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

Abstract

The invention discloses a glycosylated JAK inhibitor prodrug as well as a preparation method and application thereof. The structural general formula of the glycosylated JAK inhibitor prodrug is as shown in formula 1, and in the formula 1, JAKi represents a small molecule JAK inhibitor; in the formula 1, # imgabs0 # represents various glycosyl groups. The series of glycosylated JAK inhibitors prepared by the invention have an obvious colon targeted drug release effect, almost no raw drug components are detected in small intestines and plasma after oral administration, and a large amount of active components are released to play an anti-inflammatory role after reaching the colon, so that the targeted drug release effect is played, and the whole-body toxic and side effects of the drugs are greatly reduced; therefore, the glycosylated JAK inhibitor is an oral IBD targeted anti-inflammatory drug, and compared with an original drug small-molecule JAK inhibitor, the glycosylated JAK inhibitor has higher safety, is expected to have a more remarkable anti-inflammatory effect, and is a novel prodrug capable of locally releasing drugs and exerting drug effects. # imgabs1 #

Description

Technical Field

[0001] The present invention belongs to the field of chemical medicines, and specifically relates to a glycosylated JAK inhibitor and its pharmaceutical use, a medicine or prodrug and a preparation method. Background Art

[0002] Janus kinase (JAK) is a non-transmembrane tyrosine kinase that plays an important role in the signaling pathways of many cytokines. The JAK family consists of JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2), which are key components of JAK signal transducers and transcriptional pathway activators. They can phosphorylate cytokine receptors and signaling molecules containing src homology domains, and play a key role in the cytokine receptor signaling pathway. As a key node of signal transduction, JAK plays a key role in the occurrence and development of diseases such as inflammation, autoimmune diseases, and malignant tumors, and also plays an important regulatory role in the growth and development of hematopoietic cells. JAK inhibitors have been widely used in the treatment of many 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 related to the activation of cytokine signaling pathways. By inhibiting the activation of these signaling pathways, JAK inhibitors can slow down the progression of the disease. Currently, more than a dozen JAK inhibitors have been approved for the treatment of various diseases driven by immune mechanisms.

[0003] Inflammatory bowel disease (IBD) is a chronic nonspecific intestinal inflammatory disease, including Crohn's disease (CD) and ulcerative colitis (UC), which is characterized by a long course of disease, difficulty in cure, and easy relapse. Traditional treatments for IBD include 5-aminosalicylic acid, corticosteroids, and immunosuppressants. In recent years, more and more biological agents and new oral small molecule drugs targeting cytokines, cytokine receptors, and inflammatory signaling pathways have gradually entered the public eye. Among them, the new small molecule drug JAK inhibitor inhibits the binding of JAK kinases to cytokines by blocking the JAK / STAT pathway, reduces the expression of inflammatory factors, and effectively inhibits the occurrence and development of inflammation in IBD. However, since JAK is widely present in the signaling pathways of many cytokines, JAK inhibitors are often accompanied by adverse reactions such as dizziness, nausea, upper respiratory tract infections, and urinary tract infections. Therefore, the development of JAK inhibitors with intestinal targeting, low toxicity, and mild side effects has important academic value and economic significance for the treatment of IBD. Summary of the invention

[0004] The present invention provides a glycosylated prodrug of a small molecule JAK inhibitor based on a drug release strategy in the colorectum, which can effectively reduce the systemic toxic side effects of the drug. The colorectum, the lesion site of IBD, has a rich and diverse intestinal flora, which secretes a large number of biological enzymes, glycosidases being one of the major categories. The present invention provides a glycosylation method for a JAK inhibitor, and provides a series of glycosylated JAK inhibitor prodrugs, which can be administered orally, are stable in the stomach and small intestine, are safe and non-toxic, and cut off sugar chains under the action of the intestinal flora enzyme, glycosidase, after reaching the large intestine, to release the active molecules of the JAK inhibitor, thereby achieving the anti-inflammatory effect of colorectal targeted drug release.

[0005] The glycosylated JAK inhibitor prodrug provided by the present invention has a general structural formula as shown in Formula 1:

[0006]

[0007] In Formula 1, JAKi represents a small molecule JAK inhibitor,

[0008] Furthermore, the small molecule JAK inhibitor is a JAK inhibitor with anti-colitis effect;

[0009] Furthermore, the small molecule JAK inhibitors include but are not limited to the JAK inhibitors shown in Formula I to Formula VII;

[0010]

[0011] In formula 1, represents various sugar groups, which can be obtained from various sugar molecules (such as monosaccharides, disaccharides, trisaccharides and polysaccharides), and the sugar molecules include but are not limited to the sugar molecules shown in formula a to formula j;

[0012]

[0013] Furthermore, the glycosylated JAK inhibitor prodrug may be selected from compounds with the following structures:

[0014]

[0015]

[0016]

[0017] The mechanism of action of the glycosylated JAK inhibitor prodrug provided by the present invention is as follows:

[0018]

[0019] The present invention also provides a method for preparing the glycosylated JAK inhibitor prodrug shown in Formula 1.

[0020] The synthetic route of the glycosylated JAK inhibitor prodrug shown in Formula 1 is as follows:

[0021]

[0022] The method for preparing the glycosylated JAK inhibitor prodrug shown in Formula 1 specifically comprises the following steps:

[0023] 1) reacting a small molecule JAK inhibitor (JAKi) shown in formula 2 with formaldehyde to obtain a hydroxymethylated JAK inhibitor shown in formula 3;

[0024] 2) subjecting the hydroxymethylated JAK inhibitor of Formula 3 to a glycosylation coupling reaction with the compound of Formula 5 under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to obtain a compound of Formula 6;

[0025] 3) Deprotecting the protecting group R in the compound shown in Formula 6 to obtain the glycosylated JAK inhibitor prodrug shown in Formula 1.

[0026] In the above step 1), the small molecule JAK inhibitor represented by Formula 2 includes but is not limited to the JAK inhibitors represented by Formula I to Formula VII.

[0027] In the above step 1), water is also added to the reaction, and the feed ratio of JAKi: formaldehyde aqueous solution (mass volume concentration 37%): water is 1-1.2 mmol: 1.2-1.5 mL: 1-1.5 mL;

[0028] In the above step 1), the reaction conditions are 45 to 55 degrees Celsius for 1 to 3 hours.

[0029] In the above step 2), in the formula 5, R = Ac (acetyl) or Bz (benzoyl), or other protective groups of sugar molecules.

[0030] In the above step 2), the preparation method of the compound represented by Formula 5 is as follows: using the carbohydrate molecule represented by Formula 4 as a raw material, sequentially undergoing three steps of conventional sugar chemistry reactions of acylation, hydrolysis and trichloroacetimidation esterification to obtain the compound represented by Formula 5.

[0031] The carbohydrate molecules represented by Formula 4 include but are not limited to the carbohydrate molecules represented by Formulas a to j above.

[0032] In the above step 2), the molar ratio of the compound represented by formula 5, the hydroxymethylated JAK inhibitor represented by formula 3, and trimethylsilyl trifluoromethanesulfonate is 1:(1.0-2.0):(0.1-0.3).

[0033] In the above step 2), the reaction conditions are 0 to 25 degrees Celsius for 0.5 to 2 hours.

[0034] The present invention also protects the use of the above-mentioned glycosylated JAK inhibitor prodrug.

[0035] The application is the application of glycosylated JAK inhibitor prodrug in the preparation of anti-inflammatory drugs.

[0036] Furthermore, the anti-inflammatory drug is a drug for preventing and / or treating inflammatory bowel disease (IBD).

[0037] Furthermore, the inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC).

[0038] Furthermore, the anti-inflammatory drug has a colon-targeted drug release effect.

[0039] The invention also protects an anti-inflammatory drug with colon-targeted drug release.

[0040] The active ingredient of the drug provided by the present invention includes the glycosylated JAK inhibitor prodrug represented by Formula 1 of the present invention.

[0041] Furthermore, the anti-inflammatory drug has the efficacy of preventing and / or treating inflammatory bowel disease (IBD).

[0042] The present invention also protects a method for preventing and / or treating inflammatory bowel disease (IBD).

[0043] The method comprises the following steps: administering the glycosylated JAK inhibitor prodrug represented by formula 1 of the present invention to a recipient animal or human.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The series of glycosylated JAK inhibitors prepared by the present invention have obvious colon-targeted drug release effect. 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 components are released to exert anti-inflammatory effects. They not only exert targeted drug release effects, but also greatly reduce the systemic toxic 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the histopathological score of colitis model mice after drug treatment; the drug was administered by gavage once a day, with a dosage of 5 μmol / mg / day, for 7 consecutive days. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0048] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0049] Example 1. Synthesis of glycosylated JAK inhibitor prodrugs

[0050] Synthesis process:

[0051] 1) Under nitrogen protection, compound 2 (2-I~2-VII) (1.0mmol, 1.0eq) was added to formaldehyde aqueous solution (37%, 1.2mL) and water (1mL) and reacted at 45 degrees Celsius for 1 hour. After vacuum distillation, freeze-drying was performed in a freeze dryer to obtain a mixture of white solid powder compound 3 (3-I~3-VII), which was directly used for the next step. (This step reaction refers to step 1)

[0052] 2) Peracetyl or perbenzoyl trichloroacetimidate compound 5 (5-a to 5-j) (1.0 to 2.0 eq, which can be synthesized from sugar molecules 4 (4-a to 4-j) according to conventional methods) and compound 3 (3-I to 3-VII) (1.0 mmol, 1.0 eq) are dissolved in dry acetonitrile (or dichloromethane, acetone), trimethylsilyl trifluoromethanesulfonate (0.1 to 0.3 eq) is added, and the mixture is reacted at 25 degrees Celsius for 0.5 hour, and triethylamine is added for neutralization. The liquid is concentrated to obtain a crude product (this reaction step refers to step 2).

[0053] 3) The crude product was dissolved in a methanol-dichloromethane (1 / 1 to 1 / 4) mixed solution, 1M sodium methoxide was added dropwise to a system pH of 9 to 11, and after reacting at room temperature for 2 hours, acidic resin Resin IR120 was added and stirred to a system pH of 7.0. The resin was filtered off, and the filtrate was concentrated and passed through a silica gel column with a mobile phase of ethyl acetate: methanol: water = 7:2:0 to 7:2:1 to obtain a white powder product 1 (1-aI to 1-j-VII). (This step of reaction refers to step 3)

[0054] Compound yield and structural characterization:

[0055]

[0056] Yield: 65.4%. 1 H 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=10.8H z,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=1 0.3,6.9Hz,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 / zcalculated for C 24 H 32 F3N6O7[MH] - :560.2206,found:560.2209.

[0057]

[0058] Yield: 63.5%. 11H NMR (500 MHz, D2O): δ 8.51 (s, 1H), 7.47 (s, 1H), 7.40 (t, J = 3.0 Hz, 1H), 6.99–7.01 (m, 1H), 6.92 (t, J = 6.0 Hz, 1H), 5.50 (d, J = 11.0 Hz, 1H), 5.31 (d, J = 11.0 Hz, 1H), 4.56 (d, J = 7.7 Hz, 1H), 4.31–4.36 (m, 1H), 3.90 (d, J = 3.5 Hz, 1H), 3.60–3.91 (m, 7H), 3.51 (dd, J = 9.8, 7.4 Hz, 1H), 3.24 (dd, J = 10.0, 5.8 Hz, 1H), 2.52 (dt, J = 10.6, 5.2 Hz, 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 C 24 H 32 F3N6O7[M-H] - : 560.2206, found: 560.2208.

[0059]

[0060] Yield 62.8%. 1 1H NMR (500 MHz, D2O): δ 8.52 (s, 1H), 7.48 (s, 1H), 7.42 (t, J = 2.9 Hz, 1H), 6.99–7.02 (m, 1H), 6.94 (t, J = 6.0 Hz, 1H), 5.37 (d, J = 11.1 Hz, 1H), 5.27 (d, J = 11.1 Hz, 1H), 5.10 (d, J = 1.6 Hz, 1H), 4.30–4.35 (m, 1H), 3.95 (dd, J = 3.3, 1.8 Hz, 1H), 3.77–3.92 (m, 6H), 3.71 (dd, J = 12.4, 6.4 Hz, 1H), 3.68 (dd, J = 10.3, 6.9 Hz, 1H), 3.56–3.66 (m, 2H), 3.26 (dd, J = 10.0, 6.1 Hz, 1H), 2.55 (dt, J = 10.7, 5.2 Hz, 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 C 24 H 32 F3N6O7[M-H] - : 560.2206, found: 560.2209.

[0061]

[0062] The yield was 65.1%. 1 H NMR (500 MHz, D2O): δ 8.68 (s, 1H), 7.55 (s, 1H), 7.32 (t, J = 2.8 Hz, 1H), 6.90–6.94 (m, 1H), 6.86 (t, J = 6.0 Hz, 1H), 5.41 (d, J = 11.3 Hz, 1H), 5.30 (d, J = 11.3 Hz, 1H), 4.51 (d, J = 7.8 Hz, 1H), 4.35–4.38 (m, 1H), 3.70–3.90 (m, 7H), 3.61 (dd, J = 9.9, 3.4 Hz, 1H), 3.49 (dd, J = 9.9, 7.8 Hz, 1H), 3.26 (dd, J = 10.0, 6.1 Hz, 1H), 2.51–2.59 (m, 1H), 1.22 (d, J = 6.2 Hz, 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 C 24 H 30 F3N6O6 [M-H] - : 555.2179, found: 555.2171.

[0063]

[0064] The yield was 66.3%. 1 H NMR (500 MHz, D2O): δ 8.77 (s, 1H), 7.68 (s, 1H), 7.49 (t, J = 2.4 Hz, 1H), 7.04–7.12 (m, 1H), 6.99 (t, J = 6.0 Hz, 1H), 5.59 (d, J = 11.3 Hz, 1H), 5.40 (d, J = 11.3 Hz, 1H), 4.56 (d, J = 7.7 Hz, 1H), 4.31–4.37 (m, 1H), 3.92 (dd, J = 11.4, 5.2 Hz, 1H), 3.74–3.90 (m, 4H), 3.56–3.65 (m, 2H), 3.41 (t, J = 9.0 Hz, 1H), 3.29–3.37 (m, 3H), 2.57 (dt, J = 10.4, 5.0 Hz, 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 C 23 H28 F3N6O6[M-H] - :541.2022, found:541.2012.

[0065]

[0066] Yield 64.7%. 1 H 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 C 30 H 40 F3N6O 12 [M-H] - :733.2656, found:733.2650.

[0067]

[0068] Yield 65.9%. 11H NMR (500 MHz, D2O): δ 8.66 (s, 1H), 7.71 (s, 1H), 7.55 (s, 1H), 6.91–7.02 (m, 2H), 5.50 (d, J = 11.3 Hz, 1H), 5.40 (d, J = 3.9 Hz, 1H), 5.36 (d, J = 11.3 Hz, 1H), 4.68 (d, J = 8.0 Hz, 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, 3.1 Hz, 2H), 3.32 (t, J = 9.5 Hz, 1H), 3.27–3.22 (m, 2H), 2.50 (dt, J = 10.7, 5.2 Hz, 1H), 1.00–1.11 (m, 1H), 0.76–0.85 (m, 1H), 0.61–0.66 (m, 3H). ESI-HRMS: m / z calculated for C 30 H 40 F3N6O 12 [M-H] - : 733.2656, found: 733.2649.

[0069]

[0070] Yield 55.2%. 1 1H NMR (500 MHz, D2O): δ 8.57 (s, 1H), 7.48 (s, 1H), 7.40 (t, J = 2.9 Hz, 1H), 6.99–7.03 (m, 1H), 6.91 (t, J = 6.1 Hz, 1H), 5.50 (d, J = 11.3 Hz, 1H), 5.36 (d, J = 11.3 Hz, 1H), 4.68 (d, J = 8.0 Hz, 1H), 4.58 (d, J = 7.8 Hz, 1H), 4.31–4.35 (m, 1H), 3.59–3.91 (m, 10H), 3.48 (dt, J = 6.7, 3.1 Hz, 2H), 3.32 (t, J = 9.5 Hz, 1H), 3.22–3.27 (m, 2H), 2.54 (dt, J = 10.8, 5.4 Hz, 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 C 30 H 40 F3N6O 12 [M-H] - : 733.2656, found: 733.2651.

[0071]

[0072] The yield was 50.1%. 1 H 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 / zcalculated for C 36 H 50 F3N6O 17 [M-H] - :895.3185,found:895.3172.

[0073]

[0074] The yield was 52.9%. 1 H 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 / zcalculated for C 36 H 50 F3N6O 17 [M-H] - :895.3185,found:895.3179.

[0075]

[0076] The yield was 62.6%. 11H NMR (500 MHz, D2O): δ 8.05–8.16 (m, 1H) 7.15 (t, J = 2.9 Hz, 1H) 6.57 (s, 1H), 5.22 (t, J = 4.5 Hz, 2H), 4.86 (s, 1H), 4.77 (d, J = 7.8 Hz, 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, 4.4 Hz, 1H) 1.01 (d, J = 6.9 Hz, 3H). ESI-HRMS: m / z calculated for C 35 H 51 N6O 17 [M-H] - : 827.3311, found: 827.3317.

[0077]

[0078] Yield 60.7%. 1 1H NMR (500 MHz, D2O): δ 8.80 (s, 1H), 8.64 (s, 1H), 8.38 (s, 1H), 7.50–7.65 (m, 1H), 6.96 (dd, J = 3.4, 1.4 Hz, 1H), 5.20 (t, J = 4.2 Hz, 2H), 4.71 (d, J = 7.8 Hz, 1H), 4.54 (td, J = 9.8, 3.9 Hz, 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 C 36 H 49 N6O 16 [M-H] - : 821.3205, found: 821.3218.

[0079]

[0080] Yield 57.7%. 11H NMR (500 MHz, D2O): δ 8.90 (s, 1H), 8.68 (s, 1H), 8.45 (s, 1H), 7.60 (d, J = 4.2 Hz, 1H), 7.05 (d, J = 4.2 Hz, 1H), 5.32–3.34 (m, 2H), 4.79 (d, J = 7.8 Hz, 1H), 4.58 (d, J = 12.1 Hz, 2H), 4.22 (d, J = 12.1 Hz, 2H), 3.40–3.81 (m, 16H), 3.26 (d, J = 9.4 Hz, 1H), 3.18–3.24 (m, 2H), 1.22 (t, J = 8.0 Hz, 3H). ESI-HRMS: m / z calculated for C 38 H 48 N7O 18 S [M-H] - : 886.2777, found: 886.2768.

[0081]

[0082] Yield 42.2%. 1 1H NMR (500 MHz, D2O): δ 8.10 (s, 1H), 7.51 (d, J = 9.2 Hz, 1H), 7.08–7.25 (m, 1H), 6.64 (dd, J = 3.5, 1.8 Hz, 1H), 5.26–5.29 (m, 2H), 4.89–5.02 (m, 1H), 4.775 (d, J = 7.8 Hz, 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 = 7.5 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H). ESI-HRMS: m / z calculated for C 33 H 52 N5O 18 S [M-H] - : 838.3028, found: 838.3033.

[0083]

[0084] Yield 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=9.0 Hz,1H),1.69–1.92(m,3H),1.65(s,1H),1.25–1.29(m,3H).ESI-HRMS:m / z calculated for C 34 H 50 N5O 17 [MH] - :800.3202,found:800.3219.

[0085]

[0086] Yield: 30.6%. 1 H 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 calculatedfor C 41 H 57 N8O 16 [MH] - :917.3893,found:917.3881.

[0087] Biological Activity:

[0088] 1. Drug release of glycosylated JAK inhibitor prodrug (compound of formula 1 or compound 1) in different tissue fluids

[0089] Male SD rats (200-220 g) were killed with isoflurane and then incised in the midline. The contents of the stomach, small intestine, and large intestine were collected and diluted with an equal volume of buffer (the stomach contents were diluted with pH 2.2 acetate buffer, and the other tissue contents were diluted with pH 6.8 phosphate buffer). The tissue contents were homogenized after oscillation in a mixer for 10 minutes and stored at -80°C for later use. 500 μL of compound 1 with a concentration of 100 μM was added to 500 μL of buffer corresponding to 16 μL of tissue content homogenate. After incubation at 37°C for 2 h, 100 μL of the reaction solution was taken respectively. After high temperature treatment for 1 min, centrifugation was performed at 15000 rpm for 5 min, and 60 μL of the supernatant was taken for HPLC analysis (HPLC elution conditions: Agilent Eclipse XDB-C18 (4.6*250 nm, 5 μm); liquid A pure water (0.1% TFA), liquid B acetonitrile (0.1% TFA); flow rate 1 mL / min; detection wavelength 265 nm, gradient elution, elution time 20 min, and liquid B concentration increased from 0% to 5%.) The original drug release efficiency of compound 1 in different tissue fluids was calculated.

[0090] Table 1. Stability of compound 1 in different tissue fluids (original drug release efficiency %)

[0091]

[0092]

[0093] The stability results of glycosylated JAK inhibitors (compounds of Formula 1 or Compound 1) in different tissue fluids (stomach, small intestine, large intestine) showed that they remained basically stable in the stomach and small intestine and would not be degraded to release the original drug; while in the contents of the large intestine, most of the glycosylated JAK inhibitors would be degraded to release the original drug - small molecule JAK inhibitors, among which the release effect of L-fucose and D-xylose was not ideal.

[0094] 2. Pharmacodynamic effect of compound 1 by oral administration on the trinitrobenzenesulfonic acid (DSS)-induced ulcerative colitis mouse model.

[0095] Colitis mouse modeling: 7-week-old male BALB / c mice were divided into 20 groups, with 5 mice randomly assigned to each group. The model was established by giving 2.5% DSS in drinking water for 1 week to induce acute colitis in mice, and the colitis mouse modeling was evaluated by observing the changes in mouse weight, blood in stool, stool shape and other disease activity (DAI score). After the modeling was successful, compound 1 was administered by gavage once a day (dosage 5μmol / mg / day) for 7 consecutive days, and regular drinking water was used during the treatment. The effect of compound 1 in treating colitis mice was evaluated by the histopathological score of mice.

[0096] Table 2. Colonic histopathological scoring criteria

[0097]

[0098]

[0099] The histopathological scores of colitis model mice after drug treatment are shown in Figure 1 shown.

[0100] The results of histopathological scoring of compound 1 in the colitis mouse model showed that most glycosylated JAK inhibitors have significant anti-inflammatory effects. If the small molecule JAK inhibitors (such as III, IV and V) themselves have a weak inhibitory effect on colitis, the anti-inflammatory effect of their corresponding glycosylated prodrugs is not obvious; if the small molecule JAK inhibitors themselves have a strong inhibitory effect on colitis, the anti-inflammatory effect of their corresponding glycosylated prodrugs is relatively significant, and after such JAK inhibitors are glycosylated, the anti-inflammatory effect of the prodrug in vivo is significantly stronger than that of the original drug, indicating that the prodrug has a colon-targeted drug release effect after glycosylation, and the anti-inflammatory effect in vivo is enhanced.

[0101] 3. Oral pharmacokinetics of glycosylated JAK inhibitor 1-jI in rats

[0102] The representative compound 1-jI with the best in vivo colitis treatment effect was selected to evaluate its pharmacokinetic properties in the small intestine, colon and plasma after oral administration to rats. Compound 1-jI (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 weighed. Plasma and tissue samples were extracted with 200 μL of acetonitrile and quantified by LC-MS / MS. (LC-MS / MS condition parameters are as follows: Liquid chromatography separation adopts NX-C18 (50 mm × 2 mm, 3 μm) column, with 0.1% formic acid water and pure methanol as mobile phase, gradient elution, flow rate 0.3 mL min -1 . Using electrospray ionization source, positive ion mode, multiple reaction monitoring scanning)

[0103] Table 3. Upadacitinib concentrations in plasma and intestine after oral administration to rats

[0104]

[0105] The above experimental results show that after oral administration of 1-jI, only a small amount of the original drug upadacitinib was detected in the small intestine and plasma, indicating that the glycosylated prodrug 1-jI would not be absorbed in the small intestine and enter the blood circulation; while high concentrations of the original drug components could be detected in the colon for at least 6 hours, indicating that the glycosylated prodrug can achieve targeted drug release in the colon.

[0106] Whether the JAK inhibitor has the effect of targeted treatment of colitis after glycosylation depends mainly on two points based on the experimental results of the present invention: 1) Whether the JAK inhibitor itself has anti-colitis activity. According to the design concept of the present invention, the glycosylated JAK inhibitor enters the colon by releasing the original drug to exert an anti-inflammatory effect. Therefore, if the JAK inhibitor itself does not have an anti-colitis effect, even if the colon-targeted drug release effect is increased after glycosylation, it cannot exert an anti-colitis effect. 2) The colon drug release effect of the glycosylated JAK inhibitor prodrug. According to the experimental results of the present invention, among the selected sugars, except for L-fucose (d) and D-xylose (e), other glycosyl-modified JAK inhibitors have significant colon drug release effects. After these glycosylation modifications, the JAK inhibitors will have the effect of colon-targeted drug release and enhance their anti-inflammatory effects. Taking these two factors into consideration, it is easy to infer that the JAK inhibitors with anti-colitis effect modified with glycosyl groups with obvious colonic drug release effect can achieve targeted drug release in the colon, while reducing the systemic toxicity and side effects of the drug. Compared with the original small molecule JAK inhibitors, it has higher safety and is expected to have more significant anti-inflammatory effects. Therefore, the glycosylated JAK inhibitor prodrugs involved in the present invention are not limited to the structure described in Compound 1, and should include all glycosylated prodrug compounds obtained by modifying the JAK inhibitors with anti-colitis effect with glycosyl groups having colonic drug release effect.

[0107] In summary, the series of glycosylated JAK inhibitors prepared by the present invention have obvious colon-targeted drug release effect. 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 plays a targeted drug release effect, but also greatly reduces the systemic toxic 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.

[0108] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A glycosylated JAK inhibitor prodrug, the general structural formula of which is shown in Formula 1: In Formula 1, JAKi represents a small molecule JAK inhibitor; In formula 1, Represents various sugar groups.

2. The glycosylated JAK inhibitor prodrug according to claim 1, characterized in that: In Formula 1, the small molecule JAK inhibitor is a JAK inhibitor with anti-colitis effect; Furthermore, the JAKi includes but is not limited to the JAK inhibitors shown in Formula I to Formula VII: And / or, in Formula 1 Obtained from various sugar molecules, including but not limited to sugar molecules shown in formula a to formula j; 3. The glycosylated JAK inhibitor prodrug according to claim 1 or 2, characterized in that: The glycosylated JAK inhibitor prodrug is selected from the following compounds of any structure:

4. The method for preparing the glycosylated JAK inhibitor prodrug according to any one of claims 1 to 3, comprising the following steps: 1) reacting a small molecule JAK inhibitor (JAKi) shown in formula 2 with formaldehyde to obtain a hydroxymethylated JAK inhibitor shown in formula 3; 2) subjecting the hydroxymethylated JAK inhibitor of Formula 3 to a glycosylation coupling reaction with the compound of Formula 5 under the catalysis of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to obtain a compound of Formula 6; 3) deprotecting the protecting group R in the compound shown in Formula 6 to obtain a glycosylated JAK inhibitor prodrug shown in Formula 1; 5. The preparation method according to claim 4, characterized in that: In the step 1), the small molecule JAK inhibitor represented by formula 2 includes but is not limited to the JAK inhibitor represented by formula I to formula VII in claim 2; And / or, water is further added to the reaction, and the feed ratio of JAKi: formaldehyde aqueous solution (mass volume concentration 37%): water is 1-1.2 mmol: 1.2-1.5 mL: 1-1.5 mL; And / or, in the step 1), the reaction condition is 45 to 55 degrees Celsius for 1 to 3 hours.

6. The preparation method according to claim 4 or 5, characterized in that: In the step 2), in the formula 5, R = Ac (acetyl) or Bz (benzoyl), or other protective groups of sugar molecules; The preparation method of the compound represented by Formula 5 is as follows: using the carbohydrate molecule represented by Formula 4 as a raw material, sequentially undergoing three steps of conventional sugar chemistry reactions, namely acylation, hydrolysis and trichloroacetimidation esterification, to obtain the compound represented by Formula 5. The carbohydrate molecules represented by formula 4 include but are not limited to the carbohydrate molecules represented by formula a to formula j in claim 2.

7. The preparation method according to any one of claims 4 to 6, characterized in that: In the step 2), the molar ratio of the compound represented by Formula 5, the hydroxymethylated JAK inhibitor represented by Formula 3, and trimethylsilyl trifluoromethanesulfonate (TMSOTf) is 1: (1.0-2.0): (0.1-0.3); And / or, in the step 2), the reaction conditions of the reaction are 0 to 25 degrees Celsius for 0.5 to 2 hours.

8. Use of the glycosylated JAK inhibitor prodrug according to any one of claims 1 to 3 in the preparation of anti-inflammatory drugs.

9. An anti-inflammatory drug with colon-targeted drug release, wherein the active ingredient comprises the glycosylated JAK inhibitor prodrug according to any one of claims 1 to 3.

10. The use according to claim 8 or the medicine according to claim 9, characterized in that: The anti-inflammatory drug is a drug for preventing and / or treating inflammatory bowel disease; Furthermore, the inflammatory bowel disease includes Crohn's disease and ulcerative colitis.

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