KR20220117805A - Composition for stimulating interferon genes comprising a benzimidazole derivative as an active ingredient - Google Patents

Abstract

The present disclosure provides a compound useful as a STING agonist and pharmaceutically acceptable salts thereof. The present disclosure provides a pharmaceutical composition comprising the compound or pharmaceutically acceptable salts thereof. In addition, the present disclosure provides a pharmaceutical use for treatment or prevention of STING-mediated diseases using the compound or pharmaceutically acceptable salts thereof as an active ingredient. Moreover, the present disclosure provides a method for treating or preventing STING-mediated diseases including administration of an effective amount of the compound, salts thereof or the composition comprising the same to a subject in need of treatment for STING-mediated diseases.

Description

TECHNICAL FIELD [0002] Composition for stimulating interferon genes comprising a benzimidazole derivative as an active ingredient

The present disclosure relates to compounds useful for the treatment or prevention of STING (Stimulator of Interferon genes) mediated diseases such as various types of cancer, precancerous syndromes, infectious diseases, and the like, and uses thereof.

Since the discovery of the immune evasion mechanism by which cancer cells evade immune responses, immuno-oncology drugs that use the body's immune system to treat cancer have been in the spotlight as next-generation anticancer agents. In a tumor environment, as the signaling function of cancer cells is mainly activated, the function of immune cells is suppressed or an immune process that induces anticancer activity is avoided to form a tumor. Immune anticancer drugs target various proteins in the body that activate immune cells so that they can recognize abnormal functions of problematic areas within cells and attack and effectively remove them. In particular, immune checkpoint inhibitors that target immune checkpoints, such as PD-1, PD-L1, and CTLA-4, inhibit the mechanism by which cancer cells evade immune cells and, as a result, induce cancer cell death by T cell immune reactivity. It is an anticancer drug.

At this time, the tumor microenvironment (TME) composed of immune cells, stromal cells, extracellular matrix, etc. has a major influence on the formation of cancer and the reactivity of immuno-cancer drugs. Therefore, TME also affects the efficacy of anticancer drugs using the immune response. The immunophenotype of tumor is largely divided into 'hot tumor' and 'cold tumor' according to T cell reactivity. 'Hot tumor' refers to a case in which an immune response was present but was inhibited by the immunosuppressive action in TME. Most of them are responsive to immune checkpoint inhibitors. However, 'cold tumor' is an immune deficiency phenotype in which T cells do not penetrate, and there are patients who do not respond to immune checkpoint inhibitors. In addition, even when responding to immune checkpoint inhibitors, there is a limitation in that the anticancer activity is not great when treated alone.

Recently, STING (Stimulator of Interferon Genes) protein has been in the spotlight as a new drug target to overcome the limitation of 'cold tumor' in which no immune response exists. Activation of the innate immune process by STING activation induces priming of T cells, and as a result, it has the advantage of being able to induce an immune response by cytotoxic T cells through activation of acquired immunity. This has important significance as a new treatment strategy in that it can transform a 'cold tumor' that has had little reactivity to an immune checkpoint inhibitor into a 'hot tumor'.

In the cytoplasm of mammalian cells, the cGAS-STING signaling system is one of the first defense mechanisms of the host, which is initiated by stimuli from external factors. When cGAS (Cyclic GMP-AMP synthase) is stimulated by dsDNA derived from an external virus or cancer, cGAMP synthesized by cGAS acts as a substrate for STING. When cGAMP binds to STING, it recruits and phosphorylates TBK1 (TANK binding kinase 1), which induces phosphorylation of IRF3 (Interferon Regulatory transcription factor 3). As a result, phosphorylated IRF3 acts as a transcription factor, moves from the cytoplasm to the nucleus, binds to a specific promoter, and activates it to produce type-1 interferon and several inflammatory cytokines. Interferon and related factors activate the JAK-STAT pathway to induce transcription of ISGs (Interferon Stimulated Genes), and these proteins control the antiviral mechanism to protect the host from external infection. When the following innate immune processes are activated, T cells are primed by APC (antigen presenting cells), and cytotoxic T cells that specifically act on tumors are activated.

Various STING agonists have been reported based on the advantages of the above mechanism of action of immuno-oncology. The first known CDN-type agonist has limitations in that it has electronegative and hydrophilic properties. In addition, in the case of CDN-type ADU-S100, it is difficult to optimize the dose due to the limitations of intratumoral administration methods, and if a certain stage is exceeded, the immune response is excessively activated, and there may be side effects due to a cytokine storm.

On the other hand, International Patent Application Publication No. WO 2017/175147 discloses such STING agonist compounds, such compounds are cancer, precancerous syndrome, infectious diseases (eg, influenza, HIV, HCV, HPV or HBV infectious disease), etc. It is disclosed that it is useful for the treatment or improvement of various diseases, including. It is also disclosed that STING agonists may be useful as immunogenic compositions or vaccine adjuvants.

International Patent Application Publication No. WO 2017/175147

Accordingly, the problem to be solved by the present invention is to provide a novel compound useful as a STING agonist and a pharmaceutical use thereof.

In order to solve the above problems, one aspect of the present disclosure provides a compound of Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

(화합물 37),

(화합물 38), 또는

(화합물 39)임.In Formula 1, R ⁵ is

(Compound 37),

(Compound 38), or

(Compound 39).

, 또는

이며, 더욱 바람직하게, R⁵는

임.Preferably, in Formula 1, R ⁵ is

, or

and more preferably, R ⁵ is

lim.

Another aspect of the present invention also relates to 2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-(1-ethyl-3-methyl-1H-pyrazole- 5-carboxamido)butyl)-1H-benzo[d]imidazole-5-carboxamide (Compound 26) or a pharmaceutically acceptable salt thereof.

The compounds according to the present disclosure are small-molecular compound-based pharmaceutical ingredients of the STING protein, which stimulate the innate immune response to occur through the activation of the STING-related pathway, thereby exhibiting various pharmacological activities such as anticancer activity dependent on it. This is a low-molecular synthetic compound, which can be administered intravenously, effectively overcoming the limitations of CDN-based drugs, and shows very good activity when compared to previously reported compounds of the same class. This suggests its importance in that it can enhance the effectiveness of immunotherapy by enhancing the anticancer effect through the innate immune response by the STING agonist and the resulting acquired immune activation.

The compounds according to the present disclosure have great advantages in various aspects, such as activity, physicochemical stability, in vivo stability, pharmacokinetic properties, and safety, compared to compounds having similar structures. Specifically, the compounds according to the present disclosure show much higher activity compared to the compounds disclosed in International Patent Application Publication No. WO 2017/175147.

In addition, the excellent activity of the compounds according to the present disclosure is shown as it is in the verification of the activity for the secretion of cytokines such as IFN-beta, which is a representative marker of STING pathway activation, and when measuring the EC ₅₀ value for IFN-beta secretion, international patent Among the compounds disclosed in Application Publication No. WO 2017/175147, a representative compound (eg, Example 14 compound) showed approximately 130 nM - 200 nM values, but the compounds according to the present disclosure showed much lower values of 4 pM to 1.6 nM. indicated.

In addition, the compounds according to the present disclosure exhibited a large difference (advantage) in pharmacokinetic properties such as half-life, AUC, and clearance compared to other compounds having similar structures, and their metabolic stability in the body is remarkably excellent. For example, a representative compound (eg, Example 14 compound) among the compounds disclosed in International Patent Application Publication No. WO 2017/175147 has a half-life of 1.4 hours and an AUC of 3 μg·h ^-1 ·ml ^-1 , volume of The distribution is 0.4 L·kg ^-1 , and the clearance is 17 ml·min ^-1 ·kg ^-1 . Under the same conditions (intravenous injection, 3 mpk), Compound 37, an exemplary compound of the present invention, has a half-life of 10.5 Time, AUC of 4.2 μg·h ^-1 ·ml ^-1 , volume of distribution 17.7 L·kg ^-1 , and clearance of 2.1 ml·min ^-1 ·kg ^-1 are exceptionally excellent.

"Pharmaceutically acceptable salts" in the present invention include salts of the active compounds prepared with relatively non-toxic acids which depend on the particular substituents found on the compounds mentioned herein. Acid addition salts of a compound of the present invention may be obtained by contacting the neutral form of a compound of the present invention with a sufficient amount of the desired acid, neat or with a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts are acetic acid, propionic acid, isobutyric acid, oxalic acid, maleic, malonic, benzoic, succinic, suberic, fumaric ( fumaric), mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric acid, tartaric acid, methanesulfonic, and the like. As well as salts derived from non-toxic organic acids, hydrogen chloride, hydrogen bromide, nitric acid, carbonic acid, monohydrogencarbonic, phosphoric, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydrogen iodide or phosphorous acid ( phosphorous acid) and its analogues. Also included are salts of amino acids such as arginate and analogues thereof and analogues of organic acids such as glucuronic or galactunoric acids and analogues thereof.

As used herein, the term “compound of the present invention” is meant to include the compound(s) of Formula 1 as well as clathrates, hydrates, solvates, or polymorphs thereof. In addition, the term "compound of the present invention" is meant to include pharmaceutically acceptable salts of the compounds of the present invention unless a pharmaceutically acceptable salt thereof is mentioned.

In one embodiment, a compound of the invention is a stereoisomerically pure compound (e.g., substantially free of other stereoisomers (e.g., at least 85% ee, at least 90% ee, at least 95% ee, 97% ee or more, or 99% ee or more)). That is, in the compound of Formula 1 or a salt thereof according to the present invention, amine-imine tautomers may exist, and these are also included in the scope of the compound of the present invention.

As used herein, the term “polymorph” refers to a solid crystalline form of a compound of the present invention or a complex thereof. Different polymorphs of the same compound exhibit different physical, chemical and/or spectral properties. Differences in physical properties include, but are not limited to, stability (eg, thermal or light stability), compressibility and density (important for formulation and product manufacturing), and dissolution rate (which may affect bioavailability). doesn't happen Differences in stability may be due to changes in chemical reactivity (e.g., differential oxidation, such as a faster discoloration when composed of one polymorph than when composed of another polymorph) or mechanical properties (e.g., kinetically Tablet fragments stored as the preferred polymorph are converted to the thermodynamically more stable polymorph) or both (tablets of one polymorph are more susceptible to degradation at high humidity). Other physical properties of polymorphs can affect their processing. For example, one polymorph may be more likely to form a solvate than another polymorph, for example due to its shape or particle size distribution, or it may be more difficult to filter or wash.

As used herein, the term "solvent compound" refers to a compound of the present invention, or a pharmaceutically acceptable salt thereof, comprising a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile, non-toxic, and can be administered in trace amounts to humans.

As used herein, the term "hydrate" refers to a compound of the present invention, or a pharmaceutically acceptable salt thereof, comprising a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. .

As used herein, the term "clathrate" refers to a compound of the present invention in the form of a crystal lattice containing spaces (eg, channels) that confine guest molecules (eg, solvent or water). or salts thereof.

The term "purified" as used herein, when isolated, means that the isolate is at least 90% pure, in one embodiment at least 95% pure, in another embodiment at least 99% pure, and In another embodiment, it means at least 99.9% pure.

The term "pharmaceutically acceptable" means suitable for use as a pharmaceutical preparation, which is generally considered safe for such use, and is officially approved for such use by a national regulatory agency or It means being on the list of pharmacopeias.

One aspect of the present disclosure provides a pharmaceutical use of the compound of the present invention or a pharmaceutically acceptable salt thereof for the treatment or prevention of STING-mediated diseases.

As used herein, "STING-mediated disease" refers to a disease that can be treated due to activation of STING, and is meant to include disorders. STING-mediated diseases include various types of cancer (eg, solid cancer, non-T cell responsive cancer); precancerous syndrome; infectious diseases (eg, viral or bacterial mediated infectious diseases); In one aspect of the present invention, the infectious disease is an infectious disease caused by influenza, HIV, HCV, HPV, HBV, PIV, HRV (rhinovirus), enterovirus, poliovirus, Zika virus, dengue virus, coronavirus, etc. to be. Preferably, the STING mediated disease is cancer or a precancerous syndrome.

Accordingly, the present disclosure also provides a STING-mediated disease comprising the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient, for example, various types of cancer (eg, solid cancer, non-T-cell reactive cancer), Provided is a pharmaceutical composition for treating or preventing diseases such as cancerous syndromes and infectious diseases (eg, viral or bacterial mediated infectious diseases). Another aspect of the present disclosure is also characterized in that a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof is administered to an individual in need of treatment or prevention of a STING-mediated disease, treatment of a STING-mediated disease or a preventive method. In another embodiment, the subject is an animal. In another preferred embodiment, the subject is a human.

As used herein, "effective amount" or "effective amount" means destroying, modifying, controlling or eliminating primary, local or metastatic cancer cells or cancer tissue when the pharmaceutical use of the compounds of the present invention is cancer or a precancerous syndrome; slowing or minimizing the spread of cancer; or an amount of a compound of the invention sufficient to provide a therapeutic benefit in the treatment or management of cancer, neoplastic disease, or tumor. “Effective amount” also refers to an amount of a compound of the invention sufficient to cause cancer or neoplastic cell death. "Effective amount" also refers to an amount sufficient to activate STING, either in vitro or in vivo.

As used herein, the term “neoplastic” refers to an abnormal growth of cells or tissues (eg, boils) that may be benign or cancerous.

"Treatment" as used herein includes eradication, removal, modification, or control of primary, focal or metastatic cancer tissue when the medicinal use of the compound of the present invention is cancer or a precancerous syndrome; To minimize or delay the expansion of cancer.

As used herein, "effective amount" or "effective amount" means alleviating, controlling or eliminating symptoms caused by an infectious agent when the pharmaceutical use of the compound of the present invention is an infectious disease; control or eliminate the source of infection itself or slow or minimize its spread; or an amount of a compound of the invention sufficient to provide a therapeutic benefit in the treatment or management of an infectious disease. "Effective amount" also refers to an amount sufficient to activate STING, either in vitro or in vivo.

"Treatment" as used herein includes eradication, elimination, or control of an infectious agent when the medicinal use of a compound of the present invention is an infectious disease; Minimizing or delaying the spread of infectious disease symptoms.

As used herein, “prevention” refers to the prevention of recurrence of the STING-mediated disease in a patient, and includes prevention of expansion or onset.

The compound of the present invention or a pharmaceutically acceptable salt thereof is generally administered in a therapeutically effective amount. The compounds of the present invention may be administered by any suitable route, in the form of a pharmaceutical composition suitable for such route, and in an effective dosage for the intended treatment. An effective dosage is generally from about 0.0001 to about 200 mg/kg body weight/day, preferably from about 0.001 to about 100 mg/kg/day, more preferably from about 0.015 to 15 mg/body weight, in single or divided doses kg/day. Dosage levels below the lower limit of this range may be suitable depending on the age, species, and disease or condition being treated. In other cases, still larger doses can be used without deleterious side effects. The larger dose may be divided into several smaller doses for administration throughout the day. Methods for determining the appropriate dosage are well known in the art.

One aspect of the present disclosure provides a pharmaceutical use of a compound of the present invention or a pharmaceutically acceptable salt thereof as an immunogenic composition or vaccine adjuvant. Accordingly, the present disclosure also provides an immunogenic or vaccine composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof as an adjuvant.

Interferon is a substance that can protect cells from viral infection. For example, recombinant IFNα was the first approved biotherapeutic agent and has become an important therapy in viral infections and cancer. Interferons are known to be potent modulators of immune responses acting on cells of the immune system, as well as direct antiviral activity against cells. The compounds of the present invention are STING agonists, and not only promote IFN-beta secretion, but also promote Type 1 IFN-mediated expression of various ISG genes.

STING agonists may be used in a variety of diseases including viral infections and autoimmune diseases (eg, infectious diseases, cancer, allergic diseases, neuropathic diseases (eg, amyotrophic lateral sclerosis, multiple sclerosis, etc.), inflammatory diseases, irritable bowel disease), plays an essential role in antimicrobial host defenses, including protection against a wide range of DNA and RNA viruses and bacteria, and may be useful as a vaccine adjuvant. The content disclosed in Application Publication No. WO 2017/175147 and disclosed in International Patent Application Publication No. WO 2017/175147 is incorporated herein by reference in its entirety.

Another aspect of the present disclosure also provides a composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable additive. Such a composition may be prepared in a form for administration by various methods as follows.

Oral administration

The compound of the present invention may be administered orally, and the oral cavity is a concept including swallowing. By oral administration, the compound of the present invention may enter the gastrointestinal tract or may be absorbed directly into the bloodstream from the mouth, such as, for example, buccal or sublingual administration.

Suitable compositions for oral administration may be in solid, liquid, gel, or powder form, and may have formulations such as tablets, lozenges, capsules, granules, and powders. .

Compositions for oral administration may optionally be enteric coated and may exhibit delayed or sustained release through the enteric coating. That is, the composition for oral administration according to the present invention may be a formulation having an immediate or modified release pattern.

Liquid formulations may include solutions, syrups and suspensions, and such liquid compositions may be in the form contained within soft or hard capsules. Such formulations may contain a pharmaceutically acceptable carrier, for example, water, ethanol, polyethylene glycol, cellulose, or oil. The formulation may also contain one or more emulsifying and/or suspending agents.

In tablet formulations, the amount of drug as the active ingredient may be present in an amount of from about 0.05% to about 95% by weight relative to the total weight of the tablet, more typically from about 2% to about 50% by weight of the dosage form. Tablets may also contain from about 0.5% to about 35% by weight of a disintegrant, more typically from about 2% to about 25% by weight of the dosage form. Examples of the disintegrant include, but are not limited to, lactose, starch, sodium starch glycolate, crospovidone, croscarmellose sodium, maltodextrin, or mixtures thereof.

Suitable glidants included for the preparation of tablets may be present in an amount from about 0.1% to about 5% by weight, and include talc, silicon dioxide, stearic acid, calcium, zinc or magnesium stearate, sodium stearyl fumarate, and the like. This lubricant may be used, but the present invention is not limited to the types of these additives.

Gelatin, polyethylene glycol, sugar, gum, starch, polyvinyl pyrrolidone, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc. may be used as a binder for manufacturing tablets. In addition, suitable diluents for manufacturing tablets include mannitol, xylitol, lactose, dextrose, sucrose, sorbitol, starch, microcrystalline cellulose, etc., but the present invention is not limited to the types of these additives. .

Optionally, the solubilizer that may be included in the tablet may be used in an amount of about 0.1% to about 3% by weight based on the total weight of the tablet, for example, polysorbate, sodium lauryl sulfate, sodium dodecyl sulfate, propylene carbonate, Diethylene glycol monoethyl ether, dimethylisosorbide, polyoxyethylene glycolated natural or hydrogenated castor oil, HCOR ^™ (Nikkol), oleyl ester, Gelucire ^™ , caprylic/caprylic acid mono/ Diglyceride, sorbitan fatty acid ester, Solutol HS ^TM , etc. may be used in the pharmaceutical composition according to the present invention, but the present invention is not limited to the specific type of the solubilizer.

Parenteral Administration

The compounds of the present invention may be administered directly into the bloodstream, muscle, or intestine. Suitable methods for parenteral administration include intravenous, intra-muscular, subcutaneous intraarterial, intraperitoneal, intrathecal, intracranial injection, and the like. include Suitable devices for parenteral administration include injectors (including needle and needleless syringes) and infusion methods.

Compositions for parenteral administration may be formulations with an immediate or modified release pattern, and the modified release pattern may be a delayed or sustained release pattern.

Most parenteral formulations are liquid compositions, and the liquid composition is an aqueous solution containing the active ingredient according to the present invention, a salt, a buffer, an isotonic agent, and the like.

Parenteral formulations may also be prepared in dried form (eg, lyophilized) or as sterile non-aqueous solutions. These formulations may be used with a suitable vehicle such as sterile water. Solubility-enhancing agents may also be used in the preparation of parenteral solutions.

Topical Administration

The compounds of the present invention may be administered topically dermally or transdermally. Formulations for topical administration include lotions, solutions, creams, gels, hydrogels, ointments, foams, implants, patches, and the like. Pharmaceutically acceptable carriers for topical dosage forms may include water, alcohol, mineral oil, glycerin, polyethylene glycol, and the like. Topical administration may also be performed by electroporation, iontophoresis, phonophoresis, and the like.

Compositions for topical administration may be formulations with an immediate or modified release pattern, and the modified release pattern may be a delayed or sustained release pattern.

On the other hand, another aspect of the present disclosure also provides an immunogenic composition or vaccine composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof as an adjuvant. Such compositions may include antibody(s) or antibody fragment(s) or antigenic components including, but not limited to, proteins, DNA, RNA, live or dead bacteria and/or viruses or virus-like particles. The immunogenic or vaccine compositions of the present disclosure may contain, in addition to the adjuvants of the present disclosure, aluminum salts, oils, heat shock proteins, lipid A agents and derivatives, glycolipids, other TLR (Toll-Like Receptor) agonists (such as CpG DNA or similar agonists) , cytokines (such as GM-CSF, IL-12), including, but not limited to, one or more other ingredients having adjuvant activity.

The present disclosure discloses that various types of cancers (eg, solid cancers, non-T-cell reactive cancers), precancerous syndromes, infectious diseases (eg, viral or bacterial mediated infectious diseases) can be treated or prevented by STING agonists, etc. Provided are compounds useful for the treatment or prevention of STING-mediated diseases and their medicinal uses. The compound according to the present disclosure has various advantages as an active ingredient of a pharmaceutical.

The following drawings attached to the present specification illustrate preferred embodiments of the present invention, and serve to further understand the technical idea of the present invention together with the above-described contents of the present invention, so the present invention is limited to the matters described in such drawings It should not be construed as being limited.
1 is an ISRE reporter assay result of the compounds of one embodiment according to the present invention. Through this, the concentration dependence and STING dependence of the compound of the present invention were confirmed.
2 is a result of evaluating the MTS cell viability of the compounds of one embodiment according to the present invention.
3 is an in vitro ligand-competitive binding experiment result of the compound of one embodiment according to the present invention.
4 is a result of evaluating whether the STING binding of the compound of one embodiment of the present invention using CETSA (cellular thermal shift assay). In FIG. 4 , the upper result is a result using human immune cells (THP-1), and the lower result is a result using mouse immune cells (RAW264.7).
5 is an ELISA result for the secretion of IFN-β and IP-10 cytokines of the compound of one embodiment of the present invention and the compound 31 of a comparative example.
6 is an ELISA result for IFN-β and IP-10 cytokine secretion of the compound of one embodiment of the present invention and the compound diABZI-3, which is a comparative example.
7 is a result of verifying the expression of various ISG genes mediated by Type 1 IFN by the compound of an embodiment of the present invention.
8 is a Western blotting experiment result for confirming the activation of a sub-factor according to the STING signal transduction system.
9 is a result of evaluating the immuno-anticancer efficacy of the compound of one embodiment of the present invention in vivo (Data represents as the mean volume of xenograft tumors ± SEM (N=6). Statistical difference was analyzed by Student's t test. *P > 0.05. Arrow indicated the day of compound injection)
10 is an anticancer effect evaluation result of the compound of one embodiment of the present invention using a CT26-bearing mouse (Graphs are depicted by mean and SD. *: P <0.05. **: P <0.01 by two-way ANOVA.)
11 is a result of monitoring tumor recurrence by re-implanting CT26 in mice administered with the compound (Compound 39) in Example 1 of the present invention without additional administration of the compound.

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art to which the present invention pertains.

Synthesis of compounds (monomers) of Table 3 below

Step 1: ( S )-4-((2-hydroxy-2-phenylethyl)amino)-3-nitrobenzamide

4-Fluoro-3-methoxy-5-nitrobenzamide (1.50 g, 8.15 mmol) was dissolved in DMSO (10 mL) followed by ( S )-2-amino-1-phenylethan-1-ol (1.50 g, 8.15 mmol) and K ₂ CO ₃ (1.69 g, 12.2 mmol) were added. The reaction was stirred at 60 °C for 16 h and cooled to room temperature. The reaction mixture was diluted with water, filtered with cold water, and the solid dried under vacuum to obtain ( S )-4-((2-hydroxy-2-phenylethyl)amino)-3-nitrobenzamide (1.76 g, 5.79 mmol, 72% yield).

¹ H NMR (300 MHz, methanol- d ₄ ) δ 8.74 (d, J = 2.2 Hz, 1H), 7.94 (dd, J = 9.1, 2.2 Hz, 1H), 7.52-7.43 (m, 2H), 7.41 7.25 (m, 3H), 7.05 (d, J = 9.1 Hz, 1H), 4.98 (dd, J = 7.7, 4.4 Hz, 1H), 3.76-3.49 (m, 2H).

Step 2: ( S )-3-amino-4-((2-hydroxy-2-phenylethyl)amino)benzamide

( S )-4-((2-hydroxy-2-phenylethyl)amino)-3-nitrobenzamide (3.00 g, 9.96 mmol) was dissolved in MeOH (50 mL) followed by 10% wet Pd/C ( 106 mg, 0.996 mmol) was added and stirred for 18 hours at room temperature under a hydrogen balloon. It was then filtered through Celite®, washing with 100 mL of MeOH. The filtrate containing the product was dried under vacuum to yield ( S )-3-amino-4-((2-hydroxy-2-phenylethyl)amino)benzamide (1.51 g, 5.57 mmol, 56% yield) as a tan solid. ) was obtained.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 7.46-7.39 (m, 2H), 7.35 (ddd, J = 7.8, 6.7, 1.2 Hz, 2H), 7.30-7.23 (m, 1H), 7.12 (h) , J = 2.1 Hz, 2H), 6.75 (s, 1H), 6.45 (d, J = 8.8 Hz, 1H), 5.51 (d, J = 4.4 Hz, 1H), 4.98 (dd, J = 6.9, 4.6 Hz) , 1H), 4.80 (dt, J = 8.5, 4.4 Hz, 1H), 4.57 (s, 2H), 3.31-3.11 (m, 2H).

Step 3: ( S )-2-amino-1-(2-hydroxy-2-phenylethyl)-1 H -benzo[ d ]Imidazole-5-carboxamide

( S )-3-amino-4-((2-hydroxy-2-phenylethyl)amino)benzamide (1.00 g, 3.69 mmol) was dissolved in MeOH (6.15 mL) followed by cyanogen bromide (0.977 g, 9.23 mmol) and the reaction was stirred at 60 °C for 3 h. The residue containing the product was then removed in vacuo by removal of MeOH to ( S )-2-amino-1-(2-hydroxy-2-phenylethyl) -1H -benzo[ d ] The imidazole-5-carboxamide was obtained and used in the next reaction without further purification.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 7.77 (s, 1H), 7.69 (d, J = 1.6 Hz, 1H), 7.51-7.41 (m, 3H), 7.36-7.23 (m, 3H), 7.12-7.00 (m, 2H), 6.44 (s, 2H), 4.94 (q, J = 5.7 Hz, 1H), 4.10 (d, J = 5.7 Hz, 2H).

Step 4: ( S )-2-(acid substituent)amido-1-(2-hydroxy-2-phenylethyl)-1 H -benzo[ d ]Imidazole-5-carboxamide

( S )-2-amino-1-(2-hydroxy-2-phenylethyl) -1H -benzo[ d ]imidazole-5-carboxamide (100 mg, 0.338 mmol), substituted carboxylic acid ( 1.2 eq, 0.406 mmol), HATU (643 mg, 1.69 mmol), and triethylamine (0.565 mL, 4.06 mmol) were dissolved in NMP (1.13 mL) and heated at 80 °C for 48 hours. To the reaction was added water (30 mL) and extracted with EtOAc (4 X 40 mL). The organic layer was dried by the addition of anhydrous MgSO ₄ and the solvent was removed in vacuo. The resulting residue was purified on silica gel with CH ₂ Cl _2: MeOH=9:1. After that, the fractions containing the target compound and impurities were combined, concentrated in vacuo, and cleared by adding CH ₂ Cl ₂ (10 mL), followed by precipitation at room temperature for 10 minutes. The resulting precipitate was filtered and rinsed with CH ₂ Cl ₂ (4 X 10 mL). The solid was dried under vacuum to afford the title compounds.

Step 1: 4-Chloro-3-nitrobenzamide

Methyl 4-chloro-3-nitrobenzoate (5.00 g, 23.2 mmol) was dissolved in NH ₄ OH (28% aqueous solution, 100 mL) and stirred at room temperature for 16 hours. After stirring for 16 hours, the solid was filtered with cold water and dried to obtain 4-chloro-3-nitrobenzamide (3.43 g, 17.1 mmol, 74% yield) as a white solid.

¹ H NMR (300 MHz, methanol- d ₄ ) δ 8.43 (d, J = 2.1 Hz, 1H), 8.11 (dd, J = 8.4, 2.1 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H) .

Step 2: tert -Butyl(4-((4-carbamoyl-2-nitrophenyl)amino)butyl)carbamate

N -boc-1,4-butanediamine (9.38 g, 49.8 mmol) and 4-chloro-3-nitrobenzamide (5.00 g, 24.9 mmol) were dissolved in DMSO (40 mL) followed by K ₂ CO ₃ (17.3) g, 125 mmol) was added. The reaction was stirred at room temperature for 16 h. After stirring, the solid was filtered with cold water and dried under vacuum to form a yellow solid tert- butyl(4-((4-carbamoyl-2-nitrophenyl)amino)butyl)carbamate (5.73 g, 16.3 mmol, 65%) yield) was obtained.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 8.65 (d, J = 2.2 Hz, 1H), 8.37 (t, J = 5.8 Hz, 1H), 7.99 (dd, J = 8.9, 2.3 Hz, 2H) , 7.31-7.22 (m, 1H), 7.10 (d, J = 9.1 Hz, 1H), 6.83 (t, J = 5.8 Hz, 1H), 3.40 (q, J = 6.7 Hz, 2H), 2.95 (q, J = 6.6 Hz, 2H), 1.60 (p, J = 7.3 Hz, 2H), 1.46 (p, J = 6.7 Hz, 2H), 1.36 (s, 9H).

Step 3: tert -Butyl(4-((2-amino-4-carbamoylphenyl)amino)butyl)carbamate

tert -Butyl(4-((4-carbamoyl-2-nitrophenyl)amino)butyl)carbamate (5.00 g, 14.2 mmol) was dissolved in MeOH (284 mL) followed by 10% wet Pd/C ( 151 mg, 1.42 mmol) was added and stirred under a hydrogen balloon at room temperature for 18 hours. It was then filtered through Celite®, washing with 400 mL of MeOH. The filtrate containing the product was concentrated in vacuo to give tert -butyl(4-((2-amino-4-carbamoylphenyl)amino)butyl)carbamate as a gray solid which was used in the next reaction without further purification. was used.

¹ H NMR (500 MHz, methanol- d ₄ ) δ 7.29 (dd, J = 8.3, 2.1 Hz, 1H), 7.22 (d, J = 2.1 Hz, 1H), 6.56 (d, J = 8.4 Hz, 1H) , 3.19 (t, J = 6.9 Hz, 2H), 3.09 (t, J = 6.9 Hz, 2H), 1.68 (dq, J = 11.4, 6.7 Hz, 2H), 1.65-1.56 (m, 2H), 1.43 ( s, 9H).

Step 4: tert -Butyl(4-(2-amino-5-carbamoyl-1) H -benzo[ d ]Imidazol-1-yl)butyl)carbamate

tert -Butyl(4-((2-amino-4-carbamoylphenyl)amino)butyl)carbamate (3.33 g, 10.3 mmol) was dissolved in MeOH (17.2 mL) followed by cyanogen bromide (2.73 g, 25.8 mmol) was added, and the mixture was stirred at 60 °C for 3 hours. The residue containing the product was then removed in vacuo with MeOH removed tert -butyl(4-(2-amino-5-carbamoyl- 1H -benzo[ d ]imidazole- containing cyanogen bromide as a tan solid) 1-yl)butyl)carbamate was obtained and used in the next reaction without further purification.

¹ H NMR (500 MHz, DMSO- d ₆ ) δ 8.61 (s, 2H), 8.05 (s, 1H), 7.86 (d, J = 1.5 Hz, 1H), 7.80 (dd, J = 8.4, 1.6 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.37 (s, 1H), 6.85 (t, J = 5.8 Hz, 1H), 4.15 (t, J = 7.4 Hz, 2H), 2.94 (q, J = 6.6 Hz, 2H), 1.64 (q, J = 7.7 Hz, 2H), 1.44 (p, J = 7.2 Hz, 2H), 1.35 (s, 9H).

Step 5: tert -Butyl (4-(2-acid substituted groupamido-5-carbamoyl-1) H -benzo[ d ]Imidazol-1-yl)butyl)carbamate

tert -Butyl(4-(2-amino-5-carbamoyl- 1H -benzo[ d ]imidazol-1-yl)butyl)carbamate (200 mg, 0.576 mmol), substituted carboxylic acid (1.2 eq, 0.691 mmol), HATU (1.10 g, 2.88 mmol), and triethylamine (0.963 mL, 6.91 mmol) were dissolved in NMP (1.00 mL) and heated at 80 °C for 48 hours. To the reaction was added water (30 mL) and extracted with EtOAc (4 X 10 mL). The organic layer was dried by the addition of anhydrous MgSO ₄ and the solvent was removed in vacuo. The resulting residue was purified on silica gel with CH ₂ Cl ₂ :MeOH=9:1 to obtain the target compound.

The NMR measurement results of compounds 1 to 16 are summarized in Table 1 below.

Compound
No. IUPAC name ¹ H NMR One ( S )-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1-(2-hydroxy-2-phenylethyl) -1H -benzo[ d ]imi Dazole-5-carboxamide ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.78 (s, 1H), 7.70-7.58 (m, 3H), 7.52-7.28 (m, 5H), 7.04 (s, 1H), 6.75 (s, 2H) ), 6.61 (s, 1H), 6.19 (dd, J = 9.7, 3.7 Hz, 1H), 4.62 (dd, J = 15.4, 9.7 Hz, 1H), 4.42 (dd, J = 15.4, 3.8 Hz, 1H) , 4.19 (qd, J = 6.9, 3.5 Hz, 2H), 2.14 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H). 2 ( S )-2-(1-ethyl-3-(trifluoromethyl) -1H -pyrazole-5-carboxamido)-1-(2-hydroxy-2-phenylethyl) -1H- Benzo[ d ]imidazole-5-carboxamide ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 7.78 (s, 1H), 7.68-7.61 (m, 3H), 7.49 (dd, J = 8.3, 1.6 Hz, 1H), 7.45-7.33 (m, 4H) ), 7.27 (s, 1H), 7.05 (s, 1H), 6.76 (s, 2H), 6.24 (dd, J = 9.6, 3.6 Hz, 1H), 4.62 (dd, J = 15.3, 9.7 Hz, 1H) , 4.44 (dd, J = 15.4, 3.7 Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H). 3 ( S )-1-(2-hydroxy-2-phenylethyl)-2-(1-methyl-3-(trifluoromethyl) -1H -pyrazole-4-carboxamido) -1H- Benzo[ d ]imidazole-5-carboxamide ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.80 (s, 1H), 8.37 (s, 1H), 7.95 (d, J = 1.6 Hz, 2H), 7.72 (dd, J = 8.4, 1.7 Hz, 1H), 7.52-7.43 (m, 3H), 7.38-7.24 (m, 4H), 5.66 (d, J = 4.5 Hz, 1H), 5.08 (dt, J = 9.5, 5.1 Hz, 1H), 4.39-4.27 (m, 2H), 3.98 (s, 3H). 4 ( S )-2-(furan-3-carboxamido)-1-(2-hydroxy-2-phenylethyl) -1H -benzo[ d ]imidazole-5-carboxamide ¹ H NMR (500 MHz, methanol- d ₄ ) δ 8.11 (d, J = 1.5 Hz, 1H), 7.73 (d, J = 1.7 Hz, 1H), 7.56 (dd, J = 8.3, 1.7 Hz, 1H) , 7.52 (t, J = 1.8 Hz, 1H), 7.51-7.47 (m, 2H), 7.40-7.32 (m, 4H), 7.22 (d, J = 8.3 Hz, 1H), 6.67 (d, J = 1.8) Hz, 1H), 6.29 (dd, J = 8.6, 4.2 Hz, 1H), 4.60 (dd, J = 15.5, 8.6 Hz, 1H), 4.46 (dd, J = 15.5, 4.2 Hz, 1H). 5 ( S )-2-(benzofuran-2-carboxamido)-1-(2-hydroxy-2-phenylethyl) -1H -benzo[ d ]imidazole-5-carboxamide ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 12.82 (s, 1H), 7.98 (d, J = 19.5 Hz, 2H), 7.83 (d, J = 7.8 Hz, 1H), 7.78-7.70 (m, 2H), 7.63 (s, 1H), 7.53 (dd, J = 13.2, 8.0 Hz, 3H), 7.46 (t, J = 7.7 Hz, 1H), 7.36 (dt, J = 21.6, 7.8 Hz, 4H), 7.27 (t, J = 7.4 Hz, 1H), 5.77 (d, J = 4.7 Hz, 1H), 5.20 (dt, J = 9.1, 4.5 Hz, 1H), 4.44-4.33 (m, 2H). 6 ( S ) -N-(5-carbamoyl-1-(2-hydroxy-2-phenylethyl) -1H -benzo[ d ]imidazol-2-yl) -1H -indazole-3- carboxamide ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.17 (d, J = 8.2 Hz, 1H), 7.74 (s, 1H), 7.67-7.60 (m, 4H), 7.38 (ddt, J = 35.7, 19.9) , 7.7 Hz, 8H), 7.00 (s, 1H), 6.66 (s, 2H), 6.47 (dd, J = 9.0, 4.5 Hz, 1H), 4.81 (dd, J = 15.3, 9.0 Hz, 1H), 4.52 (dd, J = 15.3, 4.5 Hz, 1H). 7 N- (5-carbamoyl-1-(( S )-2-hydroxy-2-phenylethyl)-1H-benzo[ d ]imidazol-2-yl) -1- (tetrahydro- 2H -pyran-2-yl)- 1 H -indazole-3-carboxamide ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.17 (ddd, J = 16.2, 8.2, 3.1 Hz, 1H), 7.84 (dd, J = 8.5, 3.1 Hz, 1H), 7.74 (s, 1H), 7.64 (d, J = 8.8 Hz, 3H), 7.51 (t, J = 8.2 Hz, 1H), 7.37 (ddd, J = 19.5, 7.8, 3.3 Hz, 6H), 7.01 (s, 1H), 6.68 (s) , 2H), 6.46 (tt, J = 7.9, 3.7 Hz, 1H), 5.96 (d, J = 9.3 Hz, 1H), 4.81 (dq, J = 13.6, 4.2 Hz, 1H), 4.53 (dt, J = 15.4, 3.8 Hz, 1H), 3.91-3.82 (m, 1H), 3.79-3.70 (m, 1H), 2.43-2.28 (m, 1H), 2.11-1.92 (m, 2H), 1.74 (d, J = 12.0 Hz, 1H), 1.62 (d, J = 17.3 Hz, 2H). 8 tert -Butyl (4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-1- yl) butyl) carbamate ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.82 (s, 1H), 8.03-7.93 (m, 2H), 7.79 (dd, J = 8.4, 1.6 Hz, 1H), 7.56 (d, J = 8.4) Hz, 1H), 7.32 (s, 1H), 6.79 (d, J = 5.9 Hz, 1H), 6.66 (s, 1H), 4.62 (q, J = 7.1 Hz, 2H), 4.21 (t, J = 7.0) Hz, 2H), 2.96 (d, J = 6.6 Hz, 2H), 2.17 (s, 3H), 1.81-1.69 (m, 2H), 1.48-1.27 (m, 14H). 9 tert -Butyl (4-(5-carbamoyl-2-(1-ethyl-3-(trifluoromethyl) -1H -pyrazole-5-carboxamido) -1H -benzo[ d ] imidazol-1-yl)butyl)carbamate ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 12.77 (s, 1H), 8.02 (s, 1H), 7.97 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 12.9 Hz, 2H), 6.77 (t, J = 5.9 Hz, 1H), 4.34 (q, J = 7.2 Hz, 2H), 4.26 (d, J = 8.3 Hz, 2H), 2.99 (d, J = 6.4 Hz, 2H), 1.80-1.71 (m, 2H), 1.41 (dt, J = 23.7, 8.5 Hz, 5H), 1.33 (s, 9H). 10 tert -Butyl (4-(5-carbamoyl-2-(1-methyl-3-(trifluoromethyl) -1H -pyrazole-4-carboxamido) -1H -benzo[ d ] imidazol-1-yl)butyl)carbamate ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 12.78 (s, 1H), 8.44 (s, 1H), 8.01-7.92 (m, 2H), 7.77 (dd, J = 8.4, 1.7 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 6.80 (t, J = 5.6 Hz, 1H), 4.22 (t, J = 7.0 Hz, 2H), 3.95 (s, 3H), 2.98 (d, J = 6.4 Hz, 2H), 1.73 (p, J = 7.2 Hz, 2H), 1.42 (t, J = 7.5 Hz, 2H), 1.34 (s, 9H). 11 tert -Butyl (4-(2-(1-allyl-5-methyl- 1H -pyrazole-3-carboxamido)-5-carbamoyl- 1H -benzo[ d ]imidazole-1- yl) butyl) carbamate ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 8.03 (s, 1H), 7.95 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.54 (s, 1H), 7.28 (s, 1H), 6.80 (d, J = 5.8 Hz, 1H), 6.63 (s, 1H), 6.00 (td, J = 12.2, 11.5, 5.9 Hz, 1H), 5.19 (d, J = 10.3 Hz, 1H), 4.94 (d, J = 17.1 Hz, 1H), 4.80 (s, 2H), 4.18 (s, 2H), 2.95 (s, 2H), 2.27 (s, 3H), 1.73 (s, 2H), 1.34 (s) , 11H). 12 tert -Butyl (4-(5-carbamoyl-2-(furan-3-carboxamido) -1H -benzo[ d ]imidazol-1-yl)butyl)carbamate ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.64 (s, 1H), 8.24 (s, 1H), 7.97 (d, J = 7.6 Hz, 2H), 7.77 (d, J = 8.3 Hz, 1H) , 7.70 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.30 (s, 1H), 6.81 (d, J = 5.7 Hz, 2H), 4.21 (d, J = 7.1 Hz, 2H) , 2.99 (d, J = 6.2 Hz, 2H), 1.75 (t, J = 7.6 Hz, 2H), 1.43 (d, J = 7.6 Hz, 2H), 1.34 (s, 9H). 13 tert -Butyl (4-(5-carbamoyl-2-(furan-3-carboxamido) -1H -benzo[ d ]imidazol-1-yl)butyl)carbamate ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.81 (s, 1H), 8.04 (s, 1H), 7.98 (s, 1H), 7.79 (dd, J = 18.9, 8.0 Hz, 2H), 7.72 7.55 (m, 3H), 7.43 (t, J = 7.7 Hz, 1H), 7.36-7.27 (m, 2H), 6.80 (t, J = 5.8 Hz, 1H), 4.30 (t, J = 6.9 Hz, 2H) ), 3.01 (q, J = 6.6 Hz, 2H), 1.81 (p, J = 7.6, 7.0 Hz, 2H), 1.46 (p, J = 7.0 Hz, 2H), 1.32 (s, 9H). 14 tert -Butyl (4-(5-carbamoyl-2-(1,3-dimethyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazol-1-yl) butyl) carbamate ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.80 (s, 1H), 7.98 (d, J = 12.1 Hz, 2H), 7.79 (d, J = 8.3 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 6.79 (t, J = 5.6 Hz, 1H), 6.66 (s, 1H), 4.22 (t, J = 6.9 Hz, 2H), 4.14 (s, 3H) , 2.96 (q, J = 6.8 Hz, 2H), 2.17 (s, 3H), 1.75 (t, J = 7.7 Hz, 2H), 1.41 (t, J = 7.7 Hz, 2H), 1.34 (s, 9H) . 15 tert -Butyl (4-(5-carbamoyl-2-(1-isopropyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-1 -yl)butyl)carbamate ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 12.81 (s, 1H), 7.98 (d, J = 9.2 Hz, 2H), 7.81-7.77 (m, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 6.80 (t, J = 5.9 Hz, 1H), 6.63 (s, 1H), 5.86 (p, J = 6.7 Hz, 1H), 4.20 (t, J = 7.0 Hz, 2H), 2.96 (q, J = 6.6 Hz, 2H), 2.19 (s, 3H), 1.75 (t, J = 7.7 Hz, 2H), 1.41 (d, J = 6.6 Hz, 8H), 1.34 (s, 9H). 16 tert -butyl (4-(2-(1-( tert -butyl)-3-methyl- 1H -pyrazole-5-carboxamido)-5-carbamoyl- 1H -benzo[ d ]imi Dazol-1-yl)butyl)carbamate ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.78 (s, 1H), 8.05-7.99 (m, 1H), 7.96 (s, 1H), 7.80 (dd, J = 8.4, 1.6 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 6.78 (t, J = 5.8 Hz, 1H), 6.61 (s, 1H), 4.19 (t, J = 7.0 Hz, 2H), 2.95 (q, J = 6.6 Hz, 2H), 2.15 (s, 3H), 1.69 (s, 11H), 1.40 (t, J = 7.7 Hz, 2H), 1.34 (s, 9H).

Synthesis of the compounds of Table 4 below

tert -Butyl (3-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1H - benzo[ d ]imidazole-1- yl)propyl)carbamate (Compound 17), and

2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-1-(3-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido ) do) propyl) -1H - benzo[ d ]imidazole-5-carboxamide (Compound 27)

Step 1: tert -Butyl(4-((4-carbamoyl-2-nitrophenyl)amino)propyl)carbamate

N -boc-1,3-propanediamine (9.38 g, 49.8 mmol) and 4-chloro-3-nitrobenzamide (3.00 g, 15.0 mmol) were dissolved in DMSO (25 mL) followed by K ₂ CO ₃ (3.14) g, 18.0 mmol) was added. The reaction was stirred at 70 °C for 16 h. After stirring, the solid was filtered with cold water and dried under vacuum. tert -butyl(4-((4-carbamoyl-2-nitrophenyl)amino)propyl)carbamate as a yellow solid (4.15 g, 12.3 mmol, 82% yield) was obtained.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 8.65 (d, J = 2.2 Hz, 1H), 8.45 (t, J = 5.9 Hz, 1H), 7.99 (dd, J = 8.8, 2.3 Hz, 2H) , 7.27 (s, 1H), 7.08 (d, J = 9.1 Hz, 1H), 6.92 (d, J = 6.1 Hz, 1H), 3.42 (q, J = 6.5 Hz, 2H), 3.01 (q, J = 6.4 Hz, 2H), 1.71 (t, J = 6.6 Hz, 2H), 1.37 (s, 9H).

Step 2: tert -Butyl(4-((2-amino-4-carbamoylphenyl)amino)propyl)carbamate

tert -Butyl(4-((4-carbamoyl-2-nitrophenyl)amino)propyl)carbamate (2.00 g, 5.91 mmol) was dissolved in MeOH (118 mL) followed by 10% wet Pd/C (63 mg) , 0.59 mmol) and stirred for 18 h at room temperature under a hydrogen balloon. It was then filtered through Celite®, washing with 400 mL of MeOH. The filtrate containing the product was concentrated in vacuo to give tert -butyl(4-((2-amino-4-carbamoylphenyl)amino)propyl)carbamate (1.79 g, 5.80 mmol, 98% yield) as a purple liquid. obtained and used in the next reaction without further purification.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 7.41 (s, 1H), 7.10 (d, J = 8.2 Hz, 2H), 6.88 (t, J = 5.7 Hz, 1H), 6.72 (s, 1H) , 6.36 (d, J = 8.0 Hz, 1H), 4.88 (t, J = 5.4 Hz, 1H), 4.56 (s, 2H), 3.05 (dq, J = 12.9, 6.6 Hz, 4H), 1.70 (p, J = 6.9 Hz, 2H), 1.38 (s, 9H).

Step 3: tert -Butyl (4-(2-amino-5-carbamoyl-1) H -benzo[ d ]Imidazol-1-yl)propyl)carbamate

tert -Butyl(4-((2-amino-4-carbamoylphenyl)amino)propyl)carbamate (1.79 g, 5.80 mmol) was dissolved in MeOH (9.67 mL) followed by cyanogen bromide (1.54 g, 14.5 mmol) ), and stirred at 60 °C for 3 hours. The residue containing the product was then removed from MeOH in vacuo to tert -butyl (4-(2-amino-5-carbamoyl- 1H -benzo[ d ]imidazole) containing cyanogen bromide as a black solid. -1-yl)propyl)carbamate was obtained and used in the next reaction without further purification.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.85 (s, 1H), 8.76 (s, 2H), 8.06 (s, 1H), 7.89-7.81 (m, 2H), 7.58 (d, J = 8.3) Hz, 1H), 7.41 (s, 1H), 6.94 (t, J = 5.4 Hz, 1H), 4.14 (t, J = 7.2 Hz, 2H), 3.02 (q, J = 6.5 Hz, 2H), 1.83 ( p, J = 7.3 Hz, 2H), 1.37 (s, 9H).

Step 4: tert -Butyl (3-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1H - benzo[ d ]imidazole ) -1-yl)propyl)carbamate (compound 17)

1-Ethyl-3-methyl-1 H -pyrazole-5-carboxylic acid (463 mg, 3.00 mmol), HATU (1.14 g, 3.00 mmol), HOBt (203 mg, 1.50 mmol) and TEA (1.05 mL, 7.50 mmol) ) was dissolved in DMF (38 mL). After the reaction was stirred at room temperature for 5 min, tert -butyl (4-(2-amino-5-carbamoyl-1 H -benzo[ d ]imidazol-1-yl)propyl)carbamate (500 mg , 1.50 mmol) was added. After the reaction was stirred at room temperature for 16 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) as a light yellow solid tert -butyl (3-(5-carbamoyl-2-(1-ethyl-3-methyl)) -1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazol-1-yl)propyl)carbamate (456 mg, 0.975 mmol, 65% yield) was obtained.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.83 (s, 1H), 8.02-7.93 (m, 2H), 7.80 (dd, J = 8.4, 1.7 Hz, 1H), 7.56 (d, J = 8.4) Hz, 1H), 7.33 (s, 1H), 6.91 (t, J = 5.6 Hz, 1H), 6.68 (s, 1H), 4.62 (q, J = 7.1 Hz, 2H), 4.21 (t, J = 7.1) Hz, 2H), 3.00 (q, J = 6.6 Hz, 2H), 2.18 (s, 3H), 1.90 (q, J = 7.0 Hz, 2H), 1.36 (s, 12H).

Step 5: 1-(3-Aminopropyl)-2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-1 H -benzo[ d ]Imidazole-5-carboxamide hydrochloride)

tert -Butyl (3-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-1- MeOH (0.71 mL) was dissolved in yl)propyl)carbamate (compound 17, 200 mg, 0.426 mmol) and then 4M HCl in dioxane (2.00 mL) was slowly added. After the mixture was stirred at room temperature for 1 hour, the resulting solid was filtered, washed 3 times with Et ₂ O, and dried under vacuum to form 1-(3-aminopropyl)-2-(1-(3-aminopropyl)-2-(1-) as a white solid. Ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide hydrochloride) was obtained and used in the next reaction without further purification. .

¹ H NMR (500 MHz, DMSO- d ₆ ) δ 7.87 (d, J = 1.7 Hz, 1H), 7.72 (dd, J = 8.5, 1.7 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H) , 6.65 (s, 1H), 4.49 (q, J = 7.4 Hz, 2H), 4.22 (t, J = 6.8 Hz, 2H), 2.86 (t, J = 7.7 Hz, 2H), 2.12 (s, 3H) , 2.05 (p, J = 7.1 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H).

Step 6: 2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-1-(3-(1-ethyl-3-methyl-1 H -pyrazole-5- Carboxamido)propyl)-1H - benzo[ d ]imidazole-5-carboxamide (Compound 27)

1-Ethyl-3-methyl-1 H -pyrazole-5-carboxylic acid (37 mg, 0.243 mmol), HATU (123 mg, 0.243 mmol), HOBt (22 mg, 0.162 mmol) and TEA (0.226 mL, 1.62 mmol) ) was dissolved in DMF (4.1 mL). The reaction was stirred at room temperature for 5 min, then 1-(3-aminopropyl)-2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-1 H -benzo[ d ]imidazole-5-carboxamide hydrochloride) (60 mg, 0.162 mmol) was added. After the reaction was stirred at room temperature for 16 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) to 2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) as a white solid. -1-(3-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)propyl) -1H -benzo[ d ]imidazole-5-carboxamide (62.5 mg, 0.123 mmol, 76% yield).

¹ H NMR (500 MHz, DMSO- d ₆ ) δ 8.45 (t, J = 5.7 Hz, 1H), 8.01 (s, 2H), 7.81 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 6.58 (s, 1H), 6.55 (s, 1H), 4.58 (q, J = 7.1 Hz, 2H), 4.35 (q, J = 7.2 Hz, 2H) , 4.27 (t, J = 7.2 Hz, 2H), 3.31-3.27 (m, 2H), 2.14 (s, 3H), 2.10 (s, 3H), 2.00 (q, J = 7.2 Hz, 2H), 1.33 ( t, J = 7.1 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H).

tert -Butyl (3-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1H - benzo[ d ]imidazole-1- yl)pentyl)carbamate (Compound 18), and

2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-1-(3-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido ) do) pentyl)-1H - benzo[ d ]imidazole-5-carboxamide (Compound 28)

Step 1: tert -Butyl(4-((4-carbamoyl-2-nitrophenyl)amino)pentyl)carbamate

Dissolve N -boc-1,3-pentanediamine (3.64 g, 18.0 mmol) and 4-chloro-3-nitrobenzamide (6, 3.00 g, 15.0 mmol) in DMSO (25 mL) followed by K ₂ CO ₃ (3.14 g, 18.0 mmol) was added. The reaction was stirred at 70 °C for 16 h. After stirring, the solid was filtered with cold water and dried under vacuum. tert -butyl(4-((4-carbamoyl-2-nitrophenyl)amino)pentyl)carbamate as a yellow solid (3.20 g, 8.70 mmol, 58% yield) was obtained.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 8.65 (d, J = 2.1 Hz, 1H), 8.36 (t, J = 5.7 Hz, 1H), 8.00 (dd, J = 9.1, 2.2 Hz, 2H) , 7.26 (s, 1H), 7.09 (d, J = 9.1 Hz, 1H), 6.77 (d, J = 6.0 Hz, 1H), 3.44-3.36 (m, 2H), 2.91 (q, J = 6.4 Hz, 2H), 1.62 (p, J = 7.5 Hz, 2H), 1.36 (s, 13H).

Step 2: tert -Butyl(4-((2-amino-4-carbamoylphenyl)amino)pentyl)carbamate

tert -Butyl(4-((4-carbamoyl-2-nitrophenyl)amino)pentyl)carbamate (2.00 g, 5.46 mmol) was dissolved in MeOH (109 mL) followed by 10% wet Pd/C (59 mg , 0.55 mmol) and stirred for 18 h at room temperature under a hydrogen balloon. It was then filtered through Celite®, washing with 400 mL of MeOH. The filtrate containing the product was concentrated in vacuo to give tert -butyl(4-((2-amino-4-carbamoylphenyl)amino)pentyl)carbamate (1.70 g, 5.08 mmol, 93% yield) as a purple liquid. obtained and used in the next reaction without further purification.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 7.40 (s, 1H), 7.13-7.04 (m, 2H), 6.77 (d, J = 6.1 Hz, 1H), 6.71 (s, 1H), 6.35 ( d, J = 8.1 Hz, 1H), 4.84 (t, J = 5.3 Hz, 1H), 4.58 (s, 2H), 3.04 (q, J = 6.5 Hz, 2H), 2.92 (q, J = 6.4 Hz, 2H), 1.58 (p, J = 7.3 Hz, 2H), 1.37 (s, 14H).

Step 3: tert -Butyl (4-(2-amino-5-carbamoyl- 1H -benzo[ d ]imidazol-1-yl)pentyl)carbamate

tert -Butyl(4-((2-amino-4-carbamoylphenyl)amino)pentyl)carbamate (1.70 g, 5.05 mmol) was dissolved in MeOH (8.42 mL) followed by cyanogen bromide (1.34 g, 12.0 mmol) ), and stirred at 60 °C for 3 hours. The residue containing the product was then removed from MeOH in vacuo to tert -butyl (4-(2-amino-5-carbamoyl- 1H -benzo[ d ]imidazole) containing cyanogen bromide as a black solid. -1-yl)pentyl)carbamate was obtained and used in the next reaction without further purification.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.80 (s, 1H), 8.73 (s, 2H), 8.06 (s, 1H), 7.87-7.80 (m, 2H), 7.61 (d, J = 8.4) Hz, 1H), 7.40 (s, 1H), 6.77 (t, J = 5.7 Hz, 1H), 4.12 (t, J = 7.4 Hz, 2H), 2.89 (q, J = 6.4 Hz, 2H), 1.67 ( t, J = 7.5 Hz, 2H), 1.35 (s, 13H).

Step 4: tert -Butyl (3-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1H - benzo[ d ]imidazole ) -1-yl)pentyl)carbamate (Compound 18)

1-Ethyl-3-methyl-1 H -pyrazole-5-carboxylic acid (426 mg, 2.76 mmol), HATU (1.05 g, 2.76 mmol), HOBt (186 mg, 1.38 mmol) and TEA (0.962 mL, 6.90 mmol) ) was dissolved in DMF (34.5 mL). After the reaction was stirred at room temperature for 5 min, tert -butyl (4-(2-amino-5-carbamoyl-1 H -benzo[ d ]imidazol-1-yl)pentyl)carbamate (500 mg , 1.50 mmol) was added. After the reaction was stirred at room temperature for 16 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) as a light yellow solid tert -butyl (3-(5-carbamoyl-2-(1-ethyl-3-methyl)) -1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazol-1-yl)pentyl)carbamate (423 mg, 0.856 mmol, 62% yield) was obtained.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.81 (s, 1H), 7.99 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 8.2 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.32 (s, 1H), 6.73 (d, J = 5.8 Hz, 1H), 6.64 (s, 1H), 4.63 (q, J = 7.2 Hz, 2H), 4.19 (t, J = 7.2 Hz, 2H), 2.89 (q, J = 6.5 Hz, 2H), 2.18 (s, 3H), 1.76 (p, J = 7.4 Hz, 2H), 1.46-1.27 (m, 16H).

Step 5: 1-(3-Aminopentyl)-2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-1 H -benzo[ d ]Imidazole-5-carboxamide hydrochloride)

tert -Butyl (3-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-1- MeOH (0.67 mL) was dissolved in yl)pentyl)carbamate (compound 18, 200 mg, 0.402 mmol) and then 4M HCl in dioxane (2.00 mL) was slowly added. After the mixture was stirred at room temperature for 1 hour, the resulting solid was filtered, washed 3 times with Et ₂ O, and dried under vacuum to form 1-(3-aminopentyl)-2-(1-(3-aminopentyl)-2-(1-) as a white solid. Ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide hydrochloride) was obtained and used in the next reaction without further purification. .

¹ H NMR (500 MHz, D ₂ O) δ 7.52 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 6.42 (s, 1H), 4.36 (q, J = 7.5 Hz, 2H), 3.86 (t, J = 7.8 Hz, 2H), 2.92 (t, J = 7.7 Hz, 2H), 2.13 (s, 3H), 1.62 (p, J = 8.0) Hz, 2H), 1.54 (q, J = 7.8 Hz, 2H), 1.33 (p, J = 7.9 Hz, 2H), 1.22 (t, J = 7.3 Hz, 3H).

Step 6: 2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-1-(3-(1-ethyl-3-methyl-1 H -pyrazole-5- Carboxamido)pentyl)-1H - benzo[ d ]imidazole-5-carboxamide (Compound 28)

1-Ethyl-3-methyl-1 H -pyrazole-5-carboxylic acid (35 mg, 0.227 mmol), HATU (115 mg, 0.302 mmol), HOBt (20 mg, 0.151 mmol) and TEA (0.210 mL, 1.51 mmol) ) was dissolved in DMF (3.8 mL). After the reaction was stirred at room temperature for 5 min, 1-(3-aminopentyl)-2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-1 H -benzo[ d ]imidazole-5-carboxamide hydrochloride) (60 mg, 0.151 mmol) was added. After the reaction was stirred at room temperature for 16 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) to 2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) as a white solid. -1-(3-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)pentyl) -1H -benzo[ d ]imidazole-5-carboxamide (46.5 mg, 0.087 mmol, 58% yield).

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.82 (s, 1H), 8.30 (t, J = 5.7 Hz, 1H), 8.03-7.95 (m, 2H), 7.80 (dd, J = 8.4, 1.6) Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.33 (s, 1H), 6.63 (s, 1H), 6.48 (s, 1H), 4.62 (q, J = 7.1 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.20 (t, J = 7.2 Hz, 2H), 3.18 (q, J = 6.6 Hz, 2H), 2.14 (d, J = 3.7 Hz, 6H), 1.80 ( p, J = 7.3 Hz, 2H), 1.56 (p, J = 7.1 Hz, 2H), 1.35 (q, J = 7.1, 5.7 Hz, 5H), 1.22 (t, J = 7.1 Hz, 3H).

1-(4-aminobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1H - benzo[ d ]imidazole-5-carboxamide (compound 19)

tert -Butyl (4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-1- yl)butyl)carbamate (compound 8, 200 mg, 0.414 mmol) was dissolved in MeOH (0.69 mL) and then 4 M HCl in dioxane (4.00 mL) was slowly added. After the mixture was stirred at room temperature for 1 h, Et ₂ O was added and the resulting precipitate was filtered with Et ₂ O. The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH:aq.NH ₄ OH (28%) = 9:1:0.1) to 1-(4-aminobutyl)-2-(1-ethyl -3- Methyl - 1H -pyrazole-5-carboxamido)-1H-benzo[ d ]imidazole-5-carboxamide (55.3 mg, 0.144 mmol, 35%) was obtained as a pinkish solid.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.05-7.91 (m, 2H), 7.81-7.74 (m, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 6.63 (s, 1H), 4.62 (q, J = 7.1 Hz, 2H), 4.20 (t, J = 7.1 Hz, 2H), 2.58 (t, J = 6.9 Hz, 2H), 2.17 (s, 3H), 1.79 (p, J = 6.9 Hz, 2H), 1.37 (dt, J = 14.1, 7.5 Hz, 5H).

1-(4-acetamidobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1H - benzo[ d ]imidazole-5-carboxamide (Compound 20)

1-(4-aminobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide (compound 19, 100 mg, 0.261 mmol) was dissolved in a mixture of CH ₂ Cl ₂ :MeOH (9:1, 0.392 mL, 0.05 mL) and cooled to 0 °C. After 5 minutes, acetyl chloride (0.028 mL, 0.392 mmol) was slowly added at 0 °C. After that, the mixture was stirred at 0° C. for 3 hours and then stirred at room temperature for 3 hours. After the solvent was removed in vacuo, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) as a white solid 1-(4-acetamidobutyl)-2-(1-ethyl-3-methyl-1 H -Pyrazole-5-carboxamido)-1H-benzo[ d ]imidazole-5-carboxamide (35.4 mg, 0.0835 mmol, 32% yield) was obtained.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.82 (s, 1H), 7.99 (d, J = 6.5 Hz, 2H), 7.86-7.76 (m, 2H), 7.57 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 6.66 (s, 1H), 4.62 (q, J = 7.1 Hz, 2H), 4.21 (t, J = 6.9 Hz, 2H), 3.07 (q, J = 6.5 Hz, 2H), 2.18 (s, 3H), 1.75 (s, 5H), 1.49-1.33 (m, 5H).

2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1-(4-pivalamidobutyl)-1H - benzo[ d ]imidazole-5-carbox Amide (Compound 21)

1-(4-aminobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide (compound 19, 100 mg, 0.261 mmol) was dissolved in a mixture of CH ₂ Cl ₂ :MeOH (9:1, 0.392 mL, 0.05 mL) and cooled to 0 °C. After 5 minutes, pivaloyl chloride (0.048 mL, 0.392 mmol) was slowly added at 0 °C. After that, the mixture was stirred at 0° C. for 3 hours and then stirred at room temperature for 3 hours. After the solvent was removed in vacuo, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) to 2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) as a white solid. Obtained -1-(4- pivalamidobutyl )-1H-benzo[ d ]imidazole-5-carboxamide (55.7 mg, 0.120 mmol, 46% yield).

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.80 (s, 1H), 8.03-7.93 (m, 2H), 7.80 (dd, J = 8.4, 1.7 Hz, 1H), 7.57 (d, J = 8.4) Hz, 1H), 7.42 (t, J = 5.8 Hz, 1H), 7.33 (s, 1H), 6.66 (s, 1H), 4.62 (q, J = 7.1 Hz, 2H), 4.21 (t, J = 7.1) Hz, 2H), 3.09 (q, J = 6.4 Hz, 2H), 2.17 (s, 3H), 1.73 (p, J = 7.5 Hz, 2H), 1.46 (q, J = 7.1 Hz, 2H), 1.35 ( t, J = 7.1 Hz, 3H), 1.02 (s, 9H).

1-(4-benzamidobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1H - benzo[ d ]imidazole-5-carboxamide (Compound 22)

1-(4-aminobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide (compound 19, 100 mg, 0.261 mmol) was dissolved in a mixture of CH ₂ Cl ₂ :MeOH (9:1, 0.392 mL, 0.05 mL) and cooled to 0 °C. After 5 minutes, benzoyl chloride (0.046 mL, 0.392 mmol) was slowly added at 0 °C. After that, the mixture was stirred at 0° C. for 3 hours and then stirred at room temperature for 3 hours. After the solvent was removed in vacuo, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) as a white solid 1-(4-benzamidobutyl)-2-(1-ethyl-3-methyl- 1H ) -Pyrazole -5-carboxamido)-1H-benzo[ d ]imidazole-5-carboxamide (57.2 mg, 0.117 mmol, 45% yield) was obtained.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.81 (s, 1H), 8.45 (t, J = 5.7 Hz, 1H), 8.03-7.94 (m, 2H), 7.81-7.75 (m, 3H), 7.59 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 8.4, 6.2 Hz, 1H), 7.41 (dd, J = 8.2, 6.6 Hz, 2H), 7.33 (s, 1H), 6.66 (s) , 1H), 4.61 (q, J = 7.1 Hz, 2H), 4.25 (t, J = 7.0 Hz, 2H), 3.31 (t, J = 6.4 Hz, 2H), 2.15 (s, 3H), 1.83 (p , J = 7.2 Hz, 2H), 1.58 (dq, J = 13.7, 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H).

2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1-(4-(nicotinamido)butyl)-1H - benzo[ d ]imidazole-5-car Boxamide (Compound 23)

Nicotinic acid (64 mg, 0.522 mmol), HATU (198 mg, 0.522 mmol), HOBt (35 mg, 0.261 mmol) and TEA (0.364 mL, 2.61 mmol) were dissolved in DMF (6.5 mL). After the reaction was stirred at room temperature for 5 min, 1-(4-aminobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide (compound 19, 100 mg, 0.261 mmol) was added. After the reaction was stirred at room temperature for 16 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) to 2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido) as a light pink solid. Obtained -1-(4-(nicotinamido)butyl)-1H-benzo[ d ]imidazole-5-carboxamide (38.5 mg, 0.0783 mmol, 30% yield).

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.82 (s, 1H), 8.97 (d, J = 2.3 Hz, 1H), 8.74 (t, J = 5.6 Hz, 1H), 8.66 (dd, J = 4.8, 1.7 Hz, 1H), 8.14 (dt, J = 8.0, 2.0 Hz, 1H), 8.05-7.95 (m, 2H), 7.81 (dd, J = 8.5, 1.7 Hz, 1H), 7.60 (d, J ) = 8.4 Hz, 1H), 7.45 (dd, J = 7.9, 4.8 Hz, 1H), 7.33 (s, 1H), 6.65 (s, 1H), 4.60 (q, J = 7.1 Hz, 2H), 4.26 (t) , J = 7.0 Hz, 2H), 3.33 (t, J = 6.4 Hz, 2H), 2.14 (s, 3H), 1.84 (p, J = 7.1 Hz, 2H), 1.59 (p, J = 7.0 Hz, 2H) ), 1.34 (t, J = 7.1 Hz, 3H).

1-(4-( 1H -Pyrazole-5-carboxamido)butyl)-2-(1-ethyl-3-methyl- 1H - pyrazole-5-carboxamido) -1H- Benzo[ d ]imidazole-5-carboxamide (Compound 24)

1 H -Pyrazole-5-carboxylic acid (59 mg, 0.522 mmol), HATU (198 mg, 0.522 mmol), HOBt (35 mg, 0.261 mmol) and TEA (0.364 mL, 2.61 mmol) were mixed with DMF (6.5 mL) ) was dissolved in After the reaction was stirred at room temperature for 5 min, 1-(4-aminobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide (compound 19, 100 mg, 0.261 mmol) was added. After the reaction was stirred at room temperature for 16 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) to 1-(4-(1 H -pyrazole-5-carboxamido)butyl)-2 as a light pink solid. -(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1H-benzo[ d ]imidazole-5-carboxamide (27.0 mg, 0.0574 mmol, 22% yield) was obtained.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 13.25 (s, 1H), 12.83 (s, 1H), 8.20 (d, J = 6.0 Hz, 1H), 8.06-7.95 (m, 2H), 7.84 7.74 (m, 2H), 7.59 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 6.65 (s, 1H), 6.61 (s, 1H), 4.61 (q, J = 7.1 Hz, 2H) ), 4.24 (t, J = 6.9 Hz, 2H), 3.28 (q, J = 6.5 Hz, 2H), 2.16 (s, 3H), 1.86-1.72 (m, 2H), 1.56 (t, J = 7.6 Hz) , 2H), 1.34 (t, J = 7.1 Hz, 3H).

1-(4-(1,3-dimethyl- 1H -pyrazole-5-carboxamido)butyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido ) Figure)-1H - benzo[ d ]imidazole-5-carboxamide (compound 25)

1,3-dimethyl- 1H -pyrazole-5-carboxylic acid (73 mg, 0.522 mmol), HATU (198 mg, 0.522 mmol), HOBt (35 mg, 0.261 mmol) and TEA (0.364 mL, 2.61 mmol) ) was dissolved in DMF (6.5 mL). After the reaction was stirred at room temperature for 5 min, 1-(4-aminobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide (compound 19, 100 mg, 0.261 mmol) was added. After the reaction was stirred at room temperature for 16 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) as a light pink solid 1-(4-(1,3-dimethyl- 1H -pyrazole-5-carboxami). do)butyl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide (68.7 mg, 0.136 mmol, 52% yield).

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.82 (s, 1H), 8.34 (t, J = 5.7 Hz, 1H), 8.00 (t, J = 3.3 Hz, 2H), 7.79 (dd, J = 8.4, 1.6 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.33 (s, 1H), 6.66 (s, 1H), 6.49 (s, 1H), 4.61 (q, J = 7.1 Hz, 2H), 4.24 (t, J = 6.8 Hz, 2H), 3.92 (s, 3H), 3.25 (q, J = 6.6 Hz, 2H), 2.16 (s, 3H), 2.10 (s, 3H), 1.87- 1.74 (m, 2H), 1.53 (dt, J = 13.3, 6.6 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H).

2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-1-(4-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido ) Do)Butyl)-1H - benzo[ d ]imidazole-5-carboxamide (Compound 26)

HATU (99 mg, 0.260 mmol), HOBt (18 mg, 0.130 mmol), TEA (0.181 mL, 1.30 mmol) and 1-ethyl-3-methyl-1 H -pyrazole-5-carboxylic acid (40 mg, 0.260 mmol) was dissolved in DMF (3.3 mL, 0.04 M) and stirred at room temperature for 5 minutes, followed by 1-(4-aminobutyl)-2-(1-ethyl-3-methyl- 1H -pyrazole -5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide (compound 19, 50 mg, 0.130 mmol) was added and the mixture was further stirred at room temperature for 16 h. Then, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was washed with water and dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH = 9:1) to 2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-1- (4-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)butyl) -1H -benzo[ d ]imidazole-5-carboxamide (54.7 mg, 0.105 mmol, 81%) as a white solid.

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.84 (s, 1H), 8.34 (t, J = 5.8 Hz, 1H), 7.99 (d, J = 7.2 Hz, 2H), 7.79 (dd, J = 8.4, 1.6 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 6.66 (s, 1H), 6.46 (s, 1H), 4.61 (q, J = 7.1 Hz, 2H), 4.36 (q, J = 7.1 Hz, 2H), 4.25 (t, J = 6.9 Hz, 2H), 3.25 (q, J = 6.5 Hz, 2H), 2.16 (s, 3H), 2.11 (s, 3H), 1.81 (p, J = 7.1 Hz, 2H), 1.54 (p, J = 7.0 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H) .

tert -Butyl (4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-7-methoxy- 1H -benzo[ d ] imidazol-1-yl)butyl)carbamate (Compound 29), and

tert -Butyl ( E )-(4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-7-methoxy- 1H -benzo [ d ]imidazol-1-yl)but-2-en-1-yl)carbamate (Compound 30)

Step 1: 4-Chloro-3-methoxy-5-nitrobenzamide

Methyl 4-chloro-3-methoxy-5-nitrobenzoate (3.00 g, 12.2 mmol) was dissolved in aq.NH ₄ OH (28%) (40 mL) and stirred at room temperature for 16 hours. After stirring, the solid was filtered with cold water and dried under vacuum to give 4-chloro-3-methoxy5-nitrobenzamide (2.26 g, 9.76 mmol, 80% yield) as a tan solid.

¹ H NMR (300 MHz, Chloroform-d) δ 7.90 (d, J = 1.9 Hz, 1H), 7.76 (t, J = 1.8 Hz, 1H), 4.04 (d, J = 1.4 Hz, 3H).

Step 2: tert -Butyl ( E )-(4-((4-carbamoyl-2-methoxy-6-nitrophenyl)amino)but-2-en-1-yl)carbamate

Dissolve 4-chloro-3-methoxy-5-nitrobenzamide (1.00 g, 4.34 mmol) in EtOH (4.34 mL) in a sealed tube ( E ) -tert -butyl (4-aminobut-2-ene) -1-yl)carbamate (0.970 g, 5.21 mmol) and DIPEA (2.26 mL, 13.0 mmol) were added. The reaction was stirred at 120 °C for 3 days and cooled to room temperature. The resulting orange solid was filtered with EtOH and dried to form an orange solid tert -butyl ( E )-(4-((4-carbamoyl-2-methoxy-6-nitrophenyl)amino)but-2-ene Obtained -1-yl)carbamate (1.22 g, 3.21 mmol, 74% yield).

¹ H NMR (400 MHz, methanol- d ₄ ) δ 8.30 (d, J = 2.0 Hz, 1H), 7.54 (d, J = 2.0 Hz, 1H), 5.71-5.58 (m, 2H), 4.20 (d, J = 4.6 Hz, 2H), 3.93 (s, 3H), 3.60 (s, 2H), 1.41 (s, 9H).

Step 3: tert -Butyl ( E )-(4-((2-amino-4-carbamoyl-6-methoxyphenyl)amino)but-2-en-1-yl)carbamate

tert -Butyl ( E )-(4-((4-carbamoyl-2-methoxy-6-nitrophenyl)amino)but-2-en-1-yl)carbamate (500 mg, 1.31 mmol) was dissolved in MeOH (2.18 mL), and then aq.NH ₄ OH (28%) (0.436 mL, 13.1 mmol) and a 1.0 M aqueous sodium hydrogen sulfite solution (0.13 mL, 6.55 mmol) were sequentially added at 0 °C. . The reaction mixture was stirred at room temperature for 2 hours, and then the solvent was removed under vacuum. Then, the reaction mixture was poured into water and extracted with CH ₂ Cl ₂ . The organic layer was dried over Na ₂ SO ₄ and filtered off, and the filtered liquid was removed under vacuum to remove tert -butyl ( E )-(4-((2-amino-4-carbamoyl-6-methoxyphenyl)amino) But-2-en-1-yl)carbamate (225 mg, 0.612 mmol, 49% yield) was obtained.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 7.59 (s, 1H), 7.02-6.84 (m, 3H), 6.79 (d, J = 1.9 Hz, 1H), 5.67-5.47 (m, 2H), 4.65 (s, 2H), 3.81 (t, J = 7.1 Hz, 1H), 3.76 (s, 3H), 3.51 (q, J = 6.6 Hz, 4H), 1.37 (s, 9H).

Step 4: ( E )-2-amino-1-(4-aminobut-2-en-1-yl)-7-methoxy-1 H -benzo[ d ]Imidazole-5-carboxamide

tert -Butyl ( E )-(4-((2-amino-4-carbamoyl-6-methoxyphenyl)amino)but-2-en-1-yl)carbamate (100 mg, 0.285 mmol) was dissolved in MeOH (0.48 mL), and then cyanogen bromide (63 mg, 0.591 mmol) was added, followed by stirring at 60 °C for 3 hours. The residue containing the product was then removed in vacuo with MeOH to ( E )-2-amino-1-(4-aminobut-2-en-1-yl)-7- containing cyanogen bromide as a white solid. Methoxy- 1H -benzo[ d ]imidazole-5-carboxamide was obtained and used in the next reaction without further purification.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.91 (s, 1H), 8.65 (s, 2H), 8.09 (s, 1H), 7.54-7.40 (m, 3H), 6.96 (d, J = 5.9) Hz, 1H), 5.66 (t, J = 4.0 Hz, 2H), 4.85 (d, J = 4.1 Hz, 2H), 3.95 (s, 3H), 3.52 (d, J = 5.9 Hz, 2H), 1.34 ( s, 9H).

Step 5: tert -Butyl ( E )-(4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-7-methoxy-1 H -benzo[ d ]imidazol-1-yl)but-2-en-1-yl)carbamate (Compound 29)

( E )-2-amino-1-(4-aminobut-2-en-1-yl)-7-methoxy- 1H -benzo[ d ]imidazole-5-carboxamide (128 mg, 0.341 mmol), 1-ethyl-3-methyl-1 H -pyrazole-5-carboxylic acid (63 mg, 0.409 mmol), HATU (648 mg, 1.71 mmol), and TEA (0.570 mL, 4.09 mmol) with NMP (1.14) mL) and then heated at 80 °C for 72 hours. To the reaction was added water (30 mL) and extracted with EtOAc (4 X 10 mL). The organic layer was dried by the addition of anhydrous MgSO ₄ and the solvent was removed in vacuo. The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH=9:1) as a purple solid tert -butyl ( E )-(4-(5-carbamoyl-2-(1-ethyl-) 3-methyl- 1H -pyrazole-5-carboxamido)-7-methoxy- 1H -benzo[ d ]imidazol-1-yl)but-2-en-1-yl)carbamate (43 mg, 0.085 mmol, 25% yield).

¹ H NMR (300 MHz, methanol- d ₄ ) δ 7.57 (s, 1H), 7.34 (d, J = 1.4 Hz, 1H), 6.64 (s, 1H), 5.84-5.58 (m, 2H), 4.95 ( d, J = 5.7 Hz, 2H), 4.64 (q, J = 7.3 Hz, 2H), 3.98 (s, 3H), 3.58 (t, J = 4.8 Hz, 2H), 2.22 (s, 3H), 1.43- 1.26 (m, 12H).

Step 6: tert -Butyl (4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido)-7-methoxy-1 H -benzo [ d ]imidazol-1-yl)butyl)carbamate, (compound 30)

tert -Butyl ( E )-(4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-7-methoxy- 1H -benzo [ d ]Imidazol-1-yl)but-2-en-1-yl)carbamate (compound 29, 33 mg, 0.0645 mmol) was dissolved in MeOH (0.22 mL) followed by 10% wet Pd/C (6.0 mg, 0.00645 mmol) and stirred under a hydrogen balloon at room temperature for 3 hours. It was then filtered through Celite®, washing with 20 mL of MeOH. The filtrate containing the product was concentrated in vacuo as a purple liquid as tert -butyl (4-(5-carbamoyl-2-(1-ethyl-3-methyl-1 H -pyrazole-5-carboxamido) )-7-methoxy- 1H -benzo[ d ]imidazol-1-yl)butyl)carbamate (31 mg, 0.059 mmol, 92% yield) was obtained.

¹ H NMR (400 MHz, methanol- d ₄ ) δ 7.57 (s, 1H), 7.35 (s, 1H), 6.64 (s, 1H), 4.65 (q, J = 7.1 Hz, 2H), 4.35 (t, J = 7.2 Hz, 2H), 3.99 (s, 3H), 3.06 (t, J = 7.0 Hz, 2H), 2.22 (s, 3H), 1.79 (t, J = 7.7 Hz, 2H), 1.51 (t, J = 7.7 Hz, 2H), 1.43-1.35 (m, 12H).

Synthesis of the compounds (dimers) in Table 5 below

Step 1: 4,4'-(butane-1,4-diylbis(azanediyl))bis(3-nitrobenzamide)

Butane-1,4-diamine (0.800 g, 9.00 mmol) and 4-chloro-3-nitrobenzamide (3.00 g, 15.00 mmol) were dissolved in DMSO (15 mL) followed by K ₂ CO ₃ (3.11 g, 22.5) mmol) and stirred at room temperature for 16 hours. After stirring for 16 hours, the solid was filtered with cold water, dried under vacuum and 4,4'-(butane-1,4-diylbis(azanediyl))bis(3-nitrobenzamide) as a yellow solid (4.21 g) , 10.1 mmol, 67% yield).

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 8.65 (d, J = 2.1 Hz, 1H), 8.43 (t, J = 5.8 Hz, 1H), 8.00 (dd, J = 9.7, 2.7 Hz, 2H) , 7.30 (s, 1H), 7.13 (d, J = 9.2 Hz, 1H), 1.74 (d, J = 6.2 Hz, 2H).

Step 2: 4,4'-(Butane-1,4-diylbis(azanediyl))bis(3-aminobenzamide)

4,4′-(butane-1,4-diylbis(azanediyl))bis(3-nitrobenzamide) (1.00 g, 2.40 mmol) was dissolved in MeOH (40 mL) followed by 10% wet Pd/ C (26.0 mg, 0.240 mmol) was added and stirred for 18 hours at room temperature under a hydrogen balloon. After concentration in vacuo to remove the solvent, 4,4'-(butane-1,4-diylbis(azanediyl))bis(3-aminobenzamide) (0.775) as a green solid containing wet Pd/C g, 2.17 mmol, 91% yield).

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 7.41 (s, 1H), 7.13-7.07 (m, 2H), 6.71 (s, 1H), 6.39 (d, J = 8.1 Hz, 1H), 4.90 ( t, J = 5.2 Hz, 1H), 4.59 (s, 2H), 3.13 (d, J = 5.2 Hz, 2H), 1.73 (s, 2H).

Step 3: 1,1′-(butane-1,4-diyl)bis(2-amino-1 H -benzo[ d ]imidazole-5-carboxamide)

4,4'-(butane-1,4-diylbis(azanediyl))bis(3-aminobenzamide) (775 mg, 2.17 mmol) was dissolved in MeOH (5 mL) followed by cyanogen bromide (575 mg, 5.43 mmol) and stirred at 60 °C for 3 h. The residue containing the product was then removed in vacuo with MeOH removed as 1,1′-(butane-1,4-diyl)bis(2-amino) as a green solid containing cyanogen bromide and wet Pd/C as a tan solid. -1H -benzo[ d ]imidazole-5-carboxamide) was obtained and used in the next reaction without further purification.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.80 (s, 1H), 8.76 (s, 2H), 8.06 (s, 1H), 7.88-7.80 (m, 2H), 7.63 (d, J = 8.4) Hz, 1H), 7.43 (s, 1H), 4.19 (s, 2H), 3.16 (s, 2H), 1.79 (s, 2H).

Step 4: 1,1'-(butane-1,4-diyl)bis(2-acid substituted groupamido-1 H -benzo[ d ]imidazole-5-carboxamide)

1,1′-(butane-1,4-diyl)bis(2-amino- 1H -benzo[ d ]imidazole-5-carboxamide) (200 mg, 0.492 mmol), substituted carboxylic acid (2.2 eq, 1.08 mmol), HATU (935 mg, 2.46 mmol), and triethylamine (0.823 mL, 5.91 mmol) were dissolved in NMP (0.82 mL) and heated at 80 °C for 48 hours. Water (30 mL) was added to the reaction mixture and stirred for 10 minutes. The resulting solid was filtered and washed with CH ₂ Cl ₂ . The filtered solid was dry-loaded and purified on silica gel with 0-10% MeOH in CH ₂ Cl ₂ to obtain the target compound.

The NMR measurement results of compounds 31 to 36 are summarized in Table 2 below.

Compound
No. IUPAC name ¹ H NMR 31 1,1'-(butane-1,4-diyl)bis(2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole -5-carboxamide) ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.82 (s, 2H), 7.97 (s, 4H), 7.76 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H) , 7.34 (s, 2H), 6.59 (s, 2H), 4.56 (q, J = 7.2 Hz, 4H), 4.27 (s, 4H), 2.09 (s, 6H), 1.87 (s, 4H), 1.30 ( t, J = 7.1 Hz, 6H). 32 1,1'-(butane-1,4-diyl)bis(2-(1-allyl-5-methyl- 1H -pyrazole-3-carboxamido) -1H -benzo[ d ]imidazole- 5-carboxamide) ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.66 (s, 2H), 7.97 (d, J = 17.9 Hz, 4H), 7.73 (d, J = 8.3 Hz, 2H), 7.51 (s, 2H) , 7.29 (s, 2H), 6.58 (s, 2H), 5.93 (ddt, J = 15.7, 10.2, 5.1 Hz, 2H), 5.12 (d, J = 10.3 Hz, 2H), 4.87 (d, J = 17.2) Hz, 2H), 4.74 (s, 4H), 4.29 (s, 4H), 2.21 (s, 6H), 1.80 (s, 4H). 33 1,1'-(butane-1,4-diyl)bis(2-(benzofuran-2-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide) ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.83 (s, 2H), 7.99 (d, J = 16.2 Hz, 4H), 7.81-7.72 (m, 2H), 7.60 (dd, J = 17.3, 8.6) Hz, 8H), 7.41-7.18 (m, 6H), 4.47 (s, 4H), 2.04-1.80 (m, 4H). 34 1,1′-(butane-1,4-diyl)bis(2-(1-methyl-3-(trifluoromethyl) -1H -pyrazole-4-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide) ¹ H NMR (500 MHz, DMSO- d ₆ ) δ 12.78 (s, 2H), 8.36 (s, 2H), 7.94 (d, J = 11.8 Hz, 4H), 7.72 (d, J = 8.3 Hz, 2H) , 7.49 (d, J = 8.4 Hz, 2H), 7.30 (s, 2H), 4.28 (s, 4H), 3.87 (s, 6H), 1.85 (s, 4H). 35 Dimethyl 1,1'-(butane-1,4-diyl)bis(2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole -5-carboxylate) ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.83 (s, 2H), 8.05 (d, J = 1.6 Hz, 2H), 7.80 (dd, J = 8.4, 1.6 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 6.57 (s, 2H), 4.55 (q, J = 7.1 Hz, 4H), 4.25 (s, 4H), 3.86 (s, 6H), 2.09 (s, 6H), 1.87 ( s, 4H), 1.30 (t, J = 7.1 Hz, 6H). 36 1,1'-(butane-1,4-diyl)bis(2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole -5-carboxylic acid) ¹ H NMR (300 MHz, DMSO- d ₆ ) δ 12.83 (s, 2H), 8.07 (d, J = 1.5 Hz, 2H), 7.82 (dd, J = 8.4, 1.7 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 6.59 (s, 2H), 4.56 (q, J = 6.9 Hz, 4H), 4.27 (s, 4H), 3.17 (s, 2H), 2.09 (s, 6H), 1.88 ( s, 4H), 1.30 (t, J = 7.1 Hz, 6H).

Synthesis of compounds 37, 38 and 39 of Table 6 below

Intermediate 1

1-ethyl-3-methyl-1 H -pyrazole-5-carbonyl isothiocyanate

1-Ethyl-3-methyl-1 H -pyrazole-5-carboxylic acid (500 mg, 3.24 mmol) was dissolved in CH ₂ Cl ₂ (5.4 mL). To this mixture was added DMF (0.2 mL) followed by slow addition of oxalyl chloride (0.417 mL, 4.86 mmol). After stirring at room temperature for 1 h, the solvent was removed in vacuo and co-evaporated with CH ₂ Cl ₂ twice (30 mL each). Assuming 100% yield, 1-ethyl-3-methyl- 1H -pyrazole-5-carbonyl chloride (559 mg, 3.24 mmol, 100% yield) was used directly in the subsequent reaction as such.

KSCN (472 mg, 4.86 mmol) was dissolved in acetone (2 mL) and then cooled to 0 °C. After stirring at 0° C. for 5 minutes, 1-ethyl-3-methyl-1 H -pyrazole-5-carbonyl chloride (559 mg, 3.24 mmol) was dissolved in acetone (2 mL) and added as a solution. After addition, the reaction was stirred at 0 °C for 20 min. Then haxanes (50 mL) were added to the reaction mixture and concentrated to one third of the volume in vacuo. The process of hexanes addition and concentration was repeated twice (each of 50 mL of hexanes). After the last concentration, hexanes (50 mL) was added, the solid was filtered with hexanes (100 mL), the filtered clear pale yellow filtrate was concentrated, and the filtrate was chromatographed (30 g silica column; 0-20% EtOAc / hexanes) eluted with) to give 1-ethyl-3-methyl-1 H -pyrazole-5-carbonyl isothiocyanate (297 mg, 1.52 mmol, 47% yield) as a clear colorless liquid.

¹ H NMR (300 MHz, chloroform- d ) δ 6.72 (d, J = 0.7 Hz, 1H), 4.49 (q, J = 7.2 Hz, 2H), 2.29 (d, J = 0.6 Hz, 3H), 1.40 ( t, J = 7.2 Hz, 3H).

The acylisothiocyanate product degrades over time and is therefore immediately used directly in the subsequent reaction.

Intermediate 2

Step 1: 4-Chloro-3-methoxy-5-nitrobenzamide

Methyl 4-chloro-3-methoxy-5-nitrobenzoate (10.0 g, 40.8 mmol) was dissolved in NH ₄ OH (80 mL, 2009 mmol) and stirred at room temperature for 16 hours. After stirring, the solid was filtered with cold water and dried under vacuum to give 4-chloro-3-methoxy5-nitrobenzamide (7.79 g, 33.8 mmol, 83% yield) as a tan solid.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 8.29 (s, 1H), 8.05 (d, J = 1.8 Hz, 1H), 7.88 (d, J = 1.9 Hz, 1H), 7.79 (s, 1H) , 4.02 (s, 3H).

Step 2: tert -Butyl ( E )-(4-((4-carbamoyl-2-methoxy-6-nitrophenyl)amino)but-2-en-1-yl)carbamate

Dissolve 4-chloro-3-methoxy-5-nitrobenzamide (1.41 g, 6.13 mmol) in EtOH (6.13 mL) in a sealed tube ( E ) -tert -butyl (4-aminobut-2-ene) -1-yl)carbamate (1.37 g, 7.36 mmol) and DIPEA (2.38 mL, 18.4 mmol) were added. The reaction was stirred at 130 &lt;0&gt;C for 4 days and cooled to room temperature. The resulting orange solid was filtered with EtOH and dried to form an orange solid tert -butyl ( E )-(4-((4-carbamoyl-2-methoxy-6-nitrophenyl)amino)but-2-ene Obtained -1-yl)carbamate (1.97 g, 5.18 mmol, 84% yield).

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 8.18 (d, J = 1.9 Hz, 1H), 8.01 (s, 1H), 7.74 (t, J = 6.2 Hz, 1H), 7.55 (d, J = 1.9 Hz, 1H), 7.31 (s, 1H), 6.92 (d, J = 7.0 Hz, 1H), 5.53 (q, J = 3.1, 2.4 Hz, 2H), 4.09 (d, J = 5.6 Hz, 2H) , 3.87 (s, 3H), 3.46 (d, J = 4.2 Hz, 2H), 1.35 (s, 9H).

Step 3: ( E )-4-((4-Aminobut-2-en-1-yl)amino)-3-methoxy-5-nitrobenzamide, hydrochloride

tert -Butyl ( E )-(4-((4-carbamoyl-2-methoxy-6-nitrophenyl)amino)but-2-en-1-yl)carbamate (2.00 g, 5.26 mmol) was dissolved in MeOH (5 mL) and then 4 M HCl in dioxane (16 mL, 44.7 mmol) was added slowly. The reaction mixture was stirred at room temperature for 1 hour and 30 minutes, then the resulting solid was filtered, washed three times with Et ₂ O (100 ml X3), and dried under vacuum ( E )-4-((4-amino). But-2-en-1-yl)amino)-3-methoxy-5-nitrobenzamide, hydrochloride, was obtained and used in the next reaction without further purification.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 8.21 (d, J = 1.9 Hz, 1H), 8.05 (s, 1H), 7.90 (s, 3H), 7.58 (d, J = 2.0 Hz, 1H) , 7.35 (s, 1H), 5.93-5.81 (m, 1H), 5.62 (dd, J = 14.6, 7.3 Hz, 1H), 4.18 (d, J = 5.7 Hz, 2H), 3.89 (s, 3H), 3.44-3.35 (m, 2H).

Intermediate 3

Step 1: 4-Chloro-3-hydroxy-5-nitrobenzamide

4-Chloro-3-methoxy-5-nitrobenzamide (2.00 g, 8.68 mmol) was dissolved in CH ₂ Cl ₂ (14.5 mL) followed by BBr ₃ (36.4 mL, 1 M in CH ₂ Cl ₂ ) was added slowly and stirred overnight at room temperature under nitrogen. The reaction was poured with cold water (300 mL) and stirred vigorously for 30 minutes. The resulting solid was filtered and dried to give 4-chloro-3-hydroxy-5-nitrobenzamide (1.35 g, 6.23 mmol, 72% yield).

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 11.52 (s, 1H), 8.17 (s, 1H), 7.92 (d, J = 1.9 Hz, 1H), 7.74-7.61 (m, 2H).

Step 2: tert -Butyl (3-(5-carbamoyl-2-chloro-3-nitrophenoxy)propyl)carbamate

4-Chloro-3-hydroxy-5-nitrobenzamide (1.35 g, 6.23 mmol) was dissolved in DMF (7.79 mL) followed by tert -butyl (3-chloropropyl)carbamate (1.35 g, 6.23 mmol) and K ₂ CO ₃ (1.12 g, 8.10 mmol) were added. The reaction mixture was stirred at 70 °C for 48 h. After 48 hours, silica gel was added, DMF was removed in vacuo, and the residue was purified by dry loading silica column chromatography (CH ₂ Cl ₂ :MeOH=9:1) to form a yellow solid tert -butyl (3-( Obtained 5-carbamoyl-2-chloro-3-nitrophenoxy)propyl)carbamate (1.61 g, 4.31 mmol, 69% yield).

¹ H NMR (500 MHz, DMSO- d ₆ ) δ 8.28 (s, 1H), 8.04 (d, J = 1.7 Hz, 1H), 7.86 (d, J = 1.9 Hz, 1H), 7.77 (s, 1H) , 6.91 (t, J = 5.7 Hz, 1H), 4.23 (t, J = 6.1 Hz, 2H), 3.13 (q, J = 6.7 Hz, 2H), 1.90 (p, J = 6.4 Hz, 2H), 1.36 (s, 9H).

Synthesis of compounds 37, 38, and 39

Step 1: tert -Butyl ( E )-(3-(5-carbamoyl-2-((4-((4-carbamoyl-2-methoxy-6-nitrophenyl)amino)but-2-en-1-yl)amino) -3-nitrophenoxy)propyl)carbamate

( E )-4-((4-aminobut-2-en-1-yl)amino)-3-methoxy-5-nitrobenzamide, hydrochloride (0.930 g, 3.32 mmol) was mixed with i -PrOH (5.53 mL), DIPEA (2.31 mL, 13.3 mmol) was added, and tert -butyl (3-(5-carbamoyl-2-chloro-3-nitrophenoxy)propyl)carbamate (1.12 g) , 2.99 mmol) was added. The reaction mixture was stirred at 130 °C for 72 hours. The reaction mixture was cooled to room temperature, silica gel was added, the solvent was removed under vacuum, and the residue was purified by dry loading silica column chromatography (CH ₂ Cl ₂ :MeOH=9:1) to form a red solid tert -butyl ( E )-(3-(5-carbamoyl-2-((4-((4-carbamoyl-2-methoxy-6-nitrophenyl)amino)but-2-en-1-yl)amino )-3-nitrophenoxy)propyl)carbamate (500 mg, 0.810 mmol, 25% yield) was obtained.

¹ H NMR (300 MHz, DMSO- d ₆ ) δ 8.15 (dd, J = 1.9, 0.9 Hz, 2H), 8.01 (s, 2H), 7.70 (td, J = 6.2, 3.0 Hz, 2H), 7.51 ( t, J = 2.2 Hz, 2H), 7.31 (s, 2H), 6.91 (t, J = 5.3 Hz, 1H), 5.63 (q, J = 1.5 Hz, 2H), 4.18-3.97 (m, 6H), 3.82 (s, 3H), 3.08 (q, J = 6.4 Hz, 2H), 1.86 (q, J = 6.4 Hz, 2H), 1.35 (s, 9H).

Step 2: tert -Butyl ( E )-(3-(3-amino-2-((4-((2-amino-4-carbamoyl-6-methoxyphenyl)amino)but-2-en-1-yl)amino)-5 -carbamoylphenoxy)propyl)carbamate

tert -Butyl ( E )-(3-(5-carbamoyl-2-((4-((4-carbamoyl-2-methoxy-6-nitrophenyl)amino)but-2-ene-1 -yl)amino)-3-nitrophenoxy)propyl)carbamate (500 mg, 0.810 mmol) was dissolved in MeOH (10.2 mL) and Na ₂ S ₂ O ₄ (1.98 g) dissolved in water (5 mL) , 11.3 mmol) and stirred at room temperature for 25 min. Then solid sodium hydrogen carbonate (2.93 g, 34.8 mmol) was added and stirred at room temperature for a further 10 minutes. The solid was then filtered and filtered through Celite®, washing with MeOH. The filtrate containing the desired product was concentrated in vacuo and the residue was purified by dry loading silica column chromatography 0-10% in CH ₂ Cl ₂ (10:1 MeOH:aq.NH ₄ OH (28%)) tert -Butyl ( E )-(3-(3-amino-2-((4-((2-amino-4-carbamoyl-6-methoxyphenyl)amino)but-2-ene- as yellow solid 1-yl)amino)-5-carbamoylphenoxy)propyl)carbamate (297 mg, 0.533 mmol, 66% yield) was obtained.

¹ H NMR (500 MHz, methanol- d ₄ ) δ 6.93 (d, J = 1.8 Hz, 2H), 6.87 (dd, J = 5.5, 1.9 Hz, 2H), 5.72 (td, J = 4.8, 3.6 Hz, 2H), 3.99 (t, J = 6.1 Hz, 2H), 3.79 (s, 3H), 3.57 (dd, J = 13.2, 4.4 Hz, 4H), 3.23 (t, J = 6.7 Hz, 2H), 1.93 ( p, J = 6.4 Hz, 2H), 1.41 (s, 9H).

Step 3: tert -Butyl ( E )-(3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1) H -Pyrazole-5-carboxamido)-7-methoxy-1 H -benzo[ d ]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H -Pyrazole-5-carboxamido)-1 H -benzo[ d ]imidazol-7-yl)oxy)propyl)carbamate (Compound 37)

tert -Butyl ( E )-(3-(3-amino-2-((4-((2-amino-4-carbamoyl-6-methoxyphenyl)amino)but-2-ene- at 0° C.) 1-yl)amino)-5-carbamoylphenoxy)propyl)carbamate (100 mg, 0.179 mmol) was dissolved in DMF (1.79 mL) followed by 1-ethyl-3-methyl- 1H -pyrazole 5-carbonyl isothiocyanate (70 mg, 0.358 mmol) was added slowly and stirred for 35 min. Then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (83 mg, 0.537 mmol) was added, followed by triethyl amine (0.150 mL, 1.07 mmol), followed by stirring at room temperature overnight (~ 16 hours). The reaction was added 3:1 water:saturated aq.NH ₄ Cl solution (40 mL) and extracted with 3:1 chloroform:ethanol (4 X 40 mL). The organic layer was dried over MgSO ₄ and concentrated. The resulting residue was purified on silica gel with 2 - 20% (10:1 MeOH:aq.NH ₄ OH) in CH ₂ Cl ₂ on silica gel tert -butyl ( E )-(3-((5-car) as an off-white solid. Bamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-7-methoxy- 1H -benzo[ d ]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ] Obtained imidazol-7-yl)oxy)propyl)carbamate (35.3 mg, 0.04 mmol, 22% yield).

¹ H NMR (500 MHz, DMSO- d ₆ ) δ 12.83 (s, 2H), 7.97 (s, 2H), 7.63 (d, J = 4.2 Hz, 2H), 7.41-7.33 (m, 2H), 7.30 ( d, J = 6.1 Hz, 2H), 6.88 (t, J = 5.8 Hz, 1H), 6.50 (s, 2H), 5.82 (qd, J = 17.6, 16.8, 7.8 Hz, 2H), 4.98-4.84 (m) , 4H), 4.57-4.43 (m, 4H), 3.98 (t, J = 6.0 Hz, 2H), 3.72 (s, 3H), 3.05-2.95 (m, 2H), 2.09 (s, 6H), 1.74- 1.62 (m, 2H), 1.31 (s, 9H), 1.25 (t, J = 7.3 Hz, 6H).

Step 4: ( E )-7-(3-aminopropoxy)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1) H -Pyrazole-5-carboxamido)-7-methoxy-1 H -benzo[ d ]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H -Pyrazole-5-carboxamido)-1 H -benzo[ d ]Imidazole-5-carboxamide (Compound 38)

tert -Butyl ( E )-(3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-car) copymido)-7-methoxy- 1H -benzo[ d ]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl- 1H -pyrazole -5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl)carbamate (Compound 37, 12.0mg , 0.014mmol) was dissolved in MeOH (0.006mL) 4 M HCl in oxane (0.50 mL) was added slowly. After the mixture was stirred at room temperature for 1 hour, the resulting solid was filtered, washed 3 times with Et ₂ O, and dried under vacuum to form ( E )-7-(3-aminopropoxy)- as a white solid. 1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-7-methoxy- 1H -benzo[ d ]imidazole -1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido) -1H -benzo[ d ]imidazole- 5-carboxamide was obtained (9.8 mg, 0.013 mmol, 90%). R _f 0.16 (CH ₂ Cl ₂ :MeOH:aq.NH ₄ OH(28%)=8:2:0.2).

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.13 (s, 3H), 8.04 (s, 2H), 7.65 (d, J = 7.2 Hz, 2H), 7.41 (s, 1H), 7.36 (d, J = 9.6 Hz, 2H), 6.51 (d, J = 12.1 Hz, 2H), 5.86-5.71 (m, 2H), 4.93 (dd, J = 12.3, 5.0 Hz, 4H), 4.49 (t, J = 8.0) Hz, 4H), 4.13 (d, J = 6.3 Hz, 2H), 3.74 (s, 3H), 2.88 (q, J = 6.4 Hz, 2H), 2.10 (d, J = 6.3 Hz, 6H), 1.94 ( t, J = 6.8 Hz, 2H), 1.25 (dt, J = 11.6, 5.6 Hz, 6H).

Step 5: ( E )-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1) H -Pyrazole-5-carboxamido)-7-(3-(1-ethyl-3-methyl-1) H -Pyrazole-5-carboxamido)propoxy)-1 H -benzo[ d ]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1 H -Pyrazole-5-carboxamido)-7-methoxy-1 H -benzo[ d ]Imidazole-5-carboxamide (Compound 39)

1-ethyl-3-methyl- 1H -pyrazole-5-carboxylic acid (8.0mg, 0.051mmol), HATU (26mg, 0.051mmol), HOBt (5mg, 0.034mmol) and TEA (0.047 mL, 0.34mmol) It was dissolved in DMF (0.85 mL). After the reaction was stirred at room temperature for 5 min, ( E )-7-(3-aminopropoxy)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl- 1H- ) Pyrazole-5-carboxamido)-7-methoxy- 1H -benzo[ d ]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl) -1H- Pyrazole -5-carboxamido) -1H -benzo[ d ]imidazole-5-carboxamide (compound 38, 27.0 mg, 0.034 mmol) was added. After the reaction was stirred at room temperature for 16 h, the reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over MgSO ₄ . The residue was purified by silica gel flash column chromatography (CH ₂ Cl ₂ :MeOH:aq.NH ₄ OH(28%)=90:10:1) as ( E )-1-(4-(5-) as a white solid. Carbamoyl-2-(1-ethyl-3-methyl- 1H -pyrazole-5-carboxamido)-7-(3-(1-ethyl-3-methyl- 1H -pyrazole-5) -carboxamido )propoxy)-1H-benzo[ d ]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl- 1H -pyra Obtained sol-5-carboxamido)-7-methoxy-1 H -benzo[ d ]imidazole-5-carboxamide (15.6 mg, 0.017 mmol, 50%). R _f 0.57 (CH ₂ Cl ₂ :MeOH:aq. NH ₄ OH(28%)=80:20:2).

¹ H NMR (500 MHz, DMSO- d ₆ ) δ 12.82 (d, J = 11.8 Hz, 2H), 8.38 (t, J = 5.8 Hz, 1H), 7.97 (s, 2H), 7.63 (s, 2H) , 7.36 (d, J = 7.1 Hz, 2H), 7.29 (d, J = 3.8 Hz, 2H), 6.50 (d, J = 9.3 Hz, 3H), 5.90-5.78 (m, 2H), 4.92 (dd, J = 25.0, 4.9 Hz, 4H), 4.50 (dt, J = 14.4, 7.2 Hz, 4H), 4.30 (q, J = 7.1 Hz, 2H), 4.01 (t, J = 6.0 Hz, 2H), 3.71 ( s, 3H), 3.27 (q, J = 6.5 Hz, 2H), 2.12 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 1.80 (p, J = 6.4 Hz, 2H), 1.25 (q, J = 7.5 Hz, 6H), 1.18 (t, J = 7.1 Hz, 3H).

Cell culture and related reagents

Human monocyte cell lines, THP1-dual cell and THP1-dual STING KO cell, were purchased from Invivogen and used. The cell line was cultured in a humidified incubator at 37° C. in CO ₂ under conditions of 1X RPMI 1640 2.05 Mm L-Glutamine (HyClone), 10% Fetal Bovine Serum (FBS, HyClone) and 1% penicillin (Corning).

ISRE Reporter Assay

In order to monitor the IRSE-mediated immune response, the THP1 cell line was dispensed into a 384 well plate (Grenier) and each compound was treated. After 24 hours, 10 μl of the supernatant was taken and analyzed according to the manufacturing protocol of the QUANTI-Luc assay (Invivogen), and the reporter signal was measured through this. Relative Luminescent (RU) was measured with a microplate reader (TECAN). The measurement results were analyzed by normalization based on DMSO.

Cell viability test

Cell viability test was performed using residual cells after Luciferase assay. 24 hours compound treatment. Thereafter, cell viability was analyzed using CellTiter 96 Aqueous One Solution Proliferation Assay (MTS, Promega) according to the manufacturing protocol. The absorbance signal was measured using a microplate reader (TECAN), and the analysis results were normalized based on DMSO.

Western Blot

For Western blotting analysis, cells were washed with PBS and lysed using RIPA buffer (Bioseasang). Thereafter, centrifugation was performed at 20000g at 4°C for 20 minutes. Then, the cell lysate was separated by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane through a Trans-Blot Turbo Transfer System (Bio-Rad). After blocking the membrane in 5% BSA dissolved in TBST for 1 hour at room temperature, it was treated with a primary antibody solution overnight. All antibody solutions were diluted with TBST to the following concentrations: anti-STING (Cell Signaling/no.13647, 1:1000), anti-pSTING (Cell Signaling/no. 19781, 1:1000), anti-TBK1 (Cell Signaling/no.3504, 1:1000), anti-pTBK1 (Cell Signaling/no.5483, 1:1000), anti-IRF3 (Cell Signaling/no.11904, 1:1000), anti-pIRF3 (Cell Signaling/no.4947, 1:1000), anti-STAT1 (Cell Signaling/no.9172, 1:1000), anti-pSTAT (Cell Signaling/no.9167, 1:1000), anti-Actin (Cell Signaling/ 4970, 1:1000). After washing with TBST, a Western blot signal was detected by an anti-Rabbit-HRP-based secondary antibody (cell signal/7074, 1:3000). β-Actin was used as a control for the total protein amount. Luminescence signals were detected using ECL reagent, and immunoreactive bands were captured with ChemiDoc imaging system (Bio-Rad).

ELISA (Enzyme-Linked Immunosorbent Assay) / Enzyme-Linked Immunosorbent Assay

IFN-α and IP-10 cytokine secretion was quantified using an ELISA kit from R&D Systems. THP1 cells were seeded in 96-well plates, and cultured for 24 hours after compound treatment. 100 μl of the cell culture supernatant was extracted and then analyzed using the preparation protocol.

Gene expression analysis by rt-PCR

For cell pellets, RNA was isolated according to the manufacturing protocol using NucleoSpin RNA plus (MN). Gene expression was assessed by real-time PCR (CFX96 Real-Time PCR detection system, Bio-Rad) using IQ SYBR green Supermix (Bio-Rad). mRNA levels were normalized by GAPDH and calculated by the comparative Ct method.

The following primer sequences were used:

IFNB (F) 5'-AGGATTCTGCATTACCTGAA-3' (R) 5'-GGCTAGGAGATCTTCAGTTT-3',

CXCL10 (F) 5'-CTAGAACTGTACCGCTGTACC-3' (R) 5'-TTGATGGCCTTCGATTCTGG-3',

IFIT3 (F) 5'-AACTACGCCTGGGTCTACTATCACT-3' (R) 5'-ACACCTTCGCCCTTTCATTTC-3',

IFIM (F) 5'-GGCTTCATAGCATTCGCCTACTC-3' (R) 5'-AGATGTTCAGGCACTTGGCGGT-3'

IRF7 (F) 5'-GCTGGACGTGACCATCATGTA-3' (R) 5'-GGGCCGTATAGGAACGTGC-3',

OAS1 (F) 5'-AGGAAAGGTGCTTCCGAGGTAG-3' (R) 5'-GGACTGAGGAAGACAACCAGGT-3'

ISG15 (F) 5'-CGCAGATCACCCAGAAGATCG-3' (R) 5'-TTCGTCGCATTTGTCCACCA-3'

STING binding assay

STING WT binding assay (CisBio/ NO. 64BDSTGPEG) was used to confirm the competitive reaction between the ligand and the compound binding to STING. It was then analyzed using the manufacturing protocol.

CYP (Cytochrome P450) inhibition assay

The test drug was added to microsomes diluted with potassium phosphate buffer and incubated at 37°C for 5 minutes, then 5 kinds of substrate materials were added to NADPH and reacted at 37°C for 20 minutes. (test compound final conc.: 10 μM, microsome final conc.: 0.2 mg/mL) To terminate the reaction, cold acetonitrile containing internal standard was added, and then protein was treated. After centrifugation (4000 rpm, 4 °C, 15 min), the supernatant was analyzed by LC-MS/MS (analyzer: Mass sepctrometry (AB Sciex Qtrap 4000) with HPLC (Agilent 1260)).

hERG patch clamp assay

Thaw hERG-HEK293 cells frozen in liquid nitrogen and culture them using DMEM/F-12 medium (10% FBS, 7.5 mg/mLblasticidin S HCl, 50 mg/mL hygromycin B), and then incubate the cultured cells with trypsine- After removing with EDTA solution, it was aliquoted to contain 2x10 ⁵ cells in a 60 mm cell culture dish and cultured for 2 days in an incubator at 37 °C under 5% CO ₂ condition. After loading the cells and sealchip prepared in PatchXpress 7000A, cell settling and resistance stabilization, Voltage-clamp and data were obtained using MultiClamp 700A. For measurement of hERG channel activity and data analysis, tail currentsms DataXpress 2.0.4.5 (Axon Instruments) and Clampfit 9.2 (Axon Instruments) were used.

Plasma Stability Test

After exposing the drug to plasma for a certain period of time, the amount of the drug remaining was quantified by LC-MS/MS to measure the stability of the drug in plasma, and enalapril and procaine, which have reported stability in rat and human plasma, were used as reference materials. did. After spiking a 10 mM DMSO drug stock solution into plasma so that the drug concentration was 5 μM, the plasma sample was reacted in a shaking incubator (37° C., 4 h). To terminate the reaction, cold acetonitrile containing internal standard was added, protein treatment, and centrifugation (4000 rpm, 4℃, 15 min), the supernatant was LC-MS/MS (analytical instrument: Mass sepctrometry (Agilent 6460)) with HPLC (Agilent 1260)).

Mouse Pharmacokinetic Experiments

In the animal experiment, the drug was administered to 7-8 week old mice that had undergone the acclimatization period, and blood was collected at a set time. For sample pretreatment, blood was centrifuged to separate plasma, 9 times the volume of cold acetonitrile containing internal standard was added, and then protein was treated. After centrifugation (13000 rpm, 4℃, 10 min), the supernatant was LC-MS /MS (analysis instrument: Mass sepctrometry (Agilent 6460) with HPLC (Agilent 1260)) was analyzed. PK parameter analysis was calculated using the Phoenix WinNonlin (Pharsight ver 6.4, USA) Non-compartmental analysis model.

Structure-Activity Relationship (SAR) Study Results

To evaluate the biological activity of the synthesized compounds, luciferase gene expression was measured using THP-1 (human monocytes) engineered as an interferon sensitive immune response element (ISRE) reporter. To validate the STING-dependent immune response, the activity of the compounds was tested in both wild-type (WT) and STING knockout (KO) cells. In the reporter assay for immune responses to individual compounds, activity was first evaluated by comparing normalized fold change values from the experimental results at a single concentration (50 μM). Thereafter, EC ₅₀ values were obtained through dose-response analysis for compounds exhibiting activity, and the results are shown in Tables 3, 4, and 5 below.

In Table 3-5, the luciferase value ^a is expressed as the mean±standard deviation of the relative luciferase units. THP-1 WT and STING KO cells were treated with individual compounds (50 μM). ^b EC ₅₀ values were determined using a dose-response curve of THP-1 WT cells. N/A means not applicable. Compounds marked with N/A were not subject to calculation of EC ₅₀ values due to their low fold-change values of up to 50 μM.

In Table 3, STING action activity and cell viability were confirmed by changing the R ¹ group of Compound 1 while maintaining R ² as a (S)-2-hydroxy-phenethyl group, and cGAMP efficacy under the same experimental conditions was compared ( compounds 2-7 and cGAMP). All tested compounds showed no cytotoxicity as a result of cell viability at the same concentration. Various heterocycles were investigated, including trifluoromethyl-substituted pyrazoles (Compounds 2 and 3), furan (Compound 4), benzofuran (Compound 5) and indazole (Compounds 6 and 7), but not distinct in STING agonism. showed no effect.

When the R ² substituent of compound 1 was replaced with a tert-butyl butylcarbamate group in the (S)-2-hydroxy-phenethyl group, the resulting compound 8 showed improved efficacy in the ISRE reporter assay (compound 1 and 8). Dose dependent efficacy analysis showed that the activity of compound 8 (EC ₅₀ =6.5 μM) was approximately 15-fold and 5-fold higher than that of compound 1 (EC ₅₀ =102 μM) and cGAMP (EC ₅₀ =30.1 μM), respectively.

In addition, the activity and cell viability of the compound were investigated by changing the R ¹ group in the presence of tert-butyl butylcarbamate, which is more effective as the R ² group. Synthesis of derivatives comprising trifluoromethyl substituted pyrazoles (compounds 9 and 10), 1-allyl-5-methyl-1H-pyrazole (compound 11), furan (compound 12) and benzofuran (compound 13) However, no significant activity results were observed. Examination of other substituents at the N1 position of pyrazole showed that methyl, isopropyl and tert-butyl groups had a detrimental effect on the STING reaction, while the ethyl group provided the most potent activity (compounds 8 and 14-16). ).

In Table 4, an optimization study of compound 8 was performed by modifying the benzimidazole subcomponent through additional structure-activity relationship (SAR) studies. For the alkyl chain length of the R ² group, butyl carbamate (compound 8) was more potent and less cytotoxic than the corresponding propyl carbamate (compound 17) and pentyl carbamate (compound 18). When the amino substituent of R ² was changed, compound 19 showed no activity when the tert-butyl carbamate group was replaced with a hydrogen atom. Higher activity was confirmed in the aromatic amide compound such as compound 22 than in compounds 20 and 21, and in the case of compounds 23 and 24 having a heteroaromatic substituent as an amide substituent, reporter assay activity was not observed. On the other hand, when the nitrogen atom of pyrazole was replaced with an alkyl group, the activity was significantly improved. Compound 26 substituted with N-ethyl-3-methyl pyrazole showed significantly improved activity than compound 25 substituted with N-methyl-3-methyl pyrazole, and the chain length of compound 26 compared to compounds 27 and 28 showed better STING agonist activity and cell survival results. By confirming that the methoxy group introduced at the R ³ position also affects the efficacy, compound 29 showed submicromolar EC ₅₀ , and compound 30 compared to 29 restored luciferase activity and cell viability. The number of rotatable bonds was shown to have an effect on activity enhancement by reducing

In Table 5, the activity of the dimer compound expected to be better able to bind to the dimer structure of STING was investigated. After observing the increased efficacy of compound 31, the efficacy of compounds 32-34 in which the R ¹ group was substituted with another type of heterocyclic carboxy group was investigated. Compounds 32 and 33 did not observe improvement in enhancement compared to the corresponding monomeric compounds, and compound 34 showed a slight improvement in activity compared to the corresponding monomeric compounds. Compounds 35 and 36, in which the amide functional group at the R ⁴ position was changed to an ester and a carboxylic acid, showed no efficacy at all.

Based on the information summarized in Table 3-5, the compounds of Formula 1 below were devised, and the results of evaluation of their effects are summarized in Table 6 and FIG. 1 .

,

, 또는

임. In Formula 1, R ⁵ is

,

, or

lim.

In Table 6, luciferase values ^a are expressed as mean±standard deviation of relative luciferase units. THP-1 WT and STING KO cells were treated with individual compounds (1 μM).

The three synthesized compounds 37-39 reached saturation levels in the reporter assay, whereas compound diABZI-3 showed a lower reporter response. As a result of performing the dose-response measurement at a low concentration, the EC ₅₀ of compound 37 was 0.36 nM and the EC ₅₀ of compound 38 was 1.62 nM, showing very good efficacy, and in the case of compound 39, the EC ₅₀ of 4.0 pM was shown to mediate STING. It was confirmed that the activity increased dramatically. In addition, when the compound of the present invention was treated in the same concentration range, it was confirmed that no activity was shown in the STING KO cell line, thereby verifying that the compound of the present invention induces an immune response in a STING-dependent manner.

Cell viability was confirmed through the MTS assay, and the results are shown in FIG. 2 . As shown in FIG. 2 , even when the compounds 37 and 39 of the present invention were treated up to high concentrations ranging from 400 μM and 10,000 nM, they did not show any toxicity in cells. Considering these results and the EC ₅₀ of compounds 37 and 39 in THP1 cells were about 356.3 pM and 4.0 pM, it was confirmed that the safety window of the compounds of the present invention was very large.

In order to evaluate the binding of compound 37 to the STING protein, an in vitro ligand-competition binding experiment was performed using human STING recombinant protein and is shown in FIG. 3 . By measuring the FRET signal generated by the binding between the d2-labeled human STING ligand and the Tb3+-labeled STING antibody, it was confirmed that compound 37 has a 100-fold greater binding affinity for STING than compound 31. The IC ₅₀ value of compound 37 calculated by the HTRF (homogeneous time-resolved fluorescence) ratio was 0.06 nM, confirming the binding pattern about 100 times more strongly than that of compound 31 (6.2 nM).

It was confirmed that compound 37 could bind to STING in both human and mouse cells. CETSA (cellular thermal shift assay) was used to evaluate the change in thermodynamic stability of the STING protein when compound 37 was bound, and the results are shown in FIG. 4 . The results of CETSA confirmed that compound 37 binds to human and mouse STING. In the absence of compound 37, the thermal stability of STING decreased with increasing temperature, but incubation of THP-1 and RAW264.7 cells with compound 37 increased the stability of the protein. This is thought to be the result of the strong interaction between the protein and the compound.

Next, in order to verify the efficacy of inducing innate immune response of compounds 37 and 39 of the present invention, IFN-β, which is the most important factor among the sub-pathways by STING activation in THP-1 cell line, and IP-10 cytokine, which is a sub-factor of the type I IFN pathway. The degree of Cain secretion was measured using an enzyme adsorption immunosorbent assay (ELISA). The results are shown in FIGS. 5 and 6 .

Upon treatment with compound 37 of the present invention, secretion of IFN-β by STING activation was confirmed from 12.8 nM, which is a much lower concentration than in compound 31, and the EC ₅₀ value for IFN-β secretion was 2.047 nM, which was very high compared to the existing material. proved to be excellent. In addition, while the sub-factor IP-10 and compound 31 were insignificant even at 500 nM, it was confirmed that the compound 37 of the present invention increased dependently from a very low concentration of 0.41 nM. In this aspect, cytokine secretion was observed in both human and mouse cells in a dose-dependent manner, indicating that the efficacy of Compound 37 was very good. In addition, as a result of ELISA measurement of IFN-β production according to the compound 37 treatment of the present invention in the THP1 WT cell line and the STING KO cell line, IFN-β secretion by the compound 37 of the present invention in the STING KO cells was not observed at all. Through these results, it was verified once again that compound 37 of the present invention regulates the innate immune response according to IFN-β secretion in a STING-dependent manner.

In FIG. 6, the efficacy of diABZI-3 and compound 39, which is a previously reported STING agonist, was compared and analyzed. ELISA measurements of dose-dependent levels of secreted IFN-β and IP-10 showed that compound 39 significantly promoted cytokine secretion in THP-1 cells compared to diABZI-3.

Next, the expression of various interferon stimulated genes (ISG) according to the activation of the type 1 interferon pathway induced by compound 37 of the present invention was verified, and the results are shown in FIG. 7 . To this end, RT-PCR (real-time polymerase chain reaction) was performed in the THP1 cell line.

As confirmed in the ELISA result of FIG. 7 , it was confirmed that the genes of IFNB and CXCL10 significantly increased at 6 hours and 18 hours, respectively. In particular, it was confirmed that IFNB, one of the type I IFNs, showed a sharp increase within 6 hours, whereas the ISG genes, a subfactor by type I IFN, showed similar or higher expression at 18 hours compared to 6 hours. Based on this, it was verified that compound 37 of the present invention effectively activates the type I IFN signal transduction system by IFNB to induce an innate immune response.

When STING is activated, activation of the signaling system occurs through phosphorylation of STING itself and phosphorylation of sub-factors TBK1 and IRF3. In order to confirm whether the compound 37 of the present invention acts on the STING signaling system activity, the expression level and phosphorylation of major factors was confirmed through Western Blotting. For efficacy comparison, compound 31 was verified together as a control group. The results are shown in FIG. 8 .

Although compound 31 was treated with 1000 nM, phosphorylation of sub-factors such as p-STAT1, pTBK1, pIRF3, and pSTING was not induced, whereas in the case of compound 37 of the present invention, both pSTAT1, pTBK1, and pIRF3 at very low concentrations of 2 nM and 10 nM. , pSTING was observed. Judging from the following results, it was verified that the compound 37 of the present invention activates sub-factors of the STING signaling system even at very low concentrations, thereby proving that it acts as an effective STING agonist.

Meanwhile, the toxicity and stability of compound 37 of the present invention were evaluated, and the results are shown in Table 7 below.

(means ± SD; n = 3) compound 37 of the present invention ■ CYP inhibition (IC ₅₀ , μM) ^a 1A2 >100.0 2C9 >100.0 2C19 >100.0 2D6 >100.0 3A4 4.2 ■ Cardiotoxicity (IC ₅₀ , μM) hERG patch clamp assay > 50.0 ■ Plasma stability ^c Mouse (%) >99 Human (%) >99

Specifically, first, IC ₅₀ values for CYP (cytochrome P450) inhibition in human liver microsomes were measured. To measure the degree of inhibition of CYP metabolizing enzyme activity, the amount of metabolites produced when each CYP-specific substrate was added to human liver microsomes was quantified. Through this, it was determined that when the production of a specific metabolite by the test drug was inhibited, the CYP activity involved in the corresponding metabolism was inhibited. As a result of measuring the inhibition of enzyme activity for a total of 5 CYP isozymes, inhibition of CYP3A4 was confirmed, but weak inhibition compared to known CYP3A4 inhibitors (ketoconazole). It was confirmed that no inhibition by the compound of the present invention was observed. In the case of CYP3A4, considering that the EC50 of compound 37 of the present invention is 356.3 pM, it is thought that it does not significantly affect the inhibition of enzyme activity because it shows a difference of 11878 times or more.

Next, by using the electrophysiological patch clamp method, the possibility of inducing cardiotoxicity in vivo by drug-induced inhibition of the hERG K ⁺ channel was predicted. Compound 37 of the present invention was confirmed to be safe against the possibility of cardiotoxicity caused by inhibition of hERG K+ channel activity by not showing IC ₅₀ within 50 μM, the highest concentration tested.

On the other hand, in the plasma stability test experiment, as a result of measuring the amount of the compound of the present invention remaining after exposure of the drug to plasma of rats and humans for 4 hours, 99% or more of the compound remained. Therefore, it was confirmed that the compound is very stable against hydrolytic enzymes present in plasma.

Meanwhile, the pharmacokinetic properties of the compounds of the present invention were evaluated and shown in Table 8 below.

Parameter ^{a, b} Compound 26 Compound 37 Compound 39 T _1/2 (h) 0.59 ± 0.40 10.54 ± 4.10 1.75 ± 0.13 V _ss (L/kg) 1.80 ± 0.74 17.74 ± 5.29 1.81 ± 0.34 CL (L/h/kg) 9.48 ± 1.11 2.12 ± 0.27 0.72 ± 0.18 AUC _last (μg h/mL) 0.31 ± 0.03 4.20 ± 0.26 4.24 ± 0.96 AUC∞ (μg h/mL) 0.32 ± 0.04 4.78 ± 0.59 4.30 ± 0.97

^a Values are shown as the means ± standard deviation of at least three independent experiments. ^b Parameters from intravenous administration (3 mg/kg, BALB/c mice, n=3). AUC _last , areas under the plasma concentration-time curve from time zero to time of last measurable concentration; AUC _∞ , areas under the plasma concentration-time curve from time zero to time infinity; CL, total clearance from plasma steady; T _1/2 , terminal half-life; V _ss , state volume of distribution.

Specifically, 3 mg/kg IV of the compound of the present invention was administered to BALB/c mice to measure the change in the concentration of the compound in the blood over time. Compared to the half-life of compound 26, less than 1 hour, the half-life of compound 39 of the present invention is 1.75 hours, and the half-life of compound 37 is 10.5 hours. It can be a great advantage as an active ingredient.

On the other hand, AUC _last of compound 37 was 4.20 μg h/ml, AUC _∞ was 4.78 μg h/ml, AUC _last of compound 39 was 4.24 μg h/ml, AUC _∞ was 4.30 μg h/ml, This was also higher than other compounds with similar structures. Similarly, clearance of compound 37 was 2.12 L/h/kg and compound 39 was 0.72 L/h/kg, which was very low compared to monomeric compound 26, and the volume of distribution was also 17.7 L/kg in compound 37, similar structure It was found to be higher than other compounds having

Overall, the compounds of the present invention have a longer half-life and a lower clearance than those of a similar structure, and thus it can be seen that the AUC value is good.

In Figure 9, the effect of compound 37 on tumor growth in the mouse CT26 tumor model was confirmed. When tumors reached 50-100 mm ³ , mice were dosed with compound 37 four times at two different doses (0.015 and 1.5 mg/kg). Treatment with Compound 37 at both doses showed little weight loss in mice and significantly inhibited tumor growth (P=0.018 for 1.5 mg/kg, P=0.045 for 0.015 mg/kg). Compound 37 (1.5 mg/kg) induced 57% tumor growth inhibition in mice on day 17, confirming the anticancer efficacy of the compound in vivo mediated by activation of STING-related signaling pathways.

Compounds 26, 37, and 39 were tested for anticancer effects in CT26 bearing mice due to their high potency and good PK properties. Although the effect of cGAMP, a natural ligand of STING, has been utilized through intratumoral injection, there is a problem in that it is difficult to control the dose of cGAMP because the desired anticancer effect is observed only at low doses due to the bell-shaped response-efficiency curve. Therefore, in the present invention, compounds 26, 37 and 39 were each administered intravenously to confirm the anticancer effect in mice. The dose administered was 1.5 mg/kg for each compound. Based on the in vivo evaluation of the initially tested compound 37 ( FIG. 9 ), the number of administrations was increased to achieve sufficient immune stimulation, and treatment was stopped after 8 days, and the results are shown in FIG. 10 . Three compounds 26, 37, and 39 inhibited tumor growth without causing weight loss (P=0.0037 for compound 39, P=0.0188 for compound 37). Additionally, 1 out of 5 mice treated with compound 39 reached a tumor-free state with complete disappearance of tumors and 2 mice showed very slow tumor growth.

Thereafter, mice treated with compound 39 were re-injected with CT26 without additional compound administration to monitor tumor recurrence. Tumor growth in mice treated with compound 39 was slower than in mice that had not been previously treated with any compound, and subjects that had completely disappeared from the primary tumor test achieved tumor-free status even with a second inoculated recurrent tumor. The results are shown in FIG. 11 . These results suggest that compound 39-induced immune activation can prevent tumor recurrence through immunological memory without additional treatment.

Claims (12)

A compound of Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]

In Formula 1, R ⁵ is

,

, or

lim. The method of claim 1, wherein R ⁵ is

or

A phosphorus compound or a pharmaceutically acceptable salt thereof. The method of claim 2, wherein R ⁵ is

A phosphorus compound or a pharmaceutically acceptable salt thereof. 2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido) Butyl)-1H-benzo[d]imidazole-5-carboxamide (Compound 26) or a pharmaceutically acceptable salt thereof. A composition comprising the compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable additive. A pharmaceutical composition for the treatment or prevention of STING-mediated diseases comprising the compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition according to claim 6, wherein the STING-mediated disease is cancer, precancerous syndrome, or infectious disease. The pharmaceutical composition according to claim 7, wherein the STING-mediated disease is cancer. A vaccine composition comprising the compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof as a vaccine adjuvant. Claims 1 to 4, characterized in that the administration of a therapeutically effective amount of the compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof to an individual in need of treatment or prevention of a STING-mediated disease, treatment or Prevention methods. The method of claim 10, wherein the STING-mediated disease is cancer, precancerous syndrome, or an infectious disease. The method of claim 10 , wherein the STING-mediated disease is cancer.

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