Patent Application
20240002383
The disclosure relates to the field of biomedicine, in particular to a series of small molecule STING agonists.
Vaccines are considered the most powerful weapon to prevent the spread of infectious diseases. After the outbreak of the Corona Virus Disease 2019 (COVID-19), a variety of vaccines against the coronavirus SARS-CoV-2 have quickly entered clinical trials, including mRNA vaccines, DNA vaccines, inactivated vaccines, viral vector vaccines, etc. The main components of these vaccines are the spike protein or receptor-binding domain (RBD) of the coronavirus. At present, many of COVID-19 vaccines have proven protective effects on SARS-CoV-2 infection in human ACE2 transgenic mice and non-human primate models. Although more than a dozen vaccines now have been authorized around the globe, there are still some urgent problems that need resolving. For example, as the virus spreads in the population, the coronavirus continues to mutate. The newly mutated virus strains have brought severe challenges to the currently marketed vaccines. Studies now have shown that the SARS-CoV-2 mutant strain in the UK and the SARS-CoV-2 mutant strain in South Africa have produced some immune escape phenomena against some existing vaccines on the market (Wang et al. Mrna vaccine-elicited antibodies to SARS-CoV-2 and circulating variants, medRxiv, 2021). In addition, in the past 20 years, three highly pathogenic coronaviruses including SARS-CoV, SARS-CoV-2 and MERS-CoV have appeared and broke out one after another. At present, some coronaviruses similar to SARS from bats such as Rs3367 and WIV1 strains have been found, which suggests that SARS-related coronaviruses may still appear suddenly and spread like SARS-CoV-2 in population in the future. At present, only a few studies have shown that vaccines based on SARS-CoV-2 antigens can produce weak cross-neutralizing antibody protection against SARS-CoV and SARS-related coronavirus infections (Liu Z et. al. RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response, Signal Transduct Target Ther, 2020). It is also controversial whether the sera of patients who have recovered from the SARS-CoV infection can neutralize SARS-CoV-2. Studies have shown that the sera from patients infected with SARS-CoV can cross-neutralize SARS-CoV-2, but the neutralization activity is weak (Zhu, Y et al. Cross-reactive neutralization of SARS-CoV-2 by serum antibodies from recovered SARS patients and immunized animals. Sci Adv, 2020). Another study shows that the sera from patients who have recovered from the SARS-CoV infection cannot effectively neutralize the SARS-CoV-2 virus (Wang, Y. et al. Kinetics of viral load and antibody response in relation to COVID-19 severity. J Clin Invest, 2020). These results suggest that it is difficult to produce a broad-spectrum, long-lasting, and strong protective immune response to SARS-related coronaviruses in body through common vaccination strategies or natural infection pathways. Therefore, the development of a broad-spectrum, long-lasting and potent universal vaccine against SARS-related viruses is essential to combat the epidemic of the current SARS-CoV-2, including mutants of SARS-CoV-2, and SARS-related viruses that may appear in the future.
Adjuvants are essential to enhance the immunoprotection of protein subunit vaccines or inactivated vaccines. At present, the most commonly used adjuvant is the aluminum adjuvant. Recently, a variety of protein subunit vaccines in clinical trials use the aluminum adjuvant. Although the aluminum adjuvant is safe, the aluminum adjuvant mainly enhances the antibody humoral immune response, and the cellular immune response also plays a vital role in resisting viral infection processes. Therefore, developing new adjuvants, especially small-molecule immune enhancers, to enhance the immunoprotective effect of subunit vaccines or inactivated vaccines against SARS-CoV-2, induce more types of immune responses and extend the persistence of immunity is currently a frontier hotspot.
In recent years, STING agonists have been found to have the potential to be used as vaccine adjuvants. The present inventors previously used nano-encapsulated STING agonist cGAMP lung bionic particles as an adjuvant for influenza vaccines. Vaccination with intranasal drops can produce a strong and broad-spectrum immunoprotection against influenza viruses in mice (Wang, J. et al. Pulmonary surfactant-biomimetic nanoparticles potentiate heterosubtypic influenza immunity. Science, 2020). However, cGAMP is less effective by intramuscular injection.
In the present disclosure, a new STING small molecule agonist was used as an adjuvant for the protein subunit RBD-Fc vaccine against SARS-CoV-2. In animal models using mice, rabbits, and rhesus monkeys, the STING agonist-adjuvanted RBD-Fc strongly activated the cellular and humoral immune responses, and produced a broad-spectrum, potent, and long-lasting immunoprotection against SARS-CoV-2, its mutants, SARS-CoV, and a variety of SARS-related viruses. As compared with the traditional aluminum-adjuvanted vaccine against SARS-CoV-2, the new STING small molecule agonist can significantly improve the cellular and humoral immune responses of the vaccine, enhance the broad-spectrum protection against SARS and other coronaviruses, as well as significantly improve the persistence of the immunoprotection of the vaccine against SARS-CoV-2.
In one aspect, the present application provides a compound having formula (I) or pharmaceutically acceptable salts thereof,
wherein R1 is CR1′, wherein R1′ is H, —OMe or —O(CH2)nNR2′R3′, n is an integer of 1 to 6, preferably 2 or 3, R2′ and R3′ taken together with the nitrogen atom through which they are connected to form a substituted or unsubstituted 5-6 membered heterocycloalkyl or substituted or unsubstituted 5-6 membered heteroaryl, wherein the substituted 5-6 membered heterocycloalkyl or substituted 5-6 membered heteroaryl is independently substituted with one or more halogen, OH, amine, CN, CF3, or unsubstituted C1-C4 saturated alkyl; preferably, the heterocycloalkyl is one of:
In one embodiment, the compound has the structure:
In one aspect, the application provides a pharmaceutical composition, comprising the compound of the application or pharmaceutically acceptable salts thereof, and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable diluent.
In one aspect, the application provides use of the compound of the present application or pharmaceutically acceptable salts thereof or the pharmaceutical composition of the present application for the manufacture of an adjuvant. In one embodiment, the adjuvant is an adjuvant for a vaccine.
In one embodiment, the vaccine comprises an antigen. In one embodiment, the antigen is selected from a group consisting of a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, and a fungal antigen. In one embodiment, the vaccine is effective for preventing the infection of any one of the strains in the examples.
In one embodiment, the viral antigen is selected from a group consisting of an HIV antigen, an influenza antigen, and a coronavirus antigen. In one embodiment, the vial antigen is from one or more of HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, Varicella zoster virus (VZV) and SARS-CoV-2 such as SARS-CoV-2 Omicron mutant. In one embodiment, the vial antigen is SARS-CoV-2 RBD-Fc protein or gE protein of Varicella zoster virus.
In one aspect, the application provides a vaccine, comprising the compound of the present application or pharmaceutically acceptable salts thereof; and an antigen. In one embodiment, the vaccine is an intramuscular, an intradermal vaccine, or an inhaled vaccine. In one embodiment, the vaccine comprises an antigen. In one embodiment, the antigen is selected from a group consisting of a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, and a fungi antigen. In one embodiment, the viral antigen is selected from a group consisting of an HIV antigen, an influenza antigen and a coronavirus antigen. In one embodiment, an antigen is from one or more of HCOV-229E, HCOV-OC43, SARS-COV, HCOV-NL63, HCOV-HKU1, MERS-COV, Varicella zoster virus (VZV) and SARS-COV-2 such as SARS-CoV-2 Omicron mutant. In one embodiment, the vial antigen is SARS-CoV-2 RBD-Fc protein or gE protein of Varicella zoster virus (VZV).
In one aspect, the application provides a method for producing a vaccine of the present application, comprising mixing the compound of the present application and an antigen.
In one aspect, the application provides the compound of the present application or pharmaceutically acceptable salts thereof for use as an adjuvant. In one embodiment, the adjuvant is an adjuvant for a vaccine. In one embodiment, the vaccine comprises an antigen. In one embodiment, the antigen is selected from a group consisting of a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, and a fungi antigen. In one embodiment, the viral antigen is selected from a group consisting of an HIV antigen, an influenza antigen and a coronavirus antigen. In one embodiment, an antigen is from one or more of HCOV-229E, HCOV-OC43, SARS-COV, HCOV-NL63, HCOV-HKU1, MERS-COV, Varicella zoster virus (VZV) and SARS-COV-2 such as SARS-CoV-2 Omicron mutant. In one embodiment, the vial antigen is SARS-CoV-2 RBD-Fc protein or gE protein of Varicella zoster virus.
In one aspect, the application provides a method for treating or preventing an infectious disease or a cancer, which comprises administering an effective amount of the vaccine of the present application to a subject in need thereof. In one embodiment, the vaccine is an intramuscular, intradermal vaccine or inhaled vaccine. In one embodiment, the infectious disease is selected from a group consisting of AIDS, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), COVID-19, Varicella zoster and influenza, and the cancer is selected from a group consisting of HPV-related cancer, HBV-related cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, head and neck cancer, laryngeal cancer, lung cancer, liver cancer, pancreatic cancer, kidney cancer, bone cancer, melanoma, metastatic cancer, HTERT-related cancer, FAP antigen-related cancer, non-small cell lung cancer, blood cancer, esophageal squamous cell carcinoma, cervical cancer, bladder cancer, colorectal cancer, gastric cancer, anal cancer, synovial sarcoma, testicular cancer, recurrent respiratory system papillomatosis, skin cancer, glioblastoma, liver cancer, gastric cancer, acute myeloid leukemia, triple-negative breast cancer, and primary cutaneous T-cell lymphoma.
In one aspect, the application provides a kit comprising the compound of the application, an antigen, and instructions for treating or preventing an infectious disease or a cancer.
The benefits of the present disclosure include:
The terms “halo” or “halogen” refers to fluorine, chlorine, bromine or iodine.
The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals. The alkyl may include a designated number of carbons (e.g., C1-C10 means one to ten carbons). In embodiments, the alkyl is fully saturated. In embodiments, the alkyl is monounsaturated. In embodiments, the alkyl is polyunsaturated. Alkyl is an uncyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs, and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. In embodiments, the term “alkyl” refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing no unsaturation (i.e., saturated alkyl); having from one to ten, one to eight, one to six, or one to four carbon atoms; and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1, 1-dimethylethyl (t-butyl), and the like.
In embodiments, the term “heterocycloalkyl” means a monocyclic, bicyclic, or a multicyclic heterocycloalkyl ring system. In embodiments, heterocycloalkyl groups are fully saturated. In embodiments, a bicyclic or multicyclic heterocycloalkyl ring system refers to multiple rings fused together wherein at least one of the fused rings is a heterocycloalkyl ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heterocycloalkyl ring of the multiple rings.
In embodiments, a heterocycloalkyl is a “heterocyclyl”, which refers to a stable 3- to 15-membered ring group which consists of carbon atoms and from one to five heteroatoms selected from a group consisting of nitrogen, oxygen and sulfur. In one embodiment, the heterocyclic ring system group may be a monocyclic, bicyclic, or tricyclic ring or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen or sulfur atoms in the heterocyclic ring system group may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl group may be partially or fully saturated or aromatic. The heterocyclic ring system may be attached to the main structure at any heteroatom or carbon atom which results in the creation of a stable compound. Exemplary heterocylic radicals include, azetidinyl, benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, chromanyl, chromonyl, coumarinyl, decahydroisoquinolinyl, dibenzofuranyl, dihydrobenzisothiazinyl, dihydrobenzisoxazinyl, dihydrofuryl, dihydropyranyl, dioxolanyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrazolyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4 dithianyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, benzo[1,3]dioxol-5-yl, benzodioxolyl, 1,3-dioxolan-2-yl, dioxolanyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, tetrahydrofuran, oxazolidin-2-onyl, oxazolidinonyl, piperidinyl, piperazinyl, pyranyl, tetrahydroiuryl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl, pyrrolidinonyl, oxathiolanyl, and pyrrolidinyl.
The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. In embodiments, a fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within an aryl ring of the multiple rings. The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. In embodiments, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heteroaromatic ring of the multiple rings). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. A 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
As used herein, the symbol “” means that two atoms may be linked via a single bond or a double bond, provided that the valence states of the two atoms is suitable.
The term “antigen” refers to any substance that can be used as a target of an immune response. The immune response can be a cellular immune response or a body fluid immune response. In one embodiment, a vaccine comprises an antigen. In one embodiment, the antigen is selected from the group consisting of a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, and a fungi antigen.
In one embodiment, the viral antigen is selected from a group consisting of an HIV antigen, an influenza antigen, and a coronavirus antigen. In one embodiment, the viral antigen is an antigen from one or more of HCOV-229E, HCOV-OC43, SARS-COV, HCOV-NL63, HCOV-HKU1, MERS-COV, Varicella zoster virus and SARS-COV-2 such as SARS-CoV-2 Omicron mutant. In one embodiment, the viral antigen is a SARS-COV-2 RBD-Fc protein or gE protein of Varicella zoster virus (VZV).
In one aspect, the present disclosure provides a vaccine comprising the compound of the disclosure and an antigen. In one embodiment, the vaccine is an intramuscular, an intracellular vaccine or an inhaled vaccine. The antigen can be a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, and/or a fungi antigen. For example, the viral antigen can be selected from a group consisting of an HIV antigen, an influenza antigen and a coronavirus antigen. The viral antigen can be an antigen from one or more of HCOV-229E, HCOV-OC43, SARS-COV, HCOV-NL63, HCOV-HKU1, MERS-COV, Varicella zoster virus and and SARS-COV-2 such as SARS-CoV-2 Omicron mutant. In one embodiment, the viral antigen is a SARS-COV-2 RBD-Fc protein or gE protein of Varicella zoster virus (VZV).
The term “vaccine” herein refers to a formulation of an antigen, which typically contains certain portions of infective sources and raises immune response in a subject after its injection. The antigenic portion of the vaccine formulation can be a microorganism or a natural product purified from a microorganism, a synthetic product or a genetic engineering protein, a peptide, a polysaccharide etc. Preferably, the vaccine is an inactivated vaccine, live-attenuated vaccine, subunit vaccine, nucleic acid vaccine such as mRNA or DNA vaccine.
The term “adjuvant” as used herein refers to any substance that can increase or modify an immune response after mixing with the injected immunogen. Herein, the adjuvant can be one or more compounds as shown below:
Effective vaccines must cause appropriate responses to antigens. There are several unique types of immune responses that have different protection capabilities to resist specific diseases. For example, antibodies have a protective effect against bacterial infections, but a cell-mediated immunity is required to remove many viral infections and tumors. There are a variety of unique antibodies and cell-mediated immune response. The cell-mediated response is divided into two basic groups: 1) delayed hypersensitivity reaction, where T cells function indirectly through macrophages and other cells or cell products, and 2) cytotoxic reactions, wherein specialized T cells specifically and directly attack and kill infected cells.
There are five primary antibodies: IgM, IgG, IgE, IgA, and IgD. These antibodies have unique functions of immune responses. IgG, a type of antibody that is dominant in the blood, can be subdivided into several different subclasses or isotypes. In mice, the isotypes are IgG1, IgG2a, IgG2b, and IgG3. In human, the isotypes are IgG1, IgG2, IgG3, and IgG4. The IgG isotype has different protection capabilities against specific infections. The murine IgG2a and IgG2b can activate complements and mediate the antibody-mediated cytotoxicity and the cell-mediated cytotoxicity. They are particularly effective against many bacterial, viral and parasitic infections. The similar isotypes in human are represented by IgG1 and IgG3. In contrast, the murine IgG3 has a particularly effective protection against bacteria with a polysaccharide film, such as pneumococcus. The isotype in human may be IgG4. The isotypes such as murine IgG1 do not bind to a complement, and cannot effectively neutralize a toxin. Their effects on many bacterial and viral infections are low. Since different IgG isotypes have significantly different immune function, it is important to induce a most suitable isotype with a vaccine for a particular infection Although the names are different, the effective evidence and the prior theories have shown that the nature of an immunogen that determines the isotypes of antibodies among mammal species is similar. In other words, in a species, an immunogen which can invoke IgG antibodies in delayed type hypersensitivity or IgG antibodies in complement binding can stimulate similar responses in another species.
In some embodiments, the present application provides a kit comprising an immunogenic composition or a vaccine and instructions for use. The kit can contain immunogenic compositions or vaccines in a suitable container and various buffers well known in the art. In some embodiments, the kit comprises one or more compounds of the present application. Thus, in some embodiments, immunogenic compositions or vaccines and these compounds are in the same vial. In some embodiments, immunogenic compositions or vaccines and these compounds are in separate vials.
The container may include at least one vial, a tube, a flask, a bottle, a syringe, or other container device, which can contain an immunogenic composition or a vaccine. If other components are provided, the kit can contain other containers that hold the components. The kit can also include means for containing an immunogenic composition or a vaccine, and any other reagent container that is closed for commercial sales. Such containers can include injection or blow molding plastic containers that retain the desired vials therein. The container and/or kits can include labels with instructions and/or warnings. In some embodiments, the present application provides a kit comprising a container that includes a vaccine comprising an immunogenic composition, an optional pharmaceutically acceptable carrier, and packaging insert with instructions for the vaccination for the treatment or prevention of the disease in a subject. In some embodiments, the kit further comprises a compound of the present application and an instruction for administrating the compound for the treatment or prevention of the disease in a subject.
The present application is further illustrated by the following examples. The examples should not be construed as limiting.
The following compounds are used in the examples and the synthesis of the compounds is as follows. The starting reagents are commercial available.
To a solution of (E)-3-(4-aminobut-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (Intermediate 2, 6.00 g, 14.342 mmol, 1.00 eq, HCl), 4-fluoro-3-nitro-benzamide (2.64 g, 14.3 mmol, 1.00 eq), DIPEA (7.40 g, 57.3 mmol, 9.98 mL, 4.00 eq) and NaHCO3 (4.81 g, 57.30 mmol, 2.23 mL, 4 eq) in EtOH (60 mL) was stirred at 110° C. for 16 hrs under N2 to afford a yellow suspension. LCMS showed one main peak with desired MS peak was found. The reaction mixture was diluted with H2O (60 mL) and filtered to provide (E)-3-(4-((4-carbamoyl-2-nitrophenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (5.0 g, 9.14 mmol, 63.80% yield, 99.9% purity) was obtained as yellow solid, which was used directly in the next step without further purification.
1HNMR (400 MHz, DMSO-d6): δ (ppm) 12.83 (br s, 1H), 8.72 (d, J=1.50 Hz, 1H), 8.62 (d, J=2.00 Hz, 1H), 8.49 (t, J=5.94 Hz, 1H), 8.13 (br s, 2H), 7.86-8.01 (m, 2H), 7.51 (br s, 1H), 7.25 (br s, 1H), 6.95 (d, J=9.13 Hz, 1H), 6.59 (s, 1H), 5.86-5.96 (m, 1H), 5.72-5.83 (m, 1H), 4.81 (br d, J=4.88 Hz, 2H), 4.55 (q, J=7.05 Hz, 2H), 4.05 (br s, 2H), 2.15 (s, 3H), 1.31 (t, J=7.07 Hz, 3H). LCMS: m/z 547.2 (M+1).
To a solution of (E)-3-(4-((4-carbamoyl-2-nitrophenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (5.00 g, 9.15 mmol, 1.00 eq) in DMF (50 mL) and H2O (25 mL) was added NH3·H2O (12.8 g, 91.4 mmol, 14.0 mL, 25% purity, 10.0 eq) followed by disodium; BLAH (4.78 g, 27.4 mmol, 5.97 mL, 3.00 eq) and the reaction mixture was stirred at 25° C. for 1 hr to afford a yellow suspension. LCMS showed desired MS peak was found. The reaction mixture was diluted with H2O (500 mL) and lyophilized. The residue was diluted with DMF (400 mL) and filtered. The filtrate was concentrated in vacuum. (E)-3-(4-((2-amino-4-carbamoylphenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-4-carboxamide (4.5 g, crude) was obtained as light yellow solid, which was used directly in the next step without purification.
LCMS: m/z 517.3 (M+1).
To a solution of (E)-3-(4-((2-amino-4-carbamoylphenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (4.5 g, 8.71 mmol, 1 eq) in DMF (45 mL) and MeOH (90 mL) was added BrCN (2.77 g, 26.13 mmol, 1.92 mL, 3 eq) and the reaction mixture was stirred at 50° C. for 2 hrs under N2 to afford a yellow suspension. LCMS showed the desired MS peak. The reaction mixture was concentrated under reduced pressure. The residue was triturated with EtOAc/i-PrOH (30/10 mL) to afford (E)-3-(4-(2-amino-5-carbamoyl-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (3.20 g, 5.06 mmol, 58.1% yield, 98.5% purity, HBr) yellow solid. LCMS: m/z 542.4 (M+1).
To a solution of (E)-3-(4-(2-amino-5-carbamoyl-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (1.00 g, 1.61 mmol, 1.00 eq, HBr), 1-ethyl-3-methyl-1H-pyrazole-5-carboxylic acid (297 mg, 1.93 mmol, 1.20 eq) and DIEA (1.04 g, 8.03 mmol, 1.40 mL, 5.00 eq) in DMF (15.0 mL) was added HATU (794 mg, 2.09 mmol, 1.30 eq) and the reaction mixture was stirred at 50° C. for 16 hrs, under N2 to afford a yellow solution. LCMS showed the desired MS peak. The reaction mixture was filtered. The filter cake was washed with cold DMF (5 mL×2) and lyophilized. (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (1.40 g) as light yellow solid.
1HNMR (400 MHz, DMSO-d6): δ (ppm) 12.64-13.11 (m, 2H), 8.71 (s, 1H), 8.12 (s, 2H), 7.85-8.00 (m, 2H) 7.71 (d, J=8.13 Hz, 1H), 7.26-7.58 (m, 3H), 6.55 (s, 2H), 5.79-6.07 (m, 2H), 4.89-4.76 (m, 4H), 4.38-4.59 (m, 4H), 2.12 (s, 6H), 1.06-1.35 (m, 6H). LCMS: m/z 678.5 (M+1). HPLC: 93% purity.
4-chloro-3-methoxy-5-nitrobenzoate (25 g, 10.1 mmol) was stirred in NH4OH (250 mL, 1.9 mmol) at room temperature for 24 hrs. The reaction temperature was then increased to 50° C. for 2 hrs. An additional 50 mL (˜3.7 eq) of NH4OH was added to the vessel. After an additional 2 hrs stirring at 50° C. the reaction mixture was cooled to room temperature. The solid was filtered and rinsed with cold water. The solid was dried under house vacuum and lyophilized to give 4-chloro-3-methoxy-5-nitrobenzamide (13 g, 68% yield) as a tan solid.
1H NMR (400 MHz, DMSO-d6): δ (ppm): 8.31 (br. s., 1H), 8.06 (d, J=1.8 Hz, 1H), 7.88 (d, 1=1.8 Hz, 1H), 7.81 (br. s., 1H), 4.02 (s, 3H). LCMS: m/z 230.9 (M+1).
4-Chloro-3-methoxy-5-nitrobenzamide (16 g, 69.3 mmol) was suspended in dry DCM (250 mL) and stirred at room temperature. To the reaction was added BBr3 (280 mL, IM in DCM) dropwisely. A slurry rapidly formed which was stirred overnight at room temperature under nitrogen. The reaction was poured into ice water (3 L) and stirred vigorously for 30 min. The resulting suspension was filtered and the solids dried to afford 4-chloro-3-hydroxy-5-nitrobenzamide (11.6 g, 77% yield). 1H NMR (400 MHz, DMSO-d6): δ (ppm): 11.53 (br. s., 1H), 8.17 (br. s., 1H), 7.92 (s, 1H), 7.72 (s, 1H), 7.66 (br. s., 1H).
LC-MS: [M+H]+=217. LCMS: m/z 217.0 (M+1).
A mixture of 4-chloro-3-hydroxy-5-nitrobenzamide (11.6 g, 53.5 mmol), 4-(3-chloropropyl)morpholine (10.5 g, 64.2 mmol), K2CO3 (9.6 g, 69.6 mmol) in DMF (100 mL) was stirred at 70° C. overnight. Solvent was removed in vacuo to give a crude solid product that was purified by silica gel chromatography (MeOH:DCM=1:10) to give 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzamide (10.5 g, 57% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ (ppm): 8.30 (s, 1H), 8.05 (d, J=1.8 Hz, 1H), 7.88 (d, J=1.8 Hz, 1H), 7.80 (s, 1H), 4.28 (t, J=6.2 Hz, 2H), 3.57 (t, J=4.6 Hz, 4H), 2.41-2.47 (m, 2H), 2.37 (br. s., 4H), 1.97 (dd, J=13.94, 7.35 Hz, 2H); LCMS: m/z 344.1 (M+1).
The solution of (E)-6-((4-aminobut-2-en-1-yl)amino)-5-nitronicotinamide (Intermediate 3, 220 mg, 0.87 mmol), 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzamide (200 mg, 0.58 mmol), i-PrOH (5 mL) and DIEA (1.12 g, 8.7 mmol) in a microwave vial was irradiated at 120° C. for 6 hrs. When cool, the resulting solid was isolated by filtration, rinsed with i-PrOH (2×1 mL) and dried to afford 6-((4-((4-carbamoyl-2-(3-morpholinopropoxy)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-5-nitronicotinamide (113 mg, 35%) as a red solid.
To (E)-6-((4-((4-carbamoyl-2-(3-morpholinopropoxy)-6-nitrophenyl)amino)but-2-en-1-yl) amino)-5-nitronicotinamide (2.7 g, 4.8 mmol) in MeOH (40.0 mL) at room temperature was added sodium hydrosulfite (11.7 g, 67.2 mmol) in water (45 mL). After 15 min, solid sodium bicarbonate (24 g) was added. After 10 min., the reaction mixture was filtered and the solid was rinsed with MeOH (4×20 mL). The combined filtrates were concentrated onto Celite and purified by preparative HPLC to afford (E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(3-morpholinopropoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide (1.81 g, 3.26 mmol, 49% yield) as a dark yellow solid. 1H NMR (400 MHz, DMSO-d6): δ (ppm): 7.93 (s, 1H), 7.60 (m, 1H), 7.10 (s, 1H), 6.96 (br s, 1H), 6.85 (m, 21), 6.77 (s, 1H), 6.14 (m, 1H), 5.73 (m, 2H), 4.81 (m, 2H), 4.66 (m 2H), 3.96 (m, 4H), 3.83 (m, 111), 3.54 (m, 611), 2.39 (t, J=7.2 Hz, 2H), 2.32 (br s, 4H), 1.84 (t, J=6.4 Hz, 2H). LCMS: m/z 499.3 (M+1).
To
(E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(3-morpholinopropoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide (812 mg, 1.62 mmol) in DMF (20 mL) at 0° C. was added 0.4 M 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate in dioxane (Intermediate 4, 6 mL, 3.89 mmol). After −10 min, another portion of 0.4 M 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate in dioxane (Intermediate 3, 2 ml, 0.48 mmol) was added, followed ˜15 min later by a final portion (2 ml, 0.48 mmol). After 35 min total reaction time, EDC (1.087 g, 5.67 mmol) was added followed by triethylamine (656 mg, 6.48 mmol). The mixture was warmed to room temperature and stirred overnight. The reaction was quenched with 3:1 water:saturated aqueous NH4Cl solution (10 mL) and extracted with 3:1 chloroform:ethanol (2×40 mL). The combined organic phases were washed with water (10 mL), dried over magnesium sulfate, and concentrated. The resulting residue was purified by preparative HPLC and the desired eluents were lyophilized to give (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-eth yl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide as white solid (445 mg, yield 45%; HPLC purity: 97.7% (254 nm)).
1H-NMR (400 MHz, DMSO-d6): δ (ppm): 8.70 (s, 1H), 8.14 (s, 1H), 7.66 (s, 1H), 7.33 (s, 1H), 6.51 (d, J=12.0 Hz 2H), 5.95 (m, 1H), 5.74 (m, 1H), 4.94 (d, J=4.0 Hz, 2H), 4.79 (d, J=4.8 Hz, 2H), 4.52 (m, 4H), 4.13 (t, J=11.2 Hz 2H), 3.98 (m, 2H), 3.67 (s, 2H), 3.38 (t, J=12.4 Hz, 2H), 3.24 (m, 2H), 3.05 (m, 2H), 2.09 (s, 6H), 2.06 (m, 2H), 1.26 (t, J=14.0 Hz, 6H). LCMS: m/z 821.4 (M+1), 819.3 (M−1).
A mixture of 4-chloro-3-hydroxy-5-nitrobenzamide (prepared as example 3, 3.20 g, 14.8 mmol, 1.00 eq), tert-butyl 4-(3-chloropropyl)piperazine-1-carboxylate (4.66 g, 17.7 mmol, 1.20 eq) and K2CO3 (2.65 g, 19.2 mmol, 1.30 eq) in DMF (32 mL), the mixture was stirred at 70° C. for 16 hrs under N2 atmosphere to give a yellow solution. LC-MS showed the starting material was consumed completely and one main peak with desired MS was detected. The mixture was concentrated under reduced pressure to give a crude product. The crude product was triturated with H2O (100 mL) at 25° C. for 16 hrs. to provide tert-butyl 4-(3-(5-carbamoyl-2-chloro-3-nitrophenoxy)propyl)piperazine-1-carboxylate (6.00 g, 13.6 mmol, 91.7% yield) as a yellow solid.
HNMR (400 MHz, DMSO-d6): δ 8.34 (br s, 1H), 8.05 (s, 1H), 7.89 (s, 1H), 7.80 (br s, 1H), 4.28 (br t, J=5.4 Hz, 2H), 3.37 (br s, 12H), 2.17-2.38 (m, 5H), 1.86-2.08 (m, 2H), 1.39 (s, 121), LCMS: m/z (ES+) [M+H]+=443.5.
A solution of (E)-6-((4-aminobut-2-en-1-yl)amino)-5-nitronicotinamide (Intermediate 3, 9.74 g, 33.9 mmol, 1.50 eq, HCJ), tert-butyl 4-(3-(5-carbamoyl-2-chloro-3-nitrophenoxy)propyl)piperazine-1-carboxylate (10.0 g, 22.6 mmol, 1.00 eq), DIEA (11.7 g, 90.3 mmol, 15.7 mL, 4.00 eq) and NaHCO3 (7.59 g, 90.3 mmol, 3.51 mL, 4.00 eq) in EtOH (110 mL) was stirred at 110° C. for 16 hrs under N2 of sealed tube to afford a yellow suspension. LC-MS showed the starting material was consumed completely and one main peak with desired MS was detected. The mixture was concentrated to give crude product. The residue was purified by flash silica gel chromatography (ISCO®; 120 g SepaFlash® Silica Flash Column, Eluent of 5-10% Methanol/Dichloromethane @ 100 mL/min_Dichloromethane/Methanol=10:1, Rf of product=0.50, UV 254 nm) to produce tert-butyl (E)-4-(3-(5-carbamoyl-2-((4-((5-carbamoyl-3-nitropyridin-2-yl)amino)but-2-en-1-yl)amino)-3-nitrophenoxy)propyl)piperazine-1-carboxylate (5.50 g, 7.51 mmol, 33.2% yield, 89.8% purity) as red brown solid.
1HNMR (400 MHz, DMSO-d6): δ 8.73-8.99 (m, 3H), 7.99-8.23 (m, 3H), 7.71-7.85 (m, 1H), 7.41-7.60 (m, 2H), 7.34 (br s, 1H), 5.56-5.87 (m, 211), 4.38 (br s, 1H), 4.08-4.26 (m, 4H), 4.05 (br t, J=6.1 Hz, 2H), 3.53-3.69 (m, 1H), 3.03-3.49 (m, 15f), 2.19-2.46 (m, 6H), 1.90 (br s, 2H), 1.39 (s, 9H), 1.17-1.31 (m, 5H), 1.06 (t, J=7.0 Hz, 3H); LCMS: m/z (ES+) [M+H]+=658.2.
To a solution of tert-butyl (E)-4-(3-(5-carbamoyl-2-((4-((5-carbamoyl-3-nitropyridin-2-yl)amino)but-2-en-1-yl)amino)-3-nitrophenoxy)propyl)piperazine-1-carboxylate (5.50 g, 8.36 mmol, 1.00 eq) in MeOH (110 mL) and H2O (55.0 mL) was added NaHCO3 (42.0 g, 500 mmol, 19.5 mL, 59.8 eq) followed by disodium; BLAH (20.4 g, 117 mmol, 25.5 mL, 14.0 eq) at 0° C. and then the reaction mixture was stirred at 20° C. for 2 hrs. A light yellow suspension was obtained. LCMS showed Reactant 1 was consumed completely and one main peak with desired MS was detected. The reaction mixture was filtered and the filter cake was washed with MeOH (100 mL*2). The filtrate was concentrated. The crude product tert-butyl (E)-4-(3-(3-amino-2-((4-((3-amino-5-carbamoylpyridin-2-yl)amino)but-2-en-1-yl)amino)-5-carbamoylphenoxy)propyl)piperazine-1-carboxylate (5.00 g, 8.37 mmol, 100% yield) was used directly in the next step without purification. LCMS: m/z (ES+) [M+H]+=598.2;
To a solution of tert-butyl (E)-4-(3-(3-amino-2-((4-((3-amino-5-carbamoylpyridin-2-yl)amino)but-2-en-1-yl)amino)-5-carbamoylphenoxy)propyl)piperazine-1-carboxylate (5.00 g, 8.37 mmol, 1.00 eq) in DMF (100 mL) was added a solution of 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (Intermediate 4, 4.41 g, 22.6 mmol, 2.70 eq) in dioxane (20 mL) of 0 min (3 mL), 10 min (3 mL), 15 min (3 mL) at 0° C., respectively. The reaction mixture was stirred at 0° C. for 30 min, then EDCI (5.61 g, 29.3 mmol, 3.50 eq) and Et3N (6.77 g, 66.9 mmol, 9.31 mL, 8.00 eq) was added at 0° C., then heated to 25° C. and stirred for 2 hrs to afford a yellow solution. LC-MS showed the starting material was consumed completely and desired compound was detected. The reaction mixture was quenched with a saturated aqueous NH4Cl solution (50 mL). The solution was filtered and the filtrate was concentrated. The crude product was purified by prep-HPLC (column: Phenomenex Gemini YMC Triart C18 250*50 mm*7 um; mobile phase: [water (10 mM NH4OAc)-ACN, 30-49, 30 min) to provide tert-butyl (E)-4-(3-((5-carbamoyl-1-(4-(6-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridin-3-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl)piperazine-1-carboxylate (2.50 g, 2.42 mmol, 28.9% yield, 89.0% purity) as white solid. LCMS: m/z (ES+) [M+H]+=920.5.
To a solution of tert-butyl (E)-4-(3-((5-carbamoyl-1-(4-(6-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridin-3-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl)piperazine-1-carboxylate (900 mg, 978 μmol, 1.00 eq) in MeOH (15 mL) and dioxane (15 mL) was added HCl/dioxane (4 M, 30 mL, 123 eq) and the mixture was stirred at 25° C. for 16 hrs under N2 to afford a yellow solution. LC-MS showed the starting material was consumed completely and desired compound was detected. The mixture was concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (column: Phenomenex Gemini YMC Triart C18 250×50 mm×7 μm; mobile phase: [water (0.05% HCl)-ACN, 20-80 30 min) to provide (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-(piperazin-1-yl)propoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (150 mg, 0.176 mmol, 17.9% yield, 96.0% purity) as white solid.
1HNMR (400 MHz, DMSO-d6): δ 11.89-12.32 (m, 1H), 9.57-10.02 (m, 2H), 8.78 (d, J=1.8 Hz, 1H), 8.09-8.33 (m, 2H), 7.99 (br s, 1H), 7.66 (d, J=1.0 Hz, 1H), 7.56 (br s, 1H), 7.26-7.49 (m, 2H), 6.53 (s, 1H), 6.48 (s, 1H), 6.00 (dt, J=15.5, 5.1 Hz, 1H), 5.73 (dt, J=15.6, 5.6 Hz, 1H), 4.98 (br d, J=3.6 Hz, 2H), 4.81 (br d, J=5.3 Hz, 2H), 4.39-4.58 (m, 4H), 3.21-3.80 (m, 9H), 2.13-2.30 (m, 3H), 2.09 (d, J=4.9 Hz, 6H), 1.25 ppm (td, J=7.1, 3.9 Hz, 6H); LCMS: m/z (ES+) [M+H]+=920.4.
A mixture of 4-chloro-3-hydroxy-5-nitrobenzamide (prepared as example 3, 1.00 g, 4.62 mmol, 1.00 eq), 4-(2-chloroethyl)morpholine (1.03 g, 5.54 mmol, 1.20 eq, HCl) and K2CO3 (1.28 g, 9.23 mmol, 2.00 eq) in DMF (15.0 mL), the mixture was stirred at 70° C. for 16 hrs under N2 atmosphere to give a yellow solution. LC-MS showed the starting material was consumed completely and one main peak with desired MS was detected. The mixture was concentrated under reduced pressure to create a crude product. The crude product was triturated with H2O (50.0 mL) at 25° C. for 16 hrs. 4-chloro-3-(2-morpholinoethoxy)-5-nitrobenzamide (2.60 g, 7.65 mmol, 82.8% yield, 97.0% purity) was obtained as a yellow solid. LCMS: m/z (ES+) [M+H]+=330.1.
A solution of 4-chloro-3-(2-morpholinoethoxy)-5-nitrobenzamide (1.05 g, 3.64 mmol, 1.20 eq, HCl), NaHCO3 (1.02 g, 12.1 mmol, 472 uL, 4.00 eq) and 4-chloro-3-(2-morpholinoethoxy)-5-nitro-benzamide (Intermediate 3, 1.00 g, 3.03 mmol, 1.00 eq), DIEA (1.57 g, 12.1 mmol, 2.11 mL, 4.00 eq) in EtOH (10.0 mL) was stirred at 110° C. for 16 hrs under N2 to afford a yellow suspension. LCMS showed the starting material was consumed completely. The residue was purified by flash silica gel chromatography (ISCO®; 330 g SepaFlash® Silica Flash Column, Eluent of 2-5% Methanol/Dichloromethane @ 100 mL/min_Dichloromethane/Methanol=10:1, Rt of product=0.27, UV 254 nm). (E)-6-((4-((4-carbamoyl-2-(2-morpholinoethoxy)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-5-nitronicotinamide (1.34 g, 2.12 mmol, 69.8% yield) was obtained as a red brown solid.
1HNMR (400 MHz, DMSO-d6): δ 0.77-0.85 (m, 6H) 1.11 (t, J=7.13 Hz, 4H) 3.49 (br d, J=6.13 Hz, 2H) 3.75-4.25 (m, 12H) 5.56-5.74 (m, 2H) 6.82-6.96 (m, 1H) 7.78 (br t, J=6.00 Hz, 1H) 7.93-8.15 (m, 3H) 8.72-8.91 (m, 3H); LCMS: m/z (ES+) [M+H]+=545.2.
To a solution of (E)-6-((4-((4-carbamoyl-2-(2-morpholinoethoxy)-6-nitrophenyl)amino)but-2-en-1-yl)amino)-5-nitronicotinamide (1.00 g, 1.84 mmol, 1.00 eq) in MeOH (20 mL) and H2O (15.0 mL) was added NaHCO3 (9.00 g, 107 mmol, 4.17 mL, 58.3 eq) followed by disodium; BLAH (4.48 g, 25.7 mmol, 5.60 mL, 14.0 eq) at 0° C. and then the reaction mixture was stirred at 20° C. for 2 hrs. A light yellow suspension was obtained. LCMS showed the starting material was consumed completely and one main peak with desired MS was detected. The reaction mixture was filtered and the filter cake was washed with MeOH (50.0 mL). The filtrate was concentrated. The crude product was used directly in the next step without purification. (E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(2-morpholinoethoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide (889 mg, 1.84 mmol, 100% yield) was obtained as a yellow solid. LCMS: m/z (ES+) [M+H]+=484.3.
To a solution of the (E)-5-amino-6-((4-((2-amino-4-carbamoyl-6-(2-morpholinoethoxy)phenyl)amino)but-2-en-1-yl)amino)nicotinamide (889 mg, 1.84 mmol, 1.00 eq), in DMF (23.0 mL) was added a solution of 2-ethyl-5-methyl-pyrazole-3-carbonyl isothiocyanate (Intermediate 4, 968 mg, 4.96 mmol, 2.70 eq) in dioxane (5.30 mL) of 0 min (3.00 mL), 10 min (3.00 mL), 15 min (3.00 mL) at 0° C., respectively. The reaction mixture was stirred at 0° C. for 35 mins, then EDCI (1.23 g, 6.43 mmol, 3.50 eq) and Et3N (1.49 g, 14.7 mmol, 2.04 mL, 8.00 eq) were added at 0° C., then heated to 25° C. and stirred for 16 hrs to afford a yellow solution. LCMS showed Reactant 1 was consumed completely and desired compound was detected. The reaction mixture was quenched with a saturated aqueous NH4Cl solution (50.0 mL). The solution was filtered and the filtrate was concentrated. The crude product was purified by prep-HPLC (column: Phenomenex Gemini C18 250×50 mm×7 um; mobile phase: [water (10 mM HCl)-ACN]; B %: 10%-40%, 20 min) to provide (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(2-morpholino ethoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (52.0 mg, 0.06 mmol, 3.27% yield) as a white solid.
1HNMR (400 MHz, DMSO-d6): δ 1.19-1.36 (m, 8H) 2.03-2.25 (m, 9H) 2.26-2.35 (m, 5H) 2.68 (br s, 1H) 3.45-3.53 (m, 3H) 4.11 (br t, J=5.63 Hz, 2H) 4.45-4.61 (m, 4H) 4.79 (br s, 2H) 4.97 (br s, 2H) 5.87-5.99 (m, 2H) 6.54 (s, 2H) 7.34 (s, 2H) 7.53 (br s, 1H) 7.64 (s, 1H) 7.93 (br s, 1H) 8.12-8.19 (m, 2H) 8.71 (s, 1H); LCMS: m/z (ES+) [M+H]+=807.3.
The compound CF512 and its synthesis
To a solution of (E)-3-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-eth yl-3-methyl-1H-pyrazole-5-carboxamido)-3H-imidazo[4,5-b]pyridine-6-carboxamide (Example 4, 400 mg, 487 umol, 1.00 eq) and K2CO3 (148 mg, 1.07 mmol, 2.20 eq) in DMF (8 mL) was added a solution of Mel (3.05 g, 21.5 mmol, 1.34 mL, 44.1 eq) in DMF (0.8 mL) at 25° C. and the reaction stirred at 25° C. for 16 hrs to give a yellow solution. The mixture was concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (column: Phenomenex Gemini C18 150*25 mm*10 um; mobile phase: [water (0.05% NH3H2O+10 mM NH4HCO3)-ACN]; B %: 10%-40%, 20 min) to produce (E)-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-3-methyl-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-1-methyl-2,3-dihydro-1H-imidazo[4,5-b]pyridine-6-carboxamide (120 mg, 0.138 mmol, 28.4% yield, 97.9% h purity) as white solid.
1HNMR (400 MHz, DMSO-d6): δ 8.74 (d, J=1.6 Hz, 1H), 8.35 (d, J=1.6 Hz, 1H), 8.14 (br s, 1H), 8.04 (br s, 1H), 7.71 (s, 1H), 7.61 (br s, 1H), 7.36-7.49 (m, 2H), 6.40 (s, 1H), 6.35 (s, 1H), 5.75-5.88 (m, 1H), 5.60-5.75 (m, 1H), 4.80 (br dd, J=16.4, 5.3 Hz, 4H), 4.30-4.54 (m, 4H), 4.07 (br t, J=6.3 Hz, 2H), 3.40-3.58 (m, 10H), 2.18-2.33 (m, 6H), 2.10 (d, J=13.3 Hz, 6H), 1.73 (q, J=6.5 Hz, 2H), 1.21 (dt, J=8.9, 7.2 Hz, 6H); LCMS: m/z (ES+) [M+H]+=849.6.
1. Materials:
8-week-old SPF C57 mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. The STING agonist, 3′-3′ cGAMP (abbreviated as cGAMP below) was purchased from Invivogen. The Trizol was purchased from Takara Bio Inc. The reverse transcription kit (PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time)) was purchased from Takara BioInc. The fluorescence quantitative PCR kit (TB Green® Premix DimerEraser™ (Perfect Real lime)) was purchased from Takara BioInc.
2.1 The Detection of the Innate Immune Response in Mice Activated by Intramuscular Injection of STING Agonists
2.1.1 Experimental Methods
As shown in
2.2 Monitoring the Innate Immune Responses of Mice Activated by the Intramuscular Injection with CF501 and the SARS-CoV-2 RBD-Fc Protein at Different Times.
2.2.1 Experimental Methods
As shown in
1. Experimental Materials
6-week-old SPF Balb/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. Aluminum adjuvant was purchased from Thermo Scientific. cGAMP was purchased from Invivogen.
2. Experimental Methods.
The procedure for the vaccination of the mice is shown in
The steps are as follows.
As shown in
The titer value in the table is the mean±Standard Error of Mean; significance analysis: compared with the CF501 treatment group (RBD-Fc+CF501), One-way ANOVA. The values in the following tables are expressed in the same way.
Compared with the aluminum adjuvant, the STING agonists CF501, CF502, CF508, CF510, or CF512 can significantly improve the TH1 and TH2 immune responses in mice. Compared with other adjuvants, CF501 can significantly improve the TH1 and TH2 immune responses in mice.
1. Materials
6-week-old SPF Balb/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd., and the ELISPOT kits for mouse IFNγ, TNFα and IL-4 were purchased from Mabtech. The RBD full-length scanning peptide library is synthesized by GL Biochem (Shanghai) Ltd. Mouse IL-2 was purchased from Beijing Dakewe.
2. Experimental Steps:
The results are shown in
1. Detection of the Neutralizing Antibody Levels in Mouse Sera Using a SARS-CoV-2 Pseudovirus Detection System.
The sera were diluted in the highest dilution, 100 folds initially. If the sera could not neutralize 50/of the viruses when the sera were diluted in 100 folds, the NT50 of the sera was 50 by default.
The neutralizing titers of sera collected at Day 21 against the SARS-CoV-2 pseudovirus are shown in
2. Plaque Reduction Method for the Detection of the Inhibitory Activity Against the Live SARS-CoV-2 Virus of the Sera.
The inhibitory activities against the live SARS-CoV-2 virus of the sera from the vaccinated mice at Day 21 are shown in
The inhibitory activities against the live SARS-CoV-2 virus of the sera from the vaccinated mice at Day 35 are shown in
3. Immunofluorescence Method for the Detection of the Inhibitory Activities Against the Live SARS-CoV-2 Virus of the Sera from the Vaccinated Mice
The results are shown in
4. Inhibition of the SARS-CoV-2 S-Mediated Cell-Cell Fusion by the Sera
The results are shown in
1. Detection of the Cross-Binding Capacities to SARS-CoV RBD for Sera from Mice in the Example 2
The highest dilution at which the OD450 is greater than the OD450 of the blank control group (no serum but PBS was added)×2.1 was defined as the serum antibody titer. Sera were diluted in 100 folds initially. If the OD450 at the 100-fold dilution was still not greater than 2.1 times that of the OD450 value of the blank control group, the serum antibody titer was set to 1:50.
The results are shown in
2. Detection of the Neutralization Activities Against the SARS-CoV Pseudovirus in Sera from Vaccinated Mice
The results are shown in
3. Detection of the Neutralization Activities Against Bat-Derived SARS-Like Viruses WIV1 and Rs3367 for the Sera from the Vaccinated Mice.
The results are shown in
The results for the cross-inhibitory activities against the Rs3367 pseudovirus are consistent with the trend observed for the WIV1 pseudovirus (
1. Materials: 8-Week-Old SPF ACE2 Transgenic Mice were Purchased from the Shanghai Model Organisms Center, Inc.
The results are shown in
These results fully indicate that the vaccination of mice with CF501 and the RBD-Fc protein can effectively protect the mice from the SARS-CoV-2 infection.
1. Experimental Materials New Zealand White Rabbits were purchased from Shanghai Zeyu Biological Technology Co, Ltd.
2. Experimental Method
2.1 The Procedure for the Vaccination of the New Zealand White Rabbits is Shown in
2.2 Evaluation of the Antibody Immune Response of the New Zealand White Rabbits after Vaccination.
The results are shown in
At Day 35 after the vaccination, the titers of the SARS-CoV2 RBD-specific antibodies produced by the rabbits vaccinated with the RBD-Fc protein mixed with CF51 is still highest, and are significantly higher than those in the rabbits vaccinated with the RBD-Fc protein mixed with the aluminum adjuvant or cGAMP. It is worth noting that the other STING agonist such as CF512, CF510, CF508, and CF502 which showed similar structures to CF501 could not induce more potent binding antibodies relative to Alum in the rabbits, demonstrating that the minor change of the structure would significantly influence the adjuvant effects.
1. A Method for the Detection of the Level of Neutralizing Antibodies in Sera Using the SARS-CoV-2 Pseudovirus
The sera were diluted in 100 folds initially. If the sera could not neutralize 50% of the viruses when the sera were diluted in 100 folds, the NT50 of the sera was 50 by default.
The results are shown in
2. Plaque Reduction Test for the Detection of the Inhibitory Activities Against the Live SARS-CoV-2 Virus of the Sera from the Vaccinated Rabbits
The results are shown in
3. Immunofluorescence Test for the Detection of the Inhibitory Activities Against the SARS-CoV-2 Virus of Sera from the Vaccinated Rabbits
The results are shown in
4. Inhibition of the SARS-CoV-2 S-Mediated Cell-Cell Fusion by the Sera
The results are shown in
5. The Neutralization Activities Against SARS-CoV-2 of the Sera from the Vaccinated Rabbits after the Fourth Immunization
Sera were collected one week after the fourth immunization of the rabbits. The neutralization activities of these sera against SARS-CoV-2 were evaluated by the pseudovirus detection system. The results are shown in
All these results indicate that the most potent neutralizing antibody response can be produced when CF501 is used as an adjuvant of the RBD-Fc protein for the vaccination of rabbits. The immune response produced in the rabbits vaccinated with the RBD-Fc protein mixed with cGAMP is very week. Although the rabbits vaccinated with the RBD-Fc protein mixed with the aluminum adjuvant also produce neutralizing antibodies at a certain level, the level is still significantly lower than the level of neutralizing antibodies produced by the rabbits vaccinated with the RBD-Fc protein mixed with CF501. Therefore, the neutralizing antibodies can be produced most effectively in both the mice vaccinated with the RBD-Fc protein mixed with CF501 and the rabbits vaccinated with the RBD-Fc protein mixed with CF501 as compared with the mice or rabbits treated with other adjuvants.
1. Packaging of SARS-Related Pseudoviruses (SARS-CoV, WIV1 and Rs3367).
2. Evaluation of Inhibition of SARS-CoV, WIV1 and Rs3367 Pseudoviruses by Sera
The results are shown in
We further detected antibodies against the SARS-CoV RBD at 45 days after the vaccination of the rabbits, and found that the level of the SARS-CoV RBD specific antibodies in the sera from the rabbits vaccinated with the RBD-Fc protein mixed with CF501 is significantly higher than those in the sera from the rabbits vaccinated with the RBD-Fc protein mixed with cGAMP and from the rabbits vaccinated with the RBD-Fc protein mixed with the aluminum adjuvant (Table 19,
3. Detection of the Neutralization Activities of Antisera Against SARS-CoV-2 Mutant Viruses after the Vaccination of Rabbits
At present, mutations continue to occur in SARS-CoV-2. In order to verify whether the antibodies produced by the rabbits immunized with vaccines containing adjuvants can still have a strong neutralizing effect on the current SARS-CoV-2 mutant viruses, we conducted site-directed mutations on a plasmid of the wild-type SARS-CoV-2. Pseudoviruses of more than 40 currently discovered SARS-CoV-2 mutants were produced. For the rabbits vaccinated with the RBD-Fc protein mixed with the aluminum adjuvant, cGAMP or CF501, the neutralization activities against these mutant viruses in the sera from the rabbits were detected after 4 immunizations. The results are shown in
These results indicate that the sera from the rabbits vaccinated with the RBD-Fc protein mixed with CF501 can show broad-spectrum and potent neutralization activities against SARS-CoV-2 mutants and SARS-related viruses.
1. Materials
Nine 2-year-old rhesus monkeys were purchased from Beijing Xierxin Biological Co., Ltd, of which 5 were females and 4 were males.
2. Evaluation of the Humoral Immune Response after the Vaccination of Rhesus Monkeys with a Vaccine
2.1 The Vaccination Procedure of Rhesus Monkeys is Shown in
2.2 Detection of the SARS-CoV-2 RBD Specific Antibody Titers in Sera from the Vaccinated Rhesus Monkeys
The results are shown in
The results are shown in
1. The SARS-CoV-2 Pseudovirus was Used to Detect Neutralizing Antibody Titers Against SARS-CoV-2 in Sera from the Vaccinated Rhesus Monkeys
The results are shown in
2. The Plaque Reduction Test for the Detection of the Inhibitory Activity of Rhesus Monkey Sera Against the Live SARS-CoV-2 Virus
The results are shown in
The neutralizing antibody titers in the sera were also continuously monitored after vaccination of the rhesus monkeys. It was found that the neutralizing antibody titer against the live SARS-CoV-2 virus could still reach 4696 at 113 days post the first vaccination in the sera from the rhesus monkeys immunized with CF501/RBD-Fc. In contrast, the neutralizing antibody titer against the live SARS-CoV-2 virus was only 439 at day 113 post the first vaccination in the sera from the rhesus monkeys immunized with Alum/RBD-Fc (
The rhesus monkeys were vaccinated for the third time at 115 days after the first vaccination of the rhesus monkeys, and then the neutralizing antibody titers against the live SARS-CoV-2 virus in the sera were detected at 122 days after the first vaccination of the rhesus monkeys. It is found that the neutralizing antibody titer against the live SARS-CoV-2 virus in the sera from rhesus monkeys vaccinated with the RBD-Fc protein mixed with CF501 reaches 134827. In contrast, the neutralizing antibody titer against the live SARS-CoV-2 virus in the sera from rhesus monkeys vaccinated with the RBD-Fc protein mixed with the aluminum adjuvant reaches 9771. The neutralizing antibody titers in sera were continuously monitored by 191 days after the first vaccination of the rhesus monkeys. The neutralizing antibody titer against the live SARS-CoV-2 virus in the sera is 39746 even at Day 191 after the vaccination of the rhesus monkeys with the RBD-Fc protein mixed with CF501. In contrast, the neutralizing antibody titer against the live SARS-CoV-2 virus in the sera is 2153 after the vaccination of the rhesus monkeys with the RBD-Fc protein mixed with the aluminum adjuvant (
The pseudoviruses of SARS-CoV-2 variants or mutants were prepared as described in Example 9. The neutralization activities against pseudoviruses of 9 SARS-CoV-2 variants and 41 SARS-CoV-2 single-point mutants were detected in the sera at 28 days after vaccination of rhesus monkeys (at 7 days after the second vaccination). Results are shown in
Recently, the Omicron variant becomes the main epidemic strain. Thus, the binding ability and the neutralizing antibody titers to the Omicron pseudovirus in the sera were also detected from 28 days to 191 days after the vaccination of the rhesus monkeys. The results are shown in
The neutralizing activities against the live Omicron virus in the sera at 122 days after the first vaccination of the rhesus monkeys were also detected. The results are shown in
1. Packaging of SARS-Related Pseudoviruses (SARS-CoV, WIV1 and Rs3367).
2. Evaluation of Inhibition of SARS-CoV, WIV1, and Rs3367 Pseudoviruses by Sera
The results are shown in
The results of viral loads in nasal swabs are shown in
The results of viral loads in the lungs of the rhesus monkeys are shown in
The viral loads in nasal mucosae and turbinates of the rhesus monkeys are shown in
The Balb/c mice were divided into two groups. The first group was vaccinated with the HIV NHR trimer (N3G) (from Wang Chao who works in the Academy of Military Medical Sciences, China), and the second group was vaccinated with the HIV NHR trimer (N3G) and CF501. The mice were boosted once at 14 days after the first vaccination. The sera were collected from the mice at 7 days after the second vaccination. The specific antibodies to the HIV NHR trimer in the mice were tested by the ELISA method.
The results are shown in
The results are shown in
In particular, the ELISA test was performed as follows.
Results as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/095498 | May 2021 | WO | international |
This application is a continuation of PCT/CN2022/094090, filed May 20, 2022 which claims the priority of the PCT application No. PCT/CN2021/095498, filed on May 24, 2021 and titled “Compounds and their use as vaccine adjuvants”, both of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/094090 | May 2022 | US |
| Child | 18465513 | US |
