Patent Application
20240360126
The present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to antagonist of TLR7 and TLR8 and TLR9 useful for treating systemic lupus erythematosus or lupus nephritis.
Autoimmune connective tissue disease (CTD) include prototypical autoimmune syndromes such as Systemic Lupus Erythematosus (SLE), primary Sjögren's syndrome (pSjS), mixed connective tissue disease (MCTD), Dermatomyositis/Polymyositis (DM/PM), Rheumatoid Arthritis (RA), and systemic sclerosis (SSc). With the exception of RA, no really effective and safe therapies are available to patients. SLE represents the prototypical CTD with a prevalence of 20-150 per 100,000 and causes broad inflammation and tissue damage in distinct organs, from commonly observed symptoms in the skin and joints to renal, lung, or heart failure. Traditionally, SLE has been treated with nonspecific anti-inflammatory or immunosuppressive drugs. However, long-term usage of immunosuppressive drug, e.g. corticosteroids is only partially effective, and is associated with undesirable toxicity and side effects. Belimumab is the only FDA-approved drug for lupus in the last 50 years, despite its modest and delayed efficacy in only a fraction of SLE patients (Navarra, S. V. et al Lancet 2011, 377, 721). Other biologics, such as anti-CD20 mAbs, mAbs against or soluble receptors of specific cytokines, have failed in most clinical studies. Thus, novel therapies are required that provide sustained improvement in a greater proportion of patient groups and are safer for chronic use in many autoimmune as well as auto-inflammation diseases.
Toll like Receptors (TLR) are an important family of pattern recognition receptors (PRR) which can initiate broad immune responses in a wide variety of immune cells. As natural host defense sensors, endosomal TLRs 7, 8 and 9 recognize nucleic acids derived from viruses, bacteria; specifically, TLR7/8 and TLR9 recognize single-stranded RNA (ssRNA) and single-stranded CpG-DNA, respectively. However, aberrant nucleic acid sensing of TRL7, 8, 9 is considered as a key node in a broad of autoimmune and auto-inflammatory diseases (Krieg, A.
M. et al. Immunol. Rev. 2007, 220, 251. Jimńez-Dalmaroni, M. J. et al Autoimmun Rev. 2016, 15, 1. Chen, J. Q., et al. Clinical Reviews in Allergy & Immunology 2016, 50, 1). Anti-RNA and anti-DNA antibodies are well-established diagnostic markers of SLE, and these antibodies can deliver both self-RNA and self-DNA to endosomes. While self-RNA complexes can be recognized by TLR7 and TLR8, self-DNA complexes can trigger TLR9 activation. Indeed, defective clearance of self-RNA and self-DNA from blood and/or tissues is evident in SLE (Systemic Lupus Erythematosus) patients. TLR7 and TLR9 have been reported to be upregulated in SLE tissues, and correlate with chronicity and activity of lupus nephritis, respectively. In B cells of SLE patients, TLR7 expression correlates with anti-RNP antibody production, while TLR9 expression with IL-6 and anti-dsDNA antibody levels. Consistently, in lupus mouse models, TLR7 is required for anti-RNA antibodies, and TLR9 is required for anti-nucleosome antibody. On the other hand, overexpression of TLR7 or human TLR8 in mice promotes autoimmunity and autoinflammation. Moreover, activation of TLR8 specifically contributes to inflammatory cytokine secretion of mDC/macrophages, neutrophil NETosis, induction of Th17 cells, and suppression of Treg cells. In addition to the described role of TLR9 in promoting autoantibody production of B cells, activation of TLR9 by self-DNA in pDC also leads to induction of type I IFNs and other inflammatory cytokines. Given these roles of TLR9 in both pDC and B cells, both as key contributors to the pathogenesis of autoimmune diseases, and the extensive presence of self-DNA complexes that could readily activate TLR9 in many patients with autoimmune diseases, it may have extra benefit to further block self-DNA mediated TLR9 pathways on top of inhibition of TLR7 and TLR8 pathways. Taken together, TLR7, 8 and 9 pathways represent new therapeutic targets for the treatment of autoimmune and auto-inflammatory diseases, for which no effective steroid-free and non-cytotoxic oral drugs exist, and inhibition of all these pathways from the very upstream may deliver satisfying therapeutic effects. As such, we invented oral compounds that target and suppress TLR7, TLR8 and TLR9 for the treatment of autoimmune and auto-inflammatory diseases.
The present invention relates to novel compounds of formula (I),
wherein
Another object of the present invention is related to novel compounds of formula (I) or (Ia). 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) or (Ia) as TLR7 and TLR8 and TLR9 antagonist, and for the treatment or prophylaxis of systemic lupus erythematosus or lupus nephritis. The compounds of formula (I) or (Ia) show superior TLR7 and TLR8 and TLR9 antagonism activity. In addition, the compounds of formula (I) or (Ia) also show good cytotoxicity, phototoxicity, solubility, hPBMC, human microsome stability and SDPK profiles, as well as low CYP inhibition.
Novartis patent WO2018047081 disclosed compounds with same pyrazolo[3,4-b]pyridinyl moiety as the compounds of this invention, however the central bicyclic core of 4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine and the terminal substitution with bicyclo[2,2,2]octane/bicyclo[1,1,1]pentane moieties were essential for the TLR7/8/9 activity based on the information disclosed in Novartis patent, which also were considered as the major structural differentiation compared with the compounds of current invention. On the other hand, most of the compounds in WO2018047081 showed poor TLR9 activity. Unfortunately few compounds with relatively improved TLR9 activity, such as N79 (as Example 79) with best TLR9 activity, still suffer from poor human liver microsome stability (see Table 5) and therefore have unsatisfactory PK profile.
Another Novartis patent WO2019220390 disclosed the polymorph of compound N8 (as Example 8 in WO2018047081), which considered as the lead compound of its series and turned out to have much lower TLR9 activity and similar poor human liver microsome stability (see Table 5).
The term “C1-6alkyl” denotes a saturated, linear or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like. Particular “C1-6alkyl” groups are methyl, ethyl and n-propyl.
The term “C1-6alkoxy” denotes C1-6alkyl-O—.
The term “halogen” and “halo” are used interchangeably herein and denote fluoro, chloro, bromo, or iodo.
The term “halopiperidinyl” denotes a piperidinyl group wherein at least one of the hydrogen atoms of the piperidinyl group has been replaced by same or different halogen atoms, particularly fluoro atoms. Examples of halopiperidinyl include fluoropyrrolidinyl and difluoropiperidinyl.
The term “cis” and “trans” denote the relative stereochemistry of the molecule or moiety. For example:
Example 18 as the cis-isomer, refers to a mixture of
similarly, Example 19 as the trans-isomer, refers to a mixture of
The term “pharmaceutically acceptable salts” denotes salts which are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts.
The term “pharmaceutically acceptable acid addition salt” denotes those pharmaceutically acceptable salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicyclic acid.
The term “pharmaceutically acceptable base addition salt” denotes those pharmaceutically acceptable salts formed with an organic or inorganic base. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, and polyamine resins.
The term “A pharmaceutically active metabolite” denotes a pharmacologically active product produced through metabolism in the body of a specified compound or salt thereof. After entry into the body, most drugs are substrates for chemical reactions that may change their physical properties and biologic effects. These metabolic conversions, which usually affect the polarity of the compounds of the invention, alter the way in which drugs are distributed in and excreted from the body. However, in some cases, metabolism of a drug is required for therapeutic effect.
The term “therapeutically effective amount” denotes an amount of a compound or molecule of the present invention that, when administered to a subject, (i) treats or prevents the particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition or disorder described herein. The therapeutically effective amount will vary depending on the compound, the disease state being treated, the severity of the disease treated, the age and relative health of the subject, the route and form of administration, the judgement of the attending medical or veterinary practitioner, and other factors.
The term “pharmaceutical composition” denotes a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with pharmaceutically acceptable excipients to be administered to a mammal, e.g., a human in need thereof.
The present invention relates to (i) a compound of formula (I),
wherein
Another embodiment of present invention is (ii) a compound of formula (Ia),
wherein
A further embodiment of present invention is (iii) a compound of formula (I) or (Ia) according to (i) or (ii), or a pharmaceutically acceptable salt thereof, wherein R1 is
wherein R4 is C1-6alkyl; R5 is C1-6alkyl.
A further embodiment of present invention is (iv) a compound of formula (I) or (Ia), according to any one of (i) to (iii), or a pharmaceutically acceptable salt thereof, wherein R4 is methyl; R5 is methyl.
A further embodiment of present invention is (v) a compound of formula (I) or (Ia) according to any one of (i) to (iv), wherein A is CR6; wherein R6 is H or C1-6alkyl.
A further embodiment of present invention is (vi) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (v), wherein A is CR6; wherein R6 is H or methyl.
A further embodiment of present invention is (vii) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (vi), wherein M is CR7; wherein R7 is H.
A further embodiment of present invention is (viii) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (vii), wherein W is CH.
A further embodiment of present invention is (ix) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (viii), wherein Q is N.
A further embodiment of present invention is (x) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (ix), wherein R3 is amino-1,4-oxazepanyl, amino(C1-6alkoxy)pyrrolidinyl or piperazinyl.
A further embodiment of present invention is (xi) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (x), wherein R3 is 6-amino-1,4-oxazepan-4-yl, 3-amino-4-methoxy-pyrrolidin-1-yl or piperazin-1-yl.
A further embodiment of present invention is (xii) a compound of formula (I) or (Ia), according to any one of (i) to (xi), wherein
A further embodiment of present invention is (xiii) a compound of formula (I) or (Ia), according to any one of (i) to (xii), wherein
Another embodiment of present invention is (xiv) a compound of formula compound of formula (Ib),
wherein
A further embodiment of present invention is (xv) a compound of formula (Ib), according to (xiv), wherein R4 is methyl; R5 is methyl; R2 is methyl.
A further embodiment of present invention is (xvi) a compound of formula (Ib), according to (xiv) or (xv), wherein R3 is amino-1,4-oxazepanyl.
A further embodiment of present invention is (xvii) a compound of formula (Ib), according to any one of (xiv) to (xvi), wherein R3 is 6-amino-1,4-oxazepan-4-yl.
A further embodiment of present invention is (xviii) a compound of formula (Ib), according to any one of (xiv) to (xvii), wherein
A further embodiment of present invention is (xix) a compound of formula (Ib), according to any one of (xiv) to (xviii), wherein
Another embodiment of present invention is a compound of formula (I) or (Ia) or (Ib) selected from the following:
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 preferably ranges anywhere from about 3 to about 8. In one example, a compound of formula (I) is formulated in an acetate buffer, at pH 5. In another embodiment, the 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 TLR7, 8, 9 interaction with respective stimulatory ligands, downstream signaling mediated by MYD88, IRF7, IRF5, NFκB etc. leading to production of type I interferons and proinflammatory cytokines (e.g. TNFα, IL-6), and activation of all kind of immune cells. 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 0.001 to 1000 mg/kg, alternatively about 0.001 to 1000 mg/kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg/kg/day. In another embodiment, oral unit dosage forms, such as tablets and capsules, preferably contain from about 0.1 to about 1000 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 excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
An example of a suitable oral dosage form is a tablet containing about 0.1 to 1000 mg of the compound of the invention compounded with about 0.1 to 1000 mg anhydrous lactose, about 0.1 to 1000 mg sodium croscarmellose, about 0.1 to 1000 mg polyvinylpyrrolidone (PVP) K30, and about 0.1 to 1000 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 0.1 to 1000 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 an autoimmune disease. Another embodiment includes a pharmaceutical composition comprising a compound of Formula (I) for use in the treatment of autoimmune disease.
The following embodiments illustrate typical compositions of the present invention, but serve merely as representative thereof.
Composition A A compound of the present invention can be used in a manner known per se as the active ingredient for the production of tablets of the following composition:
Composition B A compound of the present invention can be used in a manner known per se as the active ingredient for the production of capsules of the following composition:
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 to R5, A, M, W and Q 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 routes for preparing the compound of formula (I) or (Ia) or (Ib) are shown below.
Wherein X is halogen.
The coupling of compound of formula (II) with (III) can be achieved by direct substitution at elevated temperature in the presence of a base, such as DIPEA or CsF, or via coupling reaction of Buchwald-Hartwig C—N bond formation (ref. Acc. Chem. Res. 1998, 31, 805-818; Chem. Rev. 2016, 116, 12564-12649; Topics in Current Chemistry, 2002, 219, 131-209; and references cited therein) with a catalyst, such as RuPhos Pd G2, Pd2(dba)3/XantPhos and a base, such as Cs2CO3 or t-BuONa, to provide compound of formula (IV). Subsequently, the coupling of compound of formula (IV) with R3—H can be achieved by direct coupling under Buchwald-Hartwig C—N bond formation conditions with a catalyst, such as RuPhos Pd G2, Pd2(dba)3/XantPhos and a base, such as Cs2CO3 or t-BuONa, to provide compound of formula (I). In some embodiments, the coupling of compound of formula (IV) with R3—H may give a product containing a protecting group, e.g. Boc or Cbz, originated from R3—H, which will be removed before affording the final compound of formula (I).
Wherein X is halogen.
Compound of formula (VI) can be achieved by hydrogenation reaction from compound of formula (V) in the presence of a catalyst, such as Pd(OH)2/C, Pd/C or Wilkinson catalyst under hydrogen atmosphere. The Cbz group could also be de-protected under the same condition.
Subsequently, the coupling of compound of formula (VI) with (II) can be achieved by direct substitution at elevated temperature in the presence of a base, such as DIPEA or CsF, or by Buchwald-Hartwig C—N bond formation at conditions with a catalyst, such as RuPhos Pd G2, Pd2(dba)3/XantPhos and a base, such as Cs2CO3 or t-BuONa, to provide compound of formula (I). In some embodiments, the coupling of compound of formula (VI) with (II) may give a product containing a protecting group, e.g. Boc, originated from (VI), which will be removed before affording the final compound of formula (I).
Compounds of this invention can be obtained as mixtures of diastereomers or enantiomers, which can be separated by methods well known in the art, e.g. (chiral) HPLC or SFC.
This invention also relates to a process for the preparation of a compound of formula (I) or (Ia) comprising the following steps:
A compound of formula (I) or (Ia) when manufactured according to the above process is also an object of the invention.
The present invention provides compounds that can be used as TLR7 and TLR8 and TLR9 antagonist, which inhibits pathway activation through TLR7 and/or TLR8 and/or TLR9 as well as respective downstream biological events including, but not limited to, innate and adaptive immune responses mediated through the production of all types of cytokines and all forms of auto-antibodies. Accordingly, the compounds of the invention are useful for blocking TLR7 and/or TLR8 and/or TLR9 in all types of cells that express such receptor(s) including, but not limited to, plasmacytoid dendritic cell, B cell, T cell, macrophage, monocyte, neutrophil, keratinocyte, epithelial cell. As such, the compounds can be used as a therapeutic or prophylactic agent for systemic lupus erythematosus and lupus nephritis.
The present invention provides methods for treatment or prophylaxis of systemic lupus erythematosus and lupus nephritis in a patient in need thereof.
Another embodiment includes a method of treating or preventing systemic lupus erythematosus and lupus nephritis 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.
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.
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:
Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SP1 system and the Quad 12/25 Cartridge module. ii) ISCO combi-flash chromatography instrument. Silica gel brand and pore size: i) KP-SIL 60 Å, particle size: 40-60 μm; 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 XBridge™ Prep-C18 (5 μm, OBD™ 30×100 mm) column, SunFire™ Prep-C18 (5 μm, OBD™ 30×100 mm) column, Phenomenex Synergi-C18 (10 μm, 25×150 mm) or Phenomenex Gemini-C18 (10 μm, 25×150 mm). Waters AutoP purification System (Sample Manager 2767, Pump 2525, Detector: Micromass ZQ and UV 2487, solvent system: acetonitrile and 0.1% ammonium hydroxide in water; acetonitrile and 0.1% FA in water or acetonitrile and 0.1% TFA in water). Or Gilson-281 purification System (Pump 322, Detector: UV 156, solvent system: acetonitrile and 0.05% ammonium hydroxide in water; acetonitrile and 0.225% FA in water; acetonitrile and 0.05% HCl in water; acetonitrile and 0.075% TFA in water; or acetonitrile and water).
For SFC chiral separation, intermediates were separated by chiral column (Daicel chiralpak IC, 5 μm, 30×250 mm), AS (10 μm, 30×250 mm) or AD (10 μm, 30×250 mm) using Mettler Toledo Multigram III system SFC, Waters 80Q preparative SFC or Thar 80 preparative SFC, solvent system: CO2 and IPA (0.5% TEA in IPA) or CO2 and MeOH (0.1% NH3·H2O in MeOH), back pressure 100 bar, detection UV@ 254 or 220 nm.
LC/MS spectra of compounds were obtained using a LC/MS (Waters™ Alliance 2795-Micromass ZQ, Shimadzu Alliance 2020-Micromass ZQ or Agilent Alliance 6110-Micromass ZQ), LC/MS conditions were as follows (running time 3 or 1.5 mins):
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 (MH)+.
NMR Spectra were obtained using Bruker Avance 400 MHz.
The microwave assisted reactions were carried out in a Biotage Initiator Sixty microwave synthesizer. All reactions involving air-sensitive reagents were performed under an argon or nitrogen atmosphere. Reagents were used as received from commercial suppliers without further purification unless otherwise noted.
The following examples 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:
The title compound was prepared according to the following scheme:
To a solution of 8-aminoquinoline (compound A2, CAS: 578-66-5, Vendor: Accela, 69.17 g, 479.78 mmol) in DMF (1000 mL) was added HATU (190.60 g, 501.59 mmol) and DIPEA (233 mL, 1309 mmol). The reaction was stirred at 0° C. for 1 h. Then (3S)-1-tert-butoxycarbonylpiperidine-3-carboxylic acid (compound A1, CAS: 88495-54-9, Vendor: Accela, 100.00 g, 436.17 mmol) was added. The reaction was stirred at 0° C. for 15 h. EtOAc (200 mL) and ice-water (50 mL) were added and separated. The aqueous phase was extracted with EtOAc (200 mL) twice. The combined organic layer was washed with brine (300 mL) for 4 times, dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by flash column eluting with a gradient of EA/PE (1:5) to give compound A3 (120 g) as a yellow solid. MS: calc'd 356 (MH+), measured 356 (MH+).
Step 2: Preparation of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(8-quinolylcarbamoyl)piperidine-1-carboxylate (Compound A5) and tert-butyl (3S,4S)-4-(5-chloro-2-pyridyl)-3-(8-quinolylcarbamoyl)piperidine-1-carboxylate (Compound A13)
The mixture of tert-butyl (3S)-3-(8-quinolylcarbamoyl)piperidine-1-carboxylate (compound A3, 50.00 g, 140.67 mmol), 5-chloro-2-iodo-pyridine (compound A4, CAS: 244221-57-6, Vendor: Bide Pharmatech, 77.00 g, 322.18 mmol) and Pd(OAc)2 (3.15 g, 14.07 mmol), AgOAc(46.70 g, 281.35 mmol) was degassed with N2, and then stirred at 110° C. for 48 h under N2 atmosphere. After being cooled to room temperature, the mixture was filtrated. The filtrate was concentrated under vacuum. The residue was further purified by flash column eluting with a gradient of EA/PE (1/10 to 1/1) to give compound A5 (20.00 g) and compound A13 (15.00 g) as yellow oil. MS: calc'd 467 (MH+), measured 467 (MH+).
To a solution of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(8-quinolylcarbamoyl)piperidine-1-carboxylate (compound A5, 17.00 g, 36.41 mmol) in ethanol (200 mL) was added sodium hydroxide (14.57 g, 364.22 mmol) and then stirred at 85° C. for 16 h. After being cooled to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was neutralized with HCl (3 M) to pH 6. The mixture was extracted with DCM (300 mL) three times. The organic phase was concentrated in vacuum. The residue was purified by flash column eluting with a gradient of PE/EA (5/1 to 1/1) to give compound A6 (11.50 g) as yellow oil. MS: calc'd 341 (MH+), measured 285 (M-C4H8+H+).
To a solution of (3S,4R)-1-tert-butoxycarbonyl-4-(5-chloro-2-pyridyl)piperidine-3-carboxylic acid (compound A6, 11.50 g, 33.74 mmol) in THF (50 mL) was added BH3·Me2S (13.5 mL, 134.98 mmol) at 0° C. After addition, the reaction mixture was further stirred at 25° C. under N2 atmosphere for 16 h. The volatiles were removed to give a white solid residue and then methanol (50 mL) was added. After being stirred for 30 min, the reaction mixture was concentrated and methanol (50 mL) was added again. The residue was then treated with dropwise addition of 10% citric acid. The mixture was extracted with DCM (200 mL) for three times. The combined organic layer was dried with Na2SO4, filtered and concentrated in vacuum. The residue was purified by flash column eluting with a gradient of PE/EA (3/1) to give compound A7 (8.00 g) as colorless gum. MS: calc'd 327 (MH+), measured 271 (M-C4H8+H+).
To a solution of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(hydroxymethyl)piperidine-1-carboxylate (compound A7, 4.00 g, 12.24 mmol) and TCDI (4.36 g, 24.48 mmol) in THF (60 mL) was added DMAP (299 mg, 2.45 mmol). The mixture was stirred at 25° C. for 3 h. Then the reaction mixture was concentrated and the residue was purified by flash column eluting with a gradient of PE/EA (1/1) to give compound A8 (5.00 g) as yellow oil. MS: calc'd 437 (MH+), measured 437 (MH+).
To a mixture of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-(imidazole-1-carbothioyloxymethyl)piperidine-1-carboxylate (compound A8, 5.00 g, 11.44 mmol) in toluene (250 mL) was added tris(trimethylsilyl)silane (17 mL, 47.56 mmol) and AIBN (1877 mg, 11.44 mmol). The mixture was degassed with N2 for 10 min and stirred at 85° C. for 5 h. After being cooled to room temperature, the mixture was concentrated under vacuum. The residue was purifed by flash column eluting with a gradient of PE/EA (5/1) and pre-HPLC to give compound A9 (combination of two batches, 4.00 g) as yellow oil. MS: calc'd 311 (MH+), measured 255 (M-C4H8+H+).
To a solution of tert-butyl (3S,4R)-4-(5-chloro-2-pyridyl)-3-methyl-piperidine-1-carboxylate (compound A9, 4.00 g, 12.87 mmol) in DCM (20 mL) was added TFA (31 mL, 123.08 mmol) at 0° C. The mixture was stirred at 25° C. for 16 h. The reaction mixture was concentrated under vacuum. The residue was purified by pre-HPLC to give compound A10 (2.70 g) as a white solid. MS: calc'd 211 (MH+), measured 211 (MH+).
The mixture of 4-chloro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound A12, CAS: 19867-78-8, Vendor: PharmaBlock, 4.60 g, 25.33 mmol) and cesium fluoride (19.24 mg, 126.64 mmol) in DMSO (60 mL) was stirred at 120° C. for 18 hrs. After being cooled to rt, the reaction mixture was added to water (100 mL), extracted with EA (100 mL) three times. The combined organic layer was washed with brine (200 mL) twice, dried over Na2SO4, filtered and concentrated. The residue was purified by flash column to give compound A11 (4.00 g, contained some compound A12) as a white solid. MS: calc'd 166 (MH+), measured 166 (MH+).
To a mixture of 4-fluoro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound A11, 2.12 g, 12.81 mmol) and 5-chloro-2-[(3S,4R)-3-methyl-4-piperidyl]pyridine (compound A10, 2.70 g, 12.81 mmol) in DMSO (50 mL) was added CsF (3.72 mg, 64.07 mmol). The reaction was stirred at 120° C. for 12 h under N2 atmosphere. After being cooled to room temperature, the reaction mixture was poured into ice-water (200 mL), and extracted with EA (250 mL) three times. The combined organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by flash column eluting with a gradient of PE/EA (1/1) to give a crude product which was further purified by SFC (Gradient: 55% MeOH (0.05% DEA) in CO2, Column: Daicel Chiralpak IC, 250×30 mm, 10 μm) to give Intermediate A (2.52 g, slower eluting) as a white solid. MS: calc'd 356 (MH+), measured 356 (MH+).
The title compound was prepared according to the following scheme:
To a solution of tert-butyl 3-methyl-4-oxopiperidine-1-carboxylate (compound B1, CAS: 181269-69-2, Vendor: PharmaBlock, 50.00 g, 234.44 mmol) in methanol (500 mL) was added NaBH4 (13.30 g, 351.67 mmol) in portions at 0° C. After being stirred at 0° C. for 1 h and then at 20° C. for 2.5 h, the reaction mixture was quenched with saturated ammonium chloride aqueous solution and concentrated under reduced pressure in order to remove the methanol. Then the mixture was extracted with dichloromethane, the combined organic layer was dried over sodium sulfate, filtered and concentrated to afford compound B2 (50.40 g) as light yellow oil.
To a mixture of tert-butyl 4-hydroxy-3-methyl-piperidine-1-carboxylate (compound B2, 45.00 g, 209.02 mmol) in DCE (500 mL) was added Ph3P (71.27 g, 271.73 mmol) at 20° C., followed by addition of CBr4 (110.91 g, 334.43 mmol) at 20° C. The flask was degassed and purged with N2 for four times. was After being stirred at 25° C. for 15 h under N2 atmosphere, the reaction mixture was concentrated, and the residue was purified by flash column eluting with a gradient of PE/EA (100/0 to 100/5) to give compound B3 (35.00 g) as light yellow oil.
To a 1000 mL vial equipped with a stir bar was added 2-bromo-5-chloro-3-methyl-pyridine (compound B4, CAS: 65550-77-8, Vendor: Accela, 22.00 g, 106.55 mmol), tert-butyl 4-bromo-3-methyl-piperidine-1-carboxylate (compound B3, 38.53 g, 138.52 mmol), Ir[dF(CF3)ppy]2(dtbpy)(PF6) (0.85 g, 1.07 mmol), NiCl2·dtbbpy (0.21 g, 0.530 mmol), TTMSS (26.50 g, 106.55 mmol), Na2CO3 (22.59 g, 213.11 mmol) and DME (500 mL). The vial was sealed and purged with nitrogen. The mixture was stirred and irradiated with a 34 W blue LED lamp (7 cm away), with cooling fan to keep the reaction temperature at 25° C. for 14 h. The reaction was filtered, and washed with a mixture of solvent (DCM/MeOH=10/1, 5 mL) for three times. The filtrate was concentrated and the residue was purified by flash column eluting with a gradient of PE/EA (5/1) to afford compound B5 (7.00 g) as light yellow oil. MS: calc'd 325 (MH+), measured 269 (M-C4H8+H+).
To a solution of tert-butyl-4-(5-chloro-3-methyl-2-pyridyl)-3-methyl-piperidine-1-carboxylate (compound B5, 7.00 g, 21.55 mmol) in DCM (30 mL) was added TFA (10 mL) dropwise at 0° C. was After being stirred at 20° C. for 2 h, the reaction mixture was concentrated and the residue was dissolved in DCM (50 mL) and basified by saturated NaHCO3 solution. The mixture was extracted with DCM (40 mL) for three times. The combined organic layer was washed with 50 mL brine, dried over Na2SO4 and concentrated to give compound B6 (4.80 g) as light yellow oil. MS: calc'd 225 (MH+), measured 225 (MH+).
To a mixture of 5-chloro-3-methyl-2-(3-methyl-4-piperidyl)pyridine (compound B6, 4.60 g, 20.47 mmol) and 4-fluoro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound A11, 3.72 g, 22.52 mmol) in DMSO (40 mL) was added DIPEA (2.37 g, 40.94 mmol). The reaction was stirred at 120° C. for 3 h under N2 atmosphere. After being cooled to rt, The reaction mixture was poured into ice-water (400 mL), extracted with EA (80 mL) for 3 times. The combined organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by flash column eluting with a gradient of EA/PE (1/1) to give a mixture (5.40 g) as a light brown solid. The mixture was continued to be purified by SFC (Gradient: 40% EtOH (0.1% NH3H2O) in CO2, Column: Daicel Chiralpak AD, 250×30 mm, 10 μm) to afford Intermediate B (2.10 g) as a light yellow solid. MS: calc'd 370 (MH+), measured 370 (MH+).
The title compound was prepared according to the following scheme:
To a solution of tert-butyl-3-methyl-4-oxopiperidine-1-carboxylate (compound B1, CAS: 181269-69-2, Vendor: PharmaBlock, 300 mg, 1.41 mmol) in Ethanol (10 mL) was added 4-methylbenzenesulfonohydrazide (compound C1, 341 mg, 1.83 mmol). The mixture was stirred at 70° C. for 3 h. After being cooled to rt, the mixture was concentrated to afford crude product compound C2 (537 mg) which was used in next step without further purification. MS: calc'd 382 (MH+), measured 382 (MH+).
To a solution of tert-butyl-3-methyl-4-(p-tolylsulfonylhydrazono)piperidine-1-carboxylate (compound C2, 537 mg, 1.41 mmol) in 1,4-dioxane (5 mL) was added (6-bromopyridin-3-yl)boronic acid (compound C3, CAS: 223463-14-7, Vendor: Accela, 369 mg, 1.83 mmol) and Cs2CO3 (1.38 g, 4.22 mmol). The mixture was stirred at 120° C. under N2 for 16 h. After being cooled to rt, the mixture was filtered and the solid was washed with EA (10 mL) twice. The combined organic layer was concentrated and purified by flash column eluting with a gradient of EA/PE (0% to 70%) to give the desired product C4 (50 mg) as a colorless oil. MS: calc'd 355 (MH+), measured 355 (MH+).
The mixture of tert-butyl 4-(6-bromo-3-pyridyl)-3-methyl-piperidine-1-carboxylate (compound C4, 50 mg, 141 μmol) in HCl (1M in EA, 10 ml, 10 mmol) was stirred at rt for 2 h.
The mixture was concentrated to give the desired product C5 (41 mg) as a white solid. MS: calc'd 255 (MH+), measured 255 (MH+).
To a solution of 4-chloro-1-methyl-pyrazolo[3,4-b]pyridine (compound C6, CAS: 1268520-92-8, Vendor: PharmaBlock, 35 mg, 211 μmol) and 2-bromo-5-(3-methyl-4-piperidyl)pyridine (compound C5, 41 mg, 141 μmol) in DMSO (15 mL) was added CsF (107 mg, 703 μmol). The reaction mixture was stirred at 130° C. overnight. After being cooled to room temperature, the reaction was diluted with EA, washed with water (30 mL) four times, dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by flash column eluting with a gradient of EA (with 10% MeOH)/PE (0% to 70%) to give the Intermediate C (42 mg). MS: calc'd 386 (MH+), measured 386 (MH+).
The title compound was prepared in analogy to the preparation of Intermediate A by using compound A13 instead of compound A5. Intermediate D was obtained as a white solid. MS: calc'd 356 (MH+), measured 356 (MH+). (Note: The stereo center of the 3-position on the piperidine ring is epimerized from S configuration to R configuration, when intermediate A13 was treated with sodium hydroxide in ethanol, to afford (3R,4S)-1-tert-butoxycarbonyl-4-(5-chloro-2-pyridyl)piperidine-3-carboxylic acid)
The title compound was prepared according to the following scheme:
To a mixture of 2-bromo-5-chloro-3-methyl-pyridine (compound E1, 15.00 g, 72.65 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound E2, 24.71 g, 79.91 mmol) in 1,4-dioxane (400 mL) was added sodium carbonate (15.40 g, 145.30 mmol) in water (40 mL) and Pd(dppf)Cl2·CH2Cl2 (5.93 g, 7.26 mmol). The mixture was stirred at 95° C. for 16 h under N2 atmosphere. After being cooled to room temperature, the mixture was filtered and washed with 1,4-dioxane (10 mL) for three times. The filtrate was concentrated, and the crude was purified by flash column eluting with a gradient of PE/EA (5/1 to 3/1) to give compound E3 (9.40 g) as a yellow oil. MS: calc'd 309 (MH+), measured 309 (MH+).
To the solution of tert-butyl 4-(5-chloro-3-methyl-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound E3, 6.00 g, 19.43 mmol) in tetrahydrofuran (120 mL) was added tris(triphenylphosphine)rhodium(I) chloride (4.49 g, 4.86 mmol). The flask was degassed and purged with H2 for four times. The mixture was stirred at 60° C. for 36 h under hydrogen atmosphere (45 psi). The mixture was filtered on celite and the solid was washed with methanol (5 mL). The solvent was removed under vacuum and the crude was purified by flash column eluting with a gradient of PE/EA (5/1 to 3/1) to give compound E4 (4.50 g) as an off-white solid. MS: calc'd 311 (MH+), measured 311 (MH+).
To a solution of tert-butyl 4-(5-chloro-3-methyl-2-pyridyl) piperidine-1-carboxylate (compound E4, 4.50 g, 14.48 mmol) in DCM (50 mL) was added HCl in dioxane (10 mL) at 0° C. After being stirred at 25° C. for 16 h, the reaction mixture was concentrated under reduced pressure to give compound E5 (3.56 g) as a white solid. MS: calc'd 211 (MH+), measured 211 (MH+).
To a mixture of 4-chloro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (1.15 g, 6.33 mmol) and 5-chloro-3-methyl-2-(4-piperidyl)pyridine (compound E5, 1.72 g, 6.97 mmol) in DMSO (30 mL) was added KF (3.67 g, 63.32 mmol). The reaction was stirred at 120° C. for 15 h under N2 atmosphere. After being cooled to rt, the reaction mixture was poured into ice-water (200 mL) and aq. NaHCO3 (50 mL), extracted with EA (250 mL) three times. The combined organic layer was dried over Na2SO4, filtered and concentrated under vacuum. The crude was purified by flash column eluting with a gradient of PE/EA (5/1 to 0/1) to give Intermediate E (819 mg) as a light yellow solid. MS: calc'd 356 (MH+), measured 356 (MH+).
The title compound was prepared according to the following scheme:
To the mixture of benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (compound 1a, CAS: 286961-15-7, Vendor: Accela, 100 mg, 291 μmol), tert-butyl 4-(4-bromophenyl)piperazine-1-carboxylate (compound 1b, CAS: 352437-09-3, Vendor: Accela, 99.4 mg, 291 μmol) in 1,4-dioxane (5 mL) and water (1 mL) was added K2CO3 (121 mg, 874 μmol) and PdCl2(dppf)·CH2Cl2 (24 mg, 29 μmol). The mixture was charged with N2, and stirred at 100° C. overnight. After being cooled to room temperature, the mixture was dried over Na2SO4, filtered and the solid was washed with EA (10 mL) twice. The combined organic phase was concentrated and purified by flash column eluting with a gradient of EA/PE (0% to 60%) to afford compound 1c (83 mg). MS: calc'd 478 (MH+), measured 478 (MH+).
To the mixture of tert-butyl 4-[4-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)phenyl]piperazine-1-carboxylate (compound 1c, 83 mg, 174 μmol) in MeOH (10 mL) was added Pd(OH)2 (10 wt. % in carbon, 12 mg, 87 μmol). The suspension was purged with H2 for 3 times, then stirred under hydrogen balloon at rt overnight. The mixture was filtered and concentrated to give the crude product compound 1d (60 mg) which was used in the next step without further purification. MS: calc'd 346 (MH+), measured 346 (MH+).
To the mixture of 4-chloro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound 1e, CAS: 19867-78-8, Vendor: PharmaBlock, 24 mg, 130 μmol) and tert-butyl 4-[4-(4-piperidyl)phenyl]piperazine-1-carboxylate (compound 1d, 30 mg, 87 μmol) in 1,4-dioxane (2 mL) was added RuPhos Pd G2 (7 mg, 9 μmol) and Cs2CO3 (85 mg, 261 μmol). The mixture was charged with N2, and stirred at 110° C. overnight. After being cooled to room temperature, the mixture was filtered and the solid was washed with EA (10 mL) twice. The combined organic phase was concentrated and purified by flash column eluting with a gradient of EA (with 10% MeOH)/PE (0% to 60%) to give the desired product if MS: calc'd 491 (MH+), measured 491 (MH+).
The compound 1f was dissolved in DCM (10 mL) and TFA (2 mL) and stirred at rt for 2 h. The mixture was concentrated and purified by reversed flash column eluting with a gradient of ACN/Water (with 0.5% TFA) (0% to 30%) to give Example 1 (36 mg) as a yellow solid. MS: calc'd 391 (MH+), measured 391 (MH+). 1H NMR (400 MHz, METHANOL-d4) 6=1H NMR (400 MHz, METHANOL-d4) δ=8.36 (s, 1H), 7.23-7.18 (m, 2H), 7.01-6.96 (m, 2H), 6.70 (s, 1H), 4.60 (br d, J=13.3 Hz, 2H), 4.07 (s, 3H), 3.56 (br t, J=12.7 Hz, 2H), 3.39-3.33 (m, 8H), 3.01 (tt, J=3.8, 11.9 Hz, 1H), 2.62 (s, 3H), 2.14-2.07 (m, 2H), 1.84 (dq, J=3.9, 12.7 Hz, 2H).
The title compound was prepared in analogy to the preparation of Example 1 by using tert-butyl 4-[6-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)-3-pyridyl]piperazine-1-carboxylate (compound 2b) instead of compound 1c. Example 2 (44 mg) was obtained as a white solid. MS: calc'd 392 (MH+), measured 392 (MH+). 1H NMR (400 MHz, CDCl3) δ ═8.22 (d, J=2.4 Hz, 1H), 7.90 (s, 1H), 7.15 (dd, J=2.6, 8.6 Hz, 1H), 7.04 (d, J=8.6 Hz, 1H), 6.21 (s, 1H), 4.23 (br d, J=13.1 Hz, 2H), 4.04 (s, 3H), 3.25-3.09 (m, 6H), 3.05-2.89 (m, 5H), 2.53 (s, 3H), 2.09-2.01 (m, 2H), 1.98-1.86 (m, 3H).
The compound 2b was prepared according to the following scheme:
To a solution of 2-bromo-5-chloropyridine (CAS: 40473-01-6, Vendor: Accela, 1.00 g, 5.2 mmol), benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 1a, 1.80 g, 5.24 mmol) and sodium carbonate (1.20 g, 11.32 mmol) in 1,4-dioxane (10 mL) and water (1 mL) was added PdCl2(dppf)·CH2Cl2 (380 mg, 0.52 mmol) under N2. The mixture was stirred at 80° C. for 16 h under N2. After being cooled to rt, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to remove the solvent, and the residue was purified by flash column eluting with a gradient of EA/PE (1/5 to 1/0) to give compound 2a (1.50 g) as a brown oil. MS: calc'd 329 (MH+), measured 329 (MH+).
To a solution of benzyl 4-(5-chloro-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 2a, 500 mg, 1.52 mmol), tert-butyl piperazine-1-carboxylate (566 mg, 3.04 mmol) and sodium tert-butoxide (225 mg, 2.34 mmol) in 1,4-dioxane (5 mL) was added Xphos-Pd-G3 (129 mg, 0.15 mmol) under N2. The mixture was stirred at 100° C. for 3 h under N2. After being cooled to rt, the reaction mixture was filtered on celite and the filtrate was concentrated under reduced pressure to remove the solvent, and the crude was purified by flash column eluting with a gradient of EA/PE (1/5 to 1/0) to give compound 2b (223 mg) as a yellow solid. MS: calc'd 479 (MH+), measured 479 (MH+).
The title compound was prepared in analogy to the preparation of Example 1 by using tert-butyl 4-[5-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)pyrazin-2-yl]piperazine-1-carboxylate (compound 3b) instead of compound 1c. Example 3 (23 mg) was obtained as a white solid. MS: calc'd 393 (MH+), measured 393 (MH+). 1H NMR (400 MHz, CDCl3) δ=8.09-8.06 (m, 1H), 8.01-7.98 (m, 1H), 7.93-7.91 (m, 1H), 6.24-6.22 (m, 1H), 4.29-4.21 (m, 2H), 4.07-4.04 (m, 3H), 3.55-3.50 (m, 4H), 3.27-3.18 (m, 2H), 3.02-2.97 (m, 4H), 2.97-2.89 (m, 1H), 2.57-2.54 (m, 3H), 2.07-1.93 (m, 4H).
The compound 3b was prepared according to the following scheme:
To a solution of 2,5-dichloropyrazine (CAS: 19745-07-4, Vendor: Accela, 2.00 g, 13.42 mmol) and tert-butyl piperazine-1-carboxylate (2.75 g, 14.77 mmol) in ACN (20 mL) was added potassium carbonate (2.78 g, 20.14 mmol). The mixture was stirred at 80° C. for 16 h. After being cooled to rt, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to remove the solvent, and the residue was purified by flash column eluting with a gradient of PE/EA (10/1 to 3/1) to give compound 3a (1.50 g, 5.02 mmol) as alight yellow solid. MS: calc'd 299 (MH+), measured 299 (MH+).
To a mixture of tert-butyl 4-(5-chloropyrazin-2-yl)piperazine-1-carboxylate (compound 3a, 1.45 g, 4.85 mmol) and benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 1a, 1.83 g, 5.34 mmol) in 1,4-dioxane (10 mL) and water (1 mL) was added K2CO3 (1.34 g, 9.71 mmol) and PdCl2(dppf)·CH2Cl2 (198 mg, 0.24 mmol). The flask was degassed and purged with N2 for four times. The mixture was stirred at 90° C. for 16 h under N2 atmosphere. After being cooled to rt, the mixture was diluted by water (10 mL), extracted by EA (20 mL) three times. The combined organic layer was washed by brine (50 mL), dried over Na2SO4, filtered and concentrated to give the crude product which was purified by flash column eluting with a gradient of PE/EA (5/1 to 3/1) to give compound 3b (1.50 g) as a yellow solid. MS: calc'd 480 (MH+), measured 480 (MH+).
The title compound was prepared in analogy to the preparation of Example 2 by using 5-bromo-2-iodopyrimidine (CAS: 183438-24-6, Vendor: Accela) instead of 2-bromo-5-chloropyridine. Example 4 (3.5 mg) was obtained as a white solid. MS: calc'd 393 (MH+), measured 393 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.40 (s, 2H), 8.08 (s, 1H), 6.40 (s, 1H), 5.02-4.90 (m, 2H), 4.33 (br d, J=13.2 Hz, 2H), 3.99 (s, 3H), 3.26-3.20 (m, 4H), 3.18-3.11 (m, 1H), 3.03-2.95 (m, 4H), 2.52 (s, 3H), 2.13-1.94 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 2 by using 2-bromo-5-chloro-3-methyl-pyridine (CAS: 65550-77-8, Vendor: Accela) and 4-chloro-1-methyl-pyrazolo[3,4-b]pyridine (CAS: 1268520-92-8, Vendor: PharmaBlock) instead of 2-bromo-5-chloropyridine and compound 1e. Example 5 (29 mg) was obtained as a white solid. MS: calc'd 392 (MH+), measured 392 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.16 (s, 1H), 8.10 (d, J=5.9 Hz, 1H), 7.96 (d, J=2.7 Hz, 1H), 7.21 (d, J=2.7 Hz, 1H), 6.50 (d, J=5.9 Hz, 1H), 4.41 (br d, J=13.7 Hz, 2H), 4.00 (s, 3H), 3.41-3.33 (m, 2H), 3.29-3.22 (m, 1H), 3.18-3.10 (m, 4H), 3.04-2.93 (m, 4H), 2.40 (s, 3H), 2.10-1.95 (m, 2H), 1.88 (br dd, J=1.5, 12.5 Hz, 2H).
The title compound was prepared in analogy to the preparation of Example 1 by using 4-chloro-1-methyl-pyrazolo[3,4-b]pyridine (CAS: 1268520-92-8, Vendor: PharmaBlock) and tert-butyl 4-(5-bromo-4-methyl-2-pyridyl)piperazine-1-carboxylate (CAS: 944582-92-7, Vendor: Bide Pharmatech) instead of compound 1e and compound 1b. Example 6 (35 mg) was obtained as a light yellow solid. MS: calc'd 392 (MH+), measured 392 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.45 (s, 1H), 8.06 (d, J=7.3 Hz, 1H), 7.91 (s, 1H), 7.21 (s, 1H), 6.86 (d, J 15=7.5 Hz, 1H), 4.70-4.62 (m, 2H), 4.08 (s, 3H), 3.94-3.84 (m, 4H), 3.65 (br t, J=12.1 Hz, 2H), 3.44-3.36 (m, 4H), 3.35-3.24 (m, 1H), 2.56 (s, 3H), 2.14 (br d, J=12.3 Hz, 2H), 1.90 (dq, J=3.5, 12.6 Hz, 2H).
The title compound was prepared in analogy to the preparation of Example 2 by using 2-bromo-5-chloro-3-methyl-pyridine (CAS: 65550-77-8, Vendor: Accela) instead of 2-bromo-5-chloropyridine. Example 7 (61 mg) was obtained as a white solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.07 (s, 1H), 7.96 (d, J=2.9 Hz, 1H), 7.21 (d, J=2.7 Hz, 1H), 6.40 (s, 1H), 4.37 (br d, J=13.4 Hz, 2H), 3.99 (s, 3H), 3.35 (br d, J=2.0 Hz, 1H), 3.29-3.21 (m, 2H), 3.15 (br dd, J=3.9, 6.1 Hz, 4H), 3.02-2.94 (m, 4H), 2.52 (s, 3H), 2.40 (s, 3H), 2.08-1.96 (m, 2H), 1.89-1.80 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 1 by using 4-bromo-1,6-dimethyl-pyrazolo[3,4-b]pyridine (CAS: 1783407-55-5, Vendor: Accela) and tert-butyl 4-(5-bromo-4-methyl-2-pyridyl)piperazine-1-carboxylate (CAS: 944582-92-7, Vendor: Bide Pharmatech) instead of compound 1e and compound 1b. Example 8 (32 mg) was obtained as a yellow solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.27 (s, 1H), 7.81 (s, 1H), 7.07 (s, 1H), 6.62 (s, 1H), 4.54 (br d, J=13.4 Hz, 2H), 3.98 (s, 3H), 3.82-3.73 (m, 4H), 3.51 (br t, J=12.6 Hz, 2H), 3.35-3.25 (m, 4H), 3.19-3.12 (m, 1H), 2.53 (s, 3H), 2.45 (s, 3H), 2.03 (br d, J=12.3 Hz, 2H), 1.77 (dq, J=3.5, 12.6 Hz, 2H).
The title compound was prepared in analogy to the preparation of Example 3 by using 5-bromo-2-chloro-4-methylpyridine (CAS: 778611-64-6, Vendor: Accela) instead of 2,5-dichloropyrazine, and replacing SNAr reaction (K2CO3, CAN) with Buchwald coupling reaction (XantPhos, Pd2(dba)3, t-BuONa, toluene) in the step 1. Example 9 (61 mg) was obtained as a white solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.09 (d, J=3.4 Hz, 2H), 7.18 (s, 1H), 6.41 (s, 1H), 4.37 (br d, J=13.2 Hz, 2H), 3.99 (s, 3H), 3.30-3.23 (m, 2H), 3.06-3.01 (m, 4H), 3.02-2.99 (m, 1H), 3.00-2.93 (m, 4H), 2.52 (s, 3H), 2.33 (s, 3H), 2.05-1.97 (m, 2H), 1.97-1.85 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 3 by using 3-bromo-6-fluoro-2-methylpyridine (CAS: 375368-83-5, Vendor: Accela) instead of 2,5-dichloropyrazine, and replacing SNAr reaction condition (K2CO3, CAN) with (DIPEA, DMF) in the step 1. Example 10 (7 mg) was obtained as a white solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.10 (s, 1H), 7.42 (d, J=8.8 Hz, 1H), 6.61 (d, J=8.6 Hz, 1H), 6.43 (s, 1H), 4.38 (br d, J=13.1 Hz, 2H), 4.01 (s, 3H), 3.49-3.43 (m, 4H), 3.32-3.26 (m, 2H), 3.11-3.01 (m, 1H), 2.99-2.92 (m, 4H), 2.54 (s, 3H), 2.50 (s, 3H), 1.97-1.89 (m, 2H), 1.86-1.75 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 3 by using 5-bromo-2-chloro-4-methylpyrimidine (CAS: 633328-95-7, Vendor: Accela) instead of 2,5-dichloropyrazine. Example 11 (34 mg) was obtained as a white solid. MS: calc'd 407 (MH+), measured 407 (MH+). 1H NMR (400 MHz, CDCl3) δ=8.10 (s, 1H), 7.93 (s, 1H), 6.24 (s, 1H), 4.27 (br d, J=12.8 Hz, 2H), 4.07 (s, 3H), 3.81-3.74 (m, 4H), 3.18 (dt, J=2.6, 12.5 Hz, 2H), 2.95-2.81 (m, 5H), 2.57 (s, 3H), 2.43 (s, 3H), 1.97-1.82 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 3 by using 2,5-dichloro-3-methyl-pyrazine (CAS: 107378-41-6, Vendor: Bide) instead of 2,5-dichloropyrazine. Example 12 (16 mg) was obtained as a white solid. MS: calc'd 407 (MH+), measured 407 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.09 (s, 1H), 7.92 (s, 1H), 6.41 (s, 1H), 4.37 (br d, J=13.3 Hz, 2H), 4.00 (s, 3H), 3.59-3.50 (m, 4H), 3.39-3.34 (m, 1H), 3.32-3.28 (m, 1H), 3.28-3.17 (m, 1H), 3.00-2.88 (m, 4H), 2.54 (s, 3H), 2.51 (s, 3H), 2.07-1.94 (m, 2H), 1.93-1.82 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 1 by using tert-butyl 4-[6-(1-benzyloxycarbonyl-3,6-dihydro-2H-pyridin-4-yl)-5-vinyl-3-pyridyl]piperazine-1-carboxylate (compound 13c) instead of compound 1c. Example 13 (5 mg) was obtained as a white solid. MS: calc'd 420 (MH+), measured 420 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.10 (s, 1H), 7.99 (d, J=2.8 Hz, 1H), 7.22 (d, J=2.8 Hz, 1H), 6.42 (s, 1H), 4.39 (br d, J=13.4 Hz, 2H), 4.01 (s, 3H), 3.39-3.34 (m, 1H), 3.32-3.23 (m, 2H), 3.20-3.12 (m, 4H), 3.04-2.95 (m, 4H), 2.77 (q, J=7.5 Hz, 2H), 2.54 (s, 3H), 2.18-2.02 (m, 2H), 1.85 (br d, J=11.1 Hz, 2H), 1.28 (t, J=7.5 Hz, 3H).
The compound 13c was prepared according to the following scheme:
To the mixture of benzyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 1a, 2.53 g, 7.37 mmol) and 2,3-dibromo-5-chloropyridine (CAS: 137628-17-2, Vendor: Accela, 2.00 g, 7.37 mmol) in 1,4-dioxane (10 mL) and water (1 mL) was added PdCl2(dppf)·CH2Cl2 (301 mg, 0.37 mmol) and Na2CO3 (1.56 g, 14.74 mmol).
The mixture was stirred at 80° C. for 0.5 h under N2 atmosphere. After being cooled to rt, the mixture was filtered and the solid was washed with 1,4-dioxane (15 mL) three times and concentrated. The residue was purified by flash column eluting with a gradient of PE/EA (3/1) to afford compound 13a (680 mg, 1.67 mmol) as a light yellow solid. MS: calc'd 407 (MH+), measured 407 (MH+).
To the mixture of benzyl 4-(3-bromo-5-chloro-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 13a, 100 mg, 0.25 mmol), vinylboronic acid pinacol ester (94 mg, 0.61 mmol) and Na2CO3 (20 mg, 0.490 mmol) in toluene (1 mL), ethanol (0.5 mL) and water (0.5 mL) was added PdCl2(dppf)·CH2Cl2 (20 mg, 0.02 mmol), the mixture was stirred at 100° C. for 1 h under the N2 atmosphere. This reaction was repeated four times (each for 100 mg of compound 13c). The combined reaction mixture was filtered and washed with EtOH. The filtrate was concentrated. The residue was purified by flash column eluting with a gradient of PE/EA (5/1 to 0/1) to give compound 13b (260 mg) as a light yellow oil. MS: calc'd 355 (MH+), measured 355 (MH+).
To the mixture of tert-butyl piperazine-1-carboxylate (105 mg, 0.56 mmol) and benzyl 4-(5-chloro-3-vinyl-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (compound 13b, 100 mg, 0.28 mmol) in 1,4-dioxane (2.5 mL) was added Xphos-Pd-G3 (24 mg, 0.03 mmol) and t-BuONa (67 mg, 0.70 mmol). After being stirred at 100° C. for 1.5 h under N2, the reaction mixture was combined with another same batch and worked up, and then the mixture was filtered, washed with EtOH and concentrated. The residue was purified by flash column eluting with a gradient of PE/EA (1/0 to 1/8) to afford compound 13c (90 mg) as a light yellow solid. MS: calc'd 505 (MH+), measured 505 (MH+).
The title compound was prepared in analogy to the preparation of Example 9 by using 5-bromo-2-chloro-3-methoxy-pyridine (CAS: 286947-03-3, Vendor: Accela) instead of 5-bromo-2-chloro-4-methylpyridine. Example 14 (37 mg) was obtained as a white solid. MS: calc'd 422 (MH+), measured 422 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.09 (s, 1H), 7.70 (d, J=2.3 Hz, 1H), 6.99 (d, J=2.3 Hz, 1H), 6.41 (s, 1H), 4.37 (br d, J=13.2 Hz, 2H), 4.00 (s, 3H), 3.89 (s, 3H), 3.44 (tt, J=3.9, 11.7 Hz, 1H), 3.32-3.25 (m, 2H), 3.20 (dd, J=4.0, 6.2 Hz, 4H), 3.03-2.94 (m, 4H), 2.54 (s, 3H), 2.07-1.95 (m, 2H), 1.94-1.85 (m, 2H).
The title compound was prepared according to the following scheme:
To the mixture of 4-[4-(5-chloro-3-methyl-2-pyridyl)-1-piperidyl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine (Intermediate E, 50 mg, 0.14 mmol) and tert-butyl N-[(3S,4R)-3-fluoro-4-piperidyl]carbamate (compound 15a, CAS: 1434126-99-4, Vendor: PharmaBlock, 40 mg, 0.18 mmol) in 1,4-dioxane was added RuPhos Pd G2 (11 mg, 0.014 mmol) and cesium carbonate (92 mg, 0.28 mmol). The mixture was heated to 110° C. overnight. After being cooled to rt, the solid was filtered off and washed with EA (10 mL) twice. The combined organic layer was concentrated and purified by flash column eluting with a gradient of EA (with 10% MeOH)/PE (0 to 80%) to give compound 15b which was directly used in the next step. MS: calc'd 538 (MH+), measured 538 (MH+).
The mixture of compound 15b in DCM (5 mL) and TFA (2 mL) was stirred at rt for 2 h. The mixture was concentrated and purified by reversed flash column eluting with a gradient of ACN/Water (with 0.05% TFA) (0 to 30%) to give Example 15 (56 mg) as a white solid. MS: calc'd 438 (MH+), measured 438 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.37 (s, 1H), 8.15 (d, J=2.9 Hz, 1H), 8.02 (d, J=2.7 Hz, 1H), 6.74 (s, 1H), 5.08 (d, J=49.2 Hz, 1H), 4.76-4.61 (m, 2H), 4.48-4.34 (m, 1H), 4.16-4.03 (m, 4H), 3.76-3.59 (m, 4H), 3.43-3.25 (m, 1H), 3.24-3.09 (m, 1H), 2.64 (s, 3H), 2.59 (s, 3H), 2.22-2.09 (m, 4H), 2.09-2.00 (m, 2H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl (2R)-2-(methoxymethyl)piperazine-1-carboxylate (CAS: 1023301-73-6, Vendor: PharmaBlock) instead of compound 15a. Example 16 (58 mg) was obtained as a white solid. MS: calc'd 450 (MH+), measured 450 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.37 (s, 1H), 8.13 (d, J=2.8 Hz, 1H), 7.73 (d, J=2.6 Hz, 1H), 6.73 (s, 1H), 4.70-4.62 (m, 2H), 4.08 (s, 3H), 3.98-3.90 (m, 2H), 3.72-3.57 (m, 6H), 3.52-3.46 (m, 1H), 3.45 (s, 3H), 3.36-3.27 (m, 1H), 3.20-3.06 (m, 2H), 2.63 (s, 3H), 2.53 (s, 3H), 2.14-2.03 (m, 4H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-[(3S,4S)-3-methoxy-4-piperidyl]carbamate (CAS: 907544-19-8, Vendor: PharmaBlock) instead of compound 15a. Example 17 (50 mg) was obtained as a white solid. MS: calc'd 450 (MH+), measured 450 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.37 (s, 1H), 8.17 (d, J=2.9 Hz, 1H), 8.03 (d, J=2.8 Hz, 1H), 6.75 (s, 1H), 4.74-4.65 (m, 2H), 4.39-4.29 (m, 1H), 4.08 (s, 3H), 4.03-3.91 (m, 1H), 3.75-3.60 (m, 3H), 3.55 (s, 3H), 3.41-3.32 (m, 1H), 3.26-3.14 (m, 1H), 3.00 (dt, J=2.7, 12.8 Hz, 1H), 2.72 (dd, J=10.1, 12.5 Hz, 1H), 2.64 (s, 3H), 2.60 (s, 3H), 2.21-2.09 (m, 5H), 1.79 (dq, J=4.6, 12.5 Hz, 1H).
1-methyl-4-[cis-3-methyl-4-(6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 18) and 1-methyl-4-[trans-3-methyl-4-(6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 19)
The title compounds were prepared in analogy to the preparation of Example 15 by using tert-butyl piperazine-1-carboxylate and Intermediate C instead of compound 15a and Intermediate E. The Example 18 and Example 19 were separated by prep-HPLC.
Example 18 (6 mg) was obtained as a light yellow solid. MS: calc'd 392 (MH+), measured 392 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.50 (s, 1H), 8.07-7.99 (m, 2H), 7.69 (dd, J=2.3, 8.9 Hz, 1H), 7.05 (d, J=8.9 Hz, 1H), 6.86 (d, J=7.6 Hz, 1H), 4.70 (br d, J=13.7 Hz, 1H), 4.54 (br d, J=13.4 Hz, 1H), 4.07 (s, 3H), 3.85-3.77 (m, 5H), 3.68-3.54 (m, 1H), 3.39-3.33 (m, 5H), 2.46-2.28 (m, 2H), 1.98 (br dd, J=2.8, 13.8 Hz, 1H), 0.72 (d, J=7.0 Hz, 3H).
Example 19 (8 mg) was obtained as a light yellow solid. MS: calc'd 392 (MH+), measured 392 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.41 (s, 1H), 8.08-8.02 (m, 2H), 7.70 (dd, J=2.3, 9.0 Hz, 1H), 7.07 (d, J=9.0 Hz, 1H), 6.87 (d, J=7.5 Hz, 1H), 4.65 (br d, J=13.8 Hz, 1H), 4.55 (br d, J=12.8 Hz, 1H), 4.08 (s, 3H), 3.85-3.78 (m, 4H), 3.66-3.53 (m, 1H), 3.38-3.33 (m, 4H), 3.28-3.23 (m, 1H), 2.65 (dt, J=4.0, 11.4 Hz, 1H), 2.09-1.86 (m, 3H), 0.89 (d, J=6.5 Hz, 3H). The relative configuration of the two molecules were confirmed by 2D-NMR.
1,6-dimethyl-4-[cis-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 20) and 1,6-dimethyl-4-[trans-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 21)
The title compounds were prepared according to the following scheme:
To the mixture of tert-butyl 4-(5-bromo-4-methylpyridin-2-yl)piperazine-1-carboxylate (compound 20a, CAS: 944582-92-7, Vendor: Bide Pharmatech, 100 mg, 281 μmol) and 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (107 mg, 421 μmol) in 1,4-dioxane (10 mL) was added potassium acetate (83 mg, 842 μmol) and PdCl2(dppf)·CH2Cl2 (23 mg, 28 μmol). The mixture was charged with N2, and stirred at 110° C. overnight. After being cooled to rt, the mixture was filtered off and the solid was washed with EA (10 mL) twice. The combined organic layer was concentrated to give the crude product compound 20b (113 mg), which was used in the next step without further purification. MS: calc'd 404 (MH+), measured 404 (MH+).
To the mixture of 4-chloro-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound 20c, CAS: 19867-78-8, Vendor: PharmaBlock, 511 mg, 2.82 mmol) and 3-methylpiperidin-4-one hydrochloride (compound 20d, CAS: 4629-78-1, Vendor: PharmaBlock, 351 mg, 2.35 mmol) in 1,4-dioxane (20 mL) was added RuPhos Pd G2 (182 mg, 235 μmol) and Cs2CO3 (2.29 g, 7.04 mmol). The mixture was charged with N2 and stirred at 110° C. overnight. After being cooled to rt, the solid was filtered off and washed with EA (10 mL) twice. The combined mixture was concentrated and purified by flash column eluting with a gradient of EA (with 10% MeOH)/PE (0% to 80%) to give the desired product 20e (272 mg) as a yellow oil. MS: calc'd 259 (MH+), measured 259 (MH+).
To a solution of 1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-piperidin-4-one (compound 20e, 200 mg, 774 μmol) in THF (10 mL) was added KHMDS (0.5M in THF, 2.32 mL, 1.16 mmol) in −78° C. and stirred for 0.5 h. Then 1,1,1-trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide (415 mg, 1.16 mmol) was added and the reaction mixture was stirred at −78° C. for another 2 h. After being warmed to rt, the mixture was directly purified by flash column eluting with a gradient of EA (with 10% MeOH)/PE (0% to 60%) to give the desired product compound 20f (230 mg). MS: calc'd 391 (MH+), measured 391 (MH+).
To a mixture of [1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]trifluoromethanesulfonate (compound 20f, 50 mg, 128 μmol) and tert-butyl 4-[4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]piperazine-1-carboxylate (compound 20b, 113 mg, 280 μmol) in 1,4-dioxane (5 mL) and water (1 mL) was added K2CO3 (53 mg, 384 μmol) and PdCl2(dppf)·CH2Cl2 (21 mg, 26 μmol). The mixture was charged with N2 and stirred at 100° C. for 2 h. After being cooled to rt, the mixture was dried over Na2SO4, filtered off and the solid was washed with EA (10 mL) twice. The combined organic layer was concentrated to give the crude product compound 20g (66 mg), which was used in next step without further purification. MS: calc'd 518 (MH+), measured 518 (MH+).
To a flask containing tert-butyl 4-[5-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]-4-methyl-2-pyridyl]piperazine-1-carboxylate (compound 20g, 66 mg, 128 μmol) was added Pd(OH)2 (10 wt. % in carbon, 8.99 mg, 64 μmol) and MeOH (10 mL). The suspension was purged with H2 for 3 times, then stirred under hydrogen balloon at rt overnight. The mixture was filtered and concentrated. The residue was purified by flash column eluting with a gradient of EA (with 10% MeOH)/PE (0% to 80%) to give the desired product 20 h (25 mg). MS: calc'd 520 (MH+), measured 520 (MH+).
Step 6: Preparation of 1,6-dimethyl-4-[cis-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 20) and 1,6-dimethyl-4-[trans-3-methyl-4-(4-methyl-6-piperazin-1-yl-3-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine Example 21
To the mixture of tert-butyl 4-[5-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4-piperidyl]-4-methyl-2-pyridyl]piperazine-1-carboxylate (compound 20h, 25 mg, 48 μmol) in DCM (10 mL) was added TFA (2 mL). After being stirred at rt for 2 h, the mixture was concentrated and purified by reversed flash column eluting with a gradient of ACN/Water (with 0.5% TFA) (0% to 30%) to give the desired products. The structures of Example 20 and Example 21 were confirmed by 2D-NMR.
Example 20 (9 mg) was obtained as a light yellow solid. MS: calc'd 420 (MH+), measured 420 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.45 (s, 1H), 7.81 (s, 1H), 7.20 (s, 1H), 6.76 (s, 1H), 4.71 (br d, J=13.2 Hz, 1H), 4.55 (br d, J=13.7 Hz, 1H), 4.10 (s, 3H), 3.97-3.87 (m, 4H), 3.83 (br d, J=11.9 Hz, 1H), 3.62 (br t, J=12.4 Hz, 1H), 3.53 (td, J=3.6, 12.9 Hz, 1H), 3.46-3.39 (m, 4H), 2.65 (s, 3H), 2.56 (s, 3H), 2.54-2.41 (m, 2H), 1.93-1.84 (m, 1H), 0.76 (d, J=7.0 Hz, 3H).
Example 21 (2 mg) was obtained as a light yellow solid. MS: calc'd 420 (MH+), measured 420 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.24 (s, 1H), 7.84 (s, 1H), 6.90 (s, 1H), 6.65 (s, 1H), 4.56-4.43 (m, 2H), 3.98 (s, 3H), 3.73-3.68 (m, 4H), 3.54-3.43 (m, 1H), 3.28-3.23 (m, 4H), 3.20-3.13 (m, 1H), 2.83 (dt, J=3.9, 11.5 Hz, 1H), 2.54 (s, 3H), 2.36 (s, 3H), 2.10-1.99 (m, 1H), 1.99-1.92 (m, 1H), 1.76-1.65 (m, 1H), 0.81 (d, J=6.6 Hz, 3H).
1,6-dimethyl-4-[cis-3-methyl-4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 22) and 1,6-dimethyl-4-[trans-3-methyl-4-(3-methyl-5-piperazin-1-yl-pyrazin-2-yl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 23)
The title compounds were prepared in analogy to the preparation of Example 24 and Example 25 by using tert-butyl 4-[5-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-3-methyl-3,6-dihydro-2H-pyridin-4-yl]-6-methyl-pyrazin-2-yl]piperazine-1-carboxylate (compound 22b) instead of compound 24c. The structures of Example 22 and Example 23 were confirmed by 2D-NMR.
Example 22 (1 mg) was obtained as a yellow solid. MS: calc'd 421 (MH+), measured 421 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.30 (s, 1H), 8.07 (s, 1H), 6.71 (s, 1H), 4.60 (br d, J=13.8 Hz, 1H), 4.50 (br d, J=12.3 Hz, 1H), 4.07 (s, 3H), 3.87-3.80 (m, 5H), 3.59 (br s, 1H), 3.35-3.33 (m, 4H), 3.07-3.00 (m, 1H), 2.64-2.60 (m, 4H), 2.52 (s, 4H), 1.96 (br s, 1H), 0.85 (d, J=6.5 Hz, 3H).
Example 23 (1 mg) was obtained as a yellow solid. MS: calc'd 421 (MH+), measured 421 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.37 (s, 1H), 8.09 (s, 1H), 6.70 (s, 1H), 4.51 (br d, J=12.8 Hz, 1H), 4.43 (br d, J=11.4 Hz, 1H), 4.09-4.06 (m, 3H), 3.90-3.80 (m, 5H), 3.79-3.68 (m, 1H), 3.60-3.53 (m, 1H), 3.36-3.35 (m, 1H), 3.34-3.32 (m, 3H), 2.62 (s, 3H), 2.56-2.43 (m, 5H), 1.93 (br d, J=9.9 Hz, 1H), 0.72 (d, J=7.0 Hz, 3H).
Compound 22b was prepared according to the following scheme:
To a solution of 2-bromo-5-chloro-3-methyl-pyrazine (200 mg, 0.96 mmol) in DMF (3 mL) was added tert-butyl piperazine-1-carboxylate (180 mg, 0.96 mmol) and cesium carbonate (314 mg, 0.96 mmol). The reaction mixture was stirred at 90° C. for 15 h. After being cooled to rt, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash column eluting with a gradient of MeOH/DCM (0% to 10%) to give compound 22a (77 mg) as a yellow oil. MS: calc'd 357 (MH+), measured 357 (MH+).
To a solution of 1,6-dimethyl-4-[5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridin-1-yl]pyrazolo[3,4-b]pyridine (compound 24a, 95 mg, 0.26 mmol) in 1,4-dioxane (5 mL) and water (1 mL) was added tert-butyl 4-(5-bromo-6-methyl-pyrazin-2-yl)piperazine-1-carboxylate (compound 22a, 77 mg, 0.22 mmol), Pd(dppf)Cl2·CH2Cl2 (18 mg, 0.22 mmol) and K2CO3 (45 mg, 0.32 mmol). The reaction mixture was stirred at 90° C. for 2 h under N2 atmosphere. After being cooled to rt, the reaction mixture was concentrated under reduced pressure, the residue was purified by flash column eluting with a gradient of MeOH/DCM (0% to 10%) to give compound 22b (130 mg) as a brown oil. MS: calc'd 519 (MH+), measured 519 (MH+). The structures of Example 22 and Example 23 were confirmed by 2D-NMR.
1,6-dimethyl-4-[cis-3-methyl-4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 24) and 1,6-dimethyl-4-[trans-3-methyl-4-(3-methyl-5-piperazin-1-yl-2-pyridyl)-1-piperidyl]pyrazolo[3,4-b]pyridine (Example 25)
The title compounds were prepared in analogy to the preparation of Example 20 and Example 21 by using tert-butyl 4-[6-[1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]-5-methyl-3-pyridyl]piperazine-1-carboxylate (compound 24c) instead of compound 20g. The structures of Example 24 and Example 25 were confirmed by 2D-NMR.
Example 24 (16 mg) was obtained as a light yellow solid. MS: calc'd 420 (MH+), measured 420 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.42 (s, 1H), 8.20-8.14 (m, 1H), 7.99-7.91 (m, 1H), 6.76 (s, 1H), 4.70 (br d, J=13.4 Hz, 1H), 4.53 (br d, J=13.3 Hz, 1H), 4.09 (s, 3H), 3.86 (br d, J=13.6 Hz, 2H), 3.73-3.60 (m, 5H), 3.49-3.33 (m, 4H), 2.76-2.61 (m, 4H), 2.60-2.52 (m, 4H), 2.03-1.93 (m, 1H), 0.76 (d, J=7.0 Hz, 3H).
Example 25 (8 mg) was obtained as a light yellow solid. MS: calc'd 420 (MH+), measured 420 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.33 (s, 1H), 8.17 (d, J=2.4 Hz, 1H), 7.78 (br s, 1H), 6.75 (s, 1H), 4.67 (br d, J=13.4 Hz, 1H), 4.57 (br d, J=14.6 Hz, 1H), 4.08 (s, 3H), 3.69-3.53 (m, 6H), 3.44-3.36 (m, 4H), 3.36-3.24 (m, 1H), 2.63 (s, 3H), 2.53 (s, 3H), 2.45-2.35 (m, 1H), 2.12-2.00 (m, 2H), 0.88 (d, J=6.6 Hz, 3H).
Compound 24c were prepared according to the following scheme:
To a mixture of [1-(1,6-dimethylpyrazolo[3,4-b]pyridin-4-yl)-5-methyl-3,6-dihydro-2H-pyridin-4-yl]trifluoromethanesulfonate (compound 20f, 300 mg, 0.768 mmol) and 4,44,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (292.72 mg, 1.15 mmol) in 1,4-dioxane (10 mL) was added PdCl2(dppf)·CH2Cl2 (63 mg, 0.077 mmol) and potassium acetate (226 mg, 2.31 mmol). The mixture was charged with N2 and stirred at 90° C. for 2 h. After being cooled to rt, the mixture was filtered and the solid was washed with EA (10 mL) twice. The combined organic layer was concentrated to give the crude product compound 24a (283 mg) which was used in the next step without further purification. MS: calc'd 369 (MH+), measured 369 (MH+).
To the mixture of 1,6-dimethyl-4-[5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridin-1-yl]pyrazolo[3,4-b]pyridine (compound 24a, 283 mg, 0.768 mmol) and 2-bromo-5-chloro-3-methyl-pyridine (CAS: 65550-77-8, Vendor: Accela, 175 mg, 0.845 mmol) in 1,4-dioxane (10 mL) and water (2 mL) was added PdCl2(dppf)·CH2Cl2 (31 mg, 0.038 mmol) and K2CO3 (212 mg, 1.54 mmol). The mixture was charged with N2 and stirred at 90° C. overnight. After being cooled to rt, the mixture was extracted by DCM (20 mL) three times, dried over Na2SO4, filtered and concentrated and purified by flash column eluting with a gradient of EA/PE (0% to 30%) to afford the desired product compound 24b (260 mg). MS: calc'd 368 (MH+), measured 368 (MH+).
To the mixture of 4-[4-(5-chloro-3-methyl-2-pyridyl)-5-methyl-3,6-dihydro-2H-pyridin-1-yl]-1,6-dimethyl-pyrazolo[3,4-b]pyridine (compound 24b, 130 mg, 0.353 mmol), and 1-Boc-piperazine (99 mg, 0.530 mmol) in 1,4-dioxane (5 mL) was added cesium carbonate (230 mg, 0.707 mmol) and RuPhos Pd G2 (27 mg, 0.035 mmol). The mixture was charged with N2 and stirred at 110° C. overnight. After being cooled to rt, the solid was filtered off and washed with EA (10 mL) twice. The combined organic layer was concentrated and purified by flash column eluting with a gradient of EA (with 10% MeOH)/PE (0% to 80%) to give the desired product 24c (136 mg). MS: calc'd 518 (MH+), measured 518 (MH+).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-[(3R,4R)-4-methoxypyrrolidin-3-yl]carbamate (CAS: 1932066-52-8, Vendor: PharmaBlock) and Intermediate B instead of compound 15a and Intermediate E. Example 26 (75 mg) was obtained as a white solid. MS: calc'd 450 (MH+), measured 450 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.34 (s, 1H), 7.85 (d, J=2.50 Hz, 1H), 7.58 (d, J=2.50 Hz, 1 H), 6.77 (s, 1H), 4.69 (br d, J=14.01 Hz, 1H), 4.59 (br d, J=13.01 Hz, 1H), 4.19 (dt, J=5.63, 2.94 Hz, 1H), 4.09 (s, 3H), 3.99 (dt, J=6.25, 3.38 Hz, 1H), 3.87 (dd, J=11.51, 5.50 Hz, 1H), 3.80 (dd, J=11.26, 6.25 Hz, 1H), 3.68-3.58 (m, 1H), 3.55 (dd, J=11.26, 3.25 Hz, 1H), 3.51-3.42 (m, 4H), 3.40-3.32 (m, 2H), 2.64 (s, 3H), 2.57 (s, 3H), 2.44-2.33 (m, 1H), 2.14-2.04 (m, 2H) 0.90 (d, J=6.50 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (CAS: 1251005-61-4, Vendor: PharmaBlock) and Intermediate B instead of compound 15a and Intermediate E. Example 27 (65 mg) was obtained as a white solid. MS: calc'd 462 (MH+), measured 462 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.06 (s, 1H), 7.60 (d, J=2.5 Hz, 1H), 6.78 (d, J=2.4 Hz, 1H), 6.41 (s, 1H), 4.38 (br d, J=13.8 Hz, 1H), 4.29 (br d, J=11.9 Hz, 1H), 4.00 (s, 3H), 3.91 (d, J=8.0 Hz, 2H), 3.74-3.63 (m, 4H), 3.02 (s, 3H), 2.99-2.96 (m, 1H), 2.92-2.87 (m, 1H), 2.84-2.80 (m, 2H), 2.53 (s, 3H), 2.37 (s, 3H), 2.31 (br dd, J=6.5, 11.0 Hz, 1H), 1.98-1.88 (m, 1H), 1.84-1.75 (m, 1H), 0.77 (d, J=6.5 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-[(6S)-1,4-oxazepan-6-yl]carbamate (CAS: 2306247-11-8, Vendor: PharmaBlock) and Intermediate B instead of compound 15a and Intermediate E. Example 28 (75 mg) was obtained as a light yellow solid. MS: calc'd 450 (MH+), measured 450 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.39 (s, 1H), 8.11 (d, J=3.1 Hz, 1H), 8.02 (d, J=2.6 Hz, 1H), 6.83 (s, 1H), 4.73 (br d, J=10.4 Hz, 1H), 4.63 (br d, J=12.4 Hz, 1H), 4.20-4.09 (m, 5H), 4.05-3.98 (m, 1H), 3.95-3.80 (m, 5H), 3.75-3.62 (m, 2H), 3.47-3.34 (m, 2H), 2.68 (s, 3H), 2.63 (s, 3H), 2.54-2.45 (m, 1H), 2.23-2.12 (m, 2H), 0.95 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl (2R)-2-(methoxymethyl)piperazine-1-carboxylate (CAS: 1023301-73-6, Vendor: PharmaBlock) and Intermediate B instead of compound 15a and Intermediate E. Example 29 (147 mg) was obtained as yellow solid. MS: calc'd 464 (MH+), measured 464 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.37 (s, 1H), 8.21 (d, J=2.9 Hz, 1H), 8.11 (d, J=2.6 Hz, 1H), 6.81 (s, 1H), 4.71 (br d, J=12.2 Hz, 1H), 4.65-4.58 (m, 1H), 4.15-4.04 (m, 5H), 3.77-3.70 (m, 1H), 3.70-3.62 (m, 3H), 3.54-3.49 (m, 1H), 3.48-3.41 (m, 4H), 3.38-3.31 (m, 3H), 3.25 (br dd, J=10.3, 13.7 Hz, 1H), 2.66 (s, 3H), 2.64-2.59 (m, 3H), 2.53-2.44 (m, 1H), 2.20-2.11 (m, 2H), 0.92 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate (CAS: 674792-08-6, Vendor: Accela) and Intermediate B instead of compound 15a and Intermediate E. Example 30 (42 mg) was obtained as a white solid. MS: calc'd 446 (MH+), measured 446 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.34 (s, 1H), 8.20 (d, J=2.3 Hz, 1H), 7.97 (br s, 1H), 6.76 (s, 1H), 4.73-4.65 (m, 1H), 4.59 (br d, J=13.3 Hz, 1H), 4.09 (s, 3H), 3.74-3.69 (m, 2H), 3.68-3.59 (m, 1H), 3.58-3.49 (m, 4H), 3.45-3.33 (m, 2H), 2.64 (s, 3H), 2.58 (s, 3H), 2.48-2.35 (m, 1H), 2.20-2.03 (m, 2H), 1.22-1.03 (m, 4H), 0.90 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-[(3R,4R)-3-fluoropiperidin-4-yl]carbamate (CAS: 1523530-29-1, Vendor: PharmaBlock) and Intermediate B instead of compound 15a and Intermediate E. Example 31 (44 mg) was obtained as light yellow solid. MS: calc'd 452 (MH+), measured 452 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.33 (s, 1H), 8.19 (d, J=2.7 Hz, 1H), 7.89 (d, J=2.6 Hz, 1H), 6.75 (s, 1H), 4.78-4.63 (m, 2H), 4.57 (br d, J=13.0 Hz, 1H), 4.35-4.27 (m, 1H), 4.08 (s, 3H), 3.98-3.88 (m, 1H), 3.66-3.57 (m, 1H), 3.57-3.46 (m, 1H), 3.37-3.31 (m, 2H), 3.08-2.97 (m, 2H), 2.63 (s, 3H), 2.55 (s, 3H), 2.44-2.34 (m, 1H), 2.28-2.19 (m, 1H), 2.13-2.02 (m, 2H), 1.82 (dq, J=4.3, 12.6 Hz, 1H), 0.88 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-(3-methylazetidin-3-yl)carbamate hydrochloridecarbamate (CAS: 1408076-37-8, Vendor: PharmaBlock) and Intermediate B instead of compound 15a and Intermediate E. Example 32 (66 mg) was obtained as light yellow solid. MS: calc'd 420 (MH+), measured 420 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.33 (s, 1H), 7.77 (d, J=2.7 Hz, 1H), 7.42 (d, J=2.6 Hz, 1H), 6.75 (s, 1H), 4.67 (br d, J=13.6 Hz, 1H), 4.58 (br d, J=13.1 Hz, 1H), 4.17 (d, J=8.9 Hz, 2H), 4.12-4.06 (m, 5H), 3.67-3.58 (m, 1H), 3.37-3.31 (m, 2H), 2.63 (s, 3H), 2.54 (s, 3H), 2.43-2.34 (m, 1H), 2.12-2.05 (m, 2H), 1.69 (s, 3H), 0.88 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl (4aR,7aR)-octahydropyrrolo[3,4-b]morpholine-4-carboxylate (CAS: 1932337-68-2, Vendor: PharmaBlock) and Intermediate B instead of compound 15a and Intermediate E. Example 33 (66 mg) was obtained as light yellow solid. MS: calc'd 462 (MH+), measured 462 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.33 (s, 1H), 7.83 (d, J=2.9 Hz, 1H), 7.56 (d, J=2.6 Hz, 1H), 6.75 (s, 1H), 4.68 (br d, J=13.3 Hz, 1H), 4.64-4.52 (m, 1H), 4.25 (dd, J=3.7, 13.1 Hz, 1H), 4.21-4.12 (m, 1H), 4.08 (s, 3H), 4.04-3.97 (m, 1H), 3.89-3.80 (m, 2H), 3.63 (dt, J=7.6, 10.1 Hz, 2H), 3.52 (dd, J=2.1, 13.3 Hz, 1H), 3.49-3.44 (m, 1H), 3.40 (dd, J=4.2, 12.9 Hz, 1H), 3.37-3.32 (m, 3H), 2.63 (s, 3H), 2.56 (s, 3H), 2.43-2.34 (m, 1H), 2.14-2.07 (m, 2H), 0.89 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl piperazine-1-carboxylate and Intermediate D instead of compound 15a and Intermediate E. Example 35 (5 mg) was obtained as a white solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.35 (s, 2H), 8.05-7.85 (m, 1H), 7.77-7.62 (m, 1H), 6.77 (s, 1H), 4.67 (br d, J=13.4 Hz, 1H), 4.60-4.51 (m, 1H), 4.09 (s, 3H), 3.66-3.56 (m, 5H), 3.44-3.36 (m, 4H), 3.30-3.19 (m, 1H), 3.09-2.95 (m, 1H), 2.64 (s, 3H), 2.33-2.19 (m, 1H), 2.17-1.99 (m, 2H), 0.90 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl piperazine-1-carboxylate and Intermediate A instead of compound 15a and Intermediate E. Example 34 (20 mg) was obtained as a white solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.35 (s, 1H), 8.32 (d, J=2.9 Hz, 1H), 7.82 (dd, J=2.9, 9.0 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H), 6.76 (s, 1H), 4.69-4.62 (m, 1H), 4.55 (br dd, J=2.2, 13.9 Hz, 1H), 4.09 (s, 3H), 3.63-3.53 (m, 5H), 3.47-3.36 (m, 4H), 3.29-3.22 (m, 1H), 2.95 (dt, J=4.4, 11.2 Hz, 1H), 2.64 (s, 3H), 2.30-2.17 (m, 1H), 2.15-1.98 (m, 2H), 0.89 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-[(3R,4R)-4-methoxypyrrolidin-3-yl]carbamate (CAS: 1932066-52-8, Vendor: PharmaBlock) and Intermediate D instead of compound 15a and Intermediate E. Example 36 (28 mg) was obtained as a white solid. MS: calc'd 436 (MH+), measured 436 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.35 (s, 1H), 7.99 (d, J=2.75 Hz, 1H), 7.73-7.67 (m, 1H), 7.67-7.60 (m, 1H), 6.77 (s, 1H), 4.71-4.63 (m, 1H), 4.61-4.53 (m, 1H), 4.24-4.15 (m, 1H), 4.09 (s, 3H), 4.00 (td, J=3.2, 6.3 Hz, 1H), 3.89 (dd, J=5.6, 11.1 Hz, 1H), 3.81 (dd, J=6.3, 11.3 Hz, 1H), 3.62-3.54 (m, 2H), 3.50-3.43 (m, 4H), 3.29-3.22 (m, 1H), 3.01 (dt, J=4.3, 11.4 Hz, 1H), 2.64 (s, 3H), 2.28-2.02 (m, 3H), 0.90 (d, J=6.5 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl (2R)-2-(methoxymethyl)piperazine-1-carboxylate (CAS: 1023301-73-6, Vendor: PharmaBlock) and Intermediate D instead of compound 15a and Intermediate E. Example 37 (5 mg) was obtained as a white solid. MS: calc'd 450 (MH+), measured 450 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.05 (d, J=2.8 Hz, 1H), 7.96 (s, 1H), 7.33-7.27 (m, 1H), 7.11 (d, J=8.7 Hz, 1H), 6.31 (s, 1H), 4.27 (br d, J=13.0 Hz, 1H), 4.18 (br d, J=13.6 Hz, 1H), 3.89 (s, 3H), 3.55 (br t, J=12.7 Hz, 2H), 3.46-3.34 (m, 2H), 3.31 (s, 3H), 3.17-3.09 (m, 3H), 3.01-2.92 (m, 1H), 2.88-2.72 (m, 2H), 2.62-2.47 (m, 2H), 2.43 (s, 3H), 2.09-1.98 (m, 1H), 1.86-1.75 (m, 2H), 0.69 (d, J=6.5 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl 5-oxa-2,8-diazaspiro[3.5]nonane-8-carboxylate (CAS: 1251005-61-4, Vendor: PharmaBlock) and Intermediate D instead of compound 15a and Intermediate E. Example 38 (11 mg) was obtained as a white solid. MS: calc'd 448 (MH+), measured 448 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.07 (s, 1H), 7.74 (d, J=2.8 Hz, 1H), 7.16 (d, J=8.4 Hz, 1H), 6.95 (dd, J=2.8, 8.4 Hz, 1H), 6.42 (s, 1H), 4.38 (br d, J=12.8 Hz, 1H), 4.29 (br d, J=11.0 Hz, 1H), 4.01 (s, 3H), 3.94 (d, J=8.0 Hz, 2H), 3.70 (br d, J=7.8 Hz, 4H), 3.31-3.22 (m, 1H), 3.02 (s, 2H), 2.99-2.89 (m, 1H), 2.85-2.77 (m, 2H), 2.63-2.56 (m, 1H), 2.54 (s, 3H), 2.20-2.04 (m, 1H), 1.98-1.87 (m, 2H), 0.81 (d, J=6.5 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-[(3S,4S)-3-methoxy-4-piperidyl]carbamate (CAS: 907544-19-8, Vendor: PharmaBlock) and Intermediate D instead of compound 15a and Intermediate E. Example 39 (19 mg) was obtained as a white solid. MS: calc'd 450 (MH+), measured 450 (MH+). 1H NMR (400 MHz, METHANOL-d4) δ=8.37-8.36 (m, 1H), 8.35 (s, 1H), 8.07 (dd, J=8.8, 2.9 Hz, 1H), 7.74 (d, J=9.3 Hz, 1H), 6.76 (s, 1H), 4.67 (br d, J=14.3 Hz, 1H), 4.61-4.52 (m, 1H), 4.35 (br dd, J=12.2, 3.4 Hz, 1H), 4.09 (s, 3H), 3.98 (br d, J=13.2 Hz, 1H), 3.64-3.57 (m, 1H), 3.55 (s, 3H), 3.40 (td, J=10.0, 4.4 Hz, 1H), 3.29-3.14 (m, 2H), 2.95-3.10 (m, 2H), 2.74 (dd, J=12.2, 10.3 Hz, 1H), 2.64 (s, 3H), 2.29-2.04 (m, 4H), 1.81 (qd, J=12.6, 4.7 Hz, 1H), 0.91 (d, J 20=6.4 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-[(6S)-1,4-oxazepan-6-yl]carbamate (CAS: 2306247-11-8, Vendor: PharmaBlock) and Intermediate D instead of compound 15a and Intermediate E. Example 40 (70 mg) was obtained as a white solid. MS: calc'd 436 (MH+), measured 436 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.28 (s, 1H), 8.18 (d, J=2.7 Hz, 1H), 8.00 (dd, J=2.8, 9.2 Hz, 1H), 7.75 (d, J=9.3 Hz, 1H), 6.71 (s, 1H), 4.60 (br d, J=11.7 Hz, 1H), 4.49 (br d, J=12.8 Hz, 1H), 4.10-3.97 (m, 5H), 3.95-3.88 (m, 1H), 3.84-3.78 (m, 2H), 3.77-3.68 (m, 3H), 3.64-3.47 (m, 2H), 3.27-3.21 (m, 1H), 3.01 (dt, J=3.8, 11.5 Hz, 1H), 2.56 (s, 3H), 2.25-2.14 (m, 1H), 2.14-2.07 (m, 1H), 2.06-1.94 (m, 1H), 0.85 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-[(3R,4R)-3-fluoropiperidin-4-yl]carbamate (CAS: 1523530-29-1, Vendor: PharmaBlock) and Intermediate D instead of compound 15a and Intermediate E. Example 41 (10 mg) was obtained as white solid. MS: calc'd 438 (MH+), measured 438 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.09-8.03 (m, 1H), 7.95 (s, 1H), 7.30 (dd, J=3.0, 8.6 Hz, 1H), 7.08 (d, J=8.5 Hz, 1H), 6.29 (s, 1H), 4.38-4.21 (m, 2H), 4.18-4.13 (m, 1H), 3.94-3.82 (m, 4H), 3.55 (br dd, J=1.3, 12.7 Hz, 1H), 3.24-3.19 (m, 2H), 3.18-3.09 (m, 1H), 2.92-2.78 (m, 2H), 2.77-2.66 (m, 2H), 2.49 (dt, J=4.7, 11.1 Hz, 1H), 2.09-1.89 (m, 2H), 1.86-1.76 (m, 2H), 1.52 (br dd, J=4.1, 12.4 Hz, 1H), 1.24-1.14 (m, 1H), 0.68 (d, J=6.6 Hz, 3H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl 4,7-diazaspiro[2.5]octane-4-carboxylate (CAS: 674792-08-6, Vendor: PharmaBlock) and Intermediate D instead of compound 15a and Intermediate E. Example 42 (19 mg) was obtained as white solid. MS: calc'd 432 (MH+), measured 432 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=7.99 (d, J=2.7 Hz, 1H), 7.95 (s, 1H), 7.24 (dd, J=2.9, 8.7 Hz, 1H), 7.07 (d, J=8.7 Hz, 1H), 6.30 (s, 1H), 4.26 (br d, J=13.1 Hz, 1H), 4.20-4.11 (m, 1H), 3.89 (s, 3H), 3.19-3.08 (m, 3H), 3.01-2.96 (m, 2H), 2.95 (s, 2H), 2.86-2.78 (m, 1H), 2.53-2.45 (m, 1H), 2.42 (s, 3H), 2.07-1.97 (m, 1H), 1.89-1.75 (m, 2H), 0.68 (d, J=6.6 Hz, 3H), 0.59 (br d, J=8.4 Hz, 4H).
The title compound was prepared in analogy to the preparation of Example 15 by using tert-butyl N-(3-methylazetidin-3-yl)carbamate hydrochloridecarbamate (CAS: 1408076-37-8, Vendor: PharmaBlock) and Intermediate D instead of compound 15a and Intermediate E. Example 43 (33 mg) was obtained as white solid. MS: calc'd 406 (MH+), measured 406 (MH+). 1H NMR (500 MHz, METHANOL-d4) δ=8.24 (s, 1H), 7.83 (d, J=2.7 Hz, 1H), 7.58 (d, J=8.9 Hz, 1H), 7.41 (dd, J=2.9, 8.9 Hz, 1H), 6.66 (s, 1H), 4.56 (br d, J=13.6 Hz, 1H), 4.46 (br d, J=12.7 Hz, 1H), 4.09 (d, J=9.0 Hz, 2H), 4.03-3.95 (m, 5H), 3.48 (br t, J=12.0 Hz, 1H), 3.19-3.11 (m, 1H), 2.91 (dt, J=4.2, 11.5 Hz, 1H), 2.54 (s, 3H), 2.19-1.90 (m, 3H), 1.60 (s, 3H), 0.79 (d, J=6.6 Hz, 3H).
The following tests were carried out in order to determine the activity of the compounds of formula (I) and (Ia) in HEK293-Blue-hTLR-7/8/9 cells assay.
A stable HEK293-Blue-hTLR-7 cell line was purchased from InvivoGen (Cat. #: hkb-htlr7, San Diego, California, USA). These cells were originally designed for studying the stimulation of human TLR7 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. The SEAP was induced by activating NF-κB and AP-1 via stimulating HEK-Blue hTLR7 cells with TLR7 ligands. Therefore the reporter expression was declined by TLR7 antagonist under the stimulation of a ligand, such as R848 (Resiquimod), for incubation of 20 hrs. The cell culture supernatant SEAP reporter activity was determined using QUANTI-Blue™ kit (Cat. #: rep-qbl, Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple or blue in the presence of alkaline phosphatase.
HEK293-Blue-hTLR7 cells were incubated at a density of 250,000-450,000 cells/mL in a volume of 170 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50 mg/hL streptomycin, 100 mg/mL Normocin, 2 nM L-glutamine, 10% X(v/v) heat-inactivated fetal bovine serum with addition of 20 μL test compound in a serial dilution in the presence of final DMSO at 1% and 10 μL of 20 μM R848 in above DMEM, perform incubation under 37° C. in a CO2 incubator for 20 hrs. Then 20 μL of the supernatant from each well was incubated with 180 μL Quanti-blue substrate solution at 37° C. for 2 hrs and the absorbance was read at 620-655 rim using a spectrophotometer. The signaling pathway that TLR7 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was modified for evaluating TLR7 antagonist.
HEK293-Blue-hTLR-8 cells assay:
A stable HEK293-Blue-hTLR-8 cell line was purchased from InvivoGen (Cat. #: hkb-htlr8, San Diego, California, USA). These cells were originally designed for studying the stimulation of human TLR8 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. The SEAP was induced by activating NF-κB and AP-1 via stimulating HEK-Blue hTLR8 cells with TLR8 ligands. Therefore the reporter expression was declined by TLR8 antagonist under the stimulation of a ligand, such as R848, for incubation of 20 hrs. The cell culture supernatant SEAP reporter activity was determined using QUANTT-BlueTv™ kit (Cat. #: rep-qbl, Invivogen, San Diego, Ca, USA) at a wavelength of 640 nm, a detection medium that turns purple or blue in the presence of alkaline phosphatase.
HEK293-Blue-hTLR8 cells were incubated at a density of 250,000˜450,000 cells/mL in a volume of 170 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mi penicillin, 50 mg/mL streptomycin, 100 mg/mL Normocin, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum with addition of 20 μL test compound in a serial dilution in the presence of final DMSO at 1% and 10 μL of 60 μM R848 in above DMEM, perform incubation under 37° C. in a CO2 incubator for 20 hrs. Then 20 μL of the supernatant from each well was incubated with 180 μL Quanti-blue substrate solution at 37° C. for 2 hrs and the absorbance was read at 620-655 nm using a spectrophotometer. The signaling pathway that TLR8 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was modified for evaluating TLR8 antagonist.
A stable HEK293-Blue-hTLR-9 cell line was purchased from InvivoGen (Cat. #: hkb-htlr9, San Diego, California, USA). These cells were originally designed for studying the stimulation of human TLR9 by monitoring the activation of NF-κB. A SEAP (secreted embryonic alkaline phosphatase) reporter gene was placed under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. The SEAP was induced by activating NF-κB and AP-1 via stimulating HEK-Blue hTLR9 cells with TLR9 ligands. Therefore the reporter expression was declined by TLR9 antagonist under the stimulation of a ligand, such as ODN2006 (Cat. #: tlrl-2006-1, Invivogen, San Diego, California, USA), for incubation of 20 hrs. The cell culture supernatant SEAP reporter activity was determined using QiANTI-Blue™ kit (Cat. #: rep-qbl, Invivogen, San Diego, California, USA) at a wavelength of 640 nm, a detection medium that turns purple or blue in the presence of alkaline phosphatase.
HEK293-Blue-hTLR9 cells were incubated at a density of 250,000-450,000 cells/mL in a volume of 170 μL in a 96-well plate in Dulbecco's Modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 50 U/mL penicillin, 50 mg/mL streptomycin, 100 mg/mL Normocin, mM L-glutanmine, 10% (v/v) heat-inactivated fetal bovine serum with addition of 20 μL test compound in a serial dilution in the presence of final DMSO at 1% and 10 μL of 20 μM ODN2006 in above DMEM, perform incubation under 37° C. in a C02 incubator for 20 hrs. Then 20 μL of the supernatant from each well was incubated with 180 μl. Quanti-blue substrate solution at 37° C. for 2 h and the absorbance was read at 620˜655 nm using a spectrophotometer. The signaling pathway that TLR9 activation leads to downstream NF-κB activation has been widely accepted, and therefore similar reporter assay was modified for evaluating TLR9 antagonist.
The compounds of formula (I) or (Ia) have human TLR7 and TLR8 inhibitory activities (IC50 value)<0.5 μM. Moreover, some compounds also have human TLR9 inhibitory activity <0.5 μM. Activity data of the compounds of the present invention were shown in Table 2.
hERG Channel Inhibition Assay:
The hERG channel inhibition assay is a highly sensitive measurement that identifies compounds exhibiting hERG inhibition related to cardiotoxicity in vivo. The hERG K+ channels were cloned in humans and stably expressed in a CHO (Chinese hamster ovary) cell line. CHOhERG cells were used for patch-clamp (voltage-clamp, whole-cell) experiments. Cells were stimulated by a voltage pattern to activate hERG channels and conduct IKhERG Currents (rapid delayed outward rectifier potassium current of the hERG channel). After the cells were stabilized for a few minutes, the amplitude and kinetics of IKhERG were recorded at a stimulation frequency of 0.1 Hz (6 bpm). Thereafter, the test compound was added to the preparation at increasing concentrations. For each concentration, an attempt was made to reach a steady-state effect, usually, this was achieved within 3-10 min at which time the next highest concentration was applied. The amplitude and kinetics of IKhERG are recorded in each concentration of the drug which were compared to the control values (taken as 100%). (references: Redfe W S, Carlsson L, Davis A S, Lynch W G, MacKenzie I, Palethorpe S, Siegl P K, Strang I, Sullivan A T, Wallis R, Camm A J, Hammond T G. 2003; Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. Cardiovasc. Res. 58:32-45, Sanguinetti M C, Tristani-Firouzi M. 2006; hERG potassium channels and cardiac arrhythmia. Nature 440:463-469, Webster R, Leishman D, Walker D. 2002; Towards a drug concentration effect relationship for QT prolongation and torsades de pointes. Curr. Opin. Drug Discov. Devel. 5:116-26).
Results of hERG are given in Table 3. A safety ratio (hERG IC20/EC50)>30 suggests a sufficient window to differentiate the pharmacology by inhibiting TLR7/8/9 pathways from the potential hERG related cardiotoxicity. According to the calculation of hERG IC20/TLR7/8/9 IC50 below which serves as early selectivity index to assess hERG liability, obviously reference compounds ER-887258, ER-888285 ER-888286, R1 and R2 have much narrower safety window compared to the compounds of this invention.
Unlike the HEK reporter cell lines, human peripheral blood mononuclear cell (PBMC) represents primary human immune cells in blood mainly consisting of lymphocytes, monocytes, and dendritic cells. These cells express TLR7, TLR8, or TLR9, and therefore are natural responders to respective ligand stimulation. Upon activation of these TLRs, PBMCs secrete similar cytokines and chemokines in vitro and in vivo, and therefore the in vitro potency of a TLR7/8/9 antagonist in human PBMC is readily translatable to its pharmacodynamics response in vivo.
Human peripheral blood mononuclear cells (PBMC) were isolated from freshly-drawn lithium-heparinized (Lithium Heparin Plus blood Collection tube, BD Vacutainer®) healthy donor whole blood by density gradient (Ficoll-Paque™ PLUS, GE Healthcare life Sciences). Briefly, 50 mL of blood was diluted with 25 mL PBS (without Ca2+, Mg2+) in a 50 mL conical tube with porous barrier (Leucosep tube, Greiner bio-one), where 15.5 mL Ficoll-Paque was under laid after spinning. Tubes were centrifuged for 20 minutes at 800×g (1946 rpm) with the brake in the off position, and PBMC were collected from the buffy coat. Cells were then washed twice in PBS, and red blood cells were lysed by suspension in 2 mL (Red Blood Cell Lysis Buffer, Alfa Aesar) for 5-10 minutes at room temperature. After a final wash in PBS, PBMC were resuspended at a final concentration of 2×106 cells/mL in RPMI-1640 media with GlutaMAX™ (Gibco) supplemented with 10% Fetal Bovine Serum (Sigma) and plated at 150 μL/well (3×105 cells/well) in tissue culture treated round bottom 96-well plates (Corning Incorporated). Antagonist compounds (compounds of this invention) solubilized and serial diluted in 100% DMSO were added in duplicate to cells to yield a final concentration of 1% DMSO (v/v). PBMC were incubated with antagonist compounds for 30 minutes at 37° C., 5% CO2 before adding various TLR agonist reagents in 48 μL complete media per well as follows (final concentrations indicated): CpG ODN 2216 (InvivoGen) at 1 μM for TLR9, ORN 06/LyoVec (InvivoGen) at 1p g/mL for TLR8 and R848 (InvivoGen) at 1 μg/mL for TLR7 and TLR8. PBMC were incubated overnight at 37° C. with 5% C02. Cell culture supernatants were collected, and levels of various human cytokines were assessed by Luminex assay (ProcartaPlex™ Multiplex Immunoassay, Invitrogen) or ELISA procedure according to the manufacturer's recommended protocol (eBioscience, ThermoFisher Scientific). Viability of the cells was also checked with Cell Viability Assay (CellTiter Glo® Luminescent Cell Viability Assay, Promega).
The human microsomal stability assay is used for early assessment of metabolic stability of a test compound in human liver microsomes.
Human liver microsomes (Cat. NO.: 452117, Corning, USA; Cat. NO.:H2610, Xenotech, USA) were preincubated with test compound for 10 minutes at 37° C. in 100 mM potassium phosphate buffer, pH 7.4. The reactions were initiated by adding NADPH regenerating system.
The final incubation mixtures contained 1 μM test compound, 0.5 mg/mL liver microsomal protein, 1 mM MgCl2, 1 mM NADP, 1 unit/mL isocitric dehydrogenase and 6 mM isocitric acid in 100 mM potassium phosphate buffer, pH 7.4. After incubation times of 0, 3, 6, 9, 15 and 30 minutes at 37° C., 300 μL of cold acetonitrile (including internal standard) was added to 100 μL incubation mixture to terminate the reaction. Following precipitation and centrifugation, the amount of compound remaining in the samples were determined by LC-MS/MS. Controls of no NADPH regenerating system at zero and 30 minutes were also prepared and analyzed. The compounds of present invention showed good human liver microsome stability determined in the above assay, results are shown in Table 5 below.
Phototoxicity is defined as a toxic response that is elicited after the first exposure of the skin to certain chemicals and subsequent exposure to light, or that is induced similarly by skin irradiation after systemic administration of a chemical substance. The assay used in this study is designed to detect the phototoxic potential of a chemical by using a simple in vitro cytotoxicity assay with Balb/c 3T3 mouse fibroblasts. The principle of this test is a comparison of the cytotoxicity of a chemical when tested with and without exposure to a non-toxic dose of UVA-light. Cytotoxicity is expressed as a dose dependent reduction of the growth rate of cells as determined by uptake of the vital dye Neutral Red one day after treatment.
Preparation of Stock Solution and Dosage of Test Item A small amount of substance was weighed and formulated freshly in DMSO just before the start of the exposure of the cells. This stock solution or appropriate dilutions with DMSO were added to the cell suspensions to obtain the required final concentrations. All solutions were generally prepared in Eppendorf caps and discarded after use.
The absorption spectra as such or with UV-A or with UV-B pre-irradiation were recorded between 240 nm and 400 nm with a Lambda-2 spectral photometer (Perkin Elmer).
For this study the Neutral Red uptake (NRU) assay of Borenfreund and Puemer (Borenfreund, E, Puemer J A. Toxicity determined in vitro by morphological alterations and Neutral Red absorption. Toxicology Lett. 1985; 24:119-124) modified according to INVITTOX protocol No 78 (ERGATT/FRAME data bank of in vitro techniques in toxicology. INVITTOX PROTOCOL No 78. 3T3 NRU Phototoxicity Assay. March 1994) has been adapted to examine a possible phototoxic potential of the test item. This assay is based on the active uptake of the Neutral Red dye into the lysosomes of cultured murine fibroblasts. Because lysosomal membranes are known to be a site of action of many phototoxic compounds, this assay can provide a measure of potential for phototoxic injury.
A murine fibroblasts clone A 31 (ATCC no. CCL 163—passage No. 108) were cultured in 175 cm2 tissue culture grade flasks, containing sDMEM (Dulbecco's Minimal Essential Medium, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml Penicillin and 100 μg/ml streptomycin) at 37° C. in a humidified atmosphere of 6% CO2. Before cells approach confluence they were removed from flasks by trypsinisation. Prior to use in an assay, the cells were transferred to 96-well microtiter plates at a concentration of 1×104 cells/well in 100 μl volumes of sDMEM and allowed to attach for 24 h.
For incubation with murine fibroblasts, the test item was diluted in PBS/3% DMSO (detailed concentrations see in results).
Culture medium (Dulbecco's Modified Eagle Medium (DMEM), GlutaMAX (Gibco Ref 21885-025), 10% Fetal Bovine Serum (FBS) (Gibco Ref 10270-106), 100 IU/ml Penicillin and 100 μg/ml Streptomycin (Gibco Ref 15140-122)) was removed from the wells and murine fibroblasts were washed with PBS. Afterwards 100 μL of PBS/3% DMSO containing the test item was added and target cells were incubated for 1 h at 37° C. with 6% CO2.
For each test item the microtiter plates were prepared according to Table 6. “UVA plates” were exposed to approx. 5 J/cm2 UVA light, the “Dark plates” were kept in the dark and served as cytotoxicity control. Plates with chlorpromazine hydrochloride served as positive control. UV flux was measured with a UV-meter (Dr. Gröbel RM21).
Following UV irradiation, the test item was removed from the wells (one washing step with PBS) and replaced with sDMEM. Target cells were then incubated overnight at 37° C. in 6% CO2.
Each plate contained wells with cells and solvent but without test item which were either not incubated with Neutral Red solution (0% standard—S1) or were stained with Neutral Red (100% standard—S2) for calculation of the standard cell viability curve. Wells labeled with U01-U08 contained the different test item concentrations.
The ready to use Neutral Red (NR) staining solution was freshly prepared as follows:
After the incubation the wells to be assayed were filled with 100 μL of the sDMEM containing Neutral Red. The target cells were incubated with the NR for 3 h at 37° C. in 6% CO2.
Unincorporated Neutral Red was removed from the target cells and the wells washed with at least 100 μL of PBS. 150 μL of Neutral Red desorb solution (1% glacial acetic acid, 50% ethanol in aqua bidest) was then added to quantitatively extract the incorporated dye. After at least 10 mins of vigorous shaking of the plates on a microtiter plate shaker until Neutral Red has been extracted from the cells and formed a homogeneous solution, the absorption of the resulting colored solution was measured with a SPECTRAmax PLUS microtiter plate reader (Molecular Devices) at 540 nm.
Cell viability was calculated with the SOFTmax Pro software package (Molecular Devices). First a two-point standard curve (0% and 100% viability) was calculated with the linear curve fit option of the program based on the following formula:
By this means the viability of the cells incubated with increasing concentrations of the test chemical was calculated. Chlorpromazine (HCl) served as positive control in the experiment.
All calculations were performed with the SOFTmax Pro analysis software package (Molecular Devices—for details see: http://www.mbl.edu/jbpc/files/2014/05/SoftMax_Pro_User_Guide.pdf)
For evaluation of phototoxic potential, the IC50 values determined with and without UV exposure were compared.
For discrimination between phototoxic and non-phototoxic test chemicals a cut-off factor of >5 was applied (Liebsch M, Spielmann H, Balls M, Brand M, Doring B, Dupuis J, Holzhuter H G, Klecak G, L. Eplattenier H, Lovell W, Maurer T, Moldenhauer F, Moore L, Pape W, Pfannenbecker U, Potthast J M, De Silva O, Steiling W, Willshaw A. First results of the EC/COLIPA Validation Project. In Vitro Phototoxicity Testing. In: In Vitro Skin Toxicology: Irritation, Phototoxicity, Sensitization; Vol. 10. Alternative Methods in Toxicology,-Eds. Rougier A, Maibach H I, Goldberg A M; Mary Ann Liebert Publ.: New York, USA 1994, pp. 243-251).
Test items which are not cytotoxic to murine fibroblasts even at the highest concentrations tested, but show a strong dose dependent decrease in cell viability after UV exposure are considered also phototoxic (Spielmann H, Balls M, Dupuis J, Pape W J W, Pechovitch G, Silva DeO, Holzhntter, HG, Clothier R, Desolle P, Gerberick F, Liebsch M, Lowell W W, Maurer T, Pfannenbecker U, Potthast J M, Csato M, Sladowski D, Steiling W, Brantom P. The international EU/COLIPA in vitro phototoxicity validation study: Results of phase II (blind trial). Part 1: The 3T3 NRU phototoxicity test. Toxicology in Vitro 1998, 12: 305-327).
The test results were shown below, the compounds of this invention showed very good phototoxicity profile.
The in vitro mouse Embryonic Stem Cell Test (mEST) assay is an implemented routine assay at Roche. The original EST was developed by Horst Spielmann and his group in 1997 as an in vitro model for the screening of embryotoxicity, based on a blastocyst-derived permanent embryonic mouse ESC (mESC) D3 cell line derived from mouse 129 strains and was validated by European Centre for the Validation of Alternative Methods (ECVAM).
We further optimized and modified the approach allowing for the application of the assay to pharmaceutical compounds.
The cytotoxicity (inhibition of growth) of 3T3 fibroblasts, which represents differentiated cells and the cytotoxicity of undifferentiated embryonic stem cells (D3) after 10 days of substance treatment serve as two assay endpoints. This is determined by the use of dehydrogenase enzymes, which are present in the intact mitochondria of living cells to convert yellow soluble substrate 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark blue insoluble formazan product, which gets sequestered within the cells and is detected quantitatively using an absorbance reader (570 nm) after solubilizing the cell membrane.
The third endpoint is the inhibition of differentiation of ES cells into myocards which are cardiac muscle cells after 10 days of treatment. The beating of this cells is evaluated by microscopy.
Stock solution with a concentration of 0.2 mol/L is created for all substances. Test substances are diluted in DMSO solution.
Data files of optical densities (OD570) generated by a microplate reader were copied into an EXCEL spreadsheet. Mean OD values, standard deviations and viabilities were calculated automatically. The following endpoints from the assays could be calculated graphically from the concentration-response curve in the spreadsheet:
The IC50 values of the D3 and 3T3 cells from the cytotoxicity assay and the ID50 of the D3 differentiation assay were entered into the statistical evaluation developed from the modified prediction model used by Scholz et al. 1999a:
Predictive scores between 0.5 and 0.6 are labelled borderline results.
Inconclusive results are also possible, for example, if solubility limits the dose ranges tested to an extent that no IC50 or ID50 values can be determined for one or more dose response curves (Withlow et al. 2007)
Pharmacokinetic properties of selected compounds were assessed by single dose PK studies in Male Wister-Han Rats (vendor: Beijing Vital River Laboratory Animal Technology Co., Ltd). Briefly, two groups of animals were administered a single dose of respective compound intravenously (IV, bolus) at 2 mg/kg or orally (PO, by gavage) at 10 mg/kg. Blood samples (approximately 150 μL) were collected via Jugular vein at 5 min (only for IV), 15 min, 30 min, 1 h, 2 h, 4 h, 7 h and 24 h post-dose. Blood samples were placed into tubes containing EDTA-K2 anticoagulant and centrifuged at 3000 rpm for 15 min at 4° C. to separate plasma from the samples. After centrifugation, the resulting plasma was transferred to clean tubes for bioanalysis with LC/MS/MS. The pharmacokinetic parameters were calculated using non-compartmental analysis. The volume of distribution (Vss), half-life (T1/2) and clearance (CL) were obtained based on the plasma concentration-time curve after IV dose. The peak concentration (Cmax) was recorded directly from experimental observations after PO dose. The area under the plasma concentration-time curve (AUC0-last) was calculated using the linear trapezoidal rule up to the last detectable concentration. The bioavailability (F) was calculated based on the dose normalized AUC0-last after IV and PO dose.
The Vss of a drug represents the degree to which a drug is distributed in body tissue rather than the plasma. Vss is directly proportional with the amount of drug distributed into tissue. A higher Vss indicates a greater amount of tissue distribution.
Results of PK parameters following IV and PO administration are given in Table 9.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/120406 | Sep 2021 | WO | international |
| PCT/CN2022/089033 | Apr 2022 | WO | international |
This application is a Continuation of International Application No. PCT/EP2022/076306, filed on Sep. 22, 2022, which claims benefit of priority to CN Application No. PCT/CN2021/120406 filed Sep. 24, 2021 and of CN Application No. PCT/CN2022/089033, filed Apr. 25, 2022, each of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2022/076306 | Sep 2022 | WO |
| Child | 18606123 | US |
