.23(t,J＝4.5Hz,2H),4.75(d,J＝7.8Hz,1H),4.30–4.35(m,1H),3.42–3.94(m,21H),3.20(dd,J＝10.1,6.4Hz,1H),2.53(dt,J＝10.7,5.2Hz,1H),1.01–1.12(m,1H),0.76–0.85(m,1H),0.61–0.66(m,3H).ESI-HRMS:m/z calculated for C₃₆H₅₀F₃N₆O₁₇[M-H]^-:895.3185,found:895.3179.

Yield rate 62.6％.¹H NMR(500MHz,D₂O):8.05–8.16(m,1H)7.15(t,J＝2.9Hz,1H)6.57(s,1H),5.22(t,J＝4.5Hz,2H),4.86(s,1H),4.77(d,J＝7.8Hz,1H),3.98–4.17(m,2H)3.42–3.83(m,20H),3.24–3.28(m,4H)2.34–2.45(m,1H)1.77–1.89(m,1H)1.59(td,J＝9.2,4.4Hz,1H)1.01(d,J＝6.9Hz,3H).ESI-HRMS:m/z calculated for C₃₅H₅₁N₆O₁₇[M-H]^-:827.3311,found:827.3317.

Yield rate 60.7％.¹H NMR(500MHz,D₂O):δ8.80(s,1H),8.64(s,1H),8.38(s,1H),7.50–7.65(m,1H),6.96(dd,J＝3.4,1.4Hz,1H),5.20(t,J＝4.2Hz,2H),4.71(d,J＝7.8Hz,1H),4.54(td,J＝9.8,3.9Hz,1H),3.19–3.83(m,19H),2.34–2.40(m,1H),1.70–1.81(m,1H),1.50–1.66(m,2H),1.16–1.42(m,4H).ESI-HRMS:m/z calculated for C₃₆H₄₉N₆O₁₆[M-H]^-:821.3205,found:821.3218.

Yield rate 57.7％.¹H NMR(500MHz,D₂O):δ8.90(s,1H),8.68(s,1H),8.45(s,1H),7.60(d,J＝4.2Hz,1H),7.05(d,J＝4.2Hz,1H),5.32–3.34(m,2H),4.79(d,J＝7.8Hz,1H),4.58(d,J＝12.1Hz,2H),4.22(d,J＝12.1Hz,2H),3.40–3.81(m,16H),3.26(d,J＝9.4Hz,1H),3.18–3.24(m,2H),1.22(t,J＝8.0Hz,3H).ESI-HRMS:m/z calculated for C₃₈H₄₈N₇O₁₈S[M-H]^-:886.2777,found:886.2768.

Yield rate 42.2％.¹H NMR(500MHz,D₂O):δ8.10(s,1H),7.51(d,J＝9.2Hz,1H),7.08–7.25(m,1H),6.64(dd,J＝3.5,1.8Hz,1H),5.26–5.29(m,2H),4.89–5.02(m,1H),4.775(d,J＝7.8Hz,1H),3.25–3.80(m,21H),2.84-3.01(m,2H),2.55–2.60(m,2H),2.19–2.32(m,2H),1.67(d,J＝7.5Hz,2H),0.92(t,J＝7.4Hz,3H).ESI-HRMS:m/z calculated for C₃₃H₅₂N₅O₁₈S[M-H]^-:838.3028,found:838.3033.

Yield rate 48.4％.¹H NMR(500MHz,D₂O):δ8.11(d,J＝13.0Hz,1H),7.30(dd,J＝19.0,7.4Hz,1H),6.77–7.19(m,2H),6.09(dd,J＝16.6,2.2Hz,1H),5.60–5.74(m,1H),5.25(t,J＝4.6Hz,2H),4.72(d,J＝8.0Hz,1H),3.42–3.93(m,17H),3.26(d,J＝9.0Hz,1H),1.69–1.92(m,3H),1.65(s,1H),1.25–1.29(m,3H).ESI-HRMS:m/z calculated for C₃₄H₅₀N₅O₁₇[M-H]^-:800.3202,found:800.3219.

Yield rate 30.6％.¹H NMR(500MHz,D₂O):δ8.73(s,1H),8.57(t,J＝1.8Hz,3H),8.40(d,J＝8.0Hz,1H),7.13(d,J＝8.0Hz,1H),6.93–6.96(m,1H),6.65(s,1H),6.19(s,1H),5.23(t,J＝4.5Hz,2H),4.75(d,J＝7.8Hz,1H),4.54–4.57(m,1H),3.42–3.83(m,16H),3.26(d,J＝9.4Hz,1H),2.53–2.63(m,4H),2.21(s,3H),1.71–1.93(m,8H).ESI-HRMS:m/z calculated for C₄₁H₅₇N₈O₁₆[M-H]^-:917.3893,found:917.3881.

Biological activity:

1. drug release of glycosylated JAK inhibitor prodrugs (compound of formula 1 or compound 1) in different tissue fluids

Male SD rats (200-220 g) were sacrificed by isoflurane and then midline incision. The stomach, small intestine, large intestine contents were separately collected and diluted with an equal volume of buffer (gastric contents were diluted with acetate buffer at pH 2.2 and other tissue contents were diluted with phosphate buffer at pH 6.8). Shaking for 10min to obtain tissue content homogenate, and storing at-80deg.C. Adding 500 mu L of compound 1 with the concentration of 100 mu M into 500 mu L of buffer solution corresponding to 16 mu L of tissue content homogenate, respectively shaking and incubating at 37 ℃ for 2 hours, respectively taking 100 mu L of reaction solution, centrifuging at 15000rpm for 5 minutes after high-temperature treatment for 1 minute, taking 60 mu L of supernatant for HPLC analysis (elution condition of HPLC: AGILENT ECLIPSE XDB-C18 (4.6 x 250nm,5 mu M), pure water of A solution (0.1% TFA), acetonitrile of B solution (0.1% TFA), the flow rate of 1mL/min, the detection wavelength of 265nm, gradient elution, the elution time of 20 minutes, and the concentration of B solution rising from 0% to 5%), and calculating the original drug release efficiency of the compound 1 in different tissue solutions.

TABLE I stability of Compound 1 in different tissue fluids (active principle release efficiency%)

The stability results of glycosylated JAK inhibitors (compound of formula 1 or compound 1) in different tissue fluids (stomach, small intestine, large intestine) indicate that they remain substantially stable in the stomach and small intestine portions and are not degraded to release the original drug, whereas in the large intestine contents most of the glycosylated JAK inhibitors are degraded to release the original drug, small molecule JAK inhibitors, wherein the drug release effects of L-fucose and D-xylose are not ideal.

2. Pharmacodynamic effects of compound 1 in mice model of p-trinitrobenzenesulfonic acid (DSS) induced ulcerative colitis.

Colitis mice were modeled as 7 week old male BALB/c mice, divided into 20 groups of 5 mice at random. Acute colitis was induced in mice by 1 week modeling with 2.5% DSS in drinking water, and colitis mice were evaluated for modeling by observing changes in body weight, hematochezia, stool shape, and other disease activity (DAI score). After modeling was successful, compound 1 was administered 1 time per day (5 μmol/mg/day) by gavage, for 7 consecutive days, with conventional drinking water during treatment. The effect of compound 1 on colitis mice was assessed by histopathological scoring of the mice.

Pathological scoring criteria for exterior, colon tissue

The results of histopathological scoring of the mice in the colitis model after drug treatment are shown in figure 1.

The results of histopathological scoring of compound 1 in a mouse model of colitis indicate that most glycosylated JAK inhibitors have significant anti-inflammatory effects. If the small molecule JAK inhibitor (such as III, IV and V) has weak anti-inflammatory effect on colonitis, the corresponding glycosylated prodrug shows insignificant anti-inflammatory effect, if the small molecule JAK inhibitor has strong anti-inflammatory effect on colonitis, the corresponding glycosylated prodrug shows significant anti-inflammatory effect, and after the JAK inhibitor is glycosylated, the anti-inflammatory effect of the prodrug in vivo is significantly stronger than that of the prodrug, which means that the prodrug has colon targeted drug release effect after glycosylation, and the anti-inflammatory effect in vivo is enhanced.

3. Rat oral pharmacokinetics of glycosylated JAK inhibitor 1-j-I

The representative compound 1-j-I with the best therapeutic effect on colitis in vivo was selected and evaluated for its pharmacokinetic properties in small intestine, colon and plasma after oral administration to rats. Compound 1-j-I (20 mg/kg) was orally administered to male Sprague Dawley rats (n=3/time point). At each time point (0.5, 1, 3,6, 8 and 24 hours), plasma samples were collected by cardiac puncture and small intestine and colon tissue samples were collected and small intestine and colon tissues were weighed. Plasma and tissue samples were prepared by extraction with 200 μl of acetonitrile and quantified by LC-MS/MS. (LC-MS/MS condition parameters are as follows: liquid chromatography separation is usedNX-C18 (50 mm. Times.2 mm,3 μm) column was eluted with a gradient of 0.3mL min ^-1 with 0.1% formic acid and pure methanol as mobile phases. Adopting electrospray ionization source, positive ion mode and multi-reaction monitoring scanning)

Plasma and intestinal Wu Pa Tinib concentrations after oral administration of 1-j-I in rats

The experimental results show that only a small amount of the original drug Wu Pati Ni is detected in the small intestine and blood plasma after the 1-j-I is orally taken, which indicates that the glycosylated prodrug 1-j-I cannot be absorbed in the small intestine and enter the blood circulation, and the original drug component with high concentration of at least 6 hours can be detected in the colon, which indicates that the glycosylated prodrug can realize colon targeted drug release.

Whether or not the JAK inhibitor has an effect of targeted treatment of colitis after glycosylation depends on the experimental results of the present invention, mainly on whether or not 1) the JAK inhibitor itself has an anti-colitis activity. According to the design thought of the invention, the glycosylation JAK inhibitor enters the colon part to exert the anti-inflammatory effect by releasing the original drug, so if the JAK inhibitor has no anti-colitis effect, the anti-colitis effect cannot be exerted even if the colon targeted drug release effect is increased after glycosylation. 2) Colon drug release effects of glycosylated JAK inhibitor prodrugs. According to the experimental results of the invention, among the selected glycosyl, other glycosyl modified JAK inhibitors except L-fucose (D) and D-xylose (e) have obvious colon drug release effect, and the JAK inhibitors have colon targeted drug release effect after being subjected to glycosylation modification so as to enhance the anti-inflammatory effect. By comprehensively considering the two factors, it is easy to estimate that the colon can play a role in targeted drug release by modifying the JAK inhibitor with the anti-colonitis effect by glycosyl with obvious drug release effect, and simultaneously, the systemic toxic and side effects of the drug are reduced, and compared with the original drug small molecule JAK inhibitor, the JAK inhibitor has higher safety and is expected to have more remarkable anti-inflammatory effect. Therefore, the glycosylated JAK inhibitor prodrugs according to the present invention are not limited to the structure described in compound 1, but include all glycosylated prodrug compounds obtained by glycosyl modification of JAK inhibitors having anti-colitis effect with colonic release effect.

In conclusion, the series of glycosylated JAK inhibitors prepared by the invention have obvious colon targeted drug release effect, almost no original drug ingredients can be detected in small intestine and blood plasma after oral administration, and a large amount of active ingredients can be released to play an anti-inflammatory role after reaching the colon, thus not only playing the targeted drug release effect, but also greatly reducing the systemic toxic and side effects of the drug.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1. The structural general formula of the glycosylated JAK inhibitor prodrug is shown in the formula 1:

In formula 1, JAKi represents a small molecule JAK inhibitor;

In the formula 1, the components are mixed, Representing various glycosyl groups.

2. The glycosylated JAK inhibitor prodrug of claim 1, wherein:

in the formula 1, the small molecule JAK inhibitor is a JAK inhibitor with an anti-colonitis effect;

further, the JAKi includes, but is not limited to, JAK inhibitors of formulas I-VII:

And/or, in formula 1 Obtained from a variety of carbohydrate molecules including, but not limited to, those of formulas a-j;

3. The glycosylated JAK inhibitor prodrug according to claim 1 or 2, wherein the glycosylated JAK inhibitor prodrug is selected from compounds of any structure:

4. a method of preparing a glycosylated JAK inhibitor prodrug according to any one of claims 1-3, comprising the steps of:

1) Reacting a small molecule JAK inhibitor (JAKi) represented by formula 2 with formaldehyde to obtain a methylolated JAK inhibitor represented by formula 3;

2) Carrying out glycosylation coupling reaction on a methylolated JAK inhibitor shown in a formula 3 and a compound shown in a formula 5 under the catalysis of trimethyl silicone triflate (TMSOTF) to obtain a compound shown in a formula 6;

3) Deprotection of a protecting group R in a compound of formula 6 to provide a glycosylated JAK inhibitor prodrug of formula 1;

5. The method according to claim 4, wherein in the step 1), the small molecule JAK inhibitor represented by formula 2 includes, but is not limited to, the JAK inhibitors represented by formulas I to VII in claim 2;

and/or adding water into the reaction, wherein the water solution of JAKi parts of formaldehyde (the mass volume concentration is 37%) is added in the ratio of 1-1.2 mmol to 1.2-1.5 mL to 1-1.5 mL in sequence;

And/or in the step 1), the reaction condition of the reaction is 45-55 ℃ for 1-3 hours.

6. The method according to claim 4 or 5, wherein in the step 2), R=Ac (acetyl) or Bz (benzoyl) in the formula 5, or a protecting group of other saccharide molecules;

The preparation method of the compound shown in the formula 5 comprises the following steps of taking saccharide molecules shown in the formula 4 as raw materials, and sequentially carrying out conventional reactions in three-step saccharide chemistry of acylation, hydrolysis and trichloroacetimidation to obtain the compound shown in the formula 5.

The saccharide molecules represented by formula 4 include, but are not limited to, saccharide molecules represented by formulas a to j as described in claim 2.

7. The method according to any one of claims 4 to 6, wherein in the step 2), the molar ratio of the compound represented by formula 5, the methylolated JAK inhibitor represented by formula 3, and trimethyl silicone triflate (TMSOTF) is 1 (1.0 to 2.0): 0.1 to 0.3;

and/or in the step 2), the reaction condition of the reaction is 0-25 ℃ for 0.5-2 hours.

8. Use of a glycosylated JAK inhibitor prodrug of any of claims 1-3 in the preparation of an anti-inflammatory drug.

9. A colon targeted drug delivery anti-inflammatory drug whose active ingredient comprises the glycosylated JAK inhibitor prodrug of any one of claims 1-3.

10. The use according to claim 8 or the medicament according to claim 9, wherein the anti-inflammatory medicament is a medicament for the prophylaxis and/or treatment of inflammatory bowel disease;

Still further, the inflammatory bowel disease includes crohn's disease and ulcerative colitis.