CN112724148A - Imidazopyridazine compound, modified amphiphilic functional molecule and application thereof - Google Patents

Abstract

The invention provides an imidazopyridazine compound with better PD-L1 inhibitory activity obtained after screening according to a DNA coding compound library, a modified amphiphilic functional molecule capable of being connected with an aldolase antibody and application thereof. In particular to a compound shown as a formula (I), and pharmaceutically acceptable salts, isomers and prodrugs thereof, wherein the definition of each group is described in the specification. The imidazopyridazine compound provided by the invention can treat, relieve and/or prevent related diseases mediated by PD-L1. The modified amphiphilic functional molecule of the imidazopyridazine compound has better binding interaction on PD-L1 and can carry out covalent modification and connection on an aldolase antibody, and the formed amphiphilic functional molecule is connected with the aldolase antibody, so that the specific protein targeting capability of the antibody is enhanced, and the target recognition capability of the antibody is improved.

Description

Imidazopyridazine compound, modified amphiphilic functional molecule and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to an imidazopyridazine compound, a modified amphiphilic functional molecule, a preparation method and an application thereof.

Background

Immunotherapy has been widely used for the treatment of cancer, and the treatment of various types of cancer is currently being drastically changed. Cancer immunotherapy, one of the fastest growing fields of cancer therapy, aimed at inducing the immune system against cancer tumors, is being investigated as a potential target for immunotumor therapy with the continuing success of tumor immunotherapy, where programmed death molecule (PD-L)/PD 1 ligand (PD1 ligand, PD-L1) is one of the most promising targets in the field of immunooncology. PD-L1 is a type 1 transmembrane protein of 33kDa, and the PD-1/PD-L1 pathway plays an important role in regulating the immune system, in particular hijacking the activation state of CD28/MHC, negatively regulating the immune response by regulating T lymphocyte proliferation, cytokine production and cytotoxic activity. Research shows that the tumor microenvironment can induce infiltrated T cells to over-express PD-1 protein, while ligand PD-L1 of PD-1 is over-expressed in the tumor cells, and the binding of PD-1 and ligand PD-L1 thereof leads to the down-regulation of T cell functions of cancer patients, thereby inhibiting anti-tumor immunity and leading to the failure of T cells. The PD-1/PD-L1 inhibitor can block the combination of PD-1 and PD-L1, interfere negative regulation mechanism, and enhance T cell activity, thereby enhancing immune response and treating immune tumor. The PD-1/PD-L1 interaction has become a promising target for cancer therapy. The clinical significance of PD-1/PD-L1 inhibitors has been demonstrated by the inhibition of PD-1/PD-L1 interaction by the approved monoclonal antibodies, and therefore, the development of inhibitors of PD-L1 has important implications for the development of cancer therapy.

DNA-encoded Library (DEL) compounds can efficiently infer the molecular composition of a compound through the application of combinatorial chemistry, Polymerase Chain Reaction (PCR) and high-throughput sequencing (NGS). Thus, DEL is becoming an important tool for the discovery of novel head compounds.

Furthermore, the organophilic small molecules that can link proteins and antibodies have developed rapidly in the last decade, potentially solving many prominent problems in biomedical research and turning to therapeutic applications in the future. Therefore, the potential vaccine head compound molecules which can be combined with PD-L1 and are obtained based on the DEL screening technology are subjected to fusion modification with the molecules recognized by the antibody, and the heteroamphipathic functional molecules which can be combined with PD-L1 and the antibody are developed, so that the capability of targeting specific proteins is enhanced, and the method has important significance for clinical treatment.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide an imidazopyridazine compound which has better PD-L1 inhibitory activity and has a prevention or treatment effect on various related diseases caused by immunosuppression after being screened according to DEL; and the modified amphiphilic functional molecules can be connected with PD-L1 and an aldolase antibody, so that the specific protein targeting capability of the antibody is enhanced, and the target recognition capability of the antibody is improved.

In order to achieve the above object, the present invention provides an imidazopyridazine compound, which has a structural formula shown in formula (I),

wherein, in the general formula (I), n is 0-3;

x is O or NH;

R¹is hydrogen, substituted or unsubstituted C_1-6An alkyl group;

R²is hydrogen, substituted or unsubstituted C_1-6Alkyl, alkoxy, alkylthio, haloalkyl, haloalkoxy, amino, C_3-8One or more of cycloalkyl, heterocycloalkyl, substituted or unsubstituted phenyl, five or six membered monocyclic heteroaryl, substituted or unsubstituted 8 to 12 membered fused ring aryl, substituted or unsubstituted 8 to 12 membered fused ring heteroaryl; wherein, for substitution of C_1-6The substituent of the alkyl, phenyl, aryl or heteroaryl is one or more, and is independently selected from one or more of halogen, amino, cyano, hydroxyl, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl or heteroaryl;

R³is a substituent on the imidazopyridazine ring, R³Is 0 to 3 and is independently selected from one or more of the following substituent groups: F. cl, Br, I, C_1-6Alkyl, cyano, nitro, methoxy.

Specifically, in the general formula (I), the nitrogen-containing aliphatic ring in which n is located is any one of a heterocyclic cyclobutane, a tetrahydropyrrole ring, a piperidine ring and a homopiperidine ring.

Specifically, in the general formula (I), 1-position and/or 2-position are chiral centers. The two chiral centers can be respectively and independently in R configuration or S configuration.

Specifically, the R is¹Can be selected from any one of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, amyl, isoamyl and tertiary amyl.

Preferably, the compound imidazopyridazines include the following compounds:

the invention also provides an amphipathic functional molecule modified by the imidazopyridazine compound, the structural formula of which is shown as the general formula (I),

wherein, in the general formula (I), n is 0-3;

x is O or NH;

R¹is shown in a formula (II),

wherein m in formula (II)₁Is any integer selected from 1 to 6, a polyethylene glycol chain consisting of 1 to 6 ethoxy groups; m is₂Is any integer selected from 1 to 6, a polyethylene glycol chain consisting of 1 to 6 ethoxy groups;

R²is hydrogen, substituted or unsubstituted C_1-6Alkyl, alkoxy, alkylthio, haloalkyl, haloalkoxy, amino, C_3-8One or more of cycloalkyl, heterocycloalkyl, substituted or unsubstituted phenyl, five or six membered monocyclic heteroaryl, substituted or unsubstituted 8 to 12 membered fused ring aryl, substituted or unsubstituted 8 to 12 membered fused ring heteroaryl; wherein, for substitution of C_1-6The substituent of the alkyl, phenyl, aryl or heteroaryl is one or more, and is independently selected from any one or more of halogen, amino, cyano, hydroxyl, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl or heteroaryl;

R³is a substituent on the imidazopyridazine ring, R³Is 1 to 3 and is independently selected from any one or more of the following substituents: F. cl, Br, I, C_1-6Alkyl, cyano, nitro, methoxy.

Specifically, in the general formula (I), the nitrogen-containing aliphatic ring in which n is located is any one of a heterocyclic cyclobutane, a tetrahydropyrrole ring, a piperidine ring and a homopiperidine ring.

Specifically, in the general formula (I), 1-position and/or 2-position are chiral centers. The two chiral centers can be respectively and independently in R configuration or S configuration.

Preferably, the amphiphilic functional molecule comprises the following compounds:

the imidazopyridazine compound and the modified amphiphilic functional molecule provided by the invention are obtained by the following method: performing affinity screening of a DNA coding compound library aiming at target protein, sequencing the enriched DNA coding compounds from high to low according to the calculated values and sequencing copy number of the enriched DNA coding compounds, determining the molecular sequence information of potential DNA coding seedling-end compounds and designing a synthetic path of a DNA label removal compound; the adaptor is used for coupling diketone molecules, and finally, the amphiphilic functional molecules with two ends respectively containing the head-end compound and the diketone molecules of the DNA coding compound source are formed.

The invention provides application of the imidazopyridazine compound and amphiphilic functional molecules in preparation of PD-L1 inhibitor drugs.

The invention provides application of the imidazopyridazine compound and the amphiphilic functional molecule in preparing a medicament for treating PD-L1-mediated diseases.

Preferably, the PD-L1-mediated disease includes infection, cancer, or autoimmune disease.

Preferably, the cancer is one or more of bone cancer, lung cancer, stomach cancer, colon cancer, membrane adenocarcinoma, breast cancer, prostate cancer, lung cancer, brain cancer, ovarian cancer, cancer of the shoulder, cancer of the cervix, cancer of an innocent pill, kidney cancer, cancer of the head and neck, lymphoma, leukemia, and skin cancer;

the infection is skin infection, gastrointestinal tract infection, urogenital system infection, systemic infection, or virus infection caused by one or more of influenza, hepatitis C virus, human papilloma virus, cytomegalovirus, Epstein Barr virus, poliovirus, hydrozymato-type cross-sectional virus, coxsackie virus and human immunodeficiency virus;

the autoimmune disease is one or more of rheumatoid arthritis, systemic lupus erythematosus, mixed connective tissue disease, systemic scleroderma, dermatomyositis, nodular vasculitis, nephropathy, endocrine related diseases, liver disease, psoriasis and autoimmune reaction caused by infection.

The invention provides application of the imidazopyridazine modified amphiphilic functional molecule compound in connection with an aldolase antibody.

The invention provides a pharmaceutical composition, which comprises an active component with a therapeutically effective amount and pharmaceutically acceptable auxiliary materials; the active component comprises the imidazo pyridazine compound or the imidazo pyridazine modified amphiphilic functional molecule, an isomer, a prodrug, a stable isotope derivative or a pharmaceutically acceptable salt thereof.

Preferably, the active ingredient further comprises a therapeutic agent for cancer, viral infection or autoimmune disease.

The compounds of formula (I), isomers, prodrugs, stable isotopic derivatives or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions thereof, are used in combination with one or more other therapeutic agents and/or methods for treating cancer; the other class of therapeutic agents and/or methods of treatment for cancer are one or more of tubulin inhibitors, alkylating agents, topoisomerase i/ii inhibitors, platinum-based compounds, antimetabolites, hormones and hormone analogs, signal transduction pathway inhibitors, angiogenesis inhibitors, targeted therapies, immunotherapeutic agents, pro-apoptotic agents, cell cycle signaling pathway inhibitors, and radiation therapies.

Preferably, the pharmaceutical composition is in the form of capsule, microcapsule, tablet, granule, pill, dispersion powder, liquid, soft extract, suspension, syrup, gel, aerosol, patch, liposome, oral liquid, intravenous injection or intramuscular injection.

Preferably, the dose of the imidazopyridazine compound, the isomer, the prodrug, the stable isotope derivative or the pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.05 mg/kg-90 mg/kg.

Preferably, the imidazopyridazine modified amphiphilic functional molecule is connected with the aldolase antibody, so that the specific protein targeting capacity of the antibody is enhanced, and the target recognition capacity of the antibody is improved.

The experimental result shows that the imidazopyridazine compound provided by the invention has the effect of inhibiting PD-L1, and the imidazopyridazine compound provided by the invention can treat, relieve and/or prevent related diseases mediated by PD-L1, and comprises the following components: cancer, viral or other infections or autoimmune diseases.

Experimental results show that the modified amphiphilic functional molecules of the imidazopyridazine compound have better binding interaction with PD-L1 and can carry out covalent modification and connection on an aldolase antibody, and the formed amphiphilic functional molecules are connected with the aldolase antibody, so that the specific protein targeting capacity of the antibody is enhanced, and the target recognition capacity of the antibody is improved.

Drawings

FIG. 1 is a graph showing the results of in vitro interaction of the amphiphilic functional molecule of example 9 of the present invention with PD-L1 protein and an aldolase antibody.

FIG. 2 is a schematic diagram showing the interaction results of the amphiphilic functional molecule of example 9 of the present invention with PD-L1 protein and an aldolase antibody on the surface of a cell membrane.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

Synthesis of (2-methyl- [1,1' -biphenyl ] -3-yl) methyl ((R) -1- (2- ((((R) -1- (methylamino) -1-oxo-3- (thiazol-4-yl) propan-2-ylcarbamoyl) imidazo [1,2-b ] pyridazin-6-yl) pyrrolidin-3-ylcarbamate (I-1).

Step Synthesis of 16-Chloroimidazo [1,2-b ] pyridazine-2-carboxylic acid Ethyl ester (1.1)

After 6-chloropyridazin-3-amine (50.0g,385mmol,1.0eq) was weighed and dissolved in 450mL dioxane, sodium bicarbonate (48.6g,578mmol,22.52mL,1.5eq) and ethyl 3-bromoacetone acetate (82.7g,424mmol,53.0mL,1.1eq) were added. After the addition, the reaction system is heated to 100 ℃ for reaction for 3 hours. TLC (dichloromethane: methanol 10:1, iodine color) monitored the disappearance of starting material and a new main spot was found and the reaction was stopped. The reaction system was filtered, and 700mL of water was added to the filtrate, the organic layer was washed with 3 times (500mL x 3), 1000mL of saturated saline was washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 1.1 g, 46.0g of a black colloidal crude product.

Step 2

Synthesis of ethyl (R) -6- (3- (((tert-butoxycarbonylcarbonyl) amino) pyrrolidin-1-yl) imidazo [1,2-b ] pyridazine-2-carboxylate (1.2)

1.1(25.0g,101mmol,1.0eq) was dissolved in 400mL of N, N-dimethylformamide and potassium carbonate (42.2g,305mmol,3.0eq) and (R) -3-tert-butoxycarbonylaminopyrrolidine (18.9g,101mmol,1.0eq) were added. After the addition, the reaction system was heated to 110 ℃ to react for 12 hours. TLC (petroleum ether: ethyl acetate 0:1, iodine color) monitored the disappearance of starting material and a new main spot was found and the reaction was stopped. 800mL of water was added, extracted 3 times with ethyl acetate (300mL × 3), the organic layer was washed three times with saturated brine (500mL × 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate 1:0 to 0:1) to give 1.3,19.0g of black gum with a yield of 44.8%.

Step 3

Synthesis of (R) -6- (3-aminopyrrolidin-1-yl) imidazo [1,2-b ] pyridazine-2-carboxylic acid hydrochloride (1.3)

After dissolving 1.2(9.0g,21.6mmol,1.0eq) in 100mL of dichloromethane, a hydrochloric acid/dioxane solution (4M,27.0mL,5.0eq) was added. After the addition, the reaction system was reacted at 25 ℃ for 1 hour. LC-MS monitoring showed 1.2 disappearance, m/z of the target product was detected, and the reaction was terminated. The reaction mixture was concentrated to give 1.3 g, 7.50g of crude product as a brown solid.

Step 4

Synthesis of (2-methyl- [ [1,1' -biphenyl ] -3-yl) methyl chlorocarbonate (1.6)

(2-methyl- [1,1' -biphenyl ] -3-yl) methanol (10.0g,50.4mmol,1.0eq) was weighed out and dissolved in 100mL of dichloromethane, triethanolamine (5.61g,55.48mmol,7.72mL,1.1eq) was added, then triphosgene (6.16g,20.7mmol,0.41eq) in dichloromethane (50mL) was added dropwise at-20 ℃ for 30 minutes, and the reaction was carried out at 0 ℃ for 1.5 hours. TLC (petroleum ether: ethyl acetate 10:1) monitored the disappearance of starting material and a new spot appeared and the reaction was stopped. After adding 300mL of n-hexane to the reaction system and stirring for 10 minutes, filtering, and adding dichloromethane to a filter cake: a total of 64mL of the mixed solution of n-hexane (1:2.2) was washed three times. The filtrate was concentrated to give crude 1.6,12.0g as a white solid.

Step 5

Synthesis of (R) -6- (3- (((((((((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) carbonyl) amino) pyrrolidin-1-yl) imidazo [1,2-b ] pyridazine-2-carboxylate (1.4)

1.3(6.50g,18.6mmol,1.0eq) was dissolved in 65mL of tetrahydrofuran and 26mL of water, potassium bicarbonate (4.70g,56.0mmol,2.18mL,3.0eq) was added, and then 65mL of a 1.6(9.73g,37.33mmol,2.0eq) solution in tetrahydrofuran was added dropwise at 0 ℃. After the addition, the reaction system was heated to 25 ℃ to react for 2 hours. LC-MS monitoring showed 1.3 disappearance, m/z of the target product was detected, and the reaction was terminated. 300mL of water was added, extraction was performed 3 times with ethyl acetate (200 mL. times.3), the organic layer was washed with 500mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by prep-HPLC (column: Phenomenex luna c 18250 mm: 100 mm. times.10. mu.m; mobile phase: [ water- (0.05% hydrochloric acid) -acetonitrile ]; B%: 36-66 acetonitrile%, 25min) to give 1.5,5.12g of a yellow solid with a yield of 55.1%.

Step 6

Synthesis of (R) -6- (3- (((((((((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) carbonyl) amino) pyrrolidin-1-yl) imidazo [1,2-b ] pyridazine-2-carboxylic acid (1.5)

1.4(3.10g,6.16mmol,1.0eq) was dissolved in 25mL of ethanol and 10mL of water, lithium hydroxide-monohydrate (774mg,18.4mmol,3.0eq) was added, and the reaction system was reacted at 25 ℃ for 12 hours. TLC (petroleum ether: ethyl acetate ═ 0:1) monitored the disappearance of starting material and a new spot appeared, ending the reaction. After the reaction system is concentrated to remove ethanol, 30mL of water is added, and 1M hydrochloric acid is added to adjust the pH value to 6-7. The white solid precipitated, was filtered and the filter cake was dried to give a brown solid 1.5,1.51g with a yield of 86.5%.

Step 7

Synthesis of methyl (R) -2- ((tert-butoxycarbonyl) amino) -3- (thiazol-4-yl) propionate hydrochloride (1.7)

(R) -2- ((tert-butoxycarbonyl) amino) -3- (thiazol-4-yl) propionic acid (1.0g,3.67mmol,1.0eq) was weighed out and dissolved in a solution of methanol hydrochloride (4M,20mL,21.7eq), and then the reaction system was warmed to 80 ℃ for 12 hours. LC-MS monitoring shows that the raw material disappears, m/z of the target product is detected, and the reaction is ended. The reaction was concentrated to give crude 1.7,912mg as a yellow solid.

Step 8

Synthesis of Compound I-1

1.5(0.69g,1.46mmol,1.0eq) was dissolved in 10mL of dichloromethane, N-diisopropylethylamine (378mg,2.93mmol,509uL,2.0eq), 1-hydroxybenzotriazole (296mg,2.20mmol,1.5eq) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (420mg,2.20mmol,1.5eq) were added, and the reaction mixture was reacted at 15 ℃ for 15 minutes. Then 1.6(325mg,1.46mmol,1.0eq) was added and the reaction was continued at 15 ℃ for a further 2 hours. LC-MS monitoring showed 1.5 disappearance, m/z of the target product was detected, and the reaction was terminated. The reaction mixture was concentrated and purified by prep-HPLC (column: Waters Vinris Silica 2-EP OBD 50: 150 mm: 5um, mobile phase: [ n-heptane-ethanol (0.1% ammonia water) ]; B%: 25-65% ethanol, 10min) to give I-1,510mg as a yellow solid with a yield of 54.6%.

¹H NMR(400MHz,CDCl₃)δppm 8.78(d,J＝1.6Hz,1H),8.25-8.06(m,2H),7.61(d,J＝10.0Hz,1H),7.45-7.35(m,2H),7.34-7.30(m,2H),7.29-7.24(m,1H),7.23-7.15(m,2H),7.14-7.10(m,1H),6.59(d,J＝10.0Hz,1H),5.35-5.10(m,4H),4.44(s,1H),3.80-3.75(m,1H),3.72-3.70(m,3H),3.64-3.50(m,2H),3.48-3.31(m,3H),2.37-2.26(m,1H),2.22(s,3H),2.11-1.97(m,1H).LC-MS:Rt＝0.921min,m/z＝640.1[M+H]⁺.

Example 2

Methyl group N^τ-benzyl-N^α- (6- ((R) -3- ((((((2-methyl- [1,1' -biphenyl)) s)]-3-yl) methoxy) carbonyl) amino) pyrrolidin-1-yl) imidazo [1,2-b]Synthesis of pyridazine-2-carbonyl) -L-histidine salt (I-2)

Step 1

N^τSynthesis of (2.1) benzyl-L-histidine methyl ester hydrochloride

Weighing N^τ-benzyl-N^α- (tert-butyloxycarbonyl) -L-histidine (1.0g,2.90mmol,1.0eq) was dissolved in a solution of methanol hydrochloride (4M,20mL,21.7eq), and then the reaction system was heated to 80 ℃ for 5 hours. TLC (petroleum ether: ethyl acetate ═ 0:1) monitored the disappearance of starting material and a new spot appeared, ending the reaction. The reaction was concentrated to give crude 2.1,750mg as a yellow oil.

Step 2

Synthesis of Compound I-2

2.1(500mg,1.06mmol,1.0eq), N, N-dimethylformamide (7.75mg,106umol,8.16uL,0.10eq) and triethanolamine (322mg,3.18mmol,443uL,3.0eq) were dissolved in 10mL of dichloromethane, oxalyl chloride (162mg,1.27mmol,111uL,1.20eq) was added at 0 ℃ and stirred for 30 minutes. Then, a solution of 2.1(337mg,1.06mmol,1.0eq) in methylene chloride (5mL) was added, and the reaction was warmed to 25 ℃ and stirred for 2 hours. LC-MS monitoring showed 2.1 disappearance, m/z of the target product was detected, and the reaction was terminated. The reaction mixture was extracted three times with ethyl acetate (20mL × 3) and the organic phase was washed twice with saturated brine (50mL × 2), dried over anhydrous sodium sulfate, filtered and concentrated. The concentrate was purified by prep-HPLC (column: Welch Ultimate XB-Diol 250 x 50 x 10um, mobile phase: [ n-heptane-ethanol (0.1% ammonia) ]; B%: 25-65% ethanol, 10min) to give I-2,450mg as a yellow solid with a yield of 54.8%.

¹H NMR(400MHz,CDCl₃)δppm 8.37(d,J＝8.4Hz,1H),8.12(s,1H),7.61-7.58(m,1H),7.56(s,1H),7.48-7.45(m,2H),7.41-7.39(m,3H),7.37-7.32(m,4H),7.31-7.29(m,2H),7.22-7.11(m,3H),6.72(s,1H),6.58(d,J＝10.0Hz,1H),5.22(s,2H),5.08-5.03(m,4H),4.44(s,1H),3.77-3.74(m,1H),3.66(s,3H),3.44-3.42(m,1H),3.20-3.16(m,2H),3.05-3.03(m,1H),2.34-2.32(m,1H),2.23(s,3H),2.07-2.05(m,1H).LC-MS:Rt＝0.734min,m/z＝713.3[M+H]⁺.

Example 3

Synthesis of (R) -2- (6- ((R) -3- ((((((2-methyl- [1,1' -biphenyl ] -3-yl ] methoxy) carbonyl) amino) pyrrolidin-1-yl) imidazo [1,2-b ] pyridazin-2-carboxamido) -3- (thiazol-4-yl) propionic acid (I-1-A)

I-1(510mg,786umol,1.0eq) was dissolved in 5mL of methanol and 2mL of water, and lithium hydroxide monohydrate (65.9mg,1.57mmol,2.0eq) was added, followed by reaction of the reaction system at 15 ℃ for 2 hours. TLC (dichloromethane: methanol 10:1, iodine color) monitored the disappearance of starting material and a new spot appeared and the reaction was stopped. The reaction was added with 30mL of water and extracted twice with ethyl acetate (15 mL. times.2). And then adding 1M hydrochloric acid into the water layer to adjust the pH value to 5-6, adding ethyl acetate to extract for 3 times (20mL x 3), washing the organic layer with 20mL saturated salt solution, drying the organic layer with anhydrous sodium sulfate, filtering and concentrating to obtain a white solid I-1-A,390mg and 76.3% of yield.

¹H NMR(400MHz,DMSO-d₆)δppm 9.04(d,J＝1.6Hz,1H),8.30(d,J＝8.0Hz,1H),8.15(s,1H),7.83(d,J＝10.0Hz,1H),7.75-7.60(m,1H),7.49-7.35(m,5H),7.30-7.20(m,3H),7.19-7.11(m,1H),7.00-6.90(m,1H),5.11(s,2H),4.82-4.77(m,1H),4.28-4.15(m,1H),3.70-3.65(m,1H),3.62-3.47(m,2H),3.42-3.35(m,3H),2.25-2.17(m,1H),2.15(s,3H),2.00-1.90(m,1H).LC-MS:Rt＝0.942min,m/z＝626.3[M+H]⁺.

Example 4

N^τ-benzyl-N^α- (6- ((R) -3- ((((((2-methyl- [1,1' -biphenyl)) s)]-3-yl) methoxy) carbonyl) amino) pyrrolidin-1-yl) imidazo [1,2-b]Synthesis of pyridazine-2-carbonyl) -L-histidine (I-2-A)

Referring to the synthesis of compound I-1-A, white solid I-2-A was obtained at 350mg, 82.5% yield.

¹H NMR(400MHz,DMSO-d₆)δppm 9.19(s,1H),8.73(s,1H),8.27(s,1H),7.85-7.83(m,1H),7.77-7.76(m,1H),7.50-7.47(m,1H),7.45-7.43(m,2H),7.40-7.38(m,2H),7.35-7.26(m,8H),7.25-7.21(m,1H),7.18-7.07(m,1H),5.36(s,2H),5.32(s,2H),4.80-4.76(m,1H),4.25(s,1H),3.74-3.73(m,1H),3.72-3.70(m,1H),3.64-3.54(m,1H),3.41-3.39(m,1H),3.38-3.23(m,2H),2.24-2.16(m,4H),1.99-1.95(m,1H).LC-MS:Rt＝1.017min,m/z＝699.4[M+H]⁺.

Example 5

(2-methyl- [1,1' -biphenyl)]-3-yl) methyl ((R) -1- (2- ((((R) -37- ((4- (3, 5-dioxahexyl)) phenyl) amino) -3,20,33, 37-tetraoxy-1- (thiazol-4-yl) -7,10,13,16,23,26, 29-heptaheptanol-4, 19, 32-triaza-heptanan-2-yl) carbamoyl) imidazo [1,2-b]Synthesis of pyridazin-6-yl) pyrrolidin-3-ylcarbamates (I-1-L)

Step 1

N¹- (1-amino-16-oxo-3, 6,9,12,19,22, 25-heptane-15-azaheptane-27-yl) -N⁵Synthesis of- (4- (3, 5-dioxyhexyl) phenyl glutaramide (3.2)

The t-butoxycarbonyl protected starting material (3.1,56.0mg,66.5umol,1.0eq) was weighed out and dissolved in 0.5mL of dichloromethane solution, followed by addition of hydrochloric acid/dioxane (4M,166uL,10.0eq) solution and reaction of the reaction system at 15 ℃ for 10 minutes. TLC (dichloromethane: methanol 10:1, iodine color) monitored the disappearance of starting material and a new spot appeared and the reaction was stopped. The reaction was concentrated to give crude 3.2 as a yellow oil, 50 mg.

Step 2

Synthesis of Compound I-1-L

I-1-A (40.0mg, 61.5. mu. mol,1.0eq) was dissolved in 1.5mL of methylene chloride, N-diisopropylethylamine (23.85mg, 184. mu. mol,32.1uL,2.0eq), 1-hydroxybenzotriazole (12.46mg, 92.25. mu. mol,1.5eq) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (17.68mg, 92.25. mu. mol,1.5eq) were added, and the reaction mixture was reacted at 15 ℃ for 15 minutes. Then 3.2(47.81mg,64.53umol,1.1eq) was added and the reaction was continued at 15 ℃ for another 12 hours. LC-MS monitoring showed about 9% of I-1-A remained, about 55% of m/z of the target product was detected, and the reaction was terminated. The reaction mixture was extracted twice with dichloromethane (20mL × 3) and the organic phase was washed with 5mL saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The concentrate was purified by prep-HPLC (column: Waters Xbridge 150 x 25mM x 5um, mobile phase: [ water (10mM ammonium bicarbonate) -acetonitrile ]; B%: 33-66% acetonitrile, 9min) to give I-1-L as a yellow solid, 15.0mg, 17.6% yield.

¹H NMR(400MHz,DMSO-d₆)δppm 9.78(s,1H),9.00(s,1H),8.47-8.02(m,3H),7.95-7.78(m,3H),7.73-7.65(m,1H),7.52-7.31(m,8H),7.29-7.07(m,7H),6.98-6.91(m,1H),5.11(s,2H),4.82-4.77(m,1H),4.28-4.15(m,1H),3.70-3.55(m,7H),3.54-3.45(m,22H),3.40-3.35(m,2H),3.27-3.13(m,10H),2.83-2.65(m,4H),2.34-2.23(m,5H),2.18-2.06(m,8H),2.00-1.90(m,2H),1.84-1.72(m,2H).LC-MS:Rt＝0.994min,m/z＝1348.9[M+H]⁺.

Example 6

Synthesis of (2-methyl- [1,1' -biphenyl ] -3-yl) methyl ((R) -1- (2- ((((S) -1- (1-benzyl-1H-imidazol-4-yl) -37- ((4- (3, 5-dioxahexyl) phenyl) amino) -3,20,33, 37-tetraoxy-7, 10,13,16,23,26, 29-heptyloxy-4, 19, 32-triaza-hepto-nan-2-ylcarbamoyl) imidazo [1,2-b ] pyridazin-6-yl) pyrrolidin-3-yl) carbamate (I-2-L)

Referring to the synthesis of compound I-1-L, yellow solid I-2-L,12.0mg, 6.16% yield was obtained.

¹H NMR(400MHz,DMSO-d₆)δppm 9.79(s,1H),8.26-8.20(m,2H),8.14-7.89(m,1H),7.85-7.83(m,3H),7.75-7.70(m,1H),7.64(s,1H),7.46-7.42(m,4H),7.38-7.30(m,2H),7.28-7.24(m,6H),7.20-7.14(m,5H),7.13-7.11(m,1H),6.88(s,1H),5.11(s,3H),4.67-4.62(m,1H),4.23(s,1H),3.68-3.62(m,2H),3.58-3.52(m,3H),3.48-3.44(m,22H),3.41-3.39(m,6H),3.32-3.30(m,2H),3.26-3.18(m,5H),2.85-2.78(m,2H),2.75-2.68(m,4H),2.30-2.27(m,5H),2.25-2.15(m,7H),2.11-2.07(m,1H),1.79-1.78(m,3H).LC-MS:Rt＝1.037min,m/z＝1421.9[M+H]⁺.

Example 7

Binding of DNA-ligated imidazopyridazines (imidazopyridazines on-DNA compounds) to PD-L1 was investigated.

DEL library affinity screening is carried out aiming at the target protein PD-L1, two pieces of molecular sequence information of the enriched better imidazopyridazine on-DNA compound are obtained from millions of compounds, and the on-DNA compound is synthesized again according to a library synthesis route. And the on-DNA compound was synthesized de novo and the synthesized mixture was tested for on-DNA compound binding using two test methods.

Wherein the step of synthesizing the imidazo pyridazine on-DNA compound comprises the following steps:

adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a dimethyl sulfoxide solution of N-hydroxysuccinimide into a centrifuge tube, performing vortex centrifugation, adding an N, N-dimethylacetamide solution for synthesizing a building block 1(BB1) into a reaction solution, performing vortex centrifugation, reacting in a shaker at 20 ℃ for 15 minutes, adding a compound 4.1, performing shaker reaction at 20 ℃ for 16 hours, adding ethanol to 80 ℃, performing centrifugation after 1 hour, removing a supernatant, and obtaining a crude compound 4.2 from residual solids.

Step two, dissolving the crude product compound 4.2 in double distilled water, adding 20% piperidine double distilled water solution, performing vortex centrifugation, reacting in a shaking table at 20 ℃ for 2 hours, adding ethanol to 80 ℃, centrifuging after 1 hour, removing supernatant, and performing freeze drying on residual solid; then adding double distilled water for dissolution, purifying by an ultrafiltration tube (Ultracel-3KDa,14000g,30min,20 ℃), and collecting the concentrated solution to obtain a crude compound 4.3.

Adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a dimethyl sulfoxide solution of N-hydroxysuccinimide into a centrifugal tube, performing vortex centrifugation, adding an N-methylpyrrolidone solution for synthesizing the building block 2(BB2) into a reaction solution, performing vortex centrifugation, reacting for 15 minutes in a shaking table at 20 ℃, adding a compound 4.3, performing shaking table reaction at 20 ℃ for 16 hours, adding ethanol to 80 ℃, performing centrifugation after 1 hour, removing a supernatant, and performing freeze drying on residual solids to obtain a crude compound 4.4.

Dissolving the crude product compound 4.4 in double distilled water, adding a double distilled water solution of magnesium chloride and sodium acetate, performing vortex centrifugation, reacting at 90 ℃ for 16 hours, adding ethanol to 80 ℃, centrifuging after 1 hour, removing supernatant and leaving solid; then adding double distilled water for dissolution, purifying by an ultrafiltration tube (Ultracel-3KDa,14000g,30min,20 ℃), and collecting the concentrated solution to obtain a crude compound 4.5.

Adding a synthetic block 3(BB3) and an N, N-dimethylacetamide solution of triethanolamine into a centrifugal tube, reacting for 10 minutes at 20 ℃ in a shaking table after vortex centrifugation, adding an N, N' -disuccinimidyl carbonate N, N-dimethylacetamide solution, reacting for 1.5 hours at 20 ℃ in the shaking table after vortex centrifugation, adding a crude product compound 4.5, reacting for 1.5 hours at 20 ℃ in the shaking table after vortex centrifugation, adding ethanol to 80 ℃, centrifuging for 1 hour, removing supernatant, and freeze-drying residual solids to obtain the compound; purifying with Zeba desalting column, collecting stock solution and three washing solutions, and lyophilizing to obtain white solid mixed final product. Detecting LCMS by using LTQ mass spectrum, wherein the purity of the final product I-1-DNA is 38.54%; the purity of the final product I-2-DNA was 43.38%.

One of the test methods used is affinity screening-mass spectrometry (AS-MS) for testing the binding effect and structure of imidazopyridazine on-DNA compounds.

The AS-MS test procedure refers to the test method provided in Chinese patent CN 111965295A. The test results were as follows:

TABLE 1 AS-MS test results for PD-L1 binding of imidazopyridazines on-DNA compounds

As can be seen from the results in Table 1, the imidazopyridazines on-DNA compound has better binding interaction with PD-L1, and the specific structure of the on-DNA compound can be deduced by MS analysis.

Among others, another test method used is Surface Plasmon Resonance (SPR) testing of the binding effect of imidazopyridazine on-DNA compounds.

The test step refers to SPR test small molecule and protein combination operation, target protein PD-L1 protein is coupled on the surface of a chip, imidazopyridazine on-DNA compounds with different concentrations are injected and flow through the surface of the chip to be combined with coupled PD-L1, a sensing map is recorded and analyzed, and corresponding molecular interaction information is obtained.

The test results were as follows:

TABLE 2 SPR test results for binding of PD-L1 by imidazopyridazine on-DNA compounds

Compound	KD	Rmax
			I-1-DNA	57.4μM	47.3
I-2-DNA	34.5μM	58.9

As can be seen from the results in Table 2, the imidazopyridazine on-DNA compounds have a better binding interaction with PD-L1, K_DCan reach the level of dozens of mum.

Example 8

The binding effect of the imidazopyridazine amphiphilic functional molecule on PD-L1 is studied.

The test step refers to SPR test small molecule and protein combination operation, couples target protein PD-L1 to the surface of the chip, samples imidazopyridazine amphiphilic functional molecules with different concentrations, enables the molecules to flow through the surface of the chip to be combined with coupled PD-L1, records and analyzes a sensing map, and obtains corresponding molecular interaction information.

The test results were as follows:

TABLE 3 SPR test results for PD-L1 binding by imidazopyridazine-based bifunctional molecules

Compound	KD	Rmax
			I-1-L	6.12μM	25.9
I-2-L	5.53μM	15.5

As can be seen from the results in Table 3, the imidazopyridazine bifunctional molecule has a better binding interaction with PD-L1, K_DSingle digit μ M levels can be achieved. K_DThe smaller the probability, the greater the binding affinity for the target.

Example 9

And (3) researching the affinity interaction of the imidazopyridazine amphiphilic functional molecules on the aldolase antibody.

The interaction of the imidazopyridazine amphiphilic functional molecules with aldolase antibodies was tested in vitro and at the cellular level, respectively.

In the experiment of the in vitro interaction of the amphiphilic functional molecules and the aldolase antibody, a reference group 1 only PD-L1 protein group, a reference group 2 only aldolase antibody 38C2 group, a reference group 3 protein-free group, an experiment group 1 non-amphiphilic molecule group, an experiment group 2 positive control group (BMS202-L), an experiment group 3 bifunctional molecule group (I-1-L) and an experiment group 4 bifunctional molecule group (I-2-L) are arranged. The aldolase antibody 38C2 is immobilized on Protein G immunomagnetic beads, the amphiphilic functional molecules I-1-L and I-2-L are incubated for 1 hour in a turnover mode at the temperature of 37 ℃, and then the PD-L1 substrate Protein is added for incubation for 1 hour in a turnover mode at room temperature. Then, Western Blot detection is used, and the gel diagram is shown in FIG. 1.

As can be seen from the results in FIG. 1, bands with molecular weights of the target proteins PD-L1 and aldolase antibody 38C2 can be clearly found in the gel image, indicating that the bifunctional molecules I-1-L and I-2-L can act on the target protein PL-L1 and aldolase antibody 38C2 with affinity in an in vitro assay.

Wherein, the interaction experiment between the amphiphilic functional molecule and the aldolase antibody on the surface of the cell membrane is carried out. After transfection of cells with the PD-L1 plasmid, the aldolase antibody 38C2 was dissolved in cell affinity buffer and dispensed into the reference and experimental groups, and the amphipathic molecules (I-1-L and I-2-L) were added to the experimental groups and incubated at 37 ℃ for 1 hour with inversion. Forming a 38C2 antibody-small molecule complex. 38C2 antibody-small molecule complexes were added to PD-L1 over-expressed FreeStyle 293 cells (suspension), incubated at 37 ℃ for 1 hour, washed, centrifuged, and the supernatant was discarded to collect the cells. Then, a fluorescently-labeled secondary antibody against murine 38C2 was added and incubated at normal temperature for 1 hour, followed by washing, centrifugation, and cell collection by discarding the supernatant. Cells were passed through Becton Dickinson FACSAria^TMIII flow cytometry (BD Biosciences) was performed for flow cytometry detection. Determining the cell population analyzed by using forward reflected light (FSC) and side reflected light (SSC), and detecting the expression of cell surface fluorescence after circling; the difference between the fluorescence expression of the reference group and the fluorescence expression of the experimental group is measured by taking the average fluorescence expression intensity as a contrast value, so that the fact that the cell membrane target protein acts on the aldolase antibody 38C2 through the affinity of the amphiphilic functional molecule is inferred, and the cell membrane target protein expression and the affinity action specificity are indicated by taking the cell membrane target protein expression and the affinity action specificity.

As can be seen from the results of FIG. 2, with reference to the set in which only the aldolase antibody was added, the mean fluorescence expression intensity was significantly increased in the experimental group in which the bifunctional molecules I-1-L and I-2-L were added to the PD-L1 protein, as compared to the experimental group before the addition. It was shown that the cell membrane target protein PD-L1 can act on the aldolase antibody 38C2 by the affinity of the amphipathic molecules I-1-L and I-2-L.

In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (11)

1. An imidazopyridazine compound is characterized in that the structural formula of the imidazopyridazine compound is shown as a general formula (I),

wherein, in the general formula (I), n is 0-3;

x is O or NH;

R¹is hydrogen, substituted or unsubstituted C_1-6An alkyl group;

R²is hydrogen, substituted or unsubstituted C_1-6Alkyl, alkoxy, alkylthio, haloalkyl, haloalkoxy, amino, C_3-8One or more of cycloalkyl, heterocycloalkyl, substituted or unsubstituted phenyl, five or six membered monocyclic heteroaryl, substituted or unsubstituted 8 to 12 membered fused ring aryl, substituted or unsubstituted 8 to 12 membered fused ring heteroaryl; wherein, for substitution of C_1-6The substituent of the alkyl, phenyl, aryl or heteroaryl is one or more, and is independently selected from one or more of halogen, amino, cyano, hydroxyl, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl or heteroaryl;

R³is a substituent on the imidazopyridazine ring, R³Is 0 to 3 and is independently selected from one or more of the following substituent groups: F. cl, Br, I, C_1-6Alkyl, cyano, nitro, methoxy.

2. The imidazopyridazine compound according to claim 1, wherein in the general formula (i), the nitrogen-containing aliphatic ring in which n is present is selected from any one of a azetidine ring, a tetrahydropyrrole ring, a piperidine ring and a homopiperidine ring.

3. The imidazopyridazine compound according to claim 1, wherein in the general formula (i), the 1-position and/or the 2-position is a chiral center.

4. The imidazopyridazines of claim 1, wherein R is¹Can be selected from any one of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, amyl, isoamyl and tertiary amyl.

5. The imidazopyridazine compound of claim 1, wherein said imidazopyridazine compound is selected from the group consisting of:

6. an amphiphilic functional molecule modified by an imidazopyridazine compound is characterized in that the structural formula of the modified imidazopyridazine compound is shown as a general formula (I),

wherein, in the general formula (I), n is 0-3;

x is O or NH;

R¹is shown in a formula (II),

wherein m in formula (II)₁Is selected from any integer of 1 to 6, m₂Any integer selected from 1 to 6;

R²is hydrogen, substituted or unsubstituted C_1-6Alkyl, alkoxy, alkylthio, haloalkyl, haloalkoxy, amino, C_3-8One or more of cycloalkyl, heterocycloalkyl, substituted or unsubstituted phenyl, five or six membered monocyclic heteroaryl, substituted or unsubstituted 8 to 12 membered fused ring aryl, substituted or unsubstituted 8 to 12 membered fused ring heteroaryl; wherein, for substitution of C_1-6The substituent of the alkyl, phenyl, aryl or heteroaryl is one or more, and is independently selected from any one or more of halogen, amino, cyano, hydroxyl, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl or heteroaryl;

R³is a substituent on the imidazopyridazine ring, R³Is 1 to 3 and is independently selected from any one or more of the following substituents: F. cl, Br, I, C_1-6Alkyl, cyano, nitro, methoxy.

7. The amphiphilic functional molecule of claim 6, selected from the group consisting of:

8. use of an imidazopyridazine compound according to any of claims 1-5 or an amphiphilic functional molecule according to any of claims 6-7 for the manufacture of a medicament for use as a PD-L1 inhibitor.

9. Use of an imidazopyridazine compound according to any of claims 1-5 or an amphiphilic functional molecule according to any of claims 6-7 for the preparation of a medicament for the treatment of a PD-L1-mediated disease.

10. Use of an imidazopyridazine-modified amphiphilic functional molecule compound according to any one of claims 6-7 for linking an aldolase antibody.

11. A pharmaceutical composition is characterized by comprising a therapeutically effective amount of an active component and pharmaceutically acceptable excipients; the active component comprises the imidazopyridazine compound of any one of claims 1-5 or the amphiphilic functional molecule of any one of claims 6-7, an isomer, a prodrug, a stable isotopic derivative or a pharmaceutically acceptable salt thereof.

Images