CN111757888A

CN111757888A - Application of docetaxel conjugate in preparation of medicines for preventing or treating various immune diseases

Info

Publication number: CN111757888A
Application number: CN201880058271.9A
Authority: CN
Inventors: 刘刚; 温晓明; 欧颖烨; 黄伟鑫
Original assignee: Shenzhen Salubris Pharmaceuticals Co Ltd
Current assignee: Shenzhen Salubris Pharmaceuticals Co Ltd
Priority date: 2017-09-07
Filing date: 2018-09-04
Publication date: 2020-10-09
Also published as: WO2019047811A1

Abstract

The use of a compound of formula (I) as shown below, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prevention or treatment of various immune disorders.

Description

Application of docetaxel conjugate in preparation of medicines for preventing or treating various immune diseases

Technical Field

The invention mainly relates to an application of a conjugate formed by docetaxel and a muramyl dipeptide simplified substance or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing or treating various immune diseases.

Background

The immune system protects the body from pathogen infection, cellular transformation, and physical/chemical damage. Its dysfunction, either over-or under-activation, can lead to different diseases. Dysfunction of the immune system may be caused by aging, developmental defects, disease and medical treatments (e.g. chemotherapy or immunosuppression), and it is therefore particularly urgent and critical to seek to prepare medicaments for the prevention or treatment of various immune disorders.

The taxane medicines comprise paclitaxel and docetaxel, and have the characteristic of low oral bioavailability mainly because the taxane medicines are easily discharged and pumped out by P-glycoprotein on the epithelium of the gastrointestinal tract, are easily metabolized by cytochrome P450 and have poor water solubility. Because the taxane drugs are still the first-line drugs in the treatment field, various researches around the compounds are the research hotspots of medicinal chemists, and the chemical conjugation of natural antitumor drug molecules and immunopotentiators is proposed.

The earlier patents filed protected two main classes of conjugates, one being 2' -O-MTC conjugates formed by linking muramyl dipeptide to the hydroxyl group at the 2' -position of paclitaxel (see CN1712399A), but unfortunately, 2' -O-MTC conjugates failed to demonstrate experimental results against tumor metastasis in experimental mice.

The other is MTC/MDC series conjugate formed by connecting paclitaxel/docetaxel and muramyl dipeptide derivative (see WO2011147330A1), and the MTC/MDC series conjugate has certain corresponding effect in-vitro tumor inhibition and tumor metastasis inhibition experiments, but further medicine use is limited due to the problem of druggability.

Although the above series of compounds make a great contribution to the art, the art continues to research to improve their efficacy, particularly for immune diseases.

Disclosure of Invention

The invention aims to solve the technical problem of providing an application of a conjugate formed by docetaxel and a muramyl dipeptide simplified substance or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing or treating various immune diseases.

In order to solve the technical problem of the invention, the following technical scheme is adopted: the application of the compound shown as the formula I and the pharmaceutically acceptable salt thereof in preparing the medicines for preventing or treating various immune diseases,

wherein m is a natural number from 0 to 1, i.e., m is 0 or 1;

n is selected from a natural number from 2 to 10, i.e. n is 2,3,4,5,6,7,8,9 or 10;

preferred n is selected from natural numbers from 2 to 5, i.e. n ═ 2,3,4 or 5;

R₁selected from the group consisting of substituted or unsubstituted five to ten membered aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted methyleneoxy; the substituted substituent is selected from hydroxyl, sulfydryl, halogen, amino, nitro, cyano, aldehyde group, C1-C6 alkyl and carboxylHydroxy amino, C2-C6 alkylene, C1-C4 acylamino and benzamido;

R₂is selected from hydrogen, substituted or unsubstituted alkyl of C1-C6, substituted or unsubstituted alkoxy of C1-C6, and the substituted substituent is selected from halogen.

The aforementioned uses, preferably by acting on a signaling pathway and/or factor selected from NF-. kappa.B, I.kappa.B alpha, p-ERK, p-p38, p-JNK, MMP8, MMP9, MMP2, TIMP-1, IL-1. beta., IL-6, IL-8, TNF-. alpha., CD11B, Gr-1, Ly-6G, TSP-1, NOD1, NOD2, S100A8, S100A9, prevent or treat various immune disorders.

The immunity basically comprises the inhibition of tumor growth and metastasis, anti-inflammation, such as rheumatoid arthritis and systemic lupus erythematosus; myelosuppression, and macrophage, lymphocyte defects, and the like.

NF-kB (nuclear factor-kappa B) is a transcription factor found in nuclear extracts of B lymphocytes in 1986, and is known for its ability to specifically bind to the enhancer B sequence GGGACTTTCC of immunoglobulin kappa light chain gene, promoting expression of kappa light chain gene. It is one of the members of the Rel family of eukaryotic transcription factors and is widely present in various mammalian cells. NF-kB has obvious function of inhibiting apoptosis and is closely related to a plurality of processes of generation, growth, metastasis and the like of tumors. The NF-kB signal channel activation is closely related to various diseases such as inflammatory diseases, systemic lupus erythematosus and the like. In non-stimulated cells, most of the NF-. kappa.B dimers are found in an inactive state by binding to one of the three cytoplasmic inhibitors (I.kappa.B. alpha., I.kappa.B. beta., I.kappa.B), and the NF-. kappa.B-I.kappa.Bs complex (NF-. kappa.B. alpha., I.kappa.B. beta., I.kappa.B) shuttles between the cytoplasm and the nucleus, in dynamic equilibrium. Various signals activate NF-. kappa.B by degrading I.kappa.Bs, which then enter the nucleus to bind to DNA, causing a series of cellular responses. Almost all known inducers of NF-. kappa.B are capable of rapidly and transiently activating NF-. kappa.B by degradation and phosphorylation of I.kappa.B.alpha.p-I.kappa.B.alpha..

p-ERK, p-p38, p-JNK, etc. are the phosphorylation states in which molecules of the MAPK pathway are activated. Mitogen-activated protein kinases (MAP kinases, MAPK) chain is one of the important pathways in eukaryotic signal transmission networks, and plays a key role in gene expression regulation and cytoplasmic function activities. In unstimulated cells, MAPK are in a quiescent state. Upon stimulation of cells by growth factors or other factors, MAPK is activated by receiving activation signals from MKK and mkkkk, which manifests as progressive phosphorylation. In mammals, ERK is present in a wide variety of tissues and is involved in the regulation of the proliferation and differentiation of cells. Activation of ERK is required for a variety of growth factor receptors, nutrient-related factor receptors, and the like to complete the signal transduction process. The JNK family is a key molecule of signal transduction induced by cells to various stressors and participates in stress response of the cells to radiation, osmotic pressure, temperature change and the like. p38 mediates inflammation, apoptosis, etc., and thus becomes a target for development of anti-inflammatory drugs. NF-kB is one of the downstream MAPK signaling pathways. MAPK signaling pathways affect a variety of intracellular responses, regulating inflammatory responses, cell cycle, cell growth, differentiation, senescence and apoptosis, and tumorigenesis.

Matrix Metalloproteases (MMPs) are a class of zinc-dependent endopeptidases that can degrade extracellular matrix (ECM) components and non-matrix proteins, while ECM is a major component constituting the vascular wall, and elevated MMP activity is associated with tumor growth and migration, the occurrence of arthritis, and the like. MMPs are also closely related to cardiovascular diseases such as atherosclerosis, dilated cardiomyopathy, and myocardial infarction. A tissue inhibitor of matrix metalloproteinase (TIMP-1). Expressed in a glycoprotein commonly found in tissues. The TIMPs family inhibits the MMPs family function, regulating the latter to function normally. Both maintain extracellular matrix homeostasis.

IL-1 beta, IL-6, IL-8, TNF-alpha are the major cytokines involved in inflammatory responses. IL-1 beta stimulates the production of cytokines such as colony stimulating factor, platelet growth factor, etc., and enables T cells to produce interleukin-2, playing a role in immune response and tissue repair. TNF-alpha is the earliest and most important inflammatory mediator in the inflammatory response, and can activate neutrophils and lymphocytes, increase permeability of vascular endothelial cells, regulate metabolic activity of other tissues and promote synthesis and release of other cytokines. IL-6 can induce B cell differentiation and antibody production, and induce T cell activation proliferation and differentiation, and participate in immune response of organism, and is a promoter of inflammatory reaction. IL-8 can stimulate chemotaxis of neutrophils, T lymphocytes and eosinophils, promote degranulation of neutrophils, release elastase, damage endothelial cells, and lead microcirculation blood flow to be stagnated and tissues to be necrotic, thereby causing organ function damage. These inflammatory factors are important mediators of inflammatory diseases and play important roles in infection resistance, immune response regulation and antitumor.

Myelosuppressive cells (MDSC) (CD11b, Gr-1) are a heterogeneous population of cells with potent immunosuppressive activity that can inhibit T-lymphocyte activation and proliferation. MDSCs exhibit increased recruitment in infections, autoimmune diseases, bone marrow transplantation, and are also involved in pathological processes such as immune escape, immune tolerance, inflammatory responses, and the like. Recent studies have found that MDSCs are involved in tumor growth, immune tolerance, immune escape and tumor metastasis processes.

Neutrophils (surface marker: Ly-6G) are one of the important leukocytes in peripheral blood, and play an important role in the body's immune surveillance against microbial invasion and tumors. Studies have shown that neutrophils play an important role in tumorigenesis, progression, and invasion. Neutrophilic granulocytopenia is seen in myeloproliferative syndromes, bacterial infections, certain parasitic diseases, tissue ischemia or necrosis, cancer and hodgkin's disease, after acute hemorrhage and acute hemolysis, metabolic diseases (gout crisis, diabetic acidosis, renal insufficiency), inflammatory syndromes, allergies and various toxicities.

Thrombospondin-1 (Thrombospondin), TSP-1 for short, is a member of the Thrombospondin family, is mainly present in platelet alpha granules and extracellular matrix, and macrophages are also expressed at low levels. TSP-1 is involved in a variety of physiological and pathological processes, and related studies have focused mainly on inflammation, anti-angiogenesis and tumor.

NOD1, NOD2 are important members of the intracellular pattern recognition receptor-NOD-like receptor (NLRs) family, which are important receptors of the innate immune system within the cell. After the special components of some intracellular pathogenic factors or pathogenic bacteria are combined with NOD1 and NOD2, NF-kB signal channels are mainly activated to start inflammatory reaction. As an important defense component of the innate immunity and stress system of the body, NOD1 and NOD2 are involved in the development and progression of various diseases such as cancer, pneumonia, type 2 diabetes, atherosclerosis, alzheimer disease and the like.

The S100A8 protein (CalgranulinA protein, MRP8 protein) and the S100A9 protein (CalgranulinB protein, MRP14 protein) both belong to members of the calcium binding protein S100 protein family, and both usually form a heterodimer S100A8/A9 protein complex (hereinafter referred to as S100A8/A9) in a calcium ion dependent manner, are expressed in circulating neutrophils and mononuclear macrophages, but are not expressed in normal macrophages and lymphocytes, and are also expressed in epithelia in a chronic inflammatory environment; can participate in inflammatory reaction, regulate cell growth and differentiation, inhibit growth, induce apoptosis and the like. With the continuous and intensive research on the S100A8/A9 in recent years, the S100A8/A9 is found to be closely related to various diseases, and the abnormal expression of the S100 protein in tumor tissues has close correlation with the stage and the prognosis of tumors.

The aryl group is preferably a five-ten membered aryl group and the heteroaryl group is preferably a five-ten membered heteroaryl group.

The five-ten-membered aryl is preferably selected from five-membered aryl, six-membered aryl and ten-membered condensed ring aryl;

the five-membered aryl group is selected from

The six-membered aryl is selected from

The ten-membered fused ring aryl is selected from

Said heteroaryl represents an aromatic ring system of one or more heteroatoms, preferably containing 1 to 4 heteroatoms, preferably selected from heteroaryl groups containing heteroatoms of N, O or S;

preferred heteroaryl groups are selected from five-ten membered heteroaryl groups containing 1-4 heteroatoms selected from N, O or S;

more preferred heteroaryl groups are selected from five-membered heterocyclic groups containing 1 to 4 heteroatoms selected from N, O or S, six-membered heterocyclic groups containing 1 to 4 heteroatoms selected from N, O or S, ten-membered fused heterocyclic groups containing 1 to 4 heteroatoms selected from N, O or S.

The five-membered heterocyclic group containing 1-4 heteroatoms selected from N, O or S is selected from:

the six-membered heterocyclic group containing 1 to 4 heteroatoms selected from N, O or S is selected from:

the ten-membered fused heterocyclic group containing 1 to 4 heteroatoms selected from N, O or S is selected from:

said alkyl group having C1-C6 represents a straight or branched chain alkane group having 1 to 6 carbon atoms, and may be selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclopentyl, n-hexyl, cyclohexyl, dimethylpropyl, 2-methylbutyl, 2-dimethylbutyl, and 2, 3-dimethylbutyl.

The C1-C6 alkoxy is selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexoxy, isohexoxy, methoxyethoxy, ethoxymethoxy, propoxymethoxy and propoxyethoxy.

The halogen is selected from fluorine, chlorine, bromine or iodine, the C1-C4 acylamino is selected from acetylamino, propionylamino, butyrylamino or isobutyrylamino; the alkenyl group of C2-C6 is selected from the group consisting of ethenyl, propenyl, butenyl, isobutenyl, 2-butenyl, pentenyl, isopentenyl, 2-pentenyl, hexenyl, and isohexenyl.

The R is₂Is hydrogen or hydrogen is replaced by metal or nonmetal cation to form pharmaceutically acceptable salt, and the metal or nonmetal cation is selected from Na⁺，K⁺,Ca²⁺,Mg²⁺,Zn²⁺，Al³⁺，NH₄ ⁺。

The compounds of formula I include, but are not limited to, compounds of formula IA:

R₁as defined above.

The compound is selected from the following group:

the invention provides a general method for synthesizing conjugates of docetaxel and a simplified muramyl dipeptide, which comprises the following steps:

1. liquid phase synthesis of docetaxel 2' -O-alkane diacid monoester;

2. solid-phase synthesis of muramyl dipeptide simplifier;

3. the conjugate of docetaxel and muramyl dipeptide simplified compound is synthesized by liquid phase.

General synthesis step 1: synthesis of docetaxel 2' -O-alkane diacid monoester

Reaction reagents and conditions: succinic anhydride, TEA, THF, 0 deg.C-rt, 4h

The method comprises the following operation steps: dissolving docetaxel in tetrahydrofuran, and dripping the tetrahydrofuran solution of triethylamine and alkane anhydride into the reaction system successively under ice bath. Reacting at room temperature for 4 hours after the dropwise addition is finished, monitoring by HPLC (high performance liquid chromatography), neutralizing triethylamine in a reaction system slowly by using 1.0M HCl under ice bath, adjusting the pH value to 3-5, concentrating the obtained solution at 30 ℃ under reduced pressure to 1/3 volume, slowly dropwise adding the concentrated solution into ice water with the volume being 10 times that of the ice water for crystallization, and filtering under reduced pressure to collect a solid; pulping the obtained solid in ice water, and filtering; washed with water for three times and dried in vacuum at 30 ℃ to obtain the target product white solid.

General synthesis step 2: synthesis of muramyl dipeptide simplifier

The method uses various hydroxyl resins, such as queen tree ester (loading amount is 0.83mmol/g) as solid phase carriers, and introduces Fmoc-L-Lys (Boc) -COOH, Fmoc-D-iso-Gln-COOH, Fmoc-L-Ala-COOH and various organic carboxylic acids into the resins in sequence through a polypeptide solid phase synthesis strategy. After the condensation reaction is finished, various muramyl dipeptide simplifications are obtained through the steps of fully washing resin, removing Fmoc protective groups, cracking resin and the like. The various acylation processes in the reaction are conventional amide condensation reactions, and various condensation reactions can be completed by adding excessive reaction reagents (amino acids or organic carboxylic acids) and strong condensing agents (such as DIC, DCC, HATU, HBTU, BOP, PyBOP and the like).

The specific synthetic route is as follows:

reaction reagents and conditions: (a) Fmoc-Lys (Boc) -OH, HOBt, DMAP, DIC,DCM,rt,12h；(b)Ac₂O,pyridine,DMAP,DCM,rt,3h；(c)20％piperidine/DMF；rt,1h；(d)Fmoc-D-iso-Gln-OH,HOBt,DIC,DMF rt,12h；(e)Fmoc-Ala-OH,HOBt,DIC,DMF,rt,8h；(f)RCOOH,HOBt,DIC,DMF,rt,8h；(g)90％TFA/H₂O,rt,2h.

The method comprises the following operation steps:

(a) queen resin (loading 0.83mmol/g,1.0eq.), Fmoc-Lys (Boc) -OH (2.0eq.), HOBt (2.0eq.) and DMAP (0.05eq.) were sequentially added to a solid phase reactor, and after 1 hour of vacuum-pumping under reduced pressure, anhydrous dichloromethane was added and stirred for 0.5 hour, then activator DIC (2.0eq.) was added, and after 12 hours of reaction at room temperature, Fmoc-Lys (Boc) -OH was introduced into the resin. The reaction solution is decompressed and drained, the resin is fully washed by N, N-dimethylformamide and dichloromethane for 3 times respectively, and the resin is drained and directly used in the next step;

(b) DCM, acetic anhydride (5.0eq.), pyridine (5.0eq.) and DMAP (0.05eq.) were added to the reactor in that order; blocking at room temperature for 3 hours, decompressing and draining the reaction solution, fully washing the resin with N, N-dimethylformamide and dichloromethane (1.5L for 6 times, draining the reaction solution, fully washing the resin with N, N-dimethylformamide and dichloromethane for 3 times respectively, draining, and directly using in the next step;

(c) adding 20% by volume of piperidine/N, N-dimethylformamide solution, removing the Fmoc protecting group of amino acid, reacting for 1 hour, draining the reaction solution, fully washing the resin for 6 times by using N, N-dimethylformamide and dichloromethane in sequence, draining, and directly using the resin in the next step;

(d) adding Fmoc-D-iso-Gln-OH (2.0eq.) and HOBt (2.0eq.) and N, N-dimethylformamide into a reactor, stirring for 5min until the system is uniform, adding an activator DIC (2.0eq.) into the reactor, introducing Fmoc-D-iso-Gln-OH into the resin through room temperature reaction, after 12 hours of reaction, taking a small amount of resin for detection by an indantrione method, wherein the resin is not blue and negative, indicating that the reaction is complete, draining the reaction solution, fully washing the resin by using the N, N-dimethylformamide and dichloromethane successively for 3 times, draining, and directly using the resin in the next step;

(e) adding Fmoc-Ala-OH (2.0eq.) and HOBt (2.0eq.) and N, N-dimethylformamide solvent into a reactor, stirring for 5min until the system is uniform, adding an activating agent DIC (2.0eq.) into the reactor, reacting at room temperature to introduce Fmoc-Ala-OH into resin, detecting by an indantrione method after reacting for 8 hours, taking a small amount of resin, detecting the resin by an indantrione method, indicating that the resin is not blue and negative, indicating that the reaction is complete, draining reaction liquid, fully washing the resin by N, N-dimethylformamide and dichloromethane for 3 times respectively, draining, and directly using the resin in the next step;

(f) sequentially adding organic acid RCOOH (1.5eq.) and HOBt (1.5eq.) and N, N-dimethylformamide into a reactor, uniformly stirring, then adding DIC (1.5eq.) to react at room temperature, introducing the organic acid into the resin, after reacting for 8 hours, taking a small amount of the resin for indetrione method detection, wherein the resin does not present blue and is negative, indicating that the reaction is complete, draining reaction liquid, washing the resin with N, N-dimethylformamide and dichloromethane for 3 times respectively, and draining;

(g) adding trifluoroacetic acid/water solution with the volume fraction of 90%, cracking for 2 hours at room temperature, filtering, washing the resin with dichloromethane for three times, combining the filtrate with the lysate, and evaporating to dryness under reduced pressure. Under the ice-bath condition, a large amount of anhydrous methyl tert-butyl ether is added into the residual liquid, stirred and white solid is separated out. Filtering, washing the solid with anhydrous methyl tert-butyl ether for 3 times, and vacuum drying at 30 ℃ to obtain a crude product of the target product.

General synthesis step 3: synthesis of conjugates of docetaxel and simplified muramyl dipeptide

Reaction reagents and conditions: (a) HOSu, EDC^.HCl,DMSO,rt,12h；(b)NMM,DMSO,rt,12h.

The method comprises the following operation steps: (a) adding docetaxel 2' -O-alkyl diacid monoester (1.0eq.) and HOSu (1.1eq.) and EDC into a reactor in sequence^.HCl (1.1eq.) and DMSO were stirred at room temperature for 12 h. HPLC monitoring shows that the docetaxel purity of less than 5% is considered to be complete, and is directly used for the next reaction.

Adding the muramyl dipeptide simplified oligopeptide (1.0eq.) into another reactor, adding DMSO, stirring at room temperature until the mixture is dissolved, then adding N-methylmorphine (5.0eq.) and stirring at room temperature for 5min, then slowly dripping the reaction liquid of the prepared N-hydroxysuccinimide active ester into the reaction system, reacting at room temperature for 12 hours, and monitoring by HPLC (high performance liquid chromatography), wherein the reaction is considered to be complete, and the purity of the N-hydroxysuccinimide active ester intermediate is less than 5%. Cooling the reaction system to 0-4 ℃, neutralizing the N-methylmorphinan in the reaction system by using 1M hydrochloric acid aqueous solution, and finely adjusting the pH of the reaction system to 3-5 by using 0.1M hydrochloric acid aqueous solution. Then slowly dripping the neutralized reaction solution into ice water with the volume 10 times that of the reaction solution and the temperature of 0-4 ℃ for crystallization; filtering under reduced pressure to obtain a target crude product; the crude product was washed with ice water 3 times and dried under vacuum at 30 ℃ to obtain the target crude product. And purifying the crude product by HPLC to obtain the conjugate of the target docetaxel and the simplified muramyl dipeptide.

The alkane dianhydride of the invention is selected from C4-C12 alkane dianhydride.

The preparation method of the conjugate has mild conditions, short reaction time and stable yield, is beneficial to the synthesis of the compound library by using a combinatorial chemistry method, and also belongs to the scope of the invention.

Wherein the abbreviations are each as defined below, each reagent is commercially available:

Fmoc	9-fluorenylmethoxycarbonyl group
Boc	Tert-butyloxycarbonyl radical
rt	At room temperature
eq.	Equivalent weight
succinic anhydride	Succinic anhydride
TEA	Triethanolamine
THF	Tetrahydrofuran (THF)
DIC	N, N-diisopropylcarbodiimide
DCC	Dicyclohexylcarbodiimide
HATU	2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate
HBTU	O-benzotriazole-tetramethylurea hexafluorophosphate
BOP	Benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphates

PyBOP	Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
HOBt,	1-hydroxybenzotriazoles
DMAP	4-dimethylaminopyridine
DCM	Methylene dichloride
Ac ₂O	Acetic anhydride
pyridine	Pyridine compound
piperidine	Piperidine derivatives
DMF	N, N-dimethylformamide
TFA	Trifluoroacetic acid
HOSu	N-hydroxysuccinimide
EDC	1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
DMSO	Dimethyl sulfoxide
NMM	N-methylmorpholine

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating various immune diseases, comprising the conjugate compound or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

The carrier includes, but is not limited to, various excipients suitable for pharmaceutical formulation and may be administered to a mammal, particularly a cancer patient, by the gastrointestinal or parenteral route. For gastrointestinal administration, the excipient includes various fillers such as microcrystalline cellulose and the like which can be used in tablets or granules.

The compound of the invention has the following advantages:

1. the invention provides an application of a conjugate formed by docetaxel and a muramyl dipeptide simplified substance or a pharmaceutically acceptable salt thereof in preparing medicines for preventing or treating various immune diseases.

2. The invention provides the application of a conjugate formed by docetaxel and a muramyl dipeptide simplified substance or pharmaceutically acceptable salt thereof in preparing medicaments for preventing or treating various immune diseases, particularly the application of the conjugate through the action of TIMP-1, MMP9, IL-6 and the like, has clear action mode and is beneficial to accurately treating the immune diseases caused by specific factors.

Drawings

FIG. 1 is a graph showing the effect of TIMP-1 on the conjugates of docetaxel and a simplified muramyl dipeptide or pharmaceutically acceptable salts thereof according to the present invention.

FIG. 2 is a graph showing the comparative effect of docetaxel and a muramyl dipeptide simplified substance as a conjugate of the present invention or a pharmaceutically acceptable salt thereof on MMP 9.

FIG. 3 is a graph showing the comparative effect of docetaxel in combination with a simplified muramyl dipeptide or a pharmaceutically acceptable salt thereof on IL-6 according to the present invention.

Wherein 3 is S-01, 7 is docetaxel, 5a is muramyl dipeptide MDA-1, and 6 is linker + muramyl dipeptide (MDA-1-linker).

Wherein, represents p <0.05, represents p <0.01, represents p <0.001, and all the results are compared with the control group.

Detailed Description

The various aspects and features of the invention are described in more detail below in the preferred examples of conjugate synthesis and biology of Docetaxel (Docetaxel) and Muramyl Dipeptide (MDP) simplifications. It will be understood by those skilled in the art that these examples are for illustrative purposes only and do not limit the scope of the present invention. The scope of the invention is limited only by the claims. Various modifications and improvements to the various aspects of the invention may be made by those skilled in the art without departing from the scope of the claims, and these modifications and improvements are within the scope of the invention.

In addition, it should be noted that, unless otherwise specified, various materials and reagents used in the following examples are those commonly used in the art and are commercially available in a usual manner; the intermediates used may be obtained by conventional commercial routes or prepared by well-known methods; the methods used are all conventional methods known to the person skilled in the art.

Chemical examples

Example 1: solid phase Synthesis of muramyl dipeptide simplification MDA-1 (general procedure 2)

¹H-NMR(500MHz,DMSO-d₆):12.59(1H,br.s),8.47(1H,d,J＝6.8Hz),8.24(1H,d,J＝8.1Hz),8.11(1H,d,J＝7.8Hz),7.76(1H,dd,J＝8.8,6.2Hz),7.73-7.63(3H,m),7.55(1H,dd,J＝8.8,2.6Hz),7.37-7.27(2H,m),7.13(1H,s),6.78(1H,d,J＝15.7Hz),4.42(1H,q,J＝6.9Hz),4.15(2H,m),2.77(2H,m),2.16(2H,t,J＝8.0Hz),1.97(1H,m),1.71(2H,m),1.60-1.45(3H,m),1.40-1.20(6H,m).HR-MS(ESI-TOF)m/z:Calcd for C₂₃H₃₂N₅O₆FCl[M-CF₃COO]⁺528.2020；Found 528.2023.

Example 2: solid phase Synthesis of muramyl dipeptide simplification MDA-2 (general procedure 2)

¹H-NMR(500MHz,DMSO-d₆):12.58(1H,br.s),8.38(1H,d,J＝6.8Hz),8.21(1H,d,J＝8.1Hz),8.10(1H,d,J＝7.8Hz),7.70(3H,s),7.59(2H,d,J＝8.3Hz),7.48(2H,d,J＝8.3Hz),7.40(1H,d,J＝15.8Hz),7.31(1H,s),7.11(1H,s),6.77(1H,d,J＝15.8Hz),4.40(1H,q,J＝6.7Hz),4.15(2H,m),2.76(2H,m),2.16(2H,t,J＝8.0Hz),1.96(1H,m),1.71(2H,m),1.60-1.45(3H,m),1.40-1.20(6H,m).HR-MS(ESI-TOF)m/z:Calcd for C₂₃H₃₃N₅O₆Cl[M-CF₃COO]⁺510.2114；Found 510.2116.

Example 3: solid phase Synthesis of muramyl dipeptide simplification MDA-3 (general procedure 2)

¹H-NMR(500MHz,DMSO-d₆):12.58(1H,br.s),8.54(1H,d,J＝6.1Hz),8.25(1H,d,J＝7.7Hz),8.12(1H,d,J＝7.3Hz),7.96(1H,s),7.85-7.60(4H,m),7.53(1H,d,J＝10.5Hz),7.44(1H,d,J＝15.9Hz),7.40-7.30(2H,m),7.13(1H,s),6.87(1H,d,J＝15.9Hz),4.40(1H,q,J＝6.7Hz),4.15(2H,m),2.76(2H,m),2.16(2H,t,J＝8.0Hz),1.96(1H,m),1.71(2H,m),1.60-1.45(3H,m),1.40-1.20(6H,m).HR-MS(ESI-TOF)m/z:Calcd for C₂₃H₃₂N₅O₆FCl[M-CF₃COO]⁺528.2020；Found 528.1924.

Example 4: solid phase Synthesis of muramyl dipeptide simplification MDA-4 (general procedure 2)

HR-MS(ESI-TOF)m/z:Calcd for C₂₄H₃₆N₅O₆[M-CF₃COO]⁺490.2660；Found 490.2666.

Example 5: solid phase Synthesis of muramyl dipeptide simplification MDA-5 (general procedure 2)

HR-MS(ESI-TOF)m/z:Calcd for C₂₄H₃₃N₆O₆[M-CF₃COO]⁺501.2456；Found 501.2446.

Example 6: solid phase Synthesis of muramyl dipeptide simplification MDA-6 (general procedure 2)

HR-MS(ESI-TOF)m/z:Calcd for C₂₁H₃₂N₅O₆S[M-CF₃COO]⁺482.2068；Found 482.2053.

Example 7: synthesis of S-01 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.49(1H,br.s),8.45(1H,d,J＝6.7Hz),8.25(1H,d,J＝8.0Hz),8.09(1H,d,J＝7.5Hz),7.99(2H,d,J＝7.5Hz),7.90-7.80(2H,m),7.80-7.70(2H,m),7.70-7.60(3H,m),7.54(1H,dd,J＝2.2,8.7Hz),7.45-7.30(6H,m),7.18(1H,t,J＝6.7Hz),7.11(1H,s),6.79(1H,d,J＝15.7Hz),5.79(1H,t,J＝8.2Hz),5.41(1H,d,J＝6.9Hz),5.15-4.85(6H,m),4.50-4.38(2H,m),4.20-4.10(2H,m),4.10-3.95(3H,m),3.64(1H,d,J＝6.5Hz),3.02(2H,m),2.70-2.55(2H,m),2.38(2H,t,J＝6.9Hz),2.30-2.05(6H,m),1.98(1H,m),1.85(1H,m),1.78-1.60(6H,m),1.60-1.47(5H,m),1.45-1.20(16H,m),0.99(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₀H₈₆N₆O₂₂FClNa[M+Na]⁺1439.5365；Found 1439.5358.

Example 8: synthesis of S-01-Na sodium salt

Dissolving S-01(283mg,0.2mmol) in 50mL of a mixed solvent of acetonitrile/water (1/1 (V/V), then placing a reaction bottle into a low-temperature reactor at-10 ℃, stirring for 5min, dropwise adding 100mL of 0.002mmol/mL NaOH aqueous solution into the solution, and quickly reacting the obtained solution at-20 ℃ for 4 hours after dropwise adding; freeze-drying gave 290mg of a white powdery solid.¹H-NMR(500MHz,DMSO-d₆):8.45(1H,d,J＝6.7Hz),8.25(1H,d,J＝8.0Hz),8.09(1H,d,J＝7.5Hz),7.99(2H,d,J＝7.5Hz),7.90-7.80(2H,m),7.80-7.70(2H,m),7.70-7.60(3H,m),7.54(1H,dd,J＝2.2,8.7Hz),7.45-7.30(6H,m),7.18(1H,t,J＝6.7Hz),7.11(1H,s),6.79(1H, d,J＝15.7Hz),5.79(1H,t,J＝8.2Hz),5.41(1H,d,J＝6.9Hz),5.15-4.85(6H,m),4.50-4.38(2H,m),4.20-4.10(2H,m),4.10-3.95(3H,m),3.64(1H,d,J＝6.5Hz),3.02(2H,m),2.70-2.55(2H,m),2.38(2H,t,J＝6.9Hz),2.30-2.05(6H,m),1.98(1H,m),1.85(1H,m),1.78-1.60(6H,m),1.60-1.47(5H,m),1.45-1.20(16H,m),0.99(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₀H₈₅N₆O₂₂FCl[M-Na]^-1415.5395；Found 1415.5380.

Example 9: synthesis of S-01-Ca calcium salt

S-01(283mg,0.2mmol) was dissolved in 20mL of a mixed solvent of acetonitrile/water (1/1 (V/V)), and then the reaction flask was placed in a low-temperature reactor at 0 ℃ and stirred for 5min, and 0.01mmol/mL of Ca (OH) was added dropwise to the solution₂20mL of aqueous solution, quickly pouring the obtained solution into 80mL of ice water after the dropwise addition is finished, and cooling to-20 ℃ for reaction for 4 hours; freeze-drying gave 260mg of a white powdery solid.¹H-NMR(500MHz,DMSO-d₆):8.45(1H,d,J＝6.7Hz),8.25(1H,d,J＝8.0Hz),8.09(1H,d,J＝7.5Hz),7.99(2H,d,J＝7.5Hz),7.90-7.80(2H,m),7.80-7.70(2H,m),7.70-7.60(3H,m),7.54(1H,dd,J＝2.2,8.7Hz),7.45-7.30(6H,m),7.18(1H,t,J＝6.7Hz),7.11(1H,s),6.79(1H,d,J＝15.7Hz),5.79(1H,t,J＝8.2Hz),5.41(1H,d,J＝6.9Hz),5.15-4.85(6H,m),4.50-4.38(2H,m),4.20-4.10(2H,m),4.10-3.95(3H,m),3.64(1H,d,J＝6.5Hz),3.02(2H,m),2.70-2.55(2H,m),2.38(2H,t,J＝6.9Hz),2.30-2.05(6H,m),1.98(1H,m),1.85(1H,m),1.78-1.60(6H,m),1.60-1.47(5H,m),1.45-1.20(16H,m),0.99(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₀H₈₅N₆O₂₂FCl[M-1/2Ca]^-1415.5395；Found 1415.5386.

Example 10: synthesis of S-01-Me methyl ester

Reaction reagents and conditions: (a) SOCl₂,MeOH,0℃-rt,12h；(b)HOSu,EDC^.HCl,DMSO,rt,12h；(c)NMM,DMSO,rt,12h.

Reaction operation: (a) MDA-1(6.0g,9.4mmol) was dissolved in 80mL of methanol under ice-cooling, stirred for 5min, thionyl chloride (0.75mL,10.37mmol) was slowly added dropwise to the above reactor, and the reaction was warmed to room temperature after completion of the addition, and reacted for 12 hours. The solvent was evaporated to dryness under reduced pressure at 30 ℃ and after dissolving with DCM, DCM was evaporated to dryness under reduced pressure at 30 ℃ and the solid product was dried under vacuum at room temperature for 24 hours and used directly in the next step. (b) And (c) step (see general synthesis step 3), finally 4.5g of pure S-01-Me was prepared with a yield of 34% (three-step yield).¹H-NMR(500MHz,DMSO-d₆):8.42(1H,d,J＝6.8Hz),8.20(2H,t,J＝7.8Hz),7.98(2H,d,J＝7.5Hz),7.90-7.60(7H,m),7.53(1H,dd,J＝2.6,8.8Hz),7.45-7.25(6H,m),7.18(1H,t,J＝6.8Hz),7.09(1H,s),6.78(1H,d,J＝15.7Hz),5.79(1H,t,J＝8.3Hz),5.40(1H,d,J＝7.1Hz),5.09(3H,s),4.98(1H,d,J＝7.1Hz),4.90(2H,d,J＝10.4Hz),4.50-4.35(2H,m),4.25-4.10(2H,m),4.10-3.95(3H,m),3.64(1H, d,J＝7.0Hz),3.61(3H,s),3.01(2H,q,J＝5.9Hz),2.70-2.55(2H,m),2.38(2H,t,J＝7.1Hz),2.35-2.20(4H,m),2.17(2H,t,J＝8.0Hz),1.98(1H,m),1.85(1H,m),1.78-1.60(6H,m),1.60-1.55(2H,m),1.52(3H,s),1.45-1.20(16H,m),0.99(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₁H₈₇N₆O₂₂FCl[M-H]^-1429.5551；Found 1429.5549.

Example 11: synthesis of S-02 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.42(1H,br.s),8.48(1H,d,J＝5.2Hz),8.21(1H,d,J＝7.3Hz),8.06(1H,d,J＝6.7Hz),7.96(2H,d,J＝6.6Hz),7.90-7.75(2H,m),7.75-7.55(4H,m),7.51(1H,d,J＝10.3Hz),7.45-7.20(6H,m),7.20-7.00(2H,m),6.83(1H,d,J＝16.0Hz),5.76(1H,m),5.37(1H,d,J＝6.0Hz),5.15-4.80(6H,m),4.50-4.30(2H,m),4.20-3.90(5H,m),3.61(1H,m),2.98(2H,m),2.70-2.55(2H,m),2.40-2.30(2H,m),2.30-2.05(6H,m),1.94(1H,m),1.80(1H,m),1.75-1.57(6H,m),1.57-1.43(5H,m),1.42-1.15(16H,m),0.95(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₀H₈₆N₆O₂₂FClNa[M+Na]⁺1439.5365；Found 1439.5360.

Example 12: synthesis of S-03 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.45(1H,br.s),8.28(1H,d,J＝6.6Hz),8.18(1H,d,J＝7.8Hz),8.06(1H,d,J＝7.6Hz),7.96(2H,d,J＝7.5Hz),7.90-7.75(2H,m),7.70(1H,t,J＝7.1Hz),7.63(2H,t,J＝7.3Hz),7.45-7.25(8H,m),7.20(2H,d,J＝7.6Hz),7.16(1H,t,J＝6.6Hz),7.08(1H,s),6.67(1H,d,J＝15.7Hz),5.76(1H,t,J＝8.1Hz),5.38(1H,d,J＝6.8Hz),5.06(3H,s),4.99(1H,d,J＝6.8Hz),4.95-4.80(2H,m),4.45-4.30(2H,m),4.20-4.05(2H,m),4.05-3.90(3H,m),3.62(1H,d,J＝6.3Hz),2.98(2H,m),2.70-2.55(2H,m),2.36(2H,t,J＝6.7Hz),2.30(3H,s),2.26-2.05(6H,m),1.94(1H,m),1.82(1H,m),1.75-1.57(6H,m),1.57-1.43(5H,m),1.42-1.15(16H,m),0.95(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₁H₉₁N₆O₂₂[M+H]⁺1379.6186；Found 1379.6180.

Example 13: synthesis of S-04 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.54(1H,br.s),8.99(1H,d,J＝5.2Hz),8.64(1H,d,J＝6.9Hz),8.45(1H,d,J＝5.2Hz),8.30-8.10(4H,m),8.03(2H,d,J＝5.2Hz),7.98-7.85(3H,m),7.85-7.65(4H,m),5.84(1H,m),5.46(1H,d,J＝6.0Hz),5.25-4.90(6H,m),4.70(1H,m),4.50(1H,s),4.30-4.00(5H,m),3.69(1H,d,J＝6.8Hz),3.07(2H,m),2.80-2.65(2H,m),2.50-2.35(2H,m),2.35-2.15(6H,m),2.03(1H,m),1.90(1H,m),1.75-1.57(6H,m),1.57-1.43(5H,m),1.42-1.15(16H,m),1.03(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₁H₈₇N₇O₂₂Na[M+Na]⁺1412.5802；Found 1412.5794.

Example 14: synthesis of S-05 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.47(1H,br.s),8.35(1H,d,J＝5.1Hz),8.19(1H,d,J＝7.1Hz),8.07(1H,d,J＝6.8Hz),7.99(2H,d,J＝6.6Hz),7.90-7.80(1H,m),7.78-7.50(5H,m),7.50-7.33(5H,m),7.30(1H,s),7.20-7.05(3H,m),6.50(1H,d,J＝15.6Hz),5.79(1H,t,J＝8.1Hz),5.40(1H,d,J＝6.8Hz),5.06(3H,s),4.99(1H,d,J＝6.8Hz),4.95-4.80(2H,m),4.50-4.30(2H,m),4.20-4.10(2H,m),4.10-3.90(3H,m),3.63(1H,d,J＝6.3Hz),3.01(2H,s),2.70-2.55(2H,m),2.38(2H,t,J＝6.7Hz),2.30-2.05(6H,m),1.96(1H,m),1.84(1H,m),1.75-1.57(6H,m),1.57-1.43(5H,m),1.42-1.15(16H,m),0.98(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₆₈H₈₅N₆O₂₂S[M-H]^-1369.5443；Found 1369.5445.

Example 15: synthesis of S-06 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.48(1H,br.s),8.98(1H,d,J＝7.0Hz),8.25-8.15(2H,m),8.10(1H,d,J＝7.4Hz),7.98(2H,d,J＝7.3Hz),7.93-7.80(3H,m),7.75-7.60(4H,m),7.45-7.30(4H,m),7.27(1H,s),7.18(1H,t,J＝4.8Hz),7.11(1H,s),5.78(1H,t,J＝8.1Hz),5.40(1H,d,J＝7.0Hz),5.08(3H,s),5.01(1H,d,J＝6.4Hz),4.95-4.80(2H,m),4.50-4.30(2H,m),4.19(1H,q,J＝5.4Hz),4.11(1H,q,J＝5.4Hz),4.08-3.95(3H,m),3.63(1H,d,J＝6.6Hz),3.01(2H,s),2.70-2.55(2H,m),2.38(2H,t,J＝6.8Hz),2.30-2.05(6H,m),1.95(1H,m),1.83(1H,m),1.78-1.60(6H,m),1.60-1.43(5H,m),1.42-1.15(16H,m),0.98(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₆₈H₈₃ClN₇O₂₄[M-H]^-1416.5183；Found 1416.5167.

Example 16: synthesis of S-07 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.49(1H,br.s),8.48(1H,d,J＝6.7Hz),8.24(1H,d,J＝8.1Hz),8.09(1H,d,J＝7.6Hz),7.99(2H,d,J＝7.4Hz),7.93-7.80(2H,m),7.77-7.60(4H,m),7.50-7.30(7H,m),7.18(2H,t,J＝7.2Hz),7.12(1H,s),6.82(1H,d,J＝16.0Hz),5.78(1H,t,J＝8.6Hz),5.40(1H,d,J＝7.0Hz),5.15-4.80(6H,m),4.50-4.35(2H,m),4.20-3.95(5H,m),3.63(1H,d,J＝6.7Hz),3.01(2H,m),2.70-2.55(2H,m),2.38(2H,t,J＝7.0Hz),2.30-2.05(6H,m),1.98(1H,m),1.84(1H,m),1.78-1.60(6H,m),1.60-1.45(5H,m),1.40-1.20(16H,m),0.98(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₀H₈₅N₆O₂₂F₂[M-H]^-1399.5690；Found 1399.5688.

Example 17: synthesis of S-08 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.49(1H,br.s),8.34(1H,d,J＝6.8Hz),8.23(1H,d,J＝8.1Hz),8.09(1H,d,J＝7.5Hz),7.98(2H,d,J＝7.4Hz),7.91-7.80(2H,m),7.75-7.60(4H,m),7.55-7.28(8H,m),7.18(1H,t,J＝7.1Hz),7.11(1H,s),6.75(1H,d,J＝15.8Hz),5.78(1H,t,J＝8.6Hz),5.40(1H,d,J＝7.1Hz),5.15-4.85(6H,m),4.50-4.35(2H,m),4.20-3.95(5H,m),3.63(1H,d,J＝7.0Hz),3.00(2H,m),2.70-2.55(2H,m),2.37(2H,t,J＝7.0Hz),2.30-2.05(6H,m),1.98(1H,m),1.83(1H,m),1.78-1.60(6H,m),1.60-1.45(5H,m),1.40-1.20(16H,m),0.98(6H,s).

HR-MS(ESI-TOF)m/z:Calcd for C₇₀H₈₅N₆O₂₂F₂[M-H]^-1399.5690；Found 1399.5692.

Example 18: synthesis of S-09 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.49(1H,br.s),8.35(1H,d,J＝6.8Hz),8.24(1H,d,J＝8.0Hz),8.10-7.95(3H,m),7.90-7.80(2H,m),7.78-7.60(4H,m),7.60-7.30(9H,m),7.17(1H,t,J＝7.1Hz),7.10(1H,s),6.83(1H,d,J＝15.9Hz),5.78(1H,t,J＝8.6Hz),5.40(1H,d,J＝7.0Hz),5.15-4.85(6H,m),4.50-4.35(2H,m),4.20-3.95(5H,m),3.63(1H,d,J＝7.0Hz),3.00(2H,m),2.70-2.55(2H,m),2.38(2H,t,J＝7.1Hz),2.30-2.05(6H,m),1.98(1H,m),1.82(1H,m),1.78-1.60(6H,m),1.60-1.45(5H,m),1.40-1.20(16H,m),0.98(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₀H₈₇N₆O₂₂FK[M+K]⁺1421.5495；Found 1421.5627.

Example 19: synthesis of S-10 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.48(1H,br.s),9.85(1H,s),8.20(2H,dd,J＝11.3,7.6Hz),8.08(1H,d,J＝7.8Hz),7.98(2H,d,J＝7.4Hz),7.90-7.80(2H,m),7.72(1H,t,J＝7.2Hz),7.65(2H,t,J＝7.4Hz),7.45-7.25(8H,m),7.18(1H,t,J＝7.0Hz),7.10(1H,s),6.79(2H,d,J＝8.5Hz),6.52(1H,d,J＝15.8Hz),5.78(1H,t,J＝8.6Hz),5.40(1H,d,J＝7.1Hz),5.15-4.85(6H,m),4.50-4.35(2H,m),4.20-3.95(5H,m),3.63(1H,d,J＝6.8Hz),3.01(2H,m),2.70-2.55(2H,m),2.37(2H,t,J＝7.1Hz),2.30-2.05(6H,m),1.99(1H,m),1.82(1H,m),1.78-1.60(6H,m),1.60-1.45(5H,m),1.40-1.20(16H,m),0.98(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₇₀H₈₈N₆O₂₃Na[M+Na]⁺1403.5799；Found 1403.5795.

Example 20: synthesis of S-11 (general Synthesis step 3)

¹H-NMR(500MHz,DMSO-d₆):12.49(1H,br.s),8.75(1H,s),8.56(1H,d,J＝4.1Hz),8.42(1H,d,J＝6.8Hz),8.25(1H,d,J＝8.0Hz),8.09(1H,d,J＝6.9Hz),7.98(3H,d,J＝7.7Hz),7.93-7.80(2H,m),7.80-7.60(3H,m),7.50-7.30(7H,m),7.18(1H,d,J＝6.6Hz),7.12(1H,s),6.87(1H,d,J＝16.0Hz),5.78(1H,t,J＝8.6Hz),5.40(1H,d,J＝6.9Hz),5.15-4.85(6H,m),4.50-4.35(2H,m),4.20-3.95(5H,m),3.63(1H,d,J＝6.8Hz),3.01(2H,m),2.70-2.55(2H,m),2.37(2H,t,J＝6.8Hz),2.30-2.05(6H,m),1.96(1H,m),1.81(1H,m),1.78-1.60(6H,m),1.60-1.45(5H,m),1.40-1.20(16H,m),0.98(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₆₉H₈₆N₇O₂₂[M-H]^-1364.5831；Found 1364.5820.

Example 21: synthesis of S-12 (general Synthesis step 3)

¹H-NMR(300MHz,DMSO-d₆):12.44(1H,br.s),8.62(1H,d,J＝4.0Hz),8.57(1H,d,J＝6.7Hz),8.21(1H,d,J＝8.2Hz),8.09(1H,d,J＝7.7Hz),7.99(2H,d,J＝7.1Hz),7.93-7.80(3H,m),7.78-7.62(3H,m),7.58(1H,d,J＝7.8Hz),7.50-7.28(7H,m),7.16(3H,m),5.78(1H,t,J＝8.5Hz), 5.40(1H,d,J＝7.1Hz),5.15-4.85(6H,m),4.50-4.35(2H,m),4.20-3.95(5H,m),3.63(1H,d,J＝7.1Hz),3.01(2H,m),2.70-2.55(2H,m),2.38(2H,t,J＝7.0Hz),2.30-2.05(6H,m),1.98(1H,m),1.82(1H,m),1.78-1.60(6H,m),1.60-1.45(5H,m),1.40-1.20(16H,m),0.98(6H,s).HR-MS(ESI-TOF)m/z:Calcd for C₆₉H₈₆N₇O₂₂[M-H]^-1364.5831；Found 1364.5826.

Biological examples

In vivo Activity test section

Example 22:

first, experimental animal

BALB/c mice, female, SPF grade, weight 15-17g, purchased from Guangdong province medical laboratory animal center, and certification number: 44007200015046. mice are bred in SPF-level animal laboratory of Innovative drug research center of Shenzhen Xin Tai pharmaceutical industry, Inc

Second, experiment cell

Mouse breast cancer cells (4T1, ATCC CRL2539) were cultured in 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% glutamine and 1% penicillin-streptomycin. 4T1 cells were collected in the logarithmic growth phase and adjusted to a cell concentration of 1.5X106/mL for inoculation.

II, a test method:

3.1 inoculation: mouse breast cancer cells (4T1, ATCC CRL2539) were cultured in 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% glutamine and 1% penicillin-streptomycin. Collecting 4T1 cells in logarithmic growth phase, and regulating cell concentration to 1.5X10⁶and/mL. Female BALB/c mice were inoculated with 4T1 cells in the 4 th mammary fat pad at a volume of 0.1mL/mouse, i.e., 1.5X10⁵/mouse。

3.2 grouping: of which 5 mice were not inoculated with 4T1 cells as

Groups, on day of inoculation, designated D0, day 4 (D4), were randomized into 7 groups by body weight, namely, Vehicle control, 3(5mg/kg), 3(10mg/kg), 3(20mg/kg), 7(5.7mg/kg) +5a (4.53mg/kg), 7(5.7mg/kg) +6(4.43mg/kg), 10 mice per group.

Wherein 3 is S-01, 7 is docetaxel, 5a is muramyl dipeptide MDA-1, 6 is linker + muramyl dipeptide (MDA-1-linker)

3.3 administration: dosing was started on the day of the group (dosing regimen and sample formulation methods see below). Once a week.

Table 1 grouping and dosing regimens

TABLE 2 preparation of mother liquors

Note: the crude drug mother liquor is preserved in a refrigerator at 4 ℃ in dark place, and the preparation mother liquor is prepared at present.

TABLE 3 preparation of working solution (now available)

Note: a is indicated by 7 mother liquor additions and b is indicated by 5 a. c is indicated above with 6 added amounts of mother liquor and the volume administered is 0.2ml/10g.

3.4 tumor weight: d4 tumor volume was initially measured and recorded, after which tumor major and minor diameters were measured with a vernier caliper every 2 days. According to the formula: (1/2) X major diameter X (minor diameter) 2 tumor volume is calculated.

3.5 termination of the experiment: the experiment was terminated 4 weeks after administration, the eye was removed and blood was taken, serum was separated, the tumor and lung were dissected out and weighed. The lung was fixed in Bouin's fixative for 24h, the number of lung surface metastatic nodules was counted, and statistical examination was performed on the number of lung metastatic nodules.

3.6 detecting the contents of cytokines such as TIMP-1, MMP9, IL-6 and the like in the serum of the mouse: the procedures were performed according to the ELISA kit instructions

Experimental results, as can be seen from the comparative effect of the conjugate formed by docetaxel and muramyl dipeptide simplexed or the pharmaceutically acceptable salt thereof on TIMP-1 in the invention shown in figure 1, the effect of the conjugate formed by docetaxel and muramyl dipeptide simplexed on TIMP-1 is obviously better than that of docetaxel, docetaxel + muramyl dipeptide simplexed (two substances) and docetaxel + muramyl dipeptide simplexed-linker (two substances).

As can be seen from the comparative effect of the conjugate formed by docetaxel and muramyl dipeptide simplexed of the present invention or the pharmaceutically acceptable salt thereof on MMP9 in FIG. 2, the effect of the conjugate formed by docetaxel and muramyl dipeptide simplexed on MMP9 is significantly better than that of docetaxel, docetaxel + muramyl dipeptide simplexed (two substances), and docetaxel + muramyl dipeptide simplexed-linker (two substances).

As can be seen from the comparative effect of IL-6 on the conjugate formed by docetaxel and muramyl dipeptide simplexed or the pharmaceutically acceptable salt thereof in the invention shown in figure 3, the effect of the conjugate formed by docetaxel and muramyl dipeptide simplexed on MMP9 is obviously better than that of docetaxel, docetaxel + muramyl dipeptide simplexed (two substances) and docetaxel + muramyl dipeptide simplexed-linker (two substances).

Note that in the above experiment, the structure of MDA-1-linker (Compound 6) is as follows:

the above is the preferred embodiment of the present invention, but the present invention is not limited to this, and equivalent changes or modifications on the basis of the present invention still fall within the protection scope of the present invention.

Claims

The application of the docetaxel conjugate compound shown as the formula I and the pharmaceutically acceptable salt thereof in preparing the medicines for preventing or treating various immune diseases,

wherein m is 0 or 1;

n is 2,3,4,5,6,7,8,9 or 10;

R₁selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted alkoxy of C1-C6, wherein the substituted substituent is selected from hydroxyl, sulfydryl, halogen, amino, nitro, cyano, aldehyde group, alkyl of C1-C6, carboxyl, hydroxyamino, alkylene of C2-C6, amido of C1-C4;

R₂is selected from hydrogen, substituted or unsubstituted alkyl of C1-C6, substituted or unsubstituted alkoxy of C1-C6, and the substituted substituent is selected from halogen.
The use according to claim 1, for the treatment of various immune disorders by acting on a signalling pathway and/or factors selected from NF- κ B, p-I κ B α, I κ B a, p-ERK, p-p38, p-JNK, MMP8, MMP9, MMP2, TIMP-1, IL-1 β, IL-6, IL-8, TNF- α, CD11B, Gr-1, Ly-6G, TSP-1, NOD1, NOD2, S100a8, S100a 9.
Use according to claim 1 or 2, characterized in that said immunity essentially comprises tumor growth and metastasis, anti-inflammatory, such as rheumatoid arthritis and systemic lupus erythematosus; myelosuppression, and macrophage, lymphocyte defects.
The use according to claim 1, wherein said aryl is selected from the group consisting of five-ten membered aryl and heteroaryl is selected from the group consisting of five-ten membered heteroaryl.
The use according to claim 4, wherein said five-ten membered aryl group is selected from the group consisting of five membered aryl group, six membered aryl group, nine membered fused ring aryl group, ten membered fused ring aryl group,

the five-membered aryl group is selected from
The six-membered aryl is selected from
The nine-membered fused ring aryl is selected from
The ten-membered fused ring aryl is selected from

The five-ten membered heteroaryl is selected from a five-membered heterocyclic group containing 1-4 heteroatoms selected from N, O or S, a six-membered heterocyclic group containing 1-4 heteroatoms selected from N, O or S, a ten-membered fused heterocyclic group containing 1-4 heteroatoms selected from N, O or S;

the five-membered heterocyclic group containing 1-4 heteroatoms selected from N, O or S is selected from:

the six-membered heterocyclic group containing 1 to 4 heteroatoms selected from N, O or S is selected from:

the ten-membered fused heterocyclic group containing 1 to 4 heteroatoms selected from N, O or S is selected from:
the use according to claim 1, wherein the alkyl group having 1-6 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclopentyl, n-hexyl, cyclohexyl, dimethylpropyl, 2-methylbutyl, 2-dimethylbutyl, and 2, 3-dimethylbutyl; the C1-C6 alkoxy is selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexoxy, isohexoxy, methoxyethoxy, ethoxymethoxy, propoxymethoxy and propoxyethoxy.
The use according to claim 1, wherein the halogen is selected from fluorine, chlorine, bromine or iodine; the alkenyl of C2-C6 is ethenyl, propenyl, butenyl, isobutenyl, 2-butenyl, pentenyl, isopentenyl, 2-pentenyl, hexenyl and isohexenyl; the C1-C4 acylamino is acetylamino, propionylamino, butyrylamino or isobutyrylamino.
According to claim1, wherein R is₂Is hydrogen or hydrogen is replaced by metal or nonmetal cation to form pharmaceutically acceptable salt, and the metal or nonmetal cation is selected from Na⁺，K⁺,Ca²⁺,Mg²⁺,Zn²⁺,Al³⁺And NH₄ ⁺。
The use according to claim 1, wherein the compound of formula I includes, but is not limited to, compounds of formula IA:
use according to claim 1, characterized in that said compound is selected from the group consisting of
A pharmaceutical composition for preventing or treating various immune diseases is characterized by comprising a compound shown as a formula I, pharmaceutically acceptable salts thereof and more than one pharmaceutically acceptable carrier, m, n, R₁And R₂As defined above.