Novel compounds useful as inhibitors of Toll-like receptor 7 activation

Abstract

CB-7 exhibits a weak TLR7 inhibiting effect in normal mice. The present invention provides a novel compound with a stronger TLR7 inhibiting effect than CB-7, a pharmaceutically acceptable salt of said compound, or a prodrug of said compound or salt. The present invention also provides a drug for the prevention or treatment of diseases associated with the activation of TLR7, said drug including the aforementioned TLR7 activation inhibitor.

Description

[Technical field]

[0001] The present invention relates to an activation inhibitor of Toll-like receptor 7 (TLR7) and a novel compound containing said activation inhibitor that is useful as a preventive or therapeutic agent for diseases accompanied by TLR7 activation. [Background technology]

[0002] The immune system is broadly divided into the innate immune system and the adaptive immune system. The innate immune system is a host defense mechanism that activates in the early stages of infection, in which phagocytes such as macrophages and dendritic cells play a central role. On the other hand, the adaptive immune system is an acquired host defense mechanism for dealing with countless antigens, in which lymphocytes play a central role. Adaptive immunity begins when T cells, a type of lymphocyte, receive antigens from activated dendritic cells. In other words, activation of innate immunity is essential for the induction of adaptive immunity.

[0003] TLRs expressed on the cell membrane and intracellularly of phagocytes specifically recognize components of bacteria and viruses, and activate transcription factors such as NF-κB, thereby inducing the production of inflammatory cytokines. For example, TLR7 is localized in the endosomes and lysosomes of B cells and plasmacytoid dendritic cells, and recognizes single-stranded RNA derived from viruses. Upon recognizing the ligand, TLR7 induces the production of type I interferon (IFN-α) to defend against viral infection. TLR7 also recognizes nucleic acids (autoantigens) released from damaged and dead cells, and IFN-α is one of the factors related to the onset and exacerbation of non-infectious inflammatory diseases such as autoimmune diseases. Classical dendritic cells, which are differentiated from monocytes by IFN-α, are activated by ingesting dead cells, and promote the proliferation and differentiation of autoreactive T cells through antigen presentation. IFN-α also induces the expression of BAFF (B cell activating factor) or APRIL (a proliferation inducing ligand) in dendritic cells, promoting the class switching of autoreactive B cells and their differentiation into plasma cells. This activates the production of autoantibodies in the body, and immune complexes consisting of nucleic acids and autoantibodies stimulate plasmacytoid dendritic cells, promoting the production of IFN-α. As a result of chronic production of IFN-α, the pathology progresses and worsens.

[0004] Autoimmune diseases are a group of diseases that are often characterized by the appearance of antibodies (such as antinuclear antibodies) against autoantigens and are accompanied by various characteristic inflammations. As autoimmune diseases or pathological conditions progress, they cause severe damage to various organs, including multiple organs such as the skin and kidneys, muscles, nerves, and blood vessels. A representative example of an autoimmune disease caused by abnormal TLR7 signals and the production of IFN-α is systemic lupus erythematosus. The number of systemic lupus erythematosus patients is estimated to be 1.4 million worldwide and about 100,000 in Japan (the number of patients who received a certificate for medical care for specific diseases in fiscal year 2016 was 63,792 for systemic lupus erythematosus), and the disease is particularly prevalent in women in their 20s and 30s. Systemic lupus erythematosus is an intractable immune disease of unknown cause, and is designated as a specific disease by the Ministry of Health, Labor, and Welfare. There have been several reports in mice and humans that abnormal TLR7 signals cause systemic lupus erythematosus. In BXSB, a mouse model of systemic lupus erythematosus, it has been suggested that the cause of pathogenesis is the translocation of the region containing the TLR7 gene, which is normally localized on the X chromosome, to the Y chromosome (Kumar, KR, et al. Science. 312:1665-1669, 2006). TLR7 transgenic mice develop systemic lupus erythematosus-like glomerulonephritis, and approximately half of the mice die within 20 weeks of age (Deane, JA, et al. Immunity. 27:801-810, 2007). Mice in which the 34th aspartic acid residue of Unc93B1, which is involved in the transport of TLR7 or TLR9 from the endoplasmic reticulum to endosomes, is mutated to an alanine residue exhibit autoimmune disease-like symptoms due to enhanced TLR7 responsiveness, and more than half of the mice die of hepatitis within one year (Fukui, R., et al. Immunity. 35:69-81, 2011). Compared with healthy subjects, human systemic lupus erythematosus patients have higher TLR7 gene expression in peripheral blood mononuclear cells, and produce large amounts of inflammatory cytokines in response to TLR7 ligand stimulation (Komatsuda, A., et al. Clin Exp Immunol. 152: 482-487, 2008). It is also known that TLR7 deficiency in autoimmune disease model mice improves the pathology.MRL / Mp is a mouse model of spontaneous autoimmune disease. lpr / lpr Mice are a representative animal model of human systemic lupus erythematosus, and produce autoantibodies such as antinuclear antibodies, and develop vasculitis, polyarthritis, and glomerulonephritis with age. lpr / lpr Deficiency of TLR7 in mice suppresses lymphocyte activation and alleviates the symptoms of nephritis (Christensen, SR, et al. 2006. Immunity. 25(3):417-428). lpr / lpr Deficiency of TLR9 in mice reduced anti-DNA antibody titers but did not ameliorate nephritis symptoms.

[0005] There are still many problems to be solved in the treatment of autoimmune diseases. Rheumatoid arthritis, which has the largest number of patients among all autoimmune diseases, is an inflammatory disease that mainly affects the synovium of joints, causing persistent polyarthritis, cartilage and bone damage, and progressive joint destruction. The involvement of inflammatory cytokines in autoimmune diseases has been analyzed mainly in rheumatoid arthritis model mice and rheumatoid arthritis patients. IL-1 receptor antagonists as IL-1 inhibitory therapy, anti-TNF-α antibodies as TNF-α inhibitory therapy, and anti-IL-6 receptor antibodies as IL-6 inhibitory therapy have been approved as treatments for rheumatoid arthritis and are used in clinical practice, and all of them have been more effective than conventional standard drug therapies. The usefulness of biological agents, including antibody drugs, is also expected for systemic lupus erythematosus. In fact, a recombinant human IgG1λ monoclonal antibody (trade name: belimumab) against the B cell activating factor BLyS (B lymphocyte stimulator) has been shown to be effective in treating some patients with systemic lupus erythematosus because it has the effect of suppressing the proliferation of autoreactive B cells involved in the exacerbation of the disease, and is sold in Japan and overseas. For example, in the EU, it has been approved as an additional or concomitant treatment for adult systemic lupus erythematosus patients who are autoantibody-positive and have high disease activity despite receiving standard treatment. It is also indicated for the treatment of similar patients in the United States, but its efficacy in patients with severe active lupus nephritis or central nervous system lupus has not yet been clinically evaluated, and it is prescribed only in a limited manner. A phase III trial in Northeast Asia, including Japan, announced in November 2016 reported positive results for the efficacy of belimumab, but the responder rate after 52 weeks of intravenous administration (10 mg / kg) was only 54%. In addition, a mouse-human chimeric IgG1κ monoclonal antibody (trade name: rituximab) against CD20, which is specifically expressed on the surface of B cells, is used as a treatment for malignant lymphoma and has previously attracted attention as a treatment for systemic lupus erythematosus; however, clinical trials were halted after reports of serious adverse events, including deaths.Currently, clinical trials using IFN-α and antibodies against its receptors are underway, and although good results have been obtained in safety and efficacy evaluations, the occurrence of several adverse events (anemia, lymphopenia, decreased liver function, etc.) has been reported, albeit at a low frequency. Thus, the use of biological agents is expected to be highly effective in improving pathology, but regular and long-term administration is required, which causes problems regarding the physical and medical economic burden on patients, and there is a risk that the effectiveness of this treatment method will decrease. In addition, steroids are used as the first-line drug for many autoimmune diseases for which effective treatment has not yet been established. Steroids have strong anti-inflammatory effects, but also have immunosuppressive effects, so side effects occur when taken for a long period of time. Therefore, the development of TLR7 or TLR9 inhibitors aimed at creating alternative therapeutic drugs to steroids has been promoted, and to date, oligonucleotide-based TLR7 and TLR9 inhibitors (Rommler, F., et al. J. Immunol., 191:3240-3253, 2013) and small molecule-based TLR7 and TLR9 inhibitors (Lamphier, M., et al. Mol. Pharmacol., 85:429-440, 2014) have been reported, but they have not yet been put to practical use. On the other hand, hydroxychloroquine, which was approved in Japan in 2015, is an orally administered small molecule drug, but it has not been actively prescribed due to concerns about side effects such as retinopathy.

[0006] TLR7 has also been implicated in various diseases other than systemic lupus erythematosus. Alzheimer's disease is a progressive cognitive loss, and patients have an excess of senile plaques, consisting of neurofibrillary tangles made of amyloid beta and tau proteins, in the cerebral cortex and subcortical gray matter. The main cause of the disease is thought to be the deposition of amyloid beta proteins in nerve cells. The early-onset type accounts for 2-7% of cases, while the normal type is genetic due to a mutation and occurs in people over 60 years of age, and its incidence increases with age. The Ministry of Health, Labor and Welfare announced in 2013 that there are 4.62 million dementia patients in Japan, the majority of whom are said to have Alzheimer's disease. In the United States, there are more than 5 million Alzheimer's disease patients, and the annual cost of home medical care, home care, social medical care, lost productivity, and premature death is more than 100 billion US dollars. In recent years, it has been suggested that TLR7 is involved in brain diseases caused by neurodegenerative diseases, stroke, multiple sclerosis, etc. It has been reported that let-7 (microRNA), which is abundant in the brain, induced neurotoxicity depends on TLR7 (Lehmann, SM, et al. Nat Neurosci. 15(6):827-835). Let-7 is more abundant in the cerebrospinal fluid of Alzheimer's disease patients than in healthy individuals, and is thought to be involved in neuronal degeneration.

[0007] Previously, through screening research by the inventors, they discovered that cyclobactiol and its related compounds, which are isolated from the mature seeds of the legume plant, Hollandia japonica, and are also contained in the traditional Chinese medicine, cyclobactiol, have the effect of selectively inhibiting NF-κB activation induced by TLR7 or TLR9 ligand stimulation, and together with their collaborative research partners, Teika Pharmaceutical Co., Ltd. and Tokyo Institute of Technology, they filed an international application (international publication number WO / 2017 / 047769). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Kumar KR, et al. Regulation of B cell tolerance by the lupus susceptibility gene Ly108. Science. 312:1665-1669, 2006 [Non-Patent Document 2] Deane JA, et al. Control of toll-like receptor 7 expression is essential to restrict autoimmunity and dendritic cell proliferation. Immunity. 27:801-810, 2007 [Non-Patent Document 3] Fukui R, et al. Unc93B1 restricts systemic lethal inflammation by orchestrating Toll-like receptor 7 and 9 trafficking. Immunity. 35:69-81, 2011 [Non-Patent Document 4] Komatsuda A, et al. Up-regulated expression of Toll-like receptors mRNAs in peripheral blood mononuclear cells from patients with systemic lupus erythematosus. Clin Exp Immunol. 152: 482-487, 2008 [Non-Patent Document 5] Christensen SR, et al. Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus. Immunity. 25(3):417-428, 2006 [Non-Patent Document 6] Rommler F, et al. Guanine modification of inhibitory oligonucleotides potentiates their suppressive function. J. Immunol, 191:3240-3253, 2013 [Non-Patent Document 7] Lehmann SM, et al. An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration. Nat Neurosci. 15(6):827-835, 2012 [Patent documents]

[0009] [Patent Document 1] WO / 2017 / 047769 Summary of the Invention [Problem to be solved by the invention]

[0010] In Patent Document 1, CB-7, which showed the highest TLR7 inhibitory effect, suppressed the activation of TLR7 in mouse immune cells (macrophages, dendritic cells) and human immune cells (peripheral blood mononuclear cells, dendritic cells). However, its TLR7 inhibitory effect in normal mice was weak, and its efficacy in systemic lupus erythematosus model mice was also limited. [Means for solving the problem]

[0011] The present inventors have repeatedly investigated methods and substances for creating compounds that have higher TLR7 inhibitory activity than the lead compound CB-7 and show efficacy at the animal level. As a result, they have found that several derivatives with novel structures inhibit the induction of inflammatory cytokines IL-6 and IFN-α by TLR7 activation up to about 45 times more potently than CB-7, and have completed the present invention.

[0012] According to the present invention, Provided is a compound represented by the following formula (I) or (II), a pharmacologically acceptable salt thereof, or a prodrug thereof: [ka] [ka] (In formulas (I) and (II), R1 is, an alkoxy group having 3 to 5 carbon atoms and containing at least two oxygen atoms; an alkoxy group having 2 to 4 carbon atoms and containing at least one hydroxyl group; formula: [ka] (wherein R2 and R3 each independently represent an alkyl group having 1 to 3 carbon atoms), or formula: [ka] (In the formula, Ring C is a 3- to 7-membered nitrogen-containing heterocycle; R4 is a group represented by -NH-, -O-, -CF2-, -CHF-, -C2H2F2- or -CHF-X-CHF-; X is an alkyl group having 1 to 4 carbon atoms. It is a group represented by the following formula:

[0013] Such compounds, pharmacologically acceptable salts thereof, or prodrugs thereof are capable of inhibiting at least the activation of TLR7.

[0014] Further, according to the present invention, R1 is, [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] Also provided is the above compound, a pharma- ceutical acceptable salt thereof, or a prodrug thereof, wherein:

[0015] Furthermore, according to the present invention, there is also provided the above compound, a pharmacologically acceptable salt thereof, or a prodrug thereof, which is represented by any one of the following formulas (III) to (XIII): [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0016] Furthermore, according to the present invention, there is also provided the above-mentioned compound, a pharmacologically acceptable salt thereof, or a prodrug thereof, wherein the pharmacologically acceptable salt is a hydrochloride or a formate.

[0017] The present invention also provides an inhibitor of Toll-like receptor 7 (TLR7) activation, which comprises the above compound, a pharmacologically acceptable salt thereof, or a prodrug thereof.

[0018] Furthermore, the present invention also provides the above-mentioned TLR7 activation inhibitor having an effect of suppressing the production of NF-κB, IL-6, TNF-α or IFN-α caused by TLR7 activation.

[0019] The present invention also provides a preventive or therapeutic agent for diseases accompanied by TLR7 activation, which contains the above-mentioned TLR7 activation inhibitor.

[0020] The present invention also provides the above-mentioned prophylactic or therapeutic agent, wherein the disease accompanied by activation of TLR7 is an autoimmune disease, an autoinflammatory syndrome, an autoimmune pancreatitis, arteriosclerosis, sepsis, a neurodegenerative disease, graft rejection, graft-versus-host disease, periodontal disease, viral immunodeficiency disease, IgA nephropathy, primary nephrotic syndrome, primary membranoproliferative glomerulonephritis, purpura nephritis, Langerhans cell histiocytosis, hemophagocytic lymphohistiocytosis, Rosai-Dorfman disease, obesity, type 2 diabetes mellitus, or ulcerative colitis.

[0021] The present invention also provides the above-mentioned preventive or therapeutic agent, wherein the disease accompanied by TLR7 activation is an autoimmune disease.

[0022] Furthermore, according to the present invention, the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis / dermatomyositis, mixed connective tissue disease, overlap syndrome, antiphospholipid syndrome, Behcet's disease, adult Still's disease, rheumatic fever, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, HLA-B27-associated rheumatic disease, IgG4-associated syndrome, ANCA-associated vasculitis, vasculitis syndrome, multiple sclerosis, psoriasis vulgaris, inflammatory bowel disease, and the like. The present invention also provides the above-mentioned prophylactic or therapeutic agents for the following diseases, autoimmune thyroid disease, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, primary biliary cirrhosis, primary biliary cholangitis, myasthenia gravis, Goodpasture's syndrome, Guillain-Barré syndrome, chronic atrophic gastritis, rapidly progressive glomerulonephritis, antiglomerular basement membrane nephritis, Addison's disease, type I diabetes mellitus, vitiligo, pemphigus vulgaris, pemphigoid, autoimmune neutropenia, autoimmune hepatitis, and autoimmune pancreatitis.

[0023] The present invention also provides the above-mentioned preventive or therapeutic drug, wherein the autoimmune disease is systemic lupus erythematosus. Effect of the Invention

[0024] Long-term observations in clinical studies have revealed that abnormal TLR7 signaling is one of the causes of systemic lupus erythematosus patients. It is believed that a therapeutic strategy that inhibits TLR7 activation and the associated production of inflammatory cytokines and IFN-α is important. The use of biological agents for the treatment or prevention of autoimmune diseases is not suitable as a long-term treatment for autoimmune diseases from the perspective of the physical and medical economic burden on patients. Therefore, a therapeutic method that is non-invasive and has advantages such as safety, simplicity, and cost-effectiveness is needed as an effective means of treating autoimmune diseases over the long term.

[0025] The present invention provides a TLR7 activation inhibitor and a preventive or therapeutic agent for diseases accompanied by TLR7 activation that contains a TLR7 activation inhibitor, which are non-invasive and have advantages such as safety, simplicity, cost-effectiveness, etc. Furthermore, the present invention provides a therapeutic or preventive agent for inflammatory diseases that is effective in inhibiting the activity of NF-κB, IL-6, and IFN-α. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows the modified areas (areas a and b) in lead compound CB-7. [Diagram 2] FIG. 2 is a graph showing the TLR7 inhibitory activity (suppression of IL-6 expression) of CB-7 or B2-24-4-5A·HCOOH in mouse bone marrow-derived macrophages (BMDM). [Diagram 3] FIG. 3 is a graph showing that B2-24-4-5A·HCOOH is TLR7-selective in mouse BMDM. [Figure 4] FIG. 4 is a graph showing the TLR7 inhibitory activity (inhibition of IL-6 expression) of B2-24-4-5A·HCOOH, B2-24-4·HCl, or hydroxychloroquine sulfate (HCQ) in peripheral blood mononuclear cells (PBMCs) derived from patients with systemic lupus erythematosus. [Diagram 5] FIG. 5 is a graph showing plasma concentrations after oral administration of CB-7 or B2-24-4-5A·HCOOH in mice. [Figure 6] FIG. 6 is a graph showing the plasma concentration of B2-24-4-5A·HCOOH after oral administration in systemic lupus erythematosus model mice. [Figure 7] FIG. 7 is a graph showing the TLR7 inhibitory activity (suppression of IFN-α expression) in mice by intraperitoneal administration of B2-24-4-5A·HCOOH or HCQ. [Figure 8] FIG. 8 is a graph showing the TLR7 inhibitory activity (suppression of IFN-α expression) in mice following intraperitoneal administration of B2-24-4·HCl, B2-24-4-5A, B2-24-4-5A·HCOOH, or HCQ. [Figure 9A] FIG. 9A shows photographs of the spleens of mice in which systemic lupus erythematosus-like pathology was induced (saline-administered group and B2-24-4-5A·HCOOH-administered group). [Figure 9B]FIG. 9B shows the spleen weights of mice in which systemic lupus erythematosus-like pathology was induced (saline-administered group and B2-24-4-5A·HCOOH-administered group). [Figure 9C] FIG. 9C shows the percentage of activated T cells in the spleens of mice in which systemic lupus erythematosus-like pathology was induced (saline-administered group and B2-24-4-5A·HCOOH-administered group). [Figure 9D] FIG. 9D is a photograph showing IgG deposition in the glomeruli of mice in which a systemic lupus erythematosus-like condition was induced (saline-administered group and B2-24-4-5A·HCOOH-administered group). [Figure 10A] FIG. 10A shows the survival rate curves during the administration period in systemic lupus erythematosus model mice (saline administration group, B2-24-4-5A·HCOOH administration group, B2-24-4·HCOOH administration group, and HCQ administration group). [Figure 10B] FIG. 10B shows serum urea nitrogen levels in systemic lupus erythematosus model mice (saline-administered group, B2-24-4-5A·HCOOH-administered group, B2-24-4·HCOOH-administered group, and HCQ-administered group). [Figure 10C] FIG. 10C shows serum creatinine levels in systemic lupus erythematosus model mice (saline-administered group, B2-24-4-5A·HCOOH-administered group, B2-24-4·HCOOH-administered group, and HCQ-administered group). [Figure 10D] FIG. 10D shows the urinary albumin amounts in the systemic lupus erythematosus model mice (saline-administered group, B2-24-4-5A·HCOOH-administered group, B2-24-4·HCOOH-administered group, and HCQ-administered group). [Figure 10E] Figure 10E is a photograph showing IgG deposition in the glomeruli of systemic lupus erythematosus model mice (mouse in the saline-treated group (#1), mouse in the B2-24-4-5A·HCOOH-treated group (#10), mouse in the B2-24-4·HCOOH-treated group (#4), and mouse in the HCQ-treated group (#14)). [Figure 10F]Figure 10F is a photograph showing C3 deposition in the glomeruli of systemic lupus erythematosus model mice (mouse in the saline-treated group (#1), mouse in the B2-24-4-5A·HCOOH-treated group (#10), mouse in the B2-24-4·HCOOH-treated group (#4), and mouse in the HCQ-treated group (#14)). [Figure 10G] FIG. 10G shows a photograph of stained kidney tissue section from a mouse (#1) in the saline-administered group. [Figure 10H] Figure 10H shows a photograph of a stained kidney tissue section from a mouse (#1) administered B2-24-4-5A·HCOOH. [Figure 11A] FIG. 11A shows the spleen weights of systemic lupus erythematosus model mice (saline-administered group, B2-24-4-5A·HCOOH-administered group, and HCQ-administered group). [Figure 11B] FIG. 11B shows the number of splenocytes in the systemic lupus erythematosus model mice (saline-administered group, B2-24-4-5A·HCOOH-administered group, and HCQ-administered group). [Figure 12] Figures 12(A) to (E) show the proportions of various cells in the spleen. [Figure 13] Figures 13(A) to (E) show the proportions of various cells in the spleen. [Figure 14] Figures 14(A) to (E) show the proportions of various cells in the spleen. [Figure 15] FIG. 15 is a graph showing the TLR7 inhibitory activity (suppression of IL-6 expression) of B2-24-4-5A·HCOOH in mouse Flt-3 ligand-induced dendritic cells (FLDCs). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] <Overview and definitions> Hereinafter, an embodiment of the present invention will be described in detail. Note that, in order to avoid repetition, the description of similar contents will be omitted as appropriate.

[0028] For convenience, certain terms used in this application are collected here. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0029] The numerical ranges and parameters given in the present invention are approximate values, but the numerical values ​​given in the specific examples are described as precisely as possible. However, any numerical value inherently contains a certain error that necessarily results from the standard deviation found in each test measurement. Also, the term "about" as used herein generally means within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error as considered by those skilled in the art.

[0030] The wavy line in the formula represents a covalent bond to the compound represented by formula (I) or (II).

[0031] <Embodiment> According to the present invention, there is provided a compound represented by the following formula (I) or (II), a pharmacologically acceptable salt thereof, or a prodrug thereof: [ka] [ka] (In formulas (I) and (II), R1 is, an alkoxy group having 3 to 5 carbon atoms and containing at least two oxygen atoms; an alkoxy group having 2 to 4 carbon atoms and containing at least one hydroxyl group; formula: [ka] (wherein R2 and R3 each independently represent an alkyl group having 1 to 3 carbon atoms), or formula: [ka] (In the formula, Ring C is a 3- to 7-membered nitrogen-containing heterocycle; R4 is a group represented by -NH-, -O-, -CF2-, -CHF-, -C2H2F2- or -CHF-X-CHF-; X is an alkyl group having 1 to 4 carbon atoms. It is a group represented by the following formula:

[0032] R1 is, [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] may be also possible.

[0033] The compound may be a compound represented by any one of the following formulas (III) to (XIII), a pharmacologically acceptable salt thereof, or a prodrug thereof. [ka] [ka] [ka]

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[0034] The compound of the present invention may form a salt, and the salt is preferably a pharmacologically acceptable salt. The pharmacologically acceptable salt is not particularly limited as long as it maintains the activity of the compound and does not have an adverse effect on the living body. Examples of the salt include salts with acids such as acetic acid, propionic acid, butyric acid, formic acid, trifluoroacetic acid, maleic acid, tartaric acid, citric acid, stearic acid, succinic acid, ethylsuccinic acid, malonic acid, lactobionic acid, gluconic acid, glucoheptonic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid (tosylic acid), lauryl sulfuric acid, malic acid, aspartic acid, glutamic acid, adipic acid, cysteine, N-acetylcysteine, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, hydroiodic acid, nicotinic acid, oxalic acid, picric acid, thiocyanic acid, undecanoic acid, acrylic acid polymers, and carboxyvinyl polymers; salts with inorganic bases such as lithium, sodium, potassium, and calcium; salts with organic amines such as morpholine and piperidine; and salts with amino acids. Preferred are salts with hydrochloric acid (hydrochloride salts) or salts with formic acid (formate salts).

[0035] The prodrug means a compound that is converted to any of the compounds represented by the above formulas (I) to (XIII) by a reaction with an enzyme, gastric acid, etc. under physiological conditions in a living body. For example, there are esters that are hydrolyzed in a living body to release these compounds.

[0036] The compound of the present invention or its salt may be a solvate. The solvate is preferably non-toxic and water-soluble. Suitable solvates include, for example, solvates with water and solvates with alcoholic solvents (e.g., methanol, ethanol, etc.).

[0037] The present invention also provides a TLR7 activation inhibitor containing the above-mentioned compound, a pharmacologically acceptable salt thereof, or a prodrug thereof. Examples of TLR7 activation include biological reactions such as production of cytokines such as TNF-α, IL-6, IL-12, IFN-α, or IFN-β in macrophages or dendritic cells, production of chemokines such as CCL2, CCL5, CXCL8, or CXCL10, enhancement of cell proliferation in B cells, enhanced expression of costimulatory molecules such as CD80 or CD86, induction of class switching or antibody production, and induction of immune unresponsiveness in T cells, which are known activities of TLR7. The TLR7 activation inhibitor of the present invention can suppress the above-mentioned activation (biological reaction), and preferably has an effect of suppressing production of NF-κB, IL-6, TNF-α, or IFN-α caused by TLR7 activation. Inhibition of TLR7 activation may mean reducing TLR7 activation (e.g., production of NF-κB, IL-6, TNF-α, or IFN-α) by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9%, or reducing it by a range between any two numbers selected from the above group of numbers. The TLR7 activation inhibitor of the present invention can also be used as a research reagent for analysis of TLR7 expression, function, and associated signaling mechanisms.

[0038] The TLR7 activation inhibitor of the present invention can inhibit TLR7 activation and is therefore useful as a medicament for preventing or treating a disease accompanied by TLR7 activation. That is, the present invention also provides a prophylactic or therapeutic drug for a disease accompanied by TLR7 activation, which contains the TLR7 activation inhibitor of the present invention.

[0039] Examples of diseases associated with TLR7 activation include, but are not limited to, autoimmune diseases, autoinflammatory syndromes, autoimmune pancreatitis, arteriosclerosis, sepsis, neurodegenerative diseases, graft rejection, graft-versus-host disease, periodontal disease, viral immunodeficiency, IgA nephropathy, primary nephrotic syndrome, primary membranoproliferative glomerulonephritis, purpura nephritis, Langerhans cell histiocytosis, hemophagocytic lymphohistiocytosis, Rosai-Dorfman disease, obesity, type 2 diabetes mellitus, and ulcerative colitis.

[0040] The target disease for the prophylactic or therapeutic agent of the present invention is preferably an autoimmune disease, such as systemic lupus erythematosus, Sjögren's syndrome, scleroderma, polymyositis / dermatomyositis, mixed connective tissue disease, overlap syndrome, antiphospholipid syndrome, Behcet's disease, adult Still's disease, rheumatic fever, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, HLA-B27-associated rheumatic diseases (ankylosing spondylitis, Reiter's syndrome, psoriatic arthritis, etc.), IgG4-associated syndrome, ANCA-associated vasculitis (microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis), vasculitis syndrome (rheumatic polyangiitis, eosinophilic ... Myalgia, giant cell vasculitis, polyarteritis nodosa, etc.), multiple sclerosis, psoriasis vulgaris, inflammatory bowel disease, autoimmune thyroid disease (Hashimoto's disease, Graves' disease, etc.), autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, primary biliary cirrhosis, primary biliary cholangitis, myasthenia gravis, Goodpasture's syndrome, Guillain-Barre syndrome, chronic atrophic gastritis, rapidly progressive glomerulonephritis, antiglomerular basement membrane nephritis, Addison's disease, type I diabetes mellitus, vitiligo, pemphigus vulgaris, pemphigoid, autoimmune neutropenia, autoimmune hepatitis, autoimmune pancreatitis, etc. are more preferred.

[0041] The preventive or therapeutic agent of the present invention contains a compound represented by any one of the above formulas (I) to (XIII) as an active ingredient, and can be formulated by appropriately mixing with a pharma- ceutically acceptable carrier or additive according to a known method for producing pharmaceutical preparations (e.g., a method described in the Japanese Pharmacopoeia, etc.). Specifically, for example, tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules and microcapsules), troches, syrups, liquids, emulsions, suspensions, controlled release preparations (e.g., immediate release preparations, sustained release preparations, sustained release microcapsules, etc.), aerosols, films (e.g., orally disintegrating films, oral mucosal patch films, etc.), injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), drip infusions, transdermal preparations, ointments, lotions, patches, suppositories (e.g., rectal suppositories, vaginal suppositories, etc.), pellets, nasal preparations, pulmonary preparations (inhalants), eye drops, and other oral or parenteral preparations. The blending ratio of the carrier or additive can be appropriately set based on the range normally adopted in the pharmaceutical field. The carriers or additives that can be added are not particularly limited, and examples thereof include various carriers such as water, physiological saline, other aqueous solvents, and aqueous or oily bases; and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0042] Examples of additives that can be mixed into tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth, and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, and alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose, or saccharin, and flavors such as peppermint, red pepper oil, or cherry. When the dosage unit form is a capsule, the above-mentioned materials may further contain liquid carriers such as oils and fats. Sterile compositions for injection can be prepared according to standard formulation procedures (for example, dissolving or suspending the active ingredient in a solvent such as water for injection or natural vegetable oil). As aqueous solutions for injection, for example, physiological saline, isotonic solutions containing glucose or other auxiliary agents (for example, D-sorbitol, D-mannitol, sodium chloride, etc.), etc., may be used in combination with suitable solubilizing agents such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80, HCO-50, etc.). As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. Also, a buffering agent (e.g., phosphate buffer, sodium acetate buffer, etc.), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative (e.g., benzyl alcohol, phenol, etc.), an antioxidant, etc. may be added.

[0043] The prophylactic or therapeutic agent of the present invention can be safely administered to humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.).

[0044] The prophylactic or therapeutic agent of the present invention can be produced according to a conventional method by adding the active ingredient in a proportion of usually 0.01 to 100% (w / w), preferably 0.1 to 95% (w / w), based on the total amount of the formulation, although this varies depending on the dosage form, administration method, carrier, etc.

[0045] The dosage of the prophylactic or therapeutic agent of the present invention varies depending on the subject, symptoms, administration route, etc., but in the case of oral administration, it is generally about 0.01 to 1000 mg, preferably about 0.1 to 100 mg, more preferably about 0.5 to 50 mg per day for a human weighing about 60 kg. In the case of parenteral administration, the dosage per dose varies depending on the patient's condition, symptoms, administration method, etc., but for example, an injection is usually administered intravenously at about 0.01 to 100 mg, preferably about 0.01 to 50 mg, more preferably about 0.01 to 20 mg per kg body weight. The total dosage per day may be a single dose or a divided dose.

[0046] The preventive or therapeutic agent of the present invention can be used in combination with other drugs. Examples of other drugs include drugs for treating autoimmune diseases, drugs for treating hyperlipidemia, nonsteroidal anti-inflammatory drugs, and steroid drugs. In particular, it is preferable to use the drug in combination with a drug for treating autoimmune diseases. Examples of drugs for treating autoimmune diseases include steroid drugs including glucocorticoids, nonsteroidal anti-inflammatory drugs, biological preparations such as antibody drugs, immunosuppressants, and antimalarial drugs.

[0047] The present invention includes the following inventions. A method for inhibiting TLR7 activation, comprising administering to a mammal an effective amount of a compound represented by any one of the above formulas (I) to (XIII), a pharmacologically acceptable salt thereof, or a prodrug thereof. A compound represented by any one of the above formulas (I) to (XIII), a pharmacologically acceptable salt thereof, or a prodrug thereof for inhibiting the activation of TLR7. Use of a compound represented by any one of the above formulas (I) to (XIII), a pharmacologically acceptable salt thereof, or a prodrug thereof for the production of an inhibitor of TLR7 activation. A method for preventing or treating a disease accompanied by activation of TLR7, comprising administering to a mammal an effective amount of a compound represented by any one of formulas (I) to (XIII) above, a pharmacologically acceptable salt thereof, or a prodrug thereof. A compound represented by any one of the above formulas (I) to (XIII), a pharmacologically acceptable salt thereof, or a prodrug thereof for use in the prevention or treatment of a disease accompanied by activation of TLR7. Use of a compound represented by any one of the above formulas (I) to (XIII), a pharmacologically acceptable salt thereof, or a prodrug thereof for the manufacture of a preventive or therapeutic agent for a disease involving activation of TLR7. EXAMPLES

[0048] Example 1: Synthesis of derivatives with modified areas a and b Areas a and b in CB-7 shown in Figure 1 were modified. The modification was requested from Albany Molecular Research Inc. (USA). Tables 1 and 2 show the CB-7 derivatives obtained by the modification. Note that B2-24-4·HCl was obtained as the hydrochloride salt.

[0049] [Table 1]

[0050] [Table 2]

[0051] Synthesis of each compound Reagents were purchased from commercial markets and used as received. 1 H NMR spectra were obtained on a 300 MHz Bruker AVANCE 300 spectrometer and a 400 MHz Bruker AVANCE 400 spectrometer using tetramethylsilane as an internal standard. Thin layer chromatography (TLC) was performed using Whatman No. 4500-101 (Diamond No. MK6F silica gel 60 Angstroms) plates.

[0052] The names of compounds (numbered compounds) represented by ["Compound" plus "number"] (e.g., Compound 1) or ["Compound Int-" plus "number"] (e.g., Compound Int-1) are only valid in each synthesis example. Thus, even if a numbered compound in one synthesis example is identical to a numbered compound in another synthesis example, the two compounds may be structurally different, and even if a numbered compound in one synthesis example is different from a numbered compound in another synthesis example, the two compounds may be structurally identical. In other words, the identity between numbered compounds is determined by structure.

[0053] Synthesis Example 1 Synthesis of Compounds B2-6 and B2-6A The preparation method (also referred to as scheme 1) of compounds B2-6 (also referred to as ALB-208276) and B2-6A (also referred to as ALB-208787) is as follows.

[0054] [ka]

[0055] Preparation of compound 2 To a solution of (-)-β-pinene (compound 1) (100 g, 0.73 mol) dissolved in CHCl (1.0 L), MeCN (1.0 L) and H0 (1.0 L) at 0 °C, NaIO (670 g, 3.13 mmol) and RuCl·nH0 (4.95 g, 0.02 mol) were added. The reaction mixture was stirred at room temperature for 5 h. The reaction mixture was diluted with EtOAc (1.0 L) and washed with water (500 mL) and brine (500 mL). The resulting mixture was dried over anhydrous NaSO, filtered and concentrated under reduced pressure to give compound 2 [97.0 g (crude)] as a dark brown liquid. 1H NMR (400 MHz, CDCl3) δ 2.58-2.51 (m, 3H), 2.38-2.30 (m, 1H), 2.25-2.21 (m, 1H), 2.08-2.01 (m, 1H), 1.98-1.90 (m, 1H), 1.57 (s, 1H), 1.35 (s, 3H), 0.84 (s, 3H).

[0056] Preparation of compound 3 A mixture of compound 2 (97.0 g, 0.70 mol), (PhSe)2 (108.57 g, 0.35 mol), SeO2 (93.10 g, 0.84 mol), and H2SO4 (29.8 mL, 0.559 mol) dissolved in MeOH (1.0 L) was stirred at room temperature for 5 h. The reaction mixture was quenched with HO (1.0 L) and extracted with EtOAc (1 L × 2). The organic layer was washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel (60-120 mesh) column chromatography using 5% EtOAc in hexane to give compound 3 (100 g, 48%) as a tan liquid. 1 H NMR (400 MHz, CDCl3) δ 7.61-7.58 (m, 2H),7.32-7.25 (m, 3H), 3.87 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 2.71-2.68 (m, 1H), 2.61-2.52 (m, 2H), 2.24-2.20 (m, 2H), 1.88 (s, 1H), 1.35 (s, 3H), 0.84 (m, 3H).

[0057] Preparation of compound 4 To a solution of compound 3 (32.50 g, 0.110 mol) dissolved in CHCl (400 mL), 7% HO (70.00 mL, 0.160 mmol) and pyridine (12.20 mL, 0.22 mol) were added at 0° C. and stirred at room temperature for 12 h. The reaction mixture was diluted with CHCl (500 mL) and washed with water (200 mL) and brine (250 mL). The resulting organic layer was dried over anhydrous NaSO, filtered and concentrated under reduced pressure. The crude was purified by silica gel (60-120 mesh) column chromatography using 0-5% EtOAc in hexane to give compound 4 (13.00 g, 81%) as a brown liquid. 1 H NMR (400 MHz, CDCl3) δ 7.54-7.50 (m, 1H),5.95 (d, J = 8.8 Hz, 1H), 2.86-2.83 (m, 1H), 2.73-2.57 (m, 2H), 2.13 (s, 1H), 1.51 (s, 3H), 1.04 (m, 3H).

[0058] Preparation of compound 5 A solution of compound 4 (8.00 g, 58.8 mmol) dissolved in THF (100 mL) was added to a mixture of 4-methoxyphenylmagnesium bromide (70.60 mL, 1 M in THF, 70.6 mmol) and CuI (5.60 g, 29.4 mmol) dissolved in THF (10 mL) at -78 °C, and the mixture was stirred at -78 °C for 2 h. The reaction mixture was quenched with saturated NH4Cl (250 mL) and extracted with EtOAc (200 mL x 2). The organic layer was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude was purified by silica gel (60-120 mesh) column chromatography using 0-10% EtOAc in hexane to give compound 5 (9.00 g, 63%) as a colorless liquid. 1H NMR (400 MHz, CDCl3) δ 7.17 (d, J = 8.4 Hz ,2H),6.85 (d, J = 8.4 Hz, 2H), 3.80 (s, 3H), 3.377 (t, J = 8.0 Hz, 1H), 2.81-2.61 (m, 3H), 2.57-2.51 (m, 1H), 2.39-2.36 (m, 1H), 1.89 (d, J = 10.8 Hz, 1H), 1.39 (s, 3H), 0.98 (m, 3H).

[0059] Preparation of compound 6 To a solution of compound 5 (10.70 g, 43.0 mmol) dissolved in Ac2O (100 mL), Zn(OAc)2 (8.36 g, 45.0 mmol), BF3·OEt2 (2.81 mL, 22.0 mmol) were added and stirred at room temperature for 16 h. The reaction mixture was diluted with H2O (100 mL). The resulting mixture was extracted with EtOAc (100 mL×2). The combined organic layers were washed with saturated NaHCO3 (1000 mL) and brine (500 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil, which was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 6 (7.00 g, 56%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.09 (d, J = 8.4 Hz ,2H),6.80 (d, J = 8.8 Hz, 2H), 5.45-5.44 (m, 1H), 4.67-4.59 (m, 2H), 3.77 (s, 3H), 2.96-2.89 (m, 1H), 2.70-2.64 (m, 1H), 2.39-2.24 (m, 4H), 2.11 (s, 3H), 1.49 (s, 3H).

[0060] Preparation of Compound 7 (also referred to as ALB-208224 or I-7 (intermediate)) To a solution (0° C.) of compound 6 (7.00 g, 28.0 mmol) dissolved in MeOH (100 mL) was added NaH (0.57 g, 60% in mineral oil, 14.0 mmol) in portions over 30 min and stirred at room temperature for 30 min. The reaction mixture was quenched with saturated NH4Cl (10 mL) and MeOH was evaporated under reduced pressure. The resulting solid was filtered, washed with water (250 mL) and dried to give compound 7 (ALB-208224 or I-7 (intermediate)) (5.90 g, 99%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.00 (d, J = 8.4 Hz, 2H),6.75 (d, J = 8.8 Hz, 2H), 4.56 (d, J = 10.4 Hz, 2H), 3.71 (s, 3H), 2.89-2.82 (m, 1H), 2.67-2.60 (m, 1H), 2.45 (d, J = 10 Hz, 4H), 2.04-2.00 (m,1H), 1.84-1.76 (m, 1H),1.44 (s, 3H).

[0061] Preparation of compound 8 An ice-cold solution of p-toluenesulfonylmethyl isocyanide (2.39 g, 12.2 mmol), tert-BuOK (2.75 g, 24.6 mmol) in THF (25 mL) was stirred for 10 min and a solution of compound 7 (1.50 g, 6.14 mmol) in THF (5.0 mL) was added. The reaction mixture was stirred at 0° C. for 10 min and MeOH (20 mL) was added. The resulting mixture was stirred under reflux for 1 h and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 8 (0.60 g, 38%) as a colorless liquid. 1H NMR (400 MHz, CDCl3) δ 7.05 (d, J = 8.4 Hz ,2H),6.82 (d, J = 8.8 Hz, 2H), 4.64 (d, J = 10.4 Hz, 2H), 3.78 (s, 3H), 2.96-2.89 (m, 1H), 2.74-2.68 (m, 1H), 2.53-2.49 (m, 4H), 2.11-2.07 (m,1H), 1.91-1.55 (m,1H),1.51 (s, 3H).

[0062] Preparation of compound 9 To a solution of compound 8 (2.40 g, 9.41 mmol) dissolved in 2-propanol (30 mL) was added KOH (5.20 g, 94.1 mmol) at room temperature. The reaction mixture was stirred under reflux for 16 h. The reaction mixture was diluted with cold water (100 mL) and acidified by the addition of HCl (2 N). The resulting mixture was extracted with EtOAc (50 mL×2). The organic layers were combined, washed with brine (100 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 9 (2.20 g, 85%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.17 (d, J = 8.8 Hz ,2H), 6.85 (d, J = 8.8 Hz, 2H), 4.64 (d, J = 10.4 Hz, 2H), 3.79 (s, 3H), 3.37 (t, J = 8.0 Hz, 2H), 2.81-2.54 (m, 4H), 2.39-2.36 (m, 1H), 1.89 (t, J = 5.4 Hz, 1H), 2.11-2.07 (m,1H), 1.91-1.55 (m,1H),1.51 (s, 3H).

[0063] Preparation of compound 10 To an ice-cold solution of compound 9 (2.30 g, 8.424 mmol) in CH3CN (20 mL) was added K2CO3 (3.25 g, 23.58 mmol) and MeI (1.57 mL, 25.27 mmol) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 12 h. The resulting mixture was extracted with EtOAc (100 mL × 3). The organic layers were combined, washed with brine (100 mL), dried over MgSO4, and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 10 (2.20 g, 91.6%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.04 (d, J = 8.8 Hz ,2H),6.79 (d, J = 8.4 Hz, 2H), 4.55 (m, 2H), 3.76 (s, 3H), 3.65 (s, 3H), 2.54-2.45 (m, 2H), 2.30-2.24 (m, 1H), 2.10-2.06 (m, 2H), 1.89-1.85 (m,1H), 1.62-1.58 (m,3H),1.49 (s, 3H).

[0064] Preparation of compound 11 To a solution of lithium diisopropylamide (5.20 mL, 2.0 M in THF, 10.4 mmol) dissolved in THF (6.0 mL) was added dropwise compound 10 (1.00 g, 3.40 mmol) dissolved in THF (4.0 mL) at -78 °C. The reaction mixture was stirred at -78 °C for 30 min. 2-tert-butyl bromoacetate (1.00 mL, 6.80 mmol) was added and the reaction mixture was stirred at -78 °C for 30 min and quenched with saturated NH4Cl solution (15 mL). The resulting mixture was extracted with EtOAc (50 mL x 3). The organic layers were combined, washed with brine (25 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 11 (0.50 g, 36%) as a colorless liquid.

[0065] Preparation of compound 12 To a solution (0° C.) of compound 11 (0.5 g, 1.24 mmol) dissolved in 2,2,2-trifluoroethanol (2.0 mL) was added TMSCl (0.5 mL, 3.94 mmol) dropwise. The reaction mixture was stirred at 0° C. for 2 h. Residual solvent was evaporated and the residue was diluted with EtOAc (15 mL) and washed with water (20 mL). The organic layer was washed with brine (15 mL), dried over MgSO4, and concentrated under reduced pressure to give a colorless gum. This was purified by combiflash column chromatography using 0-50% EtOAc in hexane to give compound 12 (0.40 g, 74%) as a colorless gum.

[0066] Preparation of compound 13 To an ice-cold solution of compound 12 (0.14 g, 0.40 mmol) in CHCl (20 mL) was added TEA (0.26 mL, 1.20 mmol), T3P (50% w / w in EtOAc) (0.39 mL, 1.20 mmol), followed by N,N-dimethylamine (1 M in THF) (0.4 mL, 0.80 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 h and diluted with H2O (5.0 mL). The resulting mixture was extracted with CHCl (5.0 mL × 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-60% EtOAc in hexane to give compound 13 (0.15 g, 87%) as a colorless gum. ESI MS m / z 374 [M+H] + .

[0067] Preparation of Compounds ALB-208276 (B2-6) and ALB-208787 (B2-6A) To an ice-cold solution of compound 13 (0.13 g, 0.30 mmol) dissolved in THF (5.0 mL) was added LiAlH4 (0.50 mL, 2.0 M in THF, 0.90 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and diluted with H2O and HCl (2 N). The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-70% EtOAc in hexane to give ALB-208276 (B2-6) (0.03 g, 25%) and ALB-208787 (B2-6A) (0.015 g, 13%) as an off-white colorless gum.

[0068] ALB-208276(B2-6) 1 H NMR (400 MHz, DMSO-d6) δ 7.05 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 4.78 (t, J = 5.2 Hz, 1H), 4.48 (d, 2H), 3.70 (s, 3H), ESI MS m / z 346 [M+H] + .

[0069] ALB-208787(B2-6A) 1H NMR (400 MHz, CDCl3) δ 7.06 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 4.53 (d, 2H), 3.77 (s, 3H), 3.77-3.60 (m, 2H), 2.71-2.65 (m, 1H), 2.59-2.41 (m, 2H), 2.32 (s, 6H), 2.27-2.20 (m, 1H), 1.88-1.83 (m, 2H), 1.68-1.51 (m, 3H), 1.52 (s, 3H), 1.48-1.42 (m, 3H); ESI MS m / z 332 [M+H] + .

[0070] Synthesis Example 2 Synthesis of Compound B2-5A The preparation method (also referred to as scheme 2) of compound B2-5A (also referred to as ALB-208786) is as follows.

[0071] [ka]

[0072] To an ice-cold solution of compound Int-12 (compound 12 from scheme 1) (0.10 g, 0.28 mmol) dissolved in THF (5.0 mL) was added LiBH4 (1.40 mL, 1.0 M in THF, 2.80 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min, heated at 60 °C for 12 h, and quenched with saturated NH4Cl solution (10 mL). The resulting reaction mixture was extracted with EtOAc (10 mL x 2). The extracts were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-70% EtOAc in hexanes, followed by mass triggered preparative-HPLC to give ALB-208786 (B2-5A) (0.005 g, 6%) as a colorless gum. 1H NMR (400 MHz, CDCl3) δ 7.03 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 4.57 (s, 2H), 4.31-4.27 (m, 2H), 3.78 (s, 3H), 2.53-2.46 (m, ESI MS m / z 301 [M+H] + .

[0073] Synthesis Example 3 Synthesis of Compound B2-6-7 The preparation method (also referred to as scheme 3) of compound B2-6-7 (also referred to as ALB-209871) is as follows.

[0074] [ka]

[0075] Preparation of Compound 1 To an ice-cold solution of compound Int-12 (compound 12 in scheme 1) (0.05 g, 0.14 mmol) dissolved in CHCl (3.0 mL) was added TEA (0.04 mL, 0.29 mmol), T3P (50% w / w in EtOAc) (0.14 mL, 0.43 mmol), and morpholine (0.02 g, 0.21 mmol) in that order at 0 °C. The reaction mixture was stirred at room temperature for 2 h and diluted with H2O (5.0 mL). The resulting reaction mixture was extracted with CHCl (5.0 mL × 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-70% EtOAc in hexane to give compound 1 (0.055 g, 70%) as a colorless gum. ESI MS m / z 416 [M+H] + .

[0076] Preparation of compound ALB-209871 (B2-6-7) To an ice-cold solution of compound 1 (0.03 g, 0.70 mmol) dissolved in THF (3.0 mL) was added LiAlH4 (0.10 mL, 2.0 M in THF, 0.21 mmol). The reaction mixture was stirred at 0 °C for 5 min and diluted with saturated NH4Cl solution (5.0 mL). The resulting reaction mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-70% EtOAc in hexane to give compound ALB-209871 (B2-6-7) (0.002 g, 8%) as an off-white colorless gum. 1 H NMR (400 MHz, CDCl3) δ 7.04 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 4.61 - 4.45 (m, 3H), 3.77-3.53 (m, 13H), 2.74-2.67 (m, 1H), 2.39-2.20 (m, 3H), 1.96-1.90 (m, 2H), 1.69-1.54 (m, 3H), 1.51 (s, 4H); ESI MS m / z 388 [M+H] + .

[0077] Synthesis Example 4 Synthesis of Compound B2-22 The preparation method (also referred to as scheme 4) of compound B2-22 (also referred to as ALB-210362) is as follows.

[0078] [ka]

[0079] Preparation of compound 2 To a stirred solution of compound 1 (15.0 g, 0.09 mol) in DMF (150 mL) was added imidazole (17.1 g, 0.29 mol) followed by TBDMSCl (22.2 g, 0.14 mol) in portions at 0 °C. The reaction mixture was stirred at room temperature for 16 h, diluted with EtOAc (2 × 100 mL) and washed with saturated NaHCO3 (500 mL). The organic layers were combined, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-50% EtOAc in hexane to give compound 2 (12.00 g, 48%) as an off-white solid.

[0080] Preparation of compound 3 A solution of compound Int-4 (compound 4 from scheme 1) (3.00 g, 22.00 mmol) dissolved in 1,4-dioxane (70 mL) was charged with compound 2 (8.70 g, 33.0 mmol), KOH (2 M, 2.45 g, 44.00 mmol) and degassed with argon for 15 min. [Rh(COD)Cl]2 complex (0.33 g, 0.66 mmol) was added to the reaction mixture, which was stirred and heated under reflux for 12 h. Residual solvent was evaporated. The crude was dissolved in CHCl (250 mL) and filtered through a bed of Celite. The filtrate was washed with water (250 mL), dried over anhydrous NaSO, filtered and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 3 (4.60 g, 59%) as an off-white solid. ESI MS m / z 376 [M+NH4] +

[0081] Preparation of compound 4 To a solution of 3 (4.00 g, 11.1 mmol) in Ac2O (20 mL), Zn(OAc)2 (2.24 g, 12.0 mmol), BF3·OEt2 (1.64 mL, 13.3 mmol) were added and stirred at room temperature for 12 h. The reaction mixture was diluted with H2O (100 mL). The mixture was extracted with EtOAc (100 mL×2). The combined organic layers were washed with saturated NaHCO3 solution (250 mL) and brine (100 mL). The reaction mixture was dried over MgSO4 and concentrated under reduced pressure to give a residual oil. It was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give 4 (2.50 g, 78%) as an off-white solid.

[0082] Preparation of compound 5 To a solution of compound 4 (5.00 g, 17.4 mmol) dissolved in MeOH (50 mL) was added NaH (0.70 g, 60% in mineral oil, 17.4 mmol) portionwise over 30 min at 0 °C and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was quenched with water (10 mL) at 0 °C. MeOH was evaporated and the resulting solid was filtered and washed with water and then hexane (250 mL) to give compound 5 (3.60 g, 85%) as an off-white solid. ESI MS m / z 262 [M + NH4] + .

[0083] Preparation of compound 6 To an ice-cold solution of p-toluenesulfonylmethyl isocyanide (3.90 g, 20.4 mmol) and tert-BuOK (4.59 g, 40.9 mmol) in THF (20 mL) was added compound 5 (2.50 g, 10.2 mmol) in THF (5.0 mL), stirred at 0 °C for 10 min, and MeOH (15 mL) was added. The resulting mixture was stirred under reflux for 1 h and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-50% EtOAc in hexane to give compound 6 (1.70 g, 65%) as a pale yellow liquid. ESI MS m / z 273 [M + NH4] + .

[0084] Preparation of compound 7 To a solution of compound 6 (2.60 g, 10.01 mmol) dissolved in 2-propanol (40 mL) was added KOH (5.70 g, 101.00 mmol) at room temperature. The reaction mixture was stirred under reflux for 48 h. Residual solvent was evaporated under reduced pressure. The crude was dissolved in EtOAc (50 mL) and washed with water (50 mL). The aqueous layer was neutralized by adding HCl (2 N) and extracted with EtOAc (50 mL x 2). The organic layers were combined, washed with brine (50 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 7 (1.30 g, 48%) as a pale yellow solid. ESI MS m / z 273 [MH] + .

[0085] Preparation of compound 8 To a solution of compound 7 (1.30 g, 4.50 mmol) dissolved in CH3CN:MeOH (15:5 mL) was added K2CO3 (1.88 g, 13.50 mmol) and MeI (0.83 mL, 13.50 mmol) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 12 h and extracted with EtOAc (50 mL × 2). The organic layers were combined, washed with brine (50 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 8 [1.10 g (crude)] as a brown viscous material. ESI MS m / z 271 [M + NH4] + .

[0086] Preparation of compound 9 To a stirred solution of compound 8 (0.70 g, 2.40 mol) in CHCl (10 mL) was added imidazole (0.35 g, 6.00 mmol) followed by TBDMS-Cl (0.44 g, 2.90 mmol) in portions at 0 °C. The reaction mixture was warmed to room temperature and stirred for 16 h. The reaction solution was diluted with CHCl (2 × 500 mL) and washed with saturated NaHCO (500 mL). The organic layers were combined, dried over anhydrous NaSO and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 9 (0.63 g, 64%) as a colorless oil.

[0087] Preparation of compound 10 To a solution of lithium diisopropylamide (6.30 mL, 2.0 M in THF, 12.60 mmol) dissolved in THF (15 mL) was added dropwise compound 9 (1.70 g, 4.20 mmol) dissolved in THF (5.0 mL) at -78 °C. The reaction mixture was stirred at -78 °C for 30 min, cooled to -105 °C (using MeOH, liquid N2), and phenylvinyl sulfoxide (1.12 mL, 8.40 mmol) was added. The reaction mixture was stirred at -105 °C for 30 min, and saturated NH4Cl solution (15 mL) was added to the flask. The resulting mixture was extracted with EtOAc (50 mL x 2). The organic layers were combined and washed with brine (50 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 10 (0.80 g, 34%) as a pale yellow oil. ESI MS m / z 555 [M+H] + .

[0088] Preparation of compound 11 A mixture of compound 10 (0.80 g, 1.44 mmol) and NaHCO3 (1.20 g, 14.40 mmol) in xylene (10 mL) was stirred under reflux for 18 h and diluted with H2O. The resulting mixture was extracted with EtOAc (30 mL x 3). The organic layers were combined, washed with brine (25 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil, which was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 11 (0.40 g, 60%) as a colorless gum.

[0089] Preparation of compound 12 To an ice-cold solution of compound 11 (0.40 g, 0.93 mmol) in THF (5.0 mL) was added TBAF (1.86 mL, 1 M in THF, 1.80 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 6 h and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil. This was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 12 (0.15 g, 51%) as a colorless oil. ESI MS m / z 332 [M + NH4] + .

[0090] Preparation of compound 13 To an ice-cold solution of compound 12 (0.135 g, 0.40 mmol) in CHCl (5.0 mL) was added Dess-Martin periodinane (0.40 g, 0.90 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NaSO (5.0 mL), saturated NaHCO solution (5.0 mL) and extracted with CHCl (10 mL × 2). The organic layers were combined, washed with brine (10 mL), dried over MgSO, and concentrated under reduced pressure to give compound 13 (0.11 g, 82%) as a colorless oil. ESI MS m / z 313 [M + H] + .

[0091] Preparation of compound 14 To a solution of compound 13 (0.025 g, 0.06 mmol) dissolved in MeOH (5.0 mL) and a small amount of AcOH, diethylamine (0.009 g, 0.12 mmol) was added followed by NaCNBH3 (0.016 g, 0.25 mmol) at room temperature. The reaction mixture was stirred at room temperature for 48 h. MeOH was evaporated under reduced pressure and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 14 [0.025 g (crude)] as a colorless gum. ESI MS m / z 370 [M + H] + .

[0092] Preparation of compound ALB-210362 (B2-22) To an ice-cold solution of compound 14 (0.025 g, 0.06 mmol) dissolved in THF (4.0 mL) was added LiAlH4 (0.08 mL, 2.0 M in THF, 0.16 mmol) at 0°C. The reaction mixture was stirred at 0°C for 15 min and quenched with HCl solution (2N, 3 mL). The resulting mixture was extracted with EtOAc (5.0 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by mass-triggered prep-HPLC to give compound ALB-210362 (B2-22) (0.0055 g, 26%) as a colorless gum. 1H NMR (400 MHz, CDCl3) δ 7.25 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 5.78-5.71 (m, 1H), 5.16-5.05 (m, 2H), 4.52 (d, 2H), 3.70-3.64 (m, 4H), 2.75-2.69 (m, 1H), 2.64-2.50 (m, 4H), 2.30-2.24 (m, 1H), 1.90-1.86 (m, 2H), 1.70-1.64 (m, 3H), 1.51 (s, 3H), 1.47-1.41 (m, 2H), 1.108 (t, J = 7.2 Hz, 6H); ESI MS m / z 342 [M+H] + .

[0093] Synthesis Example 5 Synthesis of Compound B2-24-1 The preparation method (also referred to as scheme 5) of compound B2-24-1 (also referred to as ALB-210798) is as follows.

[0094] [ka]

[0095] Preparation of Compound 1 To a solution of compound Int-13 (compound 13 in scheme 4) (0.025 g, 0.08 mmol) dissolved in MeOH (4.0 mL) and a small amount of AcOH, 4,4-difluropiperdine hydrochloride (0.025 g, 0.12 mmol) was added followed by NaCNBH3 (0.016 g, 0.25 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. MeOH was evaporated and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 1 [0.025 g (crude)] as a colorless viscous material. ESI MS m / z 418 [M + H] + .

[0096] Preparation of compound ALB-210798 (B2-24-1) To an ice-cold solution of compound 1 (0.025 g, 0.05 mmol) dissolved in THF (4.0 mL) was added LiAlH4 (0.07 mL, 2.0 M in THF, 0.14 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (5.0 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by mass-triggered prep-HPLC to give compound ALB-210798 (B2-24-1) (0.007 g, 26%) as an off-white solid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.17 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 5.77-5.70 (m, 1H), 5.16-5.04 (m, 2H), 4.53 (d, 2H), 3.70 (m, 2H), 3.50 (s, 2H), 2.76-2.70 (m, 1H), 2.57-2.49 (m, 4H), 2.35-2.25 (m, 1H), 2.02-1.87 (m, 6H), 1.70-1.64 (m, 2H), 1.51 (s, 3H), 1.47-1.44 (m, 2H); ESI MS m / z 390 [M+H] + .

[0097] Synthesis Example 6 Synthesis of Compound B2-24-2 The preparation method (also referred to as Scheme 6) of compound B2-24-2 (also referred to as ALB-210361) is as follows.

[0098] [ka]

[0099] Preparation of Compound 1 To a solution of compound Int-13 (compound 13 in scheme 4) (0.025 g, 0.08 mmol) dissolved in MeOH (4.0 mL) and a small amount of AcOH, morpholine (0.014 g, 0.12 mmol) was added followed by NaCNBH3 (0.016 g, 0.25 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. MeOH was evaporated and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 1 [0.025 g (crude)] as a colorless gum. ESI MS m / z 384 [M + H] + .

[0100] Preparation of compound ALB-210361 (B2-24-2) To an ice-cold solution of compound 1 (0.025 g, 0.05 mmol) dissolved in THF (4.0 mL) was added LiAlH4 (0.10 mL, 2.0 M in THF, 0.19 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (5.0 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by mass-triggered preparative HPLC to give compound ALB-210361 (B2-24-2) (0.007 g, 26%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 5.78-5.70 (m, 1H), 5.16-5.04 (m, 2H), 4.53 (d, 2H), 3.70 (m, 6H), 3.47 (s, 2H), 2.76-2.70 (m, 1H), 2.44-2.25 (m, 5H), 1.91-1.85 (m, 2H), 1.70-1.64 (m, 3H), 1.50 (s, 3H), 1.47-1.41 (m, 2H); ESI MS m / z 356 [M+H] + .

[0101] Synthesis Example 7 Synthesis of Compound B2-24-3 The preparation method (also referred to as scheme 7) of compound B2-24-3 (also referred to as ALB-210795) is as follows.

[0102] [ka]

[0103] Preparation of Compound 1 To a solution of compound Int-13 (compound 13 in scheme 4) (0.025 g, 0.08 mmol) dissolved in MeOH (4.0 mL) and a small amount of AcOH, 3,3-difluoroazitidine hydrochloride (0.02 g, 0.12 mmol) was added followed by NaCNBH3 (0.016 g, 0.25 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. MeOH was evaporated and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 1 [0.025 g (crude)] as a colorless viscous material. ESI MS m / z 390 [M + H] + .

[0104] Preparation of compound ALB-210795 (B2-24-3) To an ice-cold solution of compound 1 (0.025 g, 0.05 mmol) dissolved in THF (4.0 mL) was added LiAlH4 (0.08 mL, 2.0 M in THF, 0.15 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (5.0 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by mass-triggered prep-HPLC to give compound ALB-210795 (B2-24-3) (0.005 g, 26%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 7.16 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 5.77-5.70 (m, 1H), 5.15-5.04 (m, 2H), 4.53 (d, 2H), 3.69 (s, 2H), 3.67 (s, 2H), 3.57 (t, J = 12.0 Hz, 4H), 2.76-2.69 (m, 1H), 2.30-2.24 (m, 1H), 1.91-1.87 (m, 2H), 1.70-1.64 (m, 3H), 1.50 (s, 3H), 1.47-1.41 (m, 2H); ESI MS m / z 362 [M+H] + .

[0105] Synthesis Example 8 Synthesis of compound B2-24-4·HCl The preparation method (also referred to as scheme 8) of compound B2-24-4·HCl (also referred to as ALB-210796) is as follows.

[0106] [ka]

[0107] Preparation of Compound 1 To a solution of compound Int-13 (compound 13 in scheme 4) (0.035 g, 0.11 mmol) dissolved in MeOH (5.0 mL) and a small amount of AcOH, tert-butyl piperzine-1-carboxylate (0.041 g, 0.22 mmol) was added followed by NaCNBH3 (0.022 g, 0.30 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. MeOH was evaporated and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 1 [0.050 g (crude)] as a colorless gum. ESI MS m / z 483 [M + H] + .

[0108] Preparation of compound 2 To an ice-cold solution of compound 1 (0.050 g, 0.10 mmol) dissolved in THF (4.0 mL) was added LiAlH4 (0.15 mL, 2.0 M in THF, 0.31 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (5.0 mL × 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by mass-triggered prep-HPLC to give compound 2 (0.005 g, 26%) as a colorless gum. ESI MS m / z 455 [M + H] + .

[0109] Preparation of compound ALB-210796 (B2-24-4·HCl) To an ice-cold solution of compound 2 (0.050 g, 0.10 mmol) in CHCl (5.0 mL) was added dioxane·HCl (4 N, 0.5 mL) at 0 °C. The reaction mixture was stirred at room temperature for 3 h. Residual solvent was evaporated under reduced pressure. The crude was washed with 30% CHCl in hexanes and purified by mass-triggered preparative HPLC to give compound ALB-210796 (B2-24-4·HCl) (0.012 g, 27%) as an off-white solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.15-7.10 (m, 4H), 5.88-5.81 (m, 1H), 4.94-4.89 (m, 2H), 4.54-4.44 (d, 2H), 3.54 (s, 2H), 3.39 (s, 2H), 2.84 (t, J=4.4 Hz, 4H), 2.78-2.67 (m, 1H), 2.37-2.27 (m, 5H), 1.77-1.51 (m, 4H), 1.49 (s, 3H), 1.41-1.25 (m, 2H); ESI MS m / z 355 [M+H] + .

[0110] Synthesis Example 9 Synthesis of Compound B2-13 The preparation method (also referred to as scheme 9) of compound B2-13 (also referred to as ALB-209346) is as follows.

[0111] [ka]

[0112] Preparation of compound 2 To a suspension (0°C) of compound Int-12 (compound 12 from scheme 4) (0.025 g, 0.07 mmol) and NaH (0.012 g, 60% in mineral oil, 0.32 mmol) in DMF (1.0 mL), compound 1 (0.022 g, 0.15 mmol) was added at 0°C to form a reaction mixture. The reaction mixture was warmed to room temperature, stirred for 6 h, and quenched by the addition of HCl solution (1 N, 5.0 mL). The resulting mixture was extracted with EtOAc (10 mL x 3). The organic layers were combined, dried over MgSO4, and concentrated under reduced pressure to give a residue. This was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 2 (0.015 g, 51%) as a colorless oil. ESI MS m / z 390 [M + NH4] + .

[0113] Preparation of compound ALB-209346 (B2-13) To an ice-cold solution of compound 2 (0.015 g, 0.04 mmol) dissolved in THF (5.0 mL) was added LiAlH4 (2.0 M in THF, 0.06 mL, 0.12 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and quenched with saturated NH4Cl (10 mL). The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-50% EtOAc in hexane to give compound ALB-209346 (B2-13) (0.0085 g, 43%) as a colorless gum. 1H NMR (400 MHz, CDCl3) δ 7.23 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 5.77-5.70 (m, 1H), 5.15-5.04 (m, 2H), 4.54 (d, 2H), 4.51 (s, 2H), 3.69 (s, 2H), 3.69-3.57 (m, 4H), 3.38 (s, 3H), 2.77-2.70 (m, 1H), 2.33-2.26 (m, 1H), 1.91-1.84 (m, 2H), 1.70-1.60 (m, 2H), 1.52 (s, 3H), 1.48-1.41 (m, 2H); ESI MS m / z 362 [M+NH4] + .

[0114] Synthesis Example 10 Synthesis of Compound B2-26 The preparation method (also referred to as scheme 10) of compound B2-26 (also referred to as ALB-209873) is as follows.

[0115] [ka]

[0116] Preparation of compound 2 To a stirred solution of compound 1 (0.50 g, 3.60 mmol) in CHCl (10 mL) was added imidazole (0.53 g, 9.00 mmol) followed by TBDMSCl (0.65 g, 4.30 mmol) in portions at 0 °C. The reaction mixture was stirred at room temperature for 16 h, diluted with CHCl (50 mL), washed with saturated NaHCO (50 mL), dried over NaSO, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 2 (0.5 g, 54%) as a colorless oil.

[0117] Preparation of compound 3 To a solution of compound Int-12 (compound 12 in scheme 4) (0.04 g, 0.13 mmol) dissolved in DMF (3.0 mL), NaH (0.02 g, 60% in mineral oil, 0.51 mmol) was added at 0 °C and stirred for 15 min. Compound 2 (0.064 g, 0.25 mmol) was added to the reaction mixture at 0 °C. The reaction was carried out at room temperature for 6 h and quenched by the addition of HCl (2 N, 5.0 mL). The resulting mixture was extracted with EtOAc (10 mL × 3), dried over MgSO4, and concentrated under reduced pressure to give a residue. It was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 3 (0.025 g, 40%) as a colorless oil. ESI MS m / z 487 [M + H] + .

[0118] Preparation of compound 4 To an ice-cold solution of compound 3 (0.025 g, 0.05 mmol) dissolved in THF (3.0 mL) was added LiAlH4 (2.0 M in THF, 0.08 mL, 0.15 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and quenched with saturated NH4Cl (10 mL). The resulting mixture was extracted with EtOAc (10 mL × 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-40% EtOAc in hexane to give compound 4 (0.02 g, 85%) as a colorless oil.

[0119] Preparation of compound ALB-209873 (B2-26) To an ice-cold solution of compound 4 (0.02 g, 0.04 mmol) dissolved in THF (2.0 mL) was added TBAF (0.08 mL, 1 M in THF, 0.08 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 8 h and diluted with H2O (5.0 mL). The resulting mixture was extracted with EtOAc (10 mL x 2). The extracts were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil, which was purified by combiflash column chromatography using 0-50% EtOAc in hexane to give compound ALB-209873 (B2-26) (0.009 g, 60%) as a colorless gum. 1 H NMR (400 MHz, CDCl3) δ 7.21 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 5.77-5.70 (m, 1H), 5.15-5.04 (m, 2H), 4.54 (d, 2H), 4.47 (s, 2H), 3.78 (t, J = 5.6 Hz, 2H), 3.70 (s, 2H), 3.65 (t, J = 6.0 Hz, 2H), 2.75-2.71 (m, 1H), 2.33-2.26 (m, 1H), 1.91-1.83 (m, 4H), 1.70-1.60 (m, 2H), 1.52 (s, 3H), 1.48-1.41 (m, 2H); ESI MS m / z 362 [M+NH 4 ] + .

[0120] Synthesis Example 11 Synthesis of Compound B2-24 The preparation method (also referred to as scheme 11) of compound B2-24 (also referred to as ALB-209348) is as follows.

[0121] [ka]

[0122] Preparation of Compound 1 To an ice-cold solution of compound Int-8 (compound 8 from scheme 4) (0.28 g, 0.97 mmol) dissolved in CHCl (10 mL) was added Dess-Martin periodinane (0.82 g, 1.90 mmol) at 0 °C. The reaction mixture was warmed to room temperature and stirred at the same temperature for 2 h. The reaction mixture was quenched with saturated NaSO solution (5.0 mL), saturated NaHCO solution (5.0 mL) and extracted with CHCl (10 mL × 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO, and concentrated under reduced pressure to give compound 1 (0.2 g, 72%) as a colorless oil. ESI MS m / z 287 [M + H] + .

[0123] Preparation of compound 2 To a solution of compound 1 (0.2 g, 0.69 mmol) dissolved in MeOH (5.0 mL) and a small amount of AcOH, pyrrolidine (0.10 mL, 1.30 mmol) was added followed by NaCNBH3 (0.13 g, 2.08 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. MeOH was evaporated and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil. This was purified by combiflash column chromatography using 0-90% EtOAc in hexane to give compound 2 (0.15 g, 63%) as a colorless gum. ESI MS m / z 342 [M + H] + .

[0124] Preparation of compound 3 To a solution of lithium diisopropylamide (0.65 mL, 2.0 M in THF, 1.30 mmol) dissolved in THF (5.0 mL) was added dropwise compound 2 (0.15 g, 0.43 mmol) dissolved in THF (4.0 mL) at -78 °C. The solution was stirred at -78 °C for 30 min, then cooled to -105 °C (MeOH, liquid N2) and phenylvinyl sulfoxide (0.12 mL, 0.87 mmol) was added. The reaction mixture was stirred at -105 °C for 30 min and quenched with saturated NH4Cl solution (15 mL). The resulting mixture was extracted with EtOAc (20 mL x 2). The organic layers were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 3 [0.2 g (crude)] as a brown oil. ESI MS m / z 494 [M + H] + .

[0125] Preparation of compound 4 A solution of compound 3 [0.20 g (crude), 0.40 mmol] and NaHCO3 (0.34 g, 4.00 mmol) dissolved in xylene (5.0 mL) was stirred under reflux for 16 h and diluted with H2O. The resulting mixture was extracted with EtOAc (30 mL x 3). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 4 [0.10 g (crude)] as a brown liquid. ESI MS m / z 368 [M + H] + .

[0126] Preparation of compound ALB-209348 (B2-24) To an ice-cold solution of compound 4 (0.06 g, 0.12 mmol) dissolved in THF (2.0 mL) was added LiAlH4 (0.18 mL, 2.0 M in THF, 0.36 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (5.0 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by mass-triggered prep-HPLC to give compound ALB-209348 (B2-24) (0.007 g, 8%) as a colorless gum. 1 H NMR (400 MHz, CDCl3) δ 7.31 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 5.78-5.71 (m, 1H), 5.17-5.05 (m, 2H), 4.52 (d, 2H), 4.03-3.99 (m, 2H), 3.80-3.63 (m, 3H), 3.09-3.05 (m, 3H), 2.79-2.74 (m, 1H), 2.31-2.25 (m, 1H), 2.08-2.04 (m, 4H), 1.91-1.87 (m, 3H), 1.70-1.64 (m, 2H), 1.51 (s, 3H), 1.48-1.41 (m, 2H); ESI MS m / z 340 [M+H] + .

[0127] Synthesis Example 12 Synthesis of Compound B2-5A-9 The preparation method (also referred to as scheme 12) of compound B2-5A-9 (also referred to as ALB-210799) is as follows.

[0128] [ka]

[0129] Preparation of Compound 1 To a solution of lithium diisopropylamide (3.73 mL, 2.0 M in THF, 7.40 mmol) dissolved in THF (6.0 mL) was added compound Int-9 (compound 9 in scheme 4) [1.0 g (crude), 2.48 mmol] dissolved in THF (4.0 mL) dropwise at -78 °C. The solution was stirred at -78 °C for 30 min, and tert-butyl 2-bromoacetate (1.00 mL, 4.90 mmol) was added to the reaction mixture. The reaction mixture was stirred at -78 °C for 30 min and quenched with saturated NH4Cl solution (15 mL). The resulting mixture was extracted with EtOAc (50 mL x 2). The organic layers were combined, washed with brine (25 mL), dried over Na2SO4, and concentrated under reduced pressure to give compound 1 [0.90 g (crude)] as a brown liquid.

[0130] Preparation of compound 2 To an ice-cold solution of compound 10 (0.90 g, 1.70 mmol) in THF (15 mL) was added TBAF (5.20 mL, 1 M in THF, 5.20 mmol) at 0° C. The reaction mixture was stirred at room temperature for 12 h and diluted with H2O. The resulting mixture was extracted with EtOAc (50 mL×2). The organic layers were combined, washed with brine (25 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil, which was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 2 (0.40 g, 40%) as a colorless gum.

[0131] Preparation of compound 3 To an ice-cold solution of compound 2 (0.15 g, 0.40 mmol) in CHCl (5.0 mL) was added Dess-Martin periodinane (0.32 g, 0.80 mmol) at 0° C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated NaSO (10 mL), saturated NaHCO (10 mL) solution and extracted with CHCl (10 mL×2). The organic layers were combined, washed with brine (10 mL), dried over MgSO, and concentrated under reduced pressure to give compound 3 [0.15 g (crude)] as a colorless oil.

[0132] Preparation of compound 4 To a solution of compound 3 (0.15 g, 3.70 mmol) dissolved in MeOH (5.0 mL) and a small amount of AcOH, pyrrolidine (0.06 g, 7.50 mmol) was added followed by NaCNBH3 (0.07 g, 1.13 mmol) at room temperature. The reaction mixture was stirred at room temperature for 18 h. MeOH was evaporated and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure to give a colorless oil. This was purified by combiflash column chromatography using 0-90% EtOAc in hexane to give compound 4 (0.15 g, 88%) as a colorless gum. ESI MS m / z 456 [M + H] + .

[0133] Preparation of compound 5 To an ice-cold solution of compound 4 (0.13 g, 0.28 mmol) dissolved in THF (4.0 mL) was added LiAlH4 (2.0 M in THF, 0.90 mL, 0.85 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 3 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (10 mL × 2). The organic layers were combined, washed with brine (15 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 5 [0.08 g (crude)] as a colorless oil. ESI MS m / z 428 [M + H] + .

[0134] Preparation of compound ALB-210799 (B2-5A-9) To a solution of compound 5 (0.08 g, 0.18 mmol) dissolved in CHCl (5.0 mL) was added p-toluenesulfonic acid (0.040 g, 0.37 mmol) at room temperature. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was quenched with saturated NaHCO solution (10 mL) and extracted with CHCl (10 mL×2). The organic layers were combined, washed with brine (10 mL), dried over MgSO, and concentrated under reduced pressure. The residue was purified by mass-triggered prep-HPLC to give compound ALB-210799 (B2-5A-9) (0.00775 g, 8%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.22 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.0 Hz, 2H), 4.55 (s, 2H), 4.28 (q, J = 8.0 Hz, 2H), 3.58 (s, 2H), 2.56-2.50 (m, 5H), 2.35-2.25 (m, 3H), 1.93-1.89 (m, 2H), 1.82-1.78 (m, 5H), 1.70-1.53 ​​(m, 3H), 1.48 (s, 3H); ESI MS m / z 354 [M+H] + .

[0135] Synthesis Example 13 Synthesis of Compound B2-5A-6 The preparation method (also referred to as scheme 13) of compound B2-5A-6 (also referred to as ALB-211297) is as follows.

[0136] [ka]

[0137] Preparation of Compound 1 To a solution of compound Int-3 (compound 3 in scheme 12) (0.10 g, 0.25 mmol) dissolved in MeOH (5.0 mL) and a small amount of AcOH, diethylamine (0.05 g, 0.50 mmol) was added followed by NaCNBH3 (0.05 g, 0.75 mmol) at room temperature. The reaction mixture was stirred at room temperature for 48 h. MeOH was evaporated and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure to give a colorless oil. This was purified by combiflash column chromatography using 0-90% EtOAc in hexane to give compound 1 (0.06 g, 52%) as a colorless gum. ESI MS m / z 458 [M + H] + .

[0138] Preparation of compound 2 To an ice-cold solution of compound 1 (0.06 g, 0.13 mmol) dissolved in THF (4.0 mL) was added LiAlH4 (2.0 M in THF, 0.39 mL, 0.39 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 3 min and quenched with saturated NH4Cl (5.0 mL). The resulting mixture was extracted with EtOAc (10 mL × 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 2 [0.05 g (crude)] as a colorless oil. ESI MS m / z 430 [M + H] + .

[0139] Preparation of compound ALB-211297 (B2-5A-6) To a solution of compound 2 (0.05 g, 0.11 mmol) dissolved in CHCl (5.0 mL) was added p-toluenesulfonic acid (0.03 g, 0.23 mmol) at room temperature. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was quenched with saturated NaHCO solution (5.0 mL) and extracted with CHCl (10 mL×2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO, and concentrated under reduced pressure. The residue was purified by mass-triggered preparative HPLC to give compound ALB-211297 (B2-5A-6) (0.0075 g, 8%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.22-7.20 (d, J = 8.0 Hz, 2H), 7.05-7.03 (d, J = 8.0 Hz, 2H), 4.55 (s, 2H), 4.28 (q, J = 8.0 Hz, 2H), 3.51 (s, 2H), 2.53-2.48 (m, 5H), 2.35-2.25 (m, 3H), 1.93-1.90 (m, 2H), 1.81-1.77 (m, 1H), 1.70-1.53 ​​(m, 3H), 1.49 (s, 3H), 1.03 (t, 6H). ESI MS m / z 356 [M+H] + .

[0140] Synthesis Example 14 Synthesis of Compound B2-5A-4 The preparation method (also referred to as scheme 14) of compound B2-5A-4 (also referred to as ALB-211299) is as follows.

[0141] [ka]

[0142] Preparation of compound 2 To a suspension of compound Int-2 (compound 2 in scheme 12) (0.08 g, 0.19 mmol) and NaH (0.016 g, 60% in mineral oil, 0.39 mmol) dissolved in DMF (3.0 mL) was added compound 1 (0.037 g, 0.39 mmol) at 0 °C to form a reaction mixture. The reaction was carried out at room temperature for 6 h and quenched by the addition of saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (10 mL × 3). The organic layers were combined, dried over MgSO4, and concentrated under reduced pressure to give a residue. This was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 2 (0.04 g, 50%) as a colorless oil. ESI MS m / z 478 [M + NH4] + .

[0143] Preparation of compound 3 An ice-cold solution of compound 2 (0.04 g, 0.08 mmol) dissolved in THF (4.0 mL) was mixed with LiAlH4 (2.0 M in THF, 0.26 mL, 0.26 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 3 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (10 mL × 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 3 (0.018 g, 49%) as a colorless oil. ESI MS m / z 450 [M + NH4] + .

[0144] Preparation of compound ALB-211299 (B2-5A-4) To a solution of compound 3 (0.018 g, 0.04 mmol) dissolved in CHCl (5.0 mL) was added p-toluenesulfonic acid (0.007 g, 0.08 mmol) at room temperature. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was quenched with saturated NaHCO solution (5.0 mL) and extracted with CHCl (10 mL×2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO, and concentrated under reduced pressure. The residue was purified by mass-triggered preparative HPLC to give compound ALB-211299 (B2-5A-4) (0.012 g, 72%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 4.58 (d, J = 4.0 Hz, 2H), 4.54 (s, 2H), 4.31 (q, J = 8.0 Hz, 2H), 3.64-3.62 (m, 2H), 3.60-3.58 (m, 2H), 3.41(s, 3H), 2.60-2.53 (m, 1H), 2.37-2.29 (m, 3H), 1.96-1.89 (m, 2H), 1.84-1.80 (m, 1H), 1.70-1.60 (m, 3H), 1.50 (s, 3H); ESI MS m / z 376 [M+NH4] + .

[0145] Synthesis Example 15 Synthesis of Compound B2-29 The preparation method (also referred to as scheme 15) of compound B2-29 (also referred to as ALB-210364) is as follows.

[0146] [ka]

[0147] Preparation of compound 2 To a solution of compound 1 (0.50 g, 3.27 mmol) dissolved in CHCl (10 mL) was added imidazole (0.55 g, 8.16 mmol) followed by TBDMSCl (0.59 g, 3.92 mmol) at 0° C. The reaction mixture was stirred at room temperature for 16 h, diluted with CHCl (25 mL) and washed with saturated NaHCO (20 mL). The reaction mixture was dried over NaSO and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-40% EtOAc in hexane to give compound 2 (0.50 g, 57%) as a colorless liquid.

[0148] Preparation of compound 3 To an ice-cold solution of compound Int-10 (compound 10 from scheme 1) (0.20 g, 0.69 mmol) dissolved in THF (5.0 mL) was added LDA (1.04 mL, 2.0 M in THF, 2.08 mmol) dropwise at -78 °C. The solution was stirred at -78 °C for 30 min. Allyl bromide (0.18 g, 1.39 mmol) was added to the reaction mixture. The mixture was stirred at -78 °C for 30 min. The reaction mixture was quenched with saturated NH4Cl solution (10 mL). The resulting mixture was extracted with EtOAc (20 mL x 3). The organic layers were combined, washed with brine (10 mL), dried over MgSO4, and concentrated. The residue was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 3 (0.18 g, 79%) as a pale yellow liquid.

[0149] Preparation of compound 4 To a stirred solution of compound 3 (0.18 g, 0.55 mmol) in CHCl (5.0 mL) was added BBr3 (1 M in CHCl) (1.60 mL, 1.64 mmol) at -78 °C. The reaction mixture was gradually warmed from -78 °C to room temperature over 4 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by combiflash column chromatography using 0-60% EtOAc in hexanes to give compound 4 (0.035 g, 20%) as a colorless oil.

[0150] Preparation of compound 5 To a stirred solution of compound 4 (0.035 g, 0.11 mmol) in DMF (3.0 mL) was added NaH (0.01 g, 60% in mineral oil, 0.28 mmol) at 0 °C and stirred for 5 min. Compound 2 (0.04 g, 0.14 mmol) was added to the reaction mixture after 5 min. The mixture was stirred at room temperature for 5 h and diluted with cold H2O (20 mL). The resulting mixture was extracted with EtOAc (30 mL x 2). The extracts were combined, washed with brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-40% EtOAc in hexane to give compound 5 (0.02 g, 46%) as a colorless liquid.

[0151] Preparation of compound ALB-210364 (B2-29) To an ice-cold solution of compound 5 (0.02 g, 0.05 mmol) dissolved in THF (4.0 mL) was added LiAlH4 (0.07 mL, 2.0 M in THF, 0.16 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 30 min and diluted with H2O and Na2SO4·10H2O. The resulting mixture was extracted with EtOAc (10 mL×3). The extracts were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-60% EtOAc in hexane to give compound ALB-210364 (B2-29) (0.0039 g, 21%) as a colorless gum. 1H NMR (400 MHz, CDCl3) δ 7.03 (d, J=8.4 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 5.93-5.83 (m, 1H), 5.09-5.04 (m, 2H), 4.53 (d, J=5.6 Hz, 2H), 3.95-3.85 (m, 2H), 3.68 (t, J=4.4 Hz, 4H), 2.66-2.59 (m, 1H), 2.23-2.14 (m, 2H), 2.07-1.99 (m, 2H), 1.76 (d, J = 13.6 Hz,2H), 1.62-1.58 (m, 5H),1.49 (s, 3H), 1.38-1.28 (m, 2H), 1.01 (d, J = 7.6 Hz, 3H). ESI MS m / z 359 [M+H] + .

[0152] Synthesis Example 16 Synthesis of Compound B2-28 The preparation method (also referred to as scheme 16) of compound B2-28 (also referred to as ALB-210359) is as follows.

[0153] [ka]

[0154] Preparation of compound 2 To a solution of lithium diisopropylamide (1.04 mL, 2.0 M in THF, 2.08 mmol) dissolved in THF (5.0 mL) was added compound Int-10 (compound 10 in scheme 1) (0.20 g, 0.69 mmol) dropwise at -78 °C. The solution was stirred at -78 °C for 30 min. Compound 1 (0.09 mL, 1.04 mmol) was added to the reaction mixture at -78 °C. The reaction mixture was stirred at -78 °C for 30 min and quenched with saturated NH4Cl solution (10 mL). The resulting mixture was extracted with EtOAc (20 mL x 3). The organic layers were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 2 (0.18 g, 79%) as a pale yellow liquid. 1 H NMR (400 MHz, CDCl3) δ 7.06 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 5.71-5.63 (m, 1H), 5.02-4.96 (m, 2H), 4.53 (m, 2H), 3.77 (s, 3H), 3.74 (s, 3H), 2.62-2.57 (m, 1H), 2.32-2.19 (m, 4H), 1.73-1.69 (m, 1H), 1.53-1.50 (m, 1H), 1.46 (s, 3H), 1.33-1.25 (m, 2H).

[0155] Preparation of compound 3 To a solution of compound 2 (0.09 g, 0.27 mmol) dissolved in CHCl (5.0 mL) was added BBr3 (0.82 mL, 0.82 mmol) dissolved in CHCl at -78 °C. The reaction mixture was warmed to room temperature, stirred for 3 h, and quenched by the addition of HCl (1 N, 2.0 mL). The resulting mixture was extracted with EtOAc (30 mL x 3), dried over MgSO4, and concentrated under reduced pressure to give a residue. It was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 3 (0.03 g, 35%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.00 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 8.4 Hz, 2H), 5.71-5.65 (m, 1H), 5.02-4.97 (m, 2H), 4.52 (m, 2H), 3.74 (s, 3H), 2.62-2.55 (m, 1H), 2.32-2.19 (m, 5H), 1.72-1.68 (m, 1H), 1.45 (s, 4H), 1.33-1.23 (m, 4H).

[0156] Preparation of compound 4 To an ice-cold solution of compound 3 (0.02 g, 0.06 mmol) in DMF (1.0 mL) was added NaH (0.005 g, 0.13 mmol) at 0 °C. After 30 min, (3-bromopropoxy)(tert-butyl)dimethylsilane (0.032 g, 0.13 mmol) was added to the reaction mixture. The reaction mixture was stirred at 0 °C for 4 h and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL × 3). The organic layers were combined, washed with brine (10 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 4 (0.03 g, 97%) as a colorless oil. ESI MS m / z 387 C 29 H 46 O4Si + H] + .

[0157] Preparation of compound 5 To an ice-cold solution of compound 4 (0.03 g, 0.06 mmol) in THF (1.0 mL) was added LiAlH4 (0.18 mL, 1.0 M in THF, 0.18 mmol) at 0 °C. The mixture was stirred at 0 °C for 15 min and diluted with H2O and Na2SO4·10H2O. The resulting mixture was extracted with EtOAc (10 mL × 3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 5 (0.015 g, 68%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 6.95 (d, J = 8.8 Hz, 2H), 6.71 (d, J = 8.8 Hz, 2H), 5.85-5.79 (m, 1H), 5.02-4.97 (m, 2H), 4.46 (m, 2H), 4.06-3.94 (m, 2H), 3.72-3.49 (m, 6H), 2.58-2.49 (m, 1H), 2.18-2.11 (m, 1H), 1.89-1.68 (m, 6H), 1.48 (s, 4H), 0.81 (m, 9H), 0.00 (m, 6H).

[0158] Preparation of compound ALB-210359 (B2-28) To an ice-cold solution of compound 5 (0.015 g, 0.041 mmol) dissolved in THF (1.0 mL) was added TBAF (0.12 mL, 1.0 M in THF, 0.12 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 2 h and diluted with H2O. The resulting mixture was extracted with EtOAc (5.0 mL × 3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-60% EtOAc in hexane to give compound ALB-210359 (B2-28) (0.0065 g, 45%) as a colorless gum. 1H NMR (400 MHz, CDCl3) δ 7.03 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 5.93-5.83 (m, 1H), 5.09-5.04 (m, 2H), 4.53 (m, 2H), 4.08 (t, J = 6.0 Hz, 2H), 3.84 (t, J = 6.0 Hz, 2H), 3.70 (m, 2H), 2.66-2.59 (m, 1H), 2.33-2.19 (m, 1H), 2.04-2.00 (m, 4H), 1.78-1.58 (m, 6H), 1.49 (s, 4H).ESI MS m / z 345 C 22 H 32 O3+H] + .

[0159] Synthesis Example 17 Synthesis of Compounds B2-27 and B2-21 The preparation method (also referred to as scheme 17) of compounds B2-27 (also referred to as ALB-209872) and B2-21 (also referred to as ALB-208788) is as follows.

[0160] [ka]

[0161] Preparation of Compound 1 To a solution of lithium diisopropylamide (20.8 mL, 1.0 M in THF, 20.8 mmol) dissolved in THF (20 mL) was added compound Int-10 (compound 10 of scheme 1) (2.00 g, 6.94 mmol) dropwise at -78 °C. The solution was stirred at -78 °C for 30 min and then cooled to -105 °C (MeOH, liquid N2). Phenyl vinyl sulfoxide (2.10 g, 13.88 mmol) was added to the reaction mixture. The reaction mixture was stirred at -105 °C for 30 min and quenched with saturated NH4Cl solution (25 mL). The resulting mixture was extracted with EtOAc (50 mL x 3). The organic layers were combined and washed with brine (50 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-20% EtOAc in hexanes to give compound 1 (1.70 g, 56.6%) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 7.56-7.48 (m, ,5H), 7.03-6.99 (m, ,2H), 6.80-6.77 (m, ,2H), 4.52 (s, 2H), 3.76-3.70 (m,6H), 2.73-2.52 (m, ESI MS m / z 441 C 26 H 32 O4S+H] + .

[0162] Preparation of compound 2 A mixture of compound 11 (1.00 g, 2.27 mmol) and NaHCO3 (2.40 g, 22.72 mmol) dissolved in xylene (20 mL) was stirred under reflux for 16 h and diluted with H2O. The resulting mixture was extracted with EtOAc (30 mL × 3). The organic layers were combined, washed with brine (25 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil, which was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 2 (0.30 g, 42%) as a pale yellow liquid. 1H NMR (400 MHz, CDCl3) δ 7.07 (d, J = 8.8 Hz ,2H),6.80 (d, J = 8.4 Hz, 2H), 5.85-5.78 (m, 1H), 5.09-5.03 (m, 2H), 4.54 (m, 2H), 3.77 (s, 6H) 2.66-2.60 (m, 1H), 2.58-2.28 (m, 4H), 1.78-1.74 (m, 1H), 1.47 (s, 4H).

[0163] Preparation of compound 3 To a solution of compound 2 (0.15 g, 0.48 mmol) in CHCl (5.0 mL) was added BBr3 (0.95 mL, 0.95 mmol) in CHCl at 0 °C. The reaction mixture was warmed to room temperature and stirred for 16 h and quenched by addition of HCl solution (1 N, 5.0 mL). The resulting mixture was extracted with EtOAc (10 mL × 3), dried over NaSO and concentrated under reduced pressure to give a residue which was purified by combiflash column chromatography using 0-60% EtOAc in hexane to give compound 3 (0.07 g, 51%) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 7.03 (d, J = 8.4 Hz ,2H),6.73 (d, J = 8.4 Hz, 2H), 5.91-5.84 (m, 1H), 5.20-5.11 (m, 2H), 4.56 (m, 2H), 2.72-2.65 (m, 1H), 2.46-2.40 (m, 3H), 2.29-2.23 (m, 1H), 1.81-1.56 (m, 4H), 1.49 (s, 3H).

[0164] Preparation of compound ALB-209872 (B2-27) To an ice-cold solution of compound 13 (0.03 g, 0.10 mmol) dissolved in CH3CN (1.0 mL) was added K2CO3 (0.03 g, 0.21 mmol) and MeI (0.02 g, 0.16 mmol) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The resulting mixture was extracted with EtOAc (5.0 mL × 3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound ALB-209872 (B2-27) (0.012 g, 50%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.04 (d, J = 8.4 Hz, 2H), 6.75 (d, J = 8.4 Hz, 2H), 5.87-5.80 (m, 1H), 5.12-5.06 (m, 2H), 4.59-4.50 (m, 2H), 3.79 (s, 2H), 2.65-2.42 (m, 3H), 2.28-2.22 (m, 1H), 1.80-1.76 (m, 1H), 1.49 (s, 4H), 1.43-1.38 (m, 2H); ESI MS m / z 301 19 H 24 O3+H] + .

[0165] Preparation of compound 5 To a mixture of compound ALB-209872 (B2-27) (0.03 g, 0.10 mmol) dissolved in CH2Cl2 (1.0 mL), compound 4 (0.24 g, 0.20 mmol), Cu(OAc)2 (0.036 g, 0.20 mmol) and pyridine (0.015 g, 0.20 mmol) were added dropwise at room temperature. The reaction mixture was stirred for 48 h. The resulting mixture was extracted with EtOAc (5.0 mL×3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 5 (0.02 g, 54%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.31 (t, J = 7.6 Hz, 2H), 7.07 (t, J = 7.6 Hz, 3H),6.98 (d, J = 7.6 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 5.86-5.79 (m, 1H), 5.11-5.05 (m, 2H), 4.55 (s, 2H), 3.77 (s, 3H), 2.68-2.61 (m, 1H), 2.47-2.44 (m, 2H), 2.28-2.23 (m, 1H), 1.79-1.75 (m, 1H), 1.48 (s, 4H), 1.43 - 1.40 (m, 1H); ESI MS m / z 315 C 20 H 26 O3+H] + ESI MS m / z 376 [M+H] + .

[0166] Preparation of compound ALB-208788 (B2-21) To an ice-cold solution of compound 5 (0.02 g, 0.05 mmol) dissolved in THF (1.0 mL) was added LiAlH4 (0.08 mL, 2.0 M in THF, 0.16 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and diluted with H2O and Na2SO4·10H2O. The resulting mixture was extracted with EtOAc (5.0 mL × 3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound ALB-208788 (B2-21) (0.0095 g, 55%) as a colorless gum. 1H NMR (400 MHz, CDCl3) δ 7.33-7.29 (m, 2H), 7.11-7.05 (m, 3H), 6.99, 6.96 (dd, J = 1.2, 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 5.78-5.71 (m, 1H), 5.17-5.06 (m, 2H), 4.56 (m, 2H), 3.70 (d, J = 6.4 Hz, 2H), 2.76-2.69 (m, 1H), 2.28-2.21 (m, 1H), 1.92-1.88 (m, 2H), 1.70-1.64 (m, 2H), 1.49 (s, 4H), 1.48-1.43 (m, 2H); ESI MS m / z 349 C 24 H 28 O2+H] + .

[0167] Synthesis Example 18 Synthesis of Compound B2-19 The preparation method (also referred to as scheme 19) of compound B2-19 (also referred to as ALB-209870) is as follows.

[0168] [ka]

[0169] Preparation of compound 2 A solution of compound Int-4 (compound 4 from scheme 1) (5.00 g, 36.76 mmol) dissolved in 1,4-dioxane:water (40 mL:10 mL) was added to a mixture of compound 1 (7.58 g, 44.11 mmol) and KOH (4.10 g, 73.52 mmol) at room temperature. The reaction mixture was degassed with argon for 15 min and [Rh(COD)Cl]2 (0.54 g, 1.10 mmol) was added. The reaction mixture was stirred under reflux for 16 h. The reaction mixture was quenched with saturated NaHCO3 (30 mL) and extracted with EtOAc (20 mL×2). The organic layers were combined, washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 2 (2.00 g, quantitative) as a brown semi-solid. 1 H NMR (400 MHz, CDCl3) δ 7.80 (t, J = 8.0 Hz, 2H), 7.65 (s, 1H), 7.49-7.36 (m, 3H), 3.58 (t, J = 8.0 Hz, 1H), 2.91-2.77 (m, 2H), 2.68-2.52 (m, 3H), 1.43 (s, 3H), 1.03 (m, 3H).

[0170] Preparation of compound 3 To a solution of compound 2 (1.60 g, 6.06 mmol) in Ac2O (10 mL), Zn(OAc)2 (1.10 g, 6.06 mmol), BF3·OEt2 (0.54 mL, 3.03 mmol) were added and stirred at room temperature for 12 h. The reaction mixture was diluted with H2O (20 mL). The resulting mixture was extracted with EtOAc (20 mL×3). The combined organic layers were washed with saturated NaHCO3 (250 mL), brine (50 mL), dried over Na2SO4, and concentrated under reduced pressure. The product was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 3 (1.80 g, 78%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 7.79-7.74 (m, 3H), 7.61 (s, 1H), 7.45-7.38 (m, 2H), 7.35-7.32 (m, 1H), 5.50-5.48 (m, 1H), 4.71-4.54 (m, 2H), 3.19-3.12 (m, 1H), 2.88-2.81 (m, 1H), 2.56-2.49 (m, 2H), 2.40-2.25 (m, 2H), 2.10 (s, 3H), 1.51 (s, 3H).

[0171] Preparation of compound 4 To a solution of compound 3 (1.20 g, 3.92 mmol) dissolved in MeOH (10 mL) at 0 °C, NaH (0.078 g, 60% in mineral oil, 1.96 mmol) was added and stirred at room temperature for 30 min. The reaction mixture was diluted with saturated NH4Cl (10 mL). The resulting mixture was extracted with EtOAc (50 mL × 3). The organic layers were combined, washed with brine (50 mL), dried over Na2SO4, and concentrated to give a residual oil, which was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 4 (1.20 g, 77%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.80-7.76 (m, 3H), 7.57 (s, 1H), 7.46-7.43 (m, 2H), 7.35-7.32 (m, 1H), 4.69-4.56 (m, 2H), 3.17-3.13 (m, 1H), 2.93-2.87 (m, 1H), 2.69-2.55 (m, 4H), 2.17-2.12 (m, 4H), 1.98-1.91 (m, 1H), 1.53 (s, 3H).

[0172] Preparation of compound 5 To an ice-cold solution of p-toluenesulfonylmethyl isocyanide (0.74 g, 3.79 mmol) and t-BuOK (0.85 g, 7.57 mmol) in THF (5.0 mL) was added compound 4 (0.50 g, 1.89 mmol) in THF (5.0 mL) and stirred at 0 °C for 10 min, followed by addition of MeOH (10 mL). The resulting mixture was stirred under reflux for 1 h and concentrated under reduced pressure. The crude was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 5 (0.25 g, 48%) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 7.80-7.75 (m, 3H), 7.60 (s, 1H), 7.45-7.42 (m, 2H), 7.31-7.28 (m, 1H), 4.61 (m, J = 11.2 Hz, 1H), 4.51 (m, 1H), 3.14-3.10 (m, 1H), 2.75-2.63 (m, 2H), 2.51-2.14 (m, 2H), 1.96-1.74 (m, 4H), 1.49 (s, 3H).

[0173] Preparation of compound 6 To a solution of compound 5 (0.25 g, 0.91 mmol) dissolved in 2-propanol (5.0 mL) was added KOH (0.51 g, 9.09 mmol) at room temperature. The reaction mixture was stirred under reflux for 16 h and quenched by the addition of HCl solution (2 N). The resulting mixture was extracted with EtOAc (10 mL×3). The organic layers were combined and washed with brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure to give compound 6 (0.20 g, 75%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 7.78-7.73 (m, 3H), 7.56 (s, 1H), 7.44-7.37 (m, 2H), 7.32, 7.29 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 4.58-4.49 (m, 2H), 2.76-2.60 (m, 1H), 2.59-2.40 (m, 2H), 2.23-2.10 (m, 2H), 1.96-1.92 (m, 1H), 1.78-1.66 (m, 2H), 1.51 (s, 3H).

[0174] Preparation of compound 7 To an ice-cold solution of 6 (0.20 g, 0.68 mmol) in CH3CN:MeOH (3.0 mL:1.0 mL) was added K2CO3 (0.19 g, 1.36 mmol), CHI (0.15 g, 1.02 mmol) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 18 h and then concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-20% EtOAc in hexanes to give 7 (0.15 g, 71%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.78-7.73 (m, 3H), 7.56 (s, 1H), 7.44-7.37 (m, 2H), 7.32, 7.29 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 4.58-4.48 (m, 2H), 3.67 (s, 3H), 2.78-2.72 (m, 1H), 2.57-2.42 (m, 2H), 1.95-1.91 (m, 2H), 1.76-1.64 (m, 2H), 1.51 (s, 3H).

[0175] Preparation of compound 8 Compound 7 (0.15 g, 0.49 mmol) was added dropwise to a solution of lithium diisopropylamide (0.73 mL, 2.0 M in THF, 1.46 mmol) dissolved in THF (1.0 mL) at -78 °C. The solution was stirred at -78 °C for 30 min, then cooled to -105 °C (MeOH, liquid N2), and phenylvinyl sulfoxide (0.13 mL, 0.97 mmol) was added to the reaction mixture. The reaction mixture was stirred at -105 °C for 30 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (10 mL x 3). The organic layers were combined, washed with brine (10 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 8 (0.07 g, 31%) as a pale yellow liquid.

[0176] Preparation of compound 9 A mixture of compound 8 (0.07 g, 0.15 mmol) and NaHCO3 (0.16 g, 1.52 mmol) dissolved in xylene (2.0 mL) was stirred under reflux for 16 h and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL × 3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure to give a residual oil, which was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 9 (0.015 g, 30%) as a colorless liquid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.79-7.74 (m, 3H), 7.58 (s, 1H), 7.43-7.31 (m, 3H), 5.87-5.80 (m, 1H), 5.10-5.05 (m, 2H), 4.58-4.48 (m, 2H), 3.81 (s, 3H), 2.88-2.82 (m, 1H), 2.52-2.44 (m, 3H), 1.83-1.80 (m, 1H), 1.49 (s, 4H).

[0177] Preparation of compound ALB-209870 (B2-19) To an ice-cold solution of compound 9 (0.015 g, 0.04 mmol) dissolved in THF (1.0 mL) was added LiAlH4 (0.13 mL, 2.0 M in THF, 0.13 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and diluted with H2O and Na2SO4·10H2O. The resulting mixture was extracted with EtOAc (5.0 mL × 3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound ALB-209870 (B2-19) (0.005 g, 30%) as a colorless gum. 1 H NMR (400 MHz, CDCl3) δ 7.71-7.66 (m, 3H), 7.50 (s, 1H), 7.37-7.32 (m, 2H), 7.26, 7.24 (dd, J = 1.6 Hz, 8.8 Hz, 1H), 5.72-5.65 (m, 2H), 5.09-4.98 (m, 2H), 4.47 (m, 2H),3.67 (d, J = 4.8 Hz, 2H), 2.88-2.82 (m, 1H), 2.41-2.34 (m, 1H), 1.90-1.83 (m, 2H), 1.68-1.62 (m, 2H), 1.46 (s, 3H), 1.30-1.29 (m, 2H); ESI MS m / z 307 C 22 H 26 O+H] + .

[0178] Synthesis Example 19 Synthesis of Compound B2-18 The preparation method (also referred to as scheme 20) of compound B2-18 (also referred to as ALB-208789) is as follows.

[0179] [ka]

[0180] Preparation of compound 2 To a solution of compound Int-4 (compound 4 in scheme 1) (11.0 g, 79.00 mmol) dissolved in 1,4-dioxane (120 mL), compound 1 (16.2 g, 79.00 mmol), KOH (2 M, 8.84 g, 158.0 mmol) were added and degassed with argon for 15 min. [Rh(COD)Cl]2 complex (1.16 g, 2.30 mmol) was added to the reaction mixture. The resulting mixture was stirred and heated to reflux for 12 h. Residual solvent was evaporated under reduced pressure. The crude was dissolved in CHCl (250 mL) and filtered through a bed of Celite. The filtrate was washed with water (250 mL), dried over anhydrous NaSO, filtered and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 2 (13.00 g, 55%) as a yellow solid.

[0181] Preparation of compound 3 To a solution of compound 2 (3.50 g, 11.9 mmol) dissolved in Ac2O (20 mL), Zn(OAc)2 (1.30 g, 7.00 mmol) and BF3·OEt2 (0.40 mL, 2.90 mmol) were added and stirred at room temperature for 12 h. The reaction mixture was diluted with H2O (100 mL). The resulting mixture was extracted with EtOAc (100 mL×2). The organic layers were combined and washed with saturated NaHCO3 solution (250 mL) and brine (100 mL). The resulting mixture was dried over MgSO4 and concentrated under reduced pressure to give a residual oil. This was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 3 [3.00 g (crude)] as a brown oil. ESI MS m / z 337 [M + H] + .

[0182] Preparation of compound 4 To a solution of compound 3 (3.00 g, 6.20 mmol) dissolved in MeOH (30 mL) at 0 °C, NaH (0.13 g, 60% mineral oil, 3.00 mmol) was added and stirred at room temperature for 30 min. The reaction mixture was diluted with saturated NH4Cl solution (25 mL). The resulting mixture was extracted with EtOAc (50 mL x 2). The organic layers were combined, washed with brine (100 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil, which was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 4 (2.10 g, 61%) as an off-white solid.

[0183] Preparation of compound 5 To an ice-cold solution of p-toluenesulfonylmethyl isocyanide (2.71 g, 13.70 mmol) and t-BuOK (3.10 g, 27.40 mmol) in THF (10 mL) was added compound 4 (2.00 g, 6.85 mmol) in THF (10 mL) and stirred at 0° C. for 10 min, followed by addition of MeOH (15 mL). The resulting mixture was stirred under reflux for 1 h and concentrated. The crude material was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 5 (1.20 g, 60%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.05 (d, J = 8.4 Hz ,2H),6.82 (d, J = 8.8 Hz, 2H), 4.64 (d, J = 10.4 Hz, 2H), 3.78 (s, 3H), 2.96-2.89 (m, 1H), 2.74-2.68 (m, 1H), 2.53-2.49 (m, 4H), 2.11-2.07 (m,1H), 1.91-1.55 (m,1H),1.51 (s, 3H).

[0184] Preparation of compound 6 To a solution of compound 5 (0.48 g, 1.57 mmol) dissolved in 2-propanol (10 mL) was added KOH (0.89 g, 15.70 mmol) at room temperature. The reaction mixture was stirred under reflux for 16 h and quenched by the addition of HCl solution (2 N, 10 mL). The resulting mixture was extracted with EtOAc (10 mL×3). The organic layers were combined, washed with brine (25 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 6 [0.50 g (crude)] as an off-white solid. ESI MS m / z 323 [M + H] + .

[0185] Preparation of compound 7 To an ice-cold solution of compound 6 (0.40 g, 1.24 mmol) in CH3CN (5.0 mL) was added K2CO3 (0.51 g, 3.72 mmol) and CH3I (0.23 mL, 3.72 mmol) dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 12 h. The resulting mixture was extracted with EtOAc (10 mL × 2). The organic layers were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by column chromatography using 0-20% EtOAc in hexane to give compound 7 (0.30 g, 72%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.04 (d, J = 8.8 Hz ,2H),6.79 (d, J = 8.4 Hz, 2H), 4.55 (m, 2H), 3.76 (s, 3H), 3.65 (s, 3H), 2.54-2.45 (m, 2H), 2.30-2.24 (m, 1H), 2.10-2.06 (m, 2H), 1.89-1.85 (m, 1H), 1.62-1.58 (m, 3H),1.49 (s, 3H).

[0186] Preparation of compound 8 To a solution of compound 7 (0.08 g, 0.24 mmol) in 1,4-dioxane (10 mL) was added phenylboronic acid (0.06 g, 0.47 mmol), Na2CO3 (0.063 g, 2 M, 0.59 mmol) and degassed with argon for 15 min. Pd(dppf)Cl2·CH2Cl2 complex (0.019 g, 0.02 mmol) was added to the reaction mixture. The resulting mixture was stirred and heated to reflux for 16 h. Residual solvent was evaporated. The crude was dissolved in CHCl2 (10 mL) and filtered through a bed of Celite. The filtrate was washed with water (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using 0-10% EtOAc in hexane to give compound 8 (0.08 g, 69%) as an off-white solid. ESI MS m / z 352 [M + NH4] + .

[0187] Preparation of compound 9 To a solution of lithium diisopropylamide (0.50 mL, 2.0 M in THF, 0.89 mmol) dissolved in THF (3.0 mL) was added compound 8 (0.10 g, 0.30 mmol) dissolved in THF (3.0 mL) dropwise at -78 °C. The solution was stirred at -78 °C for 30 min, then cooled to -105 °C (MeOH, liquid N2) and phenylvinyl sulfoxide (0.091 g, 0.60 mmol) was added to the reaction mixture. The reaction mixture was stirred at -105 °C for 30 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined and washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by column chromatography using 0-10% EtOAc in hexane to give compound 9 (0.06 g, 41%) as a brown liquid. ESI MS m / z 487 [M + H] + .

[0188] Preparation of compound 10 A mixture of compound 9 (0.06 g, 0.12 mmol) and NaHCO3 (0.10 g, 1.23 mmol) in xylene (3.0 mL) was stirred under reflux for 18 h and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil, which was purified by column chromatography using 0-20% EtOAc in hexane to give compound 10 (0.03 g, 68%) as a colored oil. ESI MS m / z 361 [M + H] + .

[0189] Preparation of compound ALB-208789 (B2-18) To an ice-cold solution of compound 10 (0.03 g, 0.08 mmol) dissolved in THF (3.0 mL) was added LiAlH4 (0.12 mL, 2.0 M in THF, 0.24 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and diluted with H2O and Na2SO4·10H2O. The resulting mixture was extracted with EtOAc (5.0 mL × 2). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography using 0-30% EtOAc in hexane to give compound ALB-208789 (B2-18) (0.005 g, 19%) as an off-white solid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.51 (d, J = 7.2 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.36-7.24 (m, 5H), 7.15 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 5.70-5.63 (m, 1H), 5.09-4.97 (m, 2H), 4.52 (d, 2H), 4.48 (s, 1H), 3.66-3.61 (m, 2H), 2.75-2.61 (m, 1H), 2.28-2.13 (m, 2H), 1.85-1.80 (m, 2H), 1.66-1.60 (m, 3H), 1.44 (s, 3H), 1.41-1.25 (m, 3H).

[0190] Synthesis Example 20: Synthesis of Compound B2-23 The preparation method (also referred to as scheme 21) of compound B2-23 (also referred to as ALB-208790) is as follows.

[0191] [ka]

[0192] Preparation of compound 2 To a solution of compound Int-7 (compound 7 in scheme 20) (0.30 g, 0.89 mmol) dissolved in 1,4-dioxane (3.0 mL), compound 1 (0.27 g, 1.78 mmol), Na2CO3 (2 M, 0.19 g, 1.79 mmol) were added and degassed with argon for 15 min. Pd(dppf)Cl2·CH2Cl2 complex (0.07 g, 0.09 mmol) was added to the reaction mixture. The resulting mixture was stirred and refluxed under microwave irradiation for 1 h. Residual solvent was evaporated. The crude was dissolved in CHCl2 (10 mL) and filtered through a bed of Celite. The filtrate was washed with water (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using 0-20% EtOAc in hexane to give compound 2 (0.26 g, 80%) as an off-white solid.

[0193] Preparation of compound 3 To a solution of lithium diisopropylamide (1.20 mL, 2.0 M in THF, 2.38 mmol) dissolved in THF (5.0 mL) was added compound 2 (0.29 g, 0.79 mmol) dissolved in THF (3.0 mL) dropwise at -78 °C. The solution was stirred at -78 °C for 30 min, then cooled to -105 °C (MeOH, liquid N2) and phenylvinyl sulfoxide (0.24 g, 1.59 mmol) was added to the reaction mixture. The reaction mixture was stirred at -105 °C for 30 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined and washed with brine (5.0 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by column chromatography using 0-20% EtOAc in hexane to give compound 3 (0.16 g, 40%) as a brown liquid.

[0194] Preparation of compound 4 A mixture of compound 3 (0.16 g, 0.31 mmol), NaHCO3 (0.328 g, 3.10 mmol) in xylene (3.0 mL) was stirred under reflux for 18 h and diluted with H2O. The resulting mixture was extracted with EtOAc (10 mL x 2). The organic layers were combined, washed with brine (5.0 mL), dried over MgSO4, and concentrated to give a residual oil, which was purified by column chromatography using 0-20% EtOAc in hexane to give compound 4 (0.04 g, 33%) as a colored oil. ESI MS m / z 391 [M + H] + .

[0195] Preparation of compound ALB-208790 (B2-23) To an ice-cold solution of compound 4 (0.04 g, 0.10 mmol) dissolved in THF (4.0 mL) was added LiAlH4 (0.2 mL, 2.0 M in THF, 0.30 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and diluted with H2O and Na2SO4·10H2O. The resulting mixture was extracted with EtOAc (5.0 mL × 3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography using 0-30% EtOAc in hexane to give compound ALB-208790 (B2-23) (0.015 g, 40%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 8.8 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 5.79-5.71 (m, 1H), 5.17-5.06 (m, 2H), 4.58 (d, 2H), 3.82 (s, 3H), 2.81-2.75 (m, 1H), 2.37-2.30 (m, 2H), 1.94-1.88 (m, 2H), 1.73-1.66 (m, 2H), 1.50-1.43 (m, 5H); ESI MS m / z 363 [M+H] + .

[0196] Synthesis Example 21 Synthesis of Compound B2-5B The preparation method (also referred to as scheme 22) of compound B2-5B (also referred to as ALB-210363) is as follows.

[0197] [ka]

[0198] Preparation of Compound 1 To an ice-cold solution of compound Int-11 (compound 11 from scheme 1) (0.15 g, 0.373 mmol, IN-BSC-J-51) dissolved in THF (5.0 mL) was added LiAlH4 (0.37 mL, 2.0 M in THF, 0.746 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 6 min. The progress of the reaction was monitored by TLC, quenched with Na2SO4·10H2O, and concentrated under reduced pressure. The residue was purified by column chromatography using 0-30% EtOAc in hexane to give compound 1 (0.045 g, 32%) as a colorless gum. ESI MS m / z 375 C 23 H 24 O4+ H] + .

[0199] Preparation of compound 2 To an ice-cold solution of compound 1 (0.03 g, 0.080 mmol) dissolved in CHCl (10 mL) was added Proton Sponge (Sigma-Aldrich Co.; Trademark) (0.0859 g, 0.40 mmol) followed by MeOBF (0.059 g, 0.40 mmol) at 0°C. The reaction mixture was then stirred at room temperature for 20 h, the reaction mixture was diluted with water (25 mL) and extracted with CHCl (25 mL x 2). The organic layers were combined, dried over anhydrous NaSO, filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography using 0-6% EtOAc in hexane to give compound 2 (0.022 g, 79%) as a colorless gum.

[0200] Preparation of compound ALB-210363 (B2-5B) To a stirred solution of compound 2 (0.022 g, 0.0635 mmol) in MeOH (1.0 mL), THF (2.0 mL), and water (0.5 mL) was added LiOH·H2O (0.013 g, 0.317 mmol). The reaction was stirred at room temperature for 16 h and concentrated under reduced pressure. The residue was diluted with water (25 mL), acidified with HCl solution (2N, 3.0 mL), and extracted with EtOAc (25 mL×2). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by prep-HPLC column chromatography and the fractions were evaporated by lyophilization to give compound ALB-210363 (B2-5B) (0.006 g, 28%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.01 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 4.55 (s, 2H), 3.75 (s, 3H), 3.63 (d, 9.2 Hz, 1H), 3.56 (d, 9.2 Hz, 1H), 3.44 (s, 3H), 2.67-2.57 (m, 1H), 2.388 (s, 2H), 2.31-2.18 (m, 2H), 1.91 (t, 14.8 Hz, 2H), 1.51 (m, 1H), 1.50 (s, 3H), 1.49-1.37 (m, 3H); ESI MS m / z 331.2 C 20 H 28 O4-H] + .

[0201] Preparative HPLC conditions: Column: Gemini NX-C18 10 μm; 150×30 mm Mobile phase A: 0.05% formic acid in water; B: Acetonitrile. Gradient (T / %B): 0 / 10, 2 / 30, 10 / 75, 15 / 95, 15.5 / 98, 17.5 / 98, 18 / 20, 20 / 10 Diluent: MeOH+THF

[0202] Synthesis Example 22 Synthesis of Compound B2-5A-7 The preparation method (also referred to as scheme 23) of compound B2-5A-7 (also referred to as ALB-211303) is as follows.

[0203] [ka]

[0204] Preparation of compound 2 To a suspension of compound Int-10 (compound 10 in scheme 1) (0.75 g, 2.60 mmol), K2CO3 (0.718 g, 5.202 mmol) in NMP (8.0 mL) was added compound 1 (1.1 mL, 10.4 mmol) at room temperature. The reaction was carried out in a sealed tube at 220 °C for 5 h and quenched by the addition of HCl solution (2N, 40 mL). The resulting mixture was extracted with MTBE (60 mL × 3), dried over Na2SO4, and concentrated under reduced pressure to give a residue. This was purified by combiflash column chromatography using 0-80% EtOAc in hexane to give compound 2 (0.38 g, 56%) as a pale yellow solid. ESI MS m / z 259 [C 16 H 20 O3-H] + .

[0205] Preparation of compound 3 To a stirred solution of compound 2 (0.38 g, 1.46 mmol) in acetonitrile (20 mL) was added K2CO3 (0.604 g, 4.38 mmol) followed by methyl iodide (0.415 g, 2.92 mmol) at 0 °C. The reaction was carried out at room temperature for 16 h, filtered and concentrated under reduced pressure to give a residue which was purified by combiflash column chromatography using 0-50% EtOAc in hexanes to give compound 3 (0.33 g, 82%) as a colorless viscous liquid.

[0206] Preparation of compound 4 To a stirred solution of compound 3 (0.4 g, 1.458 mmol) in acetonitrile (20 mL) was added K2CO3 (0.603 g, 4.374 mmol) followed by (3-bromopropoxy)(tert-butyl)dimethylsilane (0.44 g, 1.74 mmol) at room temperature. The reaction was carried out at 80 °C for 48 h, filtered and concentrated under reduced pressure to give a residue which was purified by combiflash column chromatography using 0-20% EtOAc in hexane to give compound 4 (0.52 g, 79.8%) as a colorless liquid.

[0207] Preparation of compound 5 To a stirred solution of compound 4 (0.52 g, 1.164 mmol) dissolved in THF (8.0 mL) was added lithium diisopropylamide (1.74 mL, 2.0 M in THF, 3.492 mmol) dropwise at -78 °C. After the solution was stirred at -78 °C for 30 min, tert-butyl 2-bromoacetate (0.454 g, 2.32 mmol) was added to the reaction mixture. The reaction mixture was stirred at -78 °C for 30 min and quenched with saturated NH4Cl solution (10 mL). The resulting mixture was extracted with EtOAc (50 mL x 2). The organic layers were combined, washed with brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 5 (0.36 g, 55%) as a colorless viscous liquid.

[0208] Preparation of compound 6 To an ice-cold solution of compound 5 (0.18 g, 0.328 mmol) dissolved in THF (6.0 mL) was added LiAlH4 (2.0 M in THF, 0.5 mL, 0.962 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 3 min and quenched with saturated NH4Cl (5.0 mL). The resulting mixture was extracted with EtOAc (30 mL × 2). The organic layers were combined, washed with brine (10 mL), dried over Na2SO4, and concentrated under reduced pressure to give a residue. It was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 6 (0.065 g, 38%) as a colorless liquid.

[0209] Preparation of compound ALB-211303 (B2-5A-7) To a stirred solution of compound 6 (0.065 g, 0.1219 mmol) dissolved in CHCl (5.0 mL) was added p-toluenesulfonic acid monohydrate (0.696 g, 0.365 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with CHCl (50 mL) and washed with saturated NaHCO solution (30 mL) and brine (20 mL). The organic layer was dried over NaSO and concentrated under reduced pressure. The residue was purified by combiflash column chromatography on silica gel using 0-30% EtOAc in hexane to give compound ALB-211303 (B2-5A-7) (0.014 g, 33%) as a colorless gum. 1H NMR (400 MHz, CDCl3) δ 7.01 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 8.8 Hz, 2H), 4.57 (d, J = 1.2 Hz, 2H), 4.30-4.25 (q, 2H), 4.09 (t, J = 6.0 Hz, 2H), 3.85 (d, J = 4.4 Hz, 2H), 2.56-2.45 (m, 1H), 2.34 (d, J = 3.2 Hz, 2H), 2.29-2.22 (m, 1H), 2.08-2.00 (m, 2H), 1.93-1.86 (m, 2H), 1.81-1.77 (m, 2H), 1.66-1.52 (m, 3H), 1.49 (s, 3H); ESI MS m / z 345 C 21 H 28 O4+H] + .

[0210] Synthesis Example 23 Synthesis of Compound B2-5A-7-1 The preparation method (also referred to as scheme 24) of compound B2-5A-7-1 (also referred to as ALB-211355) is as follows.

[0211] [ka]

[0212] To an ice-cold solution of compound Int-5 (compound 5 of scheme 23) (0.05 g, 0.0891 mmol) dissolved in 2,2,2-trifluoroethanol (0.25 mL) was added TMSCl (0.075 mL) at 0° C. The reaction mixture was then warmed to room temperature and stirred for 2 h. The progress of the reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure to give the crude product. The crude product was purified by preparative-HPLC column chromatography and the fractions were evaporated by lyophilization to give compound ALB-211355 (B2-5A-7-1) (0.01 g, 28.7%) as a colorless viscous material. 1H NMR (400 MHz, CDCl3) δ 7.04 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 4.53 (d, J = 1.2 Hz, 2H), 4.08 (t, J = 5.8 Hz, 2H), 3.85 (t, J = 5.8 Hz, 2H), 3.759 (s, 3H), 2.86-2.75 (m, 1H), 2.63 (d, J = 15.2 Hz, 1H), 2.49 (d, J = 15.2 Hz, 1H), 2.39-2.19 (m, 4H), 2.08-1.98 (m, 2H), 1.75-1.60 (m, 2H),1.47 (s. 3H), 1.47-1.3 (m, 2H); ESI MS m / z 391 C 22 H 30 O6+H] + .

[0213] Preparative HPLC conditions: Column: Gemini NX-C18 10 μm; 150×30 mm Mobile phase: B: acetonitrile; A: 0.05% formic acid solution Gradient (min / %B): 0 / 10, 2 / 30, 10 / 55, 15 / 85, 15.5 / 98, 18.5 / 98, 19 / 10, 20 / 10 Diluent: MeOH Column flow rate: 30 mL / min

[0214] Synthesis Example 24: Synthesis of Compound B2-5A-1 The preparation method (also referred to as scheme 25) of compound B2-5A-1 (also referred to as ALB-210360) is as follows.

[0215] [ka]

[0216] Preparation of compound 2 To a solution of lithium diisopropylamide (0.24 mL, 2.0 M in THF, 0.41 mmol) dissolved in THF (2.0 mL) was added compound Int-7 (compound 7 of scheme 19) (0.05 g, 0.162 mmol) dropwise at -78 °C. The solution was stirred at -78 °C for 30 min, and then compound 1 (0.04 mg, 0.19 mmol) was added. The reaction mixture was stirred at -78 °C for 30 min and quenched with saturated NH4Cl solution (5.0 mL). The resulting mixture was extracted with EtOAc (10 mL x 3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated. The residue was purified by combiflash column chromatography using 0-10% EtOAc in hexane to give compound 2 (0.03 g, 44%) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.4 Hz, 2H), 7.57 (s, 1H), 7.45-7.30 (m, 3H), 4.58-4.47 (m, 2H), 3.81 (s, 3H), 3.05-2.98 (m, 1H), 2.59-2.43 (m, 9H), 1.75-1.70 (m, 2H), 1.68-1.67 (m, 1H), 1.45 (s, 3H), 1.39 (s, 9H).

[0217] Preparation of compound 3 To an ice-cold solution of compound 2 (0.03 g, 0.07 mmol) in THF (3.0 mL) was added LiAlH4 (0.14 mL, 1.0 M in THF, 0.14 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min and diluted with H2O and Na2SO4·10H2O. The resulting mixture was extracted with EtOAc (5.0 mL × 3). The organic layers were combined, washed with brine (5.0 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-30% EtOAc in hexane to give compound 3 (0.01 g, 36%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.74-7.71 (m, 3H), 7.55 (s, 1H), 7.43-7.36 (m, 2H), 7.31, 7.28 (dd, J = 1.6 ,8.4 Hz, 2H), 4.57-4.46 (m, 2H), 3.82 (t, J = 6.0 Hz, 2H), 3.07-2.85 (m, 1H), 2.44-2.35 (m, 2H), 2.22 (s, 2H),1.96-1.87 (m, 3H), 1.69-1.65 (m, 3H), 1.49 (s, 4H), 1.41 (s, 9H).

[0218] Preparation of compound ALB-210360 (B2-5A-1) To an ice-cold solution of compound 3 (0.01 g, 0.025 mmol) dissolved in CHCl (1.0 mL) was added p-TSA (0.009 g, 0.05 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h and diluted with HO. The resulting mixture was extracted with EtOAc (5.0 mL×3). The organic layers were combined, washed with brine (5.0 mL), dried over NaSO, and concentrated under reduced pressure. The residue was purified by combiflash column chromatography using 0-20% EtOAc in hexanes and preparative HPLC to give compound ALB-210360 (B2-5A-1) (0.0008 g) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 7.77-7.72 (m, 3H), 7.52 (s, 1H), 7.42-7.39 (m, 3H), 4.58-4.50 (m, 2H), 4.31 (s, 2H), 2.74-2.67 (m, 1H), 2.46-2.39 (m, 1H), 2.34 (d, J = 3.6 Hz, 2H), 1.96-1.72 (m, 3H), 1.66-1.60 (m, 2H), 1.49 (s, 3H); ESI MS m / z 321 C 22 H 24 O2+H] + .

[0219] Synthesis Example 25 Synthesis of Compounds B2-2A and B2-3A The preparation method of compound B2-2A (also referred to as ALB-210365) (also referred to as scheme 26) and the preparation method of compound B2-3A (also referred to as ALB-210797) are as follows.

[0220] [ka]

[0221] Preparation of Compound 1 To a solution (0 °C) of compound Int-7 (compound 7 in scheme 1) (0.02 g, 0.081 mmol) dissolved in THF (3.0 mL) and t-BuOH (2.0 mL), t-BuOK (0.0137 g, 0.122 mmol) was added followed by CH3NO2 (0.01 g, 0.1639) and stirred at 0 °C for 1 h. The reaction mixture was then stirred at room temperature for 20 h. The reaction mixture was diluted with saturated NH4Cl (20 mL), extracted with MTBE (2 x 20 mL), dried over anhydrous Na2SO4, filtered and concentrated to give the crude product. The crude product was purified by column chromatography using 0-20% EtOAc in hexane to give compound 1 (0.005 g, 20%) as a colorless liquid. ESI MS m / z 306 C 17 H 23 NO4 + H] + .

[0222] Preparation of compound 2 To a stirred solution of compound 1 (0.05 g, 0.163 mmol) dissolved in EtOH (5.0 mL) and H2O (2.0 mL), Fe (0.036 g, 0.655 mmol) was added followed by NH4Cl (0.034 g, 0.655 mmol) and stirred at 90 °C for 4 h. The reaction mixture was filtered through Celite, diluted with water (40 mL) and extracted with CHCl (50 mL × 2). The organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give compound 2 [0.055 g (crude)] as a brown liquid. The crude was used in the next step without purification.

[0223] Preparation of compound ALB-210365 (B2-2A) To a stirred solution of compound 2 (0.045 g, 0.163 mmol) dissolved in MeOH (5.0 mL) and then in AcOH (0.02 mL), formaldehyde (1.0 mL) was added at room temperature and stirred for 16 h at room temperature. NaCNBH3 (0.030 g, 0.490 mmol) was added to the reaction mixture at 0 °C. Then the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with saturated NaHCO3 (40 mL) and extracted with CHCl2 (50 mL × 2). The organic layers were combined, dried over anhydrous NaSO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by prep-HPLC column chromatography and the fractions were evaporated by lyophilization to obtain compound ALB-210365 (B2-2A) (0.02 g, 40%) as a colorless viscous liquid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.09 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 8.7 Hz, 2H), 4.58 (d, J = 15.2 Hz, 2H),3.78 (s, 3H), 3.15-3.02 (m, 1H), 2.86-2.74 (m, 1H), 2.73 (s, 6H), 2.72-2.62 (m, 1H), 2.45-2.35 (m, 1H), 2.12-1.98 (m, 3H),1.71-1.61 (m, 1H), 1.55 (s, 3H), 1.48-1.35 (m, 3H); ESI MS m / z 304 C 19 H 29 NO2+H] + .

[0224] Preparative HPLC conditions: Column: Gemini NX-C18 10 μm; 150×30 mm Mobile phase: A: acetonitrile; B: 10 mm ammonium formate solution Gradient (min / %A): 0 / 10, 2 / 20, 10 / 40, 15 / 60, 15.5 / 98, 18 / 98, 18.2 / 10, 20 / 10 Diluent: MeOH + THF

[0225] Preparation of compound ALB-210797 (B2-3A) To a stirred solution of compound Int-2 (compound 2 in scheme 26) (0.01 g, 0.036 mmol) dissolved in CHCl (5.0 mL) was added triethylamine (0.007 g, 0.072 mmol) followed by mesyl chloride (0.004 g, 0.036) at 0° C. The reaction mixture was then stirred at 0° C. for 2 h. The reaction mixture was diluted with water (20 mL) and extracted with CHCl (30 mL×2). The organic layers were combined, dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC column chromatography and the fractions were evaporated by lyophilization to obtain compound ALB-210797 (B2-3A) (0.004 g, 31%) as an off-white solid. 1 H NMR (400 MHz, CDCl 3 ) δ 7.04 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 4.74 (t, J = 6.4 Hz, 1H), 4.56 (d, J = 8.4 Hz, 2H), 3.76 (s, 3H), 3.15-3.02 (m, 2H), 2.97 (s, 3H), 2.96-2.84 (m, 1H), 2.32-2.20 (m, 1H), 1.98 (brs, 1H), 1.92-1.83 (m, 3H), 1.72-1.65 (m, 1H), 1.52 (s, 3H), 1.51-1.38 (m, 2H); ESI MS m / z 352 C 18 H 27 NO4S-H] + .

[0226] Preparative HPLC conditions: Column: Gemini NX-C18 10 μm; 150×30 mm Mobile phase: A: acetonitrile; B: 0.05% formic acid solution Gradient (min / %A): 0 / 10, 2 / 30, 10 / 60, 15 / 80, 15.2 / 98, 18 / 98, 18.2 / 10, 20 / 10 Diluent: MeOH + THF

[0227] Example 2: Evaluation of TLR7 activation inhibition in each derivative The inhibition of TLR7 activation in each derivative and CB-7 was evaluated using mouse TLR7-expressing reporter cells (Ba / F3 cells). These cells were provided by Professor Miyake Kensuke (Department of Infectious Genetics, Institute of Medical Science, University of Tokyo). A reporter gene in which the GFP gene was linked downstream of the NF-κB promoter region was introduced into these cells. Therefore, the expression of GFP was enhanced by adding a ligand for TLR7 expressed by the cells. The inhibition of TLR7 activation by each derivative was evaluated using the attenuation of the GFP fluorescence intensity by the addition of each derivative as an index.

[0228] Loxoribine (Alexis Biochemicals) was used as a ligand for mouse TLR7, and Gardiquimod (Invivogen) was used as a ligand for human TLR7.

[0229] Ba / F3 cells were cultured in RPMI1640 medium containing 10% (v / v) fetal calf serum (FCS), 50 U / mL penicillin, 50 μg / mL streptomycin, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, and 1 ng / mL interleukin-3 at a cell density of 1.0 × 10 cells per well. 5The cells were dispensed into a 96-well plate at 100 μL / cell. 50 μL of derivatives or CB-7, adjusted to final concentrations of 1, 5, 10, 25, 50, and 100 μM, were added to each well. The 96-well plate was incubated at 37°C in the presence of 5% CO2 for 30 minutes. Next, 250 μg / mL loxoribine was added to each well and cultured for 18 hours. After the culture was completed, the cells were washed with PBS (FACS buffer) containing 2.5% (v / v) FCS, and each cell was suspended in 200 μL of FACS buffer containing 25 μg / mL 7-actinomycin D. The GFP fluorescence intensity of the cells was analyzed by flow cytometry. Flow cytometry measurements were performed using a FACSCanto TM The measurements were performed using a 3D microscope (Becton Dickinson) and the data were analyzed using FlowJo software (Tree Star). The MFI (%) of stimulation with loxoribine alone was set as 100%, and the MFI (%) of stimulation with loxoribine in the presence of 1-100 μM derivatives or CB-7 was calculated. IC 50 (The compound concentration that inhibits activity by 50%) was calculated.

[0230] IC of each derivative 50 The IC of CB-7 is shown in Table 3. 50 was 3.12 μM.

[0231] [Table 3]

[0232] As shown in Table 3, the derivatives that had higher TLR7 activation inhibitory activity than CB-7 were B2-5A, B2-13, B2-22, B2-24, B2-26, B2-28, B2-29, B2-24-1, B2-24-2, B2-24-3, B2-24-4·HCl, B2-5A-4, B2-5A-6, B2-5A-7, and B2-5A-9. Among these, the derivatives that had more than twice the TLR7 activation inhibitory activity than CB-7 were B2-13, B2-22, B2-24, B2-26, B2-24-1, B2-24-2, B2-24-3, B2-24-4·HCl, B2-5A-6, and B2-5A-9. B2-24-4·HCl had the highest inhibitory activity against TLR7 activation compared with other derivatives.

[0233] It was also suggested that the introduction of a N atom into area b is important for the expression of high activity. Substitution of area a with a lactone ring did not affect the TLR7 activation inhibitory activity.

[0234] Example 3: Analysis of metabolic stability (cytochrome P450 (CYP)) of B2-24 and B2-5A using mouse liver microsomes CB-7, B2-24, and B2-5A were used in DMSO solutions at 10 mg / mL. Each compound was diluted with acetonitrile to 0.1 mg / mL, which was used as the analytical standard. Mouse liver microsome fraction was rapidly thawed in a 37°C warm bath and kept on ice. A CYP coenzyme-containing reaction buffer was prepared on ice and mixed with the microsome fraction. The reaction solution was preincubated at 37°C for 5 minutes, and then the test substance solution was added and mixed well by pipetting. The reaction solution was 200 μL, and the final concentration of the test substance was 0.1 mg / mL. The reaction solution was incubated at 37°C for 30 minutes or 2 hours, and the reaction was stopped by adding acetonitrile in an amount three times the reaction solution and vortexing. The sample after the reaction was stopped was centrifuged, the supernatant was separated, and then dried in an evaporator. 50 μL of 50% acetonitrile aqueous solution was added to the dried sample, and the sample was redissolved by vortexing for 5 minutes. The reconstituted solution was centrifuged, and the supernatant was subjected to HPLC analysis. The HPLC system was HITACHI Lachrom ELITE (Hitachi Corporation), and the column was COSMOSIL 5C. 18 -MS-II, 4.6 mm I.D. × 150 mm (manufactured by Nacalai Tesque) was used. The results are shown in Table 4.

[0235] [Table 4]

[0236] The number of peaks detected by HPLC in CB-7 was 7. On the other hand, the number of peaks detected by HPLC in B2-24 and B2-5A was 4, which was less than that of CB-7. Therefore, B2-24 and B2-5A showed improved drug metabolism, mainly by CYP, compared with CB-7. From these results, it was predicted that the hybrid derivative of B2-24 and B2-5A would have high TLR7 inhibitory activity and improved metabolic stability.

[0237] Example 4: Synthesis of hybrid derivative (B2-24-4-5A) of B2-24-4·HCl and B2-5A We commissioned Albany Molecular Research Inc. (USA) to create a hybrid derivative (B2-24-4-5A) between B2-24-4·HCl (a derivative with a similar structure to B2-24 and the highest TLR7 inhibitory activity) and B2-5A, as shown below. The structure of B2-24-4-5A is represented by the following formula (X).

[0238] [ka]

[0239] [ka] The structure of B2-24-4-5A·HCOOH is represented by the following formula (XIV).

[0240] Synthesis Example 26 Synthesis of compounds B2-24-4-5A and B2-24-4-5A·HCOOH The preparation of compounds B2-24-4-5A and B2-24-4-5A·HCOOH (also referred to as scheme 27) is as follows.

[0241] [ka]

[0242] Preparation of compound 2 To a solution (0 °C) of (-)-β-pinene (100.0 g, 0.735 mol) in CHCl (1.0 L), CHCN (1.0 L) and water (1.50 L) was added NaIO (626.4 g, 2.94 mol) and RuCl·nHO (4.56 g, 22.0 mmol). The reaction mixture was stirred at room temperature for 5 h. The reaction mixture was diluted with EtOAc (1.0 L) and washed with water (1.0 L) and brine (500 mL). The resulting mixture was dried over anhydrous NaSO, filtered and concentrated under reduced pressure to give compound 2 [96.0 g (crude), quantitative] as a brown liquid. 1H NMR (400 MHz, CDCl3) δ 2.58-2.51 (m, 3H), 2.38-2.30 (m, 1H), 2.25-2.21 (m, 1H), 2.08-2.01 (m, 1H), 1.98-1.90 (m, 1H), 1.57 (s, 1H), 1.35 (s, 3H), 0.84 (s, 3H).

[0243] Preparation of compound 3 A mixture of compound 2 (96.0 g, 695.0 mol), (PhSe)2 (108.0 g, 347.0 mol), SeO2 (91.2 g, 834.0 mol), and H2SO4 (25.9 mL, 486.0 mol) dissolved in methanol (1.0 L) was stirred at room temperature for 5 h. The reaction mixture was quenched with water (1.0 L) and extracted with EtOAc (1.0 L x 2). The organic extract was washed with brine (500 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel (60-120 mesh) column chromatography using 5% EtOAc in hexane to give compound 3 (80.0 g, 39%) as a green-brown liquid. 1 H NMR (400 MHz, CDCl3) δ 7.61-7.58 (m, 2H),7.32-7.25 (m, 1H), 3.87 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 2.71-2.68 (m, 1H), 2.61-2.52 (m, 2H), 2.24-2.20 (m, 2H), 1.88 (s, 1H), 1.35 (s, 3H), 0.84 (m, 3H).

[0244] Preparation of compound 4 To a solution of compound 3 (80.0 g, 272.0 mol) dissolved in CHCl (800 mL), 7% HO (140.0 mL, 408.0 mol) was added at 0° C., followed by pyridine (43.8 mL, 544.0 mol) and stirred at room temperature for 12 h. The reaction mixture was diluted with CHCl (1.0 L) and washed with water (1.0 L) and brine (500 mL). The resulting mixture was dried over anhydrous NaSO, filtered and concentrated under reduced pressure. The crude was purified by silica gel (60-120 mesh) column chromatography using 0-5% EtOAc in hexane to give compound 4 (30.0 g, 81%) as an oily liquid. 1 H NMR (400 MHz, CDCl3) δ 7.54-7.50 (m, 1H),5.95 (d, J = 8.8 Hz, 3H), 2.86-2.83 (m, 1H), 2.73-2.57 (m, 2H), 2.13 (s, 1H), 1.51 (s, 3H), 1.04 (m, 3H).

[0245] Preparation of compound 5 To a solution of compound 4 (10.0 g, 73.5 mmol) in 1,4-dioxane (100 mL) and water (20.0 mL), compound 4a (compound 1 in scheme 4) (14.5 g, 95.5 mmol), KOH (8.20 g, 147.0 mmol) were added and degassed for 15 min. [Rh(COD)Cl]2 complex (2.17 g, 4.40 mmol) was added to the reaction mixture, which was stirred and heated at 80 °C for 12 h. Residual solvent was evaporated under reduced pressure. The crude was dissolved in CHCl2 (500 mL) and filtered through a bed of Celite. The filtrate was washed with water (500 mL), dried over anhydrous NaSO4, filtered and concentrated under reduced pressure. The residue was purified by combiflash chromatography on silica gel using eluent 0-40% EtOAc in hexane to give compound 5 (6.15 g, 34%) as a colorless oil. ESI MS m / z 227 [M-NH4] + .

[0246] Preparation of compound 6 To a solution of compound 5 (6.15 g, 25.0 mmol) dissolved in Ac2O (60 mL), Zn(OAc)2 (5.07 g, 27.7 mmol), BF3·OEt2 (3.42 mL, 27.7 mmol) were added and stirred at room temperature for 12 h. The reaction mixture was neutralized with saturated NaHCO3 (500 mL). The resulting mixture was extracted with EtOAc (2×250 mL). The organic layers were combined, washed with brine (250 mL), dried over anhydrous MgSO4, and concentrated under reduced pressure to give a residual oil. This was purified by combiflash chromatography on silica gel using 0-30% EtOAc in hexane as eluent to give compound 6 (5.00 g, 69%) as an off-white solid. ESI MS m / z 269 [M-NH4] + .

[0247] Preparation of compound 7 To a solution of compound 6 (5.00 g, 17.4 mmol) dissolved in methanol (50 mL) was added NaH (0.70 g, 60% in mineral oil, 17.4 mmol) in portions over 30 min at 0° C. The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was quenched with saturated aqueous NH4Cl (25 mL) at 0° C. Methanol was evaporated under reduced pressure. The residual suspension was filtered and washed with water (100 mL) followed by hexanes (100 mL) to give compound 7 (4.50 g, crude) as an off-white solid. ESI MS m / z 227 [M-NH4] + .

[0248] Preparation of compound 8 To an ice-cold solution of p-toluenesulfonylmethyl isocyanide (1.59 g, 8.2 mmol) and t-BuOK (1.83 g, 16.3 mmol) in THF (15 mL) was added compound 7 (1.00 g, 4.00 mmol) in THF (15.0 mL), stirred at 0° C. for 10 min, and methanol (25 mL) was added. The resulting mixture was stirred under reflux for 1 h and concentrated under reduced pressure. The residue was purified by combiflash chromatography on silica gel using eluent 0-50% EtOAc in hexane to give compound 8 (0.80 g, 77%) as a pale yellow liquid. This step was carried out multiple times to give compound 8 in 77%.

[0249] Preparation of compound 9 To a solution of compound 8 (4.00 g, 15.0 mmol) in 2-propanol (40 mL) was added KOH (8.70 g, 156.0 mmol) at room temperature. The reaction mixture was warmed to 100 °C and stirred under reflux for 48 h. Residual solvent was evaporated under reduced pressure. The crude was dissolved in EtOAc (100 mL) and washed with water (50 mL). The aqueous layer was neutralized by adding 2N HCl solution and extracted with EtOAc (2 x 100 mL). The organic layers were combined, washed with brine (50 mL), dried over MgSO4, and concentrated under reduced pressure to give compound 9 (2.50 g, crude) as a brown gum. ESI MS m / z 273 [MH] + .

[0250] Preparation of compound 10 To a solution of compound 9 (2.50 g, 9.12 mmol) in acetonitrile:methanol (15:5 mL) was added K2CO3 (3.80 g, 27.3 mmol) and MeI (3.84 mL, 27.3 mmol) dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 16 h. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (2 x 50 mL). The organic layers were combined, washed with brine (50 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by combiflash chromatography on silica gel using eluent 0-30% EtOAc in hexane to give compound 10 (1.25 g, 48%) as an oily gum. ESI MS m / z 271 [M-NH4] + .

[0251] Preparation of compound 11 To a stirred solution of compound 10 (1.25 g, 4.30 mol) dissolved in CHCl (15 mL) was added imidazole (0.63 g, 10.8 mmol) followed by TBDMSCl (0.78 g, 5.20 mmol) in portions at 0° C. The reaction mixture was stirred at room temperature for 16 h, diluted with CHCl (100 mL) and washed with saturated aqueous NaHCO (50 mL). The resulting reaction mixture was dried over anhydrous NaSO and concentrated under reduced pressure. The residue was purified by combiflash chromatography on silica gel using eluent 0-10% EtOAc in hexane to give compound 11 (1.25 g, 72%) as a pale yellow oil.

[0252] Preparation of compound 12 To a solution of lithium diisopropylamide (5.78 mL, 2.0 M in THF, 11.5 mmol) dissolved in THF (8.0 mL) was added compound 11 (1.55 g, 3.80 mmol) dissolved in THF (8.0 mL) dropwise at -78 °C. The solution was stirred at -78 °C for 30 min and tert-butyl 2-bromoacetate (1.60 mL, 7.70 mmol) was added to the reaction mixture. The reaction mixture was stirred at -78 °C for 30 min and quenched with saturated aqueous NH4Cl (15 mL). The resulting mixture was extracted with EtOAc (2 x 50 mL). The organic layers were combined and washed with brine (25 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by combiflash chromatography on silica gel using eluent 0-10% EtOAc in hexane to give compound 12 (2.1 g, crude) as a pale yellow oil.

[0253] Preparation of compound 13 To an ice-cold solution of compound 12 (2.10 g, 4.06 mmol) dissolved in THF (20 mL) was added TBAF (8.10 mL, 1 M in THF, 8.13 mmol) at 0° C. The reaction mixture was stirred at room temperature for 8 h and diluted with water (50 mL). The resulting mixture was extracted with EtOAc (2×50 mL). The organic layers were combined, washed with brine (25 mL), dried over MgSO4, and concentrated under reduced pressure to give a residual oil, which was purified by combiflash chromatography on silica gel using 0-30% EtOAc in hexane as eluent to give compound 13 (1.48 g, 90%) as a colorless gum.

[0254] Preparation of compound 14 To an ice-cold solution of compound 13 (1.48 g, 3.60 mmol) in CHCl (15 mL) was added Dess-Martin periodinane (3.12 g, 7.36 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated aqueous NaSO (20 mL), saturated aqueous NaHCO (20 mL) at 0 °C, filtered through a bed of celite, and extracted with CHCl (2 × 50 mL). The organic layers were combined, washed with brine (50 mL), dried over MgSO, and concentrated under reduced pressure to give compound 14 [1.50 g (crude)] as a colorless oil. ESI MS m / z 401 [M + H] + .

[0255] Preparation of compound 15 To a solution of compound 14 (1.50 g, 3.75 mmol) dissolved in methanol (5.0 mL) and a small amount of AcOH (0.06 ml, 1.10 mmol), compound 14a (1.39 g, 7.50 mmol) was added followed by NaCNBH3 (0.71 g, 11.20 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. Methanol was evaporated and diluted with water (50 mL). The resulting mixture was extracted with EtOAc (2 x 50 mL). The organic layers were combined, washed with brine (25 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by combiflash chromatography on silica gel using eluent 0-40% EtOAc in hexane to give compound 15 (1.60 g, 75%) as a colorless gum. ESI MS m / z 571 [M + H] + .

[0256] Preparation of compound 16 To an ice-cold solution of compound 15 (0.10 g, 0.17 mmol) dissolved in THF (10.0 mL) was added LiAlH4 (2.0 M in THF, 0.87 mL, 1.70 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 3 min and quenched with saturated aqueous NH4Cl (5.0 mL). The resulting mixture was extracted with EtOAc (2 × 10 mL). The organic layers were combined, washed with brine (5 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was purified by combiflash chromatography on silica gel using eluent 0-60% EtOAc in hexane to give compound 16 (0.034 g) as a colorless oil. This step was carried out multiple times to give compound 16 in 36%. ESI MS m / z 543 [M + H] + .

[0257] Production of compound B2-24-4-5A·HCOOH To a solution of compound 16 (0.53 g, 0.97 mmol) dissolved in CHCl (10.0 mL) was added p-toluenesulfonic acid (0.74 g, 3.90 mmol) at room temperature and stirred at room temperature for 16 h. The reaction mixture was quenched with saturated aqueous NaHCO (10 mL) and extracted with CHCl (2×25 mL). The organic layers were combined, washed with brine (25 mL), dried over MgSO, and concentrated. The residue was purified by mass-triggered preparative HPLC to give compound B2-24-4-5A·HCOOH (0.208 g, 51%) as an off-white solid. 1 H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 7.18 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 4.55 (s, 2H), 4.28 (s, 2H), 3.49 (s, 2H), 3.09-3.03 (m, 4H), 2.60-2.50 (m, 5H), 2.39-2.25 (m, 3H), 1.92-1.88 (m, 2H), 1.82-1.78 (m, 1H), 1.68-1.52 (m, 3H) 1.46 (s, 3H). ESI MS m / z 369 [M+H] + .

[0258] Preparation of compound B2-24-4-5A Compound B2-24-4-5A·HCOOH (100 mg) dissolved in water (5 mL) was extracted with 10% methanol in CH2Cl2 (10 mL×8). The organic layers were combined, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to give compound B2-24-4-5A (45 mg). 1 H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.0 Hz, 2H), 4.55 (s, 2H), 4.28 (s, 2H), 3.45 (s, 2H), 2.94 (t, 4H), 2.57-2.42 (m, 5H), 2.35-2.25 (m, 3H), 1.92-1.75 (m, 3H), 1.68-1.52 (m, 4H) 1.48 (s, 3H). ESI MS m / z 369 [M+H] + .

[0259] Example 5: Evaluation of TLR7 activation inhibition by B2-24-4-5A·HCOOH The inhibition of TLR7 activation by B2-24-4-5A·HCOOH was evaluated by the method described in Example 2. CB-7 and B2-24-4·HCl were used as controls. The IC values ​​of CB-7 and each derivative were 50 are shown in Table 5.

[0260] [Table 5]

[0261] As shown in Table 5, the mouse TLR7 inhibitory effects of B2-24-4-5A·HCOOH and B2-24-4·HCl were almost equivalent.

[0262] Example 6: Confirmation of TLR7 inhibitory effect of B2-24-4-5A·HCOOH in mouse bone marrow-derived macrophages (BMDM) The TLR7 inhibitory effect of B2-24-4-5A·HCOOH in mouse BMDM was confirmed. Mouse bone marrow cells were cultured in RPMI1640 medium containing 10 μg / ml M-CSF, 10% (v / v) fetal calf serum (FCS), penicillin, streptomycin, L-glutamic acid, and 2-mercaptoethanol at 37°C in the presence of 5% (v / v) CO2 for 7 days. Mouse BMDMs induced to differentiate by culture were placed in each well of a 96-well plate at 1.0 × 10 5 The wells were seeded with 50 μL of CB-7 or B2-24-4-5A·HCOOH at a final concentration of 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, or 30 μM, and incubated for 30 minutes. B2-24-4-5A·HCOOH or CB-7 were used in saline or DMSO solutions at 10 mg / mL, respectively. Next, TLR7 ligand R848 (Invivogen) was added at a final concentration of 10 ng / mL and cultured for 24 hours. After the culture, all the culture medium was collected, and the IL-6 concentration in the culture medium was quantified using ELISA (kit used: Mouse IL-6 DuoSet ELISA (trade name), R&D Systems). The results are shown in Figure 2.

[0263] The R848-induced IL-6 production was attenuated by pretreatment with B2-24-4-5A·HCOOH. The TLR7 inhibitory effect of B2-24-4-5A·HCOOH was approximately 100-fold stronger than that of CB-7.

[0264] Example 7: Confirmation of TLR7 selectivity of B2-24-4-5A·HCOOH in mouse BMDM The TLR7 selectivity of B2-24-4-5A·HCOOH in mouse BMDM was confirmed. Mouse bone marrow cells were cultured in RPMI1640 medium containing 10 μg / ml M-CSF, 10% (v / v) fetal calf serum (FCS), penicillin, streptomycin, L-glutamic acid, and 2-mercaptoethanol at 37°C in the presence of 5% (v / v) CO2 for 7 days. Mouse BMDMs induced to differentiate by culture were placed in each well of a 96-well plate at 1.0 × 10 5 The cells were seeded at a concentration of 100 μL per well. 50 μL of B2-24-4-5A·HCOOH was added to each well to a final concentration of 1 nM, 10 nM, or 100 nM, and incubated for 30 minutes. TLR ligands Pam2CSK4 (Invivogen), Poly(I:C) (Invivogen), Lipid A (Sigma-Aldrich), R848 (Invivogen), or CpG-B (Invivogen) were added to each well and cultured for 24 hours. After culture, all culture medium was collected, and the IL-6 concentration in the culture medium was quantified using ELISA (kit used: Mouse IL-6 DuoSet ELISA (product name), R&D Systems). The results are shown in Figure 3.

[0265] B2-24-4-5A·HCOOH suppressed IL-6 production induced by the TLR7 ligand R848, but not by other TLR ligands, indicating that B2-24-4-5A·HCOOH is a selective inhibitor of TLR7.

[0266] Example 8: Confirmation of TLR7 inhibitory effect of B2-24-4-5A·HCOOH using peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus The TLR7 inhibitory effect of B2-24-4-5A·HCOOH was confirmed in PBMCs derived from patients with systemic lupus erythematosus. PBMCs were isolated from 7 mL of blood collected from patients with systemic lupus erythematosus. The isolated PBMCs were placed in each well of a 96-well plate containing RPMI1640 medium containing 10% (v / v) fetal calf serum (FCS), penicillin, streptomycin, L-glutamic acid, and 2-mercaptoethanol at 0.5 × 10 5 The wells were seeded with 50 μL of B2-24-4-5A·HCOOH, B2-24-4·HCl, or a control drug, hydroxychloroquine sulfate (HCQ), at a final concentration of 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, or 50 μM, and incubated for 30 minutes. Gardiquimod (Invivogen), a TLR7 ligand, was added to a final concentration of 3 μg / mL and cultured for 24 hours. After culture, all culture medium was collected, and the IL-6 concentration in the culture medium was quantified using ELISA (kit used: human IL-6 DuoSet ELISA (trade name), R&D Systems). The results are shown in Figure 4.

[0267] IL-6 production induced by guardiquimod stimulation was attenuated by pretreatment with all compounds, with the strongest effects being B2-24-4·HCl, B2-24-4-5A·HCOOH, and HCQ.

[0268] Example 9: Analysis of metabolic stability (CYP) of CB-7 and B2-24-4-5A·HCOOH using mouse liver microsomes The metabolic stability analysis of CB-7 and B2-24-4-5A·HCOOH was performed by the method of Example 5. The results for CB-7 are shown in Table 6, and the results for B2-24-4-5A·HCOOH are shown in Table 7.

[0269] [Table 6]

[0270] [Table 7]

[0271] The number of peaks detected by HPLC in CB-7 was 9 (Table 6 shows 7 out of 9 peaks). On the other hand, the number of peaks detected by HPLC in B2-24-4-5A·HCOOH was 6, which was fewer than that of CB-7. The peak area (Area) of CB-7 after 2 hours was 41.6%, while the peak area (Area) of B2-24-4-5A·HCOOH after 2 hours was 88.4%. Therefore, B2-24-4-5A·HCOOH showed a significant improvement in drug metabolism, mainly by CYP, compared with CB-7.

[0272] Example 10: Plasma concentrations after oral administration of CB-7 or B2-24-4-5A·HCOOH in mice Plasma concentrations of CB-7 or B2-24-4-5A·HCOOH after oral administration were measured using normal mice (C57BL / 6N, 11 weeks old, female). 1 mg of CB-7 or B2-24-4-5A·HCOOH was orally administered to C57BL / 6N mice. After 0.5, 1, 2, 4, or 8 hours, blood was collected from the C57BL / 6N mice to prepare plasma. The concentrations of CB-7 or B2-24-4-5A·HCOOH in plasma were measured by LC / MS / MS. The results are shown in Figure 5.

[0273] CB-7 was not detectable in plasma after 8 hours, whereas B2-24-4-5A·HCOOH was present at approximately 500 ng / ml in plasma after 8 hours, indicating that B2-24-4-5A·HCOOH remained in plasma for a longer period than CB-7.

[0274] Example 11: Plasma concentrations of B2-24-4-5A·HCOOH after oral administration in mice Using systemic lupus erythematosus model mice (NZBWF1, 20 weeks old, female), the plasma concentration after oral administration of B2-24-4-5A·HCOOH was measured. 5 mg / kg of B2-24-4-5A·HCOOH was orally administered to NZBWF1 mice. After 0.5, 1, 2, 4, 8, 16 or 24 hours, blood was collected from the NZBWF1 mice to prepare plasma. The concentration of B2-24-4-5A·HCOOH in the plasma was measured by LC / MS / MS. The results are shown in Figure 6.

[0275] B2-24-4-5A·HCOOH was present in plasma at 88.1 ng / ml 0.5 hours after administration, then disappeared, reaching a bottom level at 8 hours.

[0276] Example 12: Confirmation of TLR7 inhibitory effect by intraperitoneal administration of B2-24-4-5A·HCOOH in mice Normal mice (C57BL / 6N, 11 weeks old, female) were intraperitoneally administered with B2-24-4-5A·HCOOH (1, 5, 10, 100, and 1000 μg) or hydroxychloroquine sulfate (HCQ, 1000 μg) as a comparison drug, and 30 minutes later, 5 μg of R848 was intraperitoneally administered. Physiological saline was used as the solvent. One hour after R848 administration, blood was collected and the concentration of IFN-α in the serum was quantified using the ELISA method (kit used: Mouse IFN alpha Platinum ELISA (trade name), eBioscience). The results are shown in Figure 7. The plots in Figure 7 show the data for each individual, the thick lines show the average value of the data for each group, and the error bars show the standard error of the data for each group.

[0277] IFN-α production induced by R848 was attenuated by pretreatment with B2-24-4-5A·HCOOH, and the effect was stronger than that of HCQ. Intraperitoneal administration of 10 μg of B2-24-4-5A·HCOOH almost completely suppressed the induction of IFN-α. This result indicates that the drug was effective at 0.5 mg / kg, assuming a mouse body weight of 20 g. In other words, the in vivo TLR7 inhibitory effect was more than 100 times that of HCQ.

[0278] Example 13: Confirmation of TLR7 inhibitory effect by intraperitoneal administration of B2-24-4·HCl, B2-24-4-5A, or B2-24-4-5A·HCOOH in mice Using the same method as in Example 13, the TLR7 inhibitory effect of intraperitoneal administration of B2-24-4·HCl, B2-24-4-5A, or B2-24-4-5A·HCOOH in mice was confirmed. The dose of B2-24-4·HCl, B2-24-4-5A, and B2-24-4-5A·HCOOH was 10 μg. The results are shown in Figure 8. The plots in Figure 8 show the data for each individual, the thick lines show the average data for each group, and the error bars show the standard error for each group data.

[0279] IFN-α production induced by R848 was attenuated by pretreatment with B2-24-4·HCl, B2-24-4-5A, or B2-24-4-5A·HCOOH, with the effects being strongest in the following order: B2-24-4-5A·HCOOH, B2-24-4-5A, and B2-24-4·HCl.

[0280] Example 14: Confirmation of the therapeutic effect of B2-24-4-5A·HCOOH in drug-induced systemic lupus erythematosus model mice Systemic lupus erythematosus-like pathology was induced in normal mice (BALB / c, 7-week-old, female, n=10) by applying Veselna Cream (trade name) to the right ear three times a week for 8 weeks. These mice were then divided into a saline-treated group (control, n=5) and a B2-24-4-5A·HCOOH-treated group (n=5). The saline-treated group continued to apply Veselna Cream (trade name) in the same manner, while orally administering saline (100 μl) once a day. The B2-24-4-5A·HCOOH-treated group continued to apply Veselna Cream in the same manner, while orally administering 5 mg / kg B2-24-4-5A·HCOOH (100 μl) once a day. Six weeks after the start of administration, the mice were euthanized. After euthanasia, blood was collected from the abdominal aorta of the mice, and the spleen and kidneys were removed. The excised spleens were photographed and weighed.

[0281] The excised spleen was ground with a slide glass to prepare splenocytes, which were then stained with FITC-labeled anti-CD3 antibody and PE-labeled anti-CD69 antibody. After 20 minutes, the cells were washed with FACS buffer and suspended in 200 μL of FACS buffer containing 25 μg / mL 7-actinomycin D. The fluorescence intensity of the cells was analyzed by flow cytometry. Flow cytometry was performed using a FACSCanto TM The data was analyzed using FlowJo software (Tree Star) to calculate the proportion of CD69-positive cells (activated T cells) among CD3-positive T cells.

[0282] The excised kidneys were fixed in 4% (w / v) paraformaldehyde, and immunohistochemical staining was outsourced to Morpho Technology Co., Ltd. Blood collected from the abdominal aorta was centrifuged to prepare serum. Quantitative analysis of serum urea nitrogen and creatinine levels was outsourced to Oriental Yeast Co., Ltd. The results are shown in Figures 9A to 9D.

[0283] Figure 9A is a photograph of the excised spleen. Figure 9B shows the weight of the spleen. Figure 9C shows the ratio of activated T cells among splenic T cells. Figure 9D is a photograph showing the deposition of IgG in the glomerulus. The plots in Figures 9B and 9C show the data of each individual, the thick lines show the average data of each group, and the error bars show the standard error of the data of each group. The scale bar in Figure 9D represents 100 μm.

[0284] As is clear from Figures 9A to 9D, the B2-24-4-5A·HCOOH-treated group showed suppression of splenic swelling, a decrease in the proportion of activated T cells among splenic T cells, and suppression of IgG deposition in the glomerulus compared to the control saline-treated group.

[0285] Example 15: Synthesis of formate salt of compound B2-24-4 We commissioned Albany Molecular Research Inc. (USA) to synthesize compound B2-24-4·HCOOH. The structure of B2-24-4·HCOOH is represented by the following formula (XV).

[0286] [ka] Synthesis Example 27 Synthesis of compound B2-24-4·HCOOH The preparation method (also referred to as scheme 28) of compound B2-24-4·HCOOH (also referred to as ALB-210796) is as follows.

[0287] [ka]

[0288] Preparation of compound 2 To a stirred solution of (-)-β-pinene (100.0 g, 0.73 mol) in CHCl (1.0 L), acetonitrile (1.0 L) and water (1.0 L), NaIO (626.4 g, 2.94 mol) was added portionwise at 0 °C and the reaction mixture was stirred at the same temperature for 10 min. RuCl-nH0 (4.56 g, 22.0 mmol) was added portionwise to the reaction mixture at 0 °C and stirred at room temperature for 5 h, after which the reaction mixture was added Celite and filtered through a Celite pad and washed with CHCl (500 mL). The filtrate was diluted with H0 (1.0 L) and extracted with CHCl (2 x 500 mL). The organic layers were combined, washed with brine (500 mL), dried over anhydrous NaSO, and then concentrated under reduced pressure to give compound 2 (100.0 g, crude) as a brown liquid. 1 H NMR (400 MHz, CDCl3) δ 2.58-2.51 (m, 3H), 2.38-2.30 (m, 1H), 2.25-2.21 (m, 1H), 2.08-2.01 (m, 1H), 1.98-1.90 (m, 1H), 1.57 (s, 1H), 1.35 (s, 3H), 0.84 (s, 3H).

[0289] Preparation of compound 3 To a stirred solution of compound 2 (50.0 g, 0.362 mol) in methanol (500 mL) was added (PhSe)2 (56.5 g, 0.181 mol) and SeO2 (48.2 g, 0.434 mol), followed by dropwise addition of H2SO4 (13.5 mL, 0.253 mol) at 0 °C. The reaction mixture was stirred at room temperature for 10 h, quenched with ice-cold water (1.0 L), added with EtOAc (1.0 L) and filtered through a pad of Celite. The organic layer was separated, washed with brine (500 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel (60-120 mesh) using eluent 5-10% EtOAc in hexane to give compound 3 (50.0 g, 47%) as a wine-red liquid. 1 H NMR (400 MHz, CDCl3) δ 7.61-7.58 (m, 2H), 7.32-7.25 (m, 3H), 3.87 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 2.71-2.68 (m, 1H), 2.61-2.52 (m, 2H), 2.24-2.20 (m, 2H), 1.88 (d, J = 8.00 Hz, 1H), 1.35 (s, 3H), 0.84 (m, 3H).

[0290] Preparation of compound 4 To a stirred solution of compound 3 (45.0 g, 0.153 mol) in CHCl (450 mL) was added 30% aq. H0 (26.0 mL, 0.229 mol) dropwise at 0° C., followed by pyridine (43.8 mL, 0.544 mol) over 10 min, and the reaction mixture was stirred below 5° C. for 3 h. The reaction mixture was quenched with sodium thiosulfate (250 mL) and extracted with CHCl (2×250 mL). The combined organic layers were washed with 0.5 N hydrochloric acid (2×250 mL), water (200 mL) and brine (200 mL), dried over anhydrous NaSO and concentrated under reduced pressure below 25° C. The crude compound was purified by column chromatography on silica gel (60-120 mesh) using eluent 5-10% EtOAc in hexane to give compound 4 (15.0 g, 72%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.54-7.50 (m, 1H), 5.95 (d, J = 8.8 Hz, 1H), 2.86-2.83 (m, 1H), 2.76-2.70 (m, 1H), 2.64-2.56 (m, 1H), 2.15 (d, J = 8 Hz, 1H), 1.51 (s, 3H), 1.04 (m, 3H).

[0291] Preparation of compound 5 To a stirred solution of compound 4 (15.0 g, 110.29 mmol) in 1,4-dioxane (160 mL) and water (40.0 mL), compound 4a (compound 1 in scheme 4) (25.14 g, 165.43 mmol) and KOH (12.37 g, 220.58 mmol) were added and the reaction mixture was degassed under argon for 15 min. [Rh(COD)Cl]2 complex (1.63 g, 3.30 mmol) was added and again degassed under argon for 5 min. The resulting reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was cooled to room temperature, diluted with CHCl2 (500 mL) and filtered through a bed of Celite. The filtrate was washed with H2O (500 mL) and the organic layers were combined, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel (60-120 mesh) using eluent 20-40% EtOAc in hexane to give compound 5 (20 g, 74%) as a wine-red oil. ESI MS m / z 245 [M+H] + .

[0292] Preparation of compound 6 To a stirred solution of compound 5 (20 g, 81.91 mmol) in Ac2O (200 mL) was added Zn(OAc)2 (18 g, 98.29 mmol) and BF3·OEt2 (12.1 mL, 98.29 mmol) below 10 °C and stirred at room temperature for 16 h. The reaction mixture was neutralized with saturated NaHCO3 (500 mL) and extracted with EtOAc (2 x 250 mL). The organic layers were combined, washed with brine (250 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel (60-120 mesh) using eluent 30-40% EtOAc in hexane to give compound 6 (18.5 g, 68%) as an off-white solid. 1 H-NMR (400 MHz, CDCl3): δ 7.25 (d, J = 8.40 Hz, 2H), 7.18 (d, J = 8.00 Hz, 2H), 5.47-5.46 (m, 1H), 5.06 (s, 2H), 4.68 (s, 1H), 4.61 (s, 1H), 3.03-3.00 (m, 1H), 2.75-2.69 (m, 1H), 2.43-2.29 (m, 4H), 2.09 (s, 6H), 1.50 (s, 3H).

[0293] Preparation of compound 7 To a solution of compound 6 (18.5 g, 56.40 mmol) in methanol (185 mL) was added NaH (2.26 g, 60% in mineral oil, 56.40 mmol) portionwise at 0° C. The reaction mixture was stirred at room temperature for 1 h, then quenched with saturated aqueous aq. NH4Cl (100 mL) at 0° C., diluted with water (100 mL) and extracted with EtOAc (2×250 mL). The organic layers were combined, washed with brine (200 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound was washed with hexane (2×50 mL) and dried to give compound 7 (13.5 g, crude) as an off-white solid. 1H-NMR (400 MHz, CDCl3): δ 7.30 (d, J = 8.00 Hz, 2H), 7.16 (d, J = 8.00 Hz, 2H), 4.66 (s, 3H), 4.62 (s, 1H), 3.03-2.98 (m, 1H), 2.81-2.74 (m, 1H), 2.55-2.51 (m, 4H), 2.15-2.08 (m, 1H), 1.93-1.82 (m, 1H), 1.73-1.70 (m, 1H), 1.52 (s, 3H).

[0294] Preparation of compound 8 To an ice-cold solution of p-toluenesulfonylmethyl isocyanide (6.40 g, 32.78 mmol) in THF (80 mL) was added dropwise 1M tert-BuOK solution in THF (65.56 mL, 65.56 mmol) at 0° C. and stirred at the same temperature for 10 min. Compound 7 (4 g, 16.39 mmol) in THF (10 mL) was added to the reaction mixture below 5° C. and stirred for 15 min. Methanol (40 mL) was added to the reaction mass, stirred under reflux for 1 h, and the residual solvent was evaporated under reduced pressure. To the resulting residue was added H2O (100 mL) and extracted with EtOAc (2×100 mL). The organic layers were combined and washed with brine (50 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was purified by combiflash chromatography using eluent 30-40% EtOAc in hexane to give compound 8 (1.53 g, 36%) as a pale yellow gum, which was used in the next reaction without further purification.

[0295] Preparation of compound 9 To a stirred solution of compound 8 (2.0 g, 7.83 mmol) in 2-propanol (20 mL) was added KOH (4.4 g, 78.38 mmol) at room temperature. The reaction mixture was stirred under reflux for 48 h. Residual solvent was evaporated under reduced pressure and the crude obtained was dissolved in EtOAc (100 mL) and washed with water (50 mL). The aqueous layer was neutralized with 2 M hydrochloric acid and extracted with EtOAc (2×100 mL). The organic layers were combined and washed with brine (50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to give compound 9 (1.0 g, crude) as a grey solid. ESI MS m / z 273 [MH] + .

[0296] Preparation of compound 10 To a stirred solution of compound 9 (13.0 g, 47.44 mmol) in acetonitrile (150 mL) and methanol (30 mL), K2CO3 (19.78 g, 142.32 mmol) was added followed by dropwise addition of MeI (8.8 mL, 142.32 mmol) below 5 °C. The reaction mixture was stirred at room temperature for 16 h. Residual solvent was evaporated under reduced pressure and water (100 mL) was added to the residue and extracted with EtOAc (2 x 100 mL). The organic layers were combined and washed with water (50 mL), brine (50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound was purified by combiflash chromatography using eluent 10-20% EtOAc in hexane to give compound 10 (8.20 g, 60%) as a colorless oil. 1 H-NMR (400 MHz, CDCl3): δ 7.26 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 4.64 (d, J = 4.80 Hz, 2H), 4.56-4.53 (m, 2H), 3.65 (s, 3H), 2.59-2.46 (m, 2H), 2.36-2.30 (m, 1H), 2.12-2.08 (m, 2H), 1.91-1.86 (m, 1H), 1.67-1.60 (m, 3H), 1.52-1.49 (m, 4H).

[0297] Preparation of compound 11 To a stirred solution of compound 10 (8.20 g, 28.45 mmol) in CHCl (160 mL) was added imidazole (4.8 g, 71.12 mmol) and TBDMSCl (5.14 g, 34.14 mmol) in portions at 0° C. The reaction mixture was stirred at room temperature for 16 h, then quenched with saturated aq. NaHCO (200 mL) and extracted with CHCl (2×100 mL). The organic layers were combined and washed with water (50 mL) and brine (50 mL), dried over anhydrous NaSO, and concentrated under reduced pressure. The crude compound was purified by combiflash chromatography using eluent 10-20% EtOAc in hexane to give compound 11 (10.0 g, 87%) as a colorless oil. 1 H-NMR (400 MHz, CDCl3): δ 7.20 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 4.70 (s, 2H), 4.55 (s, 1H), 4.53 (s, 1H), 3.65 (s, 3H), 2.47-2.45 (m, 2H), 2.35-2.28 (m, 1H), 2.11-2.08 (m, 2H), 1.90-1.86 (m, 1H), 1.67-1.59 (m, 2H), 1.51 (s, 1H), 1.49 (s, 3H), 0.93 (s, 9H), 0.08 (s, 6H).

[0298] Preparation of compound 12 To a stirred solution of compound 11 (1.20 g, 2.98 mmol) in THF (60 mL) was added 2M lithium diisopropylamide solution in THF (4.47 mL, 8.94 mmol) dropwise at -78 °C. The reaction mixture was stirred at -78 °C for 30 min, then cooled to -105 °C (using MeOH, liquid N2) and phenylvinyl sulfoxide (0.6 mL, 4.47 mmol) was added. The reaction mixture was stirred at -105 °C for 30 min. The reaction mixture was quenched with saturated aqueous NH4Cl (25 mL) and extracted with EtOAc (2 x 50 mL). The organic layers were combined and washed with brine (50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound was purified by combiflash chromatography using eluent 30-40% EtOAc in hexane to give compound 12 (0.47 g, 28%) as a pale yellow oil. ESI MS m / z 555 [M+H] + .

[0299] Preparation of compound 13 To a stirred solution of compound 12 (3.40 g, 6.128 mmol) in xylene (51 mL) was added NaHCO3 (5.1 g, 61.28 mmol) at room temperature and the reaction mixture was stirred under reflux for 16 h. The reaction mixture was cooled to room temperature, diluted with H2O (50 mL) and extracted with EtOAc (3 x 50 mL). The organic layers were combined and washed with brine (50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound was purified by combiflash chromatography using eluent 10-30% EtOAc in hexane to give 13 (2.0 g, 76%) as a colorless gum. 1H-NMR (400 MHz, CDCl3): δ 7.21 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 5.87-5.78 (m, 1H), 5.09-5.04 (m, 1H), 4.70 (s, 2H), 4.54-4.52 (m, 2H), 3.77 (s, 3H), 2.69-2.61 (m, 1H), 2.43-2.41 (m, 2H), 2.33-2.27 (m, 1H), 1.80-1.75 (m, 1H), 1.58 (s, 1H), 1.51-1.39 (m, 6H), 0.93 (s, 9H), 0.08 (s, 6H).

[0300] Preparation of compound 14 To a stirred solution of compound 13 (2.0 g, 4.66 mmol) in THF (40.0 mL) was added 1M TBAF in THF (9.32 mL, 9.32 mmol) at 0° C. The reaction mixture was stirred at room temperature for 3 h, then quenched with H2O (50 mL) and extracted with EtOAc (2×50 mL). The organic layers were combined, washed with brine (50 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was purified by combiflash chromatography using eluent 20-40% EtOAc in hexane to give compound 14 (1.20 g, 82%) as a pale yellow gum. ESI MS m / z 332 [M+ H2O] + .

[0301] Preparation of compound 15 To a stirred solution of compound 14 (1.20 g, 3.81 mmol) in CHCl (25.0 mL) was added Dess-Martin periodinane (3.23 g, 7.63 mmol) at 0° C. The reaction mixture was stirred at room temperature for 2 h, quenched with saturated NaSO (25.0 mL), stirred for 10 min, and then extracted with CHCl (2×50 mL). The organic layer was washed with saturated aqueous NaHCO (25.0 mL), brine (50 mL), dried over anhydrous NaSO, and concentrated under reduced pressure. The crude compound was purified by combiflash chromatography using eluent 10-20% EtOAc in hexane to give compound 15 (1.0 g, 84%) as a pale yellow gum. ESI MS m / z 313 [M+H] + .

[0302] Preparation of compound 16 To a stirred solution of compound 15 (1.0 g, 3.20 mmol) in MeOH (20.0 mL), AcOH (0.1 mL, 1.6 mmol) was added followed by tert-butyl piperzine-1-carboxylate (1.2 g, 6.40 mmol) and the reaction mixture was stirred at room temperature for 2 h. NaCNBH3 (0.60 g, 9.6 mmol) was added in portions at 0° C. and stirred at room temperature for 16 h. The reaction mixture was quenched with H2O (25 mL) and extracted with CHCl2 (2×50 mL). The organic layers were combined, washed with brine (50 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude was purified by combiflash chromatography using eluent 30-40% EtOAc in hexane to give compound 15 (1.25 g, 81%) as a colorless gum. ESI MS m / z 483 [M+H] + .

[0303] Preparation of compound 17 To a stirred solution of compound 16 (1.0 g, 2.07 mmol) in THF (50 mL) was added 2M LiAlH4 in THF (3.1 mL, 6.2 mmol) dropwise at 0° C. The reaction mixture was stirred at 0° C. for 30 min, quenched with saturated aqueous NH4Cl (25.0 mL), and extracted with EtOAc (2×50.0 mL). The organic layers were combined, washed with brine (25.0 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was purified by combiflash chromatography using eluent 30-50% EtOAc in hexane to give compound 17 (1.0 g, 88%) as a colorless gum. ESI MS m / z 455 [M+H] + .

[0304] Preparation of compound ALB-210796 (B2-24-4·HCOOH) To a stirred solution of compound 17 (1.0 g, 2.21 mmol) in CHCl (25 mL) was added 4 M hydrochloric acid in 1,4-dioxane (10 mL) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 4 h and residual solvent was evaporated under reduced pressure. The crude was washed with hexane (2 × 20 mL) and purified by preparative HPLC eluting with CHCN in H0 using ammonium formate as buffer. The purified fractions were concentrated under reduced pressure and then lyophilized to give the formate salt of ALB-210796 (B2-24-4·HCOOH) (420 mg, 47%) as an off-white solid. 1 H-NMR (400 MHz, DMSO-d6): 8.30 (s, 1H), 7.15-7.10 (m, 4H), 5.89-5.81 (m, 1H), 4.94-4.89 (m, 2H), 4.54 (d, J = 1.60 Hz, 1H), 4.45-4.44 (m, 1H), 3.54 (s, 2H), 3.98 (s, 2H), 2.84-2.67 (m, 5H), 2.36-2.26 (m, 5H), 1.77-1.58 (m, 5H), 1.48 (s, 3H), 1.40-1.31 (m, 2H).

[0305] Example 16: Confirmation of the therapeutic effect of B2-24-4-5A·HCOOH in spontaneous systemic lupus erythematosus model mice Systemic lupus erythematosus model mice (NZBWF1, 20 weeks old, female, n=55) were divided into saline-treated group (control, n=25), B2-24-4-5A·HCOOH-treated group (n=10), B2-24-4·HCOOH-treated group (n=5), and HCQ-treated group (n=15). The saline-treated group was orally administered saline (100 μl) once daily. The B2-24-4-5A·HCOOH-treated group was orally administered 10 mg / kg B2-24-4-5A·HCOOH (100 μl) once daily. The B2-24-4·HCOOH-treated group was orally administered 10 mg / kg B2-24-4·HCOOH (100 μl) once daily. The HCQ group was orally administered 10 mg / kg HCQ (100 μl) once a day for consecutive days. After 15 weeks from the start of administration, surviving mice from the saline-treated group (control, n=17), B2-24-4-5A·HCOOH-treated group (n=10), B2-24-4·HCOOH-treated group (n=5), and HCQ-treated group (n=12) were euthanized after urine collection. After euthanasia, blood was collected from the abdominal aorta of the mice, and the kidneys and spleens were removed.

[0306] Mice that died naturally during the 15-week period from the start of administration were counted as dead mice, and survival rates were analyzed using the Kaplan Meier method. Blood was collected from the abdominal aorta of each mouse, excluding dead mice, and centrifuged to prepare serum. Quantitative analysis of serum urea nitrogen and creatinine levels was outsourced to Oriental Yeast Co., Ltd. The albumin concentration in urine was quantified using ELISA (kit used: Levis Urinary Albumin-Mouse (S-type) (product name) (manufactured by Fujifilm Wako Shibayagi Co., Ltd.)). The excised kidneys were fixed with 4% (w / v) paraformaldehyde, and immunohistochemical staining was outsourced to Morpho Technology Co., Ltd. In the PAS-stained kidney tissue sections, the histological findings of 100 glomeruli were evaluated, and among them, the increase in cells and matrix in the mesangial region, endothelial cell swelling and cell infiltration in the capillary lumen (intravascular proliferation), and glomeruli accompanied by inflammatory crescents (extravascular proliferation) were classified into grades from 0 to 2 according to the severity, and scored out of 6 points. The results are shown in Figures 10A to 10H and Table 8.

[0307] FIG. 10A shows the survival curves during the administration period. FIG. 10B shows the serum urea nitrogen levels. FIG. 10C shows the serum creatinine levels. FIG. 10D shows the urinary albumin levels. FIG. 10E shows the deposition of IgG in the glomeruli of the saline-treated mouse (#1), the B2-24-4-5A·HCOOH-treated mouse (#10), the B2-24-4·HCOOH-treated mouse (#4), and the HCQ-treated mouse (#14). FIG. 10F shows the deposition of C3 in the glomeruli of the saline-treated mouse (#1), the B2-24-4-5A·HCOOH-treated mouse (#10), the B2-24-4·HCOOH-treated mouse (#4), and the HCQ-treated mouse (#14). Figure 10G shows stained photographs of kidney tissue sections from a mouse (#1) in the saline-treated group. In the kidney tissue sections from the mouse (#1) in the saline-treated group, signs of active glomerulonephritis such as diffuse mesangial proliferation and intraluminal proliferation were observed. Figure 10H shows stained photographs of kidney tissue sections from a mouse (#1) in the B2-24-4-5A·HCOOH-treated group. In the kidney tissue sections from the mouse (#1) in the B2-24-4-5A·HCOOH-treated group, only a local increase in mesangial cells and matrix was observed. The plots in Figures 10B, 10C, and 10D show the data of each individual, the thick lines show the average data of each group, and the error bars show the standard error of the data of each group. The scale bars in Figures 10E and 10F represent 100 μm.

[0308] As is clear from Figures 10A to 10H, the B2-24-4-5A·HCOOH or B2-24-4·HCOOH administration groups showed an extension of survival rate, suppression of the increase in serum urea nitrogen level, suppression of the increase in serum creatinine level, suppression of the increase in urinary albumin level, suppression of IgG deposition in glomeruli, suppression of C3 deposition in glomeruli, and a significant reduction in the activity of lupus nephritis compared to the control saline or HCQ administration groups.

[0309] [Table 8] JPEG0007674716000085.jpg191170

[0310] While there were individual differences in the activity of nephritis in the control saline-treated group, in most of the B2-24-4-5A·HCOOH-treated groups, not only glomerulonephritis but also tubulointerstitial inflammation improved.

[0311] Example 17: Analysis of spleen tissue in NZBWF1 mice administered B2-24-4-5A·HCOOH The weight and splenocyte count of the spleen obtained in Example 16 were analyzed. The spleen weight is shown in Figure 11A, and the splenocyte count is shown in Figure 11B.

[0312] The spleen weight in the B2-24-4-5A·HCOOH group was significantly lower than that in the saline and HCQ groups. The number of splenocytes in the B2-24-4-5A·HCOOH group was significantly lower than that in the saline and HCQ groups. As is clear from Figures 11A and 11B, the spleen enlargement was suppressed in the B2-24-4-5A·HCOOH group compared with the control saline and HCQ groups.

[0313] Example 18: Analysis of spleens in NZBWF1 mice administered B2-24-4-5A·HCOOH

[0314] The spleen obtained in Example 16 was ground with a slide glass to prepare splenocytes, which were then stained with a labeled antibody. The labeled antibodies used were FITC-labeled anti-CD11b antibody, FITC-labeled anti-CD3 antibody, FITC-labeled anti-CD62L antibody, FITC-labeled anti-B220 antibody, FITC-labeled anti-CD71 antibody, PE-labeled anti-CD11c antibody, PE-labeled anti-Gr-1 antibody, PE-labeled anti-CD8 antibody, PE-labeled anti-CD44 antibody, PE-labeled anti-CD69 antibody, PE-labeled anti-CD21 antibody, PE-labeled anti-CD86 antibody, PE-labeled anti-Ter-119 antibody, PE-Cy7-labeled anti-CD23 antibody, APC-labeled anti-F4 / 80 antibody, APC-labeled anti-Ly-6G antibody, APC-labeled anti-CD4 antibody, APC-labeled anti-B220 antibody, APC-labeled anti-CD19 antibody, APC-labeled anti-CD11c antibody, or APC-labeled anti-PDCA-1 antibody. After 20 minutes, the cells were washed with FACS buffer and each cell was suspended in 200 μL of FACS buffer containing 25 μg / mL 7-actinomycin D. The fluorescence intensity of the cells was analyzed by flow cytometry. The flow cytometry was performed using a FACSCanto TM The results were analyzed using FlowJo software (Tree Star) to calculate the percentage of each type of cell in the spleen. The results are shown in Figures 12(A)-(E), 13(A)-(E), and 14(A)-(E).

[0315] In the B2-24-4-5A·HCOOH group, the percentages of plasmacytoid dendritic cells, helper T cells, cytotoxic T cells, and naive T cells were increased, and the percentages of neutrophils, erythroblasts, activated T cells, memory T cells, and CD69-positive activated B cells were decreased, compared with the saline and HCQ groups. Therefore, the administration of B2-24-4-5A·HCOOH improved the inflammatory pathology associated with the onset of systemic lupus erythematosus.

[0316] Example 19: Analysis of IFN-α production by microRNA stimulation We investigated the inhibitory effect of B2-24-4-5A·HCOOH on TLR7-stimulating microRNA in mouse Flt-3 ligand-induced dendritic cells (FLDCs). Mouse bone marrow cells were cultured in RPMI1640 medium containing 100 ng / ml Flt-3 ligand, 10% (v / v) fetal calf serum (FCS), penicillin, streptomycin, L-glutamic acid, and 2-mercaptoethanol at 37°C in the presence of 5% (v / v) CO2 for 8 days. Mouse FLDCs induced to differentiate by culture were cultured at 1.0 × 10 5 The cells were seeded at 100 μL / well. Then, 0.1, 0.3, or 1 μM of B2-24-4-5A·HCOOH was added. 30 minutes after the addition of B2-24-4-5A·HCOOH, the vehicle or the microRNA-cationic liposomal vehicle (DOTAP) complex containing 0.5 or 2 μg / ml of microRNA was added. The sequences of the microRNAs used are shown in Table 9.

[0317] [Table 9]

[0318] 24 hours after addition of the microRNA-DOTAP complex, the concentration of IFN-α in the medium was quantified using ELISA (kit used: Mouse IFN alpha Platinum ELISA (product name), eBioscience). The results are shown in Figure 15. As shown in Figure 15, B2-24-4-5A·HCOOH concentration-dependently suppressed the production of IFN-α stimulated by microRNA.

Claims

1. A compound represented by the following formula (I) or (II) or a pharmacologically acceptable salt thereof: 【Chemistry 1】 【Chemistry 2】 (In formulas (I) and (II), R 1 teeth, formula: 【Chemistry 11】 A group represented by formula: 【Chemistry 12】 A group represented by the formula: 【Chemistry 3】 (In the formula, R 2 and R 3 are each independently an alkyl group having 1 to 3 carbon atoms; formula: 【Chemistry 4】 (In the formula, Ring C is a 3- to 7-membered nitrogen-containing heterocycle; R 4 is -NH-, -O-, -CF ２ -, -CHF-, -C ２ H ２ F ２ - or a group represented by -CHF-X-CHF-, X is an alkyl group having 1 to 4 carbon atoms; 【Chemistry 7】 It is a group represented by the following formula:

2. R 1 teeth, 【Chemistry 5】 【Chemistry 6】 【Chemistry 8】 【Chemistry 9】 or 【Chemistry 10】 2. The compound according to claim 1, which is: or a pharma- cologically acceptable salt thereof.

3. The compound according to claim 1 or 2, which is represented by any one of the following formulas (III) to (XIII), or a pharmacologically acceptable salt thereof. 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemical 22】 【Chemistry 23】

4. The compound or a pharmacologically acceptable salt thereof according to any one of claims 1 to 3, wherein the pharmacologically acceptable salt is a hydrochloride or a formate.

5. 5. A Toll-like receptor 7 (TLR7) activation inhibitor comprising the compound according to any one of claims 1 to 4 or a pharmacologically acceptable salt thereof.

6. The TLR7 activation inhibitor according to claim 5, which has an effect of suppressing the production of NF-κB, IL-6, TNF-α or IFN-α caused by the activation of TLR7.

7. A preventive or therapeutic drug for a disease accompanied by TLR7 activation, comprising the TLR7 activation inhibitor according to claim 5 or 6.

8. 8. The preventive or therapeutic agent according to claim 7, wherein the disease accompanied by activation of TLR7 is an autoimmune disease, an autoinflammatory syndrome, an autoimmune pancreatitis, arteriosclerosis, sepsis, a neurodegenerative disease, graft rejection, graft-versus-host disease, periodontal disease, viral immunodeficiency, IgA nephropathy, primary nephrotic syndrome, primary membranoproliferative glomerulonephritis, purpura nephritis, Langerhans cell histiocytosis, hemophagocytic lymphohistiocytosis, Rosai-Dorfman disease, obesity, type 2 diabetes mellitus, or ulcerative colitis.

9. The preventive or therapeutic drug according to claim 8, wherein the disease accompanied by activation of TLR7 is an autoimmune disease.

10. The autoimmune disease is systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis / dermatomyositis, mixed connective tissue disease, overlap syndrome, antiphospholipid syndrome, Behcet's disease, adult Still's disease, rheumatic fever, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, HLA-B27-associated rheumatic disease, IgG4-associated syndrome, ANCA-associated vasculitis, vasculitis syndrome, multiple sclerosis, psoriasis vulgaris, inflammatory bowel disease, autoimmune diseases, and the like.

10. The prophylactic or therapeutic drug according to claim 9, which is selected from the group consisting of infectious thyroid disease, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, primary biliary cirrhosis, primary biliary cholangitis, myasthenia gravis, Goodpasture's syndrome, Guillain-Barré syndrome, chronic atrophic gastritis, rapidly progressive glomerulonephritis, antiglomerular basement membrane nephritis, Addison's disease, type I diabetes mellitus, vitiligo, pemphigus vulgaris, pemphigoid, autoimmune neutropenia, autoimmune hepatitis, and autoimmune pancreatitis.

11. The preventive or therapeutic drug according to claim 10, wherein the autoimmune disease is systemic lupus erythematosus.

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