CN109232575B - Pyrrole [1,2-b ] pyridazine compound or pharmaceutically acceptable salt thereof and application thereof - Google Patents

Abstract

The invention relates to a pyrrole [1,2-b ] pyridazine compound or pharmaceutically acceptable salt thereof and application thereof. The pyrrole [1,2-b ] pyridazine compound or pharmaceutically acceptable salt thereof has an inhibiting effect on poly (adenosine diphosphate ribose) polymerase (PARP), particularly has a selective inhibiting effect on PARP-1and PARP-2 of the PARP family, and shows good biological activity in vitro and in vivo, so that the compound can be used as a PARP inhibitor and used in pharmaceutical compositions for preventing and/or treating PARP-related diseases, such as tumors, ischemic diseases and neurodegenerative diseases.

Description

Pyrrole [1,2-b ] pyridazine compound or pharmaceutically acceptable salt thereof and application thereof

Technical Field

The invention relates to the field of medicinal chemistry, in particular to pyrrole [1,2-b ] pyridazine compounds or pharmaceutically acceptable salts thereof and application thereof.

Background

Poly (adenosine diphosphate ribose) polymerase (Poly (adenosine diphosphate ADP)]Ribose) polymerase, PARP) is a class of cellular ribozymes found in most eukaryotic cells. Its main function is to make Nicotinamide Adenine Dinucleotide (NAD)⁺) Cleavage to nicotinamide and ADP-ribose, and the formation of long-chain or branched ADP-ribose polymers on the target proteins, which regulate the function of the proteins, including histones, topoisomerases, and PARP itself (Schreiber, v.; dantzer, F; ame, J. -C.and De Murcia G.. Poly (ADP-rib): novel functions for an old molecule. Nat. Rev. mol. cell Bio,2006,7, 517-528). The ADP-ribose transferase family (ADP-ribosyltransferases, ARTs) currently found and defined subtype proteins contain a total of 18, of which only 6 subtypes have the ability to form poly ADP-riboses, which are PARP-1, PARP-2, PARP-3, PARP-4(vPARP), PARP-5a (TNKS1) and PARP-5b (TNKS2), respectively; while other ARTs 7-17 subtypes (also commonly referred to as PARPs 6-16) function only to form a single ADP-ribose modification. PARP-1, the most abundant and studied member, performs more than 90% of its functions in the PARP family. Human PARP-1 is a 113kDa molecular weight polypeptide chain consisting of three major functional domains: a DNA Binding Domain (DBD) containing two Zinc fingers (Zinc fingers) at the amino (N) -terminus, a self-modifying domain (Automodification domain), and a Catalytic domain (Catalytic domain) at the carboxy (C) -terminus. Human PARP-2 is a 62kDa polypeptide chain which is most similar in structure to PARP-1, with a catalytic domain that is 69% homologous to the catalytic domain of PARP-1and a DNA binding domain that does not contain a zinc finger (Ame, Jean-Christophe; Spenlehauer, Catherine; de Murcia, Gilbert. the PARP perfect. BioEssays 2004,26, 882) -893). PARP-1and PARP-2 play an important role in DNA damage repair, genome stability and apoptosis regulation through base excision repair, so that the PARP-1and PARP-2 play an important role in research targets of anti-tumor drugs which are most concerned in recent years (Yelamios, J.; Farres, Jordi.; Llacuna, Laura.; Ampurdanes, Coral; Martin-Caballero, Juan.; PARP-1and PARP-2: New planes in tumor depth. am. J. cancer Res.2011 1(3),328-346；Weaver,A.N.；Yang,E. S.Beyond DNA repair:additional functions of PARP-1in cancer.Front.Oncol.2013, 3,290)。

Human DNA is susceptible to DNA nicks under the action of exogenous factors (such as ultraviolet radiation, particle radiation, alkylating agents, topoisomerase and platinum drugs) or endogenous factors (such as the spontaneous reactions inherent in the normal metabolic active substances and the chemical substances of DNA) and most of the nicks are converted into DNA single-strand breaks, resulting in DNA damage. DNA damage can affect a wide variety of cellular processes and is a significant cause of tumorigenesis and other diseases. Studies have shown that the catalytic activity of PARP-1/2 is rapidly activated by DNA strand breaks. When DNA is damaged, activated PARP-1 binds to the single-stranded notch of the damaged DNA, catalyzing the formation of linear or branched poly ADP-ribose polymers on the target protein (e.g., histone or PARP-1 itself). This will result in the action between the poly ADP-ribose forming histones and the DNA becoming loose, facilitating the access of DNA repair enzymes to the damaged DNA and eventually completing the repair of the DNA. For cancer therapy, since radiotherapy and many chemotherapeutic approaches act to target DNA damage, PARP inhibitors can be used as chemo-and radiosensitizers for cancer therapy (Miwa, Masanao; Masutani, Mitsuko. PolyADP-ribosylation and cancer. cancer science 2007,98, 1528-1535).

PARP inhibitors have been shown to be useful for the specific killing of tumors deficient in the breast cancer-associated genes BRCA-1 and BRCA-2. BRCA-1/2 is a key repair factor in the Homologous Recombination (HR) repair pathway. In BRCA-1 and BRCA-2 deficient tumor cells, inhibition of PARP will lead to increased accumulation of DNA single strand breaks, which will be converted to double strand breaks due to progressive replication fork collapse, and these tumor cells will repair double strand breaks because of failure to initiate the HR pathway, eventually leading to cell death due to genomic instability. The phenomenon of co-killing of cells by PARP inhibition and BRCA-1/2 deficiency is also known as synthetic lethality. The theory of synergistic death is utilized to provide a new strategy and a new thought for the treatment of malignant tumors and open a new era of research and development of PARP inhibitors as novel antitumor drugs. (references: Farmer, H.; McCabe, N.; Lord, C.J.; Tutt, A.N.J.; Johnson, D.A.; Richardson, T.B.; Santarosa, M.; Dillon, K.J.; Hickson, I.; Knight, C.; Martin, N.M.B.; Jackson, S.P.; Smith, G.C.M.; Ashworth, A.. Targeting the DNA repair defect in BRCA mut cell as a thermal barrier. Nature, 434,917, 921; Kaelin, W.G.the concentrate of Synthetic Lethcity in the additive of the antibiotic tissue culture. Nature 2005, J.S.S. Ser. No. 9, J.S. J.; J. H.S. Ser. No. 9, J.; J.S. H.S. J.; J. H.S. Ser. No. J.; J. H.S. Ser. No. 7, S. Ser. No. 7, No. 11, No. 7, J.; J. H.S. Ser. 1, No. 3, No. 6, No. Ser. 3, No. 7, No. 6, No. 3, No. 6, No. 3, No. 6, No. C. 2, No. 6, No. 3, No. 2, No. 3, No. 6, No. 2, No. C. 3, No. C. 3, No. 2, No. 3, No. S. 3, No. 2, No. C. 3, No. 2, No. C. 2, No. 3, No. 2, No. C. 3, No. 2, No. S.2, No. 3, No. 2, No. 3, No. 2, No. 3, No. 2, No. 3, No. 2, No. 3, No. 2, No. 3, No. 2, No. 3, No. 2, No. 3, No. 2, No.

In addition to DNA repair and maintenance of genomic stability, PARP is involved in a number of biological processes including gene transcription, cell cycle progression, cell death, chromatin function. PARP inhibitors have also proven useful in a number of areas of medicinal chemistry (diseases other than malignancies) of Therapeutic interest, including stroke, myocardial ischemia, inflammation, antiviral and diabetes etc. (ref: Vir a g, L.; Szab Lou, C., The Therapeutic patent of Poly (ADP-Ribose) Polymerase inhibitors Pharmacol Rev 2002,54(3),375 a 429).

The vast majority of PARP inhibitors to date are competitive inhibitors, with NAD⁺Competitively bind to the catalytic site of the enzyme. The existing inhibitors still cannot meet the clinical requirement, and the compounds have the defects of poor solubility, lack of selectivity on PARP subtypes and the like, so that the provision of effective PARP inhibitors with novel structures is urgently needed.

The pyrrolo [1,2-b ] pyridazine structure is an advantageous structure in medicinal chemistry. Compounds containing such structures exhibit a wide variety of biological activities in the pharmaceutical field, such as hypolipidemic/hypocholesterolemic (patent CN1056690 discloses a class of compounds containing such advantageous structures and their use in the treatment of hypercholesterolemia and hyperlipidemia), anti-tumor (patent WO 2011/014817 discloses JANUS kinase inhibitors containing such structures), anti-inflammatory, anti-bacterial (Butnariu, R.M.; Mangalagiu, I.I., New pyridazine derivative: Synthesis, chemistry and biological activity. bioorg. Med. Chem 2009,17(7), 2823. 2829; Tucaliuc, R.A.; Cotema, V.V., Bucula, M. Medulus, Tuchilus, C.; Mangalu, D. Mangalagiu, I. Mandarin, New-rivazine, Synthesis, U.S. 67, C.; Man, D. Mangalagiu, I. Mangalagin. Chevriazine, U.V.; Bugular et al, U.S.S. 3. J. Biochemical activity. Chem.I., U.J. 3. Chem., m. is; poiata, a.; tuchilus, c.; florescu, M. Pyridazine and phthalazine derivatives with a potent antiviral activity. J Heterocyclic Chem 2007,44(5),1149-1152), antiviral (patent CN103288832 discloses a class of pyrrole [1,2-b ] Pyridazine derivatives useful for the treatment or prevention of HIV infection or other viral infections); WO 2004/087708A 1and WO 2007/069671 respectively disclose the application of Corticoid Releasing Factor (CRF) inhibitors containing the dominant structures in pharmacy, and WO2010022240A1 discloses derivatives containing the structures, which can effectively inhibit the activity of HIF hydroxylase so as to regulate the activities such as stability and/or activity of Hypoxia Inducible Factor (HIF).

None of the reported pyrrolo [1,2-b ] pyridazine derivatives described above are covered and relate to the compounds of the present invention and their use as PARP inhibitors.

Disclosure of Invention

Based on this, an object of the present invention is to provide a pyrrolo [1,2-b ] pyridazine compound represented by the general formula I:

wherein the content of the first and second substances,

R¹is hydrogen, C_1-4Alkyl or halogen;

R²is composed of

R³Is hydrogen, -C (═ O) R⁶、-SO₂R⁷Substituted or unsubstituted C_1-4Alkyl or substituted or unsubstituted C_3-6Cycloalkyl, wherein R is⁶And R⁷Independently is substituted or unsubstituted C_1-4Alkyl, substituted or unsubstituted C_3-6Cycloalkyl or substituted or unsubstituted C_6-10Aryl, said substitution being by a member selected from the group consisting of halogen, C_1-4At least one substituent group of alkyl, amino, nitrile, hydroxyl and nitro;

R⁴is hydrogen, halogen, C_1-4Alkyl or C_1-4An alkoxy group;

R⁵is-CH₂-or-C ═ O;

X₁s, O or NH;

X₂is CH or N;

Y₁、Y₂、Y₅and Y₆Independently is a substituted or unsubstituted methylene, substituted or unsubstituted ethylene, substituted or unsubstituted propylene or substituted or unsubstituted butylene group, said substitution being selected from the group consisting of halogen, C_1-6Alkyl radical, C_3-6Cycloalkyl and C_1-4At least one substituent in the alkoxy group;

Y₃and Y₄Independently hydrogen, substituted or unsubstituted C_1-4Alkyl or substituted or unsubstituted C_3-6Cycloalkyl, said substitution being by a group selected from halogen, C_1-6Alkyl radical, C_3-6Cycloalkyl and C_1-4At least one substituent in the alkoxy group;

Y₇is NR⁸Or CHR⁹，

Wherein, R is⁸Is hydrogen, -C (═ O) R¹⁰、-SO₂R¹¹Substituted or unsubstituted C_1-4Alkyl, substituted or unsubstituted C_3-6Cycloalkyl or substituted or unsubstituted C_6-10Aryl, wherein R is¹⁰And R¹¹Independently is substituted or unsubstituted C_1-4Alkyl, substituted or unsubstituted C_3-6Cycloalkyl, substituted or unsubstituted five-to six-membered saturated heterocyclic group, or substituted or unsubstituted C_6-10Aryl, said substitution being by a member selected from the group consisting of halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, amino, substituted by at least oneC_1-4At least one substituent of alkyl substituted amino, nitrile, hydroxyl and nitro,

the R is⁹Is hydrogen, -OR¹²Is at least one C_1-4An alkyl-substituted amino group, or a substituted or unsubstituted five-to six-membered saturated heterocyclic group, wherein R is¹²Is hydrogen, substituted or unsubstituted C_1-4Alkyl or substituted or unsubstituted C_3-6Cycloalkyl, said substitution being by a member selected from the group consisting of halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, amino, substituted by at least one C_1-4At least one substituent group of alkyl substituted amino, nitrile, hydroxyl and nitro.

Preferably, in said formula I,

R¹is hydrogen, methyl, ethyl, propyl, fluorine, chlorine or bromine;

R²is composed of

R³Is hydrogen, methyl, ethyl or propyl;

R⁴is hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, methoxy, ethoxy or propoxy;

R⁵is-CH₂-or-C ═ O;

X₁s, O or NH;

X₂is CH or N;

Y₁、Y₂、Y₅and Y₆Independently a substituted or unsubstituted methylene group, a substituted or unsubstituted ethylene group, a substituted or unsubstituted propylene group, or a substituted or unsubstituted butylene group, said substitution being substituted with at least one substituent selected from the group consisting of fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, and propoxy;

Y₃and Y₄Independently hydrogen, methyl, ethyl, propyl, isopropyl or butyl;

Y₇is NR⁸Or CHR⁹，

Wherein, R is⁸Is hydrogen, -C (═ O) R¹⁰、-SO₂R¹¹Methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or substituted or unsubstituted phenyl, wherein R is¹⁰And R¹¹Independently substituted or unsubstituted groups as follows: methyl, ethyl, propyl, isopropyl, N-butyl, sec-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl, tetrahydrofuryl, piperidinyl, piperazinyl, morpholinyl, dioxanyl or phenyl, said substitution being by at least one substituent selected from the group consisting of fluoro, chloro, bromo, methyl, ethyl, propyl, isopropyl, trifluoromethyl, amino, N-methylamino, N-ethylamino, N-dimethylamino, N-diethylamino, nitrile, hydroxyl and nitro,

the R is⁹Is hydrogen, -OR¹²N-methylamino, N-ethylamino, N-dimethylamino, N-diethylamino, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl, or dioxanyl, wherein R is¹²Is hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, said substitution being by at least one substituent selected from the group consisting of fluorine, chlorine, bromine, methyl, ethyl, propyl, isopropyl, trifluoromethyl, amino, N-methylamino, N-ethylamino, N-dimethylamino, N-diethylamino, nitrile, hydroxyl and nitro.

In one embodiment, the general formula I has a structure represented by general formula II:

preferably, said R is¹Is halogen, more preferably chlorine.

Preferably, said X₁Is S or O, more preferably S.

Preferably, the formula II has a structure represented by formula II-a:

in one embodiment, the formula I has a structure represented by formula III:

preferably, said R is¹Is halogen, more preferably chlorine.

Preferably, said R is⁴Is halogen, more preferably fluorine.

Preferably, said R is⁵is-CH₂-。

Preferably, said Y is₃And Y₄Independently hydrogen, methyl, ethyl, propyl, isopropyl or butyl, more preferably methyl.

Preferably, the formula III has a structure represented by formula III-a:

in one embodiment, the general formula I has a structure represented by general formula IV:

preferably, said R is¹Is halogen, more preferably chlorine.

Preferably, said R is⁴Is halogen, more preferably fluorine.

Preferably, said R is⁵is-CH₂-or-C ═ O.

Preferably, the formula IV has a structure represented by formula IV-a:

in one embodiment, the formula I has a structure represented by formula V:

preferably, said R is¹Is halogen, more preferably chlorine.

Preferably, said R is⁴Is hydrogen or halogen, preferably fluorine.

Preferably, the formula V has a structure represented by formula V-a:

preferably, the pyrrole [1,2-b ] pyridazine compound represented by the general formula I is one of the following compounds:

the invention further provides application of the pyrrole [1,2-b ] pyridazine compound shown in the general formula I or the pharmaceutically acceptable salt thereof in preparing PARP inhibitors.

In one embodiment, the PARP inhibitor has a selective inhibitory effect on PARP-1 and/or PARP-2.

The invention also provides a pharmaceutical composition which comprises one or more pyrrole [1,2-b ] pyridazine compounds represented by the general formula I or pharmaceutically acceptable salts thereof in a therapeutically effective amount, or one or more esters, prodrugs, hydrates or crystals of the pyrrole [1,2-b ] pyridazine compounds in a therapeutically effective amount.

In one embodiment, the pharmaceutical composition has an inhibitory effect on PARP, in particular a selective inhibitory effect on PARP-1 and/or PARP-2.

In one embodiment, the pharmaceutical composition is used for preventing and/or treating PARP-related diseases, including tumors (solid tumors such as breast cancer, ovarian cancer, liver cancer, melanoma, prostate cancer, colon cancer, gastric cancer, and the like), ischemic diseases (brain, heart, and the like), and neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, and the like).

The invention further provides a preparation method of the pyrrole [1,2-b ] pyridazine compound represented by the general formula I, which comprises the following steps:

after the pyrrole-2-methyl formate derivative is subjected to esterification reaction, iodination and amination reaction are carried out in sequence or amination and iodination reaction are carried out first and then, so that the N-amino-5-iodopyrrole-2-methyl formate derivative I-a in the following reaction formula is obtained;

aromatic aldehyde reacts with Tetrahydrofuran (THF) solution of ethynyl magnesium bromide to obtain aromatic alkynol I-b in the following reaction formula;

carrying out coupling cyclization reaction on the N-amino-5-iodopyrrole-2-methyl formate derivative I-a and the aromatic alkynol I-b in the presence of palladium acetate, cuprous iodide and DBU in a solvent under the microwave condition to obtain a compound I-0;

the compound I-0 takes a saturated methanol solution of ammonia gas as a solvent, reacts for 20 hours to 24 hours at the temperature of 90 ℃ by sealing a tube, or directly reacts with strong ammonia water, the solvent is methanol, and the compound I-0 reacts for 20 hours to 24 hours at the temperature of 90 ℃ by sealing the tube to obtain the pyrrole [1,2-b ] pyridazine-7-formamide compound represented by the general formula I;

the invention further provides a preparation method of the pyrrole [1,2-b ] pyridazine compound represented by the general formula II or the general formula II-a, and the synthetic route is as follows:

the method specifically comprises the following steps:

condensing the compound E with acyl chloride or carboxylic acid to obtain corresponding amide F;

under the action of phosphorus oxychloride, the amide F is condensed to form a ring to obtain a compound, and then the compound is subjected to imine reduction under the condition of sodium borohydride and further Boc anhydride protection of a free amino group to obtain a compound I;

reacting the compound I with n-butyllithium in a solvent THF, and further adding DMF to react to obtain a 2-formyl substituted thiophene derivative J;

reacting the compound J with a THF solution of ethynyl magnesium bromide to obtain a compound I-c; and

and (3) carrying out coupling cyclization on the compound I-c and the compound I-a, removing a protecting group by using trifluoroacetic acid, and further carrying out ammonolysis to obtain the compound shown in the general formula II.

The invention takes pyrrole [1,2-b ] pyridazine-7-formamide as a mother nucleus structure to design and synthesize a series of PARP inhibitors, and the compound related to the invention has selective inhibition effect on PARP-1and PARP-2 of PARP family, shows good biological activity in vitro and in vivo, and is expected to be developed into novel anti-tumor drugs and drugs for preventing and/or treating PARP related diseases.

Detailed Description

The structure and preparation method of the compound of the present invention and the effect of inhibiting PARP activity in vitro and in vivo are further illustrated by the following examples, but the present invention is not limited thereto.

As used herein, "alkyl" refers to straight or branched chain alkyl groups.

As used herein, "alkoxy" refers to a straight or branched chain alkoxy group.

As used herein, "substituted" refers to the substitution of one or more hydrogen atoms.

As used herein, "tumor" includes benign tumors and malignant tumors.

As used herein, the term "pyrrolo [1,2-b ] pyridazine compound" includes optical isomers.

A method for preparing a pyrrole [1,2-b ] pyridazine compound represented by the general formula I, which comprises the following steps:

(1) the preparation of methyl N-amino-5-iodopyrrole-2-carboxylate derivative I-a can be carried out by the following scheme:

scheme 1:

scheme 2:

wherein, scheme 1 includes: pyrrole-2-carboxylic acid methyl ester derivative A is heated and refluxed in a solvent (such as methanol) under the action of a base (such as 4-Dimethylaminopyridine (DMAP)) to prepare a compound B, the compound B is stirred overnight at room temperature in the solvent (such as chloroform) under the action of silver trifluoroacetate and iodine simple substance to obtain a compound C, and the compound C is stirred overnight at room temperature in the solvent (such as N-methylpyrrolidone (NMP)) under the action of the base (such as potassium tert-butoxide) and O- (4-nitrobenzoyl) hydroxylamine to prepare a compound I-a;

the scheme 2 comprises the following steps: stirring compound B in solvent (such as NMP) at room temperature overnight under the action of base (such as potassium tert-butoxide) and O- (4-nitrobenzoyl) hydroxylamine to obtain compound D, and stirring compound D in solvent (such as acetic acid) under the condition of potassium iodide and hydrogen peroxide at room temperature for 3 hr to obtain compound I-a;

(2) preparing aromatic alkynol I-b, and reacting corresponding aromatic aldehyde with Tetrahydrofuran (THF) solution of ethynyl magnesium bromide to obtain the aromatic alkynol I-b;

(3) carrying out coupling cyclization reaction on the N-amino-5-iodopyrrole-2-methyl formate derivative I-a and the aromatic alkynol I-b under the action of palladium acetate, cuprous iodide and DBU in a solvent such as toluene, tetrahydrofuran and dioxane under the microwave condition to obtain a compound I-0; and

(4) the compound I-0 takes saturated methanol solution of ammonia gas as a solvent, and reacts for 20 hours to 24 hours at the temperature of 90 ℃ by sealing a tube, or directly reacts with strong ammonia water, wherein the solvent is methanol and reacts for 20 hours to 24 hours at the temperature of 90 ℃ by sealing the tube to obtain the pyrrole [1,2-b ] pyridazine compound represented by the general formula I;

the reaction formula of the preparation method is as follows,

in all the following examples, the starting reagents, solvents and materials, except for the specific descriptions, are all supplied by the national pharmaceutical reagent group; the microwave reaction is carried out by using a CEMNULL type microwave reactor;¹h NMR was recorded by a BrucheraM-400 or GeMINI-300 nuclear magnetic resonance apparatus, chemical shifts are expressed in delta (ppm); the mass spectrum was recorded by an Agilent model 1200-6110 single quadrupole liquid chromatography mass spectrometer. The silica gel used for separation is 200-mesh and 300-mesh column chromatography silica gel produced in Qingdao ocean chemical plant. Wherein the abbreviations for English represent the following chemical agents:

NBS: n-bromosuccinimide

DMAP: n, N-dimethylpyridine

NaBH₄: sodium borohydride

BH₃THF solution of (a): tetrahydrofuran solutions of borane

EDCI: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride

HOBt: 1-hydroxybenzotriazoles

DBU: 1, 8-diazabicyclo [5.4.0] undec-7-ene

THF: tetrahydrofuran (THF)

NMP: n-methyl pyrrolidone

Example 1

Preparation and Synthesis of Compounds

Preparation method of 4-chloro-2-trichloroacetylpyrrole (compound 2)

To a 500mL round bottom flask equipped with a stirrer was added 2-trichloroacetylpyrrole 1(40 g, 190 mmol, Shanghai Shuya pharmaceutical science Co., Ltd.) and dichloromethane 200mL, sulfuryl chloride (31 g, 227mmol) was added dropwise slowly at-10 ℃ over about 45 minutes, stirring was continued for 1 hour while maintaining the temperature, and then the mixture was transferred to room temperature and stirred overnight. After the reaction was completed (thin layer chromatography tracking), the reaction solution was poured into 300mL of dichloromethane, and then ice water was added to quench, the organic phase was separated, the aqueous phase was extracted twice with 400mL of dichloromethane, the organic phases were combined, and then washed with saturated sodium bicarbonate solution and saturated brine, respectively, dried over anhydrous sodium sulfate, and concentrated to obtain 32 g of crude product with a yield of 69%.

¹H NMR(300MHz,CDCl₃)δ9.42(brs,1H),7.27(dd,J＝3.2,1.5Hz,1H), 7.11(dd,J＝3.2,1.5Hz,1H)；LC-MS(ESI):m/z 246.2[M+H]⁺。

Preparation method of 4-chloro-pyrrole-2-methyl formate (compound 3)

To a 250mL round bottom flask equipped with a stirrer was added compound 2(8.1 g, 32.8mmol) and DMAP (800 mg), 100mL of anhydrous methanol was poured, stirred at 70 ℃ for 1 hour, cooled to room temperature, and the solvent was removed under reduced pressure. An additional 100mL of ethyl acetate and 30mL of 3M HCl solution were added, the organic phase was separated by extraction, and the aqueous layer was extracted once more with 50mL of ethyl acetate. The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. Concentration in vacuo afforded 5.1 g of a white solid in 98% yield.

¹H NMR(300MHz,CDCl₃)δ9.31(brs,1H)，6.90(dd,J＝3.2,1.5Hz,1H)， 6.81(dd,J＝3.2,1.5Hz,1H)，3.86(s,3H)；LC-MS(ESI):m/z 160.1[M+H]⁺。

Preparation of 4-chloro-5-iodo-pyrrole-2-carboxylic acid methyl ester (Compound 4)

To a 100mL round-bottom flask equipped with a stirrer were added compound 3(5.3 g, 33mmol), silver trifluoroacetate (7 g, 33mmol) and 60mL of chloroform, and elemental iodine (4.7 g, 36 mmol) was added under ice bath and N was used₂The air was replaced 3 times, and the mixture was stirred at room temperature for 7 hours in the dark. After the reaction is completed, adding 25mL of saturated sodium thiosulfate solution, stirring for 15 minutes, carrying out vacuum filtration, washing a filter cake with chloroform and water, separating an organic phase of a filtrate, washing the organic phase with saturated saline solution, and drying with anhydrous sodium sulfate. Concentration column chromatography (petroleum ether: ethyl acetate: 30:1) afforded 8.3 g of a white solid in 76% yield.

¹H NMR(300MHz,CDCl₃)δ9.55(s,1H),6.80(d,J＝2.8Hz,1H),3.91(s, 3H)；LC-MS(ESI):m/z 286.1[M+H]⁺。

Preparation method of O- (4-nitrobenzoyl) hydroxylamine (compound 5)

A solution of paranitrobenzoyl chloride (13.2 g, 70mmol) in dichloromethane (50mL) was slowly added dropwise to dichloromethane (45 mL) containing N-Boc hydroxylamine (9.4 g, 70.0mmol), 5.4mL triethylamine at 0 deg.C for about 45 minutes. Stirring was continued at this temperature for 10 minutes and then transferred to room temperature for 1.5 hours. After the reaction is detected to be complete by thin layer chromatography, 60mL of water is added to quench the reaction, and the stirring is continued for 30 minutes. The organic phase was separated, washed with 48mL of 1% sodium bicarbonate solution, and separated. To the organic phase was added 9mL of methanesulfonic acid at room temperature to start the precipitation of a white solid. After standing for 6 hours, n-hexane (30 mL) was added, and a white solid was filtered off to give 5-methanesulfonate. The white solid was placed in a 500mL beaker, 200mL of methylene chloride was added, and then a saturated sodium bicarbonate solution was added dropwise until the white solid was completely dissolved. The organic phase was separated and washed with brine. Dried over anhydrous magnesium sulfate and concentrated to give 8.9 g of a white solid with a yield of 71%.

¹H NMR(300MHz,CDCl₃)δ8.35-8.28(m,2H),8.23-8.18(m,2H),6.74(s, 2H)；LC-MS(ESI)：m/z 183.1[M+H]⁺。

Preparation method of N-amino-4-chloro-pyrrole-2-methyl formate (compound 7)

The method comprises the following steps:

6 g of ammonium chloride, 10mL of ammonia water and 120mL of diethyl ether are sequentially added to a 500mL round-bottom flask, 140mL of 5% (mass percent) sodium hypochlorite solution is added dropwise to the mixed solution at-10 ℃ for about 30 minutes, the temperature is kept, stirring is carried out for 1 hour, and then an organic phase is separated and washed by 60mL of ice saturated saline. The anhydrous calcium chloride is dried and is placed at the temperature of minus 40 ℃ for drying for 1 hour for standby.

Compound 3(2.5 g, 15.7mmol) was dissolved in 30mL of anhydrous DMF (available from carbofuran chemical) under ice-bath and NaH (800 mg, 20mmol, 60% dispersed in mineral oil, available from carbofuran chemical) was added in two portions. After stirring for 30 minutes, 100mL of the prepared chloroamine solution was added, and the mixture was stirred at room temperature for 4 hours to complete the reaction by thin layer chromatography. After 30mL of saturated sodium thiosulfate solution was added and stirred for 15 minutes, 100mL of water was added, the organic phase was separated, and the aqueous layer was extracted with ethyl acetate. The organic phases were combined and washed with brine. Concentration and column chromatography (petroleum ether: ethyl acetate: 20:1) elution gave 2.3 g of a white solid in 85% yield.

The method 2 comprises the following steps:

in a 500mL round-bottomed flask equipped with a stirrer, Compound 3(2 g, 12.6mmol), ammonium chloride (4 g, 75.5mmol), 3-octylmethylammonium chloride (Aliquat-336,0.5mL), aqueous ammonia 15mL, water 5mL, K₂CO₃(4.6 g, 33.3mmol) and 45mL of methyl t-butyl ether, and then 160mL of sodium hypochlorite solution was added dropwise through a constant pressure dropping funnel over about 30 minutes. Stirring for 6-7 hours at room temperature, and adding saturated sodium thiosulfate solution after the reaction is completed50mL, after stirring for 10 minutes, the organic phase was separated and the aqueous layer was extracted with another mL of ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution, concentrated and eluted by column chromatography (petroleum ether: ethyl acetate: 20:1) to give 1.8 g of a yellow solid in 82% yield.

¹H NMR(300MHz,CDCl₃)δ6.91(d,J＝1.9Hz,1H),6.73(d,J＝2.2Hz,1H), 5.54(s,2H),3.82(s,3H)；LC-MS(ESI):m/z 143.2(100％)[M-CH₃O]⁺。

Preparation method of 1-amino-4-chloro-5-iodo-pyrrole-2-methyl formate (compound 6)

The method comprises the following steps:

potassium tert-butoxide (526 mg, 4.7mmol) was added to a solution of compound 4(857 mg, 3mmol) in N-methylpyrrolidone (10mL) under ice-cooling, and after stirring for 15 minutes, a solution of O- (4-nitrobenzoyl) -hydroxylamine (692 mg, 3.6mmol) in tetrahydrofuran 5mL was added to the mixture and the mixture was stirred at room temperature overnight. To the reaction mixture were added 50mL of saturated brine and 30mL of ethyl acetate, the organic phase was separated, and the aqueous layer was extracted with 50mL of ethyl acetate. The combined organic phases were washed with water and brine. The organic phase was concentrated and eluted by column chromatography (petroleum ether: ethyl acetate: 100:1) to give 180 mg of a white solid in 13% yield with 260 mg of recovered starting material and 50.1% conversion.

The method 2 comprises the following steps:

compound 7(625 mg, 3.6mmol) was dissolved in 10ml of glacial acetic acid, potassium iodide (900 mg, 5.4mmol) was added, and 30% H was added dropwise₂O₂2mL of the solution, gradually changing the reaction solution into a purple solution, stirring at room temperature for reaction for 3 hours, adding 5mL of saturated sodium thiosulfate solution, continuing stirring for 10 minutes, adding ethyl acetate, separating an organic phase, extracting a water layer with ethyl acetate, combining the organic phases, washing with saturated saline, concentrating, and eluting by column chromatography (petroleum ether: ethyl acetate: 30:1) to obtain 700 mg of a white solid with the yield of 78%.

¹H NMR(300MHz,CDCl₃)δ6.91(s,1H),5.74(s,2H),3.84(s,3H)；LC-MS (ESI):m/z 269.2(100％)[M-CH₃O]⁺。

Process for producing 1- (4-diethoxymethylphenyl) propiolic alcohol (Compound 9)

4-diethoxymethylbenzaldehyde 8(780 mg, 3.8mmol, available from TCI chemical) and anhydrous tetrahydrofuran (50mL) were added to a 250mL round-bottomed flask equipped with a stirrer, and then ethynylmagnesium bromide in THF (0.5M, 10mL, available from carbofuran chemical) was added through a constant pressure dropping funnel for about 15 minutes, and the mixture was further stirred at room temperature for 0.5 to 1 hour, after completion of the TLC detection reaction, 20mL of a saturated ammonium chloride solution was added, and after stirring for 10 minutes, the organic phase was separated, the aqueous layer was extracted with ethyl acetate, the organic phases were combined, and after washing with a saturated common salt solution, the mixture was concentrated, and column chromatography (petroleum ether: ethyl acetate 5:1) was performed to obtain 390 mg of an oily substance with a yield of 44%.

¹H NMR(300MHz,CDCl₃)δ7.52(q,J＝8.2Hz,4H),5.50(s,1H),5.47(dd,J ＝6.2,2.1Hz,1H),3.67-3.47(m,4H),2.68-2.65(m,1H),2.35-2.29(m,1H),1.28 -1.19(m,6H)。LC-MS(ESI):m/z 257.2[M+H]⁺。

Preparation method of 2- (4-diethoxymethylphenyl) -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxylic acid methyl ester (compound 10)

In a 25mL microwave tube equipped with a stirrer, compound 6(150 mg, 0.5mmol), compound 9(234 mg, 1mmol), palladium acetate (2.24 mg, 0.01mmol), cuprous iodide (1 mg, 0.005mmol), and 5mL of toluene were added to replace N₂After 3 times, DBU (380 mg, 2.5mmol) was added rapidly, capped with a microwave cap, placed in a microwave reactor, 9After the reaction was carried out at 0 ℃ (λ 180W) for 40 minutes and cooled, 25mL of chloroform was added to dilute the reaction solution, the diluted solution was transferred to a 100mL round-bottomed flask, concentrated and eluted by column chromatography (petroleum ether: ethyl acetate: 30:1) to obtain 62 mg of a yellow oil with a yield of 32%.

¹H NMR(300MHz,CDCl₃)δ8.06(d,J＝8.3Hz,2H),7.94(d,J＝9.4Hz,1H), 7.62(d,J＝8.4Hz,2H),7.47(s,1H),7.35(d,J＝9.5Hz,1H),5.58(s,1H),3.96(s, 3H),3.60(dd,J＝13.7,7.0Hz,4H),1.26(t,J＝7.1Hz,6H)；LC-MS(ESI):m/z 389.2[M+H]⁺。

Preparation method of 2- (4-formylphenyl) -5-chloro-pyrrolo [1,2-b ] pyridazine-7-methyl formate (compound 11)

In a 50mL round-bottom flask equipped with a stirrer, 10mL of compound 10(62 mg, 0.16mmol) and 10mL of anhydrous methanol were added, 5mL of 3N HCl solution was added, and the mixture was stirred at room temperature for 30 minutes, after completion of the reaction, the pH was adjusted to 7-8 with NaOH solution (2.5M). The organic phase was then separated, the aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give 40 mg of a yellow solid in 80% yield.

LC-MS(ESI):m/z 315[M+H]⁺。

Preparation method of 2- (4-dimethylaminomethylphenyl) -5-chloro-pyrrolo [1,2-b ] pyridazine-7-formamide (compound I-1)

Adding compound 11(50 mg, 0.16mmol), dimethylamine hydrochloride (20 mg, 0.24mmol) and a few drops of acetic acid and methanol in a 50mL round-bottom flask with a stirrer, stirring at room temperature for 5 minutes, adding sodium cyanoborohydride (50 mg, 0.8mmol), stirring for 1 hour, evaporating the solvent under reduced pressure after the reaction is completed, adding 10mL of trichloromethane and 4mL of water, extracting and separating an organic phase,extracting the water layer with chloroform for 2 times, mixing organic phases, concentrating, and eluting by column chromatography (dichloromethane: methanol 20:1) to obtain crude product 40 mg, LC-MS (ESI) M/z 344.2[ M + H ]]⁺(ii) a And (3) adding the crude product into a 25mL sealed tube, adding an ammonia methanol solution, placing the tube at 80-90 ℃ for sealed tube reaction for 18-24 hours, cooling the reaction liquid to room temperature, evaporating the solvent under reduced pressure, and eluting by column chromatography (dichloromethane: methanol: 10:1) to obtain 25 mg of yellow solid, wherein the total yield of the two steps is 47%.

¹H NMR(300MHz,CD₃OD)δ8.08(d,J＝9.5Hz,1H),8.01(d,J＝8.3Hz, 2H),7.61(d,J＝8.3Hz,2H),7.50-7.43(m,2H),4.03(s,2H),2.63(s,6H)；LC-MS (ESI):m/z 329.2[M+H]⁺。

Preparation method of 5-chloro-2- (4-methylaminomethylphenyl) pyrrole [1,2-b ] pyridazine-7-formamide (compound I-2)

Compound 11(50 mg, 0.16mmol) and methylamine alcohol solution (2mL) were used as starting materials, and with reference to the preparation of compound I-1, 20mg of a yellow solid was obtained with a total yield of 40% over two steps: .

¹H NMR(300MHz,CD₃OD)δ8.00(ddd,J＝8.4,7.2,1.6Hz,3H),7.69-7.59 (m,2H),7.42(dt,J＝10.7,1.5Hz,2H),4.28(d,J＝1.6Hz,2H),2.77(s,3H),1.96- 1.89(m,1H)；LC-MS(ESI):m/z 315.2[M+H]⁺。

Preparation method of 2- (4-diethylaminomethylphenyl) -5-chloro-pyrrolo [1,2-b ] pyridazine-7-formamide (compound I-3)

Starting from compound 11(50 mg, 0.16mmol) and diethylamine in alcohol (1mL), reference was made to the procedure for the preparation of compound I-1 to give 24 mg of a yellow solid in 42% overall yield over two steps.

¹H NMR(300MHz,CD₃OD)δ8.20(dd,J＝9.5,1.3Hz,1H),8.17-8.11(m, 2H),7.75-7.70(m,2H),7.58-7.52(m,2H),4.42(s,2H),3.22(q,J＝7.3Hz,4H), 1.37(t,J＝7.3Hz,6H)；LC-MS(ESI):m/z 357.2[M+H]⁺。

Preparation method of 2- [4- (pyrrolin-1-yl) methylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-formamide (compound I-4)

Starting from compound 11(50 mg, 0.16mmol) and pyrrolidine (1mL), the procedure for the preparation of compound I-1 gave 15 mg of a yellow solid in 26% overall yield over two steps.

¹H NMR(300MHz,CD₃OD)δ8.18(dd,J＝9.4,1.4Hz,1H),8.12(dd,J＝8.3, 1.5Hz,2H),7.73(d,J＝8.1Hz,2H),7.58-7.49(m,2H),4.46(s,2H),3.35(d,J＝ 6.0Hz,4H),2.15-2.06(m,4H)；LC-MS(ESI):m/z 355.2[M+Na]⁺。

Preparation method of 2- [4- (piperazine-1-yl) -methylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-formamide (compound I-5)

Adding 2 drops of glacial acetic acid into 5mL of methanol containing compound 11(50 mg, 0.16mmol) and N-boc piperazine (45 mg, 0.24mmol), stirring at room temperature for 5 minutes, adding sodium cyanoborohydride (50 mg), stirring at room temperature for 3 hours, directly concentrating to obtain an oily substance, adding 3mL of water and 5mL of trichloromethane into the oily substance, separating an organic phase, extracting a water layer with trichloromethane, combining the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, directly feeding the oily substance into the next step, adding 10mL of trichloromethane into the oily substance, slowly dropping 1.5mL of trifluoroacetic acid, stirring at room temperature for 1 hour, adjusting the pH value to 8-9 with 3N HCl solution under ice bath, separating the organic phase, extracting the water layer with trichloromethane, combining the organic phases, washing with saturated saline solution, directly concentrating without purification, transferring 5mL of methanol into a 25mL sealed tube, adding 8mL of saturated ammonia methanol solution, sealing the tube at 80-90 ℃ for reaction for 18-24 hours, cooling the reaction liquid to room temperature, removing the solvent under vacuum reduced pressure, and eluting by column chromatography (dichloromethane: methanol 10:1) to obtain 10 mg of yellow solid, wherein the total yield of the three steps is 17%.

¹H NMR(300MHz,CD₃OD)δ8.12-8.06(m,1H),7.92(dd,J＝8.1,2.0Hz, 2H),7.52(dd,J＝8.3,2.0Hz,2H),7.46(ddd,J＝5.9,3.8,2.3Hz,2H),3.67(d,J＝ 2.0Hz,2H),3.34(d,J＝1.6Hz,1H),3.19(t,J＝5.5Hz,4H),2.69(t,J＝4.8Hz, 4H)；LC-MS(ESI):m/z 370.2[M+H]⁺。

Preparation method of 2- [4- (3-methyl-piperazin-1-yl) methylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-6)

Starting from compound 11(50 mg, 0.16mmol) and 2-methylpiperazine (24 mg, 0.24mmol), reference was made to the preparation of compound I-1 to give 15 mg of a yellow solid in 25% yield.

¹H NMR(300MHz,CD₃OD)δ8.11(dd,J＝9.5,3.4Hz,1H),7.94(dq,J＝8.6, 2.3,1.8Hz,2H),7.54(dd,J＝8.2,3.3Hz,2H),7.51-7.45(m,2H),3.68(s,2H), 3.20-3.09(m,1H),3.05-2.93(m,2H),2.44-2.32(m,1H),2.26-2.10(m,2H), 2.02(s,1H),1.93(dd,J＝3.8,2.8Hz,1H),1.29(d,J＝3.1Hz,3H)；LC-MS(ESI): m/z 384.2[M+H]⁺。

Preparation method of 2- [4- (4-cyclopropylpiperazin-1-yl) -methylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-7)

Starting from compound 11(50 mg, 0.16mmol) and N-cyclopropylpiperazine (30 mg, 0.24mmol), reference was made to the procedure for the preparation of compound I-1 to give 24 mg of a yellow solid in 37% overall yield over two steps.

¹H NMR(300MHz,CD₃OD)δ8.10(dd,J＝9.4,1.7Hz,1H),7.95-7.88(m, 2H),7.52(dd,J＝6.8,1.6Hz,3H),7.44(dd,J＝9.5,1.6Hz,1H),3.64(s,2H),2.80- 2.65(m,4H),2.64-2.45(m,4H),0.91-0.80(m,1H),0.52-0.47(m,2H),0.45- 0.37(m,2H)；LC-MS(ESI):m/z 410.2[M+H]⁺。

Preparation method of 2- [4- (4-isopropyl-piperazin-1-yl) -methylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-8)

Starting from compound 11(50 mg, 0.16mmol) and N-isopropylpiperazine (31 mg, 0.24mmol), reference was made to the preparation of compound I-1 to give 25 mg of a yellow solid in a two-step overall yield of 38%.

¹H NMR(300MHz,CD₃OD)δ8.13(d,J＝9.5Hz,1H),7.97(d,J＝8.3Hz, 2H),7.55(d,J＝8.3Hz,2H),7.50(dd,J＝5.2,4.3Hz,2H),3.71(s,2H),3.36-3.32 (m,1H),3.22-3.10(m,4H),2.85-2.65(m,4H),1.31(d,J＝6.6Hz,6H)；LC-MS (ESI):m/z 412.2[M+H]⁺。

Preparation method of 2- [4- (4-dimethylaminopiperidin-1-yl) -methylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-9)

Starting from compound 11(50 mg, 0.16mmol) and 4-dimethylaminopiperidine (31 mg, 0.24mmol), reference was made to the preparation of compound I-1 to give 26 mg of a yellow solid in 39% overall yield over two steps.

¹H NMR(300MHz,CD₃OD)δ8.12(dd,J＝9.4,0.6Hz,1H),7.95(dd,J＝8.4, 2.2Hz,2H),7.56-7.46(m,4H),3.64(s,2H),3.11-3.02(m,2H),3.01-2.92(m, 1H),2.74(s,6H),2.23-2.12(m,2H),2.08-1.98(m,2H),1.80-1.64(m,2H)； LC-MS(ESI):m/z 412.2[M+H]⁺。

Process for the preparation of 2- {4- [4- (pyrrolidin-1-yl) -piperidin-1-yl ] -methylphenyl } -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (Compound I-10)

Starting from compound 11(50 mg, 0.16mmol) and 4-pyrrolidin-1-yl-piperidine (37 mg, 0.24mmol), reference was made to the preparation of compound I-1 to give 15 mg of a yellow solid in 21% yield.

¹H NMR(300MHz,CD₃OD)δ8.13(d,J＝9.5Hz,1H),7.99-7.93(m,2H), 7.58-7.52(m,2H),7.51(dd,J＝5.5,4.0Hz,2H),3.67(s,2H),3.42-3.35(m,3H), 3.22-3.13(m,1H),3.11-3.02(m,2H),2.25-2.11(m,4H),2.10-2.02(m,4H), 1.85-1.70(m,3H)；LC-MS(ESI):m/z 438.2[M+H]⁺。

Preparation method of 2- [4- (4-morpholinylpiperidin-1-yl) -methylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-formamide (compound I-11)

Starting from compound 11(50 mg, 0.16mmol) and 4- (4-piperidinyl) morpholine (41 mg, 0.24mmol), reference was made to the preparation of compound I-1 to give 10 mg of a yellow solid in 14% two-step overall yield.

¹H NMR(300MHz,CD₃OD)δ8.16(dd,J＝9.5,2.0Hz,1H),8.05-7.97(m, 2H),7.64-7.56(m,2H),7.55-7.48(m,2H),3.83(s,2H),3.76-3.68(m,4H),3.21- 3.10(m,2H),2.74-2.64(m,4H),2.52-2.30(m,3H),2.06-1.97(m,2H),1.73- 1.62(m,2H)；LC-MS(ESI):m/z 454.2[M+H]⁺。

Preparation method of 2- [4- (4-p-trifluoromethylphenyl-piperazin-1-yl) -methylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (Compound I-12)

Starting from compound 11(50 mg, 0.16mmol) and p-trifluoromethylphenylpiperazine (55 mg, 0.24mmol), reference was made to the preparation of compound I-1 to give 20mg of a yellow solid in 24% overall yield over two steps.

¹H NMR(300MHz,CD₃OD)δ8.17(d,J＝9.4Hz,1H),8.00(d,J＝7.8Hz, 2H),7.60(d,J＝7.9Hz,2H),7.54(dd,J＝6.5,2.8Hz,2H),7.47(d,J＝8.3Hz,2H), 7.04(d,J＝8.7Hz,2H),3.73(s,2H),3.37-3.32(m,4H),2.79-2.62(m,4H)； LC-MS(ESI):m/z 514.2[M+H]⁺。

Preparation method of 4- (4-formylphenyl) -piperazine-1-tert-butyl formate (compound 13)

A50 mL round bottom flask with a stirrer was taken, and 4-formylbenzoic acid 12(3.1 g, 20.7mmol), N-t-butyloxyformylpiperazine (3.6 g, 19.4mmol), EDCI (3.86 g, 20.2mmol), HOBt (2.68 g, 19.8mmol) in dichloromethane (25 mL) were sequentially added, DMAP (20 mg) was finally added, the mixture was stirred at room temperature for 20-24 hours, 5mL of NaOH solution (2.5M) was added after completion of the reaction, after stirring for 5 minutes, the organic phase was separated, the aqueous layer was extracted with chloroform, the organic phases were combined, concentrated, and column chromatography (petroleum ether: ethyl acetate 2:1) was performed to obtain a white solid (4.5 g, yield 73%).

¹H NMR(300MHz,CDCl₃)δ10.0(s,1H),7.93(d,J＝8.2Hz,2H),7.54(d,J＝ 8.1Hz,2H),3.82-3.67(m,2H),3.58-3.30(m,6H),1.46(s,9H)；LC-MS(ESI):m/z 319.2[M+H]⁺。

Preparation method of 4- {4- [1- (1-hydroxy-2-propynyl) ] benzoyl } piperazine-1-tert-butyl formate (compound 14)

Taking a 50mL round bottom flask with a stirrer, adding compound 13(650 mg, 2.05 mmol) and 20mL of anhydrous THF, completely dissolving, adding a THF solution (0.5M, 7mL) of ethynylmagnesium bromide, detecting complete reaction by thin layer chromatography, adding 10mL of saturated ammonium chloride solution, quenching, continuing stirring for 10 minutes, adding 10mL of ethyl acetate, separating an organic phase, continuing extracting an aqueous layer with ethyl acetate, merging the organic phase, washing with saturated saline, concentrating, and eluting by column chromatography (petroleum ether: ethyl acetate ═ 1:1) to obtain 305 mg of yellow oily matter with the yield of 50%.

¹H NMR(300MHz,CDCl₃)δ7.58(dd,J＝10.5,8.0Hz,2H),7.39(ddd,J＝10.2,8.2,1.8Hz,2H),5.52-5.44(m,1H),3.55(d,J＝96.8Hz,8H),2.67(ddd,J＝ 8.4,2.2,0.9Hz,1H),2.16(q,J＝1.6Hz,1H),1.46(s,9H)；LC-MS(ESI):m/z 367.2 [M+Na]⁺。

Preparation method of methyl 2- [4- (N-tert-butoxyformyl) piperazinocarboxoyl ] phenyl-5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxylate (compound 15)

A25 mL microwave reaction tube equipped with a stirrer was charged with Compound 6(160 mg, 0.52mmol), Compound 14(305 mg, 0.9mmol), Pd (OAc)₂(7 mg, 0.01mmol), CuI (1 mg, 0.005mmol) and 8mL of toluene, in place of N₂After 3 times, at N₂Adding DBU (380 mg, 2.5mmol) under protection, covering a microwave tube cap, placing the reaction product in a microwave reactor, reacting for 40 minutes at 90 ℃ (lambda-180W), cooling, adding 25mL of chloroform for dilution, transferring the diluted product into a 100mL round-bottom flask, concentrating, and eluting by column chromatography (petroleum ether: ethyl acetate-2: 1) to obtain 121 mg of yellow oily matter with the yield of 47%.

¹H NMR(300MHz,CDCl₃)δ8.17-8.10(m,2H),7.99(dd,J＝9.5,2.5Hz, 1H),7.59-7.52(m,2H),7.50(d,J＝3.0Hz,1H),7.35(dd,J＝9.4,3.0Hz,1H), 3.96(s,3H),3.82-3.70(m,2H),3.58-3.36(m,6H),1.49(s,9H)；LC-MS(ESI)： m/z 521.2[M+Na]⁺。

Preparation method of hydrochloride of methyl 5-chloro-2- [4- (piperazinyl-1-formyl) phenyl ] -pyrrolo [1,2-b ] pyridazine-7-carboxylate (compound 16)

Compound 15(120 mg, 0.24mmol) was placed in a 100mL round-bottomed flask, and 5mL of anhydrous dichloromethane was added, 5mL of HCl in methyl acetate was added, and the mixture was stirred at 40 ℃ for 2 hours to precipitate a white solid, which was cooled to room temperature and then filtered to obtain 100mg of a hydrochloride of methyl 5-chloro-2- [4- (N-piperazinylcarbonyl) phenyl ] -pyrrolo [1,2-b ] pyridazine-7-carboxylate.

¹H NMR(300MHz,DMSO-d₆)δ9.19-9.07(m,2H),8.29-8.20(m,3H),7.68 -7.62(m,2H),3.86(s,3H),3.55-3.48(m,4H),3.22-3.10(m,4H)；LC-MS(ESI): m/z 399.2[M+H]⁺。

Preparation method of 5-chloro-2- [4- (piperazinyl-1-formyl) phenyl ] -pyrrolo [1,2-b ] pyridazine-7-formamide (compound I-13)

10mL of Compound 16(50 mg) in methanol was dissolved and transferred to a 25mL stoppered tube, and NH was added₃The reaction solution (2) is reacted for 20-24 hours at 80-90 ℃, cooled to room temperature, transferred to a 100mL round-bottom flask, concentrated, and eluted by column chromatography (dichloromethane: methanol: 10:1) to obtain 35 mg of yellow solid.

¹H NMR(300MHz,DMSO-d₆)δ8.26(dt,J＝9.4,1.1Hz,1H),8.21(s,1H), 8.14-8.05(m,2H),7.98(s,1H),7.65-7.59(m,1H),7.59-7.53(m,2H),7.51(p,J ＝1.2Hz,1H),3.62-3.50(m,2H),3.29-3.22(m,2H),2.80-2.60(m,4H)；LC-MS (ESI):m/z 384.2[M+H]⁺。

Process for the preparation of 2- {4- [4- (cyclopropylformyl) -piperazin-1-yl ] formylphenyl } -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (Compound I-14)

15mL of anhydrous dichloromethane was added to compound 16(50 mg, 0.13mmol), and 0.5mL of NEt was added dropwise₃Cyclopropylformyl chloride (30. mu.l) stirred at room temperature for 15 minutes, after completion of the reaction by thin layer chromatography, 2mL of methanol was added, stirred at room temperature for 10 minutes, and concentrated to give a yellow solid as a crude product of 5-chloro-2- {4- [ (4-cyclopropylformyl) -piperazinyl-1-formyl chloride]Phenyl } -pyrrole [1, 2-b)]And transferring 10mL of pyridazine-7-methyl formate into a 25mL sealed tube by using 10mL of methanol, adding 8mL of ammonia methanol solution, reacting at 80-90 ℃ for 20-24 hours, cooling to room temperature, concentrating, and eluting by column chromatography (dichloromethane: methanol 20:1) to obtain 45 mg of yellow solid, wherein the total yield of the two steps is 79%.

¹H NMR(300MHz,DMSO-d₆)δ8.27(dt,J＝9.4,1.4Hz,1H),8.21(s,1H), 8.11(dt,J＝8.3,1.7Hz,2H),7.99(d,J＝3.2Hz,1H),7.71-7.57(m,3H),7.52(d,J ＝1.0Hz,1H),3.85-3.45(m,8H),1.41-1.28(m,1H),0.82-0.65(m,4H)；LC-MS (ESI):m/z 452.2[M+H]⁺。

Process for the preparation of 2- [4- (3-methyl-1-oxobutyl) -piperazin-1-yl-formyl ] -phenyl-5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (Compound I-15)

5mL of anhydrous dichloromethane was added to compound 16(50 mg, 0.16mmol), and 0.5mL of NEt was added dropwise₃Isovaleryl chloride (30 mg, 0.24mmol) was stirred at room temperature for 30 minutes, after completion of the reaction by thin layer chromatography, 3mL of methanol was added, stirred at room temperature for 5 minutes, and concentrated to give 65 mg of crude product which was 2- [ (N-isovaleryl) piperazin-1-yl formyl]-phenyl-5-chloro-pyrrolo [1,2-b ]]Pyridazine-7-carboxylic acid methyl ester; it was transferred to a 25mL stopcock with 10mL methanol, and 8mL NH added₃Reacting the methanol solution at 80-90 ℃ for 20-24 hours, cooling to room temperature, concentrating, and performing column chromatography (dichloromethane): methanol 20:1) gave 40 mg of a yellow solid in 53% two-step overall yield.

¹H NMR(300MHz,CDCl₃)δ8.69(s,1H),8.05(dd,J＝9.4,2.1Hz,1H),7.99- 7.89(m,2H),7.65(d,J＝2.1Hz,1H),7.64-7.54(m,2H),3.85-3.40(m,8H),2.30- 2.22(m,2H),2.20-2.08(m,1H),1.04-0.94(m,6H)；LC-MS(ESI):m/z 468.2 [M+H]⁺。

Preparation method of 4- (3-fluoro-4-formylphenyl) -formyl-piperazine-1-carboxylic acid tert-butyl ester (compound 17)

3-fluoro-4-formylbenzoic acid (1 g, 6mmol, preparation method thereof referring to EP2526945A1, starting material 3-fluoro-4-methylbenzoic acid purchased from Shanghai Shuyao), N-t-butoxyformylpiperazine (1.1 g, 5.66mmol), HOBt (764 mg, 5.66mmol), EDCI (1.3 g, 6.8mmol) and DMAP (20 mg) were used as starting materials, according to the preparation method of compound 13, to give 1.7 g of a white solid with a yield of 89%.

¹H NMR(300MHz,CDCl₃)δ10.38(d,J＝0.7Hz,1H),7.93(d,J＝8.3Hz,1H), 7.33-7.17(m,2H),3.85-3.23(m,8H),1.43(s,9H)；LC-MS(ESI):m/z 359.2 [M+Na]⁺。

Preparation method of 4- [ 3-fluoro-4- (1-hydroxy propyn-2-yl) benzoyl ] -piperazine-1-tert-butyl formate (compound 18)

Starting from compound 17(600 mg, 1.8mmol) and ethynylmagnesium bromide in THF (0.5M, 6.5 mL), reference was made to the preparation of compound 14 to give 420 mg of a white solid in 65% yield.

¹H NMR(300MHz,CDCl₃)δ7.72(t,J＝7.6Hz,1H),7.23-7.10(m,2H),5.75 (dd,J＝5.8,2.2Hz,1H),3.82-3.28(m,8H),2.68-2.66(m,1H)；LC-MS(ESI):m/z 385.2[M+Na]⁺。

Preparation method of 2- [ 2-fluoro- (4-tert-butyloxycarbonylpiperazin-1-ylcarbonyl) phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxylic acid methyl ester (compound 19)

Compound 6(218 mg, 0.7mmol) and compound 18(420 mg, 1.2mmol) were starting materials, according to the preparation method of compound 15, to give 160 mg of a yellow solid in 44% yield.

¹H NMR(300MHz,CDCl₃)δ8.14(t,J＝7.8Hz,1H),7.96(d,J＝9.5Hz,1H), 7.51(s,1H),7.41-7.19(m,4H),3.94(s,3H),3.80-3.65(m,2H),3.55-3.35(m, 6H),1.47(s,9H)；LC-MS(ESI):m/z 539.2[M+Na]⁺。

Preparation method of methyl 2- [ 2-fluoro-4- (piperazin-1-yl-formyl) phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxylate (compound 20)

Dissolving compound 19(150 mg, 0.3mmol) in dichloromethane, dropwise adding trifluoroacetic acid (1.5mL), detecting by TLC after reaction is completed, adding NaOH solution (2.5M) under ice bath to adjust pH to 7-8, separating out organic phase, extracting water layer with chloroform, combining organic phases, concentrating, and performing column chromatography (dichloromethane: methanol: 10:1+ few drops of NH)₃Methanol solution of (b) to give 100mg of a yellow solid in 80% yield.

LC-MS(ESI):m/z 417.2[M+H]⁺。

Preparation method of 2- [ 2-fluoro-4- (4-cyclopropylformylpiperazin-1-yl-formyl) phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-16)

Compound 20(60 mg)0.15mmol) of anhydrous dichloromethane 10mL, 0.5mL NEt was added₃And cyclopropylcarbonyl chloride (26 mg, 0.25mmol) was stirred at room temperature for 30 minutes, after completion of the TLC detection, 4mL of methanol was added and concentrated to give 65 mg of crude 5-chloro-2- [ 3-fluoro-4- (4-cyclopropylformylpiperazine-1-formyl) phenyl ] -2- [ 3-fluoro-4- (4-cyclopropylformyl-piperazine-1-formyl)]Pyrrole [1,2-b ]]Pyridazine-7-carboxylic acid methyl ester; transferring the mixture into a 25mL sealed tube by using 10mL of methanol, adding 8mL of methanol solution of ammonia, reacting at 80-90 ℃ for 20-24 hours, cooling to room temperature, concentrating, and washing by column chromatography (dichloromethane: methanol 20:1) to obtain 30mg of yellow solid, wherein the total yield of the two steps is 43%.

¹H NMR(300MHz,DMSO-d₆)δ8.83-8.48(s,1H),8.33-8.24(m,1H),8.18 (s,1H),8.04-7.92(m,2H),7.58-7.52(m,1H),7.51-7.39(m,2H),3.87-3.36(m, 8H),0.85-0.66(m,5H)；LC-MS(ESI):m/z 470.2[M+H]⁺。

Process for the preparation of 2- [ 2-fluoro-4- [4- [ (2S) -2-methylamino-1-oxo-propyl ] -piperazin-1-ylcarbonyl ] phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-17)

Compound 20(100 mg, 0.24mmol), Boc-N-methyl-L-alanine (54 mg, 0.27 mmol), EDCI (55 mg, 0.29mmol), HOBt (32 mg, 0.24mmol) and DMAP (5 mg) in dichloromethane (15mL) were stirred at room temperature for 14 hours, 3mL of NaOH (2.5M) solution was added, stirred at room temperature for 5 minutes, the organic phase was separated, the aqueous layer was extracted with chloroform, the organic phases were combined, concentrated, and column chromatography (dichloromethane: methanol: 50:1) gave 58 mg of a yellow solid, LC-MS (ESI): M/z 624.2[ M + Na 624.2]⁺(ii) a To 20mL of the above solid in dichloromethane was added CF₃COOH (1.5mL) was stirred at room temperature for 2 hours, the pH was adjusted to 8-9 with NaOH (2.5M), the organic phase was separated, the aqueous layer was extracted with chloroform, the organic phases were combined, concentrated, and eluted by column chromatography (dichloromethane: methanol 10:1) to give 40 mg of a yellow solid, LC-MS (ESI) M/z 502.2[ M + Na ] (M/z 502.2)]⁺(ii) a 10mL of methanol was transferred to a 25mL lock tube,adding 8mL of methanol solution of ammonia, reacting at 80-90 ℃ for 20-24 hours, cooling to room temperature, concentrating, and washing by column chromatography (dichloromethane: methanol 20:1) to obtain 30mg of yellow solid, wherein the total yield of the three steps is 26%.

¹H NMR(300MHz,DMSO-d₆)δ8.29(d,J＝9.5Hz,1H),8.17(s,1H),8.04- 7.95(m,2H),7.59-7.49(m,2H),7.45(ddd,J＝11.4,8.8,1.7Hz,2H),4.22-4.02 (m,1H),3.78-3.39(m,8H),2.36(s,3H),1.24-1.16(m,3H)；LC-MS(ESI):m/z 487.2[M+Na]⁺。

Process for the preparation of 2- [4- (4-isopropylsulfonylpiperazin-1-yl-formyl) phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (Compound I-18)

To 5mL of chloroform of compound I-13(10 mg, 0.024mmol), 1mL of pyridine and 250ul of isopropylsulfonyl chloride were added, stirred at room temperature for 4 hours, 3mL of 3N HCl solution was added, the organic phase was separated, the aqueous layer was extracted with chloroform, the organic phases were combined, concentrated, and eluted by column chromatography (dichloromethane: methanol ═ 20:1) to give 8 mg of a yellow solid with a yield of 67%.

¹H NMR(400MHz,CD₃OD)δ8.14(d,J＝9.4Hz,1H),8.07(d,J＝8.2Hz, 2H),7.63(d,J＝8.2Hz,2H),7.54(s,1H),7.48(d,J＝9.5Hz,1H),3.63-3.54(m, 2H),3.52-3.46(m,1H),3.43-3.37(m,6H),1.34(d,J＝6.9Hz,6H)；LC-MS(ESI): m/z 490.2[M+H]⁺。

Process for the preparation of N- (2-fluoro-5-formyl-benzoyl) -piperazin-1-yl-carboxylic acid tert-butyl ester (compound 21)

2-fluoro-5-formylbenzoic acid (850 mg, 5mmol of its preparation method reference), N-Boc piperazine (1.1 g, 6.1mmol), EDCI (1.2 g, 6.1mmol), HOBt (688 mg, 5.1mmol) and DMAP (80 mg) as starting materials, reference was made to the preparation method of Compound 13 to give 1.4 g of a white solid in 83% yield.

¹H NMR(300MHz,CDCl₃)δ9.97(s,1H),8.00-7.93(m,2H),7.28(d,J＝8.6 Hz,1H),3.81-3.74(t,J＝5.3Hz,2H),3.53(t,J＝5.3Hz,2H),3.42(t,J＝5.1Hz, 2H),3.32-3.24(t,J＝5.3Hz,2H),1.45(s,9H)；LC-MS(ESI):m/z 359.2[M+Na]⁺。

Preparation method of N- [ 2-fluoro-5- (1-hydroxy-propyn-2-yl) benzoyl ] -piperazin-1-yl-carboxylic acid tert-butyl ester (compound 22)

Starting from compound 21(500 mg, 1.5mmol) and a THF solution of ethynylmagnesium bromide (0.5M, 5mL), reference was made to the preparation of compound 14 to give 520 mg of a yellow oil in 94% yield.

¹H NMR(300MHz,CDCl₃)δ7.64-7.56(m,2H),7.11(ddd,J＝9.0,8.1,1.0 Hz,1H),5.45(dd,J＝5.9,2.2Hz,1H),3.82-3.74(m,2H),3.52(t,J＝5.3Hz,2H), 3.41(t,J＝5.0Hz,2H),3.34-3.27(m,2H),2.73(d,J＝6.2Hz,1H),2.68(d,J＝2.2 Hz,1H),1.46(s,9H)；LC-MS(ESI):m/z 385.2[M+Na]⁺。

Process for the preparation of 2- [3- (N-tert-butoxy-formyl-piperazin-1-yl-formyl) -4-fluoro-phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazinine-7-carboxylic acid methyl ester (compound 23)

Compound 6(270 mg, 0.9mmol), compound 22(520 mg, 1.5mmol) were used as starting materials in reference to the preparation of compound 15 to give 160 mg of a yellow solid in 34% yield.

¹H NMR(300MHz,CDCl₃)δ8.25-8.20(m,1H),8.05-8.02(m,1H),7.98(d, J＝8.3Hz,1H),7.48(s,1H),7.29-7.17(m,2H),3.95(s,3H)3.85-3.75(m,2H), 3.58-3.52(m,2H),3.48-3.42(m,2H),3.38-3.32(m,2H),1.47(s,9H)；LC-MS (ESI):m/z 539.2[M+Na]⁺。

Preparation method of 2- [3- (piperazin-1-yl) -formyl-4-fluoro-phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxylic acid methyl ester (compound 24)

After compound 23(108 mg, 0.23mmol) in 10mL of dichloromethane was added trifluoroacetic acid (1.5mL) at room temperature, stirring was continued for 1 hour, and the reaction was checked by thin layer chromatography, pH was adjusted to basic (8-9) with NaOH (2.5M) solution in ice bath, the organic phase was separated, the aqueous layer was extracted with chloroform, the organic phases were combined, concentrated, and eluted by column chromatography (dichloromethane: methanol 10:1) to obtain 80 mg of yellow solid with yield 83%.

LC-MS(ESI):m/z 417.2[M+H]⁺。

Preparation method of 2- [3- (N-cyclopropylformyl-piperazin-1-yl) -formyl-4-fluoro-phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-19)

Compound 24(50 mg, 0.12mmol) in 10mL of dichloromethane was added a few drops of triethylamine, cyclopropanecarbonyl chloride (20 mg, 0.18mmol), stirred at room temperature for 1 hour, methanol 5mL was added, stirring was continued for 10 minutes, and concentration was carried out to give 2- [3- (N-cyclopropanecarbonyl-piperazin-1-yl) -formyl-4-fluoro-phenyl-carbonyl-ethyl ester as an oil]-5-chloro-pyrrolo [1,2-b]Pyridazine-7-carboxylic acid methyl ester, LC-MS (ESI) M/z 485.2[ M + H [ ]]⁺(ii) a Transferring 10mL of methanol into a 25mL sealed tube, adding 8mL of methanol solution of ammonia, reacting at 80-90 ℃ for 20-24 hours, cooling to room temperature, concentrating, and eluting by column chromatography (dichloromethane: methanol 20:1) to obtain 21 mg of yellow solid, wherein the total yield of the two steps is 38%.

¹H NMR(300MHz,CDCl₃)δ7.99(d,J＝9.4Hz,1H),7.98-7.83(m,2H), 7.56(s,1H),7.27(t,J＝8.8Hz,1H),7.19(d,J＝9.4Hz,1H),3.90-3.65(m,6H), 3.44-3.33(m,2H),1.75-1.65(m,1H),0.96-0.92(m,2H),0.82-0.75(m,2H)； LC-MS(ESI):m/z 470.2[M+H]⁺。

Preparation method of 2- [3- [ N- [ (2S) -pyrrolin-2-yl ] -formyl-piperazin-1-yl ] -formyl ] -4-fluoro-phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-20)

Compound 24(50 mg, 0.12mmol), Boc-L-proline (28 mg, 0.13mmol), EDCI (28 mg, 0.15mmol) and HOBt (17 mg, 0.12mmol), and DMAP (5 mmol) were used as starting materials, in reference to the preparation of compound I-17, to give 15 mg of a yellow solid in 25% three-step overall yield.

¹H NMR(300MHz,CDCl₃)δ8.29(d,J＝9.5Hz,1H),8.25-8.11(m,3H), 8.00(s,1H),7.67-7.61(m,1H),7.59(d,J＝9.3Hz,1H),7.53(s,1H),4.47-4.30 (m,1H),3.62(dd,J＝53.7,20.3Hz,8H),3.16(d,J＝5.0Hz,2H),3.01(s,1H),2.28 (s,1H),1.76(s,4H)；LC-MS(ESI):m/z 499.2[M+H]⁺。

Process for preparing 3- (4-methoxypiperidin-1-yl-formyl) -4-fluoro-benzaldehyde (compound 25)

2-fluoro-5-formylbenzoic acid (370 mg, 2.2mmol), hydrochloride of 4-methoxypiperidine (300 mg, 2mmol, see U.S. Pat. No. 4, 2011092475A1), EDCI (460 mg, 2.4mmol), HOBt (300 mg, 2.2mmol) in 40mL of dichloromethane were added DMAP (20 mg), stirred at room temperature for 20-24 hours, after completion of the reaction by thin layer chromatography, 5mL of NaOH (2.5M) solution was added, after stirring for 10 minutes, the organic phase was separated, the aqueous layer was extracted with chloroform, the organic phases were combined, concentrated, and column chromatography (petroleum ether: ethyl acetate ═ 2:1) was performed to give 370 mg of a white solid in a yield of 70%.

1H NMR(300MHz,CDCl₃)δ9.97(t,J＝0.5Hz,1H),7.98-7.89(m,2H), 7.30-7.22(m,1H),4.05-3.97(m,1H),3.88-3.80(tt,J＝8.7,4.1Hz,1H),3.68- 3.63(m,1H),3.53-3.42(m,2H),3.36(s,3H),3.20-3.10(m,1H),3.07-2.93(m, 1H),1.98-1.92(m,1H),1.87-1.73(m,1H)；LC-MS(ESI):m/z 266.2[M+H]⁺。

Preparation method of 2-fluoro-5- (1-hydroxy-propyn-2-yl) -phenyl- (4-methoxypiperidin-1-yl) -methyl ketone (compound 26)

Compound 25(370 mg, 1.4mmol) in anhydrous THF40mL was added ethynylmagnesium bromide THF solution (0.5M, 4mL), stirred at room temperature for 1-1.5 hours, and 20mL of saturated ammonium chloride solution was added, the organic phase was separated, the aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, concentrated, and eluted by column chromatography (petroleum ether: ethyl acetate 1:1) to give 370 mg of yellow oil in 90% yield.

1H NMR(300MHz,CDCl₃)δ7.61-7.50(m,2H),7.13-7.05(m,1H),5.43(s, 1H),4.05-3.98(s,1H),3.86-3.79(m,1H),3.65-3.45(m,3H),3.36(s,3H),3.20- 3.12(m,1H),3.05-2.92(m,1H),2.66(d,J＝2.2Hz,1H),1.99-1.76(m,2H)； LC-MS(ESI):m/z 314.2[M+Na]⁺。

Preparation method of 2- [3- (4-methoxypiperidin-1-yl-formyl) -4-fluoro-phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-21)

A25-mL microwave tube equipped with a stirrer was charged with Compound 6(210 mg, 0.7mmol), Compound 26(370 mg, 1.3mmol), Palladium acetate (8 mg), cuprous iodide (1 mg), and toluene (10mL) to replace N₂After 3 times, N₂Adding DBU (532 mg) rapidly under protection, covering with a microwave cap, placing in a microwave reactor, reacting at 90 deg.C (lambda is 180W) for 40 min, cooling, adding chloroform 25ml, diluting, and transferringTransferred to a 100ml round bottom flask, concentrated and eluted by column chromatography (petroleum ether: ethyl acetate: 3: 1) to give 40 mg of a yellow solid, 2- [3- (4-methoxypiperidin-1-yl-formyl) -4-fluoro-phenyl ] -ethyl acetate]-5-chloro-pyrrolo [1,2-b]Pyridazine-7-methyl formate crude product with yield of 13%, LC-MS (ESI) M/z 446.2[ M + H]⁺(ii) a The mixture was transferred to a 25mL sealed tube with 10mL of methanol, 8mL of a methanol solution of ammonia was added, the reaction was carried out at 80 to 90 ℃ for 20 to 24 hours, the mixture was cooled to room temperature, concentrated, and eluted by column chromatography (dichloromethane: methanol 20:1) to give 20mg of a yellow solid, with a yield of 51%.

¹H NMR(300MHz,DMSO-d₆)δ8.24(d,J＝9.5Hz,1H),8.15(ddd,J＝8.7, 5.1,2.5Hz,2H),8.07(dd,J＝6.4,2.4Hz,1H),7.95(s,1H),7.62(d,J＝9.5Hz,1H), 7.55-7.46(m,2H),3.99-3.88(m,1H),3.49-3.34(m,3H),3.25(s,3H),3.18-3.00 (m,2H),1.95-1.85(m,1H),1.82-1.74(s,1H),1.51-1.41(m,1H)；LC-MS(ESI): m/z 431.2[M+H]⁺。

Process for producing 4- (4-methoxypiperidin-1-yl) -formylbenzaldehyde (compound 27)

Starting from 4-formylbenzoic acid (330 mg, 2.2mmol) and 4-methoxypiperidine hydrochloride (302 mg, 2mmol), the preparation of compound 25 gave 320 mg of a white solid in 65% yield.

¹H NMR(300MHz,CDCl₃)δ10.02(d,J＝0.6Hz,1H),7.94-7.87(d,J＝ 8.3Hz,2H),7.55-7.49(d,J＝8.3Hz,2H),4.04-3.94(m,1H),3.64-3.45(m,3H), 3.35(s,3H),3.25-3.09(m,1H),2.64-2.44(m,1H),2.02-1.90(m,1H),1.83-1.65 (m,2H)；LC-MS(ESI):m/z 248.2[M+H]⁺。

Process for the preparation of 4- (1-hydroxy-propyn-2-yl) -phenyl- (4-methoxy-piperidin-1-yl) -methylketone (compound 28)

Starting from compound 27(320 mg, 1.3mmol) and ethynylmagnesium bromide in THF (0.5M, 4mL), the procedure for the preparation of compound 26 gave 210 mg of an oil in 59% yield.

¹H NMR(300MHz,CDCl₃)δ7.61-7.55(m,2H),7.43-7.38(m,2H),5.49(dd, J＝6.1,2.3Hz,1H),4.06-4.02(m,1H),3.65-3.45(m,4H),3.36(s,3H),3.25- 3.18(m,1H),2.68(d,J＝2.2Hz,1H),2.47(d,J＝6.1Hz,1H),1.81-1.61(m,3H)； LC-MS(ESI):m/z 274.2[M+H]⁺。

Preparation method of 2- [4- (4-methoxypiperidin-1-yl-formyl) -phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-formamide (compound I-22)

Compound 28(210 mg, 0.8mmol), compound 6(150 mg, 0.5mmol), palladium acetate (8 mg), CuI (1 mg) and DBU (380 mg) were used as starting materials to prepare compound I-21 in the same manner as described above to give 40 mg of a yellow solid in 10% two-step overall yield.

¹H NMR(300MHz,DMSO-d₆)δ8.25(d,J＝9.5Hz,1H),8.21(drs,1H),8.08 (d,J＝8.0Hz,2H),7.96(drs,1H),7.65-7.57(m,2H),7.53(d,J＝15.8Hz,2H), 3.99-3.84(m,1H),3.50-3.38(m,2H),3.35-3.29(m,2H),3.25(s,3H),1.87(m, 2H),1.47(m,2H)；LC-MS(ESI):m/z 428.2[M+H]⁺。

Process for producing 2-fluoro-4- (4-methoxypiperidin-1-yl) -formylbenzaldehyde (compound 29)

2-fluoro-4-formyl-benzoic acid (370 mg, 2.2mmol), 4-methoxypiperidine hydrochloride (300 mg, 2mmol) as starting material gave 360 mg of a white solid in 50% yield according to the preparation of compound 25.

¹H NMR(300MHz,CDCl₃)δ10.37(d,J＝0.7Hz,1H),7.92(dd,J＝7.9,6.9 Hz,1H),7.29(m,2H),4.00-3.89(m,1H),3.65-3.45(m,3H),3.37(s,3H),3.25- 3.18(m,2H),1.98-1.88(m,1H),1.82-1.68(m,2H)；LC-MS(ESI):m/z 266.2 [M+H]⁺。

Preparation method of 3-fluoro-4- (1-hydroxy-propyn-2-yl) -phenyl- (4-methoxypiperidin-1-yl) -methyl ketone (compound 30)

Compound 29(360 mg, 1.4mmol) and ethynylmagnesium bromide in THF (0.5M, 2.5 mL) were used as starting materials according to the preparation of compound 26 to give 330 mg of a yellow oil in 80% yield.

¹H NMR(300MHz,CDCl₃)δ7.69(t,J＝7.6Hz,1H),7.19(dd,J＝7.7,1.5Hz, 1H),7.11(dd,J＝10.1,1.5Hz,1H),5.74(d,J＝3.8Hz,1H),4.04-3.84(m,1H), 3.60-3.42(m,3H),3.36(s,3H),3.28-3.18(m,2H),2.74(d,J＝5.8Hz,1H),2.66 (d,J＝2.2Hz,1H),1.91(d,J＝11.1Hz,3H)；LC-MS(ESI):m/z 314.2[M+H]⁺。

Preparation method of 2- [4- (4-methoxypiperidin-1-yl) -formyl-2-fluoro-phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-23)

Compound 30(330 mg, 1.2mmol), compound 6(180 mg, 0.6mmol) were used as starting materials according to the preparation method of compound I-21 to give 44 mg of a yellow solid in 17% yield over two steps.

¹H NMR(300MHz,DMSO-d₆)δ8.27(d,J＝9.4Hz,1H),8.17(s,1H),8.01- 7.92(m,2H),7.54(d,J＝1.1Hz,1H),7.49(dd,J＝11.5,1.5Hz,1H),7.45-7.37(m, 2H),3.96-3.86(m,1H),3.50-3.40(m,2H),3.25(s,3H),3.23-3.12(m,2H),1.95- 1.74(m,2H),1.45(d,J＝16.3Hz,2H)；LC-MS(ESI):m/z 431.2[M+H]⁺。

Process for producing 2-fluoro-4- (3-fluoro-4-methoxypiperidin-1-yl) formylbenzaldehyde (compound 31)

2-fluoro-4-formyl-benzoic acid (370 mg, 2.2mmol) and 3-fluoro-4-methoxypiperidine (340 mg, 2mmol) as starting materials were prepared according to the procedure for the preparation of compound 25 to give 320 mg of white solid in 56% yield.

¹H NMR(300MHz,CDCl₃)δ10.38(d,J＝0.8Hz,1H),7.93(dd,J＝7.9,6.8 Hz,1H),7.34-7.20(m,2H),4.78-4.60(m,1H),4.50-4.32(m,1H),3.62-3.50(m, 1H),3.46(s,3H),3.20-3.05(m,2H),1.98-1.90(m,2H),1.78-1.68(m,1H)； LC-MS(ESI):m/z 284.2[M+H]⁺。

Preparation method of 3-fluoro-4- (1-hydroxy-propyn-2-yl) -phenyl- (3-fluoro-4-methoxypiperidine-1-yl) methyl ketone (compound 32)

Compound 31(320 mg, 1.1mmol) and a THF solution of ethynylmagnesium bromide (0.5M, 4.2 mmol) were used as starting materials according to the preparation of compound 26 to give 190 mg of a yellow oil in 56% yield.

¹H NMR(300MHz,CDCl₃)δ7.72(t,J＝7.5Hz,1H),7.16(d,J＝10.1Hz,2H), 5.76-5.73(m,1H),4.78-4.62(m,1H),4.45-4.32(m,1H)，4.02-3.93(m,1H), 3.62-3.54(m,1H),3.45(s,3H),3.19-3.08(m,2H),2.67(d,J＝2.2Hz,1H),2.57- 2.53(m,1H),1.96-1.90(m,1H)；LC-MS(ESI):m/z 332.2[M+H]⁺。

Preparation method of 2- [ 2-fluoro-4- (3-fluoro-4-methoxy-piperidin-1-yl) -formylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-24)

Compound 32(190 mg, 0.6mmol), Compound 6(115 mg, 0.38mmol), Pd (OAc)₂Starting from (5 mg), CuI (1 mg) and DBU (300 mg), according to the preparation of compound I-21, 20mg of a yellow solid was obtained in a total yield of 12% over two steps.

¹H NMR(300MHz,DMSO-d₆)δ8.28(d,J＝9.4Hz,1H),8.17(d,J＝3.0Hz, 1H),8.03-7.92(m,2H),7.55(s,1H),7.50-7.35(m,3H),5.09-4.74(m,2H),4.41 (d,J＝20.0Hz,1H),3.83(d,J＝14.4Hz,1H),3.55(s,3H),2.98(dd,J＝10.0,5.0 Hz,1H),1.90-1.82(m,1H),1.69(d,J＝17.1Hz,2H)；LC-MS(ESI):m/z 449.2 [M+H]⁺。

Preparation method of 2-fluoro-4- (3-methoxy pyrroline-1-yl) formyl benzaldehyde (compound 33)

3-fluoro-4-formylbenzoic acid (370 mg, 2.2mmol) and 3-methoxypyrroline (274 mg, 2mmol, see U.S. Pat. No. 3, 2011092475, 1) were used as starting materials to obtain 240 mg of a white solid in 48% yield according to the method for producing compound 25.

¹H NMR(300MHz,CDCl₃)δ10.37(d,J＝0.9Hz,1H),7.91(t,J＝7.4Hz,1H), 7.44-7.29(m,2H),4.00(d,J＝28.9Hz,1H),3.88-3.68(m,2H),3.68-3.39(m, 2H),3.32(d,J＝27.5Hz,3H),2.20-1.82(m,2H)；LC-MS(ESI):m/z 252.2 [M+H]⁺。

Preparation method of 3-fluoro-4- (1-hydroxy-propyn-2-yl) -phenyl- (3-methoxy-pyrrolidine-1-yl) methyl ketone (compound 34)

Compound 33(240 mg, 1mmol) and ethynylmagnesium bromide in THF (0.5M, 3mL) were used as starting materials according to the preparation of compound 26 to give 180 mg of an oil in 65% yield.

1H NMR(300MHz,CDCl₃)δ7.68(t,J＝7.6Hz,1H),7.39-7.10(m,2H), 5.78-5.58(m,1H),4.06-3.88(m,1H),3.72(dd,J＝8.0,5.2Hz,2H),3.68-3.39 (m,2H),3.31(d,J＝29.2Hz,3H),3.18(t,J＝5.4Hz,1H),2.64(d,J＝2.3Hz,1H), 2.18-1.82(m,2H)；LC-MS(ESI):m/z 300.2[M+Na]⁺。

Preparation method of 2- [ 2-fluoro-4- (3-methoxypyrrolidin-1-yl) formylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound I-25)

Compound 34(180 mg, 0.65mmol), Compound 6(120 mg, 0.4mmol), Pd (OAc)₂Starting from (5 mg), CuI (1 mg) and DBU (300 mg), according to the preparation of compound I-21, 23 mg of a yellow solid was obtained in a total yield of 14% over two steps.

¹H NMR(500MHz,DMSO-d₆)δ8.31(dd,J＝9.5,1.4Hz,1H),8.20(d,J＝3.7 Hz,1H),8.00(tt,J＝7.9,4.6Hz,2H),7.62(dd,J＝11.1,1.6Hz,1H),7.57(d,J＝ 9.0Hz,2H),7.46(dt,J＝9.6,2.3Hz,1H),4.01(dtt,J＝39.6,4.6,2.2Hz,1H),3.72- 3.46(m,4H),3.24(d,J＝47.8Hz,3H),2.06-1.94(m,2H)；LC-MS(ESI):m/z 417.2[M+H]⁺。

Preparation method of 4- (3-methoxy pyrroline-1-yl) -formyl benzaldehyde (compound 35)

Starting from 4-formylbenzoic acid (330 mg, 2.2mmol) and 3-methoxypyrrolidine (274 mg, 2mmol), reference was made to the preparation of compound 25 to give 320 mg of a white solid in 69% yield.

LC-MS(ESI):m/z 234.2[M+H]⁺。

Preparation method of 4- (1-hydroxy-propynyl-2-yl) -phenyl- (3-methoxy pyrroline-1-yl) methyl ketone (compound 36)

Compound 35(320 mg, 1.4mmol) and a THF solution of ethynylmagnesium bromide (0.5M, 5mL) were used as starting materials in accordance with the preparation of compound 26 to give a yellow oil in yield.

¹H NMR(300MHz,CDCl₃)δ7.72-7.32(m,4H),5.48(d,J＝4.7Hz,1H), 3.97(d,J＝35.4Hz,1H),3.76-3.70(m,2H),3.64-3.40(m,2H),3.31(d,J＝32.4 Hz,3H),2.74(d,J＝6.1Hz,1H),2.67(d,J＝2.1Hz,1H),2.13-1.97(m,2H)； LC-MS(ESI):m/z 282.2[M+Na]⁺。

Preparation method of 2- [4- (3-methoxy pyrroline-1-yl) -formylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-formamide (compound I-26)

Compound 36(200 mg, 0.77mmol), compound 6(128 mg, 0.43mmol), Pd (OAc)₂(6 mg), CuI (1 mg) and DBU (327 mg) as starting materials, according to the preparation of compound I-21, 28 mg of a yellow solid was obtained in a total yield of 17% over two steps.

¹H NMR(500MHz,DMSO-d₆)δ8.28(dd,J＝9.4,1.7Hz,1H),8.24(s,1H), 8.11(dd,J＝8.1,5.9Hz,2H),8.02(s,1H),7.72(dd,J＝8.3,2.1Hz,2H),7.65(dd,J ＝9.5,3.5Hz,1H),7.54(s,1H),4.00(dtt,J＝43.0,4.6,2.2Hz,1H),3.68-3.45(m, 4H),3.23(d,J＝54.9Hz,3H),2.06-1.91(m,2H)；LC-MS(ESI):m/z 399.2[M+H]⁺。

Preparation method of 3-methyl-4- (3-fluoro-4-formylbenzene) -formyl-piperazine-1-yl-tert-butyl formate (compound 37)

3-fluoro-4-formylbenzoic acid (370 mg, 2.2mmol) and tert-butyl 3-methylpiperazine-1-carboxylate (400 mg, 2mmol) were used as starting materials, according to the preparation method of compound 25, to give 470 mg of a white solid in a yield of 67%.

¹H NMR(300MHz,CDCl₃)δ10.38(s,1H),7.94(t,J＝7.6Hz,1H),7.39-7.03 (m,2H),4.15-4.10(m,1H),3.95-3.85(m,2H),3.24-2.85(m,4H),1.47(s,9H), 1.27(d,J＝6.7Hz,3H)；LC-MS(ESI):m/z 373.2[M+Na]⁺。

Preparation method of 3-fluoro-4- (1-hydroxy-propyn-2-yl) phenyl- (2-methyl-4-tert-butyloxycarbonyl-piperazin-1-yl) methylketone (compound 38)

Starting from compound 37(470 mg, 1.4mmol) and ethynylmagnesium bromide in THF (0.5M, 5mL), reference was made to the preparation of compound 26 to give 350 mg of a yellow oil in 66% yield.

¹H NMR(300MHz,CDCl₃)δ7.72(t,J＝7.7Hz,1H),7.21-7.01(m,2H),5.74 (d,J＝5.5Hz,1H),4.05-3.84(m,3H),3.25-2.80(m,4H),2.73-2.69(m,1H), 2.67(d,J＝2.3Hz,1H),1.47(s,9H),1.26-1.21(m,3H)；LC-MS(ESI):m/z 399.2 [M+Na]⁺。

Preparation method of 2- [ 2-fluoro-4- (2-methyl-4-tert-butoxyformyl-piperazin-1-yl) -formylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxylic acid methyl ester (compound 39)

Compound 38(350 mg, 0.93mmol), compound 6(155 mg, 0.52mmol), pd (oac)2 (7 mg), CuI (1 mg) and DBU (450 mg) were starting materials according to the preparation of compound 15 to give 63 mg of a yellow solid in 23% yield.

LC-MS(ESI):m/z 553.2[M+Na]⁺。

Preparation method of 5-chloro-2- [ 2-fluoro-4- (2-methyl-4-cyclopropylformyl-piperazin-1-yl) -formylphenyl ] pyrrolo [1,2-b ] pyridazine 7-carboxamide (compound I-27)

After the reaction was completed by thin layer chromatography by adding 1.5mL of trifluoroacetic acid to 10mL of compound 39(65 mg, 0.13mmol) in dichloromethane, adjusting the pH to weak basicity (8 to 9) with a NaOH (2.5M) solution in ice bath, separating an organic phase, extracting an aqueous layer with chloroform, combining the organic phases, concentrating, and eluting with a column chromatography (dichloromethane: methanol ═ 10:1) to obtain 52 mg of a yellow solid which is (methyl 5-chloro-2- [ 2-fluoro-4- (2-methyl-piperazin-1-yl) -formylphenyl ] pyrrolo [1,2-b ] pyridazine-7-carboxylate); dissolving it in 10mL of anhydrous dichloromethane, adding a few drops of triethylamine and cyclopropylcarbonyl chloride (300 mg), stirring at room temperature for 1 hour, adding 5mL of methanol, stirring at room temperature for 10 minutes, and concentrating to give crude methyl 5-chloro-2- [ 2-fluoro-4- (2-methyl-4-cyclopropylformyl-piperazin-1-yl) -formylphenyl ] pyrrolo [1,2-b ] pyridazine 7-carboxylate; transferring 10mL of methanol into a 25mL sealed tube, adding 8mL of ammonia methanol solution, reacting at 80-90 ℃ for 20-24 hours, cooling to room temperature, concentrating, and eluting by column chromatography (dichloromethane: methanol 20:1) to obtain 40 mg of yellow solid, wherein the total yield of the three steps is 63%.

¹H NMR(300MHz,DMSO-d₆)δ8.35-8.23(m,1H),8.18(d,J＝2.9Hz,1H), 8.03-7.93(m,1H),7.55(d,J＝1.3Hz,1H),7.53-7.37(m,3H),6.78-6.68(m,1H) 4.78-4.05(m,3H),3.52-2.70(m,4H),1.95(dd,J＝20.7,9.5Hz,1H),1.35-1.03 (m,3H),0.81-0.67(m,2H),0.64-0.56(m,2H)；LC-MS(ESI):m/z 484.2[M+H]⁺。

Preparation method of 2-methyl-4- (3-fluoro-4-formylbenzene) -formyl-piperazine-1-tert-butyl formate (compound 40)

3-fluoro-4-formylbenzoic acid (370 mg, 2.2mmol) and tert-butyl 2-methylpiperazine-1-carboxylate (400 mg, 2mmol) were used as starting materials, according to the preparation method of compound 25, to give 420 mg of a white solid in a yield of 60%.

¹H NMR(300MHz,CDCl₃)δ10.38(s,1H),7.98-7.84(m,1H),7.33-7.05(m, 2H),4.62-4.21(m,1H),3.98-3.82(m,2H),3.35-2.85(m,4H),1.47(d,J＝1.2Hz, 9H),1.30-1.01(m,3H)；；LC-MS(ESI):m/z 373.2[M+Na]⁺。

Preparation method of 3-fluoro-4- (1-hydroxy-propyn-2-yl) -phenyl- (3-methyl-4-tert-butyloxycarbonyl-piperazin-1-yl) methyl ketone (compound 41)

Starting from compound 40(420 mg, 1.2mmol) and ethynylmagnesium bromide in THF (0.5M, 4.3 mL), reference was made to the preparation of compound 26 to give 365 mg of a yellow oil in 81% yield.

¹H NMR(300MHz,CDCl₃)δ7.72(t,J＝7.5Hz,1H),7.17(dd,J＝22.3,8.9 Hz,2H),5.75(d,J＝5.0Hz,1H),4.61-4.18(m,2H),3.94-3.80(m,1H),3.50- 2.89(m,4H),2.70-2.66(m,1H),2.69-2.63(m,1H),1.46(s,9H),1.11(d,J＝ 6.3Hz,3H)；LC-MS(ESI):m/z 399.2[M+Na]⁺。

Preparation method of 2- [ 2-fluoro-4- (3-methyl-4-tert-butoxyformyl-piperazin-1-yl) -formylphenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxylic acid methyl ester (compound 42)

Compound 41(364 mg, 0.97mmol), Compound 6(160 mg, 0.54mmol), Pd (OAc)₂Starting from (11 mg), CuI (1 mg) and DBU (410 mg), reference was made to the preparation of compound 15 to give 146 mg of crude yellow solid in 52% yield.

¹H NMR(300MHz,CDCl₃)δ8.14(t,J＝7.7Hz,1H),7.97(d,J＝9.5Hz,1H), 7.52(s,1H),7.40-7.27(m,3H),4.59-4.22(m,2H),3.94(s,3H),3.42-3.33(m, 1H),3.18-2.90(m,4H),1.47(s,9H),1.21-1.08(d,3H)；LC-MS(ESI):m/z 538.2 [M+Na]⁺。

Preparation method of 5-chloro-2- [ 2-fluoro-4- (3-methyl-4-cyclopropylformyl-piperazin-1-yl) -formylphenyl ] pyrrolo [1,2-b ] pyridazine 7-carboxamide (compound I-28)

Compound 42(80 mg, 0.16mmol) was used as a starting material in reference to the preparation of compound I-27 to give 45 mg of a yellow solid in 61% yield.

¹H NMR(300MHz,DMSO-d₆)δ8.30(dd,J＝9.5,0.8Hz,1H),8.19(s,1H), 8.01(dd,J＝10.4,5.3Hz,2H),7.62-7.39(m,4H),4.85-3.95(m,4H),3.79-2.79 (m,3H),1.52-1.43(tt,J＝7.4,5.1Hz,1H),1.34-1.07(m,3H),0.84-0.67(m, 4H)；LC-MS(ESI):m/z 484.2[M+H]⁺。

Preparation method of 2- (4-bromophenyl) malonic acid dimethyl ester (compound 43)

Sodium thread (4.6 g, 0.2mol) was put into 100mL of anhydrous methanol under ice bath, the temperature was controlled to prevent excessive heat generation, and after all Na was dissolved, the solvent was removed under vacuum to obtain sodium methoxide solid. Then, methyl 4-bromobenzoate (22 g; 0.1mol) in 100mL of anhydrous THF was added and stirred at room temperature for 25 minutes, dimethyl carbonate (42mL, 0.5mol) was added and stirred at room temperature for 48 hours, 2/3 volumes of solvent were removed under reduced pressure, then water and ethyl acetate were added, the mixture was sonicated to dissolve all the solids in the mixture, the organic phase was separated, the aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with a saturated sodium chloride solution, concentrated and eluted by column chromatography (petroleum ether: ethyl acetate 50:1) to give 20 g of a white solid with a yield of 70%.

¹H NMR(300MHz,CDCl₃)δ7.50(d,J＝8.5Hz,2H),7.28(d,J＝8.5Hz,2H), 4.60(s,1H),3.76(s,6H)；LC-MS(ESI):m/z 287.2[M+H]⁺,289.2[M+3H]⁺。

Preparation method of 1- (4-bromophenyl) -propane-1, 1, 3-trimethyl acid trimethyl ester (compound 44)

Under an ice bath, Na filaments (370 mg, 16mmol) were added to 100mL of anhydrous methanol, and after all were dissolved, dimethyl 2- (4-bromophenyl) malonate (10 g, 35mmol) was added, and the mixture was stirred at room temperature for 30 minutes, followed by addition of methyl acrylate (8mL), stirring at room temperature for 24 hours, and direct silica gel column chromatography (petroleum ether: ethyl acetate ═ 100:1) was added to elute 9 g of a white solid with a yield of 70%.

¹H NMR(300MHz,CDCl₃)δ7.51-7.42(d,J＝8.7Hz,2H),7.24(d,J＝8.7 Hz,2H),3.75(s,6H),3.64(s,3H),2.67-2.54(m,2H),2.32-2.25(m,2H)；LC-MS (ESI):m/z 373.2[M+H]⁺,375.2[M+3H]⁺。

Preparation method of 2- (4-bromophenyl) penta-1, 5-diacid (compound 45)

Compound 44(5.2 g, 14mmol) was added to a 250mL round bottom flask, potassium hydroxide solid (3.8 g, 67.2mmol) and 100mL water were added, stirred at 110 ℃ for 3 hours, cooled to room temperature, then concentrated sulfuric acid 10mL was added, stirred at 110 ℃ for 9 hours, cooled to room temperature, extracted 2 times with ethyl acetate, the organic phases were combined, concentrated, and eluted by column chromatography (chloroform: methanol 50:1) to give 3.6 g of oil in 90% yield.

H-NMR(300MHz,CDCl₃)δ7.47(d,J＝8.5Hz,2H),7.18(d,J＝8.5Hz,2H), 3.63(t,J＝7.5Hz,1H),2.42-2.29(m,3H),2.13-2.03(m,1H)；LC-MS(ESI):m/z 286.2[M+H]⁺,288.2[M+3H]⁺。

Preparation method of 2- (4-bromophenyl) penta-1, 5-dihydroxy-bis (4-tosylate) (compound 46)

Compound 45(3.6 g, 12.6mmol) in anhydrous THF80mL, BH was added slowly via an isobaric dropping funnel₃50mL of THF solution (1M, purchased from Shanghai Dairy chemical company), which is added in about 25 minutes, is stirred at room temperature for 1 hour, the methanol solution is slowly added dropwise until no bubbles are generated, the stirring is continued for 30 minutes, the solvent is removed under vacuum reduced pressure, 100mL of water and 100mL of ethyl acetate are added respectively, an organic phase is separated, a water layer is extracted by ethyl acetate, the organic phase is combined, the organic phase is washed by saturated saline solution, dried by anhydrous sodium sulfate, and concentrated to obtain 3.05 g of oily matter, which is directly put into the next step with the yield of 95 percent; to the crude product was added 100mL of dichloromethane and 5mL of pyridine, and after all of them were dissolved, 4-toluenesulfonyl chloride (7.5 g, 40 mmol) was added in two portions, and the mixture was stirred at room temperature overnight, 10mL of 3N HCl solution was added, and after stirring for 10 minutes, 50mL of water was added, and then the organic phase was separated, and the aqueous layer was extracted with dichloromethane, and the organic phases were combined, concentrated, and eluted by column chromatography (petroleum ether: ethyl acetate: 8:1) to give 2.8 g of a colorless oil, with a total yield of 64% in two steps.

H-NMR(300MHz,CDCl₃)δ7.74(d,J＝8.1Hz,2H),7.59(d,J＝8.3Hz,2H), 7.33(dd,J＝8.4,1.8Hz,4H),7.28(d,J＝8.1Hz,2H),6.86(d,J＝8.4Hz,2H),4.04 -3.96(m,1H),3.91(t,J＝5.9Hz,2H),2.85-2.73(m,1H),2.45(s,6H),1.83-1.68 (m,1H),1.54-1.38(m,3H)；LC-MS(ESI):m/z 567.2[M+H]⁺,569.2[M+3H]⁺。

Preparation method of 3- (4-bromophenyl) -1- (4-methoxybenzyl) -piperidine (compound 47)

To 100mL of compound 46(1.85 g, 3.2mmol) in 1, 4-dioxane was added 4-methoxybenzylamine (700 mg, 5.1mmol), stirred at 110 ℃ for 48 hours, cooled to room temperature, concentrated, and eluted by column chromatography (petroleum ether: ethyl acetate 25:1) to give 1.6 g of a yellow solid with a yield of 89%.

¹H NMR(300MHz,CDCl₃)δ7.42-7.36(m,2H),7.25-7.18(m,2H),7.08(d, J＝8.4Hz,2H),6.87-6.80(m,2H),3.79(s,3H),3.47(s,2H),2.96-2.85(m,2H), 2.78(tt,J＝11.4,3.7Hz,1H),1.92-1.84(m,2H),1.79-1.63(m,3H),1.39(qd,J＝ 12.0,4.9Hz,1H)；LC-MS(ESI):m/z 360.2[M+H]⁺,362.2[M+3H]⁺。

Preparation method of 3- (4-bromophenyl) piperidine (compound 48)

To acetonitrile 50mL of compound 47(1.8 g, 5mmol), an aqueous solution (20mL) of ceric ammonium nitrate (11 g, 20mmol) was added, the mixture was stirred at room temperature overnight, half of the volume of the solvent was removed under vacuum and reduced pressure, ethyl acetate was added for extraction, the organic phase was separated, washed with a saturated sodium chloride solution, concentrated, and eluted by column chromatography (dichloromethane: methanol 10:1) to give 1 g of a colorless liquid with a yield of 89%.

¹H NMR(300MHz,CDCl₃)δ7.42(d,J＝8.3Hz,2H),7.11(d,J＝8.3Hz,2H), 3.68(t,J＝11.0Hz,2H),3.31(dd,J＝1.9,1.3Hz,1H),3.16-2.98(m,3H),2.06(d, J＝4.2Hz,3H)；LC-MS(ESI):m/z 240.2[M+H]⁺,242.2[M+3H]⁺。

Preparation method of 3- (4-bromophenyl) piperidine-1-carboxylic acid tert-butyl ester (compound 49)

To 50mL of compound 48(600 mg, 2.5mmol) in dichloromethane was added 1mL of pyridine and a solution of boc anhydride (820 mg, 3.8mmol) in dichloromethane (10mL), stirred at room temperature for 3 hours, 10mL of 3N HCl solution was added, stirring was continued for 10 minutes, the organic phase was separated, the aqueous layer was extracted with dichloromethane, the organic phases were combined, washed with saturated sodium chloride solution, concentrated, and eluted by column chromatography (petroleum ether: ethyl acetate: 80: 1) to give 460 mg of a white solid in 54% yield.

¹H NMR(400MHz,CDCl₃)δ7.46-7.39(d,J＝8.3Hz,2H),7.13-7.06(d,J＝ 8.3Hz,2H),4.13(s,2H),2.68(dd,J＝28.8,11.4Hz,3H),2.04-1.90(m,1H),1.81- 1.68(m,1H),1.60-1.52(m,2H),1.46(s,9H)；LC-MS(ESI):m/z 340.2[M+H]⁺。

Preparation method of 3- (4-formylphenyl) -piperidine-1-carboxylic acid tert-butyl ester (compound 50)

A predried 250mL three-necked flask was charged with 50mL of compound 49(500 mg, 1.5mmol) in dry THF, Ar₂Under protection, the mixture was cooled to-78 ℃, n-BuLi (2.5M, 1mL) was slowly added dropwise, the mixture was kept at the temperature and stirred for 30 minutes, then anhydrous DMF5mL was added, the mixture was stirred for 2 hours, then 10mL of a saturated ammonium chloride solution was added, the mixture was transferred to room temperature and stirred for 30 minutes after stirring for 10 minutes, an organic phase was separated, an aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, concentrated, and eluted by column chromatography (petroleum ether: ethyl acetate 10:1) to obtain 250 mg of a yellow oily substance, and the yield was 58%.

¹H NMR(300MHz,CDCl₃)δ9.98(s,1H),7.86-7.80(m,2H),7.43-7.37(m, 2H),4.24-4.08(m,2H),2.75(d,J＝13.3Hz,3H),2.09-1.97(m,1H),1.82-1.72 (m,1H),1.66-1.58(s,2H),1.47(s,9H)；LC-MS(ESI):(m/z)290.2[M+H]⁺。

Preparation method of 3- [4- (1-hydroxy-propynyl-2-yl) phenyl ] -piperidine-1-carboxylic acid tert-butyl ester (compound 51)

To 50mL of an anhydrous THF solution of compound 50(280 mg, 1mmol), a THF solution of ethynylmagnesium bromide (0.5M, 4mL) was added, the mixture was stirred at room temperature for 1 hour, 20mL of a saturated ammonium chloride solution was added, the mixture was further stirred for 20 minutes, then, an organic phase was separated, an aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with a saturated sodium chloride solution, concentrated, and eluted by column chromatography (petroleum ether: ethyl acetate 4: 1) to give 200 mg of a white solid with a yield of 63%.

¹H NMR(300MHz,CDCl₃)δ7.50(d,J＝8.1Hz,2H),7.30-7.21(d,J＝8.1 Hz,2H),5.45(dd,J＝6.2,2.2Hz,1H),4.12(dd,J＝15.3,9.0Hz,2H),2.84-2.54 (m,4H),2.30(d,J＝6.2Hz,1H),2.04-1.94(m,1H),1.75(dt,J＝8.7,2.8Hz,1H), 1.58(d,J＝3.3Hz,1H),1.46(s,9H)；LC-MS(ESI):m/z 338.2[M+Na]⁺。

Preparation method of methyl 5-chloro-2- [4- (1-tert-butoxyformylpiperidin-3-yl) phenyl ] pyrrolo [1,2-b ] pyridazine-7-carboxylate (compound 52)

A25 mL microwave tube with a microwave cap was charged with compound 51(200 mg, 0.63mmol), compound 6(120 mg, 0.4mmol), palladium acetate (5 mg), cuprous iodide (1 mg) and 10mL of toluene, N₂After 3 times of replacement, N₂DBU (300 mg) was added under protection, the microwave cap was closed, the reaction was carried out under microwave (λ ═ 180W) at 90 ℃ for 40 min, after cooling to room temperature, chloroform was diluted and transferred to a 100ml round bottom flask, concentrated and eluted by column chromatography (petroleum ether: ethyl acetate ═ 7:1) to give 146 mg of yellow solid in 78% yield.

¹H NMR(300MHz,CDCl₃)δ8.01(d,J＝8.0Hz,2H),7.93(dd,J＝9.4,0.8Hz, 1H),7.46(d,J＝0.9Hz,1H),7.35(ddd,J＝10.4,8.9,1.1Hz,3H),4.15(d,J＝14.0 Hz,3H),3.95(s,3H),2.84-2.70(m,4H),2.08-1.98(m,1H),1.82-1.74(m,1H), 1.47(s,9H)；LC-MS(ESI):m/z 492.2[M+Na]⁺。

Preparation method of 5-chloro-2- [4- (1-tert-butoxyformylpiperidin-3-yl) phenyl ] pyrrolo [1,2-b ] pyridazine-7-carboxamide (compound 53)

Compound 52(145 mg, 0.31mmol) in 20ml of methanol was added to a 50ml closed tube, 15ml of ammonia solution in methanol was added, the tube was sealed at 90 ℃ for 20 hours, cooled to room temperature, concentrated, and eluted by column chromatography (dichloromethane: methanol 50:1) to give 110 mg of a yellow solid in 78% yield.

¹H NMR(400MHz,CDCl₃)δ8.80(d,J＝3.7Hz,1H),8.00(d,J＝9.4Hz,1H), 7.85-7.74(m,2H),7.63(d,J＝0.5Hz,1H),7.41(d,J＝8.3Hz,2H),7.23(d,J＝ 9.4Hz,1H),5.97(s,1H),4.17(s,2H),2.91-2.63(m,3H),2.07(d,J＝12.1Hz,1H), 1.84-1.76(m,1H),1.76-1.53(m,2H),1.48(s,9H)；LC-MS(ESI):m/z 477.2 [M+H]⁺。

Process for the preparation of 2- (4- [ (3S) -tert-butoxyformylpiperidin-3-yl ] -phenyl) -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (Compound 53-S)

Chiral separation of racemate 53 by chiral SFC purification, CO₂As supercritical fluid, chiral column: chiralcel OJ-H, 0.46cm i.d. x 25cm L, flow rate: 2.5mL/min, detection wavelength: UV 254 nm, column temperature: 35 ℃, mobile phase: CO 2₂: MeOH: DEA 60: 40: 0.1. the retention time of the first enantiomer was 8.68min, and the solvent was removed under reduced pressure to give a yellow solid: 2- (4- [ (3R) -tert-Butoxycarbonylpiperidin-3-yl)]-phenyl) -5-chloro-pyrrolo [1,2-b]Pyridazine-7-carboxamide (Compound 53-R, 99.9% ee). The second enantiomer had a retention time of 9.07min and the solvent was removed under reduced pressure to give a yellow solid: 2- [4- [ (3S) -tert-Butoxycarbonylpiperidin-3-yl ] amine]-phenyl radical]-5-chloro-pyrrolo [1,2-b]Pyridazine-7-carboxamide (Compound 53-S, 98% ee).

-90.4，¹H NMR(400MHz,CDCl₃)δ8.80(d,J＝3.7Hz,1H),8.00(d,J＝ 9.4Hz,1H),7.85-7.74(m,2H),7.63(d,J＝0.5Hz,1H),7.41(d,J＝8.3Hz,2H), 7.23(d,J＝9.4Hz,1H),5.97(s,1H),4.17(s,2H),2.91-2.63(m,3H)2.07(d,J＝ 12.1Hz,1H),1.84-1.76(m,1H),1.76-1.53(m,2H),1.48(s,9H)；LC-MS(ESI): m/z 477.2[M+Na]⁺. Without deletion

Process for the preparation of 2- [4- [ (3R) -tert-butoxyformylpiperidin-3-yl ] -phenyl ] -5-chloro-pyrrolo [1,2-b ] pyridazine-7-carboxamide (Compound 53-R)

93.4(c 0.004,CH₃OH),H NMR(400MHz,CDCl₃)δ8.80(d,J＝3.7Hz, 1H),8.00(d,J＝9.4Hz,1H),7.85-7.74(m,2H),7.63(d,J＝0.5Hz,1H),7.41(d,J ＝8.3Hz,2H),7.23(d,J＝9.4Hz,1H),5.97(s,1H),4.17(s,2H),2.91-2.63(m, 3H)2.07(d,J＝12.1Hz,1H),1.84-1.76(m,1H),1.76-1.53(m,2H),1.48(s,9H)； LC-MS(ESI):m/z 477.2[M+Na]⁺。

Preparation method of 5-chloro-2- [4- (piperidin-3-yl) phenyl ] pyrrole [1,2-b ] pyridazine-7-formamide (compound I-29)

To 10mL of chloroform containing compound 53(50 mg, 0.11mmol), trifluoroacetic acid (1.8 mL) was added, and the mixture was stirred at room temperature for 1 hour, then pH was adjusted to weak alkalinity (8 to 9) with NaOH solution (2.5M) in ice bath, an organic phase was separated, an aqueous layer was extracted with chloroform, the organic phases were combined, washed with saturated brine, concentrated, and eluted by column chromatography (dichloromethane: methanol 10:1, 1L eluent with 5mL of ammonia in methanol) to give 35 mg of yellow solid with a yield of 90%.

H-NMR；LC-MS(ESI):m/z 355.2[M+H]⁺。

Preparation method of 5-chloro-2- [4- ((3S) -piperidin-3-yl) phenyl ] pyrrole [1,2-b ] pyridazine-7-formamide (compound I-30)

Compound 53-S (50 mg, 0.11mmol) was used as a starting material in accordance with the preparation method of compound I-29 to obtain 35 mg of a yellow solid in a yield of 89%.

¹H NMR(400MHz,CDCl₃)δ8.79(d,J＝3.8Hz,1H),7.98(d,J＝9.4Hz,1H), 7.80(d,J＝8.2Hz,2H),7.61(s,1H),7.39(d,J＝8.3Hz,2H),7.21(d,J＝9.5Hz, 1H),6.16(d,J＝3.8Hz,1H),3.32-3.17(m,2H),2.93-2.85(m,1H),2.81-2.68(m, 2H),2.12-1.84(m,2H),1.81-1.63(m,2H)；LC-MS(ESI):m/z 355.2[M+H]⁺。

Preparation method of 5-chloro-2- [4- ((3R) -piperidine-3-yl) phenyl ] pyrrole [1,2-b ] pyridazine-7-formamide (compound I-31)

Starting from compound 53 to R (50 mg, 0.11mmol), according to the method for the preparation of compound I-31, 34 mg of a yellow solid was obtained in 88% yield.

¹H NMR(400MHz,CDCl₃)δ8.79(d,J＝3.8Hz,1H),7.98(d,J＝9.4Hz,1H), 7.80(d,J＝8.2Hz,2H),7.61(s,1H),7.39(d,J＝8.3Hz,2H),7.21(d,J＝9.5Hz, 1H),6.16(d,J＝3.8Hz,1H),3.32-3.17(m,2H),2.93-2.85(m,1H),2.81-2.68(m, 2H),2.12-1.84(m,2H),1.81-1.63(m,2H)；LC-MS(ESI):m/z 355.2[M+H]⁺。

Preparation method of 6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 54)

4,5,6, 7-tetrahydrothiophene [3,2-c ] pyridine hydrochloride (10 g, 56.9mmol) in dichloromethane 150mL, adding triethylamine 9mL, Boc anhydride (13.7 g, 62.8mmol) in dichloromethane, stirring at room temperature for 1 hour, adding saturated ammonium chloride solution 20mL, stirring for 10 minutes, separating out the organic phase, extracting the aqueous layer with dichloromethane, combining the organic phases, washing with saturated sodium chloride solution, drying with anhydrous sodium sulfate, concentrating to obtain white solid 12.5 g, yield: 92 percent.

¹H NMR(300MHz,CDCl₃)δ7.12(d,J＝5.1Hz,1H),6.78(d,J＝5.1Hz,1H), 4.50(s,2H),3.71(t,J＝5.3Hz,2H),2.84(t,J＝5.3Hz,2H),1.48(s,9H)；LC-MS (ESI):m/z 240.2[M+H]⁺。

Preparation method of 2-formyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 55)

A predried 250mL three-necked flask was charged with 50mL of compound 54(5.3 g, 22.2mmol) in dry THF, Ar₂Under protection, the mixture was cooled to-78 ℃, n-BuLi (1.6M, 22mL) was slowly added dropwise, the mixture was kept at the temperature and stirred for 30 minutes, then 5mL of anhydrous DMF was added, the mixture was stirred for 2 hours, then 10mL of a saturated ammonium chloride solution was added, the mixture was transferred to room temperature and stirred for 30 minutes after stirring for 10 minutes, an organic phase was separated, an aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with a saturated saline solution, concentrated, and eluted by column chromatography (petroleum ether: ethyl acetate 10:1) to obtain 3.8 g of a yellow oily substance with a yield of 64%.

¹H NMR(300MHz,CDCl₃)δ9.83(s,1H),7.47(s,1H),4.53(s,2H),3.74(t,J ＝5.8Hz,2H),2.92(t,J＝5.6Hz,2H),1.49(s,9H)；LC-MS(ESI):m/z 268.2 [M+H]⁺。

Preparation method of 2- (1-hydroxy-propyn-2-yl) -6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 56)

Compound 55(2.3 g, 8.4mmol) in dry THF80mL was added ethynylmagnesium bromide THF (0.5M, 25mL) and stirred at rt for 1 h, after TLC detection reaction was complete, saturated ammonium chloride solution 30mL was added and stirred for 10 min, the organic phase was separated, the aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, concentrated and eluted by column chromatography (petroleum ether: ethyl acetate: 8:1) to give 1.8 g of yellow solid with 72% yield.

H-NMR(300MHz,CDCl₃)δ6.89(d,J＝0.9Hz,1H),5.57(d,J＝2.5Hz,1H), 4.43(t,J＝1.7Hz,2H),3.75-3.65(m,2H),2.80(d,J＝6.0Hz,2H),2.68(d,J＝2.2 Hz,1H),1.48(s,9H).

Preparation method of 2- [ 5-chloro-7-methoxyformyl pyrrole [1,2-b ] pyridazine-2-yl ] -6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -carboxylic acid tert-butyl ester (compound 57)

A25 mL microwave tube with a microwave cap was charged with compound 56(470 mg, 1.6mmol), compound 6(330 mg, 1.1mmol), palladium acetate (15 mg), cuprous iodide (2 mg) and toluene 10mL, N₂After 3 times of replacement, N₂DBU (840 mg) was added under protection, the microwave cap was closed, the reaction was carried out under microwave (λ 180W) at 90 ℃ for 40 min, after cooling to room temperature, chloroform was diluted and transferred to a 100ml round bottom flask, concentrated and eluted by column chromatography (petroleum ether: ethyl acetate: 7:1) to give 135 mg of yellow solid in 28% yield.

¹H NMR(300MHz,CDCl₃)δ7.84(d,J＝9.4Hz,1H),7.41(d,J＝0.6Hz,1H), 7.35(s,1H),7.17(d,J＝9.4Hz,1H),4.55(s,2H),3.95(s,3H),3.75-3.64(m,2H), 2.93(t,J＝5.6Hz,2H),1.49(s,9H)；LC-MS(ESI):m/z 448.2[M+H]⁺。

Preparation method of methyl 5-chloro-2- (4,5,6, 7-tetrahydrothiophene [3,2-c ] pyridin-2-yl) pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 58)

After 2.5mL of trifluoroacetic acid was added to 25mL of chloroform containing compound 57(440 mg, 0.98mmol), and the mixture was stirred at room temperature for 1 hour, TLC detected completely, pH was adjusted to 8 to 9 with NaOH (2.5M) in ice bath, the organic phase was separated, the aqueous layer was extracted with chloroform, the organic phases were combined, washed with saturated sodium chloride solution, concentrated, and eluted by column chromatography (dichloromethane: methanol 50:1) to obtain 250 mg of yellow solid with 74% yield.

LC-MS(ESI):m/z 348.2[M+H]⁺。

Preparation method of methyl 2- (4,5,6, 7-tetrahydrothiophene [3,2-c ] pyridin-2-yl) -5-chloro-pyrrole [1,2-b ] pyridazine-7-carboxylate (compound I-32)

10ml of a methanol solution of compound 58(250 mg) was put into a 25ml closed tube, 8ml of a methanol solution of ammonia was added, the reaction was carried out at 90 ℃ for 20 hours, the reaction solution was cooled to room temperature, 2/3 was removed by spinning off the volume of the reaction solution under reduced pressure, and the precipitated solid was filtered and dried in vacuo to give 180 mg of a yellow solid with a yield of 75%.

¹H NMR(400MHz,DMSO-d₆)δ8.10(dd,J＝24.6,13.5Hz,3H),7.69(s,1H), 7.51(d,J＝9.5Hz,1H),7.41(s,1H),3.85(s,2H),3.06(d,J＝6.0Hz,2H),2.80(d, J＝6.0Hz,2H)；LC-MS(ESI):m/z 333.2[M+H]⁺。

Preparation method of methyl 5-chloro-2- (5-methyl-4, 5,6, 7-tetrahydrothiophene [3,2-c ] pyridine-2-yl) pyrrole [1,2-b ] pyridazine-7-carboxylate (compound I-33)

To 5mL of methanol containing Compound 58(75 mg, 0.21mmol), a few drops of acetic acid and 37% formaldehyde solution (1.5mL) were added, and after stirring at room temperature for 10 minutes, NaCNBH was added₃(50 mg) and stirred at room temperatureStirring overnight, concentrating, and eluting by column chromatography (dichloromethane: methanol 25:1) to give 55 mg of crude product, LC-MS (ESI) M/z 361.2[ M + H ]]⁺. The mixture was transferred to a 25mL lock tube with 10mL of methanol, 8mL of ammonia in methanol was added, the reaction was carried out at 90 ℃ for 20 hours, the reaction solution was cooled to room temperature, 2/3 was removed by rotation under reduced pressure, and the precipitated solid was filtered off and dried under vacuum to obtain 52 mg with a yield of 71%.

¹H NMR(300MHz,CD₃OD)δ7.86(d,J＝9.5,1H),7.42(d,J＝1.2Hz,1H), 7.31(s,1H),7.19(d,J＝9.5,1H),3.55(s,2H),2.94(d,J＝5.8Hz,2H),2.83(t,J＝5.7Hz,2H),2.50(s,3H)；LC-MS(ESI):m/z 347.2[M+H]⁺。

Preparation method of 4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 59)

To 50mL of 4,5,6, 7-tetrahydrothiophene [2,3-c ] pyridine hydrochloride (3 g, 17.1mmol) in dichloromethane was added 2mL of triethylamine, a solution of Boc anhydride (4.1 g, 18.9mmol) in dichloromethane (10mL), stirred at room temperature for 1 hour, 20mL of a saturated ammonium chloride solution was added, after stirring for 10 minutes, the organic phase was separated, the aqueous layer was extracted with dichloromethane, the organic phases were combined, washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to give 4.0 g of an oil in 98% yield.

¹H NMR(300MHz,CDCl₃)δ7.13(d,J＝5.0Hz,1H),6.79(d,J＝5.1Hz,1H), 4.62(s,2H),3.67(t,J＝5.4Hz,2H),2.70(t,J＝5.4Hz,2H),1.8(s,9H)；LC-MS (ESI):m/z 240.2[M+H]⁺。

Preparation method of 2-bromo-4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 60)

NBS (3.3 g, 18.8mmol) was added to 80mL of acetonitrile 59(4.0 g, 16.7mmol), followed by stirring at room temperature for 1 hour, concentration, and column chromatography (petroleum ether: ethyl acetate ═ 100:1) to give 4.8 g of a yellow oil in 89% yield.

LC-MS(ESI):m/z 318.2[M+H]⁺。

Preparation method of 2-formyl-4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 61)

A predried 250mL three-necked flask was charged with 50mL of compound 60(4.8 g, 15.1mmol) in anhydrous THF, Ar₂Under protection, the mixture was cooled to-78 ℃, n-BuLi (1.6M, 17mL) was slowly added dropwise, the mixture was kept at the temperature and stirred for 30 minutes, then anhydrous DMF5mL was added, the mixture was stirred for 2 hours, then saturated ammonium chloride solution 10mL was added, the mixture was transferred to room temperature and stirred for 30 minutes after stirring for 10 minutes, an organic phase was separated, an aqueous layer was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, concentrated, and eluted by column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain 3.1 g of a yellow oily substance, with a yield of 77%.

¹H NMR(300MHz,CDCl₃)δ9.84(d,J＝1.1Hz,1H),7.48(s,1H),4.68(s, 2H),3.69(t,J＝5.6Hz,2H),2.75(t,J＝5.8Hz,2H),1.48(s,9H)；LC-MS(ESI): m/z 268.2[M+H]⁺。

Preparation method of 2- (1-hydroxy-propyn-2-yl) -4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 62)

Starting from compound 61(3.1 g, 11.6mmol) and a THF solution of ethynylmagnesium bromide (0.5M, 42mL), reference was made to the preparation of compound 56 to give 2.8 g of a yellow solid in 82% yield.

¹H NMR(300MHz,CDCl₃)δ6.89(s,1H),5.58(dd,J＝6.4,2.3Hz,1H),4.58 (s,2H),3.65(t,J＝5.7Hz,2H),2.70-2.60(m,2H),2.47(d,J＝6.9Hz,1H),1.48(d, J＝2.5Hz,9H)；LC-MS(ESI):m/z 316.2[M+Na]⁺。

Preparation method of methyl 5-chloro-2- (5-tert-butyloxycarbonyl-4, 5-dihydrothiophene [2,3-c ] pyridine-2-yl) pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 63)

Compound 62(228 mg, 0,78mmol), compound 6(50 mg, 0.16mmol), Pd (OAc)₂(8 mg), CuI (1 mg) and DBU (460 mg) as starting materials, according to the preparation method of compound 57, 20mg of a yellow solid was obtained in 28% yield.

¹H NMR(300MHz,CDCl₃)δ7.84(d,J＝9.4Hz,1H),7.41(d,J＝0.6Hz,1H), 7.35(s,1H),7.17(d,J＝9.4Hz,1H),4.65(s,2H),3.95(s,3H),3.75-3.64(m,2H), 2.73(t,J＝5.6Hz,2H),1.49(s,9H)；LC-MS(ESI):m/z 448.2[M+H]⁺。

Preparation method of methyl 5-chloro-2- (4, 5-dihydrothiophene [2,3-c ] pyridine-2-yl) pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 64)

Compound 63(450 mg, 1mmol) was used as the starting material in reference to the preparation of compound 59 to give 310 mg of a yellow solid in 89% yield.

LC-MS(ESI):m/z 348.2[M+H]⁺。

Preparation method of 5-chloro-2- (4, 5-dihydrothiophene [2,3-c ] pyridine-2-yl) pyrrole [1,2-b ] pyridazine-7-formamide (compound I-34)

Compound 64(310 mg, 0.9mmol) was used as a starting material in reference to the preparation of compound I-32 to give 210 mg of a yellow solid in 70% yield.

¹H NMR(300MHz,CDCl₃)δ7.73(d,J＝9.5Hz,1H),7.25(s,1H),7.20(s, 1H),7.04(d,J＝9.5Hz,1H),3.96(s,2H),3.05(t,J＝5.8Hz 2H),2.64(t,J＝5.8Hz, 2H)；LC-MS(ESI):m/z 333.2[M+H]⁺。

Preparation method of 5-chloro-2- (6-methyl-4, 5-dihydrothiophene [2,3-c ] pyridine-2-yl) pyrrole [1,2-b ] pyridazine-7-formamide (compound I-35)

Compound I-34(75 mg, 0.21mmol) was used as a starting material in reference to the preparation of compound I-33 to give 54 mg of a yellow solid in 74% yield.

¹H NMR(300MHz,DMSO-d₆)δ8.21(d,J＝9.5Hz,1H),7.87(d,J＝2.0Hz, 1H),7.59(d,J＝9.5Hz,1H),7.45(s,1H),4.49(s,2H),3.49(d,J＝5.1Hz,2H), 3.00(d,J＝6.0Hz,2H),2.92(s,3H)；LC-MS(ESI):m/z 347.2[M+H]⁺。

Preparation method of 7-tert-butyl-4, 5-dihydrothiophene [2,3-c ] pyridine (compound 65)

To 25ml of dichloromethane of hydrochloride salt of 3-thienylethylamine (500 mg, 3.7mmol, purchased from Skychemical) were added triethylamine 1.5ml and pivaloyl chloride (665 mg, 5.5mmol), stirred at room temperature for 1 hour, methanol 5ml was added, and after stirring for 10 minutes, concentration gave an oil as crude N- (2- (thien-3-yl) ethyl) pivaloamide, LC-MS (ESI): M/z 212(M + H). Adding POCl into the oily substance₃3ml and stirred at 65 ℃ for 3 hours, after cooling to room temperature the solvent was removed under reduced pressure, then 10ml of ice-water mixture was added, the pH was adjusted to 7 with NaOH (2.5M), finally extracted with ethyl acetate (20 ml. times.2), dried over anhydrous sodium sulphate and the solvent was removed under reduced pressure to give 580 mg of a colourless oil in 97% yield over two steps.

LC-MS(ESI):m/z 194[M+H]⁺。

Preparation method of 2-bromo-7-tert-butyl-4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 66)

Add NaBH 2 portions to 5mL of compound 65(580 mg, 3mmol) in dry methanol₄(240 mg, 6.3mmol) and stirred at room temperature for 2 hours, then the solvent was spin-dried under reduced pressure, 10ml of ethyl acetate and 10ml of saturated sodium chloride solution were added, the organic phase was separated, the aqueous layer was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to give 510 mg of an oil as a crude product of 7-tert-butyl-4, 5,6, 7-tetrahydrothiophene [2,3-c ] -7-tert-butyl-4, 5,6, 7-tetrahydrothiophene]Pyridine, LC-MS (ESI): M/z 196(M + H); to the above oil was added 10ml of dichloromethane, 0.5ml of triethylamine, and a solution of Boc anhydride (654 mg, 3mmol) in dichloromethane (5 ml), stirred at room temperature for 1 hour, then ammonium chloride solution was added, stirring was continued for 10 minutes, the organic phase was separated, the aqueous layer was extracted with dichloromethane, the organic phases were combined and concentrated to give 950 mg of oil; to the oil was added 30ml of acetonitrile, followed by addition of NBS (590 mg), stirring at room temperature for 1 hour, concentration, and elution by column chromatography (petroleum ether: ethyl acetate 50:1) to give 1.0 g of a colorless oil, in a total yield of 84% in three steps.

LC-MS(ESI):m/z 396.2[M+Na]⁺。

Preparation method of 2-formyl-7-tert-butyl-4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 67)

Starting from compound 66(1.0 g, 2.7mmol), 450 mg of a yellow oil was obtained in 52% yield according to the method for the preparation of compound 61.

¹H NMR(300MHz,CDCl₃)δ9.85(d,J＝4.7Hz,1H),7.52-7.44(m,1H), 5.10(d,J＝62.0Hz,1H),4.57-4.19(m,1H),3.33-3.07(m,1H),2.86-2.55(m, 2H),1.47(s,9H),1.09(s,9H)；LC-MS(ESI):m/z 324[M+H]⁺。

Preparation method of 2- (1-hydroxy-propyn-2-yl) -7-tert-butyl-4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 68)

Starting from compound 67(420 mg, 1.3mmol) and ethynylmagnesium bromide in THF (0.5M, 5ml), reference was made to the procedure for the preparation of compound 56 to give 330 mg as a yellow oil in 73% yield.

¹H NMR(300MHz,CDCl₃)δ6.90(dd,J＝6.8,2.9Hz,1H),5.62-5.56(m, 1H),5.14-4.87(m,1H),4.53-4.12(m,1H),3.30-3.10(m,1H),2.78-2.39(m, 3H),1.45(s,9H),1.08(s,9H)；LC-MS(ESI):m/z 372.2[M+Na]⁺。

Preparation method of methyl 5-chloro-2- (6-tert-butoxyformyl-7-tert-butyl-4, 5-dihydrothiophene [2,3-c ] pyridin-2-yl) pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 69)

Compound 68(320 mg, 0.9mmol), compound 6(170 mg, 0.6mmol), Pd (OAc)₂(7 mg), CuI (1 mg) and DBU (900 mg) as starting materials, according to the preparation method of compound 62, 90 mg of a yellow solid was obtained in a yield of 30%.

¹H NMR(300MHz,CDCl₃)δ7.89-7.80(m,1H),7.42(s,1H),7.39(d,J＝6.0 Hz,1H),7.19(dd,J＝9.4,3.9Hz,1H),5.10(d,J＝53.8Hz,1H),4.60-4.14(m,1H), 3.95(s,3H),3.35-3.15(m,1H),2.80-2.60(m,2H),1.47(s,9H),1.13(s,9H)； LC-MS(ESI):m/z 504.2[M+H]⁺。

Preparation method of methyl 2- (7-tert-butyl-4, 5,6, 7-tetrahydrothiophene [2,3-c ] pyridine-2-yl) -5-chloro-pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 70)

Compound 69(90 mg, 0.18mmol) was used as the starting material in reference to the preparation of compound 64 to give 65 mg of a yellow solid in 89% yield.

¹H NMR(300MHz,CDCl₃)δ7.84(d,J＝9.4Hz,1H),7.44-7.37(m,2H), 7.20(d,J＝9.5Hz,1H),3.96(s,3H),3.40-3.26(m,1H),2.97-2.81(m,1H),2.70- 2.61(m,2H),2.52(s,1H),2.06-1.93(m,1H),1.14(s,9H)；LC-MS(ESI):m/z 404.2[M+H]⁺。

Preparation method of 2- (7-tert-butyl-4, 5,6, 7-tetrahydrothiophene [2,3-c ] pyridine-2-yl) -5-chloro-pyrrole [1,2-b ] pyridazine-7-carboxamide (compound I-36)

Compound 70(65 mg, 0.17mmol) was used as the starting material in reference to the preparation of compound I-32 to give 40 mg of a yellow solid in 64% yield.

1H NMR(300MHz,CD₃OD)δ7.98(d,J＝9.5Hz,1H),7.77(s,1H),7.77(s, 1H),7.49(s,1H),7.43(s,1H),7.38(d,J＝9.5Hz,1H),3.86-3.84(m,1H),3.37- 3.34(m,1H),2.92-2.82(m,1H),2.75-2.63(m,2H),1.13(s,9H)；LC-MS(ESI): m/z 389.2[M+H]⁺。

Preparation method of 7-isobutyl-4, 5-dihydrothiophene [2,3-c ] pyridine (compound 71)

3-Thiophenylethylamine hydrochloride (500 mg, 3.1mmol) and isovaleryl chloride (445 mg, 3.7mmol) were used as starting materials, in accordance with the preparation of compound 65, to give 560 mg of a colorless oil in a two-step overall yield of 93%.

LC-MS(ESI):m/z 194[M+H]⁺。

Preparation method of 2-bromo-7-isobutyl-4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 72)

Compound 71(560 mg, 2.9mmol) was used as a starting material, according to the preparation method of compound 66, to give 895 mg of a colorless oil with a yield of 83%.

¹H NMR(300MHz,CDCl₃)δ6.71(s,1H),5.18(d,J＝57.9Hz,1H),4.36- 3.98(m,1H),3.11-2.93(m,1H),2.74-2.55(m,1H),2.52-2.43(m,1H),1.78- 1.67(m,3H),1.47(s,9H),1.02(d,J＝6.2Hz,3H),0.95(d,J＝6.3Hz,3H)；LC-MS (ESI):m/z 374.2[M+H]⁺。

Preparation method of 2-formyl-7-isobutyl-4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 73)

Compound 72(866 mg, 2.3mmol) was used as the starting material in accordance with the preparation of compound 61 to give 375 mg of a colorless oil in 50% yield.

¹H NMR(300MHz,CDCl₃)9.83(s,1H),7.44(s,1H),5.36(d,J＝61.8Hz,1H), 4.28(d,J＝59.6Hz,1H),3.10-2.95(m,1H),2.85-2.70(m,1H),2.66-2.57(m, 1H),1.85-1.69(m,3H),1.52-1.43(m,9H),1.05(d,J＝6.3Hz,3H),0.97(d,J＝ 6.4Hz,3H)；LC-MS(ESI):m/z 324.2[M+H]⁺。

Preparation method of 2- (1-hydroxy-propyn-2-yl) -7-isobutyl-4, 5-dihydrothiophene [2,3-c ] pyridine-6 (7H) -tert-butyl formate (compound 74)

Compound 73(370 mg, 1.2mmol) and a THF solution of ethynylmagnesium bromide (0.5M, 3.5 mL) were used as starting materials in reference to the preparation of compound 56 to give 330 mg of a yellow oil in 79% yield.

1H NMR(300MHz,CDCl₃)δ6.86(s,1H),5.57(d,J＝6.6Hz,1H),5.47-5.09 (m,1H),4.44-4.02(m,1H),3.11-2.95(m,1H),2.76-2.65(m,2H),2.55-2.45(m, 1H),2.40-2.35(m,1H),1.84-1.63(m,2H),1.47(s,9H),1.03(d,J＝6.3Hz,3H), 0.96(d,J＝6.3Hz,3H)；LC-MS(ESI):m/z 350.2[M+H]⁺。

Preparation method of methyl 2- (6-tert-butoxyformyl-7-isobutyl-4, 5-dihydrothiophene [2,3-c ] pyridine-2-yl) pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 75)

Compound 74(330 mg, 0.95mmol), compound 6(180 mg, 0.6mmol), Pd (OAc)₂(7 mg), CuI (1 mg) and DBU (654 mg) as starting materials, according to the preparation of compound 62, gave 165 mg of a yellow solid in 55% yield. LC-MS (ESI) M/z 504.2[ M + H ]]⁺。

Preparation method of methyl 2- (7-isobutyl-4, 5,6, 7-tetrahydrothiophene [2,3-c ] pyridin-2-yl) -5-chloro-pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 76)

Starting from compound 75(100 mg, 0.2mmol), according to the method for the preparation of compound 59, 65 mg of a yellow solid was obtained in 80% yield. LC-MS (ESI) M/z 404.2[ M + H]⁺。

Preparation method of 2- (7-isobutyl-4, 5,6, 7-tetrahydrothiophene [2,3-c ] pyridine-2-yl) -5-chloro-pyrrole [1,2-b ] pyridazine-7-carboxamide (compound I-37)

Compound 76(65 mg, 0.17mmol) was used as the starting material in reference to the preparation of compound I-32 to give 40 mg of a yellow solid in 61% yield.

¹H NMR(300MHz,CD₃OD)δ7.97(dd,J＝9.5,1.7Hz,1H),7.67(d,J＝1.8 Hz,1H),7.45(t,J＝2.2Hz,1H),7.35(dd,J＝9.5,1.7Hz,1H),4.10-4.03(m,1H), 3.00-2.90(m,1H),2.77-2.62(m,2H),2.06-1.86(m,2H),1.70-1.60(m,2H), 1.03-0.98(m,6H)；LC-MS(ESI):m/z 389.2[M+H]⁺。

Preparation method of 4-cyclopropyl-6, 7-dihydrothiophene [3,2-c ] pyridine (compound 77)

2-Thiophenylethylamine (1.3 g, 10mmol, purchased from Yangshao Denko Chemicals) and cyclopropylcarbonyl chloride (1.3 g, 12.4mmol) were used as starting materials, in accordance with the preparation of compound 65, to give 1.2 g of crude colorless oil.

LC-MS(ESI):m/z 178.2[M+H]⁺。

Preparation method of 4-cyclopropyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 78)

To 15mL of anhydrous methanol containing Compound 77(1.2 g, 7.0mmol) was added NaBH 3 times₄(520 mg, 13.7mmol), stirred at room temperature for 2 hours, the solvent was removed under reduced pressure, ethyl acetate 20ml and water 10ml were added, the organic phase was separated, the aqueous layer was extracted with ethyl acetate 40ml, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give a colorless oil. To the above oil was added 50mL of dichloromethane, 2mL of triethylamine and a solution of Boc anhydride (2.6 g, 11.8mmol) in dichloromethane, stirred at room temperature for 2 hours, 10mL of saturated ammonium chloride solution was added, stirring was continued for 15 minutes, the organic phase was separated, the aqueous layer was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, concentrated, and column chromatography gave 1.3 g of a colorless oil, with a total yield of two steps of 59%.

¹H NMR(300MHz,CDCl₃)δ7.09(d,J＝5.2Hz,1H),6.90(d,J＝5.2Hz,1H), 4.55-4.32(m,2H),3.30-3.15(m,1H),2.95-2.81(m,1H),2.77-2.68(m,1H), 1.46(d,J＝1.1Hz,9H),1.18-1.05(m,1H),0.69-0.62(m,2H),0.58-0.39(m, 2H)；LC-MS(ESI):m/z 302.2[M+Na]⁺。

Preparation method of 2-formyl-4-cyclopropyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 79)

Compound 78(665 mg, 2.3mmol) was used as a starting material, and according to the method for producing compound 55, 260 mg of a yellow oil was obtained in a yield of 36%.

¹H NMR(300MHz,CDCl₃)δ9.83(s,1H),7.59(s,1H),4.46(s,2H),3.22(s, 1H),3.04-2.66(m,2H),1.47(s,9H),1.20-1.05(m,1H),0.82-0.21(m,4H)； LC-MS(ESI):m/z 308[M+H]⁺。

Preparation method of 2- (1-hydroxy-propyn-2-yl) -4-cyclopropyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 80)

Starting from compound 79(260 mg, 0.85mmol) and ethynylmagnesium bromide in THF (0.5M, 3ml), reference was made to the preparation of compound 57 to give 200 mg of a yellow oil in 71% yield.

¹H NMR(300MHz,CDCl₃)δ6.98(d,J＝5.3Hz,1H),5.60(dd,J＝9.1,6.6Hz, 1H),4.50-4.25(m,2H),3.29-3.10(m,1H),2.94-2.56(m,2H),2.39(dd,J＝6.8, 5.3Hz,1H),1.46(s,9H),1.20-1.02(m,1H),0.93-0.75(m,2H),0.71-0.41(m, 2H)；LC-MS(ESI):m/z 356.2[M+Na]⁺。

Preparation method of methyl 2- (4-cyclopropyl-5-tert-butoxyformyl-6, 7-dihydrothiophene [3,2-c ] pyridin-2-yl) -5-chloro-pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 81)

Compound 80(200 mg, 0.6mmol), Compound 6(120 mg, 0.4mmol), Pd (OAc)₂(6 mg), CuI (1 mg) and DBU (304 mg) as starting materials, according to the preparation method of compound 58, 80 mg of a yellow oil was obtained in a yield of 41%.

LC-MS(ESI):m/z 488.2[M+H]⁺。

Preparation method of 2- (4-cyclopropyl-4, 5,6, 7-tetrahydrothiophene [3,2-c ] pyridine-2-yl) -5-chloro-pyrrole [1,2-b ] pyridazine-7-carboxamide (compound I-38)

Adding 2ml of trifluoroacetic acid into dichloromethane of a compound 81(80 mg, 0.17mmol), stirring at room temperature for 1 hour, adjusting the pH value to 8-9 by NaOH (2.5M) under ice bath, separating an organic phase, extracting an aqueous layer by using trichloromethane, combining the organic phases, washing by using a saturated sodium chloride solution, concentrating, and eluting by using column chromatography (dichloromethane: methanol ═ 20:1) to obtain a yellow solid. Dissolved in 10mL of methanol, transferred to a 25mL sealed tube, reacted at 90 ℃ for 24 hours, cooled to room temperature, and filtered to give 35 mg of a yellow solid in 56% yield by spinning off 2/3 volumes of solvent under reduced pressure.

1H NMR(300MHz,DMSO-d6)δ8.18(d,J＝9.5Hz,1H),8.07(s,2H),7.89(s, 1H),7.68(d,J＝9.5Hz,1H),7.44(s,1H),3.58-3.46(m,2H),3.25-3.15(m,1H), 3.09-3.01(m,2H),1.21-1.06(m,1H),0.90-0.70(m,2H),0.68-0.58(m,2H)； LC-MS(ESI):m/z 373.2[M+H]⁺。

Preparation method of 4-isobutyl-6, 7-dihydrothiophene [3,2-c ] pyridine (compound 82)

2-Thiophenylethylamine (500 mg, 4mmol) and isovaleryl chloride (576 mg, 4.8mmol) were used as starting materials, in accordance with the preparation of compound 65, to give 700 mg of a colorless oil in 91% yield.

LC-MS(ESI):m/z 194(M+H)。

Preparation method of 2-bromo-4-isobutyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 83)

Compound 82(500 mg, 2.6mmol) was used as a starting material in accordance with the preparation method of compound 66 to obtain 750 mg of a colorless oil with a yield of 78%.

¹H NMR(300MHz,CDCl₃)δ6.71(s,1H),5.23-4.94(m,1H),4.48-4.07(m, 1H),3.17-2.95(m,1H),2.88-2.72(m,1H),2.64-2.47(m,1H),1.76-1.62(m, 3H),1.46(s,9H),1.03(d,J＝6.4Hz,3H),0.93(d,J＝6.5Hz,3H)；LC-MS(ESI): m/z 374.2[M+H]⁺。

Preparation method of 2-formyl-4-isobutyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 84)

Compound 83(750 mg, 2mmol) was used as the starting material in reference to the preparation of compound 61 to give 300 mg of a colorless oil in 46% yield.

¹H NMR(300MHz,CDCl₃)δ9.82(s,1H),7.45(s,1H),5.24(d,J＝57.1Hz, 1H),4.35(d,J＝62.4Hz,1H),3.23-2.72(m,3H),1.80-1.60(m,3H),1.48(s,9H), 1.07(d,J＝6.3Hz,3H),0.97(d,J＝6.5Hz,3H)；LC-MS(ESI):m/z 324.2[M+H]⁺。

Preparation method of 2- (1-hydroxy-propynyl-2-yl) -4-isobutyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 85)

Starting from compound 84(300 mg, 0.93mmol) and ethynylmagnesium bromide in THF (0.5M, 3.5 ml), reference was made to the preparation of compound 57 to give 280 mg of a yellow oil in 86% yield.

1H NMR(300MHz,CDCl₃)δ6.88-6.81(d,J＝2.4Hz,1H),5.57(d,J＝6.6 Hz,1H),5.12(d,J＝52.5Hz,1H),4.47-4.16(m,1H),3.17-2.59(m,3H),2.38- 2.31(m,1H),1.74-1.59(m,3H),1.46(s,9H),1.05(d,J＝6.3Hz,3H),0.94(d,J＝ 6.5Hz,3H)；LC-MS(ESI):m/z 372.2[M+Na]⁺。

Preparation method of methyl 2- (4-isobutyl-5-tert-butoxyformyl-6, 7-dihydrothiophene [3,2-c ] pyridine-2-yl) pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 86)

Compound 85(280 mg, 0.8mmol), Compound 6(141 mg, 0.46mmol), Pd (OAc)₂(16 mg), CuI (1 mg) and DBU (350 mg) as starting materials, according to the preparation method of compound 58, 81 mg of a yellow oil was obtained in a yield of 35%.

1H NMR(300MHz,CDCl₃)δ7.86-7.80(m,1H),7.41(s,1H),7.36-7.27(m, 1H),7.18(d,J＝9.2Hz,1H),4.48-4.30(m,1H),3.95(s,3H),3.16-2.88(m,3H), 2.78-2.68(m,1H),2.06-1.94(m,1H),1.78-1.66(m,2H),1.48(s,9H),1.09(d,J ＝5.8Hz,3H),0.97(d,J＝5.8Hz,3H)；LC-MS(ESI):m/z 504.2[M+H]⁺。

Preparation method of 2- (4-isobutyl-4, 5,6, 7-tetrahydrothiophene [3,2-c ] pyridine-2-yl) -5-chloro-pyrrole [1,2-b ] pyridazine-7-carboxamide (compound I-39)

Compound 86(80 mg, 0.16mmol) was used as a starting material in reference to the preparation of compound I-38 to give 35 mg of a yellow solid in 56% yield.

¹H NMR(300MHz,DMSO-d₆)δ8.20(d,J＝9.5Hz,1H),8.07(d,J＝3.9Hz, 2H),7.92(s,1H),7.66(d,J＝9.6Hz,1H),7.43(s,1H),4.31(d,J＝8.2Hz,1H), 3.45-3.15(m,2H),3.08-2.99(m,2H),2.02-1.94(m,1H),1.90-1.67(m,2H), 1.01(d,J＝6.2Hz,3H),0.96(d,J＝6.4Hz,3H)；LC-MS(ESI):m/z 389.2[M+H]⁺。

Preparation method of 4-tert-butyl-6, 7-dihydrothiophene [3,2-c ] pyridine (compound 87)

Starting from 2-thienylethylamine (1.5 g, 11.8mmol) and isovaleryl chloride (2.1 g, 4.8mmol), reference was made to the preparation of compound 65 to give 1300 mg of a colorless oil in 57% yield. LC-MS (ESI) M/z 194.2[ M + H]⁺。

Preparation method of 4-tert-butyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 88)

Add NaBH to Compound 87(1.3 g, 6.7mmol) in two portions in EtOH (40mL)₄(500 mg), stirred at room temperature for 1 hour, concentrated, added with 10mL of water and 20mL of ethyl acetate, separated the organic phase, the aqueous layer extracted with 10mL of ethyl acetate, combined the organic phases, dried over anhydrous sodium sulfate, concentrated to give an oil, which was used directly in the next step; 20mL of methylene chloride was added to the above oil, and NEt was added dropwise₃0.5mL and Boc₂Acid anhydride (1.5 g, 6.9mmol) in dichloromethane (8mL) was stirred at room temperature for 1 hour, concentrated, and eluted by column chromatography (petroleum ether: ethyl acetate ═ 100:1) to give 1.5 g of oil in 75% yield over two steps.

¹H NMR(300MHz,CDCl₃)δ7.08(dd,J＝10.3,5.3Hz,1H),6.91(d,J＝5.2 Hz,1H),4.96(d,J＝50.4Hz,1H),4.41(ddd,J＝70.1,13.8,6.3Hz,1H),3.45-3.17 (m,1H),2.97-2.66(m,2H),1.47-1.43(s,9H),1.03(s,9H)；LC-MS(ESI):m/z 318.2[M+Na]⁺。

Preparation method of 2-formyl-4-tert-butyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 89)

Starting from compound 88(700 mg, 2.4mmol), the preparation of compound 55 gave 250 mg of a yellow oil in 32% yield.

¹H NMR(300MHz,CDCl₃)δ9.84(s,1H),7.60(s,1H),5.00(d,J＝57.3Hz, 1H),4.45(ddd,J＝69.2,14.2,6.3Hz,1H),3.48-3.22(m,1H),2.99-2.68(m,2H), 1.47(s,9H),1.05(s,9H)；LC-MS(ESI):m/z 324.2[M+H]⁺。

Preparation method of 2- (1-hydroxy-propyne-2-yl) -4-tert-butyl-6, 7-dihydrothiophene [3,2-c ] pyridine-5 (4H) -tert-butyl formate (compound 90)

Starting from compound 89(250 mg, 0.85mmol), according to the method for the preparation of compound 57, 140 mg of an oil was obtained in 35% yield.

¹H NMR(300MHz,CDCl₃)δ7.00(s,1H),5.59(d,J＝6.0Hz,1H),4.90(d,J＝ 50.2Hz,1H),4.39(ddd,J＝69.4,13.7,6.3Hz,1H),3.44-3.17(m,1H),2.93-2.75 (m,1H),2.74-2.63(m,2H),2.53-2.39(m,1H),1.45(s,9H),1.03(s,9H)；LC-MS (ESI):m/z 372.2[M+Na]⁺。

Preparation method of methyl 2- (4-tert-butyl-5-tert-butoxyformyl-6, 7-dihydrothiophene [3,2-c ] pyridine-2-yl) pyrrole [1,2-b ] pyridazine-7-carboxylate (compound 91)

As compound 90(140 mg, 0.4mmol), compound 6(75 mg, 0.25mmol), Pd (OAc)₂(3 mg), CuI (1 mg) and DBU (190 mg) as starting materials gave 73 mg of a yellow solid in 56% yield.

¹H NMR(300MHz,CDCl₃)δ7.87-7.81(m,1H),7.48-7.37(m,2H),7.19(dd, J＝9.5,4.9Hz,1H),4.99(d,J＝50.9Hz,1H),4.42(d,J＝66.6Hz,1H),3.95(s,3H), 3.45-3.28(m,1H),2.96-2.74(m,2H),1.48(s,9H),1.08(s,9H)；LC-MS(ESI): m/z 504.2[M+H]⁺。

Preparation method of 2- (4-tert-butyl-4, 5,6, 7-tetrahydrothiophene [3,2-c ] pyridine-2-yl) -5-chloro-pyrrole [1,2-b ] pyridazine-7-carboxamide (compound I-40)

Starting from compound 91(70 mg, 0.14mmol), according to the method for producing compound I-38, 35 mg of a yellow solid was obtained in a yield of 64%.

¹H NMR(300MHz,DMSO-d₆)δ8.19-8.10(m,2H),8.05(s,1H),7.78(s,1H), 7.68(d,J＝9.5Hz,1H),7.42(s,1H),3.65(s,1H),3.20(d,J＝7.8Hz,1H),2.72(d, J＝4.9Hz,3H),0.99(s,9H)；LC-MS(ESI):m/z 388.2[M+H]⁺。

Activity test section

The compounds of the present invention were tested for their inhibitory effect on PARP-1and PARP-2 activity at the molecular level by the following pharmacological experiments

1. ELISA assay test of the molecular level of inhibition of PARP-1and PARP-2 Activity by Compounds of the invention

The experimental method comprises the following steps: Enzyme-Linked Immunosorbent Assay (ELISA) (see the Enzyme-Linked Immunosorbent Assay described in Decker P. publication; reference: Decker P, Miranda EA, de Murcia G, Muller S.an Immunopositional test to rubber a large series of new inhibition or molecules of Poly (ADP-rib) Polymerase activity for thermal applications, clinical Cancer Research, 5: 1169-99). The principle is that the substrate histone is coated on an adsorptive 96-well plate, and PARP-1 or 2 recombinase and substrate NAD are added⁺The PARP enzyme activity can be reflected by the fact that PARP-1 or PARP-2 is enzymatically reacted by activated DNA to produce a product PAR (poly-adenosyl-bis-phosphoribosyl chloride) by histone, and then an anti-PAR antibody is added to detect the strength of the product PAR on the histone coated on a 96-well plate.

Calculation of drug inhibition: inhibition (%) - (OD control well-OD administration well)/OD control well × 100% (OD is absorbance value); and calculating the concentration of the drug reaching 50% inhibition, namely IC, according to the inhibition rate by a Logit method₅₀The value is obtained. (at the molecular level IC₅₀<1000nM,+；<500nM,++；<100nM, + + +; -is of>1000nM or not), the results are shown in table 1:

2. test results

From Table 1, it can be found that the compounds of the present invention have significant inhibitory activity against PARP-1and PARP-2.

TABLE 1 partial compound inhibition of PARP-1and PARP-2 activity at molecular level

Growth inhibition of I-34 human breast cancer MDA-MB-436 nude mouse subcutaneous transplantation tumor

Test substance and positive control drug

Compound I-34 was prepared as the monohydrochloride as a yellow powder, dissolved in water for injection to a yellow turbid liquid, settled after standing, mixed well before administration, and the above compound was formulated once a week. The positive control drug AZD2281 (anticancer drug Olapariparib) was a pale yellow powder (Lot: PAR-105) and was used after formulating a suspension with 0.5% sodium carboxymethylcellulose every week.

Dose setting

Two dose groups of 100mg/kg and 20mg/kg were set for each of the I-34 hydrochloride salts. The dose of the positive control drug AZD2281 was 30 mg/kg.

Experimental animals and inoculated cells

BALB/cA nude mouse, female, 4-5 weeks old, body weight 19 + -2 g, provided by Shanghai pharmaceutical research institute of Chinese academy of sciences, production license number: SCXK (Shanghai) 2013-001. The use certificate number: SYXK (Shanghai) 2013-0049. Number of animals per group: negative control group 12, administration group 6. Human breast cancer MDA-MB-436 cell line was maintained in our home. The cell strain is used for inoculating the right armpit subcutaneous of the nude mouse, the inoculation amount of the cell is 5 multiplied by 106 per mouse, and the cell strain is used after 2 generations in the nude mouse after forming transplantation tumor.

Experimental methods

Cutting tumor tissue in vigorous growth stage into 1.5mm³And left and right, under aseptic conditions, inoculated subcutaneously in the right axilla of nude mice. Measuring the diameter of the transplanted tumor by using a vernier caliper for the subcutaneous transplanted tumor of the nude mouse until the average tumor volume grows to 200mm³Left and right, animals were randomly grouped. Compound I-34 hydrochloride 100mg/kg and 20mg/kg groups were administered orally once daily for 21 consecutive days. The positive control drug AZD 228130 mg/kg was administered orally once daily for 21 consecutive days. Solvent control an equal amount of water for injection was given. Throughout the experiment, the diameter of the transplanted tumor was measured 2 times per week, while the mouse weight was weighed. The formula for Tumor Volume (TV) is: TV 1/2 × a × b²Wherein a and b represent length and width, respectively. Calculating Relative Tumor Volume (RTV) according to the measurement result, wherein the calculation formula is as follows: RTV ═ V_t/V₀. Wherein V₀When administered separately from the cage (i.e. d)₀) Measurement of the resulting tumor volume, V_tFor the tumor volume at each measurement. The evaluation index of the antitumor activity was (1) the relative tumor proliferation rate T/C (%), and the formula was calculated as follows: T/C (%) ═ T_RTV/C_RTV)×100％，T_RTV: treatment group RTV; c_RTV: negative control group RTV; (2) the tumor volume increase inhibition rate GI% is calculated according to the following formula:

GI％＝[1-(TVt-TV₀)/(CVt-CV₀)]x 100%, TVt is the tumor volume measured for each treatment group; TV (television)₀Tumor volume obtained when cage-administered as a therapeutic component; CVt is the tumor volume measured in each time in the control group; CV of₀Tumor volume obtained when cage administration of control component was performed; (3) the tumor weight inhibition rate is calculated according to the following formula: tumor weight inhibition ratio [ (% Wc-W) ]_T) Wc × 100%, Wc: tumor weight of control group, W_T: the treated group had heavy tumor.

Results of growth inhibition of human Breast cancer MDA-MB-436 nude mouse subcutaneous transplantation tumor

The results of the experiment are shown in table 2.

The I-34 hydrochloride 100mg/kg and 20mg/kg groups were orally administered once a day for 21 consecutive days, which had extremely significant inhibitory effects on the growth of subcutaneous transplantable tumors in human breast cancer MDA-MB-436 nude mice, with T/C percentages of 1.47% and 5.70% obtained on day 21, respectively, and one of the mice in the high dose group had complete tumor regression.

AZD 228130 mg/kg, orally administered once a day for 21 days continuously, has certain inhibitory effect on the growth of human breast cancer MDA-MB-436 nude mice subcutaneous transplantation tumor, and the T/C percentage obtained on day 21 is 51.41%. The activity of the I-34 hydrochloride is obviously better than that of AZD 2281.

TABLE 2 Experimental therapeutic Effect of Compounds on human Breast cancer MDA-MB-436 nude mice subcutaneous transplantation tumors

t-test (vs solvent control), p <0.05 p <0.001 "()" number of tumor regressed mice

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (9)

1. A pyrrolo [1,2-b ] pyridazine compound represented by the general formula I:

wherein the content of the first and second substances,

R¹is hydrogen, C_1-4Alkyl or halogen;

R²is composed of

R³Is hydrogen, substituted or unsubstituted C_1-4Alkyl or substituted or unsubstituted C_3-6Cycloalkyl, said substitution being by a member selected from the group consisting of halogen, C_1-4At least one substituent of alkyl, amino, cyano, hydroxyl and nitro;

R⁴is hydrogen, halogen, C_1-4Alkyl or C_1-4An alkoxy group;

R⁵is-CH₂-or-C (═ O) -;

X₁s, O or NH;

X₂is CH or N;

Y₁、Y₂、Y₅and Y₆Independently is a substituted or unsubstituted methylene, substituted or unsubstituted ethylene, substituted or unsubstituted propylene or substituted or unsubstituted butylene group, said substitution being selected from the group consisting of halogen, C_1-6Alkyl radical, C_3-6Cycloalkyl and C_1-4At least one substituent in the alkoxy group;

Y₃and Y₄Independently hydrogen, substituted or unsubstituted C_1-4Alkyl or substituted or unsubstituted C_3-6Cycloalkyl, said substitution being by a member selected from the group consisting of halogen, C_1-6Alkyl radical, C_3-6Cycloalkyl and C_1-4At least one substituent in the alkoxy group;

Y₇is NR⁸Or CHR⁹，

Wherein, R is⁸Is hydrogen, -C (═ O) R¹⁰、-SO₂R¹¹Substituted or unsubstituted C_1-4Alkyl, substituted or unsubstituted C_3-6Cycloalkyl or substituted or unsubstituted C_6-10Aryl, wherein R is¹⁰And R¹¹Independently is substituted or unsubstituted C_1-4Alkyl, substituted or unsubstituted C_3-6Cycloalkyl, substituted or unsubstituted five-to six-membered saturated heterocyclic group, or substituted or unsubstituted C_6-10Aryl, said substitution being by a member selected from the group consisting of halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, amino, substituted by at least one C_1-4At least one substituent of the alkyl-substituted amino, cyano, hydroxyl and nitro is substituted,

the R is⁹Is hydrogen, -OR¹²Is at least one C_1-4An alkyl-substituted amino group, or a substituted or unsubstituted five-to six-membered saturated heterocyclic group, wherein R is¹²Is hydrogen, substituted or unsubstituted C_1-4Alkyl or substituted or unsubstituted C_3-6Cycloalkyl, said substitution being by a member selected from the group consisting of halogen, C_1-4Alkyl radical, C_1-4Haloalkyl, amino, substituted by at least one C_1-4At least one substituent of the alkyl-substituted amino, cyano, hydroxyl and nitro groups.

2. The pyrrolo [1,2-b ] pyridazine compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, in the general formula I,

R¹is hydrogen, methyl, ethyl, propyl, fluorine, chlorine or bromine;

R²is composed of

R³Is hydrogen or methylAlkyl, ethyl or propyl;

R⁴is hydrogen, fluorine, chlorine, bromine, methyl, ethyl, propyl, methoxy, ethoxy or propoxy;

R⁵is-CH₂-or- (C ═ O) -;

X₁s, O or NH;

X₂is CH or N;

Y₁、Y₂、Y₅and Y₆Independently a substituted or unsubstituted methylene group, a substituted or unsubstituted ethylene group, a substituted or unsubstituted propylene group, or a substituted or unsubstituted butylene group, said substitution being with at least one substituent selected from the group consisting of fluorine, chlorine, bromine, methyl, ethyl, propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, and propoxy;

Y₃and Y₄Independently hydrogen, methyl, ethyl, propyl or butyl;

Y₇is NR⁸Or CHR⁹，

Wherein, R is⁸Is hydrogen, -C (═ O) R¹⁰、-SO₂R¹¹Methyl, ethyl, propyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or substituted or unsubstituted phenyl, wherein R is¹⁰And R¹¹Independently substituted or unsubstituted groups as follows: methyl, ethyl, propyl, N-butyl, sec-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl, dioxanyl or phenyl, said substitution being by at least one substituent selected from the group consisting of fluoro, chloro, bromo, methyl, ethyl, propyl, trifluoromethyl, amino, N-methylamino, N-ethylamino, N-dimethylamino, N-diethylamino, cyano, hydroxy and nitro,

the R is⁹Is hydrogen, -OR¹²N-methylamino, N-ethylamino, N-dimethylamino, N-diethylamino, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl, or dioxyHexacyclic group, wherein R is¹²Is hydrogen, methyl, ethyl, propyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, said substitution being by at least one substituent selected from the group consisting of fluorine, chlorine, bromine, methyl, ethyl, propyl, trifluoromethyl, amino, N-methylamino, N-ethylamino, N-dimethylamino, N-diethylamino, cyano, hydroxyl and nitro.

3. The pyrrolo [1,2-b ] pyridazine compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the pyrrolo [1,2-b ] pyridazine compound is one of the following compounds:

4. use of a pyrrolo [1,2-b ] pyridazine compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof for the preparation of a PARP inhibitor.

5. Use of pyrrolo [1,2-b ] pyridazine compound or a pharmaceutically acceptable salt thereof according to claim 4 for the preparation of PARP inhibitors having a selective inhibitory effect on PARP-1 and/or PARP-2.

6. A pharmaceutical composition comprising a therapeutically effective amount of one or more pyrrolo [1,2-b ] pyridazine compounds according to any one of claims 1-3 or pharmaceutically acceptable salts thereof.

7. Use of the pharmaceutical composition of claim 6 in the preparation of PARP inhibitors, wherein said pharmaceutical composition has inhibitory effect on PARP.

8. Use of a pharmaceutical composition according to claim 7 in the preparation of a PARP inhibitor, wherein said pharmaceutical composition has a selective inhibitory effect on PARP-1 and/or PARP-2.

9. Use of the pharmaceutical composition according to claim 7 in the preparation of PARP inhibitor for the prevention and/or treatment of PARP associated diseases selected from the group consisting of tumor, ischemic diseases and neurodegenerative diseases.