WO2014154723A1 - Novel pyrrole derivatives for the treatment of cancer - Google Patents

Novel pyrrole derivatives for the treatment of cancer Download PDF

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Publication number
WO2014154723A1
WO2014154723A1 PCT/EP2014/056008 EP2014056008W WO2014154723A1 WO 2014154723 A1 WO2014154723 A1 WO 2014154723A1 EP 2014056008 W EP2014056008 W EP 2014056008W WO 2014154723 A1 WO2014154723 A1 WO 2014154723A1
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Prior art keywords
pyrrole
carboxylic acid
pyridin
hydrogen
methyl
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PCT/EP2014/056008
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French (fr)
Inventor
Xingchun Han
Min Jiang
Alexander V. Mayweg
Lisha Wang
Song Yang
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F. Hoffmann-La Roche Ag
Hoffmann-La Roche Inc.
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Application filed by F. Hoffmann-La Roche Ag, Hoffmann-La Roche Inc. filed Critical F. Hoffmann-La Roche Ag
Publication of WO2014154723A1 publication Critical patent/WO2014154723A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to organic compounds useful for therapy in a mammal, and in particular to inhibit cell proliferation and induce cell cycle arrest and apoptosis that overexpress CDK8 or Cyclin C useful for treating cancer.
  • CDK cyclin-dependent kinase
  • Dysregulation of CDKs has been linked to pathological events and both proliferative and non-pro liferative disease, including cancers, Alzhemers disease (AD), parkinson's disease, Stroke/ischemia, pain, traumatic brain injury, kidney disease, inflammation pathologies, type 2 diabetes, viral infection (HSV, HCMV, HPV, HIV).
  • CDK8 is a CyclinC-dependent CDK family kinase and functions as a transcriptional regulator.
  • RNAPII RNA polymerase II
  • CCD C-terminal domain
  • GTFs general transcription factors
  • CDK8 has also been described as a transcriptional coactivator in oncongenic signaling pathways, including the ⁇ -catenin pathway, the serum response network, the Tumor Growth Factor TGFP signaling pathway, the p53 pathway, as well as in thyroid hormone-dependent transcription. Colocalization of CDK8 and Cyclin C was also reported in neurodegenerative disease such as AD. CDK8 was found to be frequently
  • Objects of the present invention are novel compounds of formula I, their manufacture, medicaments based on a compound in accordance with the invention and their production as well as the use of compounds of formula I for the treatment of cancer.
  • C h alky alone or in combination signifies a saturated, linear- or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, for example methyl, ethyl, propyl, isopropyl, 1 -butyl, 2-butyl, tert-butyl and the like.
  • Particular "C h alky! groups are methyl, ethyl, isopropyl and tert-butyl.
  • amino alone or in combination, refers to primary (-NH 2 ), secondary (-NH-) or
  • halogen means fluorine, chlorine, bromine or iodine. Halogen is particularly fluorine or chlorine.
  • hydroxy alone or in combination refers to the group -OH.
  • the compounds according to the present invention may exist in the form of their pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases.
  • Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like.
  • Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide.
  • the chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin R.J., et al., Organic Process Research & Development 2000, 4, 427-435; or in Ansel, H., et al., In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. Particular are the sodium salts of the compounds of formula I.
  • Racemates can be separated according to known methods into the enantiomers.
  • diastereomeric salts which can be separated by crystallization are formed from the racemic mixtures by reaction with an optically active acid such as e.g. D- or L-tartaric acid, mandelic acid, malic acid, lactic acid or camphorsulfonic acid.
  • the present invention provides (i) the use of a compound of formula (I)
  • R 1 and R 3 J are independently selected from hydrogen; halogen; and C ⁇ aUcyl, which is unsubstituted or substituted by fluoro;
  • R is hydrogen, C ⁇ aUcyl or phenyl-CH 2 -;
  • R 4 is hydrogen, C ⁇ aUcyl, halogen or phenyl
  • W is nitrogen or -CR 5 ; wherein R 5 is hydrogen, C ⁇ aUcyl, amino, halogen or hydroxy; or a pharmaceutically acceptable salt thereof for the preparation of medicaments for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
  • R 1 and R 3 J are independently selected from hydrogen; halogen; and C 1-6 alkyl, which is unsubstituted or substituted by fluoro;
  • R is hydrogen, Ci ⁇ alkyl or phenyl-CH 2 -;
  • R 4 is hydrogen, Ci- 6 alkyl, halogen or phenyl
  • W is nitrogen or -CR 5 ; wherein R 5 is hydrogen, C 1-6 alkyl, amino, halogen or hydroxy; or a pharmaceutically acceptable salt thereof;
  • a further embodiment of the present invention is (iii) a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein
  • R 1 is hydrogen, chloro, methyl or ethyl
  • R is hydrogen, methyl or phenyl-CH 2 -;
  • R is hydrogen, chloro, methyl or trifluoromethyl
  • R 4 is hydrogen, methyl, chloro, bromo or phenyl
  • W is nitrogen or -CR 5 ; wherein R 5 is hydrogen, methyl, amino, chloro or hydroxy.
  • Another embodiment of the present invention is (iv) a compound of formulation (I) or a pharmaceutically acceptable salt thereof, wherein
  • R 1 is hydrogen, halogen or C 1-6 alkyl
  • R is hydrogen, Ci ⁇ alkyl or phenyl-CH 2 -;
  • R is hydrogen, Ci- 6 alkyl, halogen or trifluoromethyl
  • R 4 is hydrogen, Ci- 6 alkyl, halogen or phenyl
  • W is -CR 5 ; wherein R 5 is hydrogen, C 1-6 alkyl, amino, halogen or hydroxy.
  • R 1 is hydrogen, chloro, methyl or ethyl
  • R is hydrogen, methyl or phenyl-CH 2 -;
  • R is hydrogen, chloro, methyl or trifluoromethyl
  • R 4 is hydrogen, methyl, chloro, bromo or phenyl; -CR 5 ; wherein R 5 is hydrogen, methyl, amino, chloro or hydroxy.
  • Another embodiment of present invention is (vi) a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein
  • R 1 is hydrogen
  • R is hydrogen
  • R is C 1-6 alkyl, halogen or trifluoromethyl
  • R 4 is hydrogen or halogen
  • W is -CR 5 ; wherein R 5 is hydrogen, C 1-6 alkyl.
  • R 1 is hydrogen
  • R is hydrogen
  • R is methyl, chloro or trifluoromethyl
  • R 4 is hydrogen or chloro
  • W is -CR 5 ; wherein R 5 is hydrogen or methyl.
  • Another embodiment of present invention is (xiii) a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein
  • R 1 is hydrogen, halogen or C 1-6 alkyl
  • R is hydrogen, C 1-6 alkyl or phenyl-CH 2 -;
  • R is hydrogen
  • R 4 is hydrogen, C 1-6 alkyl, halogen or phenyl
  • W is -CR 5 ; wherein R 5 is hydrogen, C 1-6 alkyl, amino, halogen or hydroxy.
  • R 1 is hydrogen, chloro, methyl or ethyl
  • R is hydrogen, methyl or phenyl-CH 2 -;
  • R is hydrogen
  • R 4 is hydrogen, methyl, chloro, bromo or phenyl
  • W is -CR 5 ; wherein R 5 is hydrogen, methyl, amino, chloro or hydroxy.
  • R 5 is hydrogen, methyl, amino, chloro or hydroxy.
  • the compounds of the present invention can be prepared by any conventional means. Suitable processes for synthesizing these compounds as well as their starting materials are provided in the schemes below and in the examples. All substituents, in particular, R 1 , R 2 , R 3 , R 4 and W are as defined above unless otherwise indicated. Furthermore, and unless explicitly otherwise stated, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to a person of ordinary skill in organic chemistry.
  • R 2 is C ⁇ g alkyl or phenyl-CH 2 - R 1 1 is C 1 -6 alkyl. Methold 6)
  • II-l reacts with halides in the presence of NaH in in an organic solvent at 0 °C to room temperature.
  • organic solvent for example can be THF, DMF or dioxane.
  • Method 2 coupling reaction between II and bis(pinacolato)diboron affords the bronic ester intermediate III.
  • the reaction can be carried out in the presence of palladium catalyst and a suitable base such as KOAc, in a suitable solvent such as 1,4-dioxane, at 50 °C to 150 °C.
  • II-l reacts with Boc 2 0 at 0 °C to room temperature in an organic solvent such as DCM or THF, to afford intermediate II-2, which undergoes coupling reaction with bis(pinacolato)diboron to give intermediate III-l.
  • the coupling reaction can be carried out in the presence of palladium catalyst and a suitable base such as KOAc, in a suitable solvent such as 1,4-dioxane, at 50 to 150 °C.
  • 3-chloro-lH-pyrrole-2- carboxylic acid methyl ester reacts with NBS at room temperature in an organic solvent such as CH 3 CN or THF to give 4-bromo-3-chloro-lH-pyrrole-2-carboxylic acid methyl ester, which is treated with Boc 2 0 in the presence of DMAP at room temperature in an organic solvent to afford 4-bromo-3-chloro-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester.
  • the organic solvent for example can be DCM or THF.
  • the compound of formula I can be prepared according to Scheme 2.
  • Method 8 coupling reaction between compound III and IV affords the intermediate VII.
  • the reaction can be carried out in the presence of Pd catalyst such as Pd(dppf)Cl 2 , Pd(PPh 3 ) 4 or PdCl 2 (PPh 3 ) 2 , and a suitable base such as Na 2 C0 3 , K 2 C0 or Cs 2 C0 3 , in a suitable solvent such as toluene, EtOH/H 2 0 or l,4-dioxane/H 2 0, at 50 to 150 °C. Hydrolyzation of VII affords VIII.
  • Pd catalyst such as Pd(dppf)Cl 2 , Pd(PPh 3 ) 4 or PdCl 2 (PPh 3 ) 2
  • a suitable base such as Na 2 C0 3 , K 2 C0 or Cs 2 C0 3
  • a suitable solvent such as toluene, EtOH/H 2 0 or
  • the hydrolyzation reaction can be carried out in the presence of LiOH or NaOH in a suitable solvent such as EtOH/H 2 0 or MeOH/H 2 0.
  • the hydrolyzation reaction can also be carried out in hydrochloric acid at 60 to 120 °C.
  • VIII undergoes coupling reaction with NH 3 , NH 3 H 2 0 or NH 4 C1 in presence of HATU or EDCI/HOBt, and a suitable base such as TEA, DIPEA, Na 2 C0 3 or NaHC0 3 , in a suitable solvent such as DMF or THF to give compound I.
  • the intermediate VII can be prepared by Suzuki coupling of material II with bronic acid V or bronic ester VI.
  • the reaction can be carried out in the presence of Pd catalyst such as Pd(dppf)Cl 2 , Pd(PPh 3 ) 4 or PdCl 2 (PPh 3 ) 2 , and a suitable base such as Na 2 C0 3 , K 2 C0 3 or Cs 2 C0 3 , in a suitable solvent such as toluene, EtOH/H 2 0 or l,4-dioxane/H 2 0, at 50 °C to 150 °C.
  • Pd catalyst such as Pd(dppf)Cl 2 , Pd(PPh 3 ) 4 or PdCl 2 (PPh 3 ) 2
  • a suitable base such as Na 2 C0 3 , K 2 C0 3 or Cs 2 C0 3
  • a suitable solvent such as toluene, EtOH/H 2 0 or l,4-dioxane
  • the intermediate VII can be prepared by one-pot reaction.
  • Compound II reacts with bis(pinacolato)diboron in the presence of tris(dibenzylideneacetone) dipalladium and butyldi-l-adamantylphosphine at 60 °C to 130 °C, and a suitable base such as KOAc in a suitable solvent such as DME, then the compound IV, K 2 C0 3 and dioxane/H 2 0 are added and the mixture is stirred at 60 °C to 130 °C for several hours under microwave to afford the intermediate VII.
  • Scheme 3 depicts a general method to prepare compound 1-1. Suzuki coupling reaction of bronic ester III with IV-1 affords intermediate VII-1.
  • the reaction can be carried out in the presence of Pd catalyst such as Pd(dppf)Cl 2 , Pd(PPh 3 ) 4 or PdCl 2 (PPh 3 ) 2 , and a suitable base such as Na 2 C0 3 or Cs 2 C0 3 , in a suitable solvent such as ⁇ / ⁇ 3 ⁇ 40, toluene or l,4-dioxane/H 2 0, at 50 °C to 150 °C.
  • VII-1 is hydrolyzed by LiOH or NaOH in a suitable solvent such as
  • VIII-1 is treated with HCl in dioxane to afford VIII-2, which undergoes coupling reaction with NH , NH H 2 0 or NH 4 C1 in the presence of HATU or EDCI/HOBt, and a suitable base such as TEA, DIPEA, Na 2 C0 3 or NaHC0 3 , in a suitable solvent such as DMF or THF to give compound 1-1.
  • a suitable base such as TEA, DIPEA, Na 2 C0 3 or NaHC0 3
  • R 3 is C h alky!, which is unsubstituted or substituted by fluoro;
  • W is -OR 5 - wherein R 5 is hydrogen, C h alky! or halogen.
  • Scheme 4 describes a general method to prepare compound 1-2.
  • Starting material IX is treated by LDA in an organic solvent such as THF at -78 °C and followed by addition of R 3 COCl or (R 3 CO) 2 0 at -78 °C to room temperature to give intermediate X, which is heated with DMF- DMA to form XI.
  • another method is used to make XI.
  • IX is heated with C-tert-butoxy-N,N,N',N'-tetramethyl-methanediamine in an organic solvent such as DMF at 100 °C to 150 °C to form intermediate XII, which is then treated with (R 3 CO) 2 0 in the presence of TEA in an organic solvent such as DCM to give XI.
  • XI is heated with diethyl aminomalonate hydrochloride in HOAc at 100 °C to 160 °C to form compound VII-2, which is then hydrolyzed to give compound VIII-3.
  • the hydrolyzation reaction can be carried out in the presence of LiOH or NaOH in a suitable solvent such as EtOH/H 2 0, THF/H 2 0 or MeOH/H 2 0.
  • the hydrolyzation reaction can also be carried out in hydrochloric acid at 60 °C to 120 °C.
  • VIII-3 undergoes coupling reaction with NH 3 , NH 3 H 2 0 or NH 4 C1 in the presence of HATU or
  • EDCI/HOBt EDCI/HOBt
  • a suitable base such as TEA, DIPEA, Na 2 C0 3 or NaHC0 3
  • a suitable solvent such as DMF or THF
  • R 1 is C 2.6 alkyl
  • R 12 is C ⁇ alkyl .
  • Scheme 5 depicts a general method to prepare 1-3. II-3 is treated with (R CO) 2 0 in the presence of BF Et 2 0 in an organic solvent such as DCM at 0°C to room temperature to form the intermediate XIII, which reacts with Boc 2 0 to give XIV.
  • XIV reacts with bronic acid V in the presence of Pd catalyst such as Pd(dppf)Cl 2 , Pd(PPh ) 4 or PdCl 2 (PPh ) 2 , and a suitable base such as Na 2 C0 3 or Cs 2 C0 3 , in a suitable solvent such as toluene, EtOH/H 2 0 or l,4-dioxane/H 2 0, at 50 °C to 150 °C to form intermediate XV, which is reduced by TESH with BF 3 Et 2 0 and TFA at 0°C to room temperature to afford compound VII-3.
  • Pd catalyst such as Pd(dppf)Cl 2 , Pd(PPh ) 4 or PdCl 2 (PPh ) 2
  • a suitable base such as Na 2 C0 3 or Cs 2 C0 3
  • a suitable solvent such as toluene, EtOH/H 2 0 or l,4-dioxane/H 2
  • VII-3 is hydrolyzed to give compound VIII-4, which undergoes coupling reaction with NH 3 , NH 3 H 2 0 or NH 4 CI in the presence of HATU or EDCI/HOBt, and a suitable base such as TEA, DIPEA, Na 2 C0 3 or NaHC0 3 , in a suitable solvent such as DMF or THF to give compound 1-3.
  • a suitable base such as TEA, DIPEA, Na 2 C0 3 or NaHC0 3
  • a suitable solvent such as DMF or THF
  • Scheme 6 depicts a general method to prepare 1-4. VII-4 undergoes Suzuki
  • Pd catalyst such as Pd(dppf)Cl 2 or Pd(PPh ) 4 and a suitable base such as Na 2 C0 3 or Cs 2 C0 3 in a suitable solvent such as
  • VIII-5 undergoes coupling reaction with NH , NH 3 H 2 0 or NH 4 C1 in the presence of HATU or EDCI/HOBt, and a suitable base such as TEA, DIPEA, Na 2 C0 3 or NaHC0 3 , in a suitable solvent such as DMF or
  • This invention also relates to a process for the preparation of a compound of formula I comprising the reaction of
  • R 1 , R 2 , R 3 , R 4 and W are defined above unless otherwise indicated.
  • the coupling agent can be for example HATU or EDCI/HOBt.
  • the base can be for example TEA, DIPEA, Na 2 C0 3 or NaHC0 3 .
  • a compound of formula I when manufactured according to the above process is also an object of the invention.
  • the invention also relates to a compound of formula I for use as therapeutically active substance.
  • compositions or medicaments containing the compounds of the invention and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicaments.
  • compounds 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.
  • 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 particularly ranges anywhere from about 3 to about 8.
  • a compound of formula I is formulated in an acetate buffer, at pH 5.
  • the compounds of formula I are sterile.
  • the compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aque
  • 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 "effective amount" of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to inhibit CDK8 activity. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.
  • the pharmaceutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.1 to 50 mg/kg of patient body weight per day, with the typical initial range of compound used being about 0.3 to about 15 mg/kg/day.
  • oral unit dosage forms such as tablets and capsules, preferably contain from about 5 mg to about 500 mg of the compound of the invention.
  • 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 exeipient.
  • 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).
  • 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
  • An example of a suitable oral dosage form is a tablet containing about 5 mg to 500 mg of the compound of the invention compounded with about 90 mg to 30 mg anhydrous lactose, about 5 mg to 40 mg sodium croscarmellose, about 5 mg to 30 mg polyvinylpyrrolidone (PVP) K30, and about 1 mg to 10 mg magnesium stearate.
  • the powdered ingredients are first mixed together and then mixed with a solution of the PVP.
  • the resulting composition can be dried, granulated, mixed with the magnesium stearate and compressed to tablet form using conventional equipment.
  • An example of an aerosol formulation can be prepared by dissolving the compound, for example 5mg to 400 mg, of the invention in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired.
  • the solution may be filtered, e.g., using a 0.2 micron filter, to remove impurities and contaminants
  • An embodiment therefore, includes a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or excipient.
  • Another embodiment includes a pharmaceutical composition comprising a compound of Formula I for use in the treatment of a hyperproliferative disease. Another embodiment includes a pharmaceutical composition comprising a compound of Formula I for use in the treatment of cancer.
  • the compounds of the invention inhibit the kinase activity of protein. Accordingly, the compounds of the invention are useful for inhibiting cell proliferation and inducing cell cycle arrest and apoptosis in particular cancer cells.
  • Compounds of the invention are useful for inhibiting cell proliferation, inducing cell cycle arrest and apoptosis in cells that overexpress CDK8 or Cyclin C.
  • compounds of the invention are useful for inhibiting cell proliferation, inducing cell cycle arrest and apoptosis in cells in which the apoptotic pathway is disrupted or proliferation pathway is overexpressed/or immortalized, for example by deregulation of CDK8 or Cyclin C.
  • the compounds of inventions are useful as inhibitors of CDK8 or Cyclin C.
  • An embodiment of this invention includes the use of a compound for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
  • a further embodiment of this invention includes the use of a compound for the treatment of gastric cancer or colorectal cancer.
  • Another embodiment of this invention includes the use of a compound for the preparation of a medicament for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
  • a compound for the preparation of a medicament for the treatment of cancer in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
  • a further embodiment of this invention includes the use of a compound for the preparation of a medicament for the treatment of gastric cancer or colorectal cancer.
  • Another embodiment of this invention relates to a compound of formula I for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
  • a further embodiment of this invention relates to a compound of formula I for the treatment of gastric cancer or colorectal cancer.
  • Another embodiment includes a method of treating or preventing cancer in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of Formula I, a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof.
  • a therapeutically effective amount of a compound of Formula I, a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof include bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
  • the invention relates to a method of treating or preventing gastric cancer or colorectal cancer in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of Formula I, a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof.
  • Another embodiment includes a method of treating or preventing neurodegenerative disease in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of Formula I, a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof.
  • Particular neurodegenerative disease for treatment includes Alzhemers disease, parkinson's disease, Huntington's dsease and Amyotrophic lateral sclerosis (ALS).
  • the compounds of the invention can be used in combination with small molecule inhibitors such as tyrosine kinase inhibitors, Serine/Threonine kinase inhibitors, lipid kinase inhibitors, protein-protein inhibitors, etc., cytotoxic agents, radiotherapy, antibodies and cancer vaccines for the treatment of cancer.
  • small molecule inhibitors such as tyrosine kinase inhibitors, Serine/Threonine kinase inhibitors, lipid kinase inhibitors, protein-protein inhibitors, etc.
  • cytotoxic agents such as tyrosine kinase inhibitors, Serine/Threonine kinase inhibitors, lipid kinase inhibitors, protein-protein inhibitors, etc.
  • BSA bovine serum albumin
  • DIPEA N,N-diisopropylethylamine
  • EGTA ethylene glycol tetraacetic acid
  • HATU 2-(7-aza- IH-benzotriazole- 1-yl)- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate
  • HCMV human cytomegalovirus
  • HIV human immunodeficiency virus
  • HSV herpes simplex virus
  • HPV human papillomavirus
  • nM nanomoles per liter
  • PdCl 2 (PPh 3 ) 2 bis(triphenylphosphine)palladium(II) chloride
  • TR-FRET time resolved-fluorescence resonance energy transfer
  • LC/MS spectra were obtained using a MicroMass Plateform LC (WatersTM alliance 2795- ZQ2000). Standard LC/MS conditions were as follows (running time 6 minutes): Acidic condition: A: 0.1% formic acid in H 2 0; B: 0.1% formic acid in acetonitrile;
  • Mass spectra generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (M+H) + .
  • the microwave assisted reactions were carried out in a Biotage Initiator Sixty.
  • Step 1 Preparation of (4-bromo-pyridin-2-yl)-carbamic acid tert-butyl ester
  • Step 3 Preparation of 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-pyrrole-l,2- dicarboxylic acid 1-tert-butyl ester 2-methyl ester
  • Step 4 Preparation of 4-(2-tert-butoxycarbonylamino-pyridin-4-yl)-lH-pyrrole-2- carboxylic acid methyl ester
  • Step 7 Preparation of 4-(2-amino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
  • Example 2 Preparation of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
  • Step 1 Preparation of 4-bromo-l-methyl-lH-pyrrole-2-carboxylic acid methyl ester
  • Step 2 Preparation of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester
  • Step 3 Preparation of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
  • Step 4 Preparation of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
  • HATU 467 mg, 1.2 mmol
  • TEA 124 mg, 1.2 mmol
  • NH 4 C1 59 mg, 1.1 mmol
  • Step 1 Preparation of l-benzy -4-bromo-lH-pyrrole-2-carboxylic acid methyl ester
  • Step 2 Preparation of l-benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester
  • Step 3 Preparation of l-benzy -4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
  • Step 4 Preparation of l-benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
  • Step 1 Preparation of 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-lH-pyrrole-2- carboxylic acid methyl ester
  • Step 4 Preparation of 4-(3-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
  • Step 4 Preparation of 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
  • DMF dimethyl methoxysulfoxide
  • HOBt 72 mg, 0.53 mmol
  • EDCI 102 mg, 0.53 mmol
  • the mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (4 mL) was added.
  • the resulting mixture was stirred at room temperature for 2 hours and then concentrated. The residue was diluted with water (10 mL), and then extracted with EtOAc (20 mLx5).
  • Step 1 Preparation of 4-bromo- -methyl-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 2 Preparation of 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 4 Preparation of 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
  • DMF dimethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
  • HOBt 70 mg, 0.52 mmol
  • EDCI 100 mg, 0.52 mmol
  • the mixture was stirred at room temperature for 30 min, and then aqueous ammonia (4 mL) was added.
  • the resulting mixture was stirred at room temperature for 1 h, and then poured into water (30 mL). The precipitate was collected by filtering and washed with EtOAc (1 mL) to give 5- methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide (81 mg).
  • Step 3 Preparation of 5-acetyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 4 Preparation of 5-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 7 Preparation of 3-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
  • DMF dimethyl methoxysulfoxide
  • HOBt 73 mg, 0.54 mmol
  • EDCI 104 mg, 0.54 mmol
  • the mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (4 mL) was added. The resulting mixture was stirred at room temperature and was completed as monitored by HPLC.
  • Step 1 Preparation of 4-(3-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
  • Step 2 Preparation of 4-(3-chlor -pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
  • Step 3 Preparation of 4-(3-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
  • HOBt 109 mg, 0.81 mmol
  • EDCI 156 mg, 0.81 mmol
  • the mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (4 mL) was added.
  • the resulting mixture was stirred at room temperature for 5 h and then poured into water (20 mL).
  • Step 1 Preparation of 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
  • Step 3 Preparation of 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
  • Step 1 Preparation of 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
  • Step 3 Preparation of 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
  • Step 1 Preparation of l-pyridazin-4-yl-propan-2-one
  • Step 3 Preparation of 3-methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 5 Preparation of 3-methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid amide
  • DMF 3 mL
  • HOBt 159 mg, 1.18 mmol
  • EDCI 227 mg, 1.18 mmol
  • the mixture was stirred at room temperature for 30 min, and then ammonia (5 mL) was added.
  • the resulting mixture was stirred at room temperature. When the reaction was complete, the mixture was concentrated.
  • Step 1 Preparation of dimethyl-((E -2-pyridin-4-yl-vinyl)-amine
  • Step 2 Preparation of (E)-4-dimethylamino-l,l,l-trifluoro-3-pyridin-4-yl-but-3-en-2- one
  • Step 3 Preparation of 4-pyridin-4-yl-3-trifluoromethyl-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 4 Preparation of 4-pyridin-4- -3-trifluoromethyl-lH-pyrrole-2-carboxylic acid
  • Step 5 Preparation of 4-pyridin-4-yl-3-trifluoromethyl-lH-pyrrole-2-carboxylic acid amide
  • Step 1 Preparation of 4-(2-hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
  • Step 2 Preparation of 4-(2-hydrox -pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
  • Step 3 Preparation of 4-(2-hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
  • a mixture of 4-(2-hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (100 mg, 0.49 mmol), EDCI (113 mg, 0.59 mmol) and HOBt (80 mg, 0.59 mmol) in DMF (2 mL) was stirred at room temperature for 3 hours. Then aqueous ammonia (6 mL) was added. The resulting mixture was stirred for 30 minutes and purified by prep-HPLC to give 4-(2-hydroxy-pyridin-4-yl)-lH- pyrrole-2-carboxylic acid amide (14.0 mg).
  • Step 1 Preparation of 4-bromo-3-methyl-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 4 Preparation of 4-(3-chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2-carboxylic acid amide
  • Step 1 Preparation of 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
  • Step 3 Preparation of 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
  • Step 3 Preparation of 4-(2-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
  • Step 1 Preparation of 3-methyl-4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 3 Preparation of 3-methyl-4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
  • Step 2 Preparation of 3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 1 Preparation of l-pyridin-4-yl-butan-2-one
  • Step 3 Preparation of 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
  • Step 5 Preparation of 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
  • DMF dimethyl methyl-N-(2-aminoethyl)-2-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
  • HOBt 112 mg, 0.83 mmol
  • EDCI 159 mg, 0.83 mmol
  • the mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (5 mL) was added.
  • the resulting mixture was stirred at room temperature for 5 hours and then poured into water (20 mL).
  • the aqueous layer was extracted with EtOAc (20 mL x 5).
  • the biological activity of the compounds of the invention can be determined using the assay described below.
  • CDK8/Cyclin C protein was obtained from Invitrogen, cat# PV4402.
  • ULight-Glycogen Synthase (Ulight-GS) peptide with sequence PASVPPSPSLSRHSSPHQ(pS)ED, and Europium- anti-phospho Glycogen Synthase (Ser641) [Eu-anti-P-GS (Ser641)] were obtained from Perkin Elmer, cat# TRF0131-M and cat# TRF0220.
  • Adenosine-5 '-triphosphate (ATP) was obtained from Invitrogen, cat# PV3227.
  • Example 23 In vitro cell proliferation assay:
  • the compounds of the present invention were tested for their capacity to inhibit a CDK8 activity and activation as described herein.
  • the Examples were tested in the above assay and found to have IC 50 of about 0.0001 ⁇ to about 30 ⁇ .
  • Particular compounds of formula I were found to have IC 50 of about 0.0001 ⁇ to about 1 ⁇ .
  • a compound of formula I can be used in a manner known per se as the active ingredient for the production of tablets of the following composition:
  • Example B A compound of formula I can be used in a manner known per se as the active ingredient for the production of capsules of the following composition:

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Abstract

The invention provides a method for the treatment of cancer, which method comprises the general formula (I) wherein R1, R2, R3, R4 and W are as described herein.

Description

Novel pyrrole derivatives for the treatment of cancer
The present invention relates to organic compounds useful for therapy in a mammal, and in particular to inhibit cell proliferation and induce cell cycle arrest and apoptosis that overexpress CDK8 or Cyclin C useful for treating cancer.
FIELD OF THE INVENTION
The cyclin-dependent kinase (CDK) complexes are well-conserved Ser/Thr kinase family, and it has been shown to be activated by the binding of regulatory partner, generally a cyclin. There are total 20 CDK family members and 5 CDK-like proteins based on the similarities in sequence and function. CDKs regulate various key transitions of cell cycle and play an important role in the regulation of transcription, apoptosis and neuronal functions.
Dysregulation of CDKs has been linked to pathological events and both proliferative and non-pro liferative disease, including cancers, Alzhemers disease (AD), parkinson's disease, Stroke/ischemia, pain, traumatic brain injury, kidney disease, inflammation pathologies, type 2 diabetes, viral infection (HSV, HCMV, HPV, HIV).
CDK8 is a CyclinC-dependent CDK family kinase and functions as a transcriptional regulator. Several phosphorylation targets of CDK8 have been identified, including the RNA polymerase II (RNAPII) C-terminal domain (CTD), histone H3, subunits of general transcription factors (GTFs) and certain transactivators. CDK8 has also been described as a transcriptional coactivator in oncongenic signaling pathways, including the β-catenin pathway, the serum response network, the Tumor Growth Factor TGFP signaling pathway, the p53 pathway, as well as in thyroid hormone-dependent transcription. Colocalization of CDK8 and Cyclin C was also reported in neurodegenerative disease such as AD. CDK8 was found to be frequently
dysregulated in various human cancers, such as colon cancer, gastric cancer and melanoma. Inhibition of CDK8 by short hairpin RNA (shRNA) inhibits cancer cell proliferation, and induces cell cycle arrest and apoptosis in vitro and in vivo models. Although Silibinin, the major active constituent of silymarin isolated from milk thistle (Silybum marianum), has shown strong cell growth inhibition in colon cancer through downregulation CDK8 expression, there are no known direct CDK8 inhibitors under clinical development. Therefore, there is a great unmet medical need to develop CDK8 inhibitors for cancer patients.
SUMMARY OF THE INVENTION
Objects of the present invention are novel compounds of formula I, their manufacture, medicaments based on a compound in accordance with the invention and their production as well as the use of compounds of formula I for the treatment of cancer.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
As used herein, the term "Chalky!" alone or in combination signifies a saturated, linear- or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, for example methyl, ethyl, propyl, isopropyl, 1 -butyl, 2-butyl, tert-butyl and the like. Particular "Chalky!" groups are methyl, ethyl, isopropyl and tert-butyl.
The term "amino", alone or in combination, refers to primary (-NH2), secondary (-NH-) or
— N /
tertiary amino ( ^ ).
The term "halogen" means fluorine, chlorine, bromine or iodine. Halogen is particularly fluorine or chlorine.
The term "hydroxy" alone or in combination refers to the group -OH.
The compounds according to the present invention may exist in the form of their pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin R.J., et al., Organic Process Research & Development 2000, 4, 427-435; or in Ansel, H., et al., In: Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. Particular are the sodium salts of the compounds of formula I.
Compounds of the general formula I which contain one or several chiral centers can either be present as racemates, diastereomeric mixtures, or optically active single isomers. The racemates can be separated according to known methods into the enantiomers. Particularly, diastereomeric salts which can be separated by crystallization are formed from the racemic mixtures by reaction with an optically active acid such as e.g. D- or L-tartaric acid, mandelic acid, malic acid, lactic acid or camphorsulfonic acid.
INHIBITORS OF CDK8 OR CYCLIN C
The present invention provides (i) the use of a compound of formula (I)
Figure imgf000005_0001
wherein
R 1 and R 3J are independently selected from hydrogen; halogen; and C^aUcyl, which is unsubstituted or substituted by fluoro;
R is hydrogen, C^aUcyl or phenyl-CH2-;
R4 is hydrogen, C^aUcyl, halogen or phenyl;
W is nitrogen or -CR5; wherein R5 is hydrogen, C^aUcyl, amino, halogen or hydroxy; or a pharmaceutically acceptable salt thereof for the preparation of medicaments for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
Another embodiment of the present invention is (ii) compounds of formula (I), wherein R 1 and R 3J are independently selected from hydrogen; halogen; and C1-6alkyl, which is unsubstituted or substituted by fluoro;
R is hydrogen, Ci^alkyl or phenyl-CH2-;
R4 is hydrogen, Ci-6alkyl, halogen or phenyl;
W is nitrogen or -CR5; wherein R5 is hydrogen, C1-6alkyl, amino, halogen or hydroxy; or a pharmaceutically acceptable salt thereof;
with the proviso that
3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide, 3-ethyl-4-pyridin-4-yl-lH-pyrrole- 2-carboxylic acid amide and 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
are excluded.
A further embodiment of the present invention is (iii) a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, chloro, methyl or ethyl;
R is hydrogen, methyl or phenyl-CH2-;
R is hydrogen, chloro, methyl or trifluoromethyl;
R4 is hydrogen, methyl, chloro, bromo or phenyl;
W is nitrogen or -CR5; wherein R5 is hydrogen, methyl, amino, chloro or hydroxy.
Another embodiment of the present invention is (iv) a compound of formulation (I) or a pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, halogen or C1-6alkyl;
R is hydrogen, Ci^alkyl or phenyl-CH2-;
R is hydrogen, Ci-6alkyl, halogen or trifluoromethyl;
R4 is hydrogen, Ci-6alkyl, halogen or phenyl;
W is -CR5; wherein R5 is hydrogen, C1-6alkyl, amino, halogen or hydroxy.
Further embodiment of present invention is (v) a compound of formula (I) or a
pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, chloro, methyl or ethyl;
R is hydrogen, methyl or phenyl-CH2-;
R is hydrogen, chloro, methyl or trifluoromethyl;
R4 is hydrogen, methyl, chloro, bromo or phenyl; -CR5; wherein R5 is hydrogen, methyl, amino, chloro or hydroxy.
Another embodiment of present invention is (vi) a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen;
R is hydrogen;
R is C1-6alkyl, halogen or trifluoromethyl;
R4 is hydrogen or halogen;
W is -CR5; wherein R5 is hydrogen, C1-6alkyl.
Further embodiment of present invention is (vii) a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen;
R is hydrogen;
R is methyl, chloro or trifluoromethyl;
R4 is hydrogen or chloro;
W is -CR5; wherein R5 is hydrogen or methyl.
Another embodiment of present invention is (xiii) a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, halogen or C1-6alkyl;
R is hydrogen, C1-6alkyl or phenyl-CH2-;
R is hydrogen;
R4 is hydrogen, C1-6alkyl, halogen or phenyl;
W is -CR5; wherein R5 is hydrogen, C1-6alkyl, amino, halogen or hydroxy.
Further embodiment of present invention is (ix) a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, chloro, methyl or ethyl;
R is hydrogen, methyl or phenyl-CH2-;
R is hydrogen;
R4 is hydrogen, methyl, chloro, bromo or phenyl;
W is -CR5; wherein R5 is hydrogen, methyl, amino, chloro or hydroxy. Particular compounds of formula I, including their activity data, NMR data and MS data are summarized in the following Table 1 and 2.
Table 1: Structure, name and activity data of particular compounds
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
Table 2: NMR and MS data of particular compounds
MS obsd.
Example
H NMR data (ESI+) No.
[(M+H)+] 1H NMR (DMSO-d6): 511.69 (s, IH), 8.12 (s, 2H), 7.78 (d, IH),
7.60 (s, IH), 7.32 (dd, IH), 7.11 (m, IH), 7.03 (s, IH), 6.53 203 (m,lH), 6.52 (s, IH), 5.86 (s, 2H)
1H NMR (Methanol-d4): δ 8.38 (dd, 2H), 7.55 (dd, 2H), 7.50 (d,
201 IH), 7.28 (d, IH), 3.94 (s, IH)
1H NMR (DMSO-d6): δ 8.49 (d, 2H), 7.83 (d, IH), 7.64 (brs,
IH), 7.46 (d, 2H), 7.38 (d, IH), 7.30 (m, 2H), 7.25 (m, IH), 278 7.16 (d, 2H), 7.05 (brs, IH), 5.64 (s, 2H)
1H NMR (DMSO-d6): δ 8.34 (s, IH), 8.29 (d, IH), 7.62 (brs,
IH), 7.32 (d, IH), 7.27 (d, IH), 7.17 (d, IH), 7.04 (brs, IH), 202 2.37 (s, 3H)
1H NMR (DMSO-d6): 512.67 (s, IH), 8.52 (dd, 2H), 7.63 (brs,
222 IH), 7.52 (dd, 2H), 7.22 (s, IH), 7.18 (brs, IH)
1H NMR (DMSO-d6): 511.62 (s, IH), 8.45 (dd, 2H), 7.49 (s,
202 IH), 7.32 (dd, 2H), 7.09 (d, IH), 6.94 (s, IH), 2.39 (s, 3H)
1H NMR (DMSO-d6): 5 11.58 (s, IH), 8.50 (d, 2H), 7.51 (brs,
IH), 7.33 (d, 2H), 7.06 (d, IH), 6.98 (brs, IH), 2.80 (q, 2H), 216 1.18 (t, 3H)
1H NMR (DMSO-d6): 5 12.24 (brs, IH), 8.54 (dd, 2H), 7.62 (m,
222 3H), 7.51 (s, IH), 7.02 (brs, IH)
1H NMR (DMSO-d6): 5 12.02 (brs, IH), 8.58 (s, IH), 8.41 (d,
222 IH), 7.70 (brs, IH), 7.58(m, 2H), 7.41 (s,lH), 7.13 (brs, IH)
1H NMR (DMSO-d6): 5 11.99 (brs, IH), 8.71 (s, IH), 8.44 (d,
IH), 7.69 (brs, IH), 7.56-7.51 (m, 2H), 7.36 (dd,lH), 7.13 (brs, 266 IH) 1H NMR (Methanol-d4): δ 8.44 (d, IH), 8.34 (s, IH), 7.59
(d,lH) 7.46-7.42 (m, 3H), 7.33-7.31 (m, 2H), 6.91 (d, IH), 6.48 264 (s, IH)
1H NMR (DMSO-d6): ^ 11.83 (s, IH), 9.35 (d, IH), 9.09 (d,
203 IH), 7.66 (dd,lH) 7.52 (s, IH), 7.16 (s, 2H), 2.43 (s, 3H)
1H NMR (Methanol-d4): δ 8.49 (d, 2H), 7.45 (d, 2H), 7.14 (s,
256 IH)
1H NMR (Methanol-d4): δ 7.49 (d, IH), 7.38 (d, IH), 7.22 (d,
204 IH), 6.71-6.69 (dd, IH), 6.68 (s, IH)
1H NMR (Methanol-d4): δ 8.60 (s, IH), 8.44 (d, IH), 7.38 (d,
236 1H), 7.11 (s, 1H), 2.27 (s, 3H)
1H NMR (DMSO-d6): ^ 11.83 (s, IH), 8.33 (d, IH), 7.60 (brs,
IH), 7.55 (d, IH), 7.38 (s, IH), 7.31-7.27 (m, 2H), 7.09 (brs, 202 IH), 2.51 (s, 3H)
1H NMR (DMSO-d6): ^ 11.96 (s, IH), 8.29 (d, IH), 7.72-7.71
(m, IH), 7.62 (d, IH), 7.56 (brs, IH), 7.52-7.51 (m, IH), 7.32 222 (m, IH), 7.13 (brs, IH)
1H NMR (Methanol-d4): δ 8.35 (d, IH), 7.35 (s, IH), 7.30-7.28
216 (dd, IH), 7.22 (s, IH), 2.56 (s, 3H), 2.49 (s, 3H)
1H NMR (DMSO-d6): ^ 11.45 (s, IH), 8.47 (d, 2H), 7.37 (d,
202 2H), 7.26 (d, IH), 7.04 (s, 2H), 2.40 (s, 3H)
1H NMR (DMSO-d6): ^ 11.44 (s, IH), 8.49 (t, 2H), 7.37 (d,
216 2H), 7.25 (d,lH), 7.05 (s, 2H), 2.89 (q, 2H), 1.08 (t, 3H) 1H NMR (DMSO-d6): ^ 11.83 (bs, 1H), 8.83 (d, 2H), 7.56 (s,
21 188
2H), 7.46 (d, 2H), 7.25 (s, 1H), 7.07(bs, 1H)
Table 3: IC50 on HCT116 or AGS of particular compounds
Figure imgf000014_0001
More particular compounds of formula I include the following:
3- Chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide;
4- (3-Chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide;
4-(3-Bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide;
4-Pyridin-4-yl-3-trifluoromethyl- lH-pyrrole-2-carboxylic acid amide;
4-(3-Chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2-carboxylic acid amide; and
3-Methyl-4-(2-methyl-pyridin-4-yl)- lH-pyrrole-2-carboxylic acid amide;
SYNTHESIS
The compounds of the present invention can be prepared by any conventional means. Suitable processes for synthesizing these compounds as well as their starting materials are provided in the schemes below and in the examples. All substituents, in particular, R1, R2, R3, R4 and W are as defined above unless otherwise indicated. Furthermore, and unless explicitly otherwise stated, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to a person of ordinary skill in organic chemistry.
General synthetic route for intermediate (Scheme 1)
Scheme 1 Methold 1)
Figure imgf000015_0001
Methold 2)
Methold 3)
Methold 4)
Figure imgf000015_0002
Methold 5)
Figure imgf000015_0003
NHBoc
R2 is C^g alkyl or phenyl-CH2- R1 1 is C1 -6 alkyl. Methold 6)
Methold 7)
Figure imgf000016_0001
Intermediates II, II-l, II-2, III, III-l, 4-bromo-5-chloro-lH-pyrrole-2-carboxylic acid methyl ester, 4-bromo-pyridin-2-ylamine, 4-bromo-5-methyl-lH-pyrrole-2-carboxylic acid ethyl ester, and 4-bromo-3-chloro-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester can be prepared according to Scheme 1.
By Method 1), II-l reacts with halides in the presence of NaH in in an organic solvent at 0 °C to room temperature. The organic solvent for example can be THF, DMF or dioxane.
By Method 2), coupling reaction between II and bis(pinacolato)diboron affords the bronic ester intermediate III. The reaction can be carried out in the presence of palladium catalyst and a suitable base such as KOAc, in a suitable solvent such as 1,4-dioxane, at 50 °C to 150 °C.
By Method 3), II-l reacts with Boc20 at 0 °C to room temperature in an organic solvent such as DCM or THF, to afford intermediate II-2, which undergoes coupling reaction with bis(pinacolato)diboron to give intermediate III-l. The coupling reaction can be carried out in the presence of palladium catalyst and a suitable base such as KOAc, in a suitable solvent such as 1,4-dioxane, at 50 to 150 °C.
By Method 4), 4-bromo-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester reacts with NCS in an organic solvent such as CH3CN or THF at room temperature to reflux to give 4-bromo-5-chloro-lH-pyrrole-2-carboxylic acid methyl ester.
By Method 5), 4-bromo-pyridin-2-ylamine reacts with Boc20 in the presence of TEA and DMAP at room temperature in an organic solvent. The organic solvent for example can be THF or DCM. By Method 6), 5-methyl-lH-pyrrole-2-carboxylic acid ethyl ester reacts with NBS in an organic solvent such as DCM or THF at -10 °C to room temperature.
By Method 7), 5-methyl-3,4-dihydro-2H-pyrrole is treated with NCS in an organic solvent such as THF at room temperature to 55 °C to afford 4,4-dichloro-5-trichloromethyl-3,4-dihydro- 2H-pyrrole, which is converted to 3-chloro-lH-pyrrole-2-carboxylic acid methyl ester by treating with sodium methoxide in methanol at 0 °C to room temperature. Then 3-chloro-lH-pyrrole-2- carboxylic acid methyl ester reacts with NBS at room temperature in an organic solvent such as CH3CN or THF to give 4-bromo-3-chloro-lH-pyrrole-2-carboxylic acid methyl ester, which is treated with Boc20 in the presence of DMAP at room temperature in an organic solvent to afford 4-bromo-3-chloro-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester. The organic solvent for example can be DCM or THF.
General synthetic route for formula I (Scheme 2)
Scheme 2
Pd catalyst
Method 9)
Figure imgf000018_0001
The compound of formula I can be prepared according to Scheme 2. By Method 8), coupling reaction between compound III and IV affords the intermediate VII. The reaction can be carried out in the presence of Pd catalyst such as Pd(dppf)Cl2, Pd(PPh3)4 or PdCl2(PPh3)2, and a suitable base such as Na2C03, K2C0 or Cs2C03, in a suitable solvent such as toluene, EtOH/H20 or l,4-dioxane/H20, at 50 to 150 °C. Hydrolyzation of VII affords VIII. The hydrolyzation reaction can be carried out in the presence of LiOH or NaOH in a suitable solvent such as EtOH/H20 or MeOH/H20. The hydrolyzation reaction can also be carried out in hydrochloric acid at 60 to 120 °C. VIII undergoes coupling reaction with NH3, NH3 H20 or NH4C1 in presence of HATU or EDCI/HOBt, and a suitable base such as TEA, DIPEA, Na2C03 or NaHC03, in a suitable solvent such as DMF or THF to give compound I.
By Method 9), the intermediate VII can be prepared by Suzuki coupling of material II with bronic acid V or bronic ester VI. The reaction can be carried out in the presence of Pd catalyst such as Pd(dppf)Cl2, Pd(PPh3)4 or PdCl2(PPh3)2, and a suitable base such as Na2C03, K2C03 or Cs2C03, in a suitable solvent such as toluene, EtOH/H20 or l,4-dioxane/H20, at 50 °C to 150 °C.
By Method 10), the intermediate VII can be prepared by one-pot reaction. Compound II reacts with bis(pinacolato)diboron in the presence of tris(dibenzylideneacetone) dipalladium and butyldi-l-adamantylphosphine at 60 °C to 130 °C, and a suitable base such as KOAc in a suitable solvent such as DME, then the compound IV, K2C03 and dioxane/H20 are added and the mixture is stirred at 60 °C to 130 °C for several hours under microwave to afford the intermediate VII.
General synthetic route for formula 1-1 (Scheme 3)
Scheme 3
Figure imgf000020_0001
Scheme 3 depicts a general method to prepare compound 1-1. Suzuki coupling reaction of bronic ester III with IV-1 affords intermediate VII-1. The reaction can be carried out in the presence of Pd catalyst such as Pd(dppf)Cl2, Pd(PPh3)4 or PdCl2(PPh3)2, and a suitable base such as Na2C03 or Cs2C03, in a suitable solvent such as ΕίΟΗ/Ι¾0, toluene or l,4-dioxane/H20, at 50 °C to 150 °C. Then VII-1 is hydrolyzed by LiOH or NaOH in a suitable solvent such as
EtOH/H20 or MeOH/H20 to give VIII-1. VIII-1 is treated with HCl in dioxane to afford VIII-2, which undergoes coupling reaction with NH , NH H20 or NH4C1 in the presence of HATU or EDCI/HOBt, and a suitable base such as TEA, DIPEA, Na2C03 or NaHC03, in a suitable solvent such as DMF or THF to give compound 1-1.
General synthetic route for formula 1-2 (Scheme 4)
Scheme 4
Figure imgf000021_0001
1-2 VIII-3
R3 is Chalky!, which is unsubstituted or substituted by fluoro;
W is -OR5- wherein R5 is hydrogen, Chalky! or halogen.
Scheme 4 describes a general method to prepare compound 1-2. Starting material IX is treated by LDA in an organic solvent such as THF at -78 °C and followed by addition of R3COCl or (R3CO)20 at -78 °C to room temperature to give intermediate X, which is heated with DMF- DMA to form XI. For some structures, another method is used to make XI. Firstly, IX is heated with C-tert-butoxy-N,N,N',N'-tetramethyl-methanediamine in an organic solvent such as DMF at 100 °C to 150 °C to form intermediate XII, which is then treated with (R3CO)20 in the presence of TEA in an organic solvent such as DCM to give XI. And then, XI is heated with diethyl aminomalonate hydrochloride in HOAc at 100 °C to 160 °C to form compound VII-2, which is then hydrolyzed to give compound VIII-3. The hydrolyzation reaction can be carried out in the presence of LiOH or NaOH in a suitable solvent such as EtOH/H20, THF/H20 or MeOH/H20. The hydrolyzation reaction can also be carried out in hydrochloric acid at 60 °C to 120 °C. VIII-3 undergoes coupling reaction with NH3, NH3 H20 or NH4C1 in the presence of HATU or
EDCI/HOBt, and a suitable base such as TEA, DIPEA, Na2C03 or NaHC03, in a suitable solvent such as DMF or THF to give compound 1-2.
General synthetic route for formula 1-3 (Scheme 5)
Scheme 5
Figure imgf000022_0001
VIII-4 1-3
R1 is C2.6alkyl ;
R12 is C^alkyl .
Scheme 5 depicts a general method to prepare 1-3. II-3 is treated with (R CO)20 in the presence of BF Et20 in an organic solvent such as DCM at 0°C to room temperature to form the intermediate XIII, which reacts with Boc20 to give XIV. Then XIV reacts with bronic acid V in the presence of Pd catalyst such as Pd(dppf)Cl2, Pd(PPh )4 or PdCl2(PPh )2, and a suitable base such as Na2C03 or Cs2C03, in a suitable solvent such as toluene, EtOH/H20 or l,4-dioxane/H20, at 50 °C to 150 °C to form intermediate XV, which is reduced by TESH with BF3 Et20 and TFA at 0°C to room temperature to afford compound VII-3. Then VII-3 is hydrolyzed to give compound VIII-4, which undergoes coupling reaction with NH3, NH3 H20 or NH4CI in the presence of HATU or EDCI/HOBt, and a suitable base such as TEA, DIPEA, Na2C03 or NaHC03, in a suitable solvent such as DMF or THF to give compound 1-3.
General synthetic route for formula 1-4 (Scheme 6)
Scheme 6
Figure imgf000023_0001
1-4 VIII-5
Scheme 6 depicts a general method to prepare 1-4. VII-4 undergoes Suzuki
coupling with phenylboronic acid in the presence of Pd catalyst such as Pd(dppf)Cl2 or Pd(PPh )4 and a suitable base such as Na2C03 or Cs2C03 in a suitable solvent such as
EtOH or dioxane/H20 at 50 °C to 150 °C to afford VII-5, which is hydrolyzed by LiOH in a solvent such as THF/H20 to give VIII-5. Then VIII-5 undergoes coupling reaction with NH , NH3 H20 or NH4C1 in the presence of HATU or EDCI/HOBt, and a suitable base such as TEA, DIPEA, Na2C03 or NaHC03, in a suitable solvent such as DMF or
THF to give compound 1-4. This invention also relates to a process for the preparation of a compound of formula I comprising the reaction of
Figure imgf000024_0001
with NH3,or NH3 H20 or NH4C1 in the presence of a coupling agent and a base;
wherein R1, R2, R3, R4 and W are defined above unless otherwise indicated.
The coupling agent can be for example HATU or EDCI/HOBt. The base can be for example TEA, DIPEA, Na2C03 or NaHC03.
A compound of formula I when manufactured according to the above process is also an object of the invention.
PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION
The invention also relates to a compound of formula I for use as therapeutically active substance.
Another embodiment provides pharmaceutical compositions or medicaments containing the compounds of the invention and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicaments. In one example, compounds 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 particularly 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 compounds of formula I are 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 "effective amount" of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to inhibit CDK8 activity. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.
In one example, the pharmaceutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.1 to 50 mg/kg of patient body weight per day, with the typical initial range of compound used being about 0.3 to about 15 mg/kg/day. In another embodiment, oral unit dosage forms, such as tablets and capsules, preferably contain from about 5 mg to about 500 mg of the compound of the invention.
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 exeipient. 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).
An example of a suitable oral dosage form is a tablet containing about 5 mg to 500 mg of the compound of the invention compounded with about 90 mg to 30 mg anhydrous lactose, about 5 mg to 40 mg sodium croscarmellose, about 5 mg to 30 mg polyvinylpyrrolidone (PVP) K30, and about 1 mg to 10 mg magnesium stearate. The powdered ingredients are first mixed together and then mixed with a solution of the PVP. The resulting composition can be dried, granulated, mixed with the magnesium stearate and compressed to tablet form using conventional equipment. An example of an aerosol formulation can be prepared by dissolving the compound, for example 5mg to 400 mg, of the invention in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired. The solution may be filtered, e.g., using a 0.2 micron filter, to remove impurities and contaminants.
An embodiment, therefore, includes a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof. In a further embodiment includes a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or excipient.
Another embodiment includes a pharmaceutical composition comprising a compound of Formula I for use in the treatment of a hyperproliferative disease. Another embodiment includes a pharmaceutical composition comprising a compound of Formula I for use in the treatment of cancer.
INDICATIONS AND METHODS OF TREATMENT
The compounds of the invention inhibit the kinase activity of protein. Accordingly, the compounds of the invention are useful for inhibiting cell proliferation and inducing cell cycle arrest and apoptosis in particular cancer cells.
Compounds of the invention are useful for inhibiting cell proliferation, inducing cell cycle arrest and apoptosis in cells that overexpress CDK8 or Cyclin C.
Alternatively, compounds of the invention are useful for inhibiting cell proliferation, inducing cell cycle arrest and apoptosis in cells in which the apoptotic pathway is disrupted or proliferation pathway is overexpressed/or immortalized, for example by deregulation of CDK8 or Cyclin C.
The compounds of inventions are useful as inhibitors of CDK8 or Cyclin C.
An embodiment of this invention includes the use of a compound for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers. A further embodiment of this invention includes the use of a compound for the treatment of gastric cancer or colorectal cancer.
Another embodiment of this invention includes the use of a compound for the preparation of a medicament for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
A further embodiment of this invention includes the use of a compound for the preparation of a medicament for the treatment of gastric cancer or colorectal cancer.
Another embodiment of this invention relates to a compound of formula I for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
A further embodiment of this invention relates to a compound of formula I for the treatment of gastric cancer or colorectal cancer.
Another embodiment includes a method of treating or preventing cancer in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of Formula I, a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof. Particular cancers for treatment or prevention include bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers. More particularly, the invention relates to a method of treating or preventing gastric cancer or colorectal cancer in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of Formula I, a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof. Another embodiment includes a method of treating or preventing neurodegenerative disease in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of Formula I, a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof. Particular neurodegenerative disease for treatment includes Alzhemers disease, parkinson's disease, Huntington's dsease and Amyotrophic lateral sclerosis (ALS).
COMBINATION THERAPY
The compounds of the invention can be used in combination with small molecule inhibitors such as tyrosine kinase inhibitors, Serine/Threonine kinase inhibitors, lipid kinase inhibitors, protein-protein inhibitors, etc., cytotoxic agents, radiotherapy, antibodies and cancer vaccines for the treatment of cancer.
EXAMPLES
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.
Abbreviations used herein are as follows:
μL·. microliter
μιη: micrometer
μΜ: micromoles per liter
KOAc: potassium acetate
AcOH: acetic acid
Boc20 di-tert-butyl dicarbonate
BSA: bovine serum albumin
CCK-8: Cell Counting Kit-8
DCM: dichloromethane
DIPEA: N,N-diisopropylethylamine
DME: 1 ,2-dimethoxyethane
DMF: dimethylformamide DMSO-d6: deuterated dimethylsulfoxide
DTT: dithiothreitol
EtOAc: ethyl acetate
EGTA: ethylene glycol tetraacetic acid
g: gram
IC50: the half maximal inhibitory concentration
HATU: 2-(7-aza- IH-benzotriazole- 1-yl)- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate
HCMV: human cytomegalovirus
HIV: human immunodeficiency virus
HSV: herpes simplex virus
HPV: human papillomavirus
HPLC: high performance liquid chromatography
LC/MS: Liquid chromatography/mass spectrometry
MeOH: methanol
METHANOL-d4: perdeuteromethanol
M: molarity
mg: milligram
MHz: megahertz
mL: milliliter
mM: millimoles per liter
mm: millimeter
mmol: millimole
MS (ESI): mass spectroscopy (electron spray ionization)
MTBE: methyl tert-butyl ether
nM: nanomoles per liter
nm: nanometer
NCS N-chlorosuccinimide
NBS N-bromosuccinimide
NMR: nuclear magnetic resonance
N2: nitrogen
obsd.: observed
OD: optical density Pd(PPh3)4: tetrakis(triphenylphosphine)palladium
PdCl2(PPh3)2: bis(triphenylphosphine)palladium(II) chloride
Pd(dppf)Cl2: riJ'-Bis(diphenylphosphino)ferroceneldichloropalladium(II) Prep HPLC: preparative high performance liquid chromatography
TEA triethylamine
THF: tetrahvdrofurane
TLC thin layer chromatography
TR-FRET: time resolved-fluorescence resonance energy transfer
δ: chemical shift
General Experimental Conditions
Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SPl system and the Quad 12/25 Cartridge module, ii) ISCO combi-flash chromatography instrument. Silica gel Brand and pore size: i) KP-SIL 60 A, particle size: 40-60 μΜ; ii) CAS registry NO: Silica Gel: 63231-67-4, particle size: 47-60 micron silica gel; iii) ZCX from Qingdao Haiyang Chemical Co., Ltd, pore: 200-300 or 300-400.
Intermediates and final compounds were purified by preparative HPLC on reversed phase column using X Bridge™ Perp C18 (5 μηι, OBD™ 30 x 100 mm) column or SunFire™ Perp C18 (5 μηι, OBD™ 30 x 100 mm) column.
LC/MS spectra were obtained using a MicroMass Plateform LC (Waters™ alliance 2795- ZQ2000). Standard LC/MS conditions were as follows (running time 6 minutes): Acidic condition: A: 0.1% formic acid in H20; B: 0.1% formic acid in acetonitrile;
Basic condition: A: 0.01% ΝΗ3·Η20 in H20; B: acetonitrile;
Neutral condition: A: H20; B: acetonitrile.
Mass spectra (MS): generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (M+H)+.
The microwave assisted reactions were carried out in a Biotage Initiator Sixty.
NMR Spectra were obtained using Bruker Avance 400MHz.
All reactions involving air-sensitive reagents were performed under an argon atmosphere. Reagents were used as received from commercial suppliers without further purification unless otherwise noted. The following examples were prepared by the general methods outlined in the schemes above. They are intended to illustrate the meaning of the present invention but should by no means represent a limitation within the meaning of the present invention.
PREPARATIVE EXAMPLES
Example 1: Preparation of 4-(2-amino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of (4-bromo-pyridin-2-yl)-carbamic acid tert-butyl ester
Figure imgf000031_0001
To a mixture of 4-bromo-pyridin-2-ylamine (2 g, 11.6 mmol), TEA (3.51 g, 34.8 mmol) and DMAP (100 mg) in THF (25 mL) was added Boc20 (3 g, 13.9 mmol) at room temperature. The solution was stirred at room temperature for 3 h, and then concentrated. The residue was purified by column chromatography to give (4-bromo-pyridin-2-yl)-carbamic acid tert-butyl ester (1.2 g) as a white solid.
Step 2: Preparation of 4-bromo-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2- methyl ester
Figure imgf000031_0002
To a mixture of 4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (45 g, 221 mmol), DMAP (2.5 g, 22 mmol) and Et3N (22.3 g, 220 mmol) in CH2C12 (300 mL) was added a solution of Boc20 (53 g, 243 mmol) in CH2C12 (100 mL) dropwise over 30 minutes at room temperature. After being stirred at room temperature for 3 hours, the mixture was concentrated. The residue was diluted with petroleum ether (300 mL). The resulting suspension was filtered and the filtrate was washed with water (300 mLx3), followed by brine (50 mL). The organic layer was dried over anhydrous Na2S04 and then concentrated to give 4-bromo-pyrrole-l,2-dicarboxylic acid 1- tert-butyl ester 2-methyl ester (64 g) as a white solid.
Step 3: Preparation of 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-pyrrole-l,2- dicarboxylic acid 1-tert-butyl ester 2-methyl ester
Figure imgf000032_0001
A mixture of 4-bromo-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (10 g, 33 mmol), bis(pinacolato)diboron (18 g, 66 mmol), KOAc (6.2 g, 66 mmol), Pd(dppf)Cl2 (1 g, 1.5 mmol) in 1,4-dioxane (150 mL) under nitrogen protection was stirred at 100 °C for 3 hours. The mixture was filtered through a Celite pad, and the filtrate was concentrated. The residue was purified by column chromatography to give 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)- pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (4.1 g) as a white solid.
Step 4: Preparation of 4-(2-tert-butoxycarbonylamino-pyridin-4-yl)-lH-pyrrole-2- carboxylic acid methyl ester
Figure imgf000032_0002
A mixture of 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (180 mg, 0.72 mmol), (4-bromo-pyridin-2-yl)-carbamic acid tert-butyl ester (235 mg, 0.86 mmol), Cs2C03 (468 mg, 1.44 mmol) and Pd(dppf)Cl2 (73 mg, 0.1 mmol) in EtOH (4 mL) and water (1 mL) was refluxed under N2 protection for 3 hours. The reaction mixture was poured into water (30 mL). The aqueous layer was extracted with EtOAc (30 mLx3). The combined organic layers were dried over anhydrous Na2S04 and then concentrated to give crude 4-(2-tert-butoxycarbonylamino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (300 mg) as a brown solid which was used in the next step without purification.
Step 5: Preparation of 4-(2-tert-butoxycarbonylamino-pyridin-4-yl)-lH-pyrrole-2- carboxylic acid
Figure imgf000033_0001
A mixture of crude 4-(2-tert-butoxycarbonylamino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (300 mg, 0.72 mmol) and NaOH (144 mg, 3.6 mmol) in MeOH (3 mL) and water (3 mL) was refluxed for 2 hours. The reaction mixture was poured into water (30 mL). The aqueous layer was washed with EtOAc (30 mLx3) and acidified with 1 M aqueous HCl solution to pH 3. The precipitate was collected by filtration to give 4-(2-tert-butoxycarbonylamino- pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (120 mg) as a gray solid which was used in the next step without purification.
Step 6: Preparation of 4-(2-amino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
Figure imgf000033_0002
A mixture of 4-(2-tert-butoxycarbonylamino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (120 mg, 0.55 mmol) in a solution of HCl in dioxane (5 mL) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to give crude 4-(2-amino-pyridin-4-yl)-lH- pyrrole-2-carboxylic acid (70 mg) as a brown solid which was used in the next step without purification.
Step 7: Preparation of 4-(2-amino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
To a mixture of crude 4-(2-amino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (70 mg, 0.34 mmol) in DMF (2 mL) was added HOBt (55 mg, 0.41 mmol) and EDCI (79 mg, 0.41 mmol). The resulting mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (5 mL) was added. The whole mixture was stirred at room temperature for 2 hours and then concentrated. The residue was purified by preparative HPLC to give formate salt of 4-(2- amino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide (10 mg) as a white solid.
Example 2: Preparation of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide Step 1: Preparation of 4-bromo-l-methyl-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000034_0001
NaH (256 mg, 6.40 mmol) was added into DMF (5 mL) and stirred at 0 °C under nitrogen for 10 min. A solution of 4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (1 g, 4.93 mmol) in DMF (5 mL) was added in 15 min at 0 °C. The resulting mixture was stirred at 0°C for 30 min. Mel (909 mg, 6.40 mmol) was added to the reaction mixture. After being stirred for another 18 h at room temperature, the mixture was poured into water, and then extracted with MTBE twice. The combined organic layers were washed with brine, and then dried over anhydrous Na2S04, then filtered and concentrated in vacuo. The residue was purified by column chromatography to afford 4-bromo-l -methyl- lH-pyrrole-2-carboxylic acid methyl ester (986 mg) as a solid.
Step 2: Preparation of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000034_0002
A mixture of 4-bromo-l -methyl- lH-pyrrole-2-carboxylic acid methyl ester (434 mg, 2.0 mmol), pyridine-4-bronic acid (270 mg, 2.2 mmol), K2C03 (828 mg, 6.0 mmol), Pd(dppf)Cl2 (100 mg, 0.13 mmol) in EtOH/H20 (10/1, 8 mL) was stirred under microwave irradiation at 125 °C under nitrogen protection for 1 hour. The resulting mixture was diluted with EtOAc (100 mL), and then dried with anhydrous Na2S04, then filtered. The filtrate was concentrated in vacuo. The residue was purified by column chromatography to afford l-methyl-4-pyridin-4-yl-lH-pyrrole-2- carboxylic acid methyl ester (390 mg) as a solid.
Step 3: Preparation of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000034_0003
To a solution of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester (300 mg, 1.3 mmol) in EtOH (5 mL) was added aqueous NaOH solution (2 M, 2 mL, 4.0 mmol). The mixture was stirred at 50 °C for 18 hours. The reaction solution was adjusted to pH 5 with aqueous HC1 solution (6 M) and then concentrated in vacuo. The residue was washed with DCM/EtOH (5/1), and then filtered. The filtrate was concentrated in vacuo to give l-methyl-4- pyridin-4-yl-lH-pyrrole-2-carboxylic acid (265 mg) which was used in the next step directly.
Step 4: Preparation of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide To a solution of l-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (207 mg, 1.0 mmol) in DMF (5 mL) was added HATU (467 mg, 1.2 mmol) and TEA (124 mg, 1.2 mmol). After 10 min, NH4C1 (59 mg, 1.1 mmol) was added. The resulting mixture was stirred at room
temperature for 6 hours, and then poured into water, then extracted with EtOAc for 3 times. The combined organic layers were dried over anhydrous Na2S04, and then filtered and concentrated in vacuo. The residue was purified by column chromatography to afford l-methyl-4-pyridin-4-yl- lH-pyrrole-2-carboxylic acid amide (142 mg) as a solid.
Example 3: Preparation of l-benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of l-benzy -4-bromo-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000035_0001
To a mixture of 4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (13.3 g, 65 mmol) in DMF (100 mL) was added NaH (4 g, 100 mmol) portion- wise at 0°C. After being stirred for 1 hour, BnBr (14.4 g, 84 mmol) was added dropwise. Then the solution was warmed to room temperature and stirred for 3 hours. The mixture was poured into ice-water (500 mL). The aqueous layer was extracted with EtOAc (200 mLx4). The combined organic layers were washed with brine, and then dried over anhydrous Na2S04 and then concentrated to afford the crude 1- benzyl-4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (16 g).
Step 2: Preparation of l-benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000036_0001
A mixture of l-benzyl-4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (16.8 g, 55 mmol), pyridine-4-bronic acid (7.26 g, 60 mmol), CS2CO3 (36 g, 110 mmol) and Pd(dppf)Cl2 (4.4 g, 5 mmol) in EtOH/H2O=20/l (180 mL) was stirred at 100°C for 5 hours under nitrogen protection. The mixture was filtered through a Celite pad, and the filtrate was concentrated. The residue was purified by column chromatography to give l-benzyl-4-pyridin-4-yl-lH-pyrrole-2- carboxylic acid methyl ester (8.5 g) as a gray solid.
Step 3: Preparation of l-benzy -4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000036_0002
To a mixture of l-benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester (8.5 g, 29 mol) in EtOH/ H20=l/1 (50 mL) was added NaOH (5.8 g, 145 mmol). The solution was stirred at 80 °C for 2 hours and diluted with water (50 mL). The aqueous layer was washed with EtOAc (30 mLx3) and acidified with aqueous HC1 solution to pH 4. The precipitate was collected by filtration to give l-benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (7.6 g) as a gray solid.
Step 4: Preparation of l-benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
To a mixture of l-benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (100 mg, 0.36 mmol) in DMF (2 mL) was added HOBt (58 mg, 0.43 mmol) and EDCI (82.4 mg, 0.43 mmol). The mixture was stirred at room temperature for 30 minutes. Then aqueous ammonia (4 mL) was added. The resulting mixture was stirred at room temperature and the completion of the reaction was monitored by LC-MS. The reaction mixture was poured into water (30 mL). The precipitate was collected by filtration to give crude product which was recrystallized from EtOAc (2 mL) to give l-benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide (50 mg). Example 4: Preparation of 4-(3-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-lH-pyrrole-2- carboxylic acid methyl ester
Figure imgf000037_0001
To a mixture of 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (10 g, 28.5 mmol) in MeCN (50 mL) was added ZnBr2 (12.8 g, 57 mmol) at room temperature. The mixture was heated at 50 °C for 4 hours, and then quenched with water (30 mL) and then basified to pH 9 with aqueous ammonia. The aqueous layer was extracted with EtOAc (30 mLx 3). The combined organic layers were dried over anhydrous Na2S04 and then concentrated. The residue was purified by column chromatography to give 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-lH-pyrrole-2-carboxylic acid methyl ester as a white solid (2.5 g).
Step 2: Preparation of 4-(3-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000037_0002
A mixture of 4-(4,4,5,5-Tetramethyl-[l,3,2]dioxaborolan-2-yl)-lH-pyrrole-2-carboxylic acid methyl ester (502 mg, 2 mmol), 4-bromo-3-methyl-pyridin (412 mg, 2.4 mmol), CS2CO3 (1300 mg, 4 mmol) and Pd(dppf)Cl2 (146 mg, 0.2 mmol) in EtOH (10 mL) and water (3 mL) was refluxed under N2 protection for 3 hours. The reaction mixture was poured into water (30 mL) and then extracted with EtOAc (30 mLx3). The combined organic layers were concentrated to give a brown solid, which was purified by chromatography to give 4-(3-methyl-pyridin-4-yl)-lH- pyrrole-2-carboxylic acid methyl ester (150 mg) as a white solid.
Step 3: Preparation of 4-(3-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
Figure imgf000038_0001
A mixture of 4-(3-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (150 mg, 0.7 mmol) and NaOH (280 mg, 7 mmol) in EtOH (3 mL) and water (3 mL) was refluxed for 2 hours. The mixture was poured into water (10 mL). The aqueous layer was washed with EtOAc (10 mLx3) and acidified with 1 M aqueous HC1 solution to pH 5. The precipitate was collected by filtration to give 4-(3-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (100 mg) as a gray solid.
Step 4: Preparation of 4-(3-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
To a mixture of 4-(3-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (90 mg, 0.45 mmol) in DMF (2 mL) was added HOBt (72 mg, 0.53 mmol) and EDCI (102 mg, 0.53 mmol). The mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (4 mL) was added. The reaction mixture was stirred at room temperature and the completion of the reaction was monitored by TLC. Then the mixture was concentrated and the residue was purified by preparative HPLC to give 4-(3-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide (40 mg) as a white solid.
Example 5: Preparation of 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4-bromo-5-chloro-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000038_0002
To a mixture of 4-bromo-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (1000 mg, 3.2 mmol) in MeCN (20 mL) was added NCS (427 mg, 3.2 mmol). The mixture was refluxed overnight, and then poured into water (100 mL). The aqueous layer was extracted with EtOAc (100 mLx3). The combined organic layers were concentrated. The residue was purified by flash chromatography to give 4-bromo-5-chloro-lH-pyrrole-2-carboxylic acid methyl ester (400 mg) as a white solid. Step 2: Preparation of 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000039_0001
A mixture of 4-bromo-5-chloro-lH-pyrrole-2-carboxylic acid methyl ester (400 mg, 1.68 mmol), pyridine-4-bronic acid (248 mg, 1.2 mmol), CS2CO3 (1300 mg, 4 mmol) and Pd(dppf)Cl2 (146 mg, 0.2 mmol) in EtOH (10 mL) and water (3 mL) was heated at 120 °C under microwave for 30 min. The mixture was poured into water (30 mL). The aqueous layer was extracted with EtOAc (30 mLx3). The combined organic layers were concentrated to give a brown solid which was purified by column chromatography to give 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester (150 mg) as a white solid.
Step 3: Preparation of 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000039_0002
A mixture of 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester (100 mg, 0.42 mmol) and NaOH (168 mg, 4.2 mmol) in EtOH (3 mL) and water (3 mL) was refluxed for 4 hours. The reaction mixture was poured into water (10 mL). The aqueous layer was washed with EtOAc (10 mLx3) and acidified with 1 M aqueous HC1 solution to pH 5. The precipitate was collected by filtration to give 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (56 mg).
Step 4: Preparation of 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide To a mixture of 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (100 mg, 0.45 mmol) in DMF (2 mL) was added HOBt (72 mg, 0.53 mmol) and EDCI (102 mg, 0.53 mmol). The mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (4 mL) was added. The resulting mixture was stirred at room temperature for 2 hours and then concentrated. The residue was diluted with water (10 mL), and then extracted with EtOAc (20 mLx5). The combined organic layers were washed with brine (10 mL), and then dried over anhydrous Na2S04 and concentrated to give crude product. The crude product was recrystallized from EtOAc (3 mL) to give 5-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide (25 m as a yellow solid.
Example 6: Preparation of 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4-bromo- -methyl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000040_0001
To a mixture of Compound 5-methyl-lH-pyrrole-2-carboxylic acid ethyl ester (1.39 mg, 10 mmol) in DCM (20 mL) was added NBS (1.78 g, 10 mmol) at -10 °C. The mixture was stirred at -10 °C for 2 hours, and then warmed to room temperature slowly and the completion of the reaction was monitored by HPLC. The mixture was concentrated to give a yellow solid, which was purified by column chromatography to give 4-bromo-5-methyl-lH-pyrrole-2-carboxylic acid ethyl ester (1.5 g) as a white solid.
Step 2: Preparation of 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000040_0002
A mixture of 4-bromo-5-methyl-lH-pyrrole-2-carboxylic acid ethyl ester (232 mg, 1 mmol), pyridine-4-bronic acid (147.5 mg, 1.2 mmol), CS2CO3 (652 mg, 2 mmol) and Pd(dppf)Cl2 (73 mg, 0.1 mmol) in EtOH (4 mL) and water (1 mL) was refluxed for 4 h under N2 protection. The mixture was poured into water (30 mL) and then extracted with EtOAc (30 mLx3). The combined organic layers were concentrated to give a brown solid, which was purified by flash chromatography to give 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (100 mg) as a yellow solid.
Step 3: Preparation of 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000041_0001
A mixture of 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (100 mg, 0.43 mmol) and NaOH (172 mg, 4.3 mmol) in EtOH (2 mL) and water (2 mL) was refluxed for 4 hours. The mixture was poured into water (10 mL). The aqueous layer was washed with EtOAc (10 mLx3) and acidified with 1 M aqueous HCl solution to pH 3, the precipitate was collected by filtering to give 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (85 mg).
Step 4: Preparation of 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide To a mixture of 5-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (87 mg, 0.43 mmol) in DMF (2 mL) was added HOBt (70 mg, 0.52 mmol) and EDCI (100 mg, 0.52 mmol). The mixture was stirred at room temperature for 30 min, and then aqueous ammonia (4 mL) was added. The resulting mixture was stirred at room temperature for 1 h, and then poured into water (30 mL). The precipitate was collected by filtering and washed with EtOAc (1 mL) to give 5- methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide (81 mg).
Example 7: Preparation of 5-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide Step 1: Preparation of 5-acetyl-4-bromo-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000041_0002
To a mixture of 4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (1 g, 4.9 mmol) in DCM (30 mL) was added BF3 Et20 (7 g, 49 mmol), followed by dropwise addition of acetic anhydride (5 g, 49 mmol) at 0 °C. After addition, the mixture was stirred at 0 °C for 1 hour and then poured into water (200 mL). The aqueous layer was extracted with DCM (50 mLx3). The combined DCM layers were washed with brine, and then dried over anhydrous Na2S04 and concentrated to give 5-acetyl-4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (0.6 g).
Step 2: Preparation of 5-acetyl-4-bromo-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester
Figure imgf000042_0001
To a mixture of 5-acetyl-4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (3.3 g, 13.4 mmol), DMAP (1.6 g, 13.4 mmol) and Et3N (1.36 g, 13.4 mmol) in DCM (40 mL) was added a solution of Boc20 (3.22 g, 14.7 mmol) in DCM (10 mL) dropwise at 0 to 10 °C. The mixture was stirred at room temperature for 3 hours, and then diluted with DCM (150 mL). The organic layer was washed with water (50 mL) and brine (50 mL), and then dried over anhydrous Na2S04 and concentrated. The residue was purified by flash chromatography to give 5-acetyl-4-bromo- pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (1.8 g).
Step 3: Preparation of 5-acetyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000042_0002
To a mixture of 5-acetyl-4-bromo-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (1 g, 2.9 mmol), pyridine-4-bronic acid (380 mg, 3.2 mmol) and Cs2C03 (1.9 mg, 5.8 mmol) in EtOH/H20 (20/1, 15 mL) was added Pd(dppf)Cl2 (200 mg) under nitrogen protection. The mixture was stirred at 120°C under microwave radiation for 30 min, and then filtered through a Celite pad. The filtrate was concentrated and the residue was purified by flash chromatography to give 5-acetyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (200 mg) as a gray solid.
Step 4: Preparation of 5-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000042_0003
To a mixture of 5-acetyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (280 mg, 1 mmol) in TFA (10 mL) under nitrogen atmosphere was added triethyl-silane (378 mg, 3 mmol) and BF Et20 (5 mL) dropwise at 0°C. The solution was stirred at room temperature for 2 hours, and then quenched with water (100 mL). The aqueous layer was extracted with EtOAc (30 mLx3). The combined organic layers were washed with brine, and then dried over anhydrous Na2SC"4 and concentrated. The residue was purified by flash chromatography to give 5-ethyl-4- pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (200 mg).
Step 5: Preparation of 5-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000043_0001
A mixture of 5-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (190 mg, 0.8 mmol) and NaOH (200 mg, 5 mmol) in EtOH/H20 (20/1, 6 mL) was stirred at 100°C under microwave radiation for 30 minutes. Then the solution was acidified to pH 4 with 3 M HCl. The mixture was filtered. The solid was collected, and then was washed by EtOAc (15 mL) and water (15 mL) to give 5-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (170 mg) as a yellow solid.
Step 6: Preparation of 5-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide To a solution of 5-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (140 mg, 0.64 mmol) in DMF (3 mL) was added HOBt (120 mg, 0.9 mmol) and EDCI (172 mg, 0.9 mmol). After being stirred for 20 minutes, aqueous ammonia (30 mL) was added. The resulting solution was stirred at room temperature for 2 hours. The precipitate was collected and purified by preparative TLC to give 5-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide (45 mg) as a yellow solid.
Example 8: Preparation of 3-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4,4-dichlor -5-trichloromethyl-3,4-dihydro-2H-pyrrole
Figure imgf000043_0002
To a solution of 5-methyl-3,4-dihydro-2H-pyrrole (1.66 g, 20 mmol) in THF (20 mL) was added NCS (21.4 g, 160 mmol) at room temperature. The mixture was heated at 55 °C for 20 minutes, and then poured into water and extracted with petroleum ether (30 mLx3). The combined organic layers were dried over anhydrous Na2S04 and then concentrated to afford crude 4,4-dichloro-5-trichloromethyl-3,4-dihydro-2H-pyrrole (5.1 g).
Step 2: Preparation of 3-chloro-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000044_0001
To a solution of 4,4-dichloro-5-trichloromethyl-3,4-dihydro-2H-pyrrole (5.1 g, 20 mmol) in MeOH (20 mL) was added fresh prepared NaOMe (6.48 g, 120 mmol) at 0 °C. The mixture was stirred at room temperature for 30 minutes, and then poured into water and extracted with EtOAc (50 mLx3). The combined organic layer was dried over anhydrous Na2S04 and then concentrated. The residue was purified by column chromatography to afford crude 3-chloro-lH- pyrrole-2-carboxylic acid methyl ester (3.18 g) as a yellow solid.
Step 3: Preparation of 4-bromo-3-chloro-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000044_0002
To a solution of 3-chloro-lH-pyrrole-2-carboxylic acid methyl ester (3 g, 18.8 mmol) in CH3CN (50 mL) was added NBS (3.35 g, 18.8 mmol) at room temperature. The mixture was stirred at room temperature for 12 h and then concentrated. The residue was dissolved in EtOAc (500 mL) and then washed with water for three times. The organic layer was dried over anhydrous Na2S04 and then concentrated. The residue was purified by column chromatography to afford 4-bromo-3-chloro-lH-pyrrole-2-carboxylic acid methyl ester (3 g) as a white solid.
Step 4: Preparation of 4-bromo-3-chloro-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester
Figure imgf000044_0003
To a mixture of 4-bromo-3-chloro-lH-pyrrole-2-carboxylic acid methyl ester (1000 mg, 4.2 mmol), TEA (1272 mg, 12.6 mmol) and DMAP (49 mg, 0.4 mmol) in DCM (15 mL) was added a solution of Boc20 (1009 mg, 5 mmol) in DCM (5 mL) dropwise. The mixture was stirred at room temperature for 2 h, and then diluted with DCM (50 mL). The organic layer was washed with water (20 mL), and then dried over anhydrous Na2S04 and concentrated. The residue was purified by chromatography to give 4-bromo-3-chloro-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (1.2 g) as a white solid.
Step 5: Preparation of 3-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000045_0001
A mixture of 4-bromo-3-chloro-pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (1200 mg, 3.9 mmol), pyridine-4-bronic acid (578 mg, 4.7 mmol), CS2CO3 (2535 mg, 7.8 mmol) and Pd(dppf)Cl2 (292 mg, 0.4 mmol) in toluene (30 mL) was refluxed under N2 protection for 3 hours. The mixture was poured into water (100 mL). The aqueous layer was extracted with EtOAc (100 mLx4). The combined organic layers were concentrated and the residue was purified by column chromatography to give 3-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester (350 mg) as a yellow solid.
Step 6: Preparation of 3-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000045_0002
A mixture of 3-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester (350 mg, 1.48 mmol) and NaOH (600 mg, 10 mmol) in EtOH (4 mL) and water (4 mL) was refluxed for 4 hours, and then poured into water (10 mL). The aqueous layer was washed with EtOAc (10 mLx3) and acidified with 1 M aqueous HC1 solution to pH 5. The precipitate was collected to give 3-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (300 mg) which was used in next step without further purification.
Step 7: Preparation of 3-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide To a mixture of 3-chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (100 mg, 0.45 mmol) in DMF (2 mL) was added HOBt (73 mg, 0.54 mmol) and EDCI (104 mg, 0.54 mmol) at room temperature. The mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (4 mL) was added. The resulting mixture was stirred at room temperature and was completed as monitored by HPLC. The reaction mixture was poured into water (20 mL), and the aqueous layer was extracted with EtOAc (20 mLx5). The combined organic layers were concentrated and the residue was recrystallized from EtOAc (5 mL) to give 3-chloro-4-pyridin-4- yl-lH-pyrrole-2-carboxylic acid amide (76 mg) as a gray solid.
Example 9: Preparation of 4-(3-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4-(3-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000046_0001
A mixture of 4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (1200 mg, 3.9 mmol), 3- chloro-4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-pyridine (744 mg, 4.7 mmol), CS2CO3 (2535 mg, 7.8 mmol) and Pd(dppf)Cl2 (292 mg, 0.4 mmol) in EtOH (30 mL) was refluxed for 3 h under N2 protection, and then poured into water (100 mL). The aqueous layer was extracted with EtOAc (100 mLx4). The combined organic layer was concentrated and the residue was purified by column chromatography to give 4-(3-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (250 mg) as a yellow solid.
Step 2: Preparation of 4-(3-chlor -pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
Figure imgf000046_0002
A mixture of 4-(3-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (250 mg, 1.06 mmol) and NaOH (400 mg, 10 mmol) in EtOH (4 mL) and water (4 mL) was refluxed for 4 h, and then poured into water (10 mL). The aqueous layer was washed with EtOAc (10 mLx3) and acidified with 1 M aqueous HC1 solution to pH 5. The precipitate was collected to give 4-(3- chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (200 mg) as a gray solid.
Step 3: Preparation of 4-(3-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide To a mixture of 4-(3-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (150 mg, 0.67 mmol) in DMF (2 mL) was added HOBt (109 mg, 0.81 mmol) and EDCI (156 mg, 0.81 mmol). The mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (4 mL) was added. The resulting mixture was stirred at room temperature for 5 h and then poured into water (20 mL). The aqueous layer was extracted with EtOAc (20 mLx5). The combined organic layers were concentrated to give a brown solid which was recrystallized from EtOAc (5 mL) to give 4-(3-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide (110 mg) as a gray solid.
Example 10: Preparation of 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000047_0001
A mixture of 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-lH-pyrrole-2-carboxylic acid methyl ester (2.5 g, 10 mmol), 3,4-dibromo-pyridine (2.6 g, 11 mmol), CS2CO3 (6.5 g, 20 mmol) and Pd(dppf)Cl2 (732 mg, 1 mmol) in EtOH (30 mL) and water (5 mL) was refluxed for 3 hours under N2 protection, and then poured into water (100 mL). The aqueous layer was extracted with EtOAc (100 mLx5). The combined organic layers were concentrated. The residue was purified by flash chromatography to give 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (1 g) as a yellow solid.
Step 2: Preparation of 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
Figure imgf000047_0002
A mixture of 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (200 mg, 0.68 mmol) and NaOH (280 mg, 7 mmol) in EtOH (3 mL) and water (3 mL) was refluxed for 4 hours, and then poured into water (10 mL). The aqueous layer was washed with EtOAc (10 mLx3) and acidified with 1 M aqueous HCl solution to pH 5. The precipitate was collected and dried to give 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (130 mg) as a gray solid.
Step 3: Preparation of 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
To a mixture of 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (110 mg, 0.41 mmol) in DMF (2 mL) was added HOBt (68 mg, 0.49 mmol) and EDCI (94 mg, 0.49 mmol). The mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (4 mL) was added. The resulting mixture was stirred at room temperature and the completion of the reaction was monitored by LC/MS. The mixture was poured into water (20 mL). The aqueous layer was extracted with EtOAc (20 mLx5). The combined organic layers were concentrated to give a brown solid which was recrystallized from EtOAc (5 mL) to give 4-(3-bromo-pyridin-4- yl)-lH-pyrrole-2-carboxylic acid amide (50 mg) as a gray solid.
Example 11: Preparation of 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000048_0001
A mixture of 4-(3-bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (1000 mg, 3.3 mmol), phenylboronic acid (940 mg, 4 mmol), CS2CO3 (2140 mg, 6.6 mmol) and Pd(dppf)Cl2 (240 mg, 0.33 mmol) in EtOH (30 mL) was refluxed under N2 protection for 3 hours, and then poured into water (100 mL). The aqueous layer was extracted with EtOAc (100 mLx4). The combined organic layer was concentrated and the residue was purified by flash chromatography to give 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (550 mg) as a yellow solid.
Step 2: Preparation of 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
Figure imgf000048_0002
A mixture of 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (550 mg, 1.88 mmol) and NaOH (753 mg, 18.8 mmol) in EtOH (4 mL) and water (4 mL) was refluxed for 4 hours and then poured into water (50 mL). The aqueous layer was washed with EtOAc (20 mLx2) and acidified with 1 M aqueous HC1 solution to pH 5. The precipitate was collected and dried to give 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (470 mg) as a yellow solid.
Step 3: Preparation of 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide A mixture of 4-(3-phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (50 mg, 0.19 mmol), HATU (90 mg, 0.24 mmol) and NaHC03 (168 mg, 2 mmol) in DMF (1 mL) was stirred at room temperature for 20 minutes, and then NH4C1 (53 mg, 1 mmol) was added. The mixture was stirred at room temperature for 2 hours, and then purified by prep-HPLC to give 4-(3-phenyl- pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide (12 mg) as a white solid.
Example 12: Preparation of 3-methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of l-pyridazin-4-yl-propan-2-one
Figure imgf000049_0001
To a solution of 4-methyl-pyridazine (3.76 g, 40 mmol) in THF (40 mL) was added LDA (24 mL, 0.048 mol, 2.0 M) dropwise at -78 °C. After being stirred at -78 °C for 30 minutes, acetic anhydride (4.9 g, 0.048 mol) was added dropwise below -65 °C. The resulting mixture was warmed to room temperature slowly. The reaction was quenched with saturated NH4C1 solution (10 mL), and then dried over anhydrous Na2S04 and concentrated. The residue was purified by flash chromatography to give l-pyridazin-4-yl-propan-2-one (2 g) as yellow oil.
Step 2: Preparation of (E)-4-dimethylamino-3-pyridazin-4-yl-but-3-en-2-one
Figure imgf000049_0002
A mixture of l-pyridazin-4-yl-propan-2-one (2 g, 14.7 mmol) in DMF-DMA (5 mL) and MeCN (5 mL) was stirred at 120 °C under microwave radiation for 30 minutes. The mixture was concentrated to give crude (E)-4-dimethylamino-3-pyridazin-4-yl-but-3-en-2-one (2.8 g) as brown oil which was used in next step without further purification.
Step 3: Preparation of 3-methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000050_0001
A mixture of crude (E)-4-dimethylamino-3-pyridazin-4-yl-but-3-en-2-one (2.8 g, 14.7 mmol) and diethyl aminomalonate hydrochloride (3.1 g, 17.64 mmol) in AcOH (20 mL) was refluxed and monitored by TLC. When the reaction was complete, the mixture was concentrated. The residue was purified by flash chromatography to afford crude 3-methyl-4-pyridazin-4-yl-lH- pyrrole-2-carboxylic acid ethyl ester (400 mg) as a yellow solid.
Step 4: Preparation of 3-methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000050_0002
A mixture of 3-methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (300 mg, 1.47 mmol) and NaOH (600 mg, 15 mmol) in EtOH (5 mL) and water (5 mL) was refluxed for 4 hours and then poured into water (10 mL). The aqueous layer was washed with EtOAc (10 mLx2) and acidified with 1 M aqueous HC1 solution to pH 5. The precipitate was collected and dried to give 3-methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid (200 mg, 67%).
Step 5: Preparation of 3-methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid amide To a mixture of 3-methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid (200 mg, 0. 99 mmol) in DMF (3 mL) was added HOBt (159 mg, 1.18 mmol) and EDCI (227 mg, 1.18 mmol). The mixture was stirred at room temperature for 30 min, and then ammonia (5 mL) was added. The resulting mixture was stirred at room temperature. When the reaction was complete, the mixture was concentrated. And then the residue was purified by preparative HPLC to give 3- methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid amide (2.55 mg) as a yellow solid. Example 13: Preparation of 4-pyridin-4-yl-3-trifluoromethyl-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of dimethyl-((E -2-pyridin-4-yl-vinyl)-amine
Figure imgf000051_0001
A solution of 4-methyl-pyridine (2.5 g, 27 mmol) and l-tert-butoxy-N,N,N',N'- tetramethylmethanediamine (3.3 g, 33 mmol) in DMF (15 mL) was heated at 130 °C for 16 hours under N2 atmosphere, and then concentrated. Water (50 mL) was added to the residue, and the mixture was filtered. The solid was collected to give dimethyl-((E)-2-pyridin-4-yl-vinyl)-amine (2.6 g) as a light orange solid.
Step 2: Preparation of (E)-4-dimethylamino-l,l,l-trifluoro-3-pyridin-4-yl-but-3-en-2- one
Figure imgf000051_0002
To a solution of dimethyl-((E)-2-pyridin-4-yl-vinyl)-amine (2.6 g, 18 mmol) and Et3N (3.5 g, 35 mmol) in DCM (40 mL) was added trifluoroacetic anhydride (4.1 g, 20 mmol) dropwise at 0 °C. The resulting solution was stirred at room temperature overnight. The solvent was removed, and the residue was purified by column chromatography to give (E)-4-dimethylamino- l,l,l-trifluoro-3-pyridin-4-yl-but-3-en-2-one (3.5 g) as a light orange solid.
Step 3: Preparation of 4-pyridin-4-yl-3-trifluoromethyl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000051_0003
A solution of (E)-4-dimethylamino-l,l,l-trifluoro-3-pyridin-4-yl-but-3-en-2-one (2.5 g, 10 mmol) and diethyl aminomalonate hydrochloride (2.2 g, 13 mmol) in AcOH (10 mL) was heated at 140 °C for 1 hour under microwave condition. Then the solvent was concentrated and the residue was purified by flash chromatography to give 4-pyridin-4-yl-3-trifluoromethyl-lH- pyrrole-2-carboxylic acid ethyl ester (300 mg) as a light orange solid.
Step 4: Preparation of 4-pyridin-4- -3-trifluoromethyl-lH-pyrrole-2-carboxylic acid
Figure imgf000052_0001
A mixture of 4-pyridin-4-yl-3-trifluoromethyl-lH-pyrrole-2-carboxylic acid ethyl ester (200 mg, 0.7 mmol) in concentrated aqueous HC1 solution (5 mL) was heated at 80 °C for 4 hours. The solvent was removed and the residue was purified by prep-HPLC to give 4-pyridin-4- yl-3-trifluoromethyl-lH-pyrrole-2-carboxylic acid (10 mg) as a white solid.
Step 5: Preparation of 4-pyridin-4-yl-3-trifluoromethyl-lH-pyrrole-2-carboxylic acid amide
A mixture of 4-pyridin-4-yl-3-trifluoromethyl-lH-pyrrole-2-carboxylic acid (10 mg, 0.04 mmol), EDCI (15 mg, 0.08 mmol) and HOBt (10 mg, 0.07 mmol) in DMF (0.5 mL) was stirred at room temperature for 1 hour. Then a solution of NH3 in THF was added and the mixture was stirred for additional 30 minutes. The mixture was purified by Prep-TLC to give 4-pyridin-4-yl- 3-trifluoromethyl-lH-pyrrole-2-carboxylic acid amide (4.4 mg).
Example 14: Preparation of 4-(2-hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4-(2-hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000052_0002
Under a nitrogen atmosphere, 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)-lH-pyrrole- 2-carboxylic acid methyl ester (300 mg, 1.2 mmol), 4-bromopyridin-2(lH)-one (416 mg, 2.4 mmol), and Pd(PPh3)4 (140 mg, 0.12 mmol) were dissolved in dioxane (10 mL). A saturated aqueous sodium carbonate solution (4 ml) was added and the mixture was heated under reflux for 2 hours. The reaction mixture was cooled to room temperature and the solid was filtered off. The filtrate was concentrated and the residue was purified by flash chromatography to give 4- (2- hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (110 mg) as a white solid.
Step 2: Preparation of 4-(2-hydrox -pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
Figure imgf000053_0001
To a mixture of 4-(2-hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (110 mg, 0.5 mmol) in EtOH (3 mL) was added NaOH (3 mL, 3 M aqueous solution). The resulting solution was stirred at 80 °C for 1 hour. The solution was adjusted to pH 5 with cone, aqueous HC1 solution. The precipitate was collected and dried to give 4-(2-hydroxy-pyridin-4-yl)-lH- pyrrole-2-carboxylic acid (100 mg) as a white solid.
Step 3: Preparation of 4-(2-hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide A mixture of 4-(2-hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (100 mg, 0.49 mmol), EDCI (113 mg, 0.59 mmol) and HOBt (80 mg, 0.59 mmol) in DMF (2 mL) was stirred at room temperature for 3 hours. Then aqueous ammonia (6 mL) was added. The resulting mixture was stirred for 30 minutes and purified by prep-HPLC to give 4-(2-hydroxy-pyridin-4-yl)-lH- pyrrole-2-carboxylic acid amide (14.0 mg).
Example 15: Preparation of 4-(3-chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2- carboxylic acid amide
Step 1: Preparation of 4-bromo-3-methyl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000053_0002
A mixture of 3-methyl-lH-pyrrole-2-carboxylic acid ethyl ester (1.0 g, 6.5 mmol), NBS (1.22 g, 6.9 mmol) in CC14 (30 mL) was heated under reflux for 3 hours with nitrogen protection, and then cooled to room temperature and filtered. The filtrate was concentrated and the residue was purified by flash chromatography to give 4-bromo-3-methyl-lH-pyrrole-2-carboxylic acid ethyl ester (950 mg) as a white solid. Step 2: Preparation of 4-(3-chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000054_0001
Under a nitrogen atmosphere, 3-chloro-4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)- pyridine (550 mg, 2.3 mmol), 4-bromo-3-methyl-lH-pyrrole-2-carboxylic acid ethyl ester (800 mg, 3.4 mmol) and Pd(PPh3)4 (270 mg, 0.23 mmol) were dissolved in DME (20 mL). A 2M aqueous sodium carbonate solution (4 mL) was added and the mixture was heated under reflux overnight. Then the mixture was cooled to room temperature and the solid was filtered off. The filtrate was concentrated and the residue was purified by flash chromatography to give 4-(3- chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2-carboxylic acid ethyl ester (100 mg).
Step 3: Preparation of 4-(3-chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2-carboxylic acid
Figure imgf000054_0002
To a mixture of 4-(3-chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2-carboxylic acid ethyl ester (100 mg, 0.38 mmol) in EtOH was added 3 M aqueous NaOH solution (2 mL). The resulting mixture was stirred at 80 °C for 1 hour, and then cooled to room temperature and adjusted to pH 5 by hydrochloric acid. The precipitate was collected and dried to give 4-(3- chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2-carboxylic acid (45 mg).
Step 4: Preparation of 4-(3-chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2-carboxylic acid amide
A mixture of 4-(3-chloro-pyridin-4-yl)-3-methyl-lH-pyrrole-2-carboxylic acid (45 mg, 0.19 mmol), EDCI (44 mg, 0.23 mmol) and HOBt (32 mg, 0.23 mmol) in DMF (1 mL) was stirred at room temperature for 3 h. Then aqueous ammonia (3 mL) was added. The resulting mixture was stirred for 30 minutes and purified by prep-HPLC to give 4-(3-chloro-pyridin-4-yl)- 3-methyl-lH-pyrrole-2-carboxylic acid amide (4.7 mg). Example 16: Preparation of 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000055_0001
A mixture of 4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (3 g, 15 mmol), bis(pinacolato)diboron (3.9 g, 15 mmol), tris(dibenzylideneacetone)dipalladium (206 mg, 0.22 mmol), butyldi-l-adamantylphosphine (236 mg, 0.66 mmol) and potassium acetate (4.4 g, 45 mmol) in DME (50 mL) was heated at 90 °C for 6 hours, then cooled to room temperature. The above reaction mixture (10 mL), 4-bromo-2-methyl-pyridine (430 mg, 2.5 mmol), K2C03 (828 mg, 6 mmol), dioxane (5 mL) and water (2 mL) were added into a 20 mL microwave vial. The mixture was heated at 105 °C for 1.5 hours under microwave radiation, and then diluted by EtOAc (50 mL). The organic layer was washed with water (50 mLx3), and then dried over anhydrous Na2S04. The residue was purified by flash chromatography to give 4-(2-methyl- pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (200 mg).
Step 2: Preparation of 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
Figure imgf000055_0002
A mixture of 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (108 mg, 0.5 mmol) and lithium hydroxide monohydrate (63 mg, 1.5 mmol) in methanol (2 mL) and water (1 mL) was heated at 70 °C for 1 hour. The mixture was neutralized by diluted aqueous HCl solution to pH 6 and then concentrated to give crude 4-(2-methyl-pyridin-4-yl)-lH-pyrrole- 2-carboxylic acid (210 mg), which was used in next step without further purification.
Step 3: Preparation of 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide A mixture of crude 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (210 mg, 0.5 mmol), HATU (228 mg, 0.6 mmol), NH3 in dioxane (2 mL, 0.5 M, 1 mmol) and NaHC03 (168 mg, 2 mmol) in DMF 2 mL was stirred at room temperature for 1.5 hours, and then purified by prep-HPLC to give 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide (60 mg) as a white solid.
Example 17: Preparation of 4-(2-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of 4-(2-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000056_0001
A mixture of 4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (3 g, 15 mmol), bis(pinacolato)diboron (3.9 g, 15 mmol), tris(dibenzylideneacetone)dipalladium (206 mg, 0.22 mmol), butyldi-l-adamantylphosphine (236 mg, 0.66 mmol) and potassium acetate (4.4 g, 45 mmol) in DME (50 mL) was heated at 90 °C for 6 hours, then cooled to room temperature. The above reaction mixture (20 mL), 4-bromo-2-chloro-pyridine (960 mg, 5 mmol), K2C03 (1.6 mg, 12 mmol), dioxane (8 mL) and water (3 mL) were mixed together and added into two 20 mL microwave vial. The two mixtures were heated at 105 °C for 1.5 hours under microwave radiation, and then combined and diluted by EtOAc (100 mL). The organic layer was washed with water (50 mL x 3), and then dried over anhydrous Na2S04. The residue was purified by flash chromatography to give 4-(2-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (800 mg).
Step 2: Preparation of 4-(2-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid lithium salt
Figure imgf000056_0002
A mixture of 4-(2-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid methyl ester (200 mg, 0.85 mmol) and lithium hydroxide monohydrate (126 mg, 3 mmol) in methanol (3.3 mL) and water (1.7 mL) was heated at 70 °C for 3 h. The mixture was concentrated to give crude 4-(2- methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid lithium salt (370 mg), which was used in next step without further purification.
Step 3: Preparation of 4-(2-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide A mixture of 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid lithium salt (370 mg, 0.85 mmol), HATU (456 mg, 1.2 mmol), NH4C1 (265 mg, 5 mmol), NH3 in dioxane (2 mL, 0.5 M, 1 mmol) and NaHC03 (252 mg, 3 mmol) in DMF (2 mL) was stirred at room temperature for 2 h, and then purified by prep-HPLC to give 4-(2-chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide (16 mg) as a white solid.
Example 18: Preparation of 3-methyl-4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2- carboxylic acid amide
Step 1: Preparation of 3-methyl-4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000057_0001
A mixture of 4-bromo-3-methyl-lH-pyrrole-2-carboxylic acid ethyl ester (1 g, 4.3 mmol), bis(pinacolato)diboron (1.1 g, 4.3 mmol), tris(dibenzylideneacetone)dipalladium (60 mg, 0.066 mmol), butyldi-l-adamantylphosphine (70 mg, 0.2 mmol) and potassium acetate (1.5 g, 15 mmol) in DME (10 mL) was heated at 100 °C for 1 hour, then cooled to room temperature. To the above mixture was added 4-bromo-2-methyl-pyridine (800 mg, 5 mmol), K2C03 (1.6 mg, 12 mmol), dioxane (3 mL) and water (2 mL). The resulting mixture was heated at 110 °C for 1.5 h under microwave radiation, and then diluted by EtOAc (100 mL). The organic layer was washed with brine (50 mLx3), and then dried over anhydrous Na2S04. The residue was purified by flash chromatography to give 3-methyl-4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid ethyl ester (200 mg).
Step 2: Preparation of 3-methyl-4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid
Figure imgf000058_0001
A mixture of 3-methyl-4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid ethyl ester (200 mg, 0.82 mmol) and lithium hydroxide monohydrate (126 mg, 3 mmol) in methanol (3 mL) and water (1 mL) was heated at 70 °C for 4 hours. The mixture was neutralized with diluted aqueous HCl solution and then concentrated to give crude 4-(2-methyl-pyridin-4-yl)-lH-pyrrole- 2-carboxylic acid (410 mg), which was used in next step without further purification.
Step 3: Preparation of 3-methyl-4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide
A mixture of crude 4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid (410 mg, 0.82 mmol), HATU (380 mg, 1 mmol), NH3 in dioxane (5 mL, 0.5 M, 2.5 mmol) and NaHC03 (252 mg, 3 mmol) in DMF (2 mL) was stirred at room temperature for 2 hours, and then purified by prep-HPLC to give 3-methyl-4-(2-methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide (14 mg) as a white solid.
Example 19: Preparation of 3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of E-4-dimethylamino-3-pyridin-4-yl-but-3-en-2-one
Figure imgf000058_0002
A solution of l-pyridin-4-yl-propan-2-one (1.9 g, 14 mmol) and DMF-DMA (5 g, 42 mmol) in dioxane (10 mL) was sealed and stirred under microwave radiation at 110 °C for 1.5 hours. The resulting mixture was concentrated to give crude E-4-dimethylamino-3-pyridin-4-yl- but-3-en-2-one (2.66 g) which was used in the next step directly.
Step 2: Preparation of 3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000059_0001
A solution of E-4-dimethylamino-3-pyridin-4-yl-but-3-en-2-one (1.9 g, 10 mmol) and diethyl aminomalonate hydrochloride (2.11 g, 10 mmol) in HOAc (10 mL) was sealed and stirred under microwave radiation at 130 °C for 1 hour. The resulting mixture was concentrated to give the residue which was washed with solvent (petrolem ether/EtOAc/EtOH= 10/1/3) to afford 3- methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (1.2 g).
Step 3: Preparation of 3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000059_0002
A mixture of 3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (690 mg, 3 mmol) and NaOH (600 mg, 15 mmol) in EtOH (10 mL) and water (10 mL) was refluxed for 8 hours. The reaction mixture was poured into water (30 mL). The aqueous layer was washed with EtOAc (30 mL x 3) and then acidified with 1 M aqueous HC1 solution to pH 4, the precipitate was collected by filtration to give 3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (100 mg) as a gray solid.
Step 4: Preparation of 3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
To a mixture of 3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (45 mg, 0.2 mmol) in DMF (2 mL) was added HOBt (32.4 mg, 0.24 mmol) and EDCI (46 mg, 0.24 mmol). The mixture was stirred at room temperature for 30 minutes. Then aqueous ammonia (5 mL) was added. The resulting mixture was stirred at room temperature for 2 hours. The mixture was concentrated and purified by prep-HPLC to give 3-methyl-4-pyridin-4-yl-lH-pyrrole-2- carboxylic acid amide (11.1 mg) as a white solid.
Example 20: Preparation of 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
Step 1: Preparation of l-pyridin-4-yl-butan-2-one
Figure imgf000060_0001
To a solution of 4-methyl-pyridine (2.3 g, 25 mmol) in THF (40 mL) was added LDA (15 mL, 30 mmol, 2.0 M) dropwise at -78 °C. After being stirred at -78 °C for 30 minutes, propionyl chloride (3.33 g, 30 mmol) was added dropwise below -65 °C. The resulting mixture was warmed to room temperature slowly. The reaction was quenched with saturated NH4C1 (5 mL). The organic layer was dried over anhydrous Na2S04 and then concentrated. The residue was purified by flash chromatography to give l-pyridin-4-yl-butan-2-one (0.8 g).
Step 2: Preparation of (E)-l-dimethylamino-2-pyridin-4-yl-pent-l-en-3-one
Figure imgf000060_0002
A mixture of l-pyridin-4-yl-butan-2-one (800 mg, 5 mmol) in DMF-DMA (5 mL) was refluxed for 8 hours, and then concentrated to give crude (E)-l-dimethylamino-2-pyridin-4-yl- pent-l-en-3-one (1.2 g) as brown oil.
Step 3: Preparation of 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester
Figure imgf000060_0003
A mixture of crude (E)-l-dimethylamino-2-pyridin-4-yl-pent-l-en-3-one (1.2 g, 5 mmol) and diethyl aminomalonate hydrochloride (1.05 g, 6 mmol) in HOAc (20 mL) was refluxed for 14 hours, and then the reaction mixture was concentrated. The residue was purified by flash chromatography to give 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (250 mg) as a white solid.
Step 4: Preparation of 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000061_0001
A mixture of 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid ethyl ester (244 mg, 1 mmol) and NaOH (480 mg, 12 mmol) in EtOH (4 mL) and water (4 mL) was refluxed for 4 hours and then poured into water (10 mL). The aqueous layer was washed with EtOAc (10 mL x 3) and then acidified with 1 M HC1 aqueous solution to pH 5. The precipitate was collected and dried to give 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (150 mg).
Step 5: Preparation of 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide To a mixture of 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (150 mg, 0.69 mmol) in DMF (3 mL) was added HOBt (112 mg, 0.83 mmol) and EDCI (159 mg, 0.83 mmol). The mixture was stirred at room temperature for 30 minutes, and then aqueous ammonia (5 mL) was added. The resulting mixture was stirred at room temperature for 5 hours and then poured into water (20 mL). The aqueous layer was extracted with EtOAc (20 mL x 5). The combined organic layers were concentrated to give a brown solid (180 mg), which was recrystallized from EtOAc (5 mL) to give 3-ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide (146 mg) as a gray solid.
Example 21: Preparation of 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide Step 1: Preparation of 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester
Figure imgf000061_0002
A mixture of 4-bromo-lH-pyrrole-2-carboxylic acid methyl ester (820 mg, 4 mmol), pyridine-4-bronic acid (532 mg, 4.4 mmol), CS2CO3 (2.6 g, 8 mmol) and Pd(dppf)Cl2 (250 mg) in EtOH/H20 (20/1, 16 mL) was stirred at 120 °C for 30 minutes under microwave radiation. Then the mixture was concentrated. The residue was purified through flash chromatography to give 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester (150 mg) as a yellow solid.
Step 2: Preparation of 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid
Figure imgf000062_0001
To a solution of 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid methyl ester (150 mg, 0.7 mmol) in EtOH (5 mL) and water (5 mL) was added NaOH (160 mg, 4 mmol). The solution was stirred at 90 °C for 2 hours. Then the aqueous solution was acidified to pH 4 with 3 M aqueous HC1 solution. The mixture was filtered. The solid was collected and washed by EtOAc (15 mL) and water (15 mL) to give crude 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (140 mg) as a yellow solid.
Step 3: Preparation of 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
To a solution of 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid (140 mg, 0.74 mmol) in DMF (3 mL) was added HOBt (120 mg, 0.9 mmol) and EDCI (172 mg, 0.9 mmol). The mixture was stirred for 20 minutes at room temperature, and then ammonia (30 mL) solution was added. The mixture was stirred at room temperature for 2 hours. The precipitate was collected by filtration to give 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide (20 mg) as a gray solid.
BIOLOGICAL EXAMPLES
Example 22: CDK8/Cyclin C LANCE TR-FRET kinase assay:
The biological activity of the compounds of the invention can be determined using the assay described below.
CDK8/Cyclin C protein was obtained from Invitrogen, cat# PV4402. ULight-Glycogen Synthase (Ulight-GS) peptide with sequence PASVPPSPSLSRHSSPHQ(pS)ED, and Europium- anti-phospho Glycogen Synthase (Ser641) [Eu-anti-P-GS (Ser641)] were obtained from Perkin Elmer, cat# TRF0131-M and cat# TRF0220. Adenosine-5 '-triphosphate (ATP) was obtained from Invitrogen, cat# PV3227.
A mixture of (1) a compound of formula I, (2) substrate [Ulight-GS peptide (80 nM) and ATP (24 μΜ)], and (3) CDK8/Cyclin C (10 nM) in reaction buffer (50 mM Hepes, pH7.0, 10 mM MgCl2, 1 mM EGTA, 0.2 mg/mL BSA, 0.8 mM DTT) were incubated at 37 °C for 30 mins. Then, [Eu-anti-P-GS (Ser641)] (1.5 nM) was added. Following incubation at room temperature for 30 mins, the TR-FRET signals were detected using Envision reader (Ex 340 nm, Em 615 nm and 665 nm) from Perkin Elmer. The reactivity in percentage of inhibition or dose response was analyzed with GraphPad Prism 5 (GraphPad Software).
Results of CDK8/Cyclin LANCE Ultra biochemical TR-FRET kinase assay are given in Table 1.
Example 23: In vitro cell proliferation assay:
Cells were seeded on 96- well plates at 5 x 10 cells per well and precultured for 24 hours. The cells were treated with serial diluted compounds and cultured for 72 hours. Then all media was discarded and after that, 100 1: 10 (v/v) Cell Counting Kit-8 (CCK-8)-culture media solution was added to the wells. Plate was developed for 2 hours in an incubator, and the absorbance was measured at 450 nm wavelengths with SpectraMAX190 (MDS, Sunnyvale, CA). The inhibition rate (IR) of the tested compounds was determined with following formula: IR ( )= (ODDMso-ODcompound)/ODDMso xl00 . The concentration corresponding to 50% IR (IC50) was determined with plot curve of IR against tested compound concentrations with SoftMax Pro.
Results of in vitro cell proliferation assay are given in Table 3.
The compounds of the present invention were tested for their capacity to inhibit a CDK8 activity and activation as described herein. The Examples were tested in the above assay and found to have IC50 of about 0.0001 μΜ to about 30 μΜ. Particular compounds of formula I were found to have IC50 of about 0.0001 μΜ to about 1 μΜ.
Example A
A compound of formula I can be used in a manner known per se as the active ingredient for the production of tablets of the following composition:
Per tablet
Active ingredient 200 mg
Microcrystalline cellulose 155 mg
Corn starch 25 mg
Talc 25 mg
Hydroxypropylmethylcellulose 20 mg
425 mg
Example B A compound of formula I can be used in a manner known per se as the active ingredient for the production of capsules of the following composition:
Per capsule
Active ingredient 100.0 mg
Corn starch 20.0 mg
Lactose 95.0 mg
Talc 4.5 mg
Magnesium stearate 0.5 mg
220.0 mg

Claims

Claims
1. The use of a compound of formu
Figure imgf000065_0001
wherein
R 1 and R 3J are independently selected from hydrogen; halogen; and C1-6alkyl, which is unsubstituted or substituted by fluoro;
R is hydrogen, Ci^alkyl or phenyl-CH2-;
R4 is hydrogen, C1-6alkyl, halogen or phenyl;
W is nitrogen or -CR5; wherein R5 is hydrogen, C1-6alkyl, amino, halogen or hydroxy; or a pharmaceutically acceptable salt thereof for the preparation of medicaments for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
2. Compounds of formula (I), wherein
R 1 and R 3J are independently selected from hydrogen, halogen and C1-6alkyl, which is unsubstituted or substituted by fluoro;
R is hydrogen, Ci^alkyl or phenyl-CH2-;
R4 is hydrogen, Ci-6alkyl, halogen or phenyl;
W is nitrogen or -CR5; wherein R5 is hydrogen, C1-6alkyl, amino, halogen or hydroxy; or a pharmaceutically acceptable salt thereof;
with the proviso that
3-methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide, 3-ethyl-4-pyridin-4-yl-lH-pyrrole- 2-carboxylic acid amide and 4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide
are excluded.
3. A compound according to claim 2, wherein
R1 is hydrogen, chloro, methyl or ethyl;
R is hydrogen, methyl or phenyl-CH2-;
R is hydrogen, chloro, methyl or trifluoromethyl;
R4 is hydrogen, methyl, chloro, bromo or phenyl;
W is nitrogen or -CR5; wherein R5 is hydrogen, methyl, amino, chloro or hydroxy; or a pharmaceutically acceptable salt thereof.
4. A compound according to claim 2, wherein
R1 is hydrogen, halogen or C1-6alkyl;
R is hydrogen, C1-6alkyl or phenyl-CH2-;
R is hydrogen, C1-6alkyl, halogen or trifluoromethyl;
R4 is hydrogen, C1-6alkyl, halogen or phenyl;
W is -CR5; wherein R5 is hydrogen, C1-6alkyl, amino, halogen or hydroxy;
or a pharmaceutically acceptable salt thereof.
5. A compound according to any one of claims 2 to 4, wherein
R1 is hydrogen, chloro, methyl or ethyl;
R is hydrogen, methyl or phenyl-CH2-;
R is hydrogen, chloro, methyl or trifluoromethyl;
R4 is hydrogen, methyl, chloro, bromo or phenyl;
W is -CR5; wherein R5 is hydrogen, methyl, amino, chloro or hydroxy;
or a pharmaceutically acceptable salt thereof.
6. A compound according to claims 2 or 4, wherein
R1 is hydrogen;
R is hydrogen;
R is C^aUcyl, halogen or trifluoromethyl;
R4 is hydrogen or halogen;
W is -CR5; wherein R5 is hydrogen, C1-6alkyl;
or a pharmaceutically acceptable salt thereof.
7. A compound according to any one of claims 2 to 6, wherein R1 is hydrogen;
R is hydrogen;
R is methyl, chloro or trifhioromethyl;
R4 is hydrogen or chloro;
W is -CR5; wherein R5 is hydrogen or methyl;
or a pharmaceutically acceptable salt thereof.
8. A compound according to claim 2 or 4, wherein
R1 is hydrogen, halogen or C1-6alkyl;
R is hydrogen, C1-6alkyl or phenyl-CH2-;
R is hydrogen;
R4 is hydrogen, C1-6alkyl, halogen or phenyl;
W is -CR5; wherein R5 is hydrogen, C1-6alkyl, amino, halogen or hydroxy; or a pharmaceutically acceptable salt thereof.
9. A compound according to any one of claims 2, 3, 4, 5 or 8, wherein
R1 is hydrogen, chloro, methyl or ethyl;
R is hydrogen, methyl or phenyl-CH2-;
R is hydrogen;
R4 is hydrogen, methyl, chloro, bromo or phenyl;
W is -CR5; wherein R5 is hydrogen, methyl, amino, chloro or hydroxy; or a pharmaceutically acceptable salt thereof.
10. A compound according to any one of claims 2 to 9, selected from
4-(2-Amino-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide;
l-Methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide;
l-Benzyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide;
4- (3-Methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide;
5- Chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide;
5-Methyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide;
5-Ethyl-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide;
3- Chloro-4-pyridin-4-yl-lH-pyrrole-2-carboxylic acid amide;
4- (3-Chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide; 4-(3-Bromo-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide;
4-(3-Phenyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide;
3- Methyl-4-pyridazin-4-yl-lH-pyrrole-2-carboxylic acid amide;
4- Pyridin-4-yl-3-trifluoromethyl- lH-pyrrole-2-carboxylic acid amide;
4-(2-Hydroxy-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide;
4-(3-Chloro-pyridin-4-yl)-3-methyl- lH-pyrrole-2-carboxylic acid amide;
4-(2-Methyl-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide;
4-(2-Chloro-pyridin-4-yl)-lH-pyrrole-2-carboxylic acid amide; and
3-Methyl-4-(2-methyl-pyridin-4-yl)- lH-pyrrole-2-carboxylic acid amide.
11. A process for the preparation of a compound according to any one of claims 2 to 10
comprising the reaction of
Figure imgf000068_0001
with NH3, or NH3 H20 or NH4C1 in the presence of a coupling agent, and a base; wherein R1 to
R and W are defined as in any one of claims 1 to 9.
12. A compound according to any one of claims 1 to 10 for use as therapeutically active
substance.
13. A pharmaceutical composition comprising a compound in accordance with any one of claims 1 to 10 and a therapeutically inert carrier.
14. The use of a compound according to any one of claims 1 to 10 for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
15. The use of a compound according to any one of claims 1 to 10 for the treatment of gastric cancer or colorectal cancer.
16. The use of a compound according to any one of claims 1 to 10 for the preparation of a medicament for the treatment of cancer, in particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
17. The use of a compound according to any one of claims 1 to 10 for the preparation of a medicament for the treatment of gastric cancer or colorectal cancer.
18. A compound according to any one of claims 1 to 10 for the treatment of cancer, in
particular bladder, head and neck, breast, stomach, ovary, colon, lung, brain, larynx, lymphatic system, liver, skin, hematopoietic system, genitourinary tract, gastrointestinal, ovarian, prostate, gastric, bone, small-cell lung, glioma, colorectal and pancreatic cancers.
19. A compound according to any one of claims 1 to 10 for the treatment of gastric cancer or colorectal cancer.
20. A compound according to any one of claims 1 to 10 as inhibitors of CDK8 or Cyclin C.
21. A compound according to any one of claims 1 to 10, when manufactured according to a process of claim 11.
22. A method for the treatment of cancer, which method comprises administering an effective amount of a compound as defined in any one of claims 1 to 10.
23. The invention as hereinbefore described.
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