Patent Application
20230227411
The present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to compounds that modulate cGAS activity.
The invention relates in particular to a compound of formula (I)
wherein
or a pharmaceutically acceptable salt or ester thereof.
Cytokines are responsible for modulation of the innate immune response and the dysregulation of pro-inflammatory cytokines has been associated with severe systemic inflammation and autoimmune diseases, many of which lack efficient therapy as of today.
Vertebrates possess an innate and adaptive immune system as protection against pathogens and other challenges. The innate immune system is an evolutionary old system that is present beyond vertebrates. Unlike the adaptive immune system, it does not require priming or training, but works as a general physical barrier (e.g. skin) or by detection of specific patterns. One universal pattern to trigger the innate immune system is the detection of cytosolic double stranded DNA, which leads to Type I Interferon response. Sources of cytosolic dsDNA could be from bacterial or viral infection but as well accumulated self-DNA.
The cytosolic enzyme cyclic GMP-AMP Synthase (cGAS) is a sensor for cytosolic double stranded DNA. Binding of dsDNA results in the generation of the cyclic di-nucleotide 2,3-cGAMP by enzymatic linkage of ATP and GTP. 2,3-cGAMP acts as secondary messenger and binds to the Stimulator of Interferon Genes (STING), which resides in the endoplasmatic reticulum. Upon binding of 2,3-cGAMP, STING translocates to the perinuclear Golgi, where it associates with the TANK binding kinase 1 (TBK1) and recruits and phosphorylates Interferon Response Factor 3 (IRF3). Ultimately this results in the production of Type I Interferon (I IFN), other cytokines like IL-6, TNFα, IL1β and chemokines—essential factors for host defense against invading pathogens. However, inappropriate or chronic production of type I IFN and other pro-inflammatory cytokines are associated with severe systemic inflammation and autoimmune diseases. For instance, IFN signaling is involved in SLE, cutaneous skin diseases (dermatomyositis, and cutaneous lupus), interstitial pulmonary fibrosis, Sjogren syndrome, and type I diabetes (G. Trinchieri, J Exp Med. 2010 207(10): 2053-63). Other pro-inflammatory cytokine such as TNFα and IL1β play an important role in inflammatory bowel disease, NASH, juvenile inflammatory arthritis, ankylosing spondylitis and gout.
Chronic activation of cGAS/STING causes severe systemic inflammation. Evidence for its role in inflammation in the clinic comes from monogenic diseases. Patients with deficiencies in nucleic acid modifying enzymes, like Trex1, RNaseH2 and SAMHD1, suffer from Aicardi-Goutieres syndrome (AGS). The involvement of cGAS/STING was supported in Trexl deficient mice that serve as a model for AGS.
Inhibition of the cGAS pathway which is upstream from the disease mediating cytokines is therefore a novel strategy in treating patients from multiple autoimmune diseases. Indications could include those linked to IFN signaling or those driven by TNFα and IL1β.
As of today many diseases caused by dysregulation of the innate immune system lack efficient therapies.
The compound of the invention binds to and modulates cGAS activity.
The compound of formula (I) is particularly useful in the treatment or prophylaxis of e.g. systemic lupus erythrematosus (SLE), cutaneous skin diseases like dermatomyositis or cutaneous lupus, interstitial pulmonary fibrosis, Sjogren syndrome, type I diabetes, inflammatory bowel disease, non-alcoholic steatohepatitis (NASH), juvenile inflammatory arthritis, ankylosing spondylitis, gout or Aicardi-Goutieres syndrome (AGS).
In the present description the term “alkyl”, alone or in combination, signifies a straight-chain or branched-chain alkyl group with 1 to 8 carbon atoms, particularly a straight or branched-chain alkyl group with 1 to 6 carbon atoms and more particularly a straight or branched-chain alkyl group with 1 to 4 carbon atoms. Examples of straight-chain and branched-chain C1-C8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert.-butyl, sec.-butyl, the isomeric pentyls, the isomeric hexyls, the isomeric heptyls and the isomeric octyls, particularly methyl, ethyl, propyl, butyl and pentyl. Particular examples of alkyl are methyl, ethyl and propyl. Methyl and ethyl are particular examples of “alkyl” in the compound of formula (I).
The term “cycloalkyl”, alone or in combination, signifies a cycloalkyl ring with 3 to 8 carbon atoms and particularly a cycloalkyl ring with 3 to 6 carbon atoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, cycloheptyl and cyclooctyl. A particular example of “cycloalkyl” is cyclopropyl.
The term “alkoxy” or “alkyloxy”, alone or in combination, signifies a group of the formula alkyl-O— in which the term “alkyl” has the previously given significance, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert.-butoxy. A particular example of “alkoxy” is methoxy.
The term “oxy”, alone or in combination, signifies the —O— group.
The terms “halogen” or “halo”, alone or in combination, signifies fluorine, chlorine, bromine or iodine and particularly fluorine, chlorine or bromine, more particularly fluorine or chlorine. The term “halo”, in combination with another group, denotes the substitution of said group with at least one halogen, particularly substituted with one to five halogens, particularly one to four halogens, i.e. one, two, three or four halogens.
The term “haloalkyl”, alone or in combination, denotes an alkyl group substituted with at least one halogen, particularly substituted with one to five halogens, particularly one to three halogens. A particular “haloalkyl” is fluoroethyl.
The term “carbonyl”, alone or in combination, signifies the —C(O)— group.
The term “amino”, alone or in combination, signifies the primary amino group (—NH2), the secondary amino group (—NH—), or the tertiary amino group (—N—).
The term “alkylamino”, alone or in combination, denotes an amino group substituted with at least one alkyl. Particular “aminoalkyl” are aminomethyl, aminoethyl and aminopropyl. Preferred “aminoalkyl” are aminoethyl and aminopropyl.
The term “aminocarbonyl”, alone or in combination, signifies the —C(O)—NH2 group.
The term “sulfonyl”, alone or in combination, signifies the —SO2— group.
The term “pharmaceutically acceptable salts” refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. The salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, particularly hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein. In addition these salts may be prepared form addition of an inorganic base or an organic base to the free acid. Salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. The compound of formula (I) can also be present in the form of zwitterions. Particularly preferred pharmaceutically acceptable salts of compounds of formula (I) are the salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, sodium and potassium.
The term “pharmaceutically acceptable esters” means that compounds of general formula (I) may be derivatised at functional groups to provide derivatives which are capable of conversion back to the parent compounds in vivo. Examples of such compounds include physiologically acceptable and metabolically labile ester derivatives, such as methoxymethyl esters, methylthiomethyl esters and pivaloyloxymethyl esters.
Additionally, any physiologically acceptable equivalents of the compounds of general formula (I), similar to the metabolically labile esters, which are capable of producing the parent compounds of general formula (I) in vivo, are within the scope of this invention.
If one of the starting materials or compounds of formula (I) contain one or more functional groups which are not stable or are reactive under the reaction conditions of one or more reaction steps, appropriate protecting groups (as described e.g. in “Protective Groups in Organic Chemistry” by T. W. Greene and P. G. M. Wuts, 3rd Ed., 1999, Wiley, N.Y.) can be introduced before the critical step applying methods well known in the art. Such protecting groups can be removed at a later stage of the synthesis using standard methods described in the literature. Examples of protecting groups are tert-butoxycarbonyl
(Boc), 9-fluorenylmethyl carbamate (Fmoc), 2-trimethylsilylethyl carbamate (Teoc), carbobenzyloxy (Cbz) and p-methoxybenzyloxycarbonyl (Moz). A particularly preferred protecting group is tert-butoxycarbonyl (Boc).
The compound of formula (I) can contain several asymmetric centers and can be present in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
The term “asymmetric carbon atom” means a carbon atom with four different substituents. According to the Cahn-Ingold-Prelog Convention an asymmetric carbon atom can be of the “R” or “S” configuration.
The invention thus relates to:
A compound according to the invention wherein R1 is phenyl, methylcarbonylaminopropyl, methylsulfonylaminopropyl, chlorophenylmethyl, pyridinylmethyl, diaminoethyl, methoxyethyl, cyclopropyl, ethyl, trifluoroethyl, phenylmethyl or phenylethyl;
A compound according to the invention wherein R1 is phenylalkyl;
A compound according to the invention wherein R1 is phenylmethyl or phenylethyl;
A compound according to the invention wherein R2 is hydrogen;
A compound according to the invention wherein R3 is halogen; and
A compound according to the invention wherein R3 is chlorine;
A compound of formula (I) selected from
5-(2-chloro-4-methylphenyl)-1-phenyl-1H-benzo[d]imidazole-7-carboxylic acid;
1-(3-acetamidopropyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylic acid;
5-(2-chloro-4-methylphenyl)-1-(3-(methylsulfonamido)propyl)-1H-b enzo[d]imidazole-7-carboxylic acid;
5-(2-chloro-4-methylphenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-7-carboxylic acid;
5-(2-chloro-4-methylphenyl)-1-(pyridin-4-ylmethyl)-1H-benzo[d]imidazole-7-carboxylic acid;
5-(2-chloro-4-methylphenyl)-1-(pyridin-3-ylmethyl)-1H-benzo[d]imidazole-7-carboxylic acid;
6-(2-chloro-4-methylphenyl)-3-(pyridin-2-ylmethyl)benzimidazole-4-carboxylic acid;
5-(2-chloro-4-methylphenyl)-1-(2-(dimethylamino)ethyl)-1H-b enzo[d]imidazole carboxylic acid;
5-(2-chloro-4-methylphenyl)-1-(2-methoxyethyl)-1H-benzo[d]imidazole-7-carboxylic acid;
5-(2-chloro-4-methylphenyl)-1 -cyclopropyl- 1H-benzo[d]imidazole-7-carboxylic acid;
5-(2-chloro-5 -fluoro-4-methylphenyl)-1-ethyl-1 H-benzo[d]imidazole-7-carboxylic acid;
5-(2-chloro-4-methylphenyl)-1-ethyl-1H-benzo[d]imidazole-7-carboxylic acid;
5-(2-chloro-4-methylphenyl)-1-(2,2,2-trifluoroethyl)-1H-benzo[d]imidazole-7-carboxylic acid;
5-(2-chloro-4-methylphenyl)-1-phenethyl-1H-benzo[d]imidazole-7-carboxylic acid; and
1-benzyl-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylic acid;
or a pharmaceutically acceptable salt or ester thereof.
A compound of formula (I) selected from
5-(2-chloro-4-methylphenyl)-1-phenethyl-1H-benzo[d]imidazole-7-carboxylic acid; and
1-benzyl-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylic acid;
or a pharmaceutically acceptable salt or ester thereof.
The synthesis of the compound of formula (I) can, for example, be accomplished according to the following schemes.
The compound of formula (I) according to the invention, wherein R1 is phenyl, can be prepared according to scheme 1.
In scheme 1 R1 is phenyl. R2 and R3 are as defined above, R4 is alkyl, and R5 is hydrogen or alkyl.
In scheme 1, conveniently R4 is methyl.
In scheme 1, conveniently R5 is hydrogen.
Step A: The nucleophilic substitution can be accomplished by reaction of the corresponding ortho-fluoro-nitro derivative 1 with the amine 2 at 20° C. to 140° C. with or without addition of an additional base such as triethylamine, ethyldiisopropylamine or the like in a suitable solvent such as dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran, dimethoxyethane, diglyme, ethanol or methanol for 5 min to 18 h with or without microwave irradiation.
Convenient conditions are heating of the fluoro-nitro-derivative with the amine in dimethylformamide at 110° C. for 1 h.
Step B: Coupling of the bromoderivative 3 with a suitable boronic acid or boronic acid ester 4 can be accomplished by using a palladium catalyst such as palladium(II)-acetate, palladium(II)-chloride, 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex, tris(dibenzylideneacetone)dipalladium, tris(dibenzylideneacetone)dipalladium-chloroform adduct, or tetrakis(triphenylphosphine)palladium(0) in combination with a ligand such as triphenylphosphine, tricyclohexylphosphine, X-phos, Xantphos or the like, and a base such as potassium phosphate, potassium carbonate, cesium carbonate, triethylamine or diisopropylethylamine in a suitable solvent such as dioxane, toluene, dimethylacetamide, dimethylformamide, tetrahydrofuran, dimethoxyethane, diglyme, ethanol, methanol, water or mixtures of the solvents mentioned above at 20° C. to 180° C. for 5 min to 18 hrs with or without microwave irradiation.
Convenient conditions are the use of tris(dibenzylideneacetone)dipalladium-chloroform adduct, X-phos and potassium phosphate in a mixture of dioxane and water at 110° C. for 2 h.
Step C: Reduction of the nitro group can be accomplished by using hydrogen gas and a catalyst such as nickel, platinum, palladium or Pd/C (palladium on carbon 5-10%) in ethyl acetate, methanol or ethanol at atmospheric or elevated pressure at 0° C. to 70° C. Furthermore it can be accomplished by a variety of different other reducing agents such as using iron and an acid such as hydrochlorid acid in water, or iron and aqueous ammoniumchlorid solution, or tin(II)chloride in ethanol or ethylacetate, or sodium dithionite in water at room temperature or elevated temperatures.
Convenient conditions are the use of using hydrogen gas and Pd/C (10%) in ethyl acetate at atmospheric pressure at 20° C. for 18 h.
Step D: Ring closure to form the benzimidazole 7 can be accomplished by reacting the diamino compound with formic acid or a formic acid derivative such as triethyl orthoformate, trimethyl orthoformate or dimethylformamide dimethylacetal at room temperature or elevated temperatures with or without an additional solvent.
Preferred conditions are heating the diamino compound in formic acid for 5 min.
Step E: Saponification can be accomplished by reaction of the alkyl ester 7 with a base such as lithium hydroxide, sodium hydroxide, potassium hydroxide or the like in a suitable solvent such as water, tetrahydrofuran, ethanol, methanol or mixtures thereof for 1-18 h at 0° C. to 70° C. Saponification can be furthermore accomplished by reacting the alkyl ester 7 with an acid such as hydrobromic acid or hydrochlorid acid in water or acetic acid or a mixture thereof at 20° C.-110° C. for 1-24 h.
Advantageous conditions are the use of lithium hydroxide in a mixture of tetrahydrofuran and water at 65° C. for 18 h.
The compound of formula (I) according to the invention, wherein R1 is not phenyl, can be prepared according to scheme 2.
In scheme 2 R1-R3 are as defined above, R4 is alkyl, and R5 is hydrogen or alkyl.
In scheme 2, conveniently R4 is methyl.
In scheme 2, conveniently R5 is hydrogen.
Step A: Coupling of the bromoderivative 1 with a suitable boronic acid or boronic acid ester 2 can be accomplished by using a palladium catalyst such as palladium(II)-acetate, palladium(II)-chloride, 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex, tris(dibenzylideneacetone)dipalladium, tris(dibenzylideneacetone)dipalladium-chloroform adduct, or tetrakis(triphenylphosphine)palladium(0) in combination with a ligand such as triphenylphosphine, tricyclohexylphosphine, X-phos, Xantphos or the like, and a base such as potassium phosphate, potassium carbonate, cesium carbonate, triethylamine or diisopropylethylamine in a suitable solvent such as dioxane, toluene, dimethylacetamide, dimethylformamide, tetrahydrofuran, dimethoxyethane, diglyme, ethanol, methanol, water or mixtures of the solvents mentioned above at 20° C. to 180° C. for 5 min to 18 hrs with or without microwave irradiation.
Convenient conditions are the use of tris(dibenzylideneacetone)dipalladium-chloroform adduct, X-phos and potassium phosphate in a mixture of dioxane and water at 100° C. for 2-4 h.
Step B: The alkylation/benzylation can be accomplished by reacting the benzimidazole 3 with an optionally substituted alkyl halide or an optionally substituted benzyl halide 4, such as substituted alkyl chlorides, substituted alkyl bromides, substituted alkyl iodides, substituted alkyl, substituted benzyl bromides, substituted benzyl chlorides or the like and a base such as cesium carbonate, potassium carbonate, sodium carbonate, triethylamine or ethyldiisopropylamine in a solvent such as as dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran at 0° C.-150° C. for 1 h to 18 h. If regioisomeric mixtures of alkylation products are obtained they can be separated by column chromatography on silica gel using mixtures of organic solvents such as heptane, ethylacetate, methanol and dichloromethane to yield the described regioisomer as a pure compound.
The alkylation can furthermore be accomplished by reaction of the benzimidazole 3 with a suitable boronic acid or boronic acid ester 4 (X═B(OR5)2) using a copper catalyst such as copper(II) acetate, a suitable ligand such as 2,2-bipyridyl and a base such as potassium phosphate, potassium carbonate, cesium carbonate or sodium hexamethyldisilazane in a suitable solvent such as dichloroethane, dichloromethane, toluene, dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran or dimethoxyethane at 40° C.-120° C. for several hours.
Convenient conditions are the use of substituted alkyl bromides or benzyl bromides and cesium carbonate in dimethylformamide at 75° C. for 3 h or at room temperature for 18 h followed by column chromatography on silica gel.
Step C: The alkylation/benzylation can be accomplished by reacting the benzimidazole 1 an optionally substituted alkyl halide or an optionally substituted benzyl halide 4 such as substituted alkyl bromides, substituted alkyl iodides, substituted benzyl bromides, substituted benzyl chlorides or the like and a base such as cesium carbonate, potassium carbonate, sodium carbonate, triethylamine or ethyldiisopropylamine in a solvent such as as dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran at 0° C.-150° C. for 1 h to 18 h. If regioisomeric mixtures of alkylation products are obtained they can be separated by column chromatography on silica gel using mixtures of organic solvents such as heptane, ethylacetate, methanol and dichloromethane to yield the described regioisomer as a pure compound.
The alkylation can furthermore be accomplished by reaction of the benzimidazole 1 with a suitable boronic acid or boronic acid ester 4 (X═B(OR5)2) using a copper catalyst such as copper(II) acetate, a suitable ligand such as 2,2-bipyridyl and a base such as potassium phosphate, potassium carbonate, cesium carbonate or sodium hexamethyldisilazane in a suitable solvent such as dichloroethane, dichloromethane, toluene, dioxane, dimethylacetamide, dimethylformamide, tetrahydrofuran or dimethoxyethane at 40° C.-120° C. for several hours.
Convenient conditions are the use of substituted alkyl bromides or benzyl bromides in dimethylformamide at 75° C. for 3 h or at room temperature for 18 h followed by column chromatography on silica gel.
Step D: Coupling of the bromoderivative 5 with a suitable boronic acid or boronic acid ester 2 can be accomplished by using a palladium catalyst such as palladium(II)-acetate, palladium(II)-chloride, 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex, tris(dibenzylideneacetone)dipalladium, tris(dibenzylideneacetone)dipalladium-chloroform adduct, or tetrakis(triphenylphosphine)palladium(0) in combination with a ligand such as triphenylphosphine, tricyclohexylphosphine, X-phos, Xantphos or the like, and a base such as potassium phosphate, potassium carbonate, cesium carbonate, triethylamine or diisopropylethylamine in a suitable solvent such as dioxane, toluene, dimethylacetamide, dimethylformamide, tetrahydrofuran, dimethoxyethane, diglyme, ethanol, methanol, water or mixtures of the solvents mentioned above at 20° C. to 180° C. for 5 min to 18 hrs with or without microwave irradiation.
Convenient conditions are the use of tris(dibenzylideneacetone)dipalladium-chloroform adduct, X-phos and cesium phosphate in a mixture of dioxane and water at 100° C. for 2-4 h.
Step E: Saponification can be accomplished by reaction of the alkyl ester 6 with a base such as lithium hydroxide, sodium hydroxide, potassium hydroxide or the like in a suitable solvent such as water, tetrahydrofuran, ethanol, methanol or mixtures thereof for 1-18 h at 0° C. to 70° C. Saponification can be furthermore accomplished by reacting the alkyl ester 7 with an acid such as hydrobromic acid or hydrochlorid acid in water or acetic acid or a mixture thereof at 20° C.-110° C. for 1-24 h.
Advantageous conditions are the use of lithium hydroxide in a mixture of tetrahydrofuran and water at 65° C. for 18 h.
The invention thus also relates to a process for the preparation of a compound according to the invention, comprising the saponification of a compound of formula (A1)
in the presence of a base or a acid;
wherein R1, R2 and R3 are as defined above and R4 is alkyl.
R4 is conveniently methyl.
The solvent of the saponification is conveniently water, tetrahydrofuran, ethanol, methanol, acetic acid or mixtures thereof.
In the saponification the base can be for example lithium hydroxide, sodium hydroxide or potassium hydroxide.
In the saponification the acid can be for example hydrobromic acid or hydrochlorid acid.
Convenient conditions for the saponification are 0° C.-110° C. for 1-24 h.
Preferred conditions for saponification under basic conditions are the use of lithium hydroxide in a mixture of tetrahydrofuran and water at 65° C. for 18 h.
Preferred conditions for saponification under acid conditions are the use of hydrobromic acid in acetic acid at 110° C. for 18 h.
The invention also relates to a compound according to the invention when manufactured according to a process of the invention.
Another embodiment of the invention provides a pharmaceutical composition or medicament containing a compound of the invention and a therapeutically inert carrier, diluent or excipient, as well as a method of using the compounds of the invention to prepare such composition and medicament. In one example, the compound of formula (I) may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form. The pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8. In one example, a compound of formula (I) is formulated in an acetate buffer, at pH 5. In another embodiment, the compound of formula (I) is sterile. The compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous solution.
Compositions are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
The compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
The compounds of the present invention may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
A typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
The invention also relates in particular to:
The use of a compound of formula (I) for the treatment or prophylaxis of systemic lupus erythrematosus (SLE), cutaneous skin diseases like dermatomyositis or cutaneous lupus, interstitial pulmonary fibrosis, Sjogren syndrome, type I diabetes, inflammatory bowel disease, non-alcoholic steatohepatitis (NASH), juvenile inflammatory arthritis, ankylosing spondylitis, gout or Aicardi-Goutieres syndrome (AGS);
The use of a compound of formula (I) for the preparation of a medicament for the treatment or prophylaxis of systemic lupus erythrematosus (SLE), cutaneous skin diseases like dermatomyositis or cutaneous lupus, interstitial pulmonary fibrosis, Sjogren syndrome, type I diabetes, inflammatory bowel disease, non-alcoholic steatohepatitis (NASH), juvenile inflammatory arthritis, ankylosing spondylitis, gout or Aicardi-Goutieres syndrome (AGS);
A compound of formula (I) for use in the treatment or prophylaxis of systemic lupus erythrematosus (SLE), cutaneous skin diseases like dermatomyositis or cutaneous lupus, interstitial pulmonary fibrosis, Sjogren syndrome, type I diabetes, inflammatory bowel disease, non-alcoholic steatohepatitis (NASH), juvenile inflammatory arthritis, ankylosing spondylitis, gout or Aicardi-Goutieres syndrome (AGS); and
A method for the treatment or prophylaxis of systemic lupus erythrematosus (SLE), cutaneous skin diseases like dermatomyositis or cutaneous lupus, interstitial pulmonary fibrosis, Sjogren syndrome, type I diabetes, inflammatory bowel disease, non-alcoholic steatohepatitis (NASH), juvenile inflammatory arthritis, ankylosing spondylitis, gout or Aicardi-Goutieres syndrome (AGS), which method comprises administering an effective amount of a compound of formula (I) to a patient in need thereof.
The invention will now be illustrated by the following examples which have no limiting character.
Abbreviations
DCM=dichloromethane; DIPEA=diisopropylethylamine; DMSO=dimethyl sulfoxide; ESI=electrospray ionization; EtOAc=ethyl acetate; MeOH=methanol; MS=mass spectrometry; RT=room temperature; THF=tetrahydrofuran.
a) Methyl 5-bromo-3-nitro-2-(phenylamino)benzoate
In a 25 ml vial, methyl 5-bromo-2-fluoro-3-nitrobenzoate (1 g, 3.6 mmol, Eq: 1) was combined with dimethylformamide (10 ml). Aniline (1.07 g, 1.05 ml, 11.5 mmol, Eq: 3.2) was added, the vial was closed and heated to 110° C. for 1 h. The crude reaction mixture was concentrated in vacuo, poured into 100 ml 2M HCl and extracted with EtOAc (3×150 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo. The crude material was purified by chromatography (silica gel, 40 g, 0% to 40% EtOAc in heptane) to afford the title compound methyl 5-bromo-3-nitro-2-(phenylamino)benzoate (1.11 g, 3.13 mmol, 87.1% yield) as red solid, MS (ESI): 352.96 [M+H]+.
b) Methyl 2′-chloro-4′-methyl-5-nitro-4-(phenylamino)-[1,1′-biphenyl]-3-carboxylate
To methyl 5-bromo-3-nitro-2-(phenylamino)benzoate (500 mg, 1.42 mmol, Eq: 1) in dioxane (8 ml) was added (2-chloro-4-methylphenyl)boronic acid (243 mg, 1.42 mmol, Eq: 1). Potassium phosphate (604 mg, 2.85 mmol, Eq: 2) dissolved in water (2 ml) was added. The mixture was degassed during 2 min before X-Phos (33.9 mg, 71.2 μmol, Eq: 0.05) and tris(dibenzylideneacetone)dipalladium-chloroform adduct (36.8 mg, 35.6 μmol, Eq: 0.025) were added. The mixture was heated to 110° C. for 2 hours. The reaction mixture was poured into 100 ml of water and extracted with EtOAc (3×100 ml). The organic layers were dried over MgSO4 and concentrated in vacuo. The crude material was purified by chromatography (silica gel, 80 g, 0% to 30% EtOAc in heptane) to afford the title compound methyl 2′-chloro-4′-methyl-5-nitro-4-(phenylamino)-[1,1′-biphenyl]-3-carboxylate (520 mg, 966 μmol, 67.9% yield) as red solid, MS (ESI): 397.14 [M+H]+.
c) Amino-2′-chloro-4′-methyl-4-(phenylamino)-[1,1′-biphenyl]-3-carboxylate
In a 25 ml round-bottomed flask, methyl 2′-chloro-4′-methyl-5-nitro-4-(phenylamino)-[1,1′-biphenyl]-3-carboxylate (450 mg, 1.13 mmol, Eq: 1) was combined with ethyl acetate (9 ml). The air was replaced several times with argon before Pd-C (54.3 mg, 51 μmol, Eq: 0.045) was added, then argon was replaced several times with H2. The reaction was stirred at room temperature overnight. For work-up H2 was exchanged with argon and the reaction was filtered. The solution was concentrated and the residue dried in vacuo. The crude material was purified by chromatography (silica gel, 12 g, 0% to 50% EtOAc in heptane) to afford the title compound methyl 5-amino-2′-chloro-4′-methyl-4-(phenylamino)-[1,1′-biphenyl]-3-carboxylate (306 mg, 763 μmol, 67.2% yield) as light yellow solid, MS (ESI): 367.18 [M+H]+.
d) Methyl 5-(2-chloro-4-methylphenyl)-1-phenyl-1H-benzo[d]imidazole-7-carboxylate
In a 10 ml round-bottomed flask, methyl 5-amino-2′-chloro-4′-methyl-4-(phenylamino)-[1,1′-biphenyl]-3-carboxylate (50 mg, 136 μmol, Eq: 1) and formic acid (627 mg, 523 μl, 13.6 mmol, Eq: 100) were combined. The reaction mixture was heated to reflux for 5 min, then poured slowly into 20 ml saturated NaHCO3 solution and extracted with EtOAc (3×25 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo. The crude material was purified by chromatography (silica gel, 12 g, 0% to 50% EtOAc in heptane) to obtain the title compound methyl 5-(2-chloro-4-methylphenyl)-1-phenyl-1H-benzo[d]imidazole-7-carboxylate (34 mg, 83 μmol, 60.9% yield) as white solid, MS (ESI): 377.15 [M+H]+.
e) 5-(2-Chloro-4-methylphenyl)-1-phenyl-1H-benzo[d]imidazole-7-carboxylic acid
To a solution of methyl 5-(2-chloro-4-methylphenyl)-1-phenyl-1H-benzo[d]imidazole-7-carboxylate (26.6 mg, 70.6 μmol, Eq: 1) in THF (700 μl) was added LiOH (5.92 mg, 141 mol, Eq: 2) dissolved in water (350 μl). The reaction mixture was heated to 65° C. and stirred until completion of the reaction. The reaction was quenched with HCl (70.6 μl, 141 μmol, Eq: 2), 15 ml of water was added and the mixture was extracted with EtOAc (3×25 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo. The crude material was purified by chromatography (silica gel, 12 g, 0% to 100% EtOAc in heptane and then 0-7% MeOH in DCM to afford the title compound 5-(2-chloro-4-methylphenyl)-1-phenyl-1H-benzo[d]imidazole-7-carboxylic acid (15 mg, 38.8 μmol, 55% yield) as white solid, MS (ESI): 363.16 [M+H]+.
a) Methyl 6-bromo-1H-benzo[d]imidazole-4-carboxylate
In a 250 ml round-bottomed flask, 6-bromo-1H-benzo[d]imidazole-4-carboxylic acid (2.9 g, 12 mmol, Eq: 1) was combined with methanol (100 ml) to give a light brown suspension. Sulfuric acid (11.8 g, 6.41 ml, 120 mmol, Eq: 10) was added at 0° C. The reaction mixture was heated to 65° C. and stirred overnight. The crude reaction mixture was concentrated in vacuo. The reaction mixture was poured into 100 ml of saturated NaHCO3 solution and extracted with EtOAc (3×150 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo to afford the title compound methyl 6-bromo-1H-benzo[d]imidazole-4-carboxylate (1.97 g, 7.63 mmol, 63.5% yield) as off-white solid, MS (ESI): 255.04, 257.05 [M+H]+.
b) Methyl 6-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-4-carboxylate
In a 100 ml round-bottomed flask, methyl 6-bromo-1H-benzo[d]imidazole-4-carboxylate (2 g, 7.84 mmol, Eq: 1), (2-chloro-4-methylphenyl)boronic acid (1.34 g, 7.84 mmol, Eq: 1) and potassium phospohate (tribasic) (3.33 g, 15.7 mmol, Eq: 2) were combined with dioxane (40 ml) and water (10 ml). Under argon tris(dibenzylideneacetone)dipalladium-chloroform adduct (203 mg, 196 μmol, Eq: 0.025) and X-phos (187 mg, 392 μmol, Eq: 0.05) were added. The reaction was heated to 100° C. and stirred for 4 h. The reaction mixture was poured into 50 ml of saturated NaHCO3 solution and extracted with EtOAc (3×100 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo. The crude material was purified by chromatography (silica gel, 80 g, 0% to 80% EtOAc in heptane to afford the title compound methyl 6-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-4-carboxylate (852 mg, 2.69 mmol, 68.6% yield) as white solid, MS (ESI): 301.12 [M+H]+.
c) Methyl 1-(3-((tert-butoxycarbonyl)amino)propyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylate
In a 50 ml round-bottomed flask, methyl 6-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-4-carboxylate (780 mg, 2.46 mmol, Eq: 1) and cesium carbonate (1.27 g, 3.89 mmol, Eq: 1.58) were combined with dimethylformamide (20 ml). tert-butyl (3-bromopropyl)carbamate (927 mg, 3.89 mmol, Eq: 1.58) was added and the reation was stirred at room temperature overnight. The reaction mixture was poured into 150 ml of saturated NaCl solution and extracted with EtOAc (3×200 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo. The crude material was purified by chromatography (silica gel, 40 g, 0% to 100% EtOAc in heptane to afford the title compound methyl 1-(3-((tert-butoxycarbonyl)amino)propyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole carboxylate (571 mg, 1.25 mmol, 50.6% yield) as off-white solid, MS (ESI): 458.185 [M+H]+.
d) Methyl 1-(3-aminopropyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylate hydrochloride
In a 25 ml round-bottomed flask, methyl 1-(3-((tert-butoxycarbonyl)amino)propyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylate (571 mg, 1.25 mmol, Eq: 1) and HCl in dioxane (1.56 ml, 6.23 mmol, Eq: 5) were combined with dioxane (8 ml). The reaction was stirred at room temperature overnight. The reaction was concentrated in vacuo to afford the title compound methyl 1-(3-aminopropyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylate hydrochloride (315 mg, 880 μmol, 70.6% yield) as white foam which was used in the next step.
e) Methyl 1-(3-acetamidopropyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylate
In a 5 ml round-bottomed flask, methyl 1-(3-aminopropyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylate (60 mg, 168 μmol, Eq: 1) and DIPEA (65 mg, 87.9 μl, 503 μmol, Eq: 3) were combined with dichloromethane (2 ml). At 0° C. acetyl chloride (14.5 mg, 13.1 μl, 184 μmol, Eq: 1.1) was added. The reaction was stirred for 1 h at room temperature. The reaction mixture was poured into 20 ml saturated NaHCO3 solution and extracted with EtOAc (3×25 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo. The crude material was purified by chromatography (silica gel, 12 g, 0% to 10% MeOH in DCM to afford the title compound methyl 1-(3-acetamidopropyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylate (17.7 mg, 42.2 μmol, 25.2% yield) as white solid, MS (ESI): 400.25 [M+H]+.
f) 1-(3-Acetamidopropyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylic acid
To a light yellow solution of methyl 1-(3-acetamidopropyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylate (17.7 mg, 44.3 μmol, Eq: 1) in tetrahydrofuran (500 μl) was added lithium hydroxide monohydrate (3.71 mg, 88.5 μmol, Eq: 2) dissolved in water (250 μl). The mixture was heated to 65° C. and stirred overnight. The mixture was concentrated in vacuo. The residue was taken up with methyltetrahydrofuran (2 ml) and the formed crystals were filtered through a Sartorius funnel to obtain the title compound 1-(3-acetamidopropyl)-5-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-7-carboxylic acid (17.9 mg, 44.3 μmol, 100% yield) as an off-white solid, MS (ESI): 386.14 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 2 using methylsulfonyl chloride instead of acetyl chloride in step e), white solid, MS (ESI): 422.21 [M+H]+.
a) Methyl 5-(2-chloro-4-methylphenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-7-carboxylate
In a 25 ml round-bottomed flask, methyl 6-(2-chloro-4-methylphenyl)-1H-benzo[d]imidazole-4-carboxylate (see Example 2, 150 mg, 474 μmol, Eq: 1) and cesium carbonate (247 mg, 758 μmol, Eq: 1.6) were combined with dimethylformamide (3.5 ml). 3-Chlorobenzyl bromide (97.4 mg, 62.1 μl, 474 μmol, Eq: 1) was added and the reaction was stirred at 75° C. for 3 h. The reaction mixture was poured into 20 ml saturated NaCl solution and extracted with EtOAc (3×50 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo. The crude material was purified by chromatography (silica gel, 12 g, 0% to 10% MeOH in DCM to afford the title compound methyl 5-(2-chloro-4-methylphenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-7-carboxylate (102 mg, 240 μmol, 50.6% yield), MS (ESI): 425.2 [M+H]+.
b) 5-(2-Chloro-4-methylphenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-7-carboxylic acid
In a 5 ml vial, methyl 5-(2-chloro-4-methylphenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-7-carboxylate (95 mg, 223 μmol, Eq: 1) and LiOH monohydrate (14.1 mg, 335 μmol, Eq: 1.5) were combined with THF (1.2 ml) and water (600 μl). The reaction mixture was stirred at room temperature for 3 h. For work-up HCl (1M, 335 μl, 335 μmol, Eq: 1.5) was added. The reaction mixture was poured into 20 ml of water and extracted with EtOAc (3×25 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo to afford the title compound 5-(2-chloro-4-methylphenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-7-carboxylic acid (86.6 mg, 204 μmol, 91.5% yield) as white solid, (MS (ESI): 411.19 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 4 using 4-(chloromethyl)pyridine hydrochloride instead of 3-chlorobenzyl bromide in step a), white solid, MS (ESI): 378.17 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 4 using 3-(chloromethyl)pyridine hydrochloride instead of 3-chlorobenzyl bromide in step a), white solid, MS (ESI): 378.17 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 4 using 2-(chloromethyl)pyridine hydrochloride instead of 3-chlorobenzyl bromide in step a), off-white solid, MS (ESI): 378.100 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 4 using 2-bromo-N,N-dimethylethan-1-amine hydrobromide instead 3-chlorobenzyl bromide in step a), white solid, (MS (ESI): 358.23 [M+H]+.
a) Methyl 5-bromo-1-(2-methoxyethyl)-1H-benzo[d]imidazole-7-carboxylate
In a 25 ml round-bottomed flask, methyl 6-bromo-1H-benzo[d]imidazole-4-carboxylate (250 mg, 903 μmol, Eq: 1) and cesium carbonate (465 mg, 1.43 mmol, Eq: 1.58) were combined with dimethylformamide (8 ml). 1-Bromo-2-methoxyethane (198 mg, 134 μl, 1.43 mmol, Eq: 1.58) was added and the reation was stirred at room temperature overnight. The reaction mixture was poured into 50 ml saturated NaCl solution and extracted with EtOAc (3×75 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo. The crude material was purified by chromatography (silica gel, 12 g, 0% to 80% EtOAc in heptane to afford the title compound methyl 5-bromo-1-(2-methoxyethyl)-1H-benzo[d]imidazole-7-carboxylate (142.6 mg, 455 μmol, 50.4% yield) as white solid, MS (ESI): 313.0, 315.0 [M+H]+.
b) Methyl 5-(2-chloro-4-methylphenyl)-1-(2-methoxyethyl)-1H-benzo[d]imidazole-7-carboxylate
In a 5 ml vial, methyl 5-bromo-1-(2-methoxyethyl)-1H-benzo[d]imidazole-7-carboxylate (50 mg, 160 μmol, Eq: 1), (2-chloro-4-methylphenyl)boronic acid (27.2 mg, 160 μmol, Eq: 1) and potassium phosphate (tribasic) (67.8 mg, 319 μmol, Eq: 2) were combined with dioxane (1 ml) and water (250 μl). The vial was degassed with argon before X-phos (3.81 mg, 7.98 μmol, Eq: 0.05) and tris(dibenzylideneacetone)dipalladium-chloroform adduct (4.13 mg, 3.99 μmol, Eq: 0.025) were added. The vial was closed and the reaction mixture was heated to 110° C. and stirred for 2 h. For work-up the reaction mixture was poured into 20 ml of water and extracted with EtOAc (3×25 ml). The organic layers were combined, dried over MgSO4, filtered through sintered glass, concentrated and dried in vacuo. The crude material was purified by chromatography (silica gel, 12 g, 0% to 5% MeOH in DCM to afford the title compound methyl 5-(2-chloro-4-methylphenyl)-1-(2-methoxyethyl)-1H-benzo[d]imidazole-7-carboxylate (41.5 mg, 107 μmol, 66.9% yield) as off-white solid, MS (ESI): 359.21 [M+H]+.
c) 5-(2-Chloro-4-methylphenyl)-1-(2-methoxyethyl)-1H-benzo[d]imidazole-7-carboxylic acid
In a 10 ml round-bottomed flask, methyl 5-(2-chloro-4-methylphenyl)-1-(2-methoxyethyl)-1H-benzo[d]imidazole-7-carboxylate (38 mg, 106 μmol, Eq: 1) and LiOH monohydrate (6.67 mg, 159 μmol, Eq: 1.5) were combined with THF (0.75 ml) and water (375 μl). The reaction mixture was heated to 65° C. and stirred for 3 h. The reaction was quenched with HCl (0.1M, 159 μl, 159 μmol, Eq: 1.5) poured into 20 ml of water and extracted with EtOAc (3×25 ml). The organic layers were combined, concentrated and dried in vacuo to afford the title compound 5-(2-chloro-4-methylphenyl)-1-(2-methoxyethyl)-1H-benzo[d]imidazole-7-carboxylic acid (35.9 mg, 96.6 μmol, 91.2% yield) as white solid, MS (ESI): 345.12 [M+H]+.
a) Methyl 5-bromo-l-cyclopropyl-1H-benzo[d]imidazole-7-carboxylate
Methyl 6-bromo-1H-benzo[d]imidazole-4-carboxylate (302 mg, 1.18 mmol, Eq: 1), cyclopropylboronic acid (265 mg, 2.96 mmol, Eq: 2.5) and sodium carbonate (314 mg, 2.96 mmol, Eq: 2.5) were suspended in 1,2-dichloroethane (15 ml). At room temperature a solution of copper(II) acetate (263 mg, 1.42 mmol, Eq: 1.2) and 2,2-bipyridyl (224 mg, 1.42 mmol, Eq: 1.2) in 1,2-dichloroethane (20 ml, prepared at 70° C.) was added dropwise. The reaction mixture was stirred at 80° C. (oil bath temperature) overnight. For work-up the reaction mixture was diluted with dichloromethane and washed with saturated NH4Cl solution and saturated NaCl solution, successively. The organic layer was dried with Na2SO4 and concentrated in vacuo. The residue was purifieed by column chromatography (silica gel, 0% to 100% EtOAc in heptane followed by 10% MeOH in CH2Cl2) to yield the title compound methyl 5-bromo-1-cyclopropyl-1H-benzo[d]imidazole-7-carboxylate (16.4 mg, 55.5 μmol, 4.7% yield) as yellow solid, MS (ESI): 295.0, 297.0 [M+H]+.
b) Methyl 5-(2-chloro-4-methylphenyl)-1-cyclopropyl-1H-benzo[d]imidazole-7-carboxylate
Methyl 5-bromo-1-cyclopropyl-1H-benzo[d]imidazole-7-carboxylate (14.7 mg, 49.8 μmol, Eq: 1) and (2-chloro-4-methylphenyl)boronic acid (12.7 mg, 74.7 μmol, Eq: 1.5) were solved in 1,4-dioxane (800 μL) and water (400 μl). Cesium carbonate (65.6 mg, 199 μmol, Eq: 4) was added and the mixture was degassed by bubbling argon through the mixture (5 min), then 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (2.03 mg, 2.49 μmol, Eq: 0.05) was added. The reaction was stirred in a sealed tube at 90° C. (oil bath-temp.) for 15 min. For work-up the mixture was taken up in EtOAc and washed with saturated NH4Cl solution and brine successively, the organic layer was dried over Na2SO4, filtrated and evaporated. The residue was purified by column chromatography (silica gel, 0% to 100% EtOAc in heptane) to yield the title compound methyl 5-(2-chloro-4-methylphenyl)-1-cyclopropyl-1H-benzo[d]imidazole-7-carboxylate (12 mg, 35.2 μmol, 70.7% yield) as a colorless oil, MS (ESI): 341.1 [M+H]+.
c) 5-(2-Chloro-4-methylphenyl)-1-cyclopropyl-1H-benzo[d]imidazole-7-carboxylic acid
Methyl 5-(2-chloro-4-methylphenyl)-1-cyclopropyl-1H-benzo[d]imidazole-7-carboxylate (12 mg, 35.2 μmol, Eq: 1) was dissolved in THF (500 μl). At room temperature LiOH solution (1M, 105.6 μl, 105.6 μmol, Eq: 3) was added. The mixture was stirred for 2 days at 65° C. For work-up the reaction mixture was diluted with water and extracted two times with diethylether, the organic layers were washed with water and the combined aqueous layers were acidified with HCl (2M, 79.2 μl, 158 μmol, Eq: 4.5) and adjusted to pH3. It was extracted two times with 2-methyltetrahydrofuran and the combined organic layers were dried over Na2SO4, filtrated and evaporated to afford the title compound 5-(2-chloro-4-methylphenyl)-1-cyclopropyl-1H-benzo[d]imidazole-7-carboxylic acid (10 mg, 30.6 μmol, 86.9% yield) as white solid, MS (ESI): 327.1 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 9 using ethyl iodide instead of 1-bromo-2-methoxyethane in step a) and (2-chloro-5-fluoro-4-methylphenyl)boronic acid instead of (2-chloro-4-methylphenyl)boronic acid in step b), white solid, MS (ESI): 333.1 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 9 using ethyl iodide instead of 1-bromo-2-methoxyethane in step a), white solid, MS (ESI): 315.1 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 9 using 2,2,2-trifluoroethyl trifluoromethanesulfonate instead of 1-bromo-2-methoxyethane and potassium carbonate instead of cesium carbonate in step a), white solid, MS (ESI): 369.1 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 9 using phenethyl bromide instead of 1-bromo-2-methoxyethane and potassium carbonate instead of cesium carbonate in step a), white solid, MS (ESI): 391.2 [M+H]+.
The title compound was obtained in comparable yield analogous to the procedure described for Example 9 using benzyl bromide instead of 1-bromo-2-methoxyethane in step a), off-white solid, MS (ESI): 377.3 [M+H]+.
Malachite Green Assay to Measure cGAS Activity
Compounds were tested for cGAS inhibition in a coupled enzymatic assay based on Phosphate detection by Malachite Green. Final assay conditions were 20 mM TRIS pH 7.5 (Applichem), 5 mM MgCl2 (Sigma) and 0.01% BSA (Sigma) supplemented with 80 μM ATP (Sigma), 80 μM GTP (Sigma) and 100 nM Interferon Stimulating DNA (ISD) (Microsynth). Recombinantly expressed purified human cGAS (residues 161-522) was used at 25 nM.
All compounds were prepared as 10 mM stock solutions in DMSO and a 16 pt dilution series in DMSO with a dilution factor of 2.5 was prepared. 1 μL of DMSO dilution series was transferred to 32.3 μL reaction buffer, mixed by pipetting up/down, spun for 1 minute at 3000 rpm and was visually inspected for precipitation. 5 μL of 3-fold enzyme stock solution were transferred to an empty 384-well Black/Clear Flat Bottom Polystyrene NBS (Corning) rows 3-24. Rows 1-2 were filled with assay buffer. Plates were spun 10 seconds at 1000 rpm (164×g). 5 μL of compound intermediate dilution was added and mixed by pipetting up/down to rows 3-24. Rows 1-2 were filled with 3.1% DMSO assay buffer. Plates were spun 10 seconds at 1000 rpm (164×g). 5 μL 3-fold Nucleotide/DNA mix was added to all wells to start the reaction. Plates were spun 10 seconds at 1000 rpm (164×g) and incubated for 4 hour at room temperature (RT) in the dark. 5 μL 4 U/mL PPase (Sigma) were added to all wells. Plates spun 10 seconds at 1000 rpm (164×g). 10 μL BioMol green Solution (Enzo Life Sciences) was added to all wells. Plates spun 10 seconds at 1000 rpm (164×g) and incubated 30 minutes at RT in the dark. Absorbance data was collected 620 nm on an EnVision Multilable Reader (Perkin Elmer) and the following measurement settings were used: excitation filter photometric was 620 nm; excitation from the top; measurement height was 1 mm; number of flashes was 30; number of flashes integrated was 1.
All plates are checked for abnormalities and outliers in the Blank Control (no protein, row 1) and the Neutral Control (no compound, row 2) are excluded using the 3*SD rule. Data was normalized to 0 and 100% by Blank and Neutral Control and each curve was fitted and judged using the 4 parameter logistic equation to determine the IC50 for cGAS inhibition.
The results of this assay are provided in Table 1. Table 1 provides IC50 values (μM) for cGAS inhibition obtained for particular examples of the present invention as measured by the above-described assay.
Film coated tablets containing the following ingredients can be manufactured in a conventional manner:
The active ingredient is sieved and mixed with microcrystalline cellulose and the mixture is granulated with a solution of polyvinylpyrrolidone in water. The granulate is then mixed with sodium starch glycolate and magnesium stearate and compressed to yield kernels of 120 or 350 mg respectively. The kernels are lacquered with an aq. solution/suspension of the above mentioned film coat.
Capsules containing the following ingredients can be manufactured in a conventional manner:
The components are sieved and mixed and filled into capsules of size 2.
Injection solutions can have the following composition:
The active ingredient is dissolved in a mixture of Polyethylene glycol 400 and water for injection (part). The pH is adjusted to 5.0 by addition of acetic acid. The volume is adjusted to 1.0 ml by addition of the residual amount of water. The solution is filtered, filled into vials using an appropriate overage and sterilized.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20169771.1 | Apr 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/059625 | 4/14/2021 | WO |
