Patent Application
20230172940
Life has evolved to exploit the redox chemistry of iron for essential activities. Ferrous iron drives ferroptosiso a regulated cell death program genetically and biochemically distinct from apoptosiso necrosis and autophagic cell death. Ferroptosis kills malignant cells but may also be inappropriately activated in ischemic injury and neurodegeneration. This cell death mechanism is executed by (phospho)lipid hydroperoxides induced by either iron-dependent lipoxygenaseso or by an iron-catalyzed spontaneous peroxyl radical-mediated chain reaction (autoxidation). Under homeostatic conditions the ferroptotic signal is terminated by glutathione peroxidase-4 (GPx4)o a phospholipid hydroperoxidase that needs glutathione as a cofactor. While the signaling that regulates ferroptosis has been studied in deptho the role of iron load in this death signal is poorly resolved.
Redox cycling between Fe2+ and Fe3+ can contribute to cellular stress. This is mitigated by a range of storage and chaperone pathways to ensure that the labile iron pool is kept to a minimum (Hare et al.o 2013). In Caenorhabditis elegans the emergence of labile ferrous iron with age correlates with genetic effects that accelerate aging and could be a lifespan hazard. Excess iron supply has been shown to shorten lifespan in C. eleganso yet variable results have been reported with iron chelation. The iron chelator deferiprone was reported not to impact C. elegans lifespano but this study was limited by indirect measures of iron loado use of only a single dose of deferiproneo and small sample size. In contrasto use of calcium-ethylenediaminetetraacetic acid (CaEDTA)o a non-specific chelator that does not redox-silence irono caused a minor (undisclosed) increase in lifespan. Whether selective targeting of ferrous iron burden can impact on aging and lifespan is unknown.
The developmental dependence on iron for reproduction and cellular biochemistry may represent an ancient and conserved liability in late life. The load of tissue iron increases needlessly in aging nematodeso mammalso and humans. This must tax regulatory systems that prevent abnormal redox cycling of irono such as the Fe2+-glutathione complexes thought to be the dominant form of iron in the cellular labile iron pool. We hypothesized that age-dependent elevation of labile irono coupled with a reduction of glutathione levels conspire to lower the threshold for ferroptotic signalingo increasing the vulnerability of aged animals and implying that disruption to the iron-glutathione axis is fundamental to natural aging and death. To test thiso we investigated the vulnerability to ferroptosis of aging nematodes upon the natural loss of glutathione during lifespan. We examined the effects of inhibiting ferroptosis in C. elegans using two distinct treatments: a potent quenching agent for lipid peroxidation (autoxidation) as well as a small lipophilic iron chelator that prevents the initiation and amplification of lipid peroxide signals. Our analysis of these interventions indicates that post-developmental interventions to limit ferroptosis not only promotes healthy agingo but actually extends the lifespan of the organism.
Provided herein is a method of extending the lifespan of an organism comprising administering an effective amount of a ferroptosis inhibitor to the organism.
Described herein is a are methods of extending lifespan comprising administering ferroptosis inhibitors to a subject. Exemplary ferroptosis inhibitors which are suitable for use in the methods described herein include compounds of formula I:
wherein
Other ferroptosis inhibitors suitable for use in the methods described herein include compounds of formula II:
wherein
Also described herein is a pharmaceutical composition for use in extending lifespan comprising a lifespan-extending effective amount of a ferroptosis inhibitoro such as a compound of formula I or II as described aboveo and a pharmaceutically acceptable carrier and/or excipient.
Unless specifically noted otherwise hereino the definitions of the terms used are standard definitions used in the art of organic chemistry and pharmaceutical sciences. Exemplary embodimentso aspects and variations are illustrated in the figures and drawingso and it is intended that the embodimentso aspects and variationso and the figures and drawings disclosed herein are to be considered illustrative and not limiting.
While particular embodiments are shown and described hereino it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variationso changeso and substitutions will now occur to those skilled in the art. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the methods described herein. It is intended that the appended claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Unless defined otherwiseo all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art. All patents and publications referred to herein are incorporated by reference.
As used in the specification and claimso the singular form “ao” “ano” and “the” include plural references unless the context clearly dictates otherwise.
The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound described herein that is sufficient to effect the intended application including but not limited to treatment as defined below. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo)o or the subject and condition being treatedo e.g.o the weight and age of the subjecto the severity of the conditiono the manner of administration and the likeo which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cellso e.g. reduction of platelet adhesion and/or cell migration. The specific dose will vary depending on the particular compounds choseno the dosing regimen to be followedo whether it is administered in combination with other compoundso timing of administrationo the tissue to which it is administeredo and the physical delivery system in which it is carried.
The terms “treatment” “treating” “palliating” and “ameliorating” are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying condition being treated. Alsoo a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying condition such that an improvement is observed in the patiento notwithstanding that the patient may still be afflicted with the underlying condition. For prophylactic benefito the compositions may be administered to a patient at risk of developing a particular conditiono or to a patient reporting one or more of the physiological symptoms of a conditiono even though a diagnosis of this condition may not have been made.
A “therapeutic effect” as used hereino encompasses a therapeutic benefit and/or a prophylactic benefit as described above. A prophylactic effect includes delaying or eliminating the appearance of a conditiono delaying or eliminating the onset of symptoms of a conditiono slowingo haltingo or reversing the progression of a conditiono or any combination thereof.
The term “co-administrationo” “administered in combination witho” and their grammatical equivalentso as used hereino encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. Co-administration includes simultaneous administration in separate compositionso administration at different times in separate compositionso or administration in a composition in which both agents are present.
A “pharmaceutically acceptable salt” means a salt composition that is generally considered to have the desired pharmacological activityo is considered to be safeo non-toxic and is acceptable for veterinary and human pharmaceutical applications. Pharmaceutically acceptable salts may be derived from a variety of organic and inorganic counter ions well known in the art and includeo by way of example onlyo sodiumo potassiumo calciumo magnesiumo ammoniumo tetraalkylammoniumo and the like; and when the molecule contains a basic functionalityo salts of organic or inorganic acidso such as hydrochlorideo hydrobromideo tartrateo mesylateo acetateo maleateo oxalate and the like. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived includeo for exampleo hydrochloric acid hydrobromic acido sulfuric acido nitric acido phosphoric acido and the like. Organic acids from which salts can be derived includeo for exampleo acetic acido propionic acido glycolic acido pyruvic acido oxalic acido maleic acido malonic acido succinic acido fumaric acido tartaric acido citric acido benzoic acido cinnamic acido mandelic acido methanesulfonic acido ethanesulfonic acido p-toluenesulfonic acido salicylic acido and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived includeo for exampleo sodiumo potassiumo lithiumo ammoniumo calciumo magnesiumo irono zinco coppero manganeseo aluminumo and the like. Organic bases from which salts can be derived includeo for exampleo primaryo secondaryo and tertiary amineso substituted amines including naturally occurring substituted amineso cyclic amineso basic ion exchange resinso and the likeo specifically such as isopropylamineo trimethylamineo diethylamineo triethylamineo tripropylamineo and ethanolamine. In some embodimentso the pharmaceutically acceptable base addition salt is chosen from ammoniumo potassiumo sodiumo calciumo and magnesium salts.
“Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solventso dispersion mediao coatingso antibacterial and antifungal agentso isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingrediento its use in the therapeutic compositions described herein is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The terms “antagonist” and “inhibitor” are used interchangeablyo and they refer to a compound having the ability to inhibit a biological function of a target proteino whether by inhibiting the activity or expression of the target protein. Accordinglyo the terms “antagonist” and “inhibitors” are defined in the context of the biological role of the target protein. Although antagonists herein generally interact specifically with (e.g. specifically bind to) the targeto compounds that inhibit a biological activity of the target protein by interacting with other members of the signal transduction pathway of which the target protein is a member are also specifically included within the definition of “antagonist.” An exemplary biological activity inhibited by an antagonist is associated with the developmento growtho or spread of a tumoro or an undesired immune response as manifested in autoimmune disease.
The term “agonist” as used herein refers to a compound having the ability to initiate or enhance a biological function of a target proteino whether by inhibiting the activity or expression of the target protein. Accordinglyo the term “agonist” is defined in the context of the biological role of the target polypeptide. Agonists herein generally interact specifically with (e.g. specifically bind to) the targeto compounds that initiate or enhance a biological activity of the target polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member are also specifically included within the definition of “agonist.”
As used hereino “agent” or “biologically active agent” refers to a biologicalo pharmaceuticalo or chemical compound or other moiety. Non-limiting examples include simple or complex organic or inorganic moleculeo a peptideo a proteino an oligonucleotideo an antibodyo an antibody derivativeo antibody fragmento a vitamin derivativeo a carbohydrateo a toxino or a chemotherapeutic compound. Various compounds can be synthesizedo for exampleo small molecules and oligomers (e.g.o oligopeptides and oligonucleotides)o and synthetic organic compounds based on various core structures. In additiono various natural sources can provide compounds for screeningo such as plant or animal extractso and the like. A skilled artisan can readily recognize the limits to the structural nature of the agents described herein.
“Signal transduction” is a process during which stimulatory or inhibitory signals are transmitted into and within a cell to elicit an intracellular response. A modulator of a signal transduction pathway refers to a compound which modulates the activity of one or more cellular proteins mapped to the same specific signal transduction pathway. A modulator may augment (agonist) or suppress (antagonist) the activity of a signaling molecule.
The term “cell proliferation” refers to a phenomenon by which the cell number has changed as a result of division. This term also encompasses cell growth by which the cell morphology has changed (e.g.o increased in size) consistent with a proliferative signal.
The term “selective inhibition” or “selectively inhibit” as applied to a biologically active agent refers to the agent's ability to selectively reduce the target signaling activity as compared to off-target signaling activityo via direct or interact interaction with the target.
“Subject” refers to an animalo such as a mammalo for example a human. The methods described herein can be useful in both human therapeutics and veterinary applications. In some embodimentso the patient is a mammalo and in some embodimentso the patient is human.
“Prodrug” is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound described herein. Thuso the term “prodrug” refers to a precursor of a biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subjecto but is converted in vivo to an active compoundo for exampleo by hydrolysis. The prodrug compound often offers advantages of solubilityo tissue compatibility or delayed release in a mammalian organism (seeo e.g.o Bundgardo H.o Design of Prodrugs (1985)o pp. 7-9o 21-24 (Elseviero Amsterdam). A discussion of prodrugs is provided in Higuchio T.o et al.o “Pro-drugs as Novel Delivery Systemso” A.C.S. Symposium Serieso Vol. 14o and in Bioreversible Carriers in Drug Designo ed. Edward B. Rocheo American Pharmaceutical Association and Pergamon Presso 1987o both of which are incorporated in full by reference herein. The term “prodrug” is also meant to include any covalently bonded carrierso which release the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of an active compoundo as described hereino may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleavedo either in routine manipulation or in vivoo to the parent active compound. Prodrugs include compounds wherein a hydroxyo amino or mercapto group is bonded to any group thato when the prodrug of the active compound is administered to a mammalian subjecto cleaves to form a free hydroxyo free amino or free mercapto groupo respectively. Examples of prodrugs includeo but are not limited too acetateo formate and benzoate derivatives of an alcohol or acetamideo formamide and benzamide derivatives of an amine functional group in the active compound and the like.
The term “in vivo” refers to an event that takes place in a subject's body.
The term “in vitro” refers to an event that takes places outside of a subject's body. For exampleo an in vitro assay encompasses any assay run outside of a subject assay. In vitro assays encompass cell-based assays in which cells alive or dead are employed. In vitro assays also encompass a cell-free assay in which no intact cells are employed.
Unless otherwise statedo structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For exampleo compounds as described herein wherein one or more hydrogens are replaced by deuterium or tritiumo or the replacement of one or more carbon atoms by the 13C- or 14C-enriched carbon isotope. Furthero substitution with heavier isotopeso particularly deuterium (2H or D) may afford certain therapeutic advantages resulting from greater metabolic stabilityo increased in vivo half-lifeo reduced dosage requirements or an improvement in therapeutic index. It is understood that deuterium in this context is regarded as a substituent of a compound of the formula (I).
The compounds described herein may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For exampleo the compounds may be radiolabeled with radioactive isotopeso such as for example tritium (3H)o iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds described hereino whether radioactive or noto are encompassed.
“Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a “racemic” mixture. The term “(..+-..)” is used to designate a racemic mixture where appropriate. “Diastereoisomers” are stereoisomers that have at least two asymmetric atomso but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn-Ingold-Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon can be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (−) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomerso diastereomerso and other stereoisomeric forms that can be definedo in terms of absolute stereochemistryo as (R)- or (S)-. The present chemical entitieso pharmaceutical compositions and methods are meant to include all such possible isomerso including racemic mixtureso optically pure forms and intermediate mixtures. Optically active (R)- and (S)-isomers can be prepared using chiral synthons or chiral reagentso or resolved using conventional techniques. The optical activity of a compound can be analyzed via any suitable methodo including but not limited to chiral chromatography and polarimetryo and the degree of predominance of one stereoisomer over the other isomer can be determined.
When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetryo and unless specified otherwiseo it is intended that the compounds include both E and Z geometric isomers.
A “substituted” or “optionally substituted” groupo means that a group (such as alkylo arylo heterocyclylo cycloalkylo hetrocyclylalkylo arylalkylo heteroarylo or heteroarylalkyl) unless specifically noted otherwiseo may have 1o 2 or 3-H groups substituted by 1o 2 or 3 substituents selected from haloo trifluoromethylo trifluoromethoxyo methoxyo —COOHo —CHOo —NH2o —NO2 —OHo —SHo —SMeo —NHCH3o —N(CH3)2o —CNo lower alkyl and the like.
“Tautomers” are structurally distinct isomers that interconvert by tautomerization. “Tautomerization” is a form of isomerization and includes prototropic or proton-shift tautomerization which is considered a subset of acid-base chemistry. “Prototropic tautomerization” or “proton-shift tautomerization” involves the migration of a proton accompanied by changes in bond ordero often the interchange of a single bond with an adjacent double bond. Where tautomerization is possible (e.g. in solution)o a chemical equilibrium of tautomers can be reached. An example of tautomerization is keto-enol tautomerization. A specific example of keto-enol tautomerization is the interconversion of pentane-2o 4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(1H)-one tautomers.
Compounds described herein also include crystalline and amorphous forms of those compoundso includingo for exampleo polymorphso pseudopolymorphso solvateso hydrateso unsolvated polymorphs (including anhydrates)o conformational polymorphso and amorphous forms of the compoundso as well as mixtures thereof. “Crystalline formo” “polymorpho” and “novel form” may be used interchangeably hereino and are meant to include all crystalline and amorphous forms of the compound listed aboveo as well as mixtures thereofo unless a particular crystalline or amorphous form is referred to.
“Solvento” “organic solvent” and “inert solvent” each means a solvent inert under the conditions of the reaction being described in conjunction therewith includingo for exampleo benzeneo tolueneo acetonitrileo tetrahydrofuran (“THF”)o dimethylformamide (“DMF”)o chloroformo methylene chloride (or dichloromethane)o diethyl ethero methanolo N-methylpyrrolidone (“NMP”)o pyridine and the like. Unless specified to the contraryo the solvents used in the reactions described herein are inert organic solvents. Unless specified to the contraryo for each gram of the limiting reagento one cc (or mL) of solvent constitutes a volume equivalent.
Described herein is a compound of formula I:
wherein
In some embodimentso X═—CH— and Y═N. In some embodimentso X═Y═N.
Also described herein is a compound of formula II:
wherein
In some embodimentso X═Y═—CH—o and Z is —CH2—. In some embodimentso X═Y═—CH— and Z═O.
The following compounds in Table 1 have been synthesized:
Isolation and purification of the chemical entities and intermediates described herein can be effectedo if desiredo by any suitable separation or purification procedure such aso for exampleo filtrationo extractiono crystallizationo column chromatographyo thin-layer chromatography or thick-layer chromatographyo or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the examples herein. Howevero other equivalent separation or isolation procedures can also be used.
When desiredo the (R)- and (S)-isomers of the compounds described hereino if presento may be resolved by methods known to those skilled in the arto for example by formation of diastereomeric salts or complexes which may be separatedo for exampleo by crystallization; via formation of diastereomeric derivatives which may be separatedo for exampleo by crystallizationo gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagento for example enzymatic oxidation or reductiono followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environmento for example on a chiral supporto such as silica with a bound chiral ligand or in the presence of a chiral solvent. Alternativelyo a specific enantiomer may be synthesized by asymmetric synthesis using optically active reagentso substrateso catalysts or solventso or by converting one enantiomer to the other by asymmetric transformation.
The compounds described herein can be optionally contacted with a pharmaceutically acceptable acid to form the corresponding acid addition salts. Pharmaceutically acceptable forms of the compounds recited herein include pharmaceutically acceptable saltso chelateso non-covalent complexes or derivativeso prodrugso and mixtures thereof. In certain embodimentso the compounds described herein are in the form of pharmaceutically acceptable salts. In additiono if the compound described herein is obtained as an acid addition sale the free base can be obtained by basifying a solution of the acid salt. Converselyo if the product is a free baseo an addition salto particularly a pharmaceutically acceptable addition salto may be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acido in accordance with conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methodologies that may be used to prepare non-toxic pharmaceutically acceptable addition salts.
When ranges are used herein for physical propertieso such as molecular weighto or chemical propertieso such as chemical formulaeo all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error)o and thus the number or numerical range may vary fromo for exampleo between 1% and 15% of the stated number or numerical range. The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) include those embodimentso for exampleo an embodiment of any composition of mattero compositiono methodo or processo or the likeo that “consist of” or “consist essentially of” the described features.
The subject pharmaceutical compositions are typically formulated to provide a therapeutically effective amount of a compound of Formula I or II as the active ingrediento or a pharmaceutically acceptable salto estero prodrugo solvateo hydrate or derivative thereof. Where desiredo the pharmaceutical compositions contain pharmaceutically acceptable salt and/or coordination complex thereofo and one or more pharmaceutically acceptable excipientso carrierso including inert solid diluents and fillerso diluentso including sterile aqueous solution and various organic solventso permeation enhancerso solubilizers and adjuvants.
The subject pharmaceutical compositions can be administered alone or in combination with one or more other agentso which are also typically administered in the form of pharmaceutical compositions. Where desiredo a compound of Formula I or II and other agent(s) may be mixed into a preparation or both components may be formulated into separate preparations to use them in combination separately or at the same time. A compound as described herein may also be used in combination with other active agentso e.g.o an additional compound that is or is not of Formula I or IIo for extension of lifespan in an organism.
In some embodimentso the concentration of one or more of the compounds of Formula I or II in the pharmaceutical compositions described herein is less than 100%o 90%o 80%o 70%o 60%o 50%o 40%o 30%o 20%o 19%o 18%o 17%o 16%o 15%o 14%o 13%o 12%o 11%o 10%o 9%o 8%o 7%o 6%o 5%o 4%o 3%o 2%o 1%o 0.5%o 0.4%o 0.3%o 0.2%o 0.1%o 0.09%o 0.08%o 0.07%o 0.06%o 0.05%o 0.04%o 0.03%o 0.02%o 0.01%o 0.009%o 0.008%o 0.007%o 0.006%o 0.005%o 0.004%o 0.003%o 0.002%o 0.001%o 0.0009%o 0.0008%o 0.0007%o 0.0006%o 0.0005%o 0.0004%o 0.0003%o 0.0002%o or 0.0001% w/wo w/v or v/v.
In some embodimentso the concentration of one or more of the compounds of Formula I or II is greater than 90%o 80%o 70%o 60%o 50%o 40%o 30%o 20%o 19.75%o 19.50%o 19.25% 19%o 18.75%o 18.50%o 18.25% 18%o 17.75%o 17.50%o 17.25% 17%o 16.75%o 16.50%o 16.25% 16%o 15.75%o 15.50%o 15.25% 15%o 14.75%o 14.50%o 14.25% 14%o 13.75%o 13.50%o 13.25% 13%o 12.75%o 12.50%o 12.25% 12%o 11.75%o 11.50%o 11.25% 11%o 10.75%o 10.50%o 10.25% 10%o 9.75%o 9.50%o 9.25% 9%o 8.75%o 8.50%o 8.25% 8%o 7.75%o 7.50%o 7.25% 7%o 6.75%o 6.50%o 6.25% 6%o 5.75%o 5.50%o 5.25% 5%o 4.75%o 4.50%o 4.25%o 4%o 3.75%o 3.50%o 3.25%o 3%o 2.75%o 2.50%o 2.25%o 2%o 1.75%o 1.50%o 125%o 1%o 0.5%o 0.4%o 0.3%o 0.2%o 0.1%o 0.09%o 0.08%o 0.07%o 0.06%o 0.05%o 0.04%o 0.03%o 0.02%o 0.01%o 0.009%o 0.008%o 0.007%o 0.006%o 0.005%o 0.004%o 0.003%o 0.002%o 0.001%o 0.0009%o 0.0008%o 0.0007%o 0.0006%o 0.0005%o 0.0004%o 0.0003%o 0.0002%o or 0.0001% w/wo w/vo or v/v.
In some embodimentso the concentration of one or more of the compounds of Formula I or II is in the range from approximately 0.0001% to approximately 50%o approximately 0.001% to approximately 40%o approximately 0.01% to approximately 30%o approximately 0.02% to approximately 29%o approximately 0.03% to approximately 28%o approximately 0.04% to approximately 27%o approximately 0.05% to approximately 26%o approximately 0.06% to approximately 25%o approximately 0.07% to approximately 24%o approximately 0.08% to approximately 23%o approximately 0.09% to approximately 22%o approximately 0.1% to approximately 21%o approximately 0.2% to approximately 20%o approximately 0.3% to approximately 19%o approximately 0.4% to approximately 18%o approximately 0.5% to approximately 17%o approximately 0.6% to approximately 16%o approximately 0.7% to approximately 15%o approximately 0.8% to approximately 14%o approximately 0.9% to approximately 12%o approximately 1% to approximately 10% w/wo w/v or v/v.
In some embodimentso the concentration of one or more of the compounds of Formula I or II is in the range from approximately 0.001% to approximately 10%o approximately 0.01% to approximately 5%o approximately 0.02% to approximately 4.5%o approximately 0.03% to approximately 4%o approximately 0.04% to approximately 3.5%o approximately 0.05% to approximately 3%o approximately 0.06% to approximately 2.5%o approximately 0.07% to approximately 2%o approximately 0.08% to approximately 1.5%o approximately 0.09% to approximately 1%o approximately 0.1% to approximately 0.9% w/wo w/v or v/v.
In some embodimentso the amount of one or more of the compounds of Formula I or II is equal to or less than 10 go 9.5 go 9.0 go 8.5 go 8.0 go 7.5 go 7.0 go 6.5 go 6.0 go 5.5 go 5.0 go 4.5 go 4.0 go 3.5 go 3.0 go 2.5 go 2.0 go 1.5 go 1.0 go 0.95 go 0.9 go 0.85 go 0.8 go 0.75 go 0.7 go 0.65 go 0.6 go 0.55 go 0.5 go 0.45 go 0.4 go 0.35 go 0.3 go 0.25 go 0.2 go 0.15 go 0.1 go 0.09 go 0.08 go 0.07 go 0.06 go 0.05 go 0.04 go 0.03 go 0.02 go 0.01 go 0.009 go 0.008 go 0.007 go 0.006 go 0.005 go 0.004 go 0.003 go 0.002 go 0.001 go 0.0009 go 0.0008 go 0.0007 go 0.0006 go 0.0005 go 0.0004 go 0.0003 go 0.0002 go or 0.0001 g.
In some embodimentso the amount of one or more of the compounds of Formula I or II is more than 0.0001 go 0.0002 go 0.0003 go 0.0004 go 0.0005 go 0.0006 go 0.0007 go 0.0008 go 0.0009 go 0.001 go 0.0015 go 0.002 go 0.0025 go 0.003 go 0.0035 go 0.004 go 0.0045 go 0.005 go 0.0055 go 0.006 go 0.0065 go 0.007 go 0.0075 go 0.008 go 0.0085 go 0.009 go 0.0095 go 0.01 go 0.015 go 0.02 go 0.025 go 0.03 go 0.035 go 0.04 go 0.045 go 0.05 go 0.055 go 0.06 go 0.065 go 0.07 go 0.075 go 0.08 go 0.085 go 0.09 go 0.095 go 0.1 go 0.15 go 0.2 go 0.25 go 0.3 go 0.35 go 0.4 go 0.45 go 0.5 go 0.55 go 0.6 go 0.65 go 0.7 go 0.75 go 0.8 go 0.85 go 0.9 go 0.95 go 1 go 1.5 go 2 go 2.5o 3 g3.5o 4 go 4.5 go 5 go 5.5 go 6 go 6.5 go 7 go 7.5 go 8 go 8.5 go 9 go 9.5 go or 10 g.
In some embodimentso the amount of one or more of the compounds of Formula I or II is in the range of 0.0001-10 go 0.0005-9 go 0.001-8 go 0.005-7 go 0.01-6 go 0.05-5 go 0.1-4 go 0.5-4 go or 1-3 g.
The compounds of Formula I or II described herein are effective over a wide dosage range. For exampleo in the treatment of adult humanso dosages from 0.01 to 1000 mgo from 0.5 to 100 mgo from 1 to 50 mg per dayo and from 5 to 40 mg per day are examples of dosages that may be used. An exemplary dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administrationo the form in which the compound of Formula I or II is administeredo the subject to be treatedo the body weight of the subject to be treatedo and the preference and experience of the attending physician.
A pharmaceutical composition described herein typically contains an active ingredient (e.g.o a compound of Formula I or II or a pharmaceutically acceptable salt and/or coordination complex thereof)o and one or more pharmaceutically acceptable excipientso carrierso including but not limited to inert solid diluents and fillerso diluentso sterile aqueous solution and various organic solventso permeation enhancerso solubilizers and adjuvants.
Described below are non-limiting exemplary pharmaceutical compositions and methods for preparing the same.
Described herein is a pharmaceutical composition for oral administration containing a compound of formula I:
wherein
Further described herein is a pharmaceutical composition for oral administration containing a compound of formula II:
wherein
Also described herein is a solid pharmaceutical composition for oral administration containing: (i) an effective amount of a compound of Formula I or II; optionally (ii) an effective amount of a second agent; and (iii) a pharmaceutical excipient suitable for oral administration. In some embodimentso the composition further contains: (iv) an effective amount of a third agent.
In some embodimentso the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption. Pharmaceutical compositions suitable for oral administration can be presented as discrete dosage formso such as capsuleso cachetso or tabletso or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granuleso a solutiono or a suspension in an aqueous or non-aqueous liquido an oil-in-water emulsiono or a water-in-oil liquid emulsion. Such dosage forms can be prepared by any of the methods of pharmacyo but all methods include the step of bringing the active ingredient into association with the carriero which constitutes one or more necessary ingredients. In generalo the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or botho and theno if necessaryo shaping the product into the desired presentation. For exampleo a tablet can be prepared by compression or moldingo optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granuleso optionally mixed with an excipient such aso but not limited too a bindero a lubricanto an inert diluento and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
Also described herein are anhydrous pharmaceutical compositions and dosage forms comprising an active ingrediento since water can facilitate the degradation of some compounds. For exampleo water may be added (e.g.o 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. Anhydrous pharmaceutical compositions and dosage forms can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturingo packagingo and/or storage is expected. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordinglyo anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging includeo but are not limited too hermetically sealed foilso plastic or the likeo unit dose containerso blister packso and strip packs.
An active ingredient can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage formo any of the usual pharmaceutical media can be employed as carrierso such aso for exampleo watero glycolso oilso alcoholso flavoring agentso preservativeso coloring agentso and the like in the case of oral liquid preparations (such as suspensionso solutionso and elixirs) or aerosols; or carriers such as starcheso sugarso micro-crystalline celluloseo diluentso granulating agentso lubricantso binderso and disintegrating agents can be used in the case of oral solid preparationso in some embodiments without employing the use of lactose. For exampleo suitable carriers include powderso capsuleso and tabletso with the solid oral preparations. If desiredo tablets can be coated by standard aqueous or nonaqueous techniques.
Binders suitable for use in pharmaceutical compositions and dosage forms includeo but are not limited too corn starcho potato starcho or other starcheso gelatino natural and synthetic gums such as acaciao sodium alginateo alginic acido other alginateso powdered tragacantho guar gumo cellulose and its derivatives (e.g.o ethyl celluloseo cellulose acetateo carboxymethyl cellulose calciumo sodium carboxymethyl cellulose)o polyvinyl pyrrolidoneo methyl celluloseo pre-gelatinized starcho hydroxypropyl methyl celluloseo microcrystalline celluloseo and mixtures thereof.
Examples of suitable fillers for use in the pharmaceutical compositions and dosage forms disclosed herein includeo but are not limited too talco calcium carbonate (e.g.o granules or powder)o microcrystalline celluloseo powdered celluloseo dextrateso kaolino mannitolo silicic acido sorbitolo starcho pre-gelatinized starcho and mixtures thereof.
Disintegrants may be used in the compositions described herein to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which may disintegrate in the bottle. Too little may be insufficient for disintegration to occur and may thus alter the rate and extent of release of the active ingredient(s) from the dosage form. Thuso a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used may vary based upon the type of formulation and mode of administrationo and may be readily discernible to those of ordinary skill in the art. About 0.5 to about 15 weight percent of disintegranto or about 1 to about 5 weight percent of disintegranto may be used in the pharmaceutical composition. Disintegrants that can be used to form pharmaceutical compositions and dosage forms includeo but are not limited too agar-agaro alginic acido calcium carbonateo microcrystalline celluloseo croscarmellose sodiumo crospovidoneo polacrilin potassiumo sodium starch glycolateo potato or tapioca starcho other starcheso pre-gelatinized starcho other starcheso clayso other alginso other celluloseso gums or mixtures thereof.
Lubricants which can be used to form pharmaceutical compositions and dosage forms includeo but are not limited too calcium stearateo magnesium stearateo mineral oilo light mineral oilo glycerino sorbitolo mannitolo polyethylene glycolo other glycolso stearic acido sodium lauryl sulfateo talco hydrogenated vegetable oil (e.g.o peanut oilo cottonseed oilo sunflower oilo sesame oilo olive oilo corn oilo and soybean oil)o zinc stearateo ethyl oleateo ethyl laureateo agaro or mixtures thereof. Additional lubricants includeo for exampleo a syloid silica gelo a coagulated aerosol of synthetic silicao or mixtures thereof. A lubricant can optionally be addedo in an amount of less than about 1 weight percent of the pharmaceutical composition.
When aqueous suspensions and/or elixirs are desired for oral administrationo the essential active ingredient therein may be combined with various sweetening or flavoring agentso coloring matter or dyes and if so desiredo emulsifying and/or suspending agentso together with such diluents as watero ethanolo propylene glycolo glycerin and various combinations thereof.
The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For exampleo a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluento for exampleo calcium carbonateo calcium phosphate or kaolino or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil mediumo for exampleo peanut oilo liquid paraffin or olive oil.
Surfactants which can be used to form pharmaceutical compositions and dosage forms includeo but are not limited too hydrophilic surfactantso lipophilic surfactantso and mixtures thereof. That iso a mixture of hydrophilic surfactants may be employedo a mixture of lipophilic surfactants may be employedo or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.
A suitable hydrophilic surfactant may generally have an HLB value of at least 10° while suitable lipophilic surfactants may generally have an HLB value of or less than about 10. An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of non-ionic amphiphilic compounds is the hydrophilic-lipophilic balance (“HLB” value). Surfactants with lower HLB values are more lipophilic or hydrophobico and have greater solubility in oilso while surfactants with higher HLB values are more hydrophilico and have greater solubility in aqueous solutions. Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10° as well as anionico cationico or zwitterionic compounds for which the HLB scale is not generally applicable. Similarlyo lipophilic (i.e.o hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10. Howevero HLB value of a surfactant is merely a rough guide generally used to enable formulation of industrialo pharmaceutical and cosmetic emulsions.
Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants includeo but are not limited too alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acidso oligopeptideso and polypeptides; glyceride derivatives of amino acidso oligopeptideso and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.
Within the aforementioned groupo ionic surfactants includeo by way of example: lecithinso lysolecithino phospholipidso lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.
Ionic surfactants may be the ionized forms of lecithino lysolecithino phosphatidylcholineo phosphatidylethanolamineo phosphatidylglycerolo phosphatidic acido phosphatidylserineo lysophosphatidylcholineo lysophosphatidylethanolamineo lysophosphatidylglycerolo lysophosphatidic acido lysophosphatidylserineo PEG-phosphatidylethanolamineo PVP-phosphatidylethanolamineo lactylic esters of fatty acidso stearoyl-2-lactylateo stearoyl lactylateo succinylated monoglycerideso mono/diacetylated tartaric acid esters of mono/diglycerideso citric acid esters of mono/diglycerideso cholylsarcosineo caproateo caprylateo caprateo laurateo myristateo palmitateo oleateo ricinoleateo linoleateo linolenateo stearateo lauryl sulfateo teradecyl sulfateo docusateo lauroyl carnitineso palmitoyl carnitineso myristoyl carnitineso and salts and mixtures thereof.
Hydrophilic non-ionic surfactants may includeo but not limited too alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene a lkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerideso vegetable oilso hydrogenated vegetable oilso fatty acidso and sterols; polyoxyethylene sterolso derivativeso and analogues thereof; polyoxyethylated vitamins and derivatives thereof; polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof; polyethylene glycol sorbitan fatty acid esters and hydrophilic transesterification products of a polyol with at least one member of the group consisting of triglycerideso vegetable oilso and hydrogenated vegetable oils. The polyol may be glycerolo ethylene glycolo polyethylene glycolo sorbitol propylene glycolo pentaerythritolo or a saccharide.
Other hydrophilic-non-ionic surfactants includeo without limitationo PEG-10 laurateo PEG-12 laurateo PEG-20 laurateo PEG-32 laurateo PEG-32 dilaurateo PEG-12 oleateo PEG-15 oleateo PEG-20 oleateo PEG-20 dioleateo PEG-32 oleateo PEG-200 oleateo PEG-400 oleateo PEG-15 stearateo PEG-32 distearateo PEG-40 stearateo PEG-100 stearateo PEG-20 dilaurateo PEG-25 glyceryl trioleateo PEG-32 dioleateo PEG-20 glyceryl laurateo PEG-30 glyceryl laurateo PEG-20 glyceryl stearateo PEG-20 glyceryl oleateo PEG-30 glyceryl oleateo PEG-30 glyceryl laurateo PEG-40 glyceryl laurateo PEG-40 palm kernel oilo PEG-50 hydrogenated castor oilo PEG-40 castor oilo PEG-35 castor oilo PEG-60 castor oilo PEG-40 hydrogenated castor oilo PEG-60 hydrogenated castor oilo PEG-60 corn oilo PEG-6 caprate/caprylate glycerideso PEG-8 caprate/caprylate glycerideso polyglyceryl-10 laurateo PEG-30 cholesterolo PEG-25 phyto sterolo PEG-30 soya sterolo PEG-20 trioleateo PEG-40 sorbitan oleateo PEG-80 sorbitan laurateo polysorbate 20o polysorbate 80o POE-9 lauryl ethero POE-23 lauryl ethero POE-10 oleyl ethero POE-20 oleyl ethero POE-20 stearyl ethero tocopheryl PEG-100 succinateo PEG-24 cholesterolo polyglyceryl-10 oleateo Tween 40o Tween 60o sucrose monostearateo sucrose monolaurateo sucrose monopalmitateo PEG 10-100 nonyl phenol serieso PEG 15-100 octyl phenol serieso and poloxamers.
Suitable lipophilic surfactants includeo by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group consisting of glycerideso vegetable oilso hydrogenated vegetable oilso fatty acids and sterols; oil-soluble vitamins/vitamin derivatives; and mixtures thereof. Within this groupo suitable lipophilic surfactants includeo but are not limited too glycerol fatty acid esterso propylene glycol fatty acid esterso and mixtures thereofo or are hydrophobic transesterification products of a polyol with at least one member of the group consisting of vegetable oilso hydrogenated vegetable oilso and triglycerides.
In one embodimento the composition may include a solubilizer to ensure good solubilization and/or dissolution of the compound described herein and to minimize precipitation of the compound described herein. This can be especially important for compositions for non-oral useo e.g.o compositions for injection. A solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other componentso such as surfactantso or to maintain the composition as a stable or homogeneous solution or dispersion.
Examples of suitable solubilizers includeo but are not limited too the following: alcohols and polyolso such as ethanolo isopropanolo butanolo benzyl alcoholo ethylene glycolo propylene glycolo butanediols and isomers thereofo glycerolo pentaerythritolo sorbitolo mannitolo transcutolo dimethyl isosorbideo polyethylene glycolo polypropylene glycolo polyvinylalcoholo hydroxypropyl methylcellulose and other cellulose derivativeso cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000o such as tetrahydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG; amides and other nitrogen-containing compounds such as 2-pyrrolidoneo 2-piperidoneo ε-caprolactamo N-alkylpyrrolidoneo N-hydroxyalkylpyrrolidoneo N-alkylpiperidoneo N-alkylcaprolactamo dimethylacetamide and polyvinylpyrrolidone; esters such as ethyl propionateo tributylcitrateo acetyl triethylcitrateo acetyl tributyl citrateo triethylcitrateo ethyl oleateo ethyl caprylateo ethyl butyrateo triacetino propylene glycol monoacetateo propylene glycol diacetateo ε-caprolactone and isomers thereof δ-valerolactone and isomers thereofo β-butyrolactone and isomers thereof; and other solubilizers known in the arto such as dimethyl acetamideo dimethyl isosorbideo N-methyl pyrrolidoneso monooctanoino diethylene glycol monoethyl ethero and water.
Mixtures of solubilizers may also be used. Examples includeo but not limited too triacetino triethylcitrateo ethyl oleateo ethyl caprylateo dimethylacetamideo N-methylpyrrolidoneo N-hydroxyethylpyrrolidoneo polyvinylpyrrolidoneo hydroxypropyl methylcelluloseo hydroxypropyl cyclodextrinso ethanolo polyethylene glycol 200-100o glycofurolo transcutolo propylene glycolo and dimethyl isosorbide. Suitable solubilizers includeo but are not limited too sorbitolo glycerolo triacetino ethyl alcoholo PEG-400o glycofurol and propylene glycol.
The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer may be limited to a bioacceptable amounto which may be readily determined by one of skill in the art. In some circumstanceso it may be advantageous to include amounts of solubilizers far in excess of bioacceptable amountso for example to maximize the concentration of the drugo with excess solubilizer removed prior to providing the composition to a patient using conventional techniqueso such as distillation or evaporation. Thuso if presento the solubilizer can be in a weight ratio of 10%o 25%o 50%o 100%o or up to about 200% by weighto based on the combined weight of the drugo and other excipients. If desiredo very small amounts of solubilizer may also be usedo such as 5%o 2%o 1% or even less. Typicallyo the solubilizer may be present in an amount of about 1% to about 100%o more typically about 5% to about 25% by weight.
The composition can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients includeo without limitationo detackifierso anti-foaming agentso buffering agentso polymerso antioxidantso preservativeso chelating agentso viscomodulatorso tonicifierso flavorantso colorantso odorantso opacifierso suspending agentso binderso fillerso plasticizerso lubricantso and mixtures thereof.
In additiono an acid or a base may be incorporated into the composition to facilitate processingo to enhance stabilityo or for other reasons. Examples of pharmaceutically acceptable bases include amino acidso amino acid esterso ammonium hydroxideo potassium hydroxideo sodium hydroxideo sodium hydrogen carbonateo aluminum hydroxideo calcium carbonateo magnesium hydroxideo magnesium aluminum silicateo synthetic aluminum silicateo synthetic hydrocalciteo magnesium aluminum hydroxideo diisopropylethylamineo ethanolamineo ethylenediamineo triethanolamineo triethylamineo triisopropanolamineo trimethylamineo tris(hydroxymethyl)aminomethane (TRIS) and the like. Also suitable are bases that are salts of a pharmaceutically acceptable acido such as acetic acido acrylic acido adipic acido alginic acido alkanesulfonic acido amino acidso ascorbic acido benzoic acido boric acido butyric acido carbonic acido citric acido fatty acidso formic acido fumaric acido gluconic acido hydroquinosulfonic acido isoascorbic acido lactic acido maleic acido oxalic acido para-bromophenylsulfonic acido propionic acido p-toluenesulfonic acido salicylic acido stearic acido succinic acido tannic acido tartaric acido thioglycolic acido toluenesulfonic acido uric acido and the like. Salts of polyprotic acidso such as sodium phosphateo disodium hydrogen phosphateo and sodium dihydrogen phosphate can also be used. When the base is a salto the cation can be any convenient and pharmaceutically acceptable cationo such as ammoniumo alkali metalso alkaline earth metalso and the like. Examples may includeo but are not limited too sodiumo potassiumo lithiumo magnesiumo calcium and ammonium.
Suitable acids are pharmaceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acido hydrobromic acido hydriodic acido sulfuric acido nitric acido boric acido phosphoric acido and the like. Examples of suitable organic acids include acetic acido acrylic acido adipic acido alginic acido alkanesulfonic acidso amino acidso ascorbic acido benzoic acido boric acido butyric acido carbonic acido citric acido fatty acidso formic acido fumaric acido gluconic acido hydroquinosulfonic acido isoascorbic acido lactic acido maleic acido methanesulfonic acido oxalic acido para-bromophenylsulfonic acido propionic acido p-toluenesulfonic acido salicylic acido stearic acido succinic acido tannic acido tartaric acido thioglycolic acido toluenesulfonic acido uric acid and the like.
Described herein are pharmaceutical compositions for injection containing a compound of formula I:
wherein
Also described herein are pharmaceutical compositions for injection containing a compound of formula II:
wherein
The forms in which the compositions described herein may be incorporated for administration by injection include aqueous or oil suspensionso or emulsionso with sesame oilo corn oilo cottonseed oilo or peanut oilo as well as elixirso mannitol dextroseo or a sterile aqueous solutiono and similar pharmaceutical vehicles.
Aqueous solutions in saline are also conventionally used for injection. Ethanolo glycerolo propylene glycolo liquid polyethylene glycolo and the like (and suitable mixtures thereof)o cyclodextrin derivativeso and vegetable oils may also be employed. The proper fluidity can be maintainedo for exampleo by the use of a coatingo such as lecithino for the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agentso for exampleo parabenso chlorobutanolo phenolo sorbic acido thimerosalo and the like.
Sterile injectable solutions are prepared by incorporating a compound of Formula I or II in the required amount in the appropriate solvent with various other ingredients as enumerated aboveo as requiredo followed by filtered sterilization. Generallyo dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutionso certain desirable methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Also described herein is a pharmaceutical composition for transdermal delivery containing a compound of formula I:
wherein
Also described herein is a pharmaceutical composition for transdermal delivery containing a compound of formula II:
wherein
Compositions described herein can be formulated into preparations in solido semi-solido or liquid forms suitable for local or topical administrationo such as gelso water soluble jellieso creamso lotionso suspensionso foamso powderso slurrieso ointmentso solutionso oilso pasteso suppositorieso sprayso emulsionso saline solutionso or dimethylsulfoxide (DMSO)-based solutions. In generalo carriers with higher densities are capable of providing an area with a prolonged exposure to the active ingredients. In contrasto a solution formulation may provide more immediate exposure of the active ingredient to the chosen area.
The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipientso which are compounds that allow increased penetration or or assist in the delivery ofo therapeutic molecules across the stratum corneum permeability barrier of the skin. There are many of these penetration-enhancing molecules known to those trained in the art of topical formulation. Examples of such carriers and excipients includeo but are not limited too humectants (e.g.o urea)o glycols (e.g.o propylene glycol)o alcohols (e.g.o ethanol)o fatty acids (e.g.o oleic acid)o surfactants (e.g.o isopropyl myristate and sodium lauryl sulfate)o pyrrolidoneso glycerol monolaurateo sulfoxideso terpenes (e.g.o menthol)o amineso amideso alkaneso alkanolso watero calcium carbonateo calcium phosphateo various sugarso starcheso cellulose derivativeso gelatino and polymers such as polyethylene glycols.
Another exemplary formulation for use in the methods described herein employs transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of a compound of Formula I or II in controlled amountso either with or without another agent.
The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. Seeo e.g.o U.S. Pat. Nos. 5o023o252o4o992o445 and 5o001o139. Such patches may be constructed for continuouso pulsatileo or on-demand delivery of pharmaceutical agents.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptableo aqueous or organic solventso or mixtures thereofo and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. The compositions may be administered by the oral or nasal respiratory routeo for exampleo for local or systemic effect. Compositions in pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tento or intermittent positive pressure breathing machine. Solutiono suspensiono or powder compositions may be administered in any mannero such as orally or nasallyo from devices that deliver the formulation in an appropriate manner.
Pharmaceutical compositions may also be prepared from compositions described herein and one or more pharmaceutically acceptable excipients suitable for sublingualo buccalo rectalo intraosseouso intraocularo intranasalo epiduralo or intraspinal administration. Preparations for such pharmaceutical compositions are well-known in the art. Seeo e.g.o Seeo e.g.o Andersono Philip O.; Knobeno James E.; Troutmano William Go eds.o Handbook of Clinical Drug Datao Tenth Editiono McGraw-Hillo 2002; Pratt and Tayloro eds.o Principles of Drug Actiono Third Editiono Churchill Livingstono N.Y.o 1990; Katzungo ed.o Basic and Clinical Pharmacologyo Ninth Editiono McGraw Hill 2004; Goodman and Gilmano eds.o The Pharmacological Basis of Therapeuticso Tenth Editiono McGraw Hillo 2001; Remington's Pharmaceutical Scienceso 20th Ed.o Lippincott Williams & Wilkins.o 2000; Martindaleo The Extra Pharmacopoeiao Thirty-Second Edition (The Pharmaceutical Presso Londono 1999); all of which are incorporated by reference herein in their entirety.
Administration of the compounds of Formula I or II or pharmaceutical compositions described herein can be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routeso intraduodenal routeso parenteral injection (including intravenouso intraarterialo subcutaneouso intramuscularo intravascularo intraperitoneal or infusion)o topical (e.g. transdermal application)o rectal administrationo via local delivery by catheter or stent or through inhalation. Compounds can also be administered intraadiposally or intrathecally.
The amount of a compound of Formula I or II administered will be dependent on the mammal being treatedo the severity of the disorder or conditiono the rate of administrationo the disposition of the compound and the discretion of the prescribing physician. Howevero an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per dayo such as from about 1 to about 35 mg/kg/dayo in single or divided doses. For a 70 kg humano this would amount to about 0.05 to 7 g/dayo such as about 0.05 to about 2.5 g/day. In some instanceso dosage levels below the lower limit of the aforesaid range may be more than adequateo while in other cases still larger doses may be employed without causing any harmful side effecto e.g. by dividing such larger doses into several small doses for administration throughout the day.
In some embodimentso a compound of Formula I or II is administered in a single dose. Typicallyo such administration will be by injectiono e.g.o intravenous injectiono in order to introduce the agent quickly. Howevero other routes may be used as appropriate.
In some embodimentso a compound of Formula I or II is administered in multiple doses. Dosing may be about onceo twiceo three timeso four timeso five timeso six timeso or more than six times per day. Dosing may be about once a montho once every two weekso once a weeko or once every other day. In another embodiment a compound and another agent are administered together about once per day to about 6 times per day. In some caseso continuous dosing is achieved and maintained as long as necessary.
Administration of the compound(s) of Formula I or II may continue as long as necessary. In some embodimentso a compound of Formula I or II is administered for more than 1o 2o 3o 4o 5o 6o 7o 14o or 28 days. In some embodimentso a compound of Formula I or II is administered for less than 28o 14o 7o 6o 5o 4o 3o 2o or 1 day. In some embodimentso a compound of Formula I or II is administered chronically on an ongoing basiso e.g.o for the treatment of chronic effects.
An effective amount of a compound of Formula I or II may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilitieso including rectalo buccalo intranasal and transdermal routeso by intra-arterial injectiono intravenouslyo intraperitoneallyo parenterallyo intramuscularlyo subcutaneouslyo orallyo topicallyo or as an inhalant.
The compositions described herein may also be delivered via an impregnated or coated device such as a stento for exampleo or an artery-inserted cylindrical polymer. A compound of Formula I or II may be administeredo for exampleo by local delivery from the struts of a stento from a stent grafto from graftso or from the cover or sheath of a stent. In some embodimentso a compound of Formula I or II is admixed with a matrix. Such a matrix may be a polymeric matrixo and may serve to bond the compound to the stent. Polymeric matrices suitable for such useo includeo for exampleo lactone-based polyesters or copolyesters such as polylactideo polycaprolactonglycolideo polyorthoesterso polyanhydrideso polyaminoacidso polysaccharideso polyphosphazeneso poly (ether-ester) copolymers (e.g. PEO-PLLA); polydimethylsiloxaneo poly(ethylene-vinylacetate)o acrylate-based polymers or copolymers (e.g. polyhydroxyethyl methylmethacrylateo polyvinyl pyrrolidinone)o fluorinated polymers such as polytetrafluoroethylene and cellulose esters. Suitable matrices may be non-degrading or may degrade with timeo releasing the compound or compounds. A compound of Formula I or II may be applied to the surface of the stent by various methods such as dip/spin coatingo spray coatingo dip-coatingo and/or brush-coating. The compounds may be applied in a solvent and the solvent may be allowed to evaporateo thus forming a layer of compound onto the stent. Alternativelyo a compound of Formula I or II may be located in the body of the stent or grafto for example in microchannels or micropores. When implantedo the compound diffuses out of the body of the stent to contact the arterial wall. Such stents may be prepared by dipping a stent manufactured to contain such micropores or microchannels into a solution of a compound of Formula I or II in a suitable solvento followed by evaporation of the solvent. Excess drug on the surface of the stent may be removed via an additional brief solvent wash. In yet other embodimentso a compound of Formula I or II may be covalently linked to a stent or graft. A covalent linker may be used which degrades in vivoo leading to the release of a compound of Formula I. Any bio-labile linkage may be used for such a purposeo such as estero amide or anhydride linkages. A compound of Formula I or II may additionally be administered intravascularly from a balloon used during angioplasty. Extravascular administration of a compound of Formula I or II via the pericardium or via adventitial application of formulations described herein may also be performed to decrease restenosis.
A variety of stent devices which may be used as described are disclosedo for exampleo in the following referenceso all of which are hereby incorporated by reference: U.S. Pat. Nos. 5o451o233; 5o040o548; 5o061o273; 5o496o346; 5o292o331; 5o674o278; 3o657o744; 4o739o762; 5o195o984; 5o292o331; 5o674o278; 5o879o382; 6o344o053.
The compounds of Formula I or II may be administered in dosages. It is known in the art that due to inter-subject variability in compound pharmacokineticso individualization of dosing regimen is necessary for optimal therapy. Dosing for a compound of Formula I or II may be found by routine experimentation in light of the instant disclosure.
When a compound of Formula I or ll is administered in a composition that comprises one or more agentso and the agent has a shorter half-life than the compound of Formula I or II unit dose forms of the agent and the compound of Formula I or II may be adjusted accordingly.
The subject pharmaceutical composition mayo for exampleo be in a form suitable for oral administration as a tableto capsuleo pill powdero sustained release formulationso solutiono or suspensiono for parenteral injection as a sterile solutiono suspension or emulsiono for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and a compound of Formula I or II as an active ingredient. In additiono it may include other medicinal or pharmaceutical agentso carrierso adjuvantso etc.
Exemplary parenteral administration forms include solutions or suspensions of active compound in sterile aqueous solutionso for exampleo aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably bufferedo if desired.
Kits are also described herein. The kits include one or more compounds of Formula I or II as described hereino in suitable packagingo and written material that can include instructions for useo discussion of clinical studieso listing of side effectso and the like. Such kits may also include informationo such as scientific literature referenceso package insert materialso clinical trial resultso and/or summaries of these and the likeo which indicate or establish the activities and/or advantages of the compositiono and/or which describe dosingo administrationo side effectso drug interactionso or other information useful to the health care provider. Such information may be based on the results of various studieso for exampleo studies using experimental animals involving in vivo models and studies based on human clinical trials. The kit may further contain another agent. In some embodimentso a compound of Formula I or II and the agent are provided as separate compositions in separate containers within the kit. In some embodimentso the compound described herein and the agent are provided as a single composition within a container in the kit. Suitable packaging and additional articles for use (e.g.o measuring cup for liquid preparationso foil wrapping to minimize exposure to airo and the like) are known in the art and may be included in the kit. Kits described herein can be providedo marketed and/or promoted to health providerso including physicianso nurseso pharmacistso formulary officialso and the like. Kits may alsoo in some embodimentso be marketed directly to the consumer.
The compounds and pharmaceutical compositions described hereino in therapeutically effective amounts and as described aboveo are useful in methods of extending the lifespan of an organism. The methods described herein comprise the step of administeringo in an amount effective to extend the lifespan of an organismo the compound of formula I:
wherein
R1 is selected from the group consisting of Ho substituted or unsubstituted C1-C10 linear or branched alkylo substituted or unsubstituted C2-C10 linear or branched alkenylo substituted or unsubstituted C2-C10 linear or branched alkynylo substituted or unsubstituted C6-C10 arylo substituted or unsubstituted C3-C10 cycloalkylo substituted or unsubstituted C3-C10 heterocycloalkylo substituted or unsubstituted C5-C10 heteroarylo substituted or unsubstituted C6-C10 arylalkylo substituted or unsubstituted C1-C10 linear or branched alkylamino and substituted or unsubstituted C1-C10 linear or branched dialkylaminoo or R1 and its attached N together form a substituted or unsubstituted C3-C6 heterocycloalkyl or heteroaryl ring (replacing the H attached to the N);
Alternativelyo the therapeutic methods described herein comprise the step of administeringo in an amount effective to extend the lifespan of an organismo the compound of formula II:
wherein
R1 is selected from the group consisting of Ho substituted or unsubstituted C1-C10 linear or branched alkylo substituted or unsubstituted C2-C10 linear or branched alkenylo substituted or unsubstituted C2-C10 linear or branched alkynylo substituted or unsubstituted C6-C10 arylo substituted or unsubstituted C3-C10 cycloalkylo substituted or unsubstituted C3-C10 heterocycloalkylo substituted or unsubstituted C5-C10 heteroarylo substituted or unsubstituted C6-C10 arylalkylo substituted or unsubstituted C5-C10 heteroarylalkylo substituted or unsubstituted C1-C10 linear or branched alkylamino and substituted or unsubstituted C1-C10 linear or branched dialkylaminoo or R1 and its attached N together form a substituted or unsubstituted C3-C6 heterocycloalkyl or heteroaryl ring (replacing the H attached to the N);
A is selected from the group consisting of a bondo substituted or unsubstituted C6-C10 arylo substituted or unsubstituted C5-C10 heteroarylo substituted or unsubstituted C2-C10 linear or branched alkenylo substituted or unsubstituted C2-C10 linear or branched alkynylo C═Oo C═So —CH2—o —CH(OH)—o —NH—o —N(CH3)—o —O—o —S—o and SO2; and
R4 is selected from the group consisting of substituted or unsubstituted C1-C10 linear or branched alkylo substituted or unsubstituted C1-C10 linear or branched alkoxyo substituted or unsubstituted C1-C10 linear or branched alkylaminoo substituted or unsubstituted C1-C10 linear or branched dialkylaminoo substituted or unsubstituted C3-C10 cycloalkyl or heterocycloalkylo substituted or unsubstituted C6-C10 arylo substituted or unsubstituted C5-C10 heteroarylo —CN and halo;
R5
X and Y are independently selected from the group consisting of —CH— and —N—; and
Z is selected from the group consisting of C═Oo —CR9R10—o —NR9—o —O—o —S—o —S(O)— and —SO2—.
In the methods for extending lifespan described hereino administration of ferroptosis inhibitorso such as the compounds of Formula I or II or pharmaceutical compositions described herein can be effected by any method that enables delivery of the compounds to the organism. These methods include oral routeso intraduodenal routeso parenteral injection (including intravenouso intraarterialo subcutaneouso intramuscularo intravascularo intraperitoneal or infusion)o topical (e.g. transdermal application)o rectal administrationo via local delivery by catheter or stent or through inhalation. Compounds can also be administered intraadiposally or intrathecally.
The amount of the ferroptosis inhibitor to be administered will be dependent on the organism being treatedo the rate of administrationo the disposition of the compound and the discretion of the prescribing physician. Howevero an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per dayo such as from about 1 to about 35 mg/kg/dayo in single or divided doses. For a 70 kg humano this would amount to about 0.05 to 7 g/dayo such as about 0.05 to about 2.5 g/day. In some instanceso dosage levels below the lower limit of the aforesaid range may be more than adequateo while in other cases still larger doses may be employed without causing any harmful side effecto e.g. by dividing such larger doses into several small doses for administration throughout the day.
Typicallyo for extending lifespano a ferroptosis inhibitor such as a compound of Formula I or II is administered in multiple doses. Dosing may be about onceo twiceo three timeso four timeso five timeso six timeso or more than six times per day. Dosing may be about once a montho once every two weekso once a weeko or once every other day. In another embodiment a compound and another agent are administered together about once per day to about 6 times per day. In some caseso continuous dosing is achieved and maintained as long as necessary.
In the methods of extending lifespan described hereino an effective amount of a ferroptosis inhibitor may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilitieso including rectalo buccalo intranasal and transdermal routeso by intra-arterial injectiono intravenouslyo intraperitoneallyo parenterallyo intramuscularlyo subcutaneouslyo orallyo topicallyo or as an inhalant.
The compositions for extending lifespan described herein may also be delivered via an impregnated or coated device such as a stento for exampleo or an artery-inserted cylindrical polymer. A compound of Formula I or II may be administeredo for exampleo by local delivery from the struts of a stento from a stent grafto from graftso or from the cover or sheath of a stent. In some embodimentso a compound of Formula I or II is admixed with a matrix. Such a matrix may be a polymeric matrixo and may serve to bond the compound to the stent. Polymeric matrices suitable for such useo includeo for exampleo lactone-based polyesters or copolyesters such as polylactideo polycaprolactonglycolideo polyorthoesterso polyanhydrideso polyaminoacidso polysaccharideso polyphosphazeneso poly (ether-ester) copolymers (e.g. PEO-PLLA); polydimethylsiloxaneo poly(ethylene-vinylacetate)o acrylate-based polymers or copolymers (e.g. polyhydroxyethyl methylmethacrylateo polyvinyl pyrrolidinone)o fluorinated polymers such as polytetrafluoroethylene and cellulose esters. Suitable matrices may be non-degrading or may degrade with timeo releasing the compound or compounds. A compound of Formula I or II may be applied to the surface of the stent by various methods such as dip/spin coatingo spray coatingo dip-coatingo and/or brush-coating. The compounds may be applied in a solvent and the solvent may be allowed to evaporateo thus forming a layer of compound onto the stent. Alternativelyo a compound of Formula I or II may be located in the body of the stent or grafto for example in microchannels or micropores. When implantedo the compound diffuses out of the body of the stent to contact the arterial wall. Such stents may be prepared by dipping a stent manufactured to contain such micropores or microchannels into a solution of a compound of Formula I or II in a suitable solvento followed by evaporation of the solvent. Excess drug on the surface of the stent may be removed via an additional brief solvent wash. In yet other embodimentso a compound of Formula I or II may be covalently linked to a stent or graft. A covalent linker may be used which degrades in vivoo leading to the release of a compound of Formula I. Any bio-labile linkage may be used for such a purposeo such as estero amide or anhydride linkages. A compound of Formula I or II may additionally be administered intravascularly from a balloon used during angioplasty. Extravascular administration of a compound of Formula I or II via the pericardium or via adventitial application of formulations described herein may also be performed to decrease restenosis.
A variety of stent devices which may be used as described are disclosedo for exampleo in the following referenceso all of which are hereby incorporated by reference: U.S. Pat. No. 5o451o233; 5o 040o548; 5o061o 273; 5o496o346; 5o292o331; 5o674o278; 3o657o744; 4o739o762; 5o195o984; 5o292o331; 5o674o278; 5o879o382; 6o344o053.
For use in the methods of extending lifespan described hereino ferroptosis inhibitors may be administered in dosages. It is known in the art that due to inter-subject variability in compound pharmacokineticso individualization of dosing regimen is necessary for optimal therapy. Dosing for a ferroptosis inhibitors may be found by routine experimentation in light of the instant disclosure.
All reagents were purchased from commercial suppliers and used as supplied unless stated otherwise. Reactions were carried out in air unless stated otherwise. 400 MHz 1H NMR spectra were obtained on a JEOL AS 400 spectrometer. Low-resolution mass spectra (LRMS) were obtained on a JEOL JMS-T100LC DART/AccuTOF mass spectrometer. Measurement of reversal of protein aggregation may be carried out using such assays as Bis-ANS Fluorescence as described ino for exampleo W. T. Chen et al:o J. Biol. Chemo 2011o 286 (11)o 9646.
A 250 mL RBF was charged with 2o4-dichloropyrido[3o2-d]pyrimidine (2 go 10 mmol)o a stir baro THF (20 mLo 0.5 M)o DiPEA (1.25 equiv.o 2.2 mLo 12.5 mmol)o cyclopentylamine (1 equiv.o 851 mgo 10 mmol) and was stirred at RT. The reaction mixture immediately became a milky bright yellow color and stirring was continued. After 2 ho the reaction was partitioned between 50 mL of EtOAc and 50 mL of H2Oo the water layer back extracted 1×25 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide 2-chloro-N-cyclopentyl-pyrido[3,2-d]pyrimidin-4-amine (K-04) as a viscous yellow oil (2.4 go 96.5%) and the material was used in the next step without further purification. 1H NMR (CDCl3): 8.65 (to 1H)o 7.99 (ddo 1H)o 7.65 (mo 1H) 7.32 (bso 1H)o 4.63 (mo 1H)o 2.20 (mo 2H)o 2.72 (mo 6H); 13C NMR (CDCl3):
160.2o 158.4o 148.0o 145.4o 134.9o 130.6o 128.1o 52.4o 32.9o 23.7: (APCI) m/e 249.1 (M+H). Note: the reaction can also be run overnight at RT with the same result.
A 40 mL vial was charged with 2o4-dichloropyrido[3o2-d]pyrimidine (400 mgo 2 mmol)o a stir baro THF (4 mLo 0.5 M)o DiPEA (1.25 equiv.o 323 mgo 2.5 mmol)o pyrrolidine (1 equiv.o 142 mgo 2 mmol) and was stirred at RT. The reaction mixture immediately became a warm milky yellow color that quickly changed to a thick slurry and stirring was continued. After 24 ho the reaction was partitioned between 25 mL of EtOAc and 25 mL of H2Oo the water layer back extracted 1×25 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide 2-chloro-4-pyrrolidin-1-yl-pyrido[3,2-d]pyrimidine (K-05) as a yellow solid (422 mgo 89.9%) and the material was used in the next step without further purification. 1H NMR (CDCl3): 8.68 (to 1H)o 7.96 (to 1H)o 7.57 (mo 1H)o 4.46 (to 2H)o 3.87 (to 2H)o 2.11 (mo 2H)o 2.08 (mo 2H); 13C NMR (CDCl3):
158.8o 157.4o 148.1o 146.7o 134.3o 133.1o127.0o 51.7o 50.4o 27.0o 23.6: (APCI) m/e 235.0 (M+H).
A 40 mL vial was charged with 2o4-dichloropyrido[3o2-d]pyrimidine (400 m go 2 mmol)o a stir baro THF (4 mLo 0.5 M)o DiPEA (1.25 equiv.o 323 mgo 2.5 mmol)o tert-butyl amine (1.25 equiv.o 323 mgo 2.5 mmol) and was stirred at RT. After 24 ho the reaction was partitioned between 25 mL of EtOAc and 25 mL of H2Oo the water layer was back extracted 1×25 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide a yellow oil. The oil was triturated with diethyl ether to provide N-tert-butyl-2-chloro-pyrido[3,2-d]pyrimidin-4-amine (K-06) as a yellow solid (246 mgo 52%) and the material was used in the next step without further purification. 1H NMR (CDCl3): 8.60 (ddo 1H)o 7.95 (ddo 1H)o 7.58 (mo 1H) 7.33 (bso 1H)o 1.57 (so 9H); 13C NMR (CDCl3):
160.0o 157.9o 147.8o 145.2o 135.1o 131.0o 128.0o 52.8o 28.4; (APCI) m/e 237.0 (M+H).
A 250 mL RBF was charged with 2-aminopyridine (1 equiv.o 5.0 mmolo 471 mg)o tetrahydrofuran (10 mLo 0.5 M)o DiPEA (1.5 equiv.o 7.5 mmolo 1.31 mL) and then 2o4-dichloropyrido[3o2-d]pyrimidine (1 go 0.5 mmol). The reaction was stirred at room temperature for 16 h and then partitioned between 50 mL water and 50 mL EtOAc. The water layer was back extracted 2×25 mL EtOAc and the combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified on SiO2 (40 go 5-100% hexanes/EtOAC) to provide 2-chloro-N-(2-pyridyl)pyrido[3,2-d]pyrimidin-4-amine (K-08) as a pale yellow solid (285 mgo 22%). (APCI) m/e 258.0 (M+H).
A 40 mL vial was charged with 2o4-dichloropyrido[3o2-d]pyrimidine (400 m go 2 mmol)o a stir baro THF (4 mL 0.5 M)o DiPEA (1.5 equiv.o 0.52 mLo 2.5 mmol)o prop-2-yn-1-amine(1 equiv.o 110 mgo 2 mmol) and was stirred at RT. The reaction mixture immediately became a warm milky yellow color that quickly changed to a thick slurry and stirring was continued. After 2 ho the reaction was partitioned between 5 mL of EtOAc and 5 mL of H2Oo the water layer back extracted 1×5 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide 350 mg (80%) desired product that was used directly in the next step without further purification. (APCI) m/e 219.0 (M+H).
A 100 mL RBF was charged with 2o4-dichloropyrido[3o2-d]pyrimidine (1 go 5 mmol)o a stir baro THF (10 mLo 0.5 M)o DiPEA (2 equiv.o 1.75 mLo 10 mmol)o 3-methyltetrahydrofuran-3-amine (1 equiv.o 506 mgo 5 mmol) and was stirred at RT. After 16 ho the reaction was partitioned between 50 mL of EtOAc and 50 mL of H2Oo the water layer back extracted 1×25 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified on silica gel (80 go 0-60% EtOAc/hexanes) to provide 1.13 g of 2-chloro-N-(3-methyltetrahydrofuran-3-yl)pyrido[3o2-d]pyrimidin-4-amine as a yellow solid (85%). (APCI) m/e 265.0 (M+H).
A solution of 2-chloro-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (1.05 go 4.2 mmole) in anhydrous DMF (15.0 mL) was degassed 5× and then successively treated with zinc cyanide (0.993 go 8.4 mmolo 2 equiv) and then tetrakis(triphenylphosphine)palladium(0) (0.7351 go 0.63 mmolo 0.15 equiv). The reaction mixture was warmed in a microwave to 150° C. for 30 min. LC/MS analysis of the crude reaction mixture showed conversion to the desired product and full consumption of the starting material. The mixture was filtered and adsorbed onto 10 g silica. The product was purified by flash chromatography (40 g silicao 0-50% ethyl acetate/hexanes) to afford 4-(cyclopentylamino)pyrido[3,2-d]pyrimidine-2-carbonitrile (C-73) as a yellow solid (0.784 go 77.6%). 1H NMR (400 Mzo (CD3)2CO) δ 8.74 (1Ho dd)o 8.26 (1Ho dd)o 7.69 (1Ho dd)o 7.23 (1Ho bd)o 4.68 (1Ho sextet)o 3.24 (2Ho t)o 2.22 (2Ho m)o 1.75o (8Ho m)o 1.46 (2Ho sextet)o 0.97 (3Ho t); 13C NMR (400 Mzo (CD3)2CO) δ 160.5o 151.4o 144.8o 142.6o 136.7o 132.7o 129.8o 117.6o 53.5o 32.9o 24.5. MS (APCI) for C13H13N5; Calculated: 240.1 [M+H+]o Found: 240.1.
A solution of N-tert-butyl-2-chloro-pyrido[3o2-d]pyrimidin-4-amine (0.21 go 0.88 mmole) in anhydrous DMF (3 mL) was degassed 5× and then successively treated with zinc cyanide (0.21 go 1.8 mmolo 2 equiv) and then tetrakis(triphenylphosphine)palladium(0) (0.153 go 0.13 mmolo 0.15 equiv). The reaction mixture was warmed in a microwave to 150° C. for 30 min. LC/MS analysis of the crude reaction mixture showed conversion to the desired product and full consumption of the starting material. The mixture was filtered and adsorbed onto 1 g silica. The product was purified by flash chromatography (12 g silicao 0-50% ethyl acetate/hexanes) to afford 4-(tert-butylamino)pyrido[3,2-d]pyrimidine-2-carbonitrile (C-87) as a pale yellow solid (0.167 go 83.2%). MS (APCI) for C12H13N5; Calculated: 228.1 [M+H+]o Found: 228.1.
A solution of 4-(cyclopentylamino)pyrido[3o2-d]pyrimidine-2-carbonitrile (0.574 go 2.4 mmol) in anhydrous THF (10 mL) was cooled to −78° C. and then treated with sodium hydride (0.138 go 3.6 mmolo 1.5 equiv) and the mixture was left stirring for 30 min. The mixture was then successively treated with copper (I) bromide (52 mgo 0.36 mmolo 0.15 equiv) and then butylmagnesium bromide (2M in diethyl ethero 1.6 mLo 5.3 mmolo 2.2 equiv). After stirring for 20 mino the reaction mixture was slowly warmed to 30° C. LC/MS analysis after four hours showed partial conversion to the desired product. The mixture was then warmed to 0° C. After an additional 4 hrs.o LC/MS showed clean conversion to the desired product. The reaction mixture was quenched with satd. aq. ammonium chloride (10 mL) and poured onto ethyl acetate (50 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×50 mL). The combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residual oil was purified by flash chromatography (24 g silicao 0-50% ethyl acetate/hexanes) to afford 1-[4-(cyclopentylamino)pyrido[3,2-d]pyrimidin-2-yl]pentan-1-one (C-76) as a yellow oil (0.630 go 88.0%). 1H NMR (400 Mzo (CD3)2CO) δ 8.74 (1Ho dd)o 8.02 (1Ho dd)o 7.78 (1Ho dd)o 4.52 (1Ho pent)o 2.03o (2Ho m)o 1.71 (4Ho m)o 1.58 (2Ho m); 13C NMR (400 Mzo (CD3)2CO) δ 160.5o 151.4o 144.8o 142.6o 136.7o 132.7o 129.8o 117.6o 53.5o 32.9o 24.5. MS (APCI) for C17H22N4O; Calculated: 299.2 [M+H+]o Found: 299.1.
A solution of (0.405 go 1.7 mmole) in anhydrous THF (8 mL) was treated with copper (I) bromide (37 mgo 0.25 mmolo 0.15 equiv) and then cooled to −78° C. After 10 min.o the reaction mixture was treated dropwise with methylmagnesium bromide (3M in diethyl ethero 1.3 mLo 3.7 molo 2.2 equiv) after the addition was complete the reaction was stirred for an additional 10 min and then warmed to 0° C. After 2 hr.o LC/MS analysis showed complete conversion of the starting material to the desired product. The reaction was quenched with satd. aq. ammonium chloride (3 mL) and then warmed to room temperature. The biphasic mixture was diluted with ethyl acetate (30 mL) and the layers were separated. The aqueous layer was further extracted with ethyl acetate (2×30 mL). The combined organic layers were dried (Na2SO4) and the solvent was removed in vacuo. The residual solid was purified by flash chromatography (adsorbed onto 2 g silica pre-columno 24 g silicao 0-50% ethyl acetate/hexanes) to afford 1-[4-(cyclopentylamino)pyrido[3,2-d]pyrimidin-2-yl]ethanone (C-89) as an off-white solid (0.138 go 31.3%). MS (APCI) for C14H16N4O; Calculated: 257.1 [M+H+]o Found: 257.0.
A solution of 4-(cyclopentylamino)pyrido[3o2-d]pyrimidine-2-carbonitrile (0.203 go 0.85 mmol) in THF (3 mL) was treated with copper (I) bromide (18 mgo 0.13 mmolo 0.15 equiv) and then cooled to −78° C. After 10 min.o the reaction mixture was treated dropwise with ethylmagnesium bromide (1M in THFo 1.3 mLo 3.7 molo 2.2 equiv) after the addition was complete the reaction was stirred for an additional 10 min and then warmed to 0° C. After 2 hr.o LC/MS analysis showed complete conversion of the starting material to the desired product. The reaction was quenched with satd. aq. ammonium chloride (3 mL) and then warmed to room temperature. The biphasic mixture was diluted with ethyl acetate (20 mL) and the layers were separated. The aqueous layer was further extracted with ethyl acetate (2×20 mL). The combined organic layers were dried (Na2SO4) and the solvent was removed in vacuo. The residual solid was purified by flash chromatography (adsorbed onto 1 g silica pre-columno 24 g silicao 0-50% ethyl acetate/hexanes) to afford 1-[4-(cyclopentylamino)pyrido[3,2-d]pyrimidin-2-yl]propan-1-one (C-90) as an off-white solid (0.145 go 63.2%). MS (APCI) for C15H18N4O; Calculated: 271.1 [M+H+]o Found: 271.0.
A solution of 4-(cyclopentylamino)pyrido[3o2-d]pyrimidine-2-carbonitrile (0.21 go 0.88 mmol) in anhydrous THF (3 mL) was treated with copper (I) bromide (19 mgo 0.13 mmolo 0.15 equiv) and then cooled to −78° C. After 10 mino the mixture was then treated with phenylmagnesium chloride (2M in THFo 1.1 mLo 2.2 mmolo 2.5 equiv). After stirring for 10 mino the reaction mixture was slowly warmed to 0° C. LC/MS analysis after one hour showed clean conversion to the desired product. The reaction mixture was quenched with satd. aq. ammonium chloride (3 mL) and poured onto ethyl acetate (10 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residual solid was purified by flash chromatography (24 g silicao 0-50% ethyl acetate/hexanes) to afford [4-(cyclopentylamino)pyrido[3,2-d]pyrimidin-2-yl]-phenyl-methanone (A-02) as a pale yellow solid (0.21 go 75.2%). MS (APCI) for C19H18N4O; Calculated: 319.2 [M+H+]o Found: 319.1.
A solution of 4-(cyclopentylamino)pyrido[3o2-d]pyrimidine-2-carbonitrile (0.21 go 0.88 mmol) in anhydrous THF (3 mL) was treated with copper (I) bromide (19 mgo 0.13 mmolo 0.15 equiv) and then cooled to −78° C. After 10 mino the mixture was then treated with 4-fluorophenylmagnesium bromide (2M in diethyl ethero 1.1 mLo 2.2 mmolo 2.5 equiv). After stirring for 10 mino the reaction mixture was slowly warmed to 0° C. LC/MS analysis after one hour showed clean conversion to the desired product. The reaction mixture was quenched with satd. aq. ammonium chloride (3 mL) and poured onto ethyl acetate (10 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residual solid was purified by flash chromatography (24 g silicao 0-50% ethyl acetate/hexanes) to afford [4-(cyclopentylamino)pyrido[3,2-d]pyrimidin-2-yl]-(4-fluorophenyl)methanone (A-03) as a pale yellow solid (0.287 go 97.2%). MS (APCI) for C19H17FN4O; Calculated: 337.1 [M+H+]o Found: 337.0.
A solution of 4-(cyclopentylamino)pyrido[3o2-d]pyrimidine-2-carbonitrile (0.20 go 0.84 mmol) in anhydrous THF (3 mL) was treated with copper (I) bromide (18 mgo 0.13 mmolo 0.15 equiv) and then cooled to −78° C. After 10 mino the mixture was then treated with tert-butylmagnesium chloride (2M in diethyl ethero 1.1 mLo 2.2 mmolo 2.6 equiv). After stirring for 10 mino the reaction mixture was slowly warmed to 0° C. LC/MS analysis after one hour showed conversion to the desired product with some residual starting material. After 2 hrso no further progress was noted and an additional 0.7 mL of tert-butylmagnesium chloride solution was added. Two hours after the second additiono the LC/MS analysis showed full consumption of the starting material and formation of the bis tert-butyl addition product. The reaction mixture was quenched with satd. aq. ammonium chloride (3 mL) and poured onto ethyl acetate (10 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residual solid was purified by flash chromatography (24 g silicao 0-50% ethyl acetate/hexanes) to afford 1-[4-(cyclopentylamino)pyrido[3,2-d]pyrimidin-2-yl]-2,2-dimethyl-propan-1-one (A-04) as a pale yellow film (0.080 go 32.3%). MS (APCI) for C17H22N4O; Calculated: 299.2 [M+H+]o Found: 299.1.
A solution of 4-(tert-butylamino)pyrido[3o2-d]pyrimidine-2-carbonitrile (0.167 go 0.74 mmol) in anhydrous THF (3 mL) was treated with copper (I) bromide (16 mgo 0.11 mmolo 0.15 equiv) and then cooled to −78° C. After 10 mino the mixture was then treated with butylmagnesium chloride (2M in diethyl ethero 1.0 mLo 1.8 mmolo 2.5 equiv). After stirring for 10 mino the reaction mixture was slowly warmed to 0° C. LC/MS analysis after one hour showed clean conversion to the desired product. The reaction mixture was quenched with satd. aq. ammonium chloride (3 mL) and poured onto ethyl acetate (10 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residual solid was purified by flash chromatography (24 g silicao 0-50% ethyl acetate/hexanes) to afford 1-[4-(tert-butylamino)pyrido[3,2-d]pyrimidin-2-yl]pentan-1-one (C-99) as a pale yellow solid (0.167 go 79.4%). 1H NMR (400 Mzo CDCl3) δ 8.70 (1Ho dd)o 8.25 (1Ho dd)o 7.65 (1Ho dd)o 7.30 (1Ho bs)o 3.20 (2Ho t)o 1.75 (2Ho pent)o 1.63 (9Ho s)o 1.43 (2Ho sextet)o 0.93 (3Ho t); 13C NMR (400 Mzo CDCl3) δ 201.7o 159.5o 156.7o 149.0o 144.1o 137.5o 131.9o 127.8o 52.4o 39.3o 28.5o 26.3o 22.5o 13.9. MS (APCI) for C16H22N4O; Calculated: 287.2 [M+H+]o Found: 287.1.
A solution of 4-pyrrolidin-1-ylpyrido[3o2-d]pyrimidine-2-carbonitrile (0.190 go 0.84 mmol) in anhydrous THF (3 mL) was treated with copper (I) bromide (19 mgo 0.13 mmolo 0.15 equiv) and then cooled to −78° C. After 10 mino the mixture was then treated with butylmagnesium chloride (2M in diethyl ethero 1.1 mLo 2.1 mmolo 2.5 equiv). After stirring for 10 mino the reaction mixture was slowly warmed to 0° C. LC/MS analysis after one hour showed clean conversion to the desired product. The reaction mixture was quenched with satd. aq. ammonium chloride (3 mL) and poured onto ethyl acetate (10 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×10 mL). The combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residual solid was purified by flash chromatography (24 g silicao 0-50% ethyl acetate/hexanes) to afford 1-[4-(tert-butylamino)pyrido[3,2-d]pyrimidin-2-yl]pentan-1-one (A-01) as a pale yellow solid (0.073 go 30.4%). MS (APCI) for C16H20N4O; Calculated: 285.2 [M+H+]o Found: 285.0.
A solution of a mixture of 1-[4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]pentan-1-ol and 1-[4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]pentan-1-one (0.203 go 0.67 mmol) in methylene chloride (3 mL) was treated with Dess-Martin Periodinane (0.34 go 0.80 mmolo 1.2 equiv). After stirring for 2 hrs.o LC/MS analysis showed clean conversion to the desired product. The reaction mixture was dried and the residue was purified by flash chromatography (12 g silicao 0-20% acetonitrile/ethyl acetate) to afford 1-[4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]pentan-1-oneo 170 mgo as a yellow solid. 1H NMR (400 Mzo (CD3)2CO) δ 4.59 (1Ho m)o 3.42 (2Ho m)o 3.12 (2Ho t)o 2.98 (2Ho t)o 2.04 (2Ho m)o 1.95 (2Ho m)o 1.78 (4Ho m)o 1.64 (4Ho m)o 1.37 (2Ho m)o 0.90 (3Ho t); 13C NMR (400 Mzo (CD3)2CO) δ 195.7o 151.4o 141.2o 128.5o 116.6o 54.7o 54.6o 40.9o 37.4o 32.9o 26.8o 25.1o 24.7o 23.0o 19.8o 14.1
A solution of 2-chloro-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (0.60 go 2.4 mmol) in ethanol (12 mL) was treated with TFA (0.18 mLo 2.4 mmolo 1 equiv) and then degassed with nitrogen with 5 cycles. The reaction mixture was then treated with platinum(IV)oxide (0.164 go 0.72 mmolo 0.3 equiv) and the solution was bubbled with hydrogen gas via balloon for 10 min. The needle was removed from the solution and the reaction mixture was stirred overnight under an balloon pressure of hydrogen gas. LC/MS analysis showed complete consumption of the starting material to two productso desired as major and tetrahydropyridine ring with replacement of the chloride for hydrogen as a minor product. The reaction mixture was filtered through Celite and the solvent was removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) and then 0-10% methanol/ethyl acetate) to afford 2-chloro-N-cyclopentyl-5o6o7o8-tetahydropyrido[3o2-d]pyrimidin-4-amine (0.346 g) as an off-white solid. LCMS: (APCI) m/e 253.1 (M+H).
Isolated by-product from C-80 (80 mg)
1H NMR (400 Mzo (CD3)2CO) δ 11.52 (1Ho bs)o 8.47 (1Ho dd)o 7.40 (1Ho m)o 7.19 (1Ho m)o 7.09 (1Ho m)o 6.96 (1Ho t)o 6.73 (1Ho d)o 6.31 (1Ho d)o 5.03 (1Ho bs)o 4.03 (3Ho s)o 2.07 (2Ho m)o 1.78 (2Ho m)o 1.64 (4Ho m); 13C NMR (400 Mzo (CD3)2CO) δ 152.9o 148.3o 144.3o 125.0o 53.1o 53.0o 47.2o 33.6o 24.3o 22.5.
A solution of 1-[4-(cyclopentylamino)pyrido[3o2-d]pyrimidin-2-yl]pentan-1-one (0.20 go 0.67 mmol) in ethanol (3 mL) was successively treated with nickel (II) chloride (17 mgo 0.13 mmolo 0.2 equiv) and then slowly with sodium borohydride (76 mgo 2.0 mmolo 3 equiv). The reaction mixture slowly released a gas and changed colors to brownish-black. After stirring overnighto LC/MS analysis showed clean conversion to the desired product. The reaction mixture was poured onto satd. aqueous sodium bicarbonate (5 mL) and then extracted with ethyl acetate (3×25 mL). The combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residual solid was purified by flash chromatography (12 g silicao 0-20% methanol/methylene chloride) to afford 1-[4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]pentan-1-olo 0.15 go as a reddish-brown solid. LCMS: (APCI) m/e 305.1 (M+H).
A solution of 1-[4-(cyclopentylamino)pyrido[3o2-d]pyrimidin-2-yl]ethanone (0.138 go 0.54 mmol) in ethanol (5 mL) was treated with TFA (40 uLo 0.54 mmoo 1.0 equiv) and then degassed by bubbling N2 through the reaction mixture. After 10 min.o the reaction mixture was treated with PtO2 (25 mgo 0.11 mmolo 0.2 equiv) and then the reaction was subjected to bubbling of H2 gas with a needle exhaust. After 20 min.o the needle introducing the H2 gas was raised above the reaction and the mixture was stirred overnight under balloon pressure. LC/MS analysis showed complete consumption of the starting material and 80% conversion to the desired product with additional 20% conversion to the over reduced product where the ketone is also reduced to the alcohol. The reaction mixture was degassed with N2 gas and then the reaction mixture was filtered through Celite. The solvent was removed in vacuo and the residual solid purified by flash chromatography (adsorbed mixture onto 2 g silica pre-columno 12 g silicao 0-30% methanol/methylene chloride) to afford 1-[4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]ethanoneo 0.123 go as a yellow solid. LCMS: (APCI) m/e 261.1 (M+H).
A solution of 1-[4-(tert-butylamino)pyrido[3o2-d]pyrimidin-2-yl]pentan-1-one (0.14 go 0.49 mmol) in ethanol (3 mL) was treated with TFA (36 uLo 0.49 mmoo 1.0 equiv) and then degassed by bubbling N2 through the reaction mixture. After 10 min.o the reaction mixture was treated with PtO2 (22 mgo 0.098 mmolo 0.2 equiv) and then the reaction was subjected to bubbling of H2 gas with a needle exhaust. After 20 min.o the needle introducing the H2 gas was raised above the reaction and the mixture was stirred overnight balloon pressure. LC/MS analysis showed complete consumption of the starting material and >90% conversion to the over reduced product where the ketone is also reduced to the alcohol. Crude LC/MS does not show a separate peak for the ketone product. The reaction mixture was degassed with N2 gas and then the reaction mixture was filtered through Celite. The solvent was removed in vacuo and the residual solid purified by flash chromatography (12 g silicao 0-30% methanol/methylene chloride) to afford 1-[4-(tert-butylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]pentan-1-olo 0.107 go as a viscous yellow oil. In additiono 13 mg of the ketone was isolated as a yellow solid (D-06). LCMS: (APCI) m/e 293.1 (M+H).
A solution of 1-[4-(tert-butylamino)pyrido[3o2-d]pyrimidin-2-yl]pentan-1-one (0.14 go 0.49 mmol) in ethanol (3 mL) was treated with TFA (36 uLo 0.49 mmoo 1.0 equiv) and then degassed by bubbling N2 through the reaction mixture. After 10 min.o the reaction mixture was treated with PtO2 (22 mgo 0.098 mmolo 0.2 equiv) and then the reaction was subjected to bubbling of H2 gas with a needle exhaust. After 20 min.o the needle introducing the H2 gas was raised above the reaction and the mixture was stirred overnight under balloon pressure. LC/MS analysis showed complete consumption of the starting material and >90% conversion to the over reduced product where the ketone is also reduced to the alcohol. Crude LC/MS does not show a separate peak for the ketone product. The reaction mixture was degassed with N2 gas and then the reaction mixture was filtered through Celite. The solvent was removed in vacuo and the residual solid purified by flash chromatography (12 g silicao 0-30% methanol/methylene chloride) to afford 13 mg of the ketone isolated as a yellow solid. In additiono 1-[4-(tert-butylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]pentan-1-olo 0.107 go as a viscous yellow oil. (D-00). LCMS: (APCI) m/e 291.1 (M+H); 1H NMR (400 Mzo CDCl3) δ 4.52 (2Ho bs)o 3.31 (2Ho dd)o 3.11 (2Ho dd)o 2.85 (2Ho dd)o 1.95 (3Ho pentet)o 1.21 (2Ho pentet)o 1.51 (9Ho s)o 1.41 (2Ho sextet)o 0.93 (3Ho t); 13C NMR (400 Mzo CDCl3) δ 200.9o 151.7o 131.8o 126.1o 123.8o 51.9o 42.0o 38.6o 29.3o 29.0o 26.8o 22.7o 21.6o 13.9.
1-[4-(2-pyridylamino)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidin-2-yl]pentan-1-ol (G-63) was prepared following a procedure similar to A-00 to provide 26 mg (18%). LCMS: (APCI) m/e 314.1 (M+H).
1-[4-(2-pyridylamino)-5,6,7,8-tetrahydropyrido[3,2-d]pyrimidin-2-yl]pentan-1-one (G-65) was prepared following a procedure similar to A-06 to provide 9 mg (6%). LCMS: (APCI) m/e 312.1 (M+H).
A solution of 1-(4-pyrrolidin-1-ylpyrido[3o2-d]pyrimidin-2-yl)pentan-1-one (73 mgo 0.26 mmol) in ethanol (1 mL) was treated with TFA (19 uLo 0.26 mmolo 1.0 equiv) and then degassed by bubbling N2 through the reaction mixture. After 10 min.o the reaction mixture was treated with PtO2 (6 mgo 26 umolo 0.1 equiv) and then the reaction was subjected to bubbling of H2 gas with a needle exhaust. After 20 min.o the needle introducing the H2 gas was raised above the reaction and the mixture was stirred overnight under balloon pressure. LC/MS analysis showed complete consumption of the starting material and 80% conversion to the desired product with additional 20% conversion to the over reduced product where the ketone is also reduced to the alcohol. The reaction mixture was degassed with N2 gas and then the reaction mixture was filtered through Celite. The solvent was removed in vacuo and the residual solid purified by flash chromatography (adsorbed mixture onto 2 g silica pre-columno 12 g silicao 0-30% methanol/methylene chloride) to afford 1-(4-pyrrolidin-1-yl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl)pentan-1-olo 0.123 go as a yellow solid. LCMS: (APCI) m/e 291.1 (M+H).
A solution of [4-(cyclopentylamino)pyrido[3o2-d]pyrimidin-2-yl]phenyl-methanone (0.21 go 0.66 mmol) in ethanol (3 mL) was treated with TFA (76 uLo 0.66 mmoo 1.0 equiv) and then degassed by bubbling N2 through the reaction mixture. After 10 min.o the reaction mixture was treated with PtO2 (15 mgo 0.066 mmolo 0.1 equiv) and then the reaction was subjected to bubbling of H2 gas with a needle exhaust. After 20 min.o the needle introducing the H2 gas was raised above the reaction and the mixture was stirred overnight under balloon pressure. LC/MS analysis showed complete consumption of the starting material and conversion to the over reduced product. The reaction mixture was degassed with N2 gas and then the reaction mixture was filtered through Celite. The solvent was removed in vacuo and the residual solid purified by flash chromatography (adsorbed mixture onto 2 g silica pre-columno 12 g silicao 0-30% methanol/methylene chloride) to afford [4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]phenyl-methanolo 0.185 go as a pale yellow solid. LCMS: (APCI) m/e 325.1 (M+H).
A solution of [4-(cyclopentylamino)pyrido[3o2-d]pyrimidin-2-yl]-(4-fluorophenyl)methanone (0.287 go 0.85 mmol) in ethanol (3 mL) was treated with TFA (98 uLo 0.85 mmoo 1.0 equiv) and then degassed by bubbling N2 through the reaction mixture. After 10 min.o the reaction mixture was treated with PtO2 (20 mgo 0.085 mmolo 0.1 equiv) and then the reaction was subjected to bubbling of H2 gas with a needle exhaust. After 20 min.o the needle introducing the H2 gas was raised above the reaction and the mixture was stirred overnight under balloon pressure. LC/MS analysis showed complete consumption of the starting material and conversion to the alcohol. The reaction mixture was degassed with N2 gas and then the reaction mixture was filtered through Celite. The solvent was removed in vacuo and the residual solid purified by flash chromatography (adsorbed mixture onto 2 g silica pre-columno 12 g silicao 0-30% methanol/methylene chloride) to afford [4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]-(4-fluorophenyl)methanolo 0.245 go as a pale yellow solid. LCMS: (APCI) m/e 343.1 (M+H).
A solution of a mixture of 1-[4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]phenyl-methanol (0.069 go 24 mmol) in methylene chloride (1 mL) was treated with Dess-Martin Periodinane (0.12 go 0.28 mmolo 1.2 equiv). After stirring for 2 hrs.o LC/MS analysis showed clean conversion to the desired product. The reaction mixture was dried and the residue was purified by flash chromatography (12 g silicao 0-20% acetonitrile/ethyl acetate) to afford 1-[4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]phenyl-methanoneo 170 mgo as a yellow solid. 1H NMR (400 Mzo (CD3)2CO) δ 4.59 (1Ho m)o 3.42 (2Ho m)o 3.12 (2Ho t)o 2.98 (2Ho t)o 2.04 (2Ho m)o 1.95 (2Ho m)o 1.78 (4Ho m)o 1.64 (4Ho m)o 1.37 (2Ho m)o 0.90 (3Ho t); 13C NMR (400 Mzo (CD3)2CO) δ 195.7o 151.4o 141.2o 128.5o 116.6o 54.7o 54.6o 40.9o 37.4o 32.9o 26.8o 25.1o 24.7o 23.0o 19.8o 14.1
A solution of a mixture of [4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]-(4-fluorophenyl)methanol (0.069 go 24 mmol) in methylene chloride (1 mL) was treated with Dess-Martin Periodinane (0.12 go 0.28 mmolo 1.2 equiv). After stirring for 2 hrs.o LC/MS analysis showed clean conversion to the desired product. The reaction mixture was dried and the residue was purified by flash chromatography (12 g silicao 0-20% acetonitrile/ethyl acetate) to afford [4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]-(4-fluorophenyl)methanoneo 170 mgo as a yellow solid. 1H NMR (400 Mzo (CD3)2CO) δ 4.59 (1Ho m)o 3.42 (2Ho m)o 3.12 (2Ho t)o 2.98 (2Ho t)o 2.04 (2Ho m)o 1.95 (2Ho m)o 1.78 (4Ho m)o 1.64 (4Ho m)o 1.37 (2Ho m)o 0.90 (3Ho t); 13C NMR (400 Mzo (CD3)2CO) δ 195.7o 151.4o 141.2o 128.5o 116.6o 54.7o 54.6o 40.9o 37.4o 32.9o 26.8o 25.1o 24.7o 23.0o 19.8o 14.1
A solution of 1-(4-pyrrolidin-1-yl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl)pentan-1-ol (43 mgo 0.15 mmol) in acetone (1 mL) was successively treated with Dess-Martin reagent (63 mgo 0.15 mmolo 1.0 equiv). After stirring for 2 hrs.o LC/MS analysis showed complete and clean conversion to the desired ketone. The solvent was removed in vacuo and the residual solid was purified by flash chromatography (12 g silicao 0-10% methanol/methylene chloride) to afford 1-(4-pyrrolidin-1-yl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl)pentan-1-oneo mgo as a yellow gum. LCMS: (APCI) m/e 289.1 (M+H); 1H NMR (d6-DMSO): δ 5.07 (bso 2H)o 3.56 (mo 3H)o 3.28 (mo 2H)o 3.00 (mo 2H)o 2.72 (mo 2H)o 1.85 (mo 6H)o 1.32 (mo 4H)o 0.86 (t. 3H).
A solution of 1-[4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]-2o2-dimethyl-propan-1-ol (33 mgo 0.11 mmol) in acetone (1 mL) was treated with Dess-Martin periodinane (51 mgo 0.12 mmolo 1.1 equiv) and the reaction was stirred at RT. After 16 ho the reaction was complete by crude LCMS. The reaction mixture was partitioned between 20 mL DCM and 20 mL 1M NaOH (aq); and stirred for 10 minutes. The aqueous layer was extracted extract with DCM (3×20 mL). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified on silica gel (40 go 0-30% EtOAc/hexanes) to provide 30 mg of 1-[4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-2-yl]-2o2-dimethyl-propan-1-one (91%). LCMS: (APCI) m/e 303.1 (M+H).
A solution of 1-[4-(tert-butylamino)pyrido[3o2-d]pyrimidin-2-yl]pentan-1-one (0.14 go 0.49 mmol) in ethanol (3 mL) was treated with TFA (36 uLo 0.49 mmoo 1.0 equiv) and then degassed by bubbling N2 through the reaction mixture. After 10 min.o the reaction mixture was treated with PtO2 (22 mgo 0.098 mmolo 0.2 equiv) and then the reaction was subjected to bubbling of H2 gas with a needle exhaust. After 20 min.o the needle introducing the H2 gas was raised above the reaction and the mixture was stirred overnight under balloon pressure. LC/MS analysis showed complete consumption of the starting material and >90% conversion to the over reduced product where the ketone is also reduced to the alcohol. Crude LC/MS does not show a separate peak for the ketone product. The reaction mixture was degassed with N2 gas and then the reaction mixture was filtered through Celite. The solvent was removed in vacuo and the residual solid purified by flash chromatography (12 g silicao 0-30% methanol/methylene chloride) to afford N-cyclopentyl-2-pentyl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (A-63)o 0.107 go as a viscous yellow oil. LCMS: (APCI) m/e 289.1 (M+H).
In a 25 mL microwave vialo a solution of 2-chloro-N-cyclopentyl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (0.75 go 2.97 mmole)o zinc cyanide (0.7 go 5.93 mmolo 2 eq)o and benzaldehyde (0.332 mLo 3.26 mmolo 1.2 eq) in anhydrous DMF (10 mL) was degassed 4× (until no more bubbling) and then treated with tetrakis(triphenylphosphine)palladium(0) (0.686 go 0.593 mmolo 0.2 equiv). The reaction mixture was warmed in a microwave to 150° C. for 45 min. LC/MS analysis of the crude reaction mixture showed conversion to the desired product and full consumption of the starting material. The mixture was filtered and adsorbed onto silica. The product was purified by flash chromatography to afford 4-(cyclopentylamino)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidine-2-carbonitrileo 0.62 go as a yellow/beige solid. LCMS: (APCI) m/e 244.1 (M+H).
A slurry of 2-chloro-N-cyclopentyl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (0.15 go 0.59 mmol) in triethylamine (2.0 mL) was treated with phenylacetylene (0.1 mLo 0.89 mmolo 1.5 equiv) and then degassed with bubbling nitrogen. After 10 min.o the reaction mixture was successively treated with palladium (II) acetate (35 mgo 0.15 mmolo 0.25 equiv) and then triphenylphosphine (82 mgo 0.31 mmolo 0.52 equiv). The reaction mixture was then microwaved at 100° C. for 1 hr. LC/MS analysis showed approx. 10% of the desired product had formed. The reaction mixture was diluted with methylene chlorideo filtered through Celite® and the solvent was removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford N-cyclopentyl-2-(2-phenylethynyl)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amineo 6.2 mgo as a yellowish-red film. LCMS: (APCI) m/e 319.1 (M+H).
A solution of 2-chloro-N-cyclopentyl-5o67o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (0.1 go 0.48 mmol) in acetonitrile (0.75 mL) and water (1.5 mL) was successively treated with 4o4o5o5-tetramethyl-2-prop-1-ynyl-1o3o2-dioxaborolane (0.085 mLo 0.48 mmolo1.2 equiv) and cesium carbonate (0.387 go 1.2 mmoleo 3.0 equiv) and then degassed with bubbling nitrogen. After 10 min.o the reaction mixture was successively treated with palladium(II) acetate (9 mgo 40 umolo 0.1 equiv) and Triphenylphosphine-3o3′o3″-trisulfonic acid trisodium salt (90 mgo 1.6 mmolo 0.4 equiv). The reaction mixture was then microwaved at 160° C. for 1 hr. LC/MS analysis showed 50% product formation. The reaction mixture was diluted with methylene chlorideo filtered through Celite® and the solvent was removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-10% methanol/methylene chloride) to afford N-cyclopentyl-2-prop-1-ynyl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amineo 14 mgo as a yellow film. LCMS: (APCI) m/e 257.1 (M+H).
A solution of 2-chloro-N-cyclopentyl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (0.12 go 0.48 mmol) in 1o4-dioxane (2.0 mL) was successively treated with 4o4o5o5-tetramethyl-2-prop-1-ynyl-1o3o2-dioxaborolane (0.8 mLo 0.48 mmolo 1.0 equiv) and sodium carbonate (0.13 go 1.2 mmoleo 2.5 equiv) and then degassed with bubbling nitrogen. After 10 min.o the reaction mixture was successively treated with tetrakis(triphenylphosphine)palladium (0.11 go 95 umolo 0.2 equiv). The reaction mixture was then microwaved at 160° C. for 1 hr. LC/MS analysis showed approx. 10% of the desired product had formed. The reaction mixture was diluted with methylene chlorideo filtered through Celite® and the solvent was removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford N-cyclopentyl-2-(2-phenylethynyl)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amineo 6.2 mgo as a yellowish-red film. LCMS: (APCI) m/e 285.1 (M+H).
A solution of 2-azido-N-cyclopentyl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (75 mgo 2.9 mmol) in DMSO (1 mL) was degassed with bubbling N2 via balloon for 20 min. The reaction mixture was then treated with phenylacetylene (48 uLo 4.3 mmolo 1.5 equiv) and then copper (I) iodide (12 mgo 58 umolo 0.2 equiv) and then the reaction mixture was warmed to 60° C. After 1 hr.o LC/MS analysis showed clean conversion to the desired product. The reaction mixture was diluted with water 10 mL and the mixture was extracted with ethyl acetate (4×10 mL). The combined organic extracts were dried (Na2SO4) and solvent was removed in vacuo. The residual solid was purified by flash chromatography (12 g silicao 0-10% methylene chloride/methanol) to afford N-cyclopentyl-2-(4-phenyltriazol-1-yl)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amineo 44 mgo as a yellow solid. LCMS: (APCI) m/e 362.1 (M+H).
A microwave tube containing 2-chloro-N-cyclopentyl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (0.2 go 0.79 mmol)o cesium carbonate (1.0 go 3.2 mmolo 4 equiv)o p-tolylboronic acid (0.27 go 2.0 mmolo 2.5 equiv)o palladium (II) acetate (18 mgo 79 umolo 0.1 equiv) and triphenylphosphine-3o3′o3″-trisulfonic acid trisodium salt (0.18 g03.2 mmolo 0.4 equiv) was purged with N2 gas for 2 min and then sealed. The mixture was then diluted with water (1.5 mL) and acetonitrile (0.75 mL). The reaction mixture was then microwaved at 175° C. for 2 hr. LC/MS analysis showed approx. 50% of the desired product had formed. The reaction mixture was diluted with methylene chloride (5 mL) and the layers were separated. The aqueous phase was extracted with methylene chloride (2×10 mL) and the combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-10% methanol/methylene chloride) to afford N-cyclopentyl-2-(p-tolyl)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amineo 19.6 mgo as a yellowish solid. LCMS: (APCI) m/e 309.1 (M+H); 1H NMR (d6-DMSO): δ 8.15 (do 2H)o 7.08 (do 2H)o 5.56 (bso 1H)o 4.45 (bso 1H)o 3.17 (mo 2H)o 2.67 (mo 1H)o 2.23 (so 3H)o 1.94 (mo 6H)o 1.92 (mo 6H).
A microwave tube containing 2-chloro-N-cyclopentyl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (0.2 go 0.79 mmol)o cesium carbonate (1.0 go 3.2 mmolo 4 equiv)o 4-pyridylboronic acid (0.24 go 2.0 mmolo 2.5 equiv)o palladium (II) acetate (18 mgo 79 umolo 0.1 equiv) and triphenylphosphine-3o3′o3″-trisulfonic acid trisodium salt (0.18 go3.2 mmolo0.4 equiv) was purged with N2 gas for 2 min and then sealed. The mixture was then diluted with water (1.5 mL) and acetonitrile (0.75 mL). The reaction mixture was then microwaved at 150° C. for 2 hr. LC/MS analysis showed approx. 50% of the desired product had formed. The reaction mixture was diluted with methylene chloride (5 mL) and the layers were separated. The aqueous phase was extracted with methylene chloride (2×10 mL) and the combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-10% methanol/methylene chloride) to afford N-cyclopentyl-2-(4-pyridyl)-5o67o8-tetrahydropyrido[3o2-d]pyrimidin-4-amineo 47.5 mgo as a yellowish solid. LCMS: (APCI) m/e 296.1 (M+H); 1H NMR (CDCl3): δ 8.71 (do 1H)o 8.69 (bso 1H)o 8.17 (bso 1H)o 7.73 (do 1H)o 5.86 (bso 1H)o 4.56 (bso 1H)o 3.32 (mo 1H)o 2.76 (mo 3H)o 2.73 (mo 3H)o 2.03 (mo 3H)o 1.94 (mo 2H)o 1.26 (mo 2H).
A 250 mL RBF was charged with 2o4-dichloro-5-nitro-pyrimidine (500 mgo 2.58 mmol)o THF (25 mLo 0.1 M) and cooled to −78° C. in a dry ice bath. The cooled reaction mixture was then treated carefully with DiPEA (3 eq.o 7.74 mmolo 1.4 mL). The reaction mixture was then treated with N-benzylcyclopentanamine;hydrochloride (1 eq.o 2.58 mmolo 546 mg) as a solid. The reaction was purged with nitrogen and allowed to gradually warm to RT. After 16 ho the reaction was partitioned between water (50 mL) and EtOAc (50 mL)o the water layer was back extracted 3×50 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide a red oil (850 mgo 99%) and used directly in the next step. (APCI) m/e 333.0 (M+H).
A 40 mL vial fitted with a stirbar was charged with N4-cyclopentyl-2-(p-tolyppyrimidine-4o5-diamine (F-68o 0.065 go 0.242 mmol)o MEK (1.3 eq.o 0.028 mLo 0.315 mmol)o TFA (2 eq.o 0.036 mLo 2.47 mmol)o and isopropyl acetate (3.25 mL). The reaction was stirred at RT for 15 mino and treated carefully with sodium triacetoxyborohydride (0.0565 go 0.266 mmol)o purged with N2 and allowed to stir at RT for 3 days. The reaction mixture was partitioned between sat. NaHCO4 (10 mL) and EtOAc (10 mL). The aqueous layer was back extracted 3×10 mL EtOAc and the combined organic later was dried over Na2SO4o concentrated under reduced pressure and the residue was purified on silica gel (24 go Hexane/Ethyl Acetate). LCMS: (APCI) m/e 325.1 (M+H); 1H NMR (CDCl3): δ 8.17 (do 2H)o 7.63 (so 1H)o 7.17 (to 2H)o 4.43 (bso 1H)o 3.13 (bso 1H)o 2.32 (so 3H)o 2.10 (mo 2H)o 1.62 (mo 10H)o 0.91 (mo 3H)o 0.88 (to 3H).
A 40 mL vial fitted with a stirbar was charged with N4-cyclopentyl-2-(3-pyridyl)pyrimidine-4o5-diamine (F-76o0.150 go 0.588 mmol)o MEK (3 eq.o 0.158 mLo 1.76 mmol)o TFA (2 eq.o 0.0873 mLo 1.18 mmol)o and isopropyl acetate (7.5 mL).The reaction was stirred at RT for 15 min and treated carefully with sodium triacetoxyborohydride (1.1 eqo 0.138 go 0.646 mmol)o purged with N2 and allowed to stir at RT for 3 days. The reaction mixture was partitioned between sat. NaHCO4 (10 mL) and EtOAc (10 mL). The aqueous layer was back extracted 3×10 mL EtOAc and the combined organic later was dried over Na2SO4o concentrated under reduced pressure and the residue was purified on silica gel (24 go Hexane/Ethyl Acetate). LCMS: (APCI) m/e 312.1 (M+H); 1H NMR (CDCl3): δ 9.35 (bso 1H)o 8.46 (mo 2H)o 7.39 (bso 1H)o 6.63 (do 2H)o 4.96 (bso 1H)o 4.53 (mo 1H)o 3.42 (mo 1H)o 2.09 (mo 2H)o 1.60 (mo 8H)o 1.17 (mo 3H)o 0.93 (to 3H).
A 40 mL vial fitted with a stirbar was charged with N4-cyclopentyl-2-pyrimidin-5-yl-pyrimidine-4o5-diamine (F-79o0.300 go 1.17 mmol)o MEK (3 eq.o 0.315 mLo 3.51 mmol)o TFA (2 eq.o 0.174 mLo 2.34 mmol)o and isopropyl acetate (15 mL). The reaction was stirred at RT for 15 min and treated carefully with sodium triacetoxyborohydride (1.1 eqo 0.273 go 1.29 mmol)o purged with N2 and allowed to stir at RT for 3 days. The reaction mixture was partitioned between sat. NaHCO4 (10 mL) and EtOAc (10 mL). The aqueous layer was back extracted 3×10 mL EtOAc and the combined organic later was dried over Na2SO4o concentrated under reduced pressure and the residue was purified on silica gel (24 go Hexane/Ethyl Acetate). LCMS: (APCI) m/e 313.1 (M+H); 1H NMR (CDCl3): δ 9.41 (bso 2H)o 9.12 (bso 1H)o 7.64 (so 1H)o 6.84 (do 1H)o 5.08 (mo 1H)o 4.46 (mo 1H)o 2.02 (mo 2H)o 1.65 (mo 9H)o 1.16 (mo 3H)o 0.91 (to 3H).
A 40 mL vial fitted with a stirbar was charged with N4-cyclopentyl-2-pyrimidin-5-yl-pyrimidine-4o5-diamine (F-79o 0.300 go 1.17 mmol)o oxetanone (3 eq.o 0.206 mLo 3.51 mmol)o TFA (2 eq.o 0.174 mLo 2.34 mmol)o and isopropyl acetate (15 mL). The reaction was stirred at RT for 15 min and treated carefully with sodium triacetoxyborohydride (1.1 eqo 0.273 go 1.29 mmol)o purged with N2 and allowed to stir at RT for 3 days. The reaction mixture was partitioned between sat. NaHCO4 (10 mL) and EtOAc (10 mL). The aqueous layer was back extracted 3×10 mL EtOAc and the combined organic later was dried over Na2SO4o concentrated under reduced pressure and the residue was purified on silica gel (24 go DCM/Methanol). LCMS: (APCI) m/e 313.1 (M+H).
A 40 mL vial fitted with a stirbar was charged with N4-cyclopentyl-2-(4-pyridyl)pyrimidine-4o5-diamine (F-84o 0.123 go 0.482 mmol)o MEK (3 eq.o 0.13 mLo 1.45 mmol)o TFA (2 eq.o 0.072 mLo 0.964 mmol)o and isopropyl acetate (6.5 mL). The reaction was stirred at RT for 15 min and treated carefully with sodium triacetoxyborohydride (1.1 eqo 0.112 go 0.53 mmol)o purged with N2 and allowed to stir at RT for 24 hours. The reaction mixture was partitioned between sat. NaHCO4 (10 mL) and EtOAc (10 mL). The aqueous layer was back extracted 3×10 mL EtOAc and the combined organic later was dried over Na2SO4o concentrated under reduced pressure and the residue was purified on silica gel (24 go Hexane/Ethyl Acetate). LCMS: (APCI) m/e 312.1 (M+H); 1H NMR (CDCl3): δ 8.55 (to 2H)o 8.06 (to 2H)o 7.54 (so 1H)o 6.63 (do 1H)o 5.11 (do 1H)o 4.43 (mo 1H)o 3.42 (mo 2H)o 2.08 (mo 1H)o 1.60 (mo 8H)o 1.13 (mo 3H)o 0.91 (to 3H).
A 20 mL microwave vial fitted with a stirbar was charged with the 6-chloro-N4-cyclopentyl-2-methyl-pyrimidine-4o5-diamine (F-98o 1 go 4.41 mmol)o n-butanol (12 mL)o water (1.2 mL)o 2o2-dimethylethenylboronic acid (2.5 eq.o 1.1 go 11 mmol) and potassium acetate (3.5 eq.o 1.52 go 15.4 mmol). The vial was then evacuated and backfilled with nitrogen (2×) and treated with tetrakis(triphenylphosphine)palladium(0) (0.01 eq; 35 mgo 0.0441 mmol)o the vial sealed and then heated in the microwave at 110° C. for 15 minutes. LC indicates primarily the desired product with trace starting material. The reaction mixture was filtered through a PTFE 0.45 um syringe filter into a 250 ml RBF and concentrated under reduced pressure. The residue was dissolved in 3 mL DCM and absorbed on silica gel concentrated under reduced pressure and the solid material was heated at 100° C. overnight. The solid was purified directly on silica gel (50 go Hexane/Ethyl Acetate) to provide the desired product (F-99). LCMS: (APCI) m/e 247.1 (M+H).
In a 40-mL vial equipped with stir baro 2o6-dichloro-3-nitro-pyridine (0.500 go 2.59 mmol) was dissolved in THF (5 mL). To this was added DIEA (0.554 mLo 3.24 mmol 1.25 equiv) followed by cyclopentylamine (0.256 mLo 2.59 mmolo 1 equiv). The reaction was allowed to stir at room temperature for 2 hourso at which time LCMS analysis suggested formation of desired product. The reaction mixture was poured into water (˜20 mL) and extracted with ethyl acetate (3ט25 mL). The organic extracts were combinedo dried over anhydrous magnesium sulfateo filteredo and rotavapped down. The resulting orange oil was purified via flash chromatography (hexanes/EtOAc). Desired product fractions were combinedo rotavapped downo and dried overnight at 40° C. under vacuum to yield 6-chloro-N-cyclopentyl-3-nitro-pyridin-2-amine as an orange oil (375 mgo 60.0%). 1H-NMR (400 MHzo CDCl3): δ 8.31 (do 1H)o 6.56 (do 1H)o 4.53 (mo 1H)o 2.13 (mo 2H)o 1.76 (mo 2H)o 1.67 (mo 2H)o 1.54 (mo 2H). LCMS: (APCI) m/e 242 (M+H).
A 250 mL RBF was charged with 2o6-dichloro-3-nitro-pyridine (1.0 go 5.18 mmol)o a stir baro THF (10 mLo 0.5M)o DiEA (2 eq.o 1.8 mLo 10.4 mmol) 2-methylpropan-2-amine (1 eq.o 5.18 mmolo 380 mg) in 4 mL of THF (1 eq.o 5.18 mmolo 380 mg) and the reaction was stirred at RT overnight. The reaction was then partitioned between 75 mL of water and 75 mL EtOAc. The water layer was extracted 3×50 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide 1.15 of an oil that was >70% pure by LCMS and was purified on silica gel (40 go 0-50% EtOAc/hexanes) to provide 550 mg as a yellow oil (46%). LCMS: (APCI) m/e 230.1 (M+H).
A 250 mL RBF was charged with 2o6-dichloro-3-nitro-pyridine (1.0 go 5.18 mmol)o a stir baro THF (8 mLo 0.5M)o DiEA (2 eq.o 1.8 mLo 10.4 mmol)o 3-methyloxetan-3-amine in 2 mL of THF (1 eq.o 5.18 mmolo 451 mg) and the reaction was stirred at RT overnight. The reaction was then partitioned between 75 mL of water and 75 mL EtOAc. The water layer was extracted 3×50 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide 1.5 of an oil that was >70% pure by LCMS and was purified on silica gel (80 go 0-50% EtOAc/hexanes) to provide 740 mg as a yellow solid (58%). LCMS: (APCI) m/e 244.0 (M+H).
A 40 mL vial was charged with 2-chloro-6-methyl-3-nitro-pyridine (1.0 go 5.79 mmol)o a stir baro DMF (5 mLo 1 M)o DiEA (3 eq.o 3.1 mLo 17.4 mmol)o N-benzylcyclopentanamine;hydrochloride (1.1 eq.o 6.37 mmolo 1.35 g)o 80° C. overnight. After 16 ho the starting material had been consumed and the desired product was confirmed in the crude LCMS. The reaction mixture was partitioned between 75 mL of water and 75 mL EtOAc. The water layer was back extracted 3×50 mL EtOAc and the combined organic layer was dried over Na2SO4. The residue was purified on silica gel (80 go 0-30% EtOAc/hexanes) to provide 1.2 g (85%) as a yellow solid. LCMS: (APCI) m/e 312.1 (M+H).
A 40 mL vial was charged with 2-chloro-6-methyl-3-nitro-pyridine (1.0 go 5.79 mmol)o a stir baro DMF (5 mLo 1 M)o DiEA (3 eq.o 3.1 mLo 17.4 mmol)o 3o3-difluorocyclobutanamine;hydrochloride (1 eq.o 5.79 mmolo 937 mg) and the reaction was stirred at 80° C. overnight. The reaction was then heated for 24 h at 75° C. and the THF evaporated under reduced pressure. The residue was directly purified on silica gel (80 go 0-30% EtOAc/hexanes) to provide 1.2 g (85%) as a yellow solid. LCMS: (APCI) m/e 244.1 (M+H).
A 250 mL RBF was charged with 2o6-dichloro-3-nitro-pyridine (1.0 go 5.18 mmol)o a stir baro DMF (8 mLo 0.5M)o DiEA (3 eq.o 2.7 mLo 15.5 mmol)o 3o3-difluoro-1-methyl-cyclobutanamine;hydrochloride (1 eq.o 5.18 mmolo 817 mg) and the reaction was stirred at RT for 3 d. The reaction was then partitioned between 75 mL of water and 75 mL EtOAc. The water layer was extracted 3×50 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide an oil that was >70% pure by LCMS and was purified on silica gel (80 go 0-40% EtOAc/hexanes) to provide 1.07 g of 6-chloro-N-(3o3-difluoro-1-methyl-cyclobutyI)-3-nitro-pyridin-2-amine as a yellow solid (74%). LCMS: (APCI) m/e 278.1 (M+H).
A 250 mL RBF was charged with 2o6-dichloro-3-nitro-pyridine (1.0 go 5.18 mmol)o a stir baro THF (8 mLo 0.5M)o DiEA (2 eq.o 1.8 mLo 10.4 mmol)o 3-methyltetrahydrofuran-3-amine in 2 mL of THF (1 eq.o 5.18 mmolo 524 mg) and the reaction was stirred at RT for 3 d. The reaction was then partitioned between 75 mL of water and 75 mL EtOAc. The water layer was extracted 3×50 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide an oil that was >70% pure by LCMS and was purified on silica gel (80 go 0-40% EtOAc/hexanes) to provide 770 mg as a yellow solid (48%). LCMS: (APCI) m/e 358.0 (M+H).
N-cyclopentyl-3-nitro-6-(p-tolyl)pyridin-2-amine (H-54) (Represents general procedure followed for all boronic acid couplings in this series)
In a 2.0-5.0 mL microwave vialo 6-chloro-N-cyclopentyl-3-nitro-pyridin-2-amine (0.750 go 3.10 mmol) was dissolved in DMF (5 mL). To this were added cesium carbonate (2.53 go 7.76 mmolo 2.5 equiv) and p-tolylboronic acid (0.844 go 6.20 mmolo 2 equiv). The mixture was then purged with nitrogen. tetrakis(triphenylphosphine)palladium(0) (0.538 go 0.466 mmolo 0.15 equiv) was then added. The vial was sealed and heated in the microwave reactor for 20 min at 120° C. The reaction mix was then filteredo loaded onto silica and purified by flash chromatography (hexanes/EtOAc). Desired product fractions 13-21 combinedo rotavapped down and dried at 40° C. overnight to yield N-cyclopentyl-3-nitro-6-(p-tolyl)pyridin-2-amine as a yellow-orange solid (354 mgo 38.3%). LCMS: (APCI) m/e 298 (M+H).
In a 40-mL vialo 6-chloro-N-(3o3-difluoro-1-methyl-cyclobutyl)-3-nitro-pyridin-2-amine (0.400 go 1.44 mmol)o 3-pyridylboronic acid (0.354 go 2.88 mmolo 2 equiv) and potassium carbonate (0.597 go 4.32 mmolo 3 equiv) were stirred in THF (4 mL) and water (2 mL). tetrakis(triphenylphosphine)palladium(0) (0.166 go 0.144 mmolo 0.1 equiv) was addedo and the vial capped and stirred at 60° C. After overnight reactiono LCMS analysis of crude reaction mixture suggests predominant formation of desired product. Reaction mixture was poured onto water (˜25 mL)o and extracted with EtOAc (4ט30 mL). Organic extracts were combinedo dried over anhydrous Mg sulfateo and rotavapped down to a deep red oil. This was subsequently dried under vacuum for ˜1 hr at 40° C. Resulting mass is greater than expected yieldo which is presumably due to the presence of tetrakis byproduct(s) (also suggested by LCMS). This material was used in the next step (H-71) without further purificationo assuming quantitative yield. LCMS: (APCI) m/e 321.0 (M+H).
A 40 mL vial was charged with 6-chloro-N-(3-methyltetrahydrofuran-3-yI)-3-nitro-pyridin-2-amine (518 mgo 2.01 mmol)o THF (4 mL)o water (2 mL)o (4-fluorophenyl)boronic acid (2 eq.o 563 mgo 4.02 mmol)o sodium carbonate (4 eq.o 852 mgo 8.04 mmol) and then fitted with a stir baro and septa. The solution was degassed using a stream of nitrogen directly in the solution and an exit needle for 10 min. The reaction mixture was then treated with tetrakis(triphenylphosphine)palladium(0) (0.1 eq.o 232 mgo 0.201 mmol) and fitted with a nitrogen balloon and stirred at 60° C. After 2 ho crude LCMS confirmed complete consumption of the starting material and the major product exhibited the correct MS for the desired product. The reaction mixture was allowed to cool to RT and then partitioned between 20 mL of EtOAC and 20 mL water. The aqueous layer was back extracted 2×20 mL EtOAc and the combined organic layer dried over Na2SO4. The solvent was removed under reduced pressure and the resulting residue was purified on silica gel (80 go 0-30% EtOAc/hexanes) to provide 6-(4-fluorophenyl)-N-(3-methyltetrahydrofuran-3-yl)-3-nitro-pyridin-2-amine as a yellow solid confirmed (500 mgo 78%). LCMS: (APCI) m/e 318.1 (M+H).
A 40 mL vial was charged with 6-chloro-N-(3-methyltetrahydrofuran-3-yI)-3-nitro-pyridin-2-amine (550 mgo 2.13 mmol)o THF (4 mL)o water (2 mL)o [4-(dimethylcarbamoyl)phenyl]boronic acid (2 eq.o 824 mgo 4.27 mmol) sodium carbonate (4 eq.o 905 mgo 8.54 mmol) and then fitted with a stir baro and septa. The solution was degassed using a stream of nitrogen directly in the solution and an exit needle for 10 min. The reaction mixture was then treated with tetrakis(triphenylphosphine)-palladium(0) (0.1 eq.o 247 mgo 0.213 mmol) and fitted with a nitrogen balloon and stirred at 60° C. After 4 ho crude LCMS confirmed complete consumption of the starting material and the major product exhibited the correct MS for the desired product. The reaction mixture was allowed to cool to RT and then partitioned between 20 mL of EtOAC and 20 mL water. The aqueous layer was back extracted 2×20 mL EtOAc and the combined organic layer dried over Na2SO4. The solvent was removed under reduced pressure and the resulting residue was purified on silica gel (80 go 0-30% EtOAc/hexanes) to provide NoN-dimethyl-4-[6-[(3-methyltetrahydrofuran-3-yl)amino]-5-nitro-2-pyridyl]benzamide as a yellow solid (700 mgo 85%). LCMS: (APCI) m/e 371.1 (M+H).
A 40 mL vial was charged with 6-chloro-N-(3o3-difluoro-1-methyl-cyclobutyI)-3-nitro-pyridin-2-amine (550 mgo 2.33 mmol)o THF (4 mL)o water (2 mL)o [4-(dimethylcarbamoyl)phenyl]boronic acid (2 eq.o 898 mgo 4.65 mmol)o sodium carbonate (4 eq.o 986 mgo 9.31 mmol) and then fitted with a stir baro and septa. The solution was degassed using a stream of nitrogen directly in the solution and an exit needle for 10 min. The reaction mixture was then treated with tetrakis(triphenylphosphine)-palladium(0) (0.1 eq.o 269 mgo 0.233 mmol) and fitted with a nitrogen balloon and stirred at 60° C. After 16 ho crude LCMS confirmed complete consumption of the starting material and the major product exhibited the correct MS for the desired product. The reaction mixture was allowed to cool to RT and then partitioned between 50 mL of EtOAC and 50 mL water. The aqueous layer was back extracted 2×50 mL EtOAc and the combined organic layer dried over Na2SO4. The solvent was removed under reduced pressure and the resulting residue was purified on silica gel (80 go 0-40% EtOAc/hexanes) to provide 4-[6-[(3o3-difluoro-1-methyl-cyclobutyl)amino]-5-nitro-2-pyridyl]-NoN-dimethyl-benzamide as a yellow solid (830 mgo 91%). LCMS: (APCI) m/e 391.1 (M+H).
N2-cyclopentyl-6-(p-tolyl)pyridine-2,3-diamine (H-59) (Represents general procedure for all nitro reduction reactions in this series)
In a 40-mL vial equipped with stir baro N-cyclopentyl-3-nitro-6-(p-tolyl)pyridin-2-amine (0.354 go 1.19 mmol)o ammonium chloride (0.063 go 1.19 mmol) and iron filings (0.332 go 5.95 mmol) were stirred in 5 mL ethanol:water 4:1. The vial was sealed and the mixture stirred at 80° C. in a reaction block. After 2 hourso LCMS showed clean conversion to desired product. The reaction was cooled to room temperature and the iron filtered off. The filtrate was poured into water and extracted with ethyl acetate (×3). Combined organic extracts were dried over magnesium sulfateo filteredo and rotavapped down and dried under vacuum at 40° C. overnight to yield N2-cyclopentyl-6-(p-tolyl)pyridine-2o3-diamine as a dark brown solid (0.3032 go 95.3%). LCMS: (APCI) m/e 268 (M+H).
In a 40-mL vial equipped with stir baro N-(3o3-difluoro-1-methyl-cyclobutyl)-3-nitro-6-(3-pyridyl)pyridin-2-amine (M-03o 0.299 go 0.933 mmol)o ammonium chloride (0.0499 go 0.933 mmol) and iron filings (0.260 go 4.66 mmol) were stirred in 5 mL ethanol:water 4:1. The vial was sealed and the mixture stirred at 80° C. in a reaction block for 8 hours. LC-MS suggests reaction has gone to completion. Reaction was cooled to room temperatureo diluted with methanol and filtered through a plug of Celite®. Filtrate was rotavapped down and dried under vacuum at 40° C. overnight to provide a quantitative yield. The material was used directly in the next step without further purification. LCMS: (APCI) m/e 291.1 (M+H).
A 20 mL microwave vial was charged with NoN-dimethyl-4-[6-[(3-methyltetrahydrofuran-3-yl)amino]-5-nitro-2-pyridyl]benzamide (700 mgo 1.89 mmol)o EtOH (5 mL)o water (1.25 mL)o ammonium chloride (1 eq.o 1.89 mmolo 102 mg)o iron shavings (5 eq.o 9.45 mmolo 528 mg)o fitted with a stir baro was purged with nitrogeno sealed and stirred at 80° C. After 16 ho the reaction was cooled to RT and filtered using an ISCO sample cartridge with wet Celite® (MeOH) and washed several times with MeOH. The yellow solution dried over Na2SO4o filtered and was concentrated under reduced pressure to provide 850 mg. The residue was dissolved in 50 ml 0.1 M HCl and 50 mL EtOAC. The aq. layer was extracted 2×50 mL EtOAc and the combined organic layer was discarded. The acidic layer was made pH 12 with the addition of 5 N NaOH and then extracted 4×50 mL DCMo dried over Na2SO4 and concentrated under reduced pressure to provide 590 mg of 4-[5-amino-6-[(3-methyltetrahydrofuran-3-yl)amino]-2-pyridyl]-NoN-dimethyl-benzamide (91%) as a pale green solid. The material was pure by LCMS and was used directly in the next step. LCMS: (APCI) m/e 341.1 (M+H).
A 20 mL microwave vial was charged with 6-(4-fluorophenyl)-N-(3-methyltetrahydrofuran-3-yl)-3-nitro-pyridin-2-amine (500 mgo 1.58 mmol) EtOH (4 mL)o water (1 mL)o ammonium chloride (1 eq.o 1.58 mmolo 86 mg)o iron shavings (5 eq.o 7.88 mmolo 440 mg)o fitted with a stir baro was purged with nitrogeno sealed and stirred at 80° C. After 3 ho the reaction was cooled to RT and filtered using an ISCO sample cartridge with wet Celite® (MeOH) and washed several times with MeOH. The yellow solution dried over Na2SO4o filtered and was concentrated under reduced pressure to provide 950 mg. The residue was dissolved in 50 ml 0.1 M HCl and 50 mL EtOAC. The aq. layer was extracted 2×50 mL EtOAc and the combined organic layer was discarded. The acidic layer was made pH 12 with the addition of 5 N NaOH and then extracted 4×50 mL DCMo dried over Na2SO4 and concentrated under reduced pressure to provide 420 mg of 6-(4-fluorophenyl)-N2-(3-methyltetrahydrofuran-3-yl)pyridine-2o3-diamine (92%) as a grey solid. The material was pure by LCMS and was used directly in the next step. LCMS: (APCI) m/e 288.1 (M+H).
To a vial containing N2-cyclopentyl-6-(p-tolyl)pyridine-2o3-diamine (0.3032 go 1.13 mmol) and a stir baro 2-butanone (0.112 mLo 1.25 mmolo 1.1 equiv)o TFA (0.168 mLo 2 equiv) and isopropyl acetate (4 mL) were added. To this was added sodium triacetoxyborohydride (0.288 go 1.36 mmolo 1.2 equiv) over ˜5 min. An additional 1 mL isopropyl acetate was added to facilitate mixing. The reaction was then allowed to stir at room temperature for 1.5 hours. The reaction mixture was then filteredo the filtrate poured onto water and extracted with EtOAc (×3). Combined organic extracts were dried over anhydrous magnesium sulfateo filtered and concentrated by rotavap. Material was then loaded onto silica and purified by flash chromatography (24 g columno hexanes/EtOAc). Desired product fractions were combined and dried down to provide N2-cyclopentyl-6-(p-tolyl)-N3-sec-butyl-pyridine-2o3-diamine as a red-brown oil (42.2 mgo 11.5%). 1H-NMR (400 MHzo DMSO-d6): δ 7.80 (do 2H)o 7.15 (do 2H)o 6.97 (do 1H)o 6.55 (do 1H)o 5.71 (do 1H (NH))o 4.74 (do 1H)o 4.36 (mo 1H)o 2.29 (so 3H)o 2.07 (mo 2H)o 1.71 (mo 2H)o 1.58 (mo 2H)o 1.52 (mo 2H)o 1.43 (mo 2H)o 1.14 (do 3H)o 0.91 (to 3H). 13C-NMR (400 MHzo DMSO-d6): 146.53o 139.92o 137.60o 135.21o 129.30o 128.89 (2C)o 124.64 (2C)o 113.28o 107.91o 52.62o 48.87o 32.73 (2C)o 28.51o 23.91 (2C)o 20.75o 19.74o 10.62. LCMS: (APCI) m/e 324 (M+H).
A solution of N2-cyclopentyl-6-(p-tolyl)pyridine-β-diamine (0.238 go 0.89 mmol) in dichloromethane (2.5 mL) was treated with tert-butyl 2o2o2-trichloroethanimidate (0.39 go 1.8 mmolo 2 equiv) and then borontrifluoride etherate (22 uLo 0.18 mmolo 0.2 equiv). After stirring for 3 hrs.o LC/MS analysis showed partial conversion to the desired product and a significant amount of starting material. The reaction mixture was treated with an additional amount of tert-butyl 2o2o2-trichloroethanimidate (0.39 go 1.8 mmolo 2 equiv) and borontrifluoride etherate (22 uLo 0.18 mmolo 0.2 equiv). After stirring overnighto LC/MS analysis showed 50% conversion to the desired product and 50% starting material. Purification on silica gel provided 23 mg (8%) of N3-tert-butyl-N2-cyclopentyl-6-(p-tolyl)pyridine-2,3-diamine (A-98). LCMS: (APCI) m/e 324.1 (M+H).
To a vial containing N2-cyclopentyl-6-pentyl-pyridine-β-diamine (0.268 go 1.08 mmol) and a stir baro 2-butanone (0.107 mLo 1.19 mmolo 1.1 equiv)o TFA (0.161 mLo 2.17 mmolo 2 equiv) and isopropyl acetate (5 mL) were added. To this was added sodium triacetoxyborohydride (0.276 go 1.30 mmolo 1.2 equiv) over ˜5 min. The reaction was then allowed to stir at room temperature. After 45 min reaction timeo LCMS suggests conversion to desired product. Reaction was filteredo the filtrate poured onto water and extracted with EtOAc (×3). Combined organic extracts were dried over anhydrous magnesium sulfateo rotavapped downo loaded onto silica and purified by column chromatography (40 g columno hexanes/EtOAc). Desired product fractions were combined and dried down to provide N2-cyclopentyl-6-pentyl-N3-sec-butyl-pyridine-2o3-diamine (40.3 mgo 12.3%). 1H-NMR (400 MHzo DMSO-d6): δ 6.40 (do 1H)o 6.20 (do 1H)o 5.44 (do 1H (NH))o 4.30 (do 1H (NH))o 4.23 (mo 1H)o 4.21 (mo 1H)o 2.39 (to 2H)o 1.97 (mo 4H)o 1.67 (mo 2H)o 1.55 (mo 4H)o 1.41 (mo 2H)o 1.26 (mo 4H)o 1.09 (do 3H)o 0.89 (to 3H)o 0.85 (to 3H). LCMS: (APCI) m/e 304 (M+H).
To a vial containing N2-cyclopentyl-6-(3-pyridyl)pyridine-β-diamine (0.316 go 1.24 mmol) and a stir baro 2-butanone (0.122 mLo 1.37 mmolo 1.1 equiv)o TFA (0.185 mLo 2.48 mmolo 2 equiv) and isopropyl acetate (5 mL) were added. To this was added sodium triacetoxyborohydride (0.316 go 1.49 mmolo 1.2 equiv) over ˜5 min. The reaction was then allowed to stir at room temperature. After 45 mino LCMS suggested conversion to desired product. Reaction was filteredo the filtrate poured onto water and extracted with EtOAc (×3). Combined organic extracts were dried over anhydrous magnesium sulfateo rotavapped downo and loaded onto silica. The product was purified by column chromatography (hexanes/ethyl acetate). Desired product fractions were combined and dried down to afford N2-cyclopentyl-6-pentyl-N3-sec-butyl-pyridine-2o3-diamine (0.1443 go 37.4%) as a light brown solid. 1H-NMR (400 MHzo DMSO-d6): δ 9.13 (do 1H)o 8.39 (ddo 1H)o 8.23 (mo 1H)o 7.36 (mo 1H)o 7.11 (do 1H)o 6.58 (do 1H)o 5.86 (do 1H (NH))o 4.93 (do 1H)o 4.37 (mo 1H)o 2.08 (mo 2H)o 1.71 (mo 2H)o 1.60 (mo 2H)o 1.53 (mo 2H)o 1.45 (mo 2H)o 1.15 (do 3H)o 0.93 (to 3H). 13C-NMR (400 MHzo DMSO-d6): δ 147.00o 146.70o 146.33o 136.86o 135.48o 131.68o 130.27o 123.47o 112.71o 109.02o 52.69o 48.84o 32.67 (2C)o 28.47o 23.91 (2C)o 19.71o 10.64. LCMS: (APCI) m/e 311 (M+H).
To a vial containing N2-cyclopentyl-6-(3-pyridyl)pyridine-2o3-diamine (0.316 go 1.24 mmol) and a stir baro oxetan-3-one (0.087 mLo 1.37 mmolo 1.1 equiv)o TFA (0.185 mLo 2.48 mmolo 2 equiv) and isopropyl acetate (5 mL) were added. To this was added sodium triacetoxyborohydride (0.316 go 1.49 mmolo 1.2 equiv) over ˜5 min. The reaction was then allowed to stir at room temperature. After 45 min reaction timeo LCMS suggested conversion to desired product. The reaction mixture was poured onto water and extracted with EtOAc (×3). Combined organic extracts were dried over anhydrous magnesium sulfateo rotavapped downo loaded onto silicao and purified by column chromatography (hexanes/ethyl acetate). Desired product fractions were combined and dried down to afford N2-cyclopentyl-N3-(oxetan-3-yl)-6-(3-pyridyl)pyridine-2o3-diamine as an off-white solid (89 mgo 23.1%). 1H-NMR (400 MHzo DMSO-d6): δ 9.13 (do 1H)o 8.42 (ddo 1H)o 8.24 (mo 1H)o 7.37 (mo 1H)o 7.08 (do 1H)o 6.34 (do 1H)o 5.85 (mo 2H (NHs))o 4.91 (to 2H)o 4.54 (mo 1H)o 4.46 (to 2H)o 4.38 (mo 1H)o 2.09 (mo 2H)o 1.73 (mo 2H)o 1.61 (mo 2H)o 1.54 (mo 2H). 13C-NMR (400 MHzo DMSO-d6): 147.48o 147.22o 146.59o 138.95o 135.26o 131.99o 129.07o 123.50o 113.62o 108.77o 77.44 (2C)o 52.59o 47.60o 32.74 (2C)o 23.86 (2C).LCMS: (APCI) m/e 311 (M+H).
To a vial containing N2-cyclopentyl-6-(4-pyridyl)pyridine-2o3-diamine (0.1391 go 0.547 mmol) and a stir baro 2-butanone (0.108 mLo 1.204 mmol)o TFA (0.081 mLo 1.09 mmol)o and isopropyl acetate (5 mL) were added. To this was added sodium triacetoxyborohydride (0.139 go 0.656 mmol) over ˜2 min. The reaction was then allowed to stir at room temperature overnight. The resulting reaction mixture was then poured onto water and extracted with ethyl acetate (×3). Combined organic extracts were dried over anhydrous magnesium sulfateo filteredo rotavapped downo loaded onto silica and purified by column chromatography (24 g columno hexanes/EtOAc). Desired product fractions were combined and dried down to yield N2-cyclopentyl-6-(4-pyridyl)-N3-sec-butyl-pyridine-2o3-diamine as a brown solid (39.6 mgo 23.3%). 1H-NMR (400 MHzo DMSO-d6): δ 8.48 (do 2H)o 7.86 (do 2H)o 7.23 (do 1H)o 6.58 (do 1H)o 5.91 (do 1H (NH))o 5.10 (do 1H (NH))o 4.37 (mo 1H)o 3.40 (mo 1H)o 2.09 (mo 2H)o 1.72 (mo 2H)o 1.59 (mo 2H)o 1.52 (mo 2H)o 1.45 (mo 2H)o 1.15 (do 3H)o 0.92 (to 3H). 13C-NMR (400 MHzo DMSO-d6): δ 149.73 (2C)o 147.00o 146.42o 136.20o 131.40o 118.88 (2C)o 112.10o 110.22o 52.71o 48.85o 32.66o 28.46o 23.96 (2C)o 19.68o 10.65. LCMS: (APCI) m/e 311 (M+H).
To a vial containing N2-cyclopentyl-6-(4-pyridyl)pyridine-2o3-diamine (0.1375 go 0.541 mmol) and a stir baro oxetan-3-one (0.076 mLo 1.19 mmol)o TFA (0.080 mLo 1.08 mmol)o and isopropyl acetate (5 mL) were added. To this was added sodium triacetoxyborohydride (0.137 go 0.649 mmol) over ˜2 min. The reaction was then allowed to stir at room temperature overnight. The resulting reaction mixture was poured onto water and extracted with ethyl acetate (×3). Combined organic extracts were dried over anhydrous magnesium sulfateo filteredo rotavapped down and loaded onto silica. The material was purified by column chromatography (24 g columno DCM/MeOH). Desired product fractions were combined and dried down to afford N2-cyclopentyl-N3-(oxetan-3-yl)-6-(4-pyridyl)pyridine-2o3-diamine as a pale yellow solid (8.4 mgo 5.01%). 1H-NMR (400 MHzo DMSO-d6): δ 8.49 (do 2H)o 7.86 (do 2H)o 7.18 (do 1H)o 6.32 (do 1H)o 6.01 (do 1H (NH))o 5.88 (do 1H (NH))o 4.89 (to 2H)o 4.54 (mo 1H)o 4.45 (mo 2H)o 4.37 (mo 1H)o 2.09 (mo 2H)o 1.71 (mo 2H)o 1.60 (mo 2H)o 1.52 (mo 2H). 13C-NMR (400 MHzo DMSO-d6): 149.81 (2C)o 146.96o 146.79o 138.25o 130.19o 119.15 (2C)o 113.04o 109.87o 77.34 (2C)o 52.59o 47.55o 32.72 (2C)o 23.89 (2C).LCMS: (APCI) m/e 311 (M+H).
In a 2.0-5.0 mL capacity microwave vial equipped with stir baro 6-chloro-N2-cyclopentyl-N3-sec-butyl-pyridine-2o3-diamine (byproduct recovered from H-72o 0.1559 g)o potassium acetate (0.171 go 3 equiv)o and pyrimidin-5-ylboronic acid (0.159 go 2.2 equiv) were combined in n-butanol (3 mL) and water (0.3 mL). The reaction mixture was flushed with nitrogen. Dichlorobis{[4-(NoN-dimethylamino)phenyl]di-t-butylphenylphosphino}palladium(II) (8.2 mgo 0.02 equiv) was then added and the vial sealed. The vial was then placed in the microwave reactor for 20 min at 110° C. The resulting mixture was poured onto water and extracted with ethyl acetate (×3). Organic extracts were combined and dried over anhydrous magnesium sulfate. Material was then filteredo concentratedo loaded onto silica and purified via flash chromatography (hexanes/ethyl acetate). Desired product fractions were combined and dried down to yield N2-cyclopentyl-6-pyrimidin-5-yl-N3-sec-butyl-pyridine-2o3-diamine as a light brown solid (73.8 mgo 40.7%). 1H-NMR (400 MHzo DMSO-d6): δ 9.24 (so 2H)o 8.98 (so 1H)o 7.19 (do 1H)o 6.57 (do 1H)o 5.94 (do 1H (NH))o 5.05 (do 1H (NH))o 4.35 (mo 1H)o 3.38 (mo 1H)o 2.07 (mo 2H)o 1.69 (mo 2H)o 1.57 (mo 2H)o 1.49 (mo 2H)o 1.44 (mo 2H)o 1.13 (do 3H)o 0.90 (to 3H). 13C-NMR (400 MHzo DMSO-d6): δ 155.88o 152.76 (2C)o 146.82o 133.80o 132.97o 130.98o 112.31o 109.62o 52.70o 48.82o 32.59 (2C)o 28.43o 23.88 (2C)o 19.65o 10.59. LCMS: (APCI) m/e 312 (M+H). LCMS: (APCI) m/e 312 (M+H).
To a vial containing N2-cyclopentyl-6-(p-tolyl)pyridine-2o3-diamine (0.278 go 1.04 mmol) and a stir baro oxetan-3-one (0.100 mLo 1.56 mmolo 1.5 equiv)o TFA (0.154 mLo 2.08 mmolo 2 equiv) and isopropyl acetate (5 mL) were added. To this was added sodium triacetoxyborohydride (0.331 go 1.56 mmolo 1.5 equiv) over ˜2 min. The reaction was then allowed to stir at room temperature. After 2 hourso the reaction mixture was poured onto water and extracted with EtOAc (×3). Combined organic extracts were dried over anhydrous magnesium sulfateo filtered and concentrated by rotavap. Material was then loaded onto silica and purified by flash chromatography (24 g columno hexanes/EtOAc). Desired product fractions were combined and dried down to yield N2-cyclopentyl-N3-(oxetan-3-yl)-6-(p-tolyl)pyridine-2o3-diamine as a pale purple solid (59.6 mgo 17.7%). 1H-NMR (400 MHzo DMSO-d6): δ 7.81 (do 2H)o 7.16 (do 2H)o 6.94 (do 1H)o 6.30 (do 1H)o 5.70 (mo 2H (NHs))o 4.90 (to 2H)o 4.50 (mo 1H)o 4.45 (to 2H)o 4.38 (mo 1H)o 2.29 (so 3H)o 2.08 (mo 2H)o 1.72 (mo 2H)o 1.60 (mo 2H)o 1.53 (mo 2H). 13C-NMR (400 MHzo DMSO-d6): δ 147.03o 141.89o 137.36o 135.68o 128.92 (2C)o 128.10o 124.90 (2C)o 114.06o 107.69o 77.51 (2C)o 52.51o 47.70o 32.79 (2C)o 23.84 (2C)o 20.76.LCMS: (APCI) m/e 324 (M+H).
To a vial containing N2-cyclopentyl-6-pyrimidin-5-yl-pyridine-2o3-diamine (0.213 go 0.834 mmol) and a stir baro oxetan-3-one (0.081 mLo 1.25 mmolo 1.5 equiv)o TFA (0.124 mLo 1.67 mmolo 2 equiv) and isopropyl acetate (5 mL) were added. To this was added sodium triacetoxyborohydride (0.212 go 1.00 mmolo 1.2 equiv) over ˜2 min. The reaction was then allowed to stir at room temperature overnight. The reaction was stoppedo poured onto watero and extracted with ethyl acetate (×4). Combined organic extracts were dried over anhydrous magnesium sulfateo filteredo concentrated by rotavap and loaded onto silica. Material was purified by column chromatography (hexanes/ethyl acetate). Desired product fractions were combined and dried down to afford N2-cyclopentyl-N3-(oxetan-3-yl)-6-pyrimidin-5-yl-pyridine-2o3-diamine as a yellow oil (21.4 mgo 8.24%). 1H-NMR (400 MHzo DMSO-d6): δ 9.26 (so 2H)o 9.01 (so 1H)o 7.17 (do 1H)o 6.33 (do 1H)o 5.99 (do 1H (NH))o 5.93 (do 1H (NH))o 4.89 (to 2H)o 4.53 (mo 1H)o 4.45 (to 2H)o 4.36 (mo 1H)o 2.07 (mo 2H)o 1.71 (mo 2H)o 1.60 (mo 2H)o 1.52 (mo 2H). 13C-NMR (400 MHzo DMSO-d6): 156.28o 153.09 (2C)o 147.34o 135.88o 132.79o 129.81o 113.22o 109.36o 77.34 (2C)o 52.61o 47.54o 32.66 (2C)o 23.84 (2C). LCMS: (APCI) m/e 312 (M+H).
To a vial containing N2-cyclopentyl-6-(4-methoxyphenyl)pyridine-2o3-diamine (0.250 go 0.882 mmol) and a stir baro isopropyl acetate (5 mL)o TFA (0.131 mLo 1.76 mmol) and 2-butanone (0.119 mLo 1.32 mmol) were added. To the stirring mixture was added sodium triacetoxyborohydride (0.224 go 1.06 mmol) over ˜2 min. The reaction was then allowed to stir at room temperature. After 45 mino saturated sodium bicarbonate (aq) was addedo and the organic layer isolated and loaded onto silica. The material was then purified by column chromatography (hexanes/EtOAc). Desired product fractions were combined and rotavapped down to afford N2-cyclopentyl-6-(4-methoxyphenyl)-N3-sec-butyl-pyridine-2o3-diamine as a viscous brown oil (0.2594 go 86.6%). 1H-NMR (400 MHzo DMSO-d6): δ 7.83 (do 2H)o 6.91 (do 2H)o 6.90 (do 1H)o 6.53 (do 1H)o 5.68 (do 1H (NH))o 4.66 (do 1H)o 4.33 (mo 1H)o 3.74 (so 3H)o 2.07 (mo 2H)o 1.69 (mo 2H)o 1.56 (mo 2H)o 1.50 (mo 2H)o 1.41 (mo 2H)o 1.12 (do 3H)o 0.90 (to 3H). 13C-NMR (400 MHzo DMSO-d6): 158.10o 146.59o 139.95o 133.08o 128.88o 125.88 (2C)o 113.69o 113.55o 107.37o 55.02o 52.63o 48.89o 32.74 (2C)o 28.53o 23.90 (2C)o 19.75o 10.62. LCMS: (APCI) m/e 340 (M+H).
To a vial containing N2-cyclopentyl-6-(4-methoxyphenyl)pyridine-2o3-diamine (0.250 go 0.882 mmol) and a stir baro isopropyl acetate (5 mL)o TFA (0.131 mLo 1.76 mmol)o and oxetanone (0.0851 mLo 1.32 mmol) were added. To the stirring mixture was added sodium triacetoxyborohydride (0.224 go 1.06 mmol) over ˜2 min. The reaction was then allowed to stir at room temperature for 3 hours. At this timeo saturated sodium bicarbonate (aq) was addedo and the organic layer isolated and loaded onto silica. The material was purified by column chromatography (hexanes/EtOAc). Desired product fractions were combinedo rotavapped downo and dried under vacuum at 40° C. to afford N2-cyclopentyl-6-(4-methoxyphenyl)-N3-(oxetan-3-yl)pyridine-2o3-diamine as a fluffy tan solid (169.3 mgo 56.5%). 1H-NMR (400 MHzo DMSO-d6): δ 7.85 (do 2H)o 6.93 (do 2H)o 6.89 (do 1H)o 6.29 (do 1H)o 5.65 (mo 2H (NH))o 4.89 (to 2H)o 4.49 (mo 1H)o 4.45 (mo 2H)o 4.37 (mo 1H)o 3.76 (so 3H)o 2.07 (mo 2H)o 1.72 (mo 2H)o 1.60 (mo 2H)o 1.52 (mo 2H). 13C-NMR (400 MHzo DMSO-d6): 158.38o 147.07o 141.87o 132.80o 127.69o 126.17 (2C)o 114.26o 113.74 (2C)o 107.16o 77.55 (2C)o 55.06o 52.53o 47.73o 32.81 (2C)o 23.85 (2C). LCMS: (APCI) m/e 340 (M+H).
A solution of N2-tert-butyl-6-(p-tolyl)pyridine-2o3-diamine (0.147 go 0.58 mmol) in isopropylacetate (3.0 mL) was successively treated with 2-butanone (63 mgo 0.86 mmolo 1.5 equiv) and then TFA (85 ulo 1.1 mmolo 2.0 equiv). After 30 mino the reaction was then treated with sodium triacetoxyborohydride (0.147 go 0.68 mmolo 1.2 euiv). After 1 hr.o LC/MS analysis showed clean conversion to the desired product. The reaction mixture was quenched with satd. aq. NaCl (5 mL) and extracted with ethyl acetate (3×10 mL). The combined extracts were dried (Na2SO4) and the solvent removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford N2-tert-butyl-6-(p-tolyl)-N3-sec-butyl-pyridine-2o3-diamine (0.154 go 86%) as a blue oil. LCMS: (APCI) m/e 312.2 (M+H).
A solution of N2-cyclopentyl-6-(p-tolyl)pyridine-2o3-diamine (0.238 go 0.89 mmol) in dichloromethane (2.5 mL) was treated with tert-butyl 2o2o2-trichloroethanimidate (0.39 go 1.8 mmolo 2 equiv) and then borontrifluoride etherate (22 uLo 0.18 mmolo 0.2 equiv). After stirring for 3 hrs.o LC/MS analysis showed partial conversion to the desired product and a significant amount of starting material. The reaction mixture was treated with an additional amount of tert-butyl 2o2o2-trichloroethanimidate (0.39 go 1.8 mmolo 2 equiv) and borontrifluoride etherate (22 uLo 0.18 mmolo 0.2 equiv). After stirring overnighto LC/MS analysis showed 50% conversion to the desired product and 50% starting material. After stirring overnighto LC/MS showed only slight increase in conversion to the desired product. The reaction mixture was quenched with satd. aq. ammonium chloride (5 mL) and the mixture was extracted with methylene chloride (3×15 mL). The combined organic extracts were dried (Na2SO4) and the solvent was removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford N2
A solution of N2-cyclopentyl-6-(p-tolyl)pyridine-2o3-diamine (0.238 go 0.89 mmol) in dichloromethane (2.5 mL) was treated with tert-butyl 2o22-trichloroethanimidate (0.39 go 1.8 mmolo 2 equiv) and then borontrifluoride etherate (22 uLo 0.18 mmolo 0.2 equiv). After stirring for 3 hrs.o LC/MS analysis showed partial conversion to the desired product and a significant amount of starting material. The reaction mixture was treated with an additional amount of tert-butyl 2o2o2-trichloroethanimidate (0.39 go 1.8 mmolo 2 equiv) and borontrifluoride etherate (22 uLo 0.18 mmolo 0.2 equiv). After stirring overnighto LC/MS analysis showed 50% conversion to the desired product and 50% starting material. The reaction was quenched with satd. aq. ammonium chloride (5 mL) and the mixture was extracted with ethyl acetate (3×15 mL methylene chloride). The combined organic extracts were dried (Na2SO4) and the solvent removed in vacuo. The residue was purified by flash chromatography (0-100% ethyl acetate/hexanes). The product co-eluted with an impurity from an unknown source. The product was re-purified by RP-HPLC to afford N3-tert-butyl-N2-cyclopentyl-6-(4-pyridyl)pyridine-2o3-diamine (7 mgo 4%) as a red solid. LCMS: (APCI) m/e 311.1 (M+H).
A solution of 6-(2-pyridyl)-N2-tetrahydrofuran-3-yl-pyridine-2o3-diamine (87 mgo 034 mmol) in methanol (1 mL) was successively treated with 2-butanone (38 mgo 0.51 mmolo 1.5 equiv) and then acetic acid (40 uLo 0.68 mmolo 2.0 equiv). After stirring for 30 mino the reaction mixture was then treated with sodium cyanoborohydride (33 mgo 0.51 mmolo 1.5 equiv). After stirring overnighto LC/MS analysis showed partial conversion to the desired product. Additional 1.5 equiv of 2-butanone and sodium cyanoborohydride was added to drive the reaction to product. LC/MS analysis showed clean conversion to the desired product. The reaction mixture was adsorbed onto a 12 g cartridge and purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford 6-(2-pyridyl)-N3-sec-butyl-N2-tetrahydrofuran-3-yl-pyridine-2o3-diamine (0.101 go 95%) as an orangish-yellow solid. LCMS: (APCI) m/e 313.1 (M+H).
A solution of 6-(2-pyridyl)-N2-tetrahydrofuran-3-yl-pyridine-2o3-diamine (77 mgo 0.30 mmol) in methanol (1 mL) was successively treated with tetrahydrofuran-3-one (39 mgo 0.45 mmolo 1.5 equiv) and then acetic acid (35 uLo 0.60 mmolo 2.0 equiv). After stirring for 30 mino the reaction mixture was then treated with sodium cyanoborohydride (29 mgo 0.45 mmolo 1.5 equiv). After stirring overnighto LC/MS analysis showed partial conversion to the desired product. Additional 1.5 equiv of tetrahydrofuran-3-one and sodium cyanoborohydride was added to drive the reaction to product. LC/MS analysis showed clean conversion to the desired product. The reaction mixture was adsorbed onto a 12 g cartridge and purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford 6-(2-pyridyl)-N2
A solution of N2-(3-methyltetrahydrofuran-3-yl)-6-(2-pyridyl)pyridine-2o3-diamine (79 mgo 0.29 mmol) in methanol (1 mL) was successively treated with 2-buantone (32 mgo 0.44 mmolo 1.5 equiv) and then acetic acid (33 uLo 0.58 mmolo 2.0 equiv). After stirring for 30 mino the reaction mixture was then treated with sodium cyanoborohydride (28 mgo 0.44 mmolo 1.5 equiv). After stirring overnighto LC/MS analysis showed partial conversion to the desired product. Additional 1.5 equiv of 2-butanone and sodium cyanoborohydride was added to drive the reaction to product. LC/MS analysis showed clean conversion to the desired product. The reaction mixture was adsorbed onto a 12 g cartridge and purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford N2-(3-methyltetrahydrofuran-3-yl)-6-(2-pyridyl)-N3-sec-butyl-pyridine-2o3-diamine (0.079 go 83%) as an orangish-yellow solid. LCMS: (APCI) m/e 327.2 (M+H); 1H NMR (CDCl3): δ 8.35 (bso 1H)o 8.22 (bso 1H)o 7.77 (do 2H)o 7.15 (mo 1H)o 6.82 (do 1H)o 3.87 (mo 4H)o 2.82 (mo 3H)o 2.02 (mo 4H)o 1.67 (so 3H)o 1.07 (mo 4H).
A solution of N2-(3-methyltetrahydrofuran-3-yl)-6-(2-pyridyl)pyridine-2o3-diamine (83 mgo 0.31 mmol) in methanol (1 mL) was successively treated with tetrahydrofuran-3-one (40 mgo 0.46 mmolo 1.5 equiv) and then acetic acid (35 uLo 0.61 mmolo 2.0 equiv). After stirring for 30 mino the reaction mixture was then treated with sodium cyanoborohydride (29 mgo 0.46 mmolo 1.5 equiv). After stirring overnighto LC/MS analysis showed partial conversion to the desired product. Additional 1.5 equiv of tetrahydrofuran-3-one and sodium cyanoborohydride was added to drive the reaction to product. LC/MS analysis showed clean conversion to the desired product. The reaction mixture was adsorbed onto a 12 g cartridge and purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford N2-(3-methyltetrahydrofuran-3-yl)-6-(2-pyridyl)-N3-tetrahydrofuran-3-yl-pyridine-2o3-diamine (0.082 go 79%) as a brown solid. LCMS: (APCI) m/e 341.1 (M+H); 1H NMR (CDCl3): δ 8.54 (do 1H)o 8.27 (do 1H)o 7.76 (mo 2H)o 7.25 (mo 1H)o 6.85 (do 1H)o 3.69 (mo 8H)o 2.02 (mo 5H)o 1.66 (so 3H).
A 40 mL vial was charged with N2-(3o3-difluoro-1-methyl-cyclobutyl)-6-(3-pyridyl) pyridine-2o3-diamine (0.271 go 0.933 mmol). A stir baro 3o3-difluorocyclobutanone (1.6 eq.o 0.158 go 1.49 mmol) TFA (1.2 eq.o 0.083 mLo 1.12 mmol)o and isopropyl acetate (6 mL) were added. To this was added sodium triacetoxyborohydride (1.5 eq.o 0.297 go 1.40 mmol). The reaction was stirred at 25° C. overnighto after which LCMS analysis suggested bulk of material had converted to desired product. The reaction was partitioned between water and ethyl acetate. The organic layer was isolatedo and the water layer extracted three times with ethyl acetate. Organic extracts were combined and dried over anhydrous magnesium sulfateo filteredo and concentrated via rotavap. The resulting concentrate was loaded onto silica and purified by column chromatography (hexanes/ethyl acetate)o to afford N3-(3o3-difluorocyclobutyl)-N2-(3o3-difluoro-1-methyl-cyclobutyl)-6-(3-pyridyl)pyridine-2o3-diamine (58.7 mgo 16.5%) as a pale peach-colored solid. LCMS: (APCI) m/e 381 (M+H); 1H NMR (DMSO-d6): δ 8.65 (mo 1H)o 8.42 (mo 1H)o 8.21 (mo 1H)o 7.38 (mo 1H)o 7.19 (do 1H)o 6.60 (do 1H)o 6.13 (bso 1H (NH))o 5.53 (do 1H (NH))o 3.81 (mo 1H)o 3.12 (mo 2H)o 2.96 (mo 2H)o 2.83 (mo 2H)o 2.53 (mo 2H)o 1.65 (so 3H).
A 40 mL vial was charged with 6-(4-fluorophenyl)-N2-(3-methyltetrahydrofuran-3-yl) pyridine-2o3-diamine (0.200 go 0.696 mmol). A stir baro 3o3-difluorocyclobutanone (1.2 eq.o 0.089 go 0.835 mmol)o TFA (1.2 eq.o 0.062 mLo 0.835 mmol)o and isopropyl acetate (6 mL) were added. To this was added sodium triacetoxyborohydride (1.5 eq.o 0.221 go 1.04 mmol). The reaction was stirred at 25° C. overnight. LCMS after overnight reaction suggested conversion to desired product. The reaction was partitioned between water and ethyl acetate. The organic layer was isolatedo and water layer extracted three times with ethyl acetate. Combined organic extracts were dried over anhydrous magnesium sulfateo filteredo and concentrated via rotavap. The resulting concentrate was loaded onto silica and purified by column chromatography (hexanes/ethyl acetate)o to afford N3-(3o3-difluorocyclobutyl)-6-(4-fluorophenyl)-N2-(3-methyltetrahydrofuran-3-yl) pyridine-2o3-diamine (102 mgo 38.8%) as a pale tan solid. LCMS: (APCI) m/e 378 (M+H); 1H NMR (DMSO-d6): δ 7.90 (mo 2H)o 7.19 (mo 2H)o 7.03 (do 1H)o 6.55 (do 1H)o 5.67 (bso 1H (NH))o 5.54 (do 1H (NH))o 4.00 (do 1H)o 3.91 (do 1H)o 3.82 (mo 2H)o 3.77 (mo 1H)o 3.10 (mo 2H)o 2.54 (mo 1H)o 2.41 (mo 1H)o 2.01 (mo 1H)o 1.58 (so 3H).
A 40 mL vial was charged with 4-[5-amino-6-[(3-methyltetrahydrofuran-3-yl)amino]-2-pyridyl]-NoN-dimethyl-benzamide (279 go 0.820 mmol) and a stir baro 3o3-difluorocyclobutanone (1.2 eq.o 104 mgo 0.983 mmol) TFA (1.2 eq.o 0.74 mLo 0.983 mmol) and isopropyl acetate (5 mL. 0.2 M) were added. To this was added sodium triacetoxyborohydride (1.5 eq.o 261 mgo 1.23 mmol) over ˜2 min. The reaction was then allowed to stir at room temperature. After 16 ho the reaction was complete by LCMS and was partitioned between 25 mL of water and 25 mL of EtOAc. The water layer was extracted 3×25 mL EtOAco dried over Na2SO4o filtered and concentrated under reduced pressure. The residue was purified on silica gel (40 go 0-50% EtOAc/hexanes) to provide 110 mg of 4-[5-[(3o3-difluorocyclobutyl)amino]-6-[(3-methyltetrahydrofuran-3-yl)amino]-2-pyridyl]-NoN-dimethyl-benzamide (31%) as a yellow film. LCMS (APCI) m/e 431.1 (M+H); 1H NMR (CDCl3): δ 7.92 (do 2H)o 7.41 (do 2H)o 7.06 (do 1H)o 6.58 (do 1H)o 4.56 (bso 1H)o 4.00 (mo 2H)o 3.95 (mo 2H)o 3.92 (bso 1H)o 3.00 (mo 6H)o 2.47 (mo 3H)o 2.02 (mo 2H)o 1.66 (mo 3H)o 1.22 (to 2H).
A 100 mL 14/22 RBF was charged with ethyl 2o6-dichloro-5-nitro-pyrimidine-4-carboxylate (500 mgo 1.88 mmol)o THF (4 mL)o fitted with a balloon of nitrogen and cooled to −78° C. The reaction was then treated with DiPEA (1.5 eq.o 2.8 mmolo 0.5 mL) and then treated dropwise with a solution of cyclopentanamine (1.0 eq.o 1.88 mmolo 160 mg) in THF (3 mL) over a 15 min period. The reaction mixture was allowed to gradually warn to RT overnight. After 16 ho the reaction was partitioned between 25 mL of EtOAc and 25 mL of H2O the water layer back extracted 2×25 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide ethyl 2-chloro-6-(cyclopentylamino)-5-nitro-pyrimidine-4-carboxylate (K-19) as a viscous yellow oil (450 mgo 76%) and the material was used in the next step without further purification. 1H NMR (CDCl3): δ 8.50 (bso 1H)o 4.50 (mo 1H)o 4.46 (q 2H) 2.18 (mo 2H)o 1.72 (mo 3H)o 1.56 (mo 3H)o 1.40 (to 3H); LCMS (APCI) m/e 315.0 (M+H).
A 40 mL vial was charged with the chloropyrimidine (500 mgo 1.6 mmol)o THF (3 mL)o water (1.5 mL)o p-tolylboronic acid (2 eq.o 432 mgo 3.2 mmol)o sodium carbonate (4 eq.o 674 mgo 6.4 mmol) and then fitted with a stir baro and septa. The solution was degassed using a stream of nitrogen directly in the solution and an exit needle for 20 min. The reaction mixture was then treated with tetrakis(triphenylphosphine)palladium(0) (0.1 eq.o 184 mgo 0.159 mmol) and fitted with a nitrogen balloon and stirred at 60° C. After 0.5 ho LCMS confirmed complete consumption of the starting material and the major product exhibited the correct MS for the desired product. The reaction mixture was allowed to cool to RT and then partitioned between 20 mL of EtOAc and 20 mL water. The aqueous layer was back extracted 2×20 mL EtOAc and the combined organic layer dried over Na2SO4. The solvent was removed under reduced pressure and the resulting residue was purified on silica gel (40 go 0-30% EtOAc/hexanes) to provide ethyl 6-(cyclopentylamino)-5-nitro-2-(p-tolyl)pyrimidine-4-carboxylate (K-20) as a yellow solid (400 mgo 68%). 1H NMR (CDCl3): δ 8.35 (bso 1H)o 8.23 (do 2H)o 7.18 (do 2H) 4.65 (mo 1H)o 4.41 (qo 2H)o 2.32 (so 3H)o 2.21 (mo 2H)o 1.65 (mo 4H)o 1.47 (mo 2H)o 1.32 (to 3H); LCMS (APCI) m/e 371.1 (M+H).
A 40 mL vial was charged with ethyl 6-(cyclopentylamino)-5-nitro-2-(p-tolyl)pyrimidine-4-carboxylate (520 mgo 1.4 mmol)o EtOH (8 mL)o water (2 mL)o ammonium chloride (1 eq.o 1.4 mmolo 75 mg)o iron powder (5 eq.o 7 mmolo 392 mg)o fitted with a stir baro purged with nitrogeno sealed and stirred at 80° C. After 16 ho the reaction was cooled to RT and filtered using a syringe filter. The reaction residue was washed 3×5 mL of EtOH allowed to settle and filtered. The yellow solution was concentrated under reduced pressure to provide ethyl 5-amino-6-(cyclopentylamino)-2-(p-tolyl)pyrimidine-4-carboxylate (K-33) (270 mgo 55%) as a brown powder. The material was pure by LCMS and was used directly in the hydrolysis step. LCMS (APCI) m/e 341.1 (M+H).
A 20 mL vial was charged with ethyl 5-amino-6-(cyclopentylamino)-2-(p-tolyl)pyrimidine-4-carboxylate (118 mgo 0.9347 mmol)o THF (1 mL)o methanol (0.5 mL)o H2O (0.5 mL)o LiOH—H2O (1.5 eq.o 0.52 mmolo 22 mg)o fitted with a stir bar and stirred at RT. After 3 do crude LCMS confirmed complete consumption of the starting ethyl ester. The reaction mixture was partitioned between 25 mL of water and 25 mL of EtOAc. The water layer was back extracted 2×25 mL of EtOAc but the water layer remained yellow with a pH=8. The water layer was treated with 1 mL of 1 N HCl and a precipitate formed and the pH=4. The acidic aqueous layer was extracted 3×20 mL DCM and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide 5-amino-6-(cyclopentylamino)-2-(p-tolyl)pyrimidine-4-carboxylic acid (K-35) as a reddish solid (40 mgo 37%). The material was pure by LCMS and was used directly in the amide coupling step. LCMS (APCI) m/e 313.1 (M+H).
A 4 mL vial was charged with 5-amino-6-(cyclopentylamino)-2-(p-tolyl)pyrimidine-4-carboxylic acid (40 mgo 0.128 mmol)o DMF 1 mL)o DiPEA (3 eq.o 0.384 mmolo 50 mg)o 1-[Bis(dimethylamino)methylene]-1H-1o2o3-triazolo[4o5-b]pyridinium 3-oxide hexafluorophosphate (HATUo 1.5 eq.o 0.192 mmolo 73 mg) and the reaction was stirred for 20 minutes at RT. The reaction was then treated with methylamine hydrochloride (1.5 eq.o 0.192 mmolo 13 mg) and the reaction was stirred at RT. After 16 ho crude LCMS complete consumption of the starting carboxylic acid. The reaction mixture was treated with 2 mL water the afforded 5-amino-6-(cyclopentylamino)-N-methyl-2-(p-tolyl)pyrimidine-4-carboxamide (K-31) as an off-white precipitate that was isolated by filtration (40.0 mgo 96%). The material was pure by LCMS and was used directly in the cyclization step. LCMS (APCI) m/e 326.1 (M+H).
A 20 ml microwave vial was charged with a solution of 5-amino-6-(cyclopentylamino)-N-methyl-2-(p-tolyl)pyrimidine-4-carboxamide in acetoneo p-Toluenesulfonic acid monohydrate (0.25 eq.o 190.22 MWo 0.031 mmolo 69 mg)o glacial acetic acid (1 mL) sealed and heated at 70° C. After 16 ho the starting material was consumed and a major and minor product with the correct M+H+ was observed in the crude LCMS. The reaction mixture was cooled to RT and partitioned between 15 mL of water and 15 mL of EtOAc. There was a precipitate in the EtOAc layer. The aqueous later was back extracted 2×10 mL of EtOAC and the combined organic layer was concentrated under reduced pressure without drying over Na2SO4 and concentrated under reduced pressure to provide 45 mg of a yellow solid. The solid was triturated 5×3 mL of ether to provide a yellow powder that was dried under reduced pressure to afford 9-cyclopentyl-N,8,8-trimethyl-2-(p-tolyI)-5,7-dihydro-4H-purine-6-carboxamide (K-34) (23.0 mgo 50.9%). LCMS (APCI) m/e 366.1 (M+H). The ether layer was further purified as described for K-36.
The combined ether layer from K-34 was concentrated under reduced pressure to provide 12 mg of a 1:1 mixture of the major and minor products from K-34. A 5-inch pipette was plugged with cotton and filled ¾ with silica gel. The silica gel was washed with 3 column volumes of 10% EtOAc/hexanes. The crude residue was dissolved in the smallest amount of DMC possible and loaded onto the column. The material was eluted using 12 column volumes of 10% EtOAc/hexanes via a pipette bulb collecting 2 fractions per column volumeo then 8 column volumes of 50% EtOAc/hexanes was flushed through the columno which resulted in the elution of 8-(cyclopentylamino)-2,2,3-trimethyl-6-(p-tolyl)-1H-pyrimido[5,4-d]pyrimidin-4-one (K-36) as a residue (2 mgo 3.7%). LCMS (APCI) m/e 366.1 (M+H).
A 40 mL vial was charged with 2-chloro-6-methyl-3-nitro-pyridine (1.0 go 5.79 mmol)o a stir baro DMF (5 mLo 1 M)o DiEA (3 eq.o 3.1 mLo 17.4 mmol)o N-benzylcyclopentanamine: hydrochloride (1.1 eq.o 6.37 mmolo 1.35 g)o 80° C. overnight. After 16 ho the starting material had been consumed and the desired product was confirmed in the crude LCMS. The reaction mixture was partitioned between 75 mL of water and 75 mL EtOAc. The water layer was back extracted 3×50 mL EtOAc and the combined organic layer was dried over Na2SO4. The residue was purified on silica gel (80 go 0-30% EtOAc/hexanes) to provide 1.2 g of N-benzyl-N-cyclopentyl-6-methyl-3-nitro-pyridin-2-amine (85%) as a yellow solid. LCMS (APCI) m/e 312.1 (M+H).
A solution of N-benzyl-N-cyclopentyl-6-methyl-3-nitro-pyridin-2-amine (0.69 go 2.2 mmol) in 1o4-dioxane (10 mL) was treated with selenium dioxide (0.370 go 3.3 mmolo 1.5 equiv) and then warmed to 100 C. After stirring overnighto LC/MS analysis showed partial conversion to the desired product. Additional selenium dioxide was added and the reaction was progressed an additional 8 hrs. LC/MS analysis showed no further progress of the reaction. The mixture was dried onto silica (10 g) and purified by flash chromatography (24 g silicao 0-50% methylene chloride/hexanes) to afford 6-[benzyl(cyclopentyl)amino]-5-nitro-pyridine-2-carbaldehyde (0.498 go 69%) as a orangish-yellow solid. LCMS (APCI) m/e 326.1 (M+H).
A solution of 6-[benzyl(cyclopentyl)amino]-5-nitro-pyridine-2-carbaldehyde (0.243 go 0.75 mmol) in anhydrous dichloromethane (3 mL) was cooled to −78° C. and then successively treated with allyltrimethylsilane (0.142 mLo 90 mmolo 1.2 equiv) and then dropwise titanium tetrachloride (40 uLo 0.37 mmolo 0.5 equiv). After 1 hr.o LC/MS analysis showed complete and clean conversion to the desired product as two peakso consistent with one being the Ti-complexed and the other as the non-complexed product. The reaction mixture was quenched with satd. aq. ammonium chloride (10 mL) and then diluted with methylene chloride (10 mL). The layers were separated and the aqueous layer was further extracted with methylene chloride (2×15 mL). The combined organic extracts were dried (Na2SO4) and the solvent removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford the 1-[6-[benzyl(cyclopentyl)amino]-5-nitro-2-pyridyl]but-3-en-1-ol (0.20 go 73%) in two different peaks as a yellow solid. LCMS (APCI) m/e 368.1 (M+H).
A solution of 1-[6-[benzyl(cyclopentyl)amino]-5-nitro-2-pyridyl]but-3-en-1-ol (0.20 go 0.54 mmol) in methanol (2 mL) was degassed with nitrogen balloon for 15 min. The reaction mixture was then treated with Pd/C (58 mgo 54 umolo 0.1 equiv) and then charged with hydrogen via balloon. After stirring overnighto LC/MS analysis showed only reduction of the nitro and olefin with the benzyl moiety being retained. The sample was filtered through Celite® and the 1-[5-amino-6-[benzyl(cyclopentyl)amino]-2-pyridyl]butan-1-ol (0.16 go 87%) was carried forward without any further purification. LCMS (APCI) m/e 340.1 (M+H).
A solution of 1-[5-amino-6-[benzyl(cyclopentyl)amino]-2-pyridyl]butan-1-ol (0.16 go 0.47 mmolo) in anhydrous methanol (2 mL) was treated with 2-butanone (68 mgo 0.94 mmolo 2.0 equiv) and then acetic acid (57 uLo 0.94 mmolo 2.0 equiv). After 1 hr.o the reaction was treated with sodium cyanoborohydride (45 mgo 0.71 mmolo 1.5 equiv). After stirring overnighto LC/MS analysis showed conversion to the desired product. In additiono the mixture had two peaks consistent with the formation of the product from acetone and acetaldehyde. The mixture was dissolved onto silica and purified by flash chromatography (12 g silicao 0-100% ethyl acetate/hexanes) to afford 1-[6-[benzyl(cyclopentyl)amino]-5-(sec-butylamino)-2-pyridyl]butan-1-ol (44 mgo 24%) as a red oil. LCMS (APCI) m/e 396.1 (M+H).
A solution of 1-[6-[benzyl(cyclopentyl)amino]-5-(sec-butylamino)-2-pyridyl]butan-1-ol (45 mgo 0.11 mmol) in acetone (0.5 mL) was treated with Dess-Martin reagent (58 mgo 0.14 mmolo 1.2 equiv). After stirring overnighto LC/MS analysis showed clean conversion to the desired ketone. The reaction mixture was filtered through a plug of silica (1 go ethyl acetate) and the filtrated was dried in vacuo. The residue was carried forward without any further purification. LCMS (APCI) m/e 394.1 (M+H).
A solution of 1-[6-[benzyl(cyclopentyl)amino]-5-(sec-butylamino)-2-pyridyl]butan-1-ol (44 mgo 0.11 mmol)o in anhydrous methanol (0.5 mL) was degassed with N2 balloon. After 15 min.o the reaction was treated with 20% palladium hydroxide on carbon (50% wettedo 32 mgo 23 umolo 0.2 equiv). The reaction mixture was then subjected to bubbling H2 via balloon and then left to react. After stirring overnighto LC/MS analysis showed conversion to the desired product and the formation of some bi-products. The sample was filtered through Celite and dried in vacuo. The sample was then purified by RP-HPLC to provide 1-[6-(cyclopentylamino)-5-(sec-butylamino)-2-pyridyl]butan-1-one (5.5 mgo 16%) as a yellow film. LCMS (APCI) m/e 304.1 (M+H).
A 40 mL vial was charged with 2-chloro-6-methyl-3-nitro-pyridine (400 mgo 2.32 mmol)o a stir baro DMF (5 mLo 0.5 M)o DiPEA (2 eq.o 0.8 mLo 4.64 mmol)o N-benzyl-3o3-difluoro-cyclobutanamine (2 eq.o 4.64 mmolo 0.914 g)o and stirred at 80° C. for 72 h. Crude LCMS confirmed the reaction was complete. The reaction mixture was partitioned between 50 mL of water and 50 mL of EtOAc. The water layer was back extracted 3×50 mL EtOAC and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified on silica gel (80 go 0-30% EtOAc/hexanes) to provide 700 mg of N-benzyl-N-(3o3-difluorocyclobutyI)-6-methyl-3-nitro-pyridin-2-amine (90%) as a yellow solid. LCMS (APCI) m/e 334.1 (M+H).
A 40 mL vial was charged with N-benzyl-N-(3o3-difluorocyclobutyI)-6-methyl-3-nitro-pyridin-2-amine (700 mgo 2.10 mmol)o dioxane (0.4 Mo 5 mL)o SeO2 (2 eq.o 4.2 mmolo 466 mg)o purged with nitrogen and stirred at 100° C. After 16 ho the reaction was complete by crude LCMS and was directly purified on silica gel (40 go 0-50% EtOAc/hexanes) to provide 540 mg of 6-[benzyl-(3o3-difluorocyclobutyl)amino]-5-nitro-pyridine-2-carbaldehyde (74%) as a yellow oil. LCMS (APCI) m/e 348.1 (M+H).
A solution of 6-[benzyl-(3o3-difluorocyclobutyl)amino]-5-nitro-pyridine-2-carbaldehyde (540 mgo 1.55 mmol) in anhydrous dichloromethane (6 mLo 0.25 M) was cooled to −78° C. and then successively treated with allyltrimethylsilane (0.25 mLo 1.86 mmolo 1.2 equiv.) and then dropwise titanium tetrachloride (85 μLo 0.78 mmolo 0.5 equiv). After 2 hr.o LC/MS analysis showed complete and clean conversion to the desired product as two peakso consistent with one being the Ti-complexed and the other as the non-complexed product. The reaction mixture was quenched with satd. aq. ammonium chloride (20 mL) and then diluted with methylene chloride (20 mL). The layers were separated and the aqueous layer was further extracted with methylene chloride (2×30 mL). The combined organic extracts were dried (Na2SO4) and the solvent removed in vacuo. The residue was purified by flash chromatography (80 g silicao 0-40% ethyl acetate/hexanes) to afford the 11-[6-[benzyl-(3o3-difluorocyclobutyl)amino]-5-nitro-2-pyridyl]but-3-en-1-ol (0.320 go 52%) as a yellow oil. LCMS (APCI) m/e 390.1 (M+H).
A 40 mL vial was charged with 1-[6-[benzyl-(3o3-difluorocyclobutyl)amino]-5-nitro-2-pyridyl]but-3-en-1-ol (320 mgo 0.822 mmol)o DCM (8 mLo 0.1 M)o sodium bicarbonate (10 eq.o 8.22 mmolo 690 mg) and stirred for 5 min. The reaction mixture was then treated with Dess-Martin Periodinane (1.5 eq.o 1.23 mmolo 523 mg) and stirred at RT. After 3 h. the reaction was 50% complete. The reaction was treated with Dess-Martin Periodinane (1.5 eq.o 1.23 mmolo 523 mg) and stirred at RT overnight. After 16 ho the reaction was complete by crude LCMS. The reaction mixture was partitioned between 20 mL DCM and 20 mL 1M NaOH (aq); stir for 10 minutes. The aqueous layer was extracted extract with DCM (3×20 mL). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified on silica gel (40 go 0-30% EtOAc/hexanes) to provide 156 mg of 1-[6-[benzyl-(3o3-difluorocyclobutyl)amino]-5-nitro-2-pyridyl]but-3-en-1-one (318 mgo 49%) as a yellow solid. LCMS (APCI) m/e 388.1 (M+H).
156 mgs of 1-[6-[benzyl-(3o3-difluorocyclobutyl)amino]-5-nitro-2-pyridyl]but-3-en-1-oneo (K-96) was dissolved in 10 ml of MeOH. The solution was degassed and flushed with nitrogen. The solution was charged with 50 mg of 20% Pd(OH)2 on carbon followed by a hydrogen balloon. The reaction was stirred at RT for 18 h. LC-MS showed one peak with the mass of the desired product. There was no evidence of any starting material. The reaction was worked up by filtration. The MeOH was evaporated to give 97 mgs (90%) of a brown solid. LC-MS and NMR confirms the structure and purity. LCMS (APCI) m/e 270.1 (M+H); 1H NMR (d6-DMSO): δ 7.20 (do 1H)o 6.73 (do 1H)o 6.30 (do 1H)o 5.65 (bso 2H)o 4.18 (bso 1H)o 3.03 (mo 2H)o 2.91 (to 2H)o 2.48 (mo 2H)o 1.59 (q 2H)o 0.951 (to 3H).
1-[5o6-bis[(3o3-difluorocyclobutyl)amino]-2-pyridyl]butan-1-one (P-47) was prepared using the standard reductive amination conditions (similar to L-29). LCMS (APCI) m/e 360.1 (M+H); 1H NMR (d6-DMSO): δ 7.37 (do1H)o 6.61 (do 1H)o 6.37 (do 1H)o 6.05 (do 1H)o 4.23 (bso 1H)o 3.86 (bso 1H)o 3.35 (mo 2H)o 3.11 (mo 4H)o 2.93 (mo 2H)o 2.43 (mo 2H)o 1.46 (mo 2H)o0.960 (to 3H).
2-Chloro-4-(cyclopentylamino)-5H-pyrimido[4o5-b][1o4]oxazin-6-one (550 mgo 2.05 mmol) was dissolved in dry THF under argon. A 1 M solution of BH3-THF complex (10.0 equiv) was slowly added. The mixture was stirred for 1 h. The mixture was diluted with water. The aqueous phase was extracted with ethyl acetate. The organic layer was dried over Na2SO4o filteredo and concentrated under reduced pressure. The product was purified by silica gel chromatography (hexane/ethyl acetate as eluent) to provide the title compound as a solid (350 mgo 67.1%). LC-MS m/z: ES+ [M+H]+:255.1; tR=2.23 min.
A mixture of ethyl 8-hydroxy-1o7-naphthyridine-6-carboxylate (300 mg) in POCl3 (7 ml) was stirred for 30 mins at 110° C. When all starting material was converted to the producto the mixture was cooled downo concentratedo then the residue obtained was poured onto crushed ice and stirred for 15 mins. The pH of the aqueous mixture was basified to pH 8 at 0° C. by careful addition of aq. sat. sodium carbonate. The product was extracted three times with DCMo the organic phases were combinedo washed with brineo driedo filtered then concentrated. The residue obtained was purified by silica-gel column chromatography (12 g) using a gradient 0-50% Ethyl acetate in hexanes. The desired product was isolated in 58% yield (189 mg). LC-MS m/z: ES+ [M+H]+:237.1; (B05) tR=1.99 mins.
A mixture of pyrido[3o2-d]pyrimidine-2o4-diol (1 g)o POCl3 (10 ml) and PCl5 (5.11 g) was heated at 120° C. for 12 h under argon. The reaction mixture was cooled down to rto POCl3 was evaporated under reduced pressureo and the residue obtained was taken up in DCM. Ice and water was added the mixture was cooled down to 0° C.o and the pH was adjusted to 8 by slow addition of aq saturated NaHCO3. The aqueous phase was extracted three times with DCMo the organic phases were combined then washed successively with water and brine. The organic phase was filteredo concentratedo and the residue obtained was purified by silica-gel column chromatography (40 g) using a gradient 0-20% EtOAc in hexanes providing 2o4-dichloropyrido[3o2-d]pyrimidine in 42% yield (510 mg). 1H NMR (500 MHzo CDCl3) δ 9.15 (ddo J=4.1o 1.4 Hzo 1H)o 8.33 (ddo J=8.6o1.4 Hzo 1H)o 7.92 (ddo J=8.6o4.2 Hzo 1H); LC-MS m/z: ES+ [M+H]+:200.1; (B05) tR=2.0 m.
A mixture composed of 2-chloro-4-(tetrahydrofuran-3-ylamino)-5H-pyrimido[4o5-b][1o4]oxazin-6-one (45.0 mgo 0.166 mmol)o [(E)-pent-1-enyl]boronic acid (56.8 mgo 0.498 mmol)o and Potassium carbonate (68.9 mgo 0.499 mmol) in Toluene (0.800 mL)o Ethanol (0.20 ml)o and water (0.20 ml) was degassed for 10 mins by bubbling argon. Tetrakis(triphenylphosphine)palladium(0) (38.4 mgo 0.0332 mmol) was addedo the vial was sealed then stirred at 100° C. for 16 h. The mixture was cooled down to rto diluted with ethyl acetate and aq. Sat. NaHCO3. The organic phase was separated and the aqueous phase was further extracted twice with EtOAc. The organic phases were combinedo washed with brineo dried over sodium sulfateo filteredo and concentrated. The residue obtained was purified by silica-gel column chromatography using a gradient 0-10% MeOH in DCM to afford the title compound (23.0 mgo 46%). LC-MS m/z: ES+[M+H]+:305.2o LCMS; tR=4.14 mins (10 mins run).
To a solution of 4-(cyclopentylamino)-2-[(E)-pent-1-enyl]-5H-pyrimido[4o5-b][1o4]thiazin-6-one (150 mgo 0.471 mmol) in dry Tetrahydrofuran (10.0 mL) under argon was added BH3·THF (0.405 go 4.71 mmol) dropwise. Then the mixture was stirred for 1 h at rto diluted with water and ethyl acetateo and the organic phase was separated. The organic layer was washed with brineo dried over Na2SO4o filteredo concentrated and the residue obtained was purified by silica-gel column chromatography using a gradient 0-100% ethyl acetate in hexanes as eluent to afford the title compound (102 mgo 71%). 1H NMR (500 MHzo CD3OD) δ 4.38 (po J=6.8 Hzo 1H)o 3.52-3.47 (mo 2H)o 3.10-3.05 (mo 2H)o 2.51 (to J=7.5 Hzo 2H)o 2.04 (de J=14.1o 6.5 Hzo 2H)o 1.74 (do J=6.5 Hzo 2H)o 1.70-1.59 (mo 4H)o 1.49 (tdo J=13.7o 7.1 Hzo 2H)o 1.38-1.25 (mo 4H)o 0.89 (to J=6.9 Hzo 3H). LC-MS m/z: ES+ [M+H]+:307.2; tR=3.70 min.
A mixture of 2-chloro-4-(cyclopentylamino)-5H-pyrimido[4o5-b][1o4]thiazin-6-one (250 mg)o 1-Pentenylboronic acid (100 mg)o and potassium carbonate (364 mg) in Toluene (1.5 ml)o Ethanol (0.7 ml)o and water (0.7 ml) was degassed for 10 mins by bubbling argon. Pd(PPh3)4 was addedo the vial was sealed and the mixture was stirred at 100° C. for 12 h. The mixture was cooled down to rt and the product was partitioned between aq. sat. NaHCO3 and EtOAc. The separated organic layer was separatedo washed with brineo dried over Na2SO4o filteredo concentrated and the residue obtained was purified by silica-gel column chromatography using a gradient 0-100% EtOAc in Hexane as an eluent to afford the title compound (155 mgo 56%). 1H NMR (500 MHzo CD3OD) δ 7.02-6.92 (mo 1H)o 6.22 (do J=15.4 Hzo 1H)o 4.44 (po J=6.7 Hzo 1H)o 3.53 (so 2H)o 2.21 (qo J=7.2 Hzo 2H)o 2.08 (dto J=12.3o 6.1 Hzo 2H)o 1.82-1.71 (mo 2H)o 1.66 (ddo J=14.9o 7.9 Hzo 2H)o 1.53 (tdo J=14.6o 7.2 Hzo 4H)o 0.96 (to J=7.4 Hzo 3H). LC-MS m/z: ES+ [M+H]+:319.2; tR=4.82 mins.
To a solution of 1-[1-benzyl-8-(2-pyridylamino)-3o4-dihydro-2H-quinolin-6-yl]pentan-1-one in anhydrous EtOAc (5 mL) under argon atmosphere and Pd—C was added carefully. The flasko was connected a H2 balloon. The resulting suspension was stirred at room temperature for 6 h and after this time the reaction was stopped filtering the mixture through Celite®. The solvent was evaporated under reduced pressure obtaining a reaction crude that was purified by flash chromatography (0-50% EtOAc/hexane). 1H NMR (500 MHzo CD3OD) δ 7.95 (do J=4.3 Hzo 1H)o 7.60 (do J=1.8 Hzo 1H)o 7.52 (so 1H)o 7.51-7.46 (mo 1H)o 6.69-6.65 (mo 1H)o 6.49 (do J=8.5 Hzo 1H)o 3.37-3.33 (mo 2H)o 3.29 (dto J=2.9o 1.5 Hzo 2H)o 2.85-2.79 (mo 4H)o 1.90 (dto J=11.9o 6.1 Hzo 2H)o 1.62 (dto J=20.8o 7.6 Hzo 2H)o 1.41-1.32 (mo 2H)o 0.92 (to J=7.4 Hzo 3H). LC-MS m/z: ES+ [M+H]+:310.2o tR: 3.29 min
Additional Synthetic Schema for Heterocycloalkyl Aromatics
A 40 mL vial was charged with 2-chloro-3-nitro-6-(trifluoromethyl)pyridine (0.5 go 2.21 mmol)o a stir baro THF (3 mLo 0.5 M)o DiEA (2 eq.o 0.8 mLo 4.41 mmol)o cyclopentanamine in 2 mL of THF (1 eq.o 2.21 mmolo 188 mg) and the reaction was stirred at RT. After 2 ho the reaction was complete by LCMS and the reaction was then partitioned between 50 mL of water and 50 mL EtOAc. The water layer was extracted 3×30 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide 370 mg of an oil (60%) that was >90% pure by LCMS and was used in the next step without further purification. LCMS: (APCI) m/e 276.0 (M+H).
A 40 mL vial was charged with 2-chloro-3-nitro-6-(trifluoromethyl)pyridine (0.5 go 2.21 mmol) a stir baro THF (3 mLo 0.5 M)o DiEA (2 eq.o 0.8 mLo 4.41 mmol)o 3-methyltetrahydrofuran-3-amine in 2 mL of THF (1.1 eq.o 2.43 mmolo 246 mg) and the reaction was stirred at RT. After 24 ho the reaction was ˜60% complete an additional 0.5 eq. of the amine was added (1.22 mmol 123 mg). After 48 ho the reaction was complete by LCMS and the reaction was then partitioned between 50 mL of water and 50 mL EtOAc. The water layer was extracted 3×30 mL EtOAc and the combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to provide a yellow residue that was purified on silica gel (80 go 0-30% EtOAc/hexanes) to afford 410 mg of N-(3-methyltetrahydrofuran-3-yl)-3-nitro-6-(trifluoromethyl)pyridin-2-amine as a yellow oil (63%). LCMS: (APCI) m/e 292.0 (M+H).
A 20 mL microwave vial was charged with N-cyclopentyl-3-nitro-6-(trifluoromethyl)pyridin-2-amine (370 mgo 1.34 mmol)o EtOH (8 mL)o water (2 mL)o ammonium chloride (1 eq.o 1.34 mmolo 72 mg)o iron shavings (5 eq.o 6.72 mmolo 375 mg)o fitted with a stir baro was bubbled with nitrogen for 10 mino sealed and stirred at 80° C. After 4 ho the reaction was cooled to RT and filtered using a ISCO sample cartridge with wet Celite® (MeOH) and washed several times with MeOH. The yellow solution dried over Na2SO4 and was concentrated under reduced pressure to provide 320 mg (97%) as a yellow oil. The material was pure by LCMS and was used directly in the next step. LCMS: (APCI) m/e 246.1 (M+H).
A 20 mL microwave vial was charged with N-(3-methyltetrahydrofuran-3-yl)-3-nitro-6-(trifluoromethyl)pyridin-2-amine (410 mgo 1.41 mmol)o EtOH (8 mL)o water (2 mL)o ammonium chloride (1 eq.o 1.41 mmolo 75 mg)o iron shavings (5 eq.o 7.042 mmolo 393 mg)o fitted with a stir baro was purged with nitrogeno sealed and stirred at 80° C. After 3 ho the reaction was cooled to RT and filtered using an ISCO sample cartridge with wet Celite® (MeOH) and washed several times with MeOH. The yellow solution dried over Na2SO4o filtered and was concentrated under reduced pressure to provide 350 mg (95%) of N2-(3-methyltetrahydrofuran-3-yl)-6-(trifluoromethyl)pyridine-2o3-diamine as an orange film. The material was pure by LCMS and was used directly in the next step. LCMS: (APCI) m/e 262.1 (M+H).
A 40 mL vial was charged with N2-cyclopentyl-6-(trifluoromethyl)pyridine-2o3-diamine (320 go 1.30 mmol) and a stir baro 2-butanone (1.1 eq.o 103 mgo 1.44 mmol)o TFA (2 eq.o 0.194 mLo 2.61 mmol)o and isopropyl acetate(4 mLo 0.3 M) were added. To this was added sodium triacetoxyborohydride (1.2 eq.o 332 mgo 1.57 mmol) over ˜2 min. The reaction was then allowed to stir at room temperature. After 2 ho the reaction was complete by LCMS and was partitioned between 25 mL of water and 25 mL of EtOAc. The water layer was extracted 3×25 mL EtOAco dried over Na2SO4o filtered and concentrated under reduced pressure. The residue was purified on silica gel (40 go 0-50% EtOAc/hexanes) to provide 276 mg (70%) as a yellow oil. 1H NMR (CDCl3): δ 6.97 (do 1H)o 6.67 (do 1H)o 4.31 (to 1H) 3.96 (bso 1H)o 3.34 (qo 1H)o 2.16 (to 2H)o 1.64 (mo 6H)o 1.52 (mo 3H)o 1.21 (mo 3H)o 0.99 (mo 3H); LCMS (APCI) m/e 302.1 (M+H).
N2-(3-methyltetrahydrofuran-3-yl)-N3-tetrahydrofuran-3-yl-6-(trifluoromethyl)pyridine-2,3-diamine (K-67)
A 40 mL vial was charged with N2-(3-methyltetrahydrofuran-3-yl)-6-(trifluoromethyl)pyridine-3-diamine (350 mgo 1.34 mmol) and a stir baro tetrahydrofuran-3-one (1.1 eq.o 127 mgo 1.47 mmol)o TFA (2 eq.o 0.306 mLo 2.68 mmol)o and isopropyl acetate (4 mLo 0.3 M) were added. To this was added sodium triacetoxyborohydride (1.2 eq.o 341 mgo 1.61 mmol). The reaction was then allowed to stir at room temperature. After 1 ho the reaction was complete by LCMS and was partitioned between 25 mL of water and 25 mL of EtOAc. The water layer was extracted 3×25 mL EtOAco dried over Na2SO4o filtered and concentrated under reduced pressure. The residue was purified on silica gel (40 go 0-100% EtOAc/hexanes) to provide 270 mg (61%) of N2-(3-methyltetrahydrofuran-3-yl)-N3-tetrahydrofuran-3-yl-6-(trifluoromethyl)pyridine-2o3-diamine as an orange foam. LCMS: (APCI) m/e 232.1 (M+H); 1H NMR (CDCl3): δ 6.96 (do 1H)o 6.68 (do 1H)o 3.94 (mo 10H) 2.46 (mo 1H)o 2.28 (mo 1H)o 2.04 (mo 1H)o 2.01 (mo 1H)o 1.60 (so 3H)o 1.09 (bso 1H).
To a solution of 2-chloro-4-(cyclopentylamino)-5H-pyrimido[4o5-b][1o4]oxazin-6-one (550 mgo 2.05 mmol) in dry THF (10 mL) under argono was slowly added a 1 M solution of BH3·THF (20.5 mLo 20.5 mmolo) and the reaction mixture was stirred for 1 h at rt. The mixture was diluted with water and the aqueous layer was extracted with EtOAc. The organic layer was dried (Na2SO4)o filtered then concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (350 mgo 67%) as a solid. LCMS m/z: ES+ [M+H]+=255.1; tR=2.23 min.
A mixture of ethyl 8-hydroxy-1o7-naphthyridine-6-carboxylate (300 mgo 1.37 mmol) in POCl3 (7 mL) was stirred for 30 min at 110° C. The mixture was cooled to rt and concentrated under reduced pressure. The residue was poured onto crushed ice and stirred for 15 min. The pH was adjusted to 8 at 0° C. by careful addition of aqueous saturated aqueous sodium carbonate. The aqueous layer was extracted with DCMo and the combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The residue obtained was purified by column chromatography on silica gel (12 g) using a gradient of 0-50% EtOAc in hexane to afford title compound (189 mgo 58%) as a solid. 1H NMR (500 MHzo CDCl3) δ 9.22 (ddo J=4.2o 1.6 Hzo 1H)o 8.51 (so 1H)o 8.34 (ddo J=8.3o 1.6 Hzo 1H)o 7.77 (ddo J=8.3o 4.2 Hzo 1H)o 4.52 (qo J=7.1 Hzo 2H)o 1.46 (to J=7.1 Hzo 3H). LCMS m/z: ES+ [M+H]+=237.1; (B05) tR=1.99 min.
A mixture of pyrido[3o2-d]pyrimidine-2o4-diol (1.0 go 6.13 mmol)o POCl3 (10.1 mLo 110 mmol) and PCl5 (5.11 go 24.5 mmol) was heated at 120° C. for 12 h under argon. The mixture was cooled to rto and the volatiles were evaporated under reduced pressure. The residue was diluted with DCMo ice and water were addedo and the mixture was cooled to 0° C. The pH was adjusted to 8 by slow addition of aqueous saturated aqueous NaHCO3. The aqueous layer was extracted with DCMo and the combined organic layers were washed with water and brine. The organic layer was dried (Na2SO4)o filteredo concentrated under reduced pressure. The material was purified by column chromatography on silica gel (40 g) using a gradient of 0-20% EtOAc in hexane to afford title compound (510 mgo 42%) as a solid. 1H NMR (500 MHzo CDCl3) δ 9.15 (ddo J=4.1o 1.4 Hzo 1H)o 8.33 (ddo J=8.6o 1.4 Hzo 1H)o 7.92 (ddo J=8.6o 4.2 Hzo 1H); LCMS m/z: ES+ [M+H]+=200.1; tR=2.00 min.
To a suspension of 3-methylpyridine-2-carbonitrile (10 go 84.6 mmol) in tert-butanol (30 mL) at 70° C.o was added dropwise sulfuric acid (10 mLo 186 mmol). The mixture was stirred for 30 min at 75° C.o diluted with water (150 mL) then cooled to rt. The volatiles were evaporatedo and the aqueous layer was extracted with EtOAc (3×50 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (120 g) using 0.5% EtOAc in hexanes to afford title compound (10.44 go 64%) as a solid. 1H NMR (500 MHzo CDCl3) δ 8.34 (do J=2.2 Hzo 1H)o 8.03 (so 1H)o 7.55 (do J=7.6 Hzo 1H)o 7.26 (ddo J=7.4o 4.5 Hzo 1H)o 2.72 (so 3H)o 1.47 (do J=1.9 Hzo 9H). LCMS m/z: ES+ [M+H]+=193.2 ; tR=2.00 min.
A solution of n-BuLi in hexane (1.6 M in hexaneo 23.6 mLo 37.8 mmol) was added dropwise to a stirred solution of N-tert-butyl-3-methylpyridine-2-carboxamide (3.3 go 17.2 mmol) in THF (48 mL) at −78° C. under argon. NoNoN′oN′-tetramethylethylenediamine (2.57 mLo 17.2 mmol) was then added dropwise and the resulting solution was stirred for 30 min at −78° C. A solution of diethyl oxalate (4.65 mLo 34.3 mmol) in THF (48 mL) was added dropwise to the reaction mixture and the resulting solution was stirred for 1 h at −78° C. The reaction was diluted with saturated aqueous NH4Cl and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were dried (Na2SO4)o filteredo and concentrated under reduced pressure to afford title compound (5.5 g) as a solido which was used in the next step without further purification. LCMS m/z: ES+ [M+H]+=293.2; tR=2.45 min.
A mixture of ethyl 3-[2-(tert-butylcarbamoyl)-3-pyridyl]-2-oxo-propanoate (5.30 go 18.1 mmol) and ammonium acetate (2.88 go 36.3 mmol) in acetic acid (50 mL) was stirred at 110° C. The mixture was concentrated under vacuum and the material was purified by column chromatography on silica gel using a gradient of 0-4% MeOH in DCM to afford title compound (2.17 go 55% over 2 steps) as a solid. 1H NMR (500 MHzo CDCl3) δ 10.30 (so 1H)o 8.86 (do J=3.7 Hzo 1H)o 7.98 (do J=8.0 Hzo 1H)o 7.57 (ddo J=8.0o 4.4 Hzo 1H)o 7.26 (do J=5.1 Hzo 1H)o 4.36 (qo J=7.1 Hzo 2H)o 1.33 (to J=7.1 Hzo 3H). LC-MS m/z: ES+ [M+H]+=219.1; tR=1.65 min.
A mixture of ethyl 8-hydroxy-1o7-naphthyridine-6-carboxylate (300 mgo 1.37 mmol) in POCl3 (7 mL) was stirred for 30 min at 110° C. The mixture was cooled to rto concentratedo and then was poured onto crushed ice and stirred for 15 min. The pH of the aqueous mixture was basified to pH 8 at 0° C. by careful addition of saturated aqueous NaHCO3. The aqueous layer was extracted with DCM (3×15 mL)o and the combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (12 g) using a gradient of 0-50% EtOAc in hexane to afford title compound (189 mgo 58%) as a solid. LCMS m/z: ES+ [M+H]+=237.1; tR=1.99 min.
A mixture of ethyl 8-chloro-1o7-naphthyridine-6-carboxylate (350 mgo 1.48 mmol)o cyclopentylamine (126 mgo 1.48 mmol) and Cs2CO3 (482 mgo 1.48 mmol) in anhydrous DMF (3 mL) under argono was sealed and the resulting mixture was heated at 100° C. for 12 h. The mixture was cooled to rto diluted with water (10 mL) and the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic layers were dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (255 mgo 54%) as a solid. LCMS m/z: ES+ [M+H]+=286.2; tR=2.24 min.
A) A solution of ethyl 8-(cyclopentylamino)-1o7-naphthyridine-6-carboxylate (350 mgo 1.22 mmol) in a mixture composed of THF:MeOH:H2O (15 mLo 3:1:1)o was added LiOH (59 mgo 2.45 mmol) and the mixture was stirred at rt for 4 h. The volatiles were evaporatedo and the aqueous layer was washed once with EtOAc and then the pH was adjusted to 2 by adding of 1 N HCl. The aqueous layer was extracted with EtOAc (3×15 mL)o and the combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure to afford 8-(cyclopentylamino)-1o7-naphthyridine-6-carboxylic acid as a solid which was used in the next step without further purification. LCMS m/z: ES+ [M+H]+=258.1; tR=1.61 min.
B) To a solution of above material (200 mgo 0.77 mmol) in anhydrous DMF (10 mL) was successively added NoO-dimethylhydroxylamineo HCl (91 mgo 0.933 mmol)o HATU (355 mgo 0.933 mmol) and DIPEA (0.314 mLo 2.31 mmol) and the resulting mixture was stirred for 8 h at rt. The mixture was diluted with EtOAc (15 mL) and 0.1 N HCl (3 mL). The layers were separatedo and the aqueous layer was extracted with EtOAc (2×15 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-60% EtOAc in hexane to afford title compound (190 mgo 82%) as a solid. LCMS m/z: ES+ [M+H]+=301.2; tR=1.95 min.
To a solution of 8-(cyclopentylamino)-N-methoxy-N-methyl-1o7-naphthyridine-6-carboxamide (145 mgo 0.483 mmol) in THF (10 mL) was added n-BuMgCl (2 M in THFo 0.3 mLo 0.579 mmol) at 0° C. and the reaction mixture was warmed up to rt and stirred for 2 h. The mixture was quenched with saturated aqueous NH4Cl and then the aqueous layer was extracted with EtOAc (3×10 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-20% EtOAc in hexane to afford title compound (70 mgo 50%) as an oil. LCMS m/z: ES+ [M+H]+=298.2o tR=2.83 min.
A mixture of 1-[8-(cyclopentylamino)-1o7-naphthyridin-6-yl]pentan-1-one (40 mgo 0.135 mmol) and PtO2 (15 mgo 0.068 mmol) in anhydrous EtOH (10 mL) and TFA (1 drop) was hydrogenated under hydrogen atmosphere at rt for 6 h. The mixture was filtered through Celite® washed with EtOH (2×20 mL) and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (22 mgo 54%) as a solid. 1H NMR (500 MHzo CD3OD) δ 7.54 (so 1H)o 4.29 (ddo J=11.9o 5.9 Hzo 1H)o 3.54-3.46 (mo 2H)o 2.94 (to J=7.3 Hzo 2H)o 2.85 (to J=5.8 Hzo 2H)o 2.28-2.19 (mo 2H)o 1.99-1.93 (mo 2H)o 1.88-1.81 (mo 2H)o 1.72 (qdo J=15.1o 7.3 Hzo 6H)o 1.40 (dto J=13.3o 6.7 Hzo 2H)o 0.95 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=302.3o tR=3.63 min.
A solution of ethyl 8-chloro-1o7-naphthyridine-6-carboxylate (900 mgo 3.80 mmol)o DIPEA (2 mLo 11.68 mmol) and 2-methylpropan-2-amine (3.2 mLo 30.4 mmol) in dry DMF (4.0 mL) and were heated in a microwave at 170° C. for 2 h. Note: the reaction was performed 5 times for a total of 4.5 g. The vials were combinedo and the volatiles were evaporated under reduced procedure and then used in next step without further purification. To the above material in a mixture of THF/MeOH/Water (125 mLo 3:1:1) at rto was added LiOH·H2O (1.6 go 38 mmol) and the reaction mixture was stirred at rt for 18 h. The volatiles were evaporated under reduced pressure and then water (250 mL) was added. The mixture was acidified to pH 1 using 1 N aqueous HCl and then the aqueous layer was extracted with CHCl3 (3×150 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was triturated with ether (25 mL) and the resulting precipitate was filteredo then dried to afford title compound (2 go 43%) as a solid. LCMS m/z: ES+ [M+H]+=246.1. tR=1.63 min.
To a solution of 8-(tert-butylamino)-1o7-naphthyridine-6-carboxylic acid (1.00 go 4.08 mmol) and HATU (1.66 go 4.36 mmol) in acetonitrile (10 mL) at rto was added DIPEA (1.40 mLo 8.15 mmol) and then N-methoxymethanamine;hydrochloride (0.437 go 4.48 mmol) was added and the resulting mixture was stirred for 1 h. The mixture was diluted with EtOAc (50 mL) and 0.1N aqueous HCl (10 mL). The layers were separatedo and the aqueous layer was extracted with EtOAc (2×25 mL). The combined organic phases were washed with brineo then dried (MgSO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (40 g) using a gradient 0-60% EtOAc in hexane to afford title compound (652 mgo 56%) as a solid. LCMS m/z: ES+ [M+H]+=289.5. tR=2.31 min.
To a solution of 8-(tert-butylamino)-N-methoxy-N-methyl-1o7-naphthyridine-6-carboxamide 3 (452 mgo 1.57 mmol) in THF (10.0 mL) at 0° C.o was added n-BuMgCl (2N in THFo 3.14 mLo 6.27 mmol) and the reaction mixture was stirred at rt for 3 h. The mixture was diluted with water (20 mL) and the pH was adjusted to 3 using 1N aqueous HCl. The aqueous layer was extracted with Et2O (2×25 mL) and the combined organic layers were dried (MgSO4)o filtered and concentrated under reduced pressure to afford title compound 4 (290 mgo 65%) as a solid. 1H NMR (500 MHzo CDCl3) δ 8.78 (ddo J=4.3o 1.4 Hzo 1H)o 8.15 (do J=5.7 Hzo 1H)o 7.68 (so 1H)o 7.57 (ddo J=8.0o 4.2 Hzo 1H)o 7.13 (so 1H)o 3.30-3.14 (mo 2H)o 1.79-1.70 (mo 2H)o 1.65 (so 9H)o 1.49-1.42 (mo 2H)o 0.96 (to J=7.4 Hzo 3H). LCMS m/z: ES+ [M+H]+=287.8. tR=2.94 min.
A solution of 1-[8-(tert-butylamino)-1o7-naphthyridin-6-yl]pentan-1-one (180 mgo 0.63 mmol)o PtO2 (14.3 mgo 0.06 mmol) and TFA (0.23 mLo 3.15 mmol) in EtOH (7.00 mL) was hydrogenated under hydrogen atmosphere for 3 h at rt. The mixture was filtered on Celiteo washed and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (12 g) using a gradient of 0-100% EtOAc in hexane and was further purified by preparative HPLC (BEH C18 30×100; using 66-86% 10 mM ammonium formate in water and MeCN) to afford title compound (31.0 mgo 17%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.19 (so 1H)o 3.47 (bro 2H)o 3.29 (so 2H)o 3.01 (to J=7.0 Hzo 2H)o 2.64 (to J=6.1 Hzo 2H)o 1.85-1.78 (mo 2H)o 1.61 (ddo J=15.1o 7.7 Hzo 2H)o 1.45 (so 9H)o 1.34 (dqo J=14.7o 7.4 Hzo 2H)o 0.86 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=290.3. tR=1.89 min.
To a mixture of NaH (60.0%o 9.28 go 242 mmol) in DMF (130.0 mL) at 0° C.o was added slowly a solution of ethyl 2-cyanoacetate (17.4 mLo 163 mmol) in DMF (20.0 mL) and the mixture was stirred for 15 min. A solution 3-chloro-5-(trifluoromethyl)pyridine-2-carbonitrile (25.0 go 121 mmol) in DMF (20.0 mL) was slowly added and the reaction mixture was then heated to 70° C. and stirred for 2 h. The mixture was cooled to rt and diluted with EtOAc and 1N aqueous HCl. The layers were separatedo and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (330 g) using a gradient 0-100% EtOAc in hexane to afford title compound (31.0 go 91%) as an oil. LCMS m/z: ES− [M−H]−=282.6; tR=2.38 min.
To a solution of ethyl 2-cyano-2-[2-cyano-5-(trifluoromethyl)-3-pyridyl]acetate (31.0 go 109 mmol) in DMSO (100.0 mL)o was added a solution of lithium Sulfate (20.1 go 183 mmol) and NaOH (0.438 go 10.9 mmol) in water (28.0 mL) and the resulting mixture was stirred at 135° C. for 1 h. The mixture was cooled to rt diluted with water (100.0 mL). The aqueous layer was extracted with EtOAc (3×350.0 mL)o and the combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a mixture of DCM/Ethyl acetate/hexane (1:1:6) to afford title compound (9.60 go 42%) as an oil. 1H NMR (500 MHzo CDCl3) δ 8.98 (do J=0.8 Hzo 1H)o 8.27 (do J=1.0 Hzo 1H)o 4.14 (so 2H). LCMS: m/z: ES− [M−H]−=210.1; tR=2.21 min.
To a solution of 3-(cyanomethyl)-5-(trifluoromethyl)pyridine-2-carbonitrile (4.00 go 18.9 mmol) in DCM (100.0 mL) at 0° C.o was added dropwise HBr (5.00 Mo 11.4 mLo 56.8 mmolo 30% in AcOH) and the reaction mixture was warned to rt and stirred for 30 min. The mixture was diluted with water and stirred vigorously for 15 min. The layers were separatedo and the aqueous layer was extracted with DCM (75.0 mL). The combined organics layers were washed with saturated aqueous NaHCO3 (2×60.0 mL)o then dried (Na2SO4)o filtered and concentrated to afford title compound (4.50 go 82%) as a solid. LCMS m/z: ES+ [M+H]+=292.0; tR=2.41 min.
To 8-bromo-3-(trifluoromethyl)-1o7-naphthyridin-6-amine (360 mgo 1.23 mmol) at 0° C.o was slowly added concentrated HCl (12.0 Mo 3.39 mLo 40.7 mmol) and the resulting mixture was stirred for 30 min at 0° C. NaNO2 (0.170 go 2.47 mmol) was then added slowly and the mixture was stirred for another 10 min at 0° C. and then for 1.5 h at rt. The mixture was diluted with DCM and water at 0° C. Saturated aqueous Na2CO3 was slowly added and the layers were separated. The aqueous layer was extracted DCM (2×)o and the organic combined layers were washed with saturated aqueous NaHCO3o then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-20% EtOAc in hexane to afford title compound (105 mgo 32%) as a solid. 1H NMR (500 MHzo CDCl3) δ 9.26 (do J=2.1 Hzo 1H)o 8.43 (do J=0.8 Hzo 1H)o 7.80 (so 1H). LCMS m/z: ES+ [M+H]+=267.0o LCMS; tR=2.61 min.
A solution of 6o8-dichloro-3-(trifluoromethyI)-1o7-naphthyridine (2.10 go 7.86 mmol)o tert-butylamine (0.690 go 9.44 mmol) and DIPEA (1.62 mLo 9.44 mmol) in anhydrous DMF (10.2 mL) was heated at 170° C. in a microwave for 1 h. The mixture was diluted with EtOAc (150.0 mL) and the organic layer was washed with saturated aqueous NaHCO3 (50.0 mL) and brine (50.0 mL)o then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% DCM in hexane to afford title compound (2.05o 86%) as a solid. 1H NMR (500 MHzo CDCl3) δ 8.79 (do J=2.0 Hzo 1H)o 8.08 (do J=1.0 Hzo 1H)o 7.06 (bso 1H)o 6.81 (so 1H)o 1.59 (so 9H). LCMS m/z: ES+ [M+H]+=304.1; tR=3.36 min.
A mixture of N-tert-butyl-6-chloro-3-(trifluoromethyl)-1o7-naphthyridin-8-amine (1.75 go 5.76 mmol)o Zn(CN)2 (1.27 go 10.8 mmol) and BrettPhos (0.579 go 1.08 mmol) in DMF (23.1 mL) was degassed by bubbling argon for 10 min. (Note: the mixture was transferred under argon into 3 microwave vials. Each vial was processed as follows: Pd2(dba)3 (0.166 go 0.180 mmol) was addedo the mixture was degassed for 5 min after and then the vial was sealed and heated at 160° C. in a microwave for 1 h). The mixtures were combinedo diluted with EtOAc (100.0 mL) and saturated aqueous NaHCO3 (50.0 mL). The layers were separatedo and the organic layer was washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (40 g) using a gradient 0-100% DCM in hexane to afford title compound (1.65 go 92%) as a solid. 1H NMR (500 MHzo CDCl3) δ 8.97 (do J=2.0 Hzo 1H)o 8.23 (so 1H)o 7.28 (so 1H)o 7.17 (so 1H)o 1.59 (so 9H). LCMS m/z: ES+ [M+H]+=314.1o LCMS; tR=2.56 min.
To a solution of 8-(tert-butylamino)-3-(trifluoromethyl)-1o7-naphthyridine-6-carbonitrile (1.54 go 5.23 mmol) in ethanol (120.0 mL)o was added aqueous NaOH (5.00 Mo 41.9 mLo 209 mmol) and the reaction mixture was heated at 100° C. for 2 h then cooled to rt. The volatiles were evaporated under reduced pressure and the residue diluted with water and then the aqueous layer was washed with EtOAc. The aqueous layer was acidified to pH 2˜4 by slow addition of 1N aqueous HCl (approx. 250 mL). The aqueous layer was extracted with EtOAc (3×150.0 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure to afford title compound (1.03 go 63%) as a solid. 1H NMR (500 MHzo MeOD) δ 9.07 (do J=2.1 Hzo 1H)o 8.65 (do J=1.0 Hzo 1H)o 7.81 (so 1H)o 1.63 (so 9H). LCMS m/z: ES+ [M+H]+=314.1o LCMS; tR=2.56 min.
To a solution of 8-(tert-butylamino)-3-(trifluoromethyl)-1o7-naphthyridine-6-carboxylic acid (1030 mgo 3.29 mmol) in ethanol (41.0 mL)o was added TFA (0.122 mLo 1.64 mmol). platinum(IV)oxide (0.224 go 0.986 mmol) was added and the resulting mixture was hydrogenated under hydrogen atmosphere for 10 h. The mixture was filtered on Celiteo washed and the filtrate was concentrated under reduced pressure to afford title compound (976 mgo 94%) as a solido which was used in the next step without further purification. 1H NMR (500 MHzo MeOD) δ 7.25 (so 1H)o 3.68 (dddo J=12.3o 3.5o 2.3 Hzo 1H)o 3.35-3.27 (mo 1H)o 3.03 (dddo J=16.9o 5.0o 1.8 Hzo 1H)o 2.91 (ddo J=16.8o 10.3 Hzo 1H)o 2.84-2.71 (mo 1H)o 1.56 (so 9H). LCMS m/z: ES+ [M+H]+=318.2o LCMS; tR=1.91 min.
To a solution of 8-(tert-butylamino)-3-(trifluoromethyl)-1o2o3o4-tetrahydro-1o7-naphthyridine-6-carboxylic acid (1.03 go 3.25 mmol) in DMF (17.6 mL) was added morpholine (0.341 mLo 3.90 mmol)o followed by bis(dimethylamino)methylene-(triazolo[4o5-b]pyridin-3-yl) oxonium;hexafluorophosphate (1.48 go 3.90 mmol) and DIPEA (1.67 mLo 9.74 mmol). The reaction mixture was stirred for 3 h at rt. The mixture was diluted with brine (10 mL)o and the aqueous layer was extracted with EtOAc (3×50.0 mL). The combined organic layers were washed with saturated aqueous NaHCO3 (10 mL) and brine (10.0 mL)o then dried (Na2SO4)o filteredo concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (850 mgo 68%) as a solid. 1H NMR (500 MHzo CDCl3) δ 6.84 (so 1H)o 4.06 (so 1H)o 3.80 (mo 6H)o 3.72-3.64 (mo 2H)o 3.64-3.56 (mo 1H)o 3.20-3.05 (mo 2H)o 2.88 (dddo J=16.7o 5.5o 1.7 Hzo 1H)o 2.80 (ddo J=16.7o 10.8 Hzo 1H)o 2.62-2.43 (mo 1H)o 1.44 (so 9H). LCMS m/z: ES+ [M+H]+=387.2o LCMS; tR=2.48 min.
To a solution of [8-(tert-butylamino)-3-(trifluoromethyl)-1o2o3o4-tetrahydro-1o7-naphthyridin-6-yl]-morpholino-methanone (39.0 mgo 0.101 mmol) in anhydrous THF (0.767 mL) at 0° C.o was added n-BuLi (2.50 M in hexaneo 0.121 mLo 0.303 mmol). The mixture was stirred for 15 min at 0° C. and then warmed to rt and stirred for 1 h. The mixture was cooled to 0° C. then diluted with water (0.3 mL)o EtOAc (1.0 mL) and 1M aqueous HCl (0.2 mL). The layers were separatedo and the organic layer was dried (Na2SO4)o filtered and concentrated reduced pressure. The material was purified by reverse phase chromatography on C18 (5.5 g) using a gradient 10-100% acetonitrile in water (contains 0.1% formic acid) and was further purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (9.5 mgo 27%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.28 (so 1H)o 3.86 (bso 1H)o 3.65 (do J=12.0 Hzo 1H)o 3.43 (bso 1H)o 3.22 (to J=11.4 Hzo 1H)o 3.12-3.05 (mo 2H)o 2.92 (dddo J=16.6o 5.2o 1.7 Hzo 1H)o 2.84 (ddo J=16.6o 11.0 Hzo 1H)o 2.62-2.51 (mo 1H)o 1.73-1.65 (mo 2H)o 1.52 (so 9H)o 1.45-1.36 (mo 2H)o 0.93 (to J=7.4 Hzo 3H). LCMS m/z: ES+ [M+H]+=358.2o LCMS; tR=6.20 mins (10 mins run).
To a solution of 8-(tert-butylamino)-3-(trifluoromethyl)-1o7-naphthyridine-6-carboxylic acid (0.880 go 2.81 mmol) in anhydrous DMF (15.0 mL) was successively added NoO-dimethylhydroxylamine hydrochloride (0.329 go 3.37 mmol)o HATU (674 mgo 1.77 mmol) and DIPEA (0.77 mLo 4.43 mmol). The mixture was stirred for 8 h at rt then diluted with EtOAc (100 mL) and 0.1N aqueous HCl (6 mL). The layers were separatedo and the aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-60% EtOAc in hexane to afford title compound (850 mgo 85%) as a solid. LCMS m/z: ES+ [M+H]+=357.2; tR=2.69 min.
To a solution of 8-(tert-butylamino)-N-methoxy-N-methyl-3-(trifluoromethyl)-1o7-naphthyridine-6-carboxamide (850 mgo 2.39 mmol) in THF (15.0 mL) at −78° C.o was added n-butyl magnesium chloride (2.00 Mo 4.77 mLo 9.54 mmol) and the reaction mixture was stirred −78° C. for 5 mino and then warmed to rt and stirred for 1 h. The reaction was diluted with saturated aqueous NH4Cl (50 mL) at −78° C.o warmed to rt and the aqueous layer was extracted with EtOAc (3×50 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% DCM in hexane to afford title compound (350 mgo 42%) as a solid. LCMS m/z: ES+ [M+H]+=354.2; tR=3.43 min.
A solution of 1-[8-(tert-butylamino)-3-(trifluoromethyl)-1o7-naphthyridin-6-yl]pentan-1-one (60.0 mgo 0.170 mmol) in ethanol (3.0 mL) was added platinum(IV)oxide (0.0416 go 0.170 mmol) and 3 drops of TFA and the reaction mixture was hydrogenated under hydrogen atmosphere for 50 min. The mixture was diluted with EtOAc and filtered through Celite. The volatiles were evaporated under reduced pressure and the material was purified by reverse phase chromatography on C18 using 10-100% MeCN in water to afford title compound (61 mgo 25%) as a solid. 1H NMR (300 MHzo MeOD) δ 6.39 (so 1H)o 4.48 (ddo J=7.1o 5.4 Hzo 1H)o 3.59 (dddo J=12.3o 3.3o 1.9 Hzo 1H)o 3.11 (dddo J=12.2o 10.3o 1.8 Hzo 1H)o 2.88 (dddo J=17.1o 6.1o 1.9 Hzo 1H)o 2.80 (ddo J=16.6o 10.4 Hzo 1H)o 2.72-2.52 (mo 1H)o 1.97-1.80 (mo 1H)o 1.77-1.59 (mo 1H)o 1.50 (do J=2.2 Hzo 9H)o 1.44-1.25 (mo 5H)o 0.99-0.84 (mo 3H). LCMS: m/z: ES+ [M+H]+=360.3; tR=2.37 min.
To a solution of tert-butyl N-(1o 1-dimethyl-2-oxo-ethyl)carbamate (150 mgo 0.80 mmol) in anhydrous THF (2.5 mL) under argon at rto was added triphenylcarbethoxy methylenephosphorane (558 mgo 1.60 mmol) in one portion and the reaction mixture was stirred for 5 h at rt. The mixture was diluted with saturated aqueous NH4Cl and the aqueous layer was extracted with EtOAc (3×). The combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by reverse phase chromatography on C18 using a gradient 10-100% MeCN in water (contains 0.1% formic acid) to afford title compound (173 mgo 84%) as an oil. 1H NMR (500 MHzo CDCl3) δ 6.92 (do J=15.9 Hzo 1H)o 5.75 (do J=15.9 Hzo 1H)o 4.80 (so 1H)o 4.10 (ddo J=5.0o 10.0 Hzo 2H)o 1.33 (so 9H)o 1.32 (so 6H)o 1.19 (to J=7.1 Hzo 3H; LCMS m/z: ES+ [M+Na]+280.1; tR=2.42 min.
To a solution of ethyl (E)-4-(tert-butoxycarbonylamino)-4-methyl-pent-2-enoate (510 mgo 1.98 mmol) in DCM (5.0 mL) was added TFA (637 μLo 9.91 mmol)o and the mixture was stirred for 3 h at rt. The volatiles were concentrated under reduced pressure to afford title compound (538 mg) as a solido which was used in the next step without further purification. 1H NMR (500 MHzo CDCl3) δ 7.95 (so 2H)o 6.99 (do J=16.1 Hzo 1H)o 6.06 (do J=16.1 Hzo 1H)o 4.23 (qo J=7.2 Hzo 2H)o 1.57 (so 6H)1.31 (ddo J=11.7o 4.5 Hzo 3H). LCMS (ES+): m/z [M+H]+ 158.5; tR=0.86 min.
To a solution of ethyl (E)-4-amino-4-methyl-pent-2-enoate;2o2o2-trifluoroacetic acid (4.14 g; 8.06 mmol) in anhydrous acetonitrile (25.0 mL) under argon at rto was added Cs2CO3 (9.19 go 1.6 mmolo 28.2 mmol) followed by ethyl 2-bromoacetate (1.34 mLo 12.1 mmol) and the resulting mixture was stirred for 16 h at rt. The mixture was filteredo and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (0.874 go 45%) as an oil. LCMS (ES+): m/z [M+H]+ 244.7; tR=1.44 min.
To a solution of ethyl (E)-4-[(2-ethoxy-2-oxo-ethyl)amino]-4-methyl-pent-2-enoate (0.878 go 3.61 mmol)) in anhydrous DCM (3.0 mL) under argon at 0° C.o was added anhydrous pyridine (3.81 mLo 72.2 mmol) followed by Trifluoroacetic anhydride (0.752 mLo 5.41 mmol) and the reaction mixture was stirred for 30 min at 0° C. The mixture was diluted with watero and the aqueous layer was extracted with EtOAc (3×150 mL). The organic combined layers were washed with 1M aqueous HCl and brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-50% EtOAc in hexane to afford title compound (60%o 733 mg) as an oil. 1H NMR (500 MHzo CDCl3) δ 7.10 (do J=16.0 Hzo 1H)o 5.89 (do J=16.0 Hzo 1H)o 4.29-4.16 (mo 6H)o 1.54 (so 6H)o 1.33-1.26 (mo 6H). LCMS (ES+): m/z [M+H]+ 339.7; tR=2.62 min.
A mixture of ethyl (E)-4-[(2-ethoxy-2-oxo-ethyl)-(2o2o2-trifluoroacetyl)amino]-4-methyl-pent-2-enoate (0.743 go 2.19 mmol) and Pd/C (0.233 go 0.219 mmol) in EtOAc (5.0 mL) was hydrogenated under hydrogen atmosphere for 2 h at rt. The mixture was filtered through Celiteo was washed with EtOAc and the filtrate was concentrated under reduced pressure to afford title compound (0.787 go 100%) as an oilo which was used in the next without further purification. 1H NMR (500 MHzo CDCl3) δ 4.23 (qo J=7.2 Hzo 4H)o 4.14-4.08 (mo 4H)o 2.25 (so 4H)o 1.31-1.22 (mo 12H). LCMS (ES+): m/z [M+H]+ 342.8; tR=2.70 min.
To a solution of ethyl 4-[(2-ethoxy-2-oxo-ethyl)-(2o2o2-trifluoroacetyl)amino]-4-methyl-pentanoate (0.454 go 1.33 mmol) in anhydrous THF (5.0 mL) at 0° C.o was added NaH (61.2 mgo 1.60 mmol) and the reaction mixture was warmed to rt and stirred for 1 h. The mixture was diluted with water (5.0 mL)o and the aqueous layer was extracted with EtOAc (3×15.0 mL). The combined organic layers were washed with water and brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography in silica gel using a gradient of 0-50% EtOAc in hexane to afford title compound (0.115 go 29%) as a solid. LCMS (ES+): m/z [M+H]+ 296.1; tR=2.71 min.
To a solution of ethyl 6o6-dimethyl-3-oxo-1-(2o2o2-trifluoroacetyl)piperidine-2-carboxylate (255 mgo 0.86 mmol) in MeOH (2.0 mL) was addedo hexanamidine;hydrochloride (195 mgo 1.3 mmol) and the reaction mixture was heated at 110° C. for 48 h. The mixture was concentratedo and the material was purified by column chromatography on silica gel using a gradient 0-20% MeOH in DCM to afford title compound (23 mgo 11%) as a solid. 1H NMR (500 MHzo CDCl3) δ 3.83 (so 2H)o 2.60 (to J=6.5 Hzo 2H)o 2.41 (so 2H)o 1.77-1.70 (mo 2H)o 1.25 (so 6H)o 1.20 (so 6H)o 0.90 (to J=6.9 Hzo 3H). LCMS (ES+): m/z [M+H]+250.2; tR=1.67 min.
To a solution of 4-chloro-6o6-dimethyl-2-pentyl-7o8-dihydro-5H-pyrido[3o2-d]pyrimidine (11.0 mgo 0.041 mmol) and cyclopentanamine (16.0 μLo 0.16 mmol) in anhydrous n-butanol (1.0 mL)o was added DIPEA (28.0 μLo 0.16 mmol) and the reaction mixture was heated to 95° C. for 16 h. The mixture was concentrated under reduced pressureo and the material was purified by reverse phase chromatography on C18 using a gradient 10-60% acetonitrile in water (contains 0.1% formic acid) to afford title compound (1.8 mgo 14%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.51 (ddo J=14.8o 7.4 Hzo 1H)o 4.01 (so 2H)o 2.66 (to J=7.6 Hzo 2H)o 2.52 (so 2H)o 2.11-2.02 (mo 2H)o 1.83-1.75 (mo 4H)o 1.69-1.61 (mo 2H)o 1.58-1.51 (mo 2H)o 1.40 (so 6H)o 1.38-1.34 (mo 4H)o 0.92 (to J=6.7 Hzo 3H). LCMS (ES+): m/z [M+H]+317.3; tR=3.31 min.
To a suspension of 1-[8-(cyclopentylamino)-1o7-naphthyridin-6-yl]pentan-1-one (40.0 mgo 0.135 mmol) in anhydrous EtOH (10.0 mL) under argono was added platinum oxide (0.0189 go 0.161 mmol) and 1 drop of TFAo was hydrogenated under hydrogen atmosphere for 6 hat rt. The mixture was filtered on Celiteo and the filtrate was evaporated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-50% EtOAc in hexane to afford title compound (22 mgo 54%) as a solid. 1H NMR (300 MHzo MeOD) δ 7.55 (so 1H)o 4.35-4.25 (mo 1H)o 3.54-3.48 (mo 2H)o 2.96 (to J=7.4 Hzo 2H)o 2.87 (to J=6.2 Hzo 2H)o 2.32-2.18 (mo 2H)o 2.02-1.93 (mo 2H)o 1.91-1.65 (mo 7H)o 1.43 (ddo J=14.6o 9.5o 6.4 Hzo 2H)o 0.97 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=302.3o LCMS; tR=3.66 min.
B-626o FER-1o was purchased from Combi Blockso San Diegoo WZ9339.
To a solution of 2o4o6-trichloro-5-nitro-pyrimidine (100 mgo 0.438 mmol) in iPrOH (2.0 mL) at −78° C. under argono was added a solution of tetrahydrofuran-3-amine (38.1 mgo 0.438 mmol) in iPrOH (1.0 mL) over 15 min and the reaction mixture was stirred at 30 min at −78° C. and then warmed to rt and stirred for 1 h. DIPEA (0.150 mLo 0.876 mmol) was then added and the resulting mixture was stirred for 2 h at rt. The volatiles were evaporated under reduced pressure and the material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (80 mgo 66%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.87 (so 1H)o 4.95-4.77 (mo 1H)o 4.02 (ddo J=15.6o 7.7 Hzo 1H)o 3.96 (ddo J=9.8o 5.4 Hzo 1H)o 3.86 (tdo J=8.6o 6.0 Hzo 1H)o 3.79 (ddo J=9.8o 2.3 Hzo 1H)o 2.43 (tdo J=14.6o 7.5 Hzo 1H)o 1.98-1.89 (mo 1H). LCMS m/z: ES+ [M+H]+=279.5; tR=2.27 min.
To a solution of 2o6-dichloro-5-nitro-N-tetrahydrofuran-3-yl-pyrimidin-4-amine (0.205 go 0.734 mmol) and methyl 2-hydroxyacetate (99 mgo 1.10 mmol) in iPrOH (8.0 mL) and DCM (2.0 mL) under argon at 0° C.o was added sodium tert-butoxide (2.00 Mo 0.404 mLo 8.08 mmol) in THF (0.50 mL) and the reaction mixture was stirred for 1 h at rt. The mixture was diluted with water and the aqueous layer extracted with DCM (3×20.0 mL). The combined organic layers were dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (158 mgo 64%) as a solid. LCMS m/z: ES+ [M+H]+=331.1o tR: 2.27 min.
To a solution of methyl 2-[2-chloro-5-nitro-6-(tetrahydrofuran-3-ylamino)pyrimidin-4-yl]oxyacetate (150 mgo 0.451 mmol) in THF (6.0 mL) and 10% aqueous HCl (3.0 mL)o was added Zn (88.5 mgo 1.35 mmol) and the reaction mixture was heated to 70° C. for 30 min. The mixture was diluted with saturated aqueous NaHCO3 and the aqueous layer was extracted with EtOAc (3×20.0 mL). The combined organics were dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (50.0 mgo 41%) as a solid. LCMS m/z: ES+ [M+H]+=271.1o tR: 1.80 min.
A mixture of 2-chloro-4-(tetrahydrofuran-3-ylamino)-5H-pyrimido[4o5-b][1o4]oxazin-6-one (45.0 mgo 0.166 mmol)o [(E)-pent-1-enyl]boronic acid (56.8 mgo 0.498 mmol)o and potassium carbonate (68.9 mgo 0.500 mmol) in toluene (0.80 mL)o ethanol (0.20 mL)o and water (0.20 mL) was degassed for 10 min by bubbling argon. Tetrakis(triphenylphosphine)palladium(0) (38.4 mgo 0.0332 mmol) was addedo the vial was sealed then stirred at 100° C. for 16 h. The mixture was cooled to rto diluted with EtOAc and saturated aqueous NaHCO3. The layers were separatedo and the aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-10% MeOH in DCM to afford title compound (23.0 mgo 46%) as a solid. LCMS m/z: ES+ [M+H]+=305.2 LCMS; tR=4.14 mins (10 mins run).
To a solution of 2-[(E)-pent-1-enyl]-4-(tetrahydrofuran-3-ylamino)-5H-pyrimido[4o5-b][1o4]oxazin-6-one (19.0 mgo 0.0624 mmol) in THF (0.25 mL) at 0° C.o was added BH3. THF (1.00 Mo 0.624 mLo 0.624 mmol) and the reaction mixture was warmed and stirred at rt for 2 h. The mixture was diluted with saturated aqueous NaHCO3 and the aqueous layer was extracted with EtOAc (3×2.0 mL). The combined organic layers were dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-10% MeOH in DCM to afford title compound (8.0 mgo 44%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.53 (do J=6.9 Hzo 1H)o 3.45 (dto J=8.1o 4.1 Hzo 2H)o 2.67 (to J=7.4 Hzo 2H)o 2.13-2.02 (mo 2H)o 1.84-1.72 (mo 4H)o 1.66 (ddo J=14.3 10.1 Hzo 2H)o 1.62-1.52 (mo 2H)o 1.38-1.29 (mo 4H)o 0.91 (to J=6.5 Hzo 3H). LCMS m/z: ES+ [M+H]+=293.2o LCMS; tR=2.96 min.
To a solution of 2o4-dichloropyrido[3o2-d]pyrimidine (125 mgo 0.625 mmol) in THF (5.0 mL) and water (3.0 mL)o was added cyclopentanamine (62 μLo 0.625 mmol) followed by and CH3COONa (0.0513 go 0.625 mmol) and the reaction mixture was stirred at rt for 12 h. The mixture was diluted with EtOAc and the layers were separated. The organic layer was washed with water (3×)o then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a mixture of 20% EtOAc in hexane to afford title compound (130 mgo 84%) as a solid. 1H NMR (500 MHzo CDCl3) δ 8.64 (do J=3.8 Hzo 1H)o 8.00 (do J=8.4 Hzo 1H)o 7.63 (ddo J=8.4o 4.1 Hzo 1H)o 7.29 (bso 1H)o 4.68-4.55 (mo 1H)o 2.21-2.16 (mo 2H)o 1.90-1.77 (mo 2H)o 1.76-1.66 (mo 2H)o 1.67-1.54 (mo 2H). LCMS m/z: ES+ [M+H]+=249.1; tR=2.44 min.
To a solution of 2-chloro-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (90 mgo 0.362 mmol)o 1-cyclopentylboronic acid (122 mgo 1.09 mmol)o and potassium carbonate (150 mgo 1.09 mmol) in toluene (1.5 mL)o ethanol (0.35 mL)o and water (0.35 mL) was degassed for 10 min by bubbling argon. Pd(PPh3)4 (83 mgo 0.724 mmol) was then addedo and the vial was sealed and heated at 100° C. for 8 h. The mixture was cooled to rt and the mixture was diluted with saturated aqueous. NaHCO3 and EtOAc. The layers were separatedo and the aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-70% EtOAc in hexane to afford title compound (35 mgo 35%) as a solid. 1H NMR (500 MHzo CDCl3) δ 8.57 (ddo J=4.2o 1.4 Hzo 1H)o 8.05 (ddo J=8.5o 1.4 Hzo 1H)o 7.57 (ddo J=8.5o 4.2 Hzo 1H)o 7.08-7.01 (mo 1H)o 6.98 (do J=6.5 Hzo 1H)o 4.68-4.55 (mo 1H)o 2.91 (tdo J=7.7o 2.1 Hzo 2H)o 2.60 (ddto J=10.0o 4.8o 2.4 Hzo 2H)o 2.19 (dto J=13.0o 6.3 Hzo 2H)o 2.11-2.02 (mo 2H)o 1.87-1.75 (mo 2H)o 1.76-1.57 (mo 4H). LCMS m/z: ES+ [M+H]+=281.2.; tR=1.91 min.
To a solution of 2-(cyclopenten-1-yl)-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (30 mgo 0.107 mmol) in ethanol (2.0 mL) under argon was added PtO2 (7.2 mgo 0.0321 mmol) followed by 3 drops of TFA and the reaction mixture was hydrogenated under hydrogen atmosphere at rt fro 2 h. The mixture was filtered on Celiteo washed and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-30% MeOH in DCM to afford title compound (30 mgo 97%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.51 (po J=6.8 Hzo 1H)o 3.37-3.25 (mo 2H)o 3.11 (po J=7.8 Hzo 1H)o 2.75 (to J=6.4 Hzo 2H)o 2.17-1.99 (mo 4H)o 1.99-1.81 (mo 6H)o 1.78 (ddo J=9.0o 5.7 Hzo 2H)o 1.74-1.49 (mo 6H). LCMS m/z: ES+ [M+H]+=287.3; tR=3.45 min.
A solution of 2-chloro-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (50 mgo 0.201 mmol)o 1-pentenylboronic acid (30 mgo 0.261 mmol)o and potassium carbonate (84 mgo 0.603 mmol) in toluene (1.5 mL)o ethanol (0.35 mL)o and water (0.35 mL) was degassed for 10 min by bubbling argon. Pd(dppf)Cl2 (30 mgo 0.0402 mmol) and PPh3 (21 mgo 0.0804 mmol) were addedo and the vial was sealed and heated at 100° C. overnight. The mixture was cooled to rt and the diluted with saturated aqueous NaHCO3. The aqueous layer was extracted with EtOAc (2×15.0 mL) and the combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-70% EtOAc in hexane to afford title compound (35 mgo 62%) as a solid. LCMS m/z: ES+ [M+H]+=283.3; tR=2.00 min.
To a mixture of N-cyclopentyl-2-[(E)-pent-1-enyl]pyrido[3o2-d]pyrimidin-4-amine (30 mgo 0.105 mmol) and PtO2 (7 mgo 0.0315 mmol) in ethanol (2.0 mL)o was added TFA (15.6 μLo 0.0210 mmol) and the resulting mixture was hydrogenated under hydrogen atmosphere for 2 h at rt. The mixture was filtered on Celiteo washed and the filtrate was concentrated under reduced pressure. The material was purified by reverse phase chromatography on C18 (5.5 g) using a gradient 10-100% MeCN and water (contains 0.1% formic acid) to afford title compound (30 mgo 99%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.55 (po J=7.0 Hzo 1H)o 3.36-3.31 (mo 2H)o 2.75 (to J=6.4 Hzo 2H)o 2.69 (to J=7.5 Hzo 2H)o 2.17-2.03 (mo 2H)o 2.01-1.92 (mo 2H)o 1.83-1.74 (mo 4H)o 1.68 (dto J=8.4o 7.6 Hzo 2H)o 1.62-1.53 (mo 2H)o 1.41-1.31 (mo 4H)o 0.91 (to J=6.8 Hzo 3H). LCMS m/z: ES+ [M+H]+=289.3; tR=3.89 mins (10 mins run).
To a solution of 1-butanol (55 mgo 0.754 mmol) in anhydrous THF (10.0 mL) under argon at 0° C.o was added NaH (48 mgo 2.01 mmol) and the mixture was stirred for 10 min at rt. And theno a solution of 2-chloro-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (125 mgo 0.502 mmol) in THF (2.0 mL) was added and the resulting mixture was stirred at 65° C. for 30 min. The mixture was cooled to rt and diluted with saturated aqueous NH4Cl. The aqueous layer was extracted EtOAc (3×10.0 mL) and the combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-30% methanol in DCM to afford title compound (73 mgo 50%) as a solid. LCMS m/z: ES+ [M+H]+=287.2.; tR1.78 min.
To a mixture of 2-butoxy-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (50 mgo 0.175 mmol) and PtO2 (3.97 mgo 0.0175 mmol) in anhydrous EtOH (10.0 mL) under argon atmosphereo was TFA (13 μLo 0.0175 mmol) and the resulting mixture was hydrogenated under hydrogen atmosphere for 6 h at rt. The mixture was filtered on Celiteo washed and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-30% MeOH in DCM to afford title compound (15 mgo 30%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.50-4.45 (mo 1H)o 4.42 (to J=6.5 Hzo 2H)o 3.26-3.21 (mo 2H)o 2.63 (to J=6.4 Hzo 2H)o 2.13-2.04 (mo 2H)o 1.90 (ddo J=11.3o 5.9 Hzo 2H)o 1.83-1.73 (mo 4H)o 1.69-1.58 (mo 4H)o 1.47 (dto J=13.2o 6.6 Hzo 2H)o 0.97 (to J=7.4 Hzo 3H). LCMS m/z: ES+ [M+H]+=291.3; tR=3.59 min.
To a solution of 2-chloro-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (100 mgo 0.402 mmol) in anhydrous 1o4-dioxane (8.0 mL)o was added n-butylamine (52 μLo 0.523 mmol) followed by triethylamine (0.112 mLo 0.804 mmol) and the reaction mixture was stirred at reflux for 12 h. The mixture was cooled to rto and then diluted with water and EtOAc. The layers were separatedo and the aqueous layer was extracted with EtOAc (3×20.0 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-25% MeOH in DCM to afford title compound (42 mgo 37%) as a solid. 1H NMR (500 MHzo CDCl3) δ 8.35-8.17 (mo 1H)o 7.65 (do J=7.1 Hzo 1H)o 7.39 (ddo J=8.5o 4.2 Hzo 1H)o 6.90 (do J=6.0 Hzo 1H)o 4.98 (so 1H)o 4.47 (ddo J=13.6 6.8 Hzo 1H)o 3.48 (ddo J=13.0o 6.9 Hzo 2H)o 2.12 (ddo J=12.1o 5.7 Hzo 2H)o 1.84-1.73 (mo 2H)o 1.74-1.50 (mo 7H)o 1.52-1.34 (mo 2H)o 0.95 (to J=7.4 Hzo 3H). LCMS m/z: ES+ [M+H]+=286.3; tR=1.87 min.
To a mixture of N-cyclopentyl-2-[(E)-pent-1-enyl]pyrido[3o2-d]pyrimidin-4-amine (10 mgo 0.0350 mmol) and PtO2 (3 mgo 0.0105 mmol) in ethanol (5.0 mL) was added 3 drops of TFA and the resulting mixture was hydrogenated under hydrogen atmosphere for 2 h at rt. The mixture was filtered on Celiteo washed and the filtrate was concentrated under reduced pressure. The material was purified by reverse phase chromatography on C18 (5.5 g) using a gradient 10-100% MeCN in water (contains 0.1% formic acid) to afford title compound (3 mgo 99%) as a solid. 1H NMR (500 MHzo CDCl3) δ 9.62 (so 1H)o 8.66 (so 1H)o 5.80 (do J=6.2 Hzo 1H)o 4.42-4.29 (mo 1H)o 3.36 (ddo J=12.4o 6.5 Hzo 2H)o 3.14-3.05 (mo 2H)o 2.67 (to J=6.5 Hzo 2H)o 2.09 (tdo J=12.4o 6.6 Hzo 2H)o 1.87-1.79 (mo 2H)o 1.78-1.55 (mo 6H)o 1.49 (tdo J=13.1o 6.6 Hzo 2H)o 1.43-1.33 (mo 2H)o 0.92 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=290.3.; tR=3.45 min.
To a solution of 2-chloro-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (150 mgo 0.603 mmol) in anhydrous DMFo was added N-methylbutylamine (52.6 mgo 0.603 mmol) followed by Cs2CO3 (393 mgo 1.21 mmol) and the mixture was degassed for 5 min by bubbling N2. Xantphos (41.9 mgo 0.0724 mmol) was then addedo followed by Pd2dba3 (69.4 mgo 0.121 mmol) and the resulting mixture was degassed for 5 min and then heated to 100° C. for 12 h. The mixture was diluted with water (10.0 mL) and the organic layer was extracted with EtOAc (2×). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (90 mgo 49.8%) as a solid. LCMS m/z: ES+ [M+H]+=300.3o tR=1.90 min.
To a mixture of 2-butoxy-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (50 mgo 0.167 mmol) and PtO2 (3.80 mgo 0.0167 mmol) in anhydrous EtOH (10.0 mL) was added 3 drops of TFA and the resulting mixture was hydrogenated under hydrogen atmosphere for 6 h at rt. The mixture was filtered on Celiteo washed and the filtrate was concentrated under reduced pressure. The material was purified by flash chromatography on silica gel using 0-100% EtOAc in hexane to afford title compound (15 mgo 29%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.42 (po J=6.7 Hzo 1H)o 3.64-3.58 (mo 2H)o 3.29 (so 3H)o 3.21-3.16 (mo 2H)o 2.65 (to J=6.4 Hzo 2H)o 2.10-2.01 (mo 2H)o 1.93-1.86 (mo 2H)o 1.77 (do J=6.2 Hzo 2H)o 1.68-1.56 (mo 6H)o 1.40-1.32 (mo 2H)o 0.96 (to J=7.4 Hzo 3H). LCMS m/z: ES+ [M+H]+=304.3; tR=3.62 min.
To a solution of 2-Methoxyethanol (0.0594 mLo 0.754 mmol) in anhydrous THF (10.0 mL) at 0° C.o was added NaH (60% oil dispersiono 77 mgo 2.01 mmol) and the mixture was stirred for 10 min at rt. 2-chloro-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (125 mgo 0.503 mmol) was then added and the resulting the mixture was stirred at 65° C. for 30 min. The mixture was cooled to rt and diluted with saturated aqueous NH4Cl. The aqueous layer was extracted EtOAc and the combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-30% methanol in DCM to afford title compound (130 mgo 90%) as a solid. LCMS m/z: ES+ [M+H]+=289.2.; tR=1.73 min.
To a mixture of N-cyclopentyl-2-(2-methoxyethoxy)pyrido[3o2-d]pyrimidin-4-amine (30 mgo 0.104 mmol) and PtO2 (7.1 mgo 0.0312 mmol) in ethanol (2 mL)o was added TFA (1.55 μLo 0.0208 mmol) and the resulting mixture was hydrogenated under hydrogen atmosphere for 2 h at rt. The mixture was filtered on Celiteo rinsed with EtOH and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on C18 (5.5 g) using a gradient 10-100% MeCN in water (contains 0.1% formic acid) to afford title compound (30 mgo 99%) as a solid. 1H NMR (500 MHzo CDCl3) δ 5.22 (bso 1H)o 4.48-4.32 (mo 3H)o 3.73 (to J=5.1 Hzo 2H)o 3.41 (so 3H)o 3.17 (bso 2H)o 2.66 (to J=5.9 Hzo 2H)o 2.06 (dto J=12.4o 6.2 Hzo 2H)o 1.92-1.82 (mo 2H)o 1.78-1.67 (mo 2H)o 1.66-1.56 (mo 2H)o 1.51-1.39 (mo 2H). LCMS m/z: ES+ [M+H]+=293.2.; tR=2.72 min.
A mixture of 2-chloro-N-cyclopentyl-C7-dihydro-5H-pyrimido[4o5-b][1o4]oxazin-4-amine (150 mgo 0.589 mmol) 1-pentenylboronic acid (67.1 mgo 0.589 mmol) and potassium carbonate (244 mgo 1.77 mmol) in toluene (1.5 mL)o ethanol (0.7 mL)o and water (0.7 mL) was degassed for 10 min by bubbling argon. Pd(PPh3)4 (136 mgo 0.118 mmol) was then added the resulting mixture was heated at 100° C. for 12 h. The mixture was cooled to rt and diluted with saturated aqueous NaHCO3 and EtOAc. The layers were separatedo and the organic layer was dried (Na2SO4) filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (25 mgo 15%) as a solid. 1H NMR (500 MHzo CD3OD) δ 6.85-6.77 (mo 1H)o 6.13 (do J=15.4 Hzo 1H)o 4.42 (po J=6.7 Hzo 1H)o 4.25 (do J=3.5 Hzo 2H)o 3.30 (do J=2.2 Hzo 2H)o 2.18 (qo J=7.1 Hzo 2H)o 2.06 (ddo J=12.2o 5.8 Hzo 2H)o 1.77-1.73 (mo 2H)o 1.65-1.61 (mo 2H)o 1.50 (dto J=14.6o 7.5 Hzo 4H)o 0.95 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=289.2; QC tR=3.63 min.
A mixture of N-cyclopentyl-2-[(E)-pent-1-enyl]-6o7-dihydro-5H-pyrimido[4o5-b][1o4]oxazin-4-amine (150 mgo 0.520 mmol) and Pd/C (20% wto 55 mgo 0.520 mmol) in MeOH (10 mL) was hydrogenated under hydrogen atmosphere for 2 h at rt. The mixture was filtered on Celiteo washed and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (155 mgo 99%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.53 (po J=6.9 Hzo 1H)o 4.45 (ddo J=13.8 9.7 Hzo 2H)o 3.45 (dto J=8.1o 4.1 Hzo 2H)o 2.67 (to J=7.4 Hzo 2H)o 2.13-2.02 (mo 2H)o 1.84-1.72 (mo 4H)o 1.66 (ddo J=14.3o 10.1 Hzo 2H)o 1.62-1.52 (mo 2H)o 1.36 (do J=3.4 Hzo 4H)o 0.91 (to J=6.5 Hzo 3H). LCMS m/z: ES+ [M+H]+=291.2; tR=1.94 min.
To a solution of 2-chloro-N-cyclopentyl-6o7-dihydro-5H-pyrimido[4o5-b][1o4]oxazin-4-amine (150 mgo 0.589 mmol) in DMF (10.0 mL)o was added Zn(CN)2 (0.138 go 1.18 mmol) followed by Pd(PPh3)4 (204 mgo 0.177 mmol) and the mixture was degassed by bubbling argon for 5 min and then heated at 100° C. for 12 h. The mixture was cooled to rto saturated aqueous NH4Cl was addedo and the aqueous layer was extracted with EtOAc. The organic layer was washed with brineo then dried (Na2SO4)o filteredo concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (100 mgo 69%) as a solid. LCMS (ES+): m/z [M+H]+ 246.1; tR=2.23 min.
To a solution of 4-(cyclopentylamino)-6o7-dihydro-5H-pyrimido[4o5-b][1o4]oxazine-2-carbonitrile (40.0 mgo 0.163 mmol) in THF (1.5 mL)o was added n-butylmagnesium chloride solution (2 M in THFo 0.16 mLo 0.326 mmol) at 0° C. and the reaction mixture was warmed to rt and stirred for 2 h. The mixture was diluted with saturated aqueous NH4Cl and the aqueous layer was extracted with EtOAc (3×20.0 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (2.5 mgo 5%) as a solid. LCMS m/z: ES+ [M+H]+=305.2; tR=4.74 min.
To a solution of 2o4o6-trichloro-5-nitro-pyrimidine (100 mgo 0.438 mmol) in 2-propanol (3 mL) at −78° C.o was added a solution of cyclopentanamine (43 μLo 0.438 mmol) in 2-propanol (1 mL) over 15 min and the resulting mixture was stirred at 30 min at −78° C. and then warmed to rt and stirred 1 h. DIPEA (0.15 mLo 0.876 mmol) was then added dropwise and the mixture was stirred for 2 h at rt. The volatiles were evaporated under reduced pressure and the material was purified by column chromatography on silica gel (12 g) using a gradient of 0-100% EtOAc in hexane to afford title compound (100 mgo 83%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.76 (so 1H)o 4.50 (ddo J=13.9 7.0 Hzo 1H)o 2.12 (tto J=13.5o 6.7 Hzo 2H)o 1.88-1.61 (mo 4H)o 1.52 (tdo J=13.2o 6.6 Hzo 2H). LCMS m/z: ES+ [M+H]+=277.5.; tR=2.72 min.
To a solution of 2o6-dichloro-N-cyclopentyl-5-nitro-pyrimidin-4-amine (500 mgo 1.80 mmol) in THF (15.0 mL) at 0° C.o was added methyl thioglycolate (0.192 go 1.80 mmol) followed by DIPEA (0.309 mLo 1.80 mmol) and the reaction mixture was stirred at 0° C. for 1 h. The mixture was diluted with water (10 mL) and EtOAc (25 mL). The separated organic layer was dried (Na2SO4)o filteredo concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (403 mgo 65%) as a solid. LCMS m/z: ES+ [M+H]+=347.1; tR=2.95 min.
To a solution of methyl 2-[2-chloro-6-(cyclopentylamino)-5-nitro-pyrimidin-4-yl]sulfanylacetate (250 mgo 0.721 mmol) in a mixture THF (6 mL) 10% aqueous HCl (3.0 mL)o was added zinc (141 mgo 2.16 mmol) and the resulting suspension was heated to 70° C. for 30 min. The mixture was diluted slowly with saturated aqueous NaHCO3 and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (100 mgo 49%) as a solid. LCMS (ES+): m/z [M+H]+285.1; tR=2.36 min.
A mixture of 2-chloro-4-(cyclopentylamino)-5H-pyrimido[4o5-b][1o4]thiazin-6-one (250 mgo 0.79 mmol)o 1-pentenylboronic acid (100 mgo 0.88 mmol)o and potassium carbonate (364 mgo 2.63 mmol) in toluene (1.5 mL)o ethanol (0.7 mL)o and water (0.7 mL) was degassed for 10 min by bubbling argon. Pd(PPh3)4 (46 mgo 0.04 mmol) was addedo and the mixture was heated at 100° C. for 12 h. The mixture was cooled rt and diluted saturated aqueous NaHCO3 and EtOAc. The separated organic layer was washed with brineo then dried (Na2SO4)o filteredo concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (155 mgo 56%) as a solid. 1H NMR (500 MHzo CD3OD) δ 7.02-6.92 (mo 1H)o 6.22 (do J=15.4 Hzo 1H)o 4.44 (po J=6.7 Hzo 1H)o 3.53 (so 2H)o 2.21 (qo J=7.2 Hzo 2H)o 2.08 (dto J=12.3o 6.1 Hzo 2H)o 1.82-1.71 (mo 2H)o 1.66 (ddo J=14.9o 7.9 Hzo 2H)o 1.53 (tqo J=14.6o 7.2 Hzo 4H)o 0.96 (to J=7.4 Hzo 3H). LCMS m/z: ES+ [M+H]+=319.2; tR=4.82 min.
To a solution of 4-(cyclopentylamino)-2-[(E)-pent-1-enyl]-5H-pyrimido[4o5-b][1o4]thiazin-6-one (150 mgo 0.471 mmol) in dry tetrahydrofuran (10 mL)o was added BH3·THF (1 M in THF; 4.71 mLo 4.71 mmol) and the reaction mixture was stirred for 1 h at rt. The mixture was diluted with water and EtOAco and the layers were separated. The organic layer was washed with brineo then dried (Na2SO4)o filteredo concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (102 mgo 71%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.38 (po J=6.8 Hzo 1H)o 3.52-3.47 (mo 2H)o 3.10-3.05 (mo 2H)o 2.51 (to J=7.5 Hzo 2H)o 2.04 (dto J=14.1o 6.5 Hzo 2H)o 1.74 (do J=6.5 Hzo 2H)o 1.70-1.59 (mo 4H)o 1.49 (tdo J=13.7o 7.1 Hzo 2H)o 1.38-1.25 (mo 4H)o 0.89 (to J=6.9 Hzo 3H). LCMS m/z: ES+ [M+H]+=307.2; tR=3.70 min.
To a solution of N-cyclopentyl-2-[(E)-pent-1-enyl]-6o7-dihydro-5H-pyrimido[4o5-b][1o4]thiazin-4-amine (40 mgo 0.131 mol) in AcOH (3 mL)o was added slowly H2O2 (31 μLo 0.393 mmol; 30% solution) and the reaction mixture was stirred at 60° C. for 1 h. The mixture was cooled to rt and diluted with saturated aqueous NaHCO3. The aqueous layer was extracted EtOAco and the combined organic layers were dried (Na2SO4)o filteredo concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (19 mgo 43%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.43 (po J=6.8 Hzo 1H)o 3.87-3.81 (mo 2H)o 3.43-3.37 (mo 2H)o 2.60 (to J=7.5 Hzo 2H)o 2.12-2.04 (mo 2H)o 1.75 (do J=7.3 Hzo 2H)o 1.70 (ddo J=14.5o 7.3 Hzo 2H)o 1.66 (ddo J=14.4o 7.5 Hzo 2H)o 1.57-1.48 (mo 2H)o 1.39-1.27 (mo 4H)o 0.89 (to J=6.9 Hzo 3H). LCMS m/z: ES+ [M+H]+=339.2; tR=5.01 min.
To a solution of 4-aminobenzonitrile (5.0 go 42.3 mmol) and 2-methylbut-3-yn-2-ol (5.29 mLo 63.5 mmol) in anhydrous toluene (40 mL) was bubbled argon for 5 mino and then CuCl2 (570 mgo 4.23 mmol) and CuCl (419 mgo 4.23 mmol) were added and the resulting mixture was stirred at 110° C. for 48 h. The mixture was cooled to rt and diluted with EtOAc and brine. The layers were separatedo and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (80 g) using a gradient of 0-100% EtOAc in hexane to afford title compound (4.65 go 60%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.18 (ddo J=8.3o 1.7 Hzo 1H)o 7.08 (do J=1.5 Hzo 1H)o 6.33 (do J=8.3 Hzo 1H)o 6.19 (do J=9.9 Hzo 1H)o 5.50 (do J=9.8 Hzo 1H)o 4.11 (so 1H)o 1.34 (so 6H). LCMS m/z: ES+ [M+H]+=185.1. tR=2.50 min.
A mixture of 2o2-dimethyl-1H-quinoline-6-carbonitrile (2.06 go 11.2 mmol) in 12 N HCl (25.0 mL) was was heated at 90° C. for 3 h. The mixture was concentrated under reduced pressureo diluted with watero and then cooled to 0° C. The pH was adjusted to 3 by slow addition of saturated aqueous NaHCO3. The aqueous layer was extracted with EtOAco and the combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure to afford title compound (1.94 g 86%) as a solid which was used in the next step without further purification. LCMS m/z: ES+ [M+H]+=204.1; (B05) tR=2.20 min.
To a solution of 2o2-dimethyl-1H-quinoline-6-carboxylic acid (1.54 go 7.58 mmol) in anhydrous DMF (30 mL)o was added NoO-dimethylhydroxylamine hydrochloride (1.11 go 11.4 mmol)o followed by HATU (3.46 go 9.09 mmol) and DIPEA (3.89 mLo 22.7 mmol) and the resulting mixture was stirred for 18 h at rt. The mixture was diluted with EtOAc and brine. The layers were separatedo and the aqueous layer was extracted with EtOAc (2×). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (80 g) using a gradient of 0-100% EtOAc in hexane to afford title compound (1.9 go 38%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.44 (ddo J=8.3o 1.2 Hzo 1H)o 7.36 (so 1H)o 6.34 (do J=8.3 Hzo 1H)o 6.26 (do J=9.8 Hzo 1H)o 5.46 (do J=9.8 Hzo 1H)o 3.95 (so 1H)o 3.58 (so 3H)o 3.32 (so 3H)o 1.32 (so 6H); LCMS m/z: ES+ [M+H]+=247.2; QC tR=4.28 min.
To a solution of n-BuLi (1.50 M in hexaneo 1.89 mLo 2.84 mmol) in anhydrous THF (2 mL) at −10° C.o was added a −10° C. solution of N-methoxy-No2o2-trimethyl-1H-quinoline-6-carboxamide (700 mgo 2.84 mmol) in anhydrous THF (7.à mL) and the resulting mixture was stirred 15 min at −10° C. The mixture was diluted with brineo and the aqueous layer was extracted with EtOAc (3×50 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (12 g) using a gradient of 0-30% EtOAc in hexane to afford title compound (95 mgo 14%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.61 (ddo J=8.5o1.3 Hzo 1H)o 7.50 (so 1H)o 6.34 (do J=8.4 Hzo 1H)o 6.26 (do J=9.8 Hzo 1H)o 5.46 (do J=9.9 Hzo 1H)o 4.32 (so 1H)o 2.81 (to J=7.5 Hzo 2H)o 1.70-1.61 (mo 2H)o 1.40 (do J=7.4 Hzo 2H)o 1.32 (so 6H)o 0.92 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=244.2; QC tR=5.0 min.
To a solution of N-methoxy-No2o2-trimethyl-1H-quinoline-6-carboxamide (219 mgo 0.889 mmol) in mixture of DCM (4 mL) and pyridine (0.281 mLo 5.33 mmol)o was added trifluoroacetic anhydride (0.162 mLo 1.16 mmol) and the resulting mixture was stirred for 4 h at rt. The mixture was diluted with DCM and the organic layer was washed subsequently with 1 M aqueous HClo watero saturated aqueous NaHCO3 and brine. The organic layer was dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on slica gel (12 g) using a gradient of 0-50% EtOAc in hexane to afford title compound (255 mgo 84%) as a solid. LCMS m/z: ES+ [M+H]+=343.2; tR=2.73 min.
To a solution of N-methoxy-No2o2-trimethyl-8-(2o2o2-trifluoroacetyl)-1H-quinoline-6-carboxamide (210 mgo 0.613 mmol) in absolute ethanol (5 mL) at rto was added H2SO4 (12 μLo 0.123 mmol) and the reaction mixture was heated at 85° C. for 12 h. The mixture was cooled to rt and the volatiles were concentrated under reduced pressure. The material was purified by reverse phase chromatography on C18 (5.5 g) using a gradient of 10-100% MeCN in water (contains 0.1% formic acid) to afford title compound (110 mgo 55%) as a solid. 1H NMR (500 MHzo CDCl3) δ 9.09 (so 1H)o 8.09 (so 1H)o 7.64 (so 1H)o 6.47 (do J=10.1 Hzo 1H)o 5.75 (do J=10.2 Hzo 1H)o 4.23 (qo J=7.0 Hzo 2H)o 1.40 (so 6H)o 1.25 (to J=7.1 Hzo 3H). LCMS m/z: ES+ [M+H]+=328.1; QC tR=5.81 min.
To a solution of methyl 1o2o3o4-tetrahydroquinoline-6-carboxylate (1.0 go 4.82 mmol) in anhydrous DCM (27 mL) at re was added NBS (945 mgo 5.31 mmol) and the reaction mixture was stirred for 30 min. The mixture was diluted with saturated aqueous NaHCO3 and the layers were separated. The aqueous layer was extracted with DCM. The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a mixture of 15% EtOAc in hexane to afford title compound (1.12 go 86%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.94 (so 1H)o 7.58 (so 1H)o 5.30 (bso 1H)o 3.83(so 3H)o 3.45-3.43(mo 2H)o 2.80-2.77 (mo 2H)o 1.93-1.92 (mo 2H). LCMS m/z: ES+ [M+H]+=270.1; tR=2.60 min.
To a solution of methyl 1o2o3o4-tetrahydroquinoline-8-bromo-6-carboxylate (2.9 go 10.1 mmol) in THFo MeOH and H2O (3:1:1; 30 mL)o was added LiOH (851 mgo 20.3 mmol) and the reaction mixture was stirred at 50° C. for 4 h. The volatiles were evaporated under reduced pressure and diluted with EtOAc. The pH was adjusted to ˜2 with in 1 N aqueous HCl and the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure to afford title compound as a solido which was used in the next step without further purification. LC-MS m/z: ES+ [M+H]+=256.0; tR=2.20 min.
To a solution of 8-bromo-1o2o3o4-tetrahydroquinoline-6-carboxylic acid (900 mgo 3.51 mmol)o NoO-dimethylhydroxylamine;hydrochloride (411 mgo 4.22 mmol) and HATU (1.66 go 4.22 mmol) in anhydrous DMF (25 mL) was added DIPEA (1.84 mLo 10.5 mmol) and the reaction mixture was stirred overnight at rt. The mixture was diluted with saturated aqueous NaHCO3 and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brineo then dried (Na2SO4o)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (895 mgo 85%) as a solid. LCMS m/z: ES+ [M+H]+=301.1; tR=2.32 mins
To a solution of 8-bromo-N-methoxy-N-methyl-1o2o3o4-tetrahydroquinoline-6-carboxamide (400 mgo 1.34 mmol) in DMF (10 mL)o was added Cs2CO3 (871 mgo 2.67 mmol) followed by benzyl chloride (154 μLo 1.34 mmol) and the reaction mixture stirred at 90° C. for 12 h. The mixture was cooled to rt and diluted with H2O (15 mL). The aqueous layer was extracted with EtOAc (2×15 mL) and the combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a mixture of 15% EtOAc in hexane to afford title compound (75 mgo 15%) as a solid. LCMS m/z: ES+ [M+H]+=389.1; tR=2.74 min.
To a solution of 1-benzyl-8-bromo-N-methoxy-N-methyl-3o4-dihydro-2H-quinoline-6-carboxamide (700 mgo 1.80 mmol) in THF (20.0 mL) at 0° C.o was added n-BuMgCl (2 M in THFo 1.18 mLo 2.36 mmol) and the reaction mixture was warmed up to rt and then stirred for 6 h. The mixture was diluted with saturated aqueous NH4Cl and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-20% EtOAc in hexane to afford title compound (600 mgo 73% yield) as a solid. LCMS m/z: ES+ [M+H]+=386.1o LCMS; tR=2.70 min.
To a solution of 1-(1-benzyl-8-bromo-3o4-dihydro-2H-quinolin-6-yl)pentan-1-one (70 mgo 0.181 mmol) in ammonium hydroxide (1 mL) and DMF (1 mL)o was added 2o4-pentanedione (5.4 mgo 0.054 mmol)o followed by cesium carbonate (177 mgo 0.544 mmol)o and CuI (8.60 mgo 0.045 mmol) and the reaction mixture was heated at 110° C. for 6 h. The mixture was cooled to rto diluted with EtOAc (10 mL) was added. The organic layer was washed with water (10 mL) and brine (5 mL)o then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (7.0 mgo 12%) as a solid. LCMS m/z: ES+ [M+H]+=323.2; LCMS; tR=2.97 min.
To a solution of 1-(8-amino-1-benzyl-3o4-dihydro-2H-quinolin-6-yl)pentan-1-one (30 mgo 0.0930 mmol) in DCM (3 mL) at 0° C.o was added DMAP (2.4 mgo 0.02 mmol) followed by triethylamine (14.2 μLo 0.102 mmol) and a solution of isobutanesulfonyl chloride (14.6 mgo 0.093 mmol) in DCM (0.5 mL)o and the reaction mixture was stirred at rt for 12 h. The mixture was diluted with saturated aqueous NaHCO3 and the aqueous layer was extracted with DCM. The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a mixture of 5% EtOAc in hexane to afford title compound (7 mgo 17%) as a solid. LCMS m/z: ES+ [M+H]+=443.2o tR=3.07 min.
To a mixture of N-(1-benzyl-6-pentanoyl-3o4-dihydro-2H-quinolin-8-yl)-2-methyl-propane-1-sulfonamide (10.0 mgo 0.0226 mmol) and 10% Pd/C (24 mgo 0.226 mmol) in anhydrous EtOAc (5 mL) was hydrogenated under hydrogen atmosphere for 6 h at rt. The mixture was filtered on Celiteo rinsed with EtOAc and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-50% EtOAc in hexane to afford title compound (4 mgo 53%) as a solid. 1H NMR (500 MHzo CD3OD) δ 7.60 (so 1H)o 7.54 (so 1H)o 3.42-3.37 (mo 2H)o 2.99 (do J=6.4 Hzo 2H)o 2.86-2.81 (mo 2H)o 2.79 (to J=6.2 Hzo 2H)o 2.25 (dto J=13.3o 6.7 Hzo 1H)o 1.91-1.84 (mo 2H)o 1.63 (dto J=15.2o 7.5 Hzo 2H)o 1.39 (dto J=15.0o 7.4 Hzo 2H)o 1.08 (do J=6.7 Hzo 6H)o 0.93 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=353.2 tR=5.58 min.
A solution of 8-bromo-N-methoxy-N-methyl-1o2o3o4-tetrahydroquinoline-6-carboxamide (900 mgo 3.01 mmol)o di-tert-butyl dicarbonate (788 mgo 3.61 mmol) and DMAP (110 mgo 0.903 mmol) in THF (25 mL) was heated to 68° C. for 12 h. The reaction was cooled to re diluted with saturated aqueous NaHCO3 (10. mL) and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated to afford title compound (1.02 go 85%) as a solid. LCMS m/z: ES+ [M-Boc]: 399.1; tR=2.72 min.
To a solution of tert-butyl 8-bromo-6-pentanoyl-3o4-dihydro-2H-quinoline-1-carboxylate (500 mgo 1.25 mmol) in THF (15 mL) was added n-BuMgCl (2 Mo 0.95 mLo 1.87 mmol) at 0° C.o the reaction mixture was warmed to rt and stirred for 2 h. The mixture was diluted with saturated aqueous NH4Cl and the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (400 mgo 80%) as an oil. LCMS m/z: ES+ [M-Boc]: 296.1o tR=2.90 min.
To a solution of tert-butyl 8-bromo-6-pentanoyl-3o4-dihydro-2H-quinoline-1-carboxylate (500 mgo 1.26 mmol) in DCM (10 mL)o was added TFA (2.34 mLo 31.5 mmol) and the mixture was stirred at rt for 2 h. The volatiles were evaporated under reduced pressureo and the residue was dissolved in 2 mL of water and pH was adjusted to 7 with saturated aqueous NaHCO3 at 0° C. The aqueous layer was extracted with EtOAc (3×10 mL) and the combined organic layers were dried (Na2SO4)o filtered and concentrated to afford title compound (345 mgo 92%) as an oil. LCMS m/z: ES+ [M+H]+=296.1; tR=2.86 min.
To a solution of 1-(8-bromo-1o2o3o4-tetrahydroquinolin-6-yl)pentan-1-one (500 mgo 1.69 mmol) in DCM (15 mL) at 0° C.o were successively added triethylamine (342 mgo 3.38 mmol) DMAP (412 mgo 0.338 mmol) and trifluoroacetic anhydride (0.307 mLo 2.19 mmol) and the reaction mixture was stirred at rt for 6 h. The mixture was poured into saturated aqueous NaHCO3 and the layers were separated. The aqueous layer was extracted with DCM. The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-50% EtOAc in hexane to afford title compound (545 mgo 82%) as an oil. 1H NMR (500 MHzo CD3OD) δ 8.06 (so 1H)o 7.87 (so 1H)o 4.30 (so 1H)o 3.49-3.36 (mo 1H)o 3.00 (to J=7.3 Hzo 2H)o 2.92-2.73 (mo 2H)o 2.24 (so 1H)o 2.00 (so 1H)o 1.70-1.58 (mo 2H)o 1.46-1.31 (mo 2H)o 1.01-0.90 (mo 3H). LCMS m/z: ES+ [M+H]+=392.1o tR=2.93 min.
To a solution of 1-[8-bromo-1-(2o2o2-trifluoroacetyl)-3o4-dihydro-2H-quinolin-6-yl]pentan-1-one (150 mgo 0.382 mmol) in ammonium hydroxide (2 mL) and DMF (2 mL)o was added pentane-2o4-dione (11.4 mgo 0.114 mmol) followed by Cs2CO3 (249 mgo 0.765 mmol) and CuI (18 mgo 0.096 mmol) and the reaction mixture was heated at 120° C. for 3 h. The mixture was cooled to rt and diluted with EtOAc (100 mL) and water (20 mL). The layers were separatedo and the organic layer was washed with brine (2×20 mL)o then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (40 mgo 45%) as a solid. 1H NMR (500 MHzo CD3OD) δ 7.18 (do J=3.7 Hzo 2H)o 3.42-3.34 (mo 2H)o 2.87-2.78 (mo 2H)o 2.75 (to J=6.2 Hzo 2H)o 1.89 (dto J=11.9o 6.1 Hzo 2H)o 1.64-1.57 (mo 2H)o 1.41-1.33 (mo 2H)o 0.96-0.90 (mo 3H). LCMS m/z: ES+ [M+H]+=233.1; tR=3.82 min.
To a solution of 1-(8-amino-1o2o3o4-tetrahydroquinolin-6-yl)pentan-1-one (12 mgo 0.052 mmol) in dry pyridine (2 mL) at 0° C.o was added isobutanesulfonyl chloride (7.41 μLo 0.057 mmol) and the reaction mixture was stirred for 12 h at rt. The mixture was diluted with water (20 mL) and the aqueous layer was extracted with EtOAc (2×10 mL). The combined organic layers were washed with 0.5 M aqueous HCl (5 mL) and brine (10 mL)o then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a mixture of 50% EtOAc in hexane to afford title compound (8 mgo 44%) as a solid. 1H NMR (500 MHzo CD3OD) δ 7.60 (so 1H)o 7.53 (so 1H)o 3.42-3.36 (mo 2H)o 2.99 (do J=6.4 Hzo 2H)o 2.84 (to J=7.5 Hzo 2H)o 2.78 (to J=6.2 Hzo 2H)o 2.27-2.23 (mo 1H)o 1.88 (dto J=11.9o 6.1 Hzo 2H)o 1.63 (dto J=15.1o 7.5 Hzo 2H)o 1.37 (dto J=13.4o 6.7 Hzo 2H)o 1.08 (do J=6.6 Hzo 6H)o 0.93 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=353.2 ; tR=5.23 min.
To a solution of valeryl chloride (562 μLo 559 mgo 4.64 mmolo) in anhydrous DCM (4 mL) at −10° C.o was added AlCl3 (619 mgo 4.64 mmol) in portion and the mixture was stirred 15 min. The mixture was then added to a solution of 8-bromochromane (989 mgo 4.64 mmol) in anhydrous DCM (2.5 mL) and the resulting mixture was stirred for 1.5 h. The mixture was poured into a mixture of ice and 12 N HCl. The aqueous layer was extracted with DCM. The combined organic layers were washed with brineo then dried (MgSO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (12 g) using a gradient of 0-45% EtOAc in hexane to afford title compound (801 mgo 58%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.94 (do J=1.8 Hzo 1H)o 7.61 (so 1H)o 4.54-4.23 (mo 2H)o 2.86-2.81 (mo 4H)o 2.14-1.92 (mo 2H)o 1.71-1.56 (mo 2H)o 1.44-1.27 (mo 2H)o 0.92 (to J=7.4 Hzo 3H). LCMS m/z: ES+ [M+H]+=299.1; tR=3.00 min.
A mixture of 1-(8-bromochroman-6-yl)pentan-1-one (100 mgo 0.336 mmol)o D-proline (39 mgo 0.336 mmol)o cyclopentylamine (60.0 μLo 0.707 mmol)o and K2CO3 (93 mgo 0.673 mmol) in anhydrous DMF (0.75 mL) was degassed by bubbling argon for 5 min. CuI (32 mgo 0.168 mmol) was then added and the resulting mixture was stirred overnight at 120° C. The mixture was cooled to rt and diluted with brine and EtOAc. The layers were separatedo and the aqueous layer was extracted with EtOAc. The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by reverse phase chromatography on C18 (15 g) using a gradient 15-100% MeCN and water (contains 0.1% formic acid) to afford title compound (40 mgo 40%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.06 (so 1H)o 4.43-4.19 (mo 1H)o 4.14 (so 1H)o 3.83 (po J=6.3 Hzo 1H)o 2.96-2.84 (mo 1H)o 2.78 (to J=6.4 Hzo 1H)o 2.13-1.95 (mo 2H)o 1.82-1.58 (mo 3H)o 1.49 (qdo J=7.2o 3.8 Hzo 1H)o 1.38 (dto J=14.7o 7.4 Hzo 1H)o 0.94 (to J=7.3 Hzo 1H). LCMS m/z: ES+ [M+H]+=302.3; QC tR=6.80 min.
To a solution of 6-bromo-8-fluoro-quinoline (1.5 go 6.63 mmol) in DMF (30 mL) was addedo Zn(CN)2 (1.55 go 13.26 mmol) followed by Pd(PPh3)4 (383 mgo 0.331 mmol) and the mixture was degassed by bubbling argon for 5 min and then heated at 100° C. for 3 h. The mixture was cooled to rt and diluted with saturated aqueous NH4Cl. The aqueous layer was extracted with EtOAc (3×30 mL) and the combined organic layers were washed with brineo then dried (Na2SO4)o filteredo concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (1.2 go 100%) as a solid. LCMS (ES+): m/z [M+H]+173.1; tR=2.23 min.
A solution of 8-fluroquniloine 6-carbonitrile (1.2 go 6.93 mmol) in 12 N HCl (25.0 mL) was heated at 90° C. for 2 h. The mixture was cooled to rt and the volatiles were evaporated under reduced pressure. The residue was diluted with watero cooled to 0° C. and the pH was adjusted to 3 by addition of saturated aqueous sodium carbonate (NaHCO3). The aqueous layer was extracted with EtOAc (3×30 mL) and the combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure to afford title compound (1.0 go 75%) as a solid which was used in the next step without further purification. LCMS m/z: ES+ [M+H]+=192.02; tR=1.54 mins.
To a solution of 8-fluoroquinoline-6-carboxylic acid (700 mgo 3.66 mmol) in anhydrous dimethylformamide (25 mL)o was added NoO-dimethylhydroxylamine hydrochloride (428 mgo 4.39 mmol) followed by HATU (1.66 go 4.39 mmol) and DIPEA (1 mLo 5.49 mmol) and the reaction mixture was stirred overnight at rt. The mixture was diluted with saturated aqueous NaHCO3 and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (750 mgo 87%) as a solid. LCMS m/z: ES+ [M+H]+=235.1; tR=2.31 min.
To a solution of 8-fluoro-N-methoxy-N-methyl-quinoline-6-carboxamide (750 mgo 3.20 mmol) in THF (20 mL) at 0° C.o was added n-BuMgCl (2 M in THFo 2.4 ml: 4.80 mmol) and the reaction mixture was warmed to rt and stirred for 2 h. The mixture was diluted with saturated aqueous NH4Cl and the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (565 mgo 65%) as a solid. LCMS m/z: ES+ [M+H]+=232.1o tR=2.83 min.
To a suspension of NaH (60% oil dispersiono 151 mgo 4.5 mmol) in anhydrous DMF (10 mL) was added a solution of cyclopentanol (0.294 mLo 3.0 mmol) in DMF (2.0 mL) at 0° C. and the mixture was stirred at rt for 15 min. (8-fluoro-6-quinolyl)pentan-1-one (231 mgo 1.0 mmol) was then added and the reaction mixture was heated to 80° C. for 4 h. The mixture was diluted with saturated aqueous NH4Cl and the aqueous layer was extracted with EtOAc (3×15 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (195 mgo 65%) as a solid. LCMS m/z: ES+ [M+H]+=298.1; tR=2.15 min.
To a solution of 1-[8-(cyclopentoxy)-6-quinolyl]pentan-1-one (149 mgo 0.5 mmol) in DCM (8 mL) at rto was added Fe(ClO4)2 (63 mgo 0.25 mmol) followed by Hantzsch ester (253 mgo 1.0 mmol) and the reaction mixture was stirred for 24 h at rt. The volatiles were evaporated under reduced pressure and the material was purified by column chromatography on silica gel using a gradient of 0-15% MeOH in DCM to afford title compound (35 mgo 24%) as a solid. 1H NMR (500 MHzo MeOD); δ 7.25 (so 1H)o 7.05 (so 1H)o 4.14-4.07 (mo 1H)o 4.02-3.90 (mo 2H)o 3.82 (tdo J=8.1o 5.4 Hzo 1H)o 3.70 (ddo J=9.0o 3.2 Hzo 1H)o 3.41-3.34 (mo 4H)o 2.85 (to J=7.5 Hzo 2H)o 2.74 (to J=6.2 Hzo 2H)o 2.31-2.26 (mo 1H)o 1.94-1.82 (mo 3H)o 1.65-1.61 (mo 2H)o 1.45-1.32 (mo 2H)o 0.95 (to 3H). LCMS m/z: ES+ [M+H]+=302.2; tR=3.88 min.
To a solution of tert-butyl 8-bromo-6-pentanoyl-3o4-dihydro-2H-quinoline-1-carboxylate (200 mgo 0.506 mmol)o 4-pyridinylboronic acid (74 mgo 0.606 mmol) and NaHCO3 (85 mgo 1.01 mmol) in toluene (6 mL) and water (1 mL) was degassed for 10 min by bubbling argon. Pd(dppf)Cl2 (49 mgo 0.067 mmol) was then addedo degassed for 5 min with N2 and the resulting mixture was heated at 110° C. for 12 h. The mixture was cooled to re diluted with EtOAc and filtered on celite. The filtrate was concentrated under reduced pressure and the material was purified by column chromatography on silica using a gradient of 0-100% EOAc in hexane to afford title compound (110 mgo 55%) as a solid. LCMS m/z: ES+ [M+H]+=395.1; tR=2.53 min.
To a solution of tert-butyl 6-pentanoyl-8-(4-pyridyl)-3o4-dihydro-2H-quinoline-1-carboxylate (80 mgo 0.202 mmol) in DCM (0 mL) was added TFA (1.0 mL) and the reaction mixture was stirred at rt for 2 h. The volatiles were evaporated under reduced pressure and the residue was diluted with water (2 mL) and saturated aqueous NaHCO3 (10 mL). The aqueous layer was extracted with EtOAc (3×10 mL) and the combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (38 mgo 63%) as a solid. 1H NMR (500 MHzo MeOD) δ 7.75 (do J=7.2 Hzo 2H)o 7.67 (so 1H)o 7.54 (so 1H)o 7.32 (ddo J=7.4 Hzo 2H)o 3.42-3.30 (mo 2H)o 2.87 (ddo J=12.2o 6.4 Hzo 4H)o 1.93 (dto J=11.4o 6.4 Hzo 2H)o 1.60 (ddo J=12.1o 7.4 Hzo 2H)o 1.41-1.30 (mo 2H)o 0.95 (do J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=295.1o QC tR=3.74 min.
To a mixture of tert-butyl 8-bromo-6-pentanoyl-3o4-dihydro-2H-quinoline-1-carboxylate (400 mgo 1.01 mmol)o imidazole (109 mgo 1.60 mmol)o Pd2dba3 (122 mgo 0.134 mmol)o BINAP (83 mgo 0.134 mmol)o and sodium t-butoxide (193 mgo 2.01 mmol) in toluene (5 mL) was degassed for 10 min with nitrogen and the resulting mixture was heated at 100° C. for 12 h. The mixture was cool to rto diluted with EtOAc and filtered on Celite. The filtrate was concentrated under reduced pressure and the material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (133 mgo 34%) as a solid. LCMS m/z: ES+ [M+H]+=384. 2; tR=2.48 min.
To a solution of tert-butyl 8-imidazol-1-yl-6-pentanoyl-3o4-dihydro-2H-quinoline-1-carboxylate (40 mgo 0.104 mmol) in DCM (3 mL) was added TFA (1.0 mL) and the reaction mixture was stirred at rt for 2 h. The volatiles were evaporated under reduced pressure and the residue was diluted in water (2.0 mL) and saturated aqueous NaHCO3 (10 mL). The aqueous layer was extracted with EtOAc (3×10 mL) and the combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (11.5 mgo 40%) as a solid. 1H NMR (500 MHzo MeOD) δ 7.75 (so 1H)o 7.67 (so 1H)o 7.54 (so 1H)o 7.20 (do J=7.2 Hzo 2H)o 3.31-3.29 (mo 2H)o 2.85 (ddo J=14.2o 6.9 Hzo 4H)o 1.90 (dto J=11.8o 6.1 Hzo 2H)o 1.61 (ddo J=15.1o 7.5 Hzo 2H)o 1.40-1.30 (mo 2H)o 0.93 (do J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=284.1o QC tR=3.62 min.
To a mixture of tert-butyl 8-bromo-6-pentanoyl-3o4-dihydro-2H-quinoline-1-carboxylate (200 mgo 0.506 mmol)o 2-pyrrolidinone (43 mgo 0.506 mmol)o NoN′-dimethylethylenediamine (8.9 mgo 0.101 mmol)o K2CO3 (139 mgo 1.01 mmol) and CuI (48 mgo 0.253 mmol) in dioxane (5 mL) was heated at 110° C. overnight. The mixture was cooled to re filtered on Celite and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (81 mgo 40%) as a solid. LCMS m/z: ES+ [M+H]+=401.2; tR=2.44 min.
To a solution of tert-butyl 8-(2-oxopyrrolidin-1-yl)-6-pentanoyl-3o4-dihydro-2H-quinoline-1-carboxylate (80 mgo 0.199 mmol) in DCM (3 mL)o was added TFA (1.0 mL) was stirred at rt for 2 h. The volatiles were evaporated under reduced pressure and the residue was diluted with water (2 mL) and saturated aqueous NaHCO3 (10 mL). The aqueous layer was extracted with EtOAc (3×10 mL) and the combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-100% EtOAc in hexane to afford title compound (48 mgo 80%) as a solid. 1H NMR (500 MHzo MeOD); δ 7.23 (so 1H)o 7.06 (so 1H)o 4.02-3.92 (mo 2H)o 3.70 (to J=9.0o 3.2 Hzo 2H)o 3.41-3.34 (mo 4H)o 2.85 (to J=7.5 Hzo 2H)o 2.74 (to J=6.2 Hzo 2H)o 1.94-1.61 (mo 4H)o 1.45-1.32 (mo 2H)o 0.95 (mo 3H). LCMS m/z: ES+ [M+H]+=302. 2; tR=3.88 min. LCMS m/z: ES+ [M+H]+=301.1o QC tR=3.58 min.
A solution of ethyl 4-aminobenzoate (1.00 go 6.05 mmol) and 2-methylbut-3-yn-2-ol (0.76 mLo 9.08 mmol) in anhydrous toluene (10 mL) was sparged with bubbling argon for 5 min. CuCl2 (81 mgo 0.605 mmol) was added followed by CuCl (60 mgo 0.605 mmol) and the resulting mixture was stirred at 110° C. for 48 h. The mixture was cooled to rt and diluted with EtOAc and brine. The layers were separatedo and the aqueous layer was extracted with EtOAc (2×150 mL). The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica (12 g) using a gradient of 0-100% EtOAc in hexane to afford title compound (727 mgo 52%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.66 (ddo J=8.3o 1.9 Hzo 1H)o 7.56 (do J=1.6 Hzo 1H)o 6.34 (do J=8.4 Hzo 1H)o 6.27 (do J=9.8 Hzo 1H)o 5.46 (do J=9.8 Hzo 1H)o 4.29 (do J=7.1 Hzo 2H)o 4.06 (so 1H)o 1.41-1.28 (mo 9H). LCMS m/z: ES+ [M+H]+=232.2; (B05) tR=2.60 min.
A mixture of ethyl 2o2-dimethyl-1H-quinoline-6-carboxylate (727 mgo 3.14 mmol) and Pd/C (10% on carbono 335 mgo 3.14 mmol) in ethanol (10 mL) was hydrogenated under hydrogen atmosphere for 1 h. The mixture was filtered on Celiteo rinsed with EtOH and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel (12 g) using a gradient of 0-100% EtOAc in hexane to afford title compound (615 mgo 84%) as a solid. 1H NMR (500 MHzo CDCl3)o 7.70 (so 1H)o 7.65 (ddo J=8.4o 1.8 Hzo 1H)o 6.38 (do J=8.4 Hzo 1H)o 4.29 (qo J=7.2 Hzo 2H)o 4.11 (so 1H)o 2.79 (to J=6.7 Hzo 2H)o 1.70 (to J=6.7 Hzo 2H)o 1.35 (to J=7.1 Hzo 3H)o 1.22 (so 6H). LCMS m/z: ES+ [M+H]+=234.2; tR=2.65 min.
A solution of HNO3 (0.0284 mLo 0.675 mmol) in H2SO4 (0.50 mL) was added dropwise to a solution of ethyl 2o2-dimethyl-3o4-dihydro-1H-quinoline-6-carboxylate (150 mgo 0.643 mmol) in H2SO4 (1.50 mL) at 0° C. and the reaction mixture was stirred for 30 min at 0° C. The mixture was added slowly onto crushed ice and the resulting solid that formed was collected by filtration and dried under high vacuum to afford title compound (146 mgo 74%) as a solid which was used in the next step without purification. LCMS m/z: ES+ [M+H]+=279.2; tR=2.69 min.
To a solution of crude ethyl 2o2-dimethyl-8-nitro-3o4-dihydro-1H-quinoline-6-carboxylate (131 mgo 0.471 mmol) in methanol (5 mL) was added ammonium formate (297 mgo 4.71 mmol) followed by Pd/C (10% on carbono 50 mgo 0.471 mmol) and the reaction mixture was stirred at 50° C. overnight. The mixture was filtered on Celiteo rinsed with methanolo and the filtrate was concentrated under reduced pressure. The material was purified by reverse phase chromatography on C18 (5.5 g) using a gradient 10-100% MeCN in water (contains 0.1% formic acid) to afford title compound (20 mgo 18%) as a solid. 1H NMR (500 MHzo DMSO) δ 7.24 (so 1H)o 6.20-6.15 (mo 3H)o 5.64 (so 1H)o 4.09 (qo J=7.1 Hzo 2H)o 2.52-2.48 (mo 2H)o 1.50 (to J=6.6 Hzo 2H)o 1.21 (to J=7.1 Hzo 3H)o 1.09 (so 6H). LCMS m/z: ES+ [M+H]+=249.2; QC tR=4.99 min.
To a mixture of ethyl 8-amino-2o2-dimethyl-3o4-dihydro-1H-quinoline-6-carboxylate (10 mgo 0.04 mmol) and cyclopentanone (21 μLo 0.242 mmol) in anhydrous DCM (0.5 mL) was successively added sodium triacetoxyborohydride (51 mgo 0.242 mmol) and TFA (2.3 μLo 0.040 mmol) and the reaction mixture was stirred overnight at rt. The mixture was diluted with DCM (5 mL) and saturated aqueous NaHCO3 (10 mL). The layers were separatedo and the aqueous layer was extracted with DCM. The combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by reverse phase chromatography on C18 (5.5 g) using a gradient 10-100% MeCN in water (contains 0.1% formic acid) to afford title compound (65 mgo 51%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.62 (bso 1H)o 7.56 (so 1H)o 5.63 (so 1H)o 4.23 (qo J=7.2 Hzo 2H)o 4.08 (bso 1H)o 3.72 (so 1H)o 2.66 (to J=6.6 Hzo 2H)o 2.06-1.90 (mo 2H)o 1.74 (ddo J=12.2o 8.1 Hzo 2H)o 1.67 (to J=6.7 Hzo 2H)o 1.57 (dto J=15.8o 6.4 Hzo 4H)o 1.33 (to J=7.1 Hzo 3H)o 1.21 (so 6H). LCMS m/z: ES+ [M+H]+=317.3; QC tR=6.83 min.
To a solution of 1-(1-benzyl-8-bromo-3o4-dihydro-2H-quinolin-6-yl)pentan-1-one (50 mgo 0.129 mmol) in anhydrous DMF (1.0 mL)o was added 2-aminopyridine (12.2 mgo 0.130 mmol) and Cs2CO3 (84.3 mgo 0.259 mmol) and the mixture was degassed for 5 min by bubbling argon. Xantphos (9.0 mgo 0.0155 mmol) and Pd2dba3 (14.9 mgo 0.0259 mmol) were added and the mixture was degassed for another 5 min and then the reaction mixture was stirred at 100° C. for 12 h. The mixture was cooled to rt and diluted with water (1 mL) and EtOAc (10 mL). The separated organic layer was washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a mixture of 5% MeOH in DCM to afford title compound (20 mgo 40%) as a solid. LCMS m/z: ES+ [M+H]+=400.3 tR=2.17 min.
A mixture of 1-[1-benzyl-8-(2-pyridylamino)-3o4-dihydro-2H-quinolin-6-yl]pentan-1-one (17 mgo 0.043 mmol) and Pd/C (10% on carbono 45 mgo 0.43 mmol) in EtOAc (5 mL) was hydrogenated under hydrogen atmosphere for 6 h at rt. The mixture was filtered on Celiteo rinsed with EtOAc and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using 0-50% EtOAc in hexane to afford title compound (9 mgo 70%) as a solid. 1H NMR (500 MHzo CD3OD) δ 7.95 (do J=4.3 Hzo 1H)o 7.60 (do J=1.8 Hzo 1H)o 7.52 (so 1H)o 7.51-7.46 (mo 1H)o 6.69-6.65 (mo 1H)o 6.49 (do J=8.5 Hzo 1H)o 3.37-3.33 (mo 2H)o 3.29 (dto J=2.9o 1.5 Hzo 2H)o 2.85-2.79 (mo 4H)o 1.90 (dto J=11.9o 6.1 Hzo 2H)o 1.62 (dto J=20.8o 7.6 Hzo 2H)o 1.41-1.32 (mo 2H)o 0.92 (to J=7.4 Hzo 3H). LCMS m/z: ES+ [M+H]+=310.2 QC tR=3.29 min.
To a solution of 1-[8-(2-pyridylamino)-1o2o3o4-tetrahydroquinolin-6-yl]pentan-1-one (20 mgo 0.065 mmol) in MeOH (5 mL) at 0° C.o was added NaBH4 (4.89 mgo 0.129 mmol) and the reaction mixture was stirred for 30 min at 0° C.o and then warmed to rt and stirred for 1 h. The mixture was diluted with water and the aqueous layer was extracted with EtOAc (2×10 mL). The combined organic layers were washed with brine (10 mL)o then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 1-5% MeOH in DCM to afford title compound (12 mgo 60%) as a solid. 1H NMR (500 MHzo CD3OD) δ 7.95 (ddo J=5.1o 1.2 Hzo 1H)o 7.45 (dddo J=8.6o 7.1o 1.8 Hzo 1H)o 6.90 (do J=1.6 Hzo 1H)o 6.80 (so 1H)o 6.64 (ddo J=6.5o 5.4 Hzo 1H)o 6.49 (do J=8.3 Hzo 1H)o 4.38 (to J=6.8 Hzo 1H)o 3.27-3.23 (mo 2H)o 2.78 (to J=6.3 Hzo 2H)o 1.94-1.85 (mo 2H)o 1.77-1.67 (mo 1H)o 1.67-1.56 (mo 1H)o 1.35-1.27 (mo 3H)o 1.18 (ddto J=10.8o 7.4o 5.2 Hzo 1H)o 0.90-0.84 (mo 3H). LCMS m/z: ES+ [M+H]+=312.2 tR: 3.08 min.
To a solution of 1-[8-(2-pyridylamino)-1o2o3o4%-tetrahydroquinolin-6-yl]pentan-1-ol (16 mgo 0.051 mmol) in DCM (5 mL)o was added (Et)3SiH (0.017 mLo 0.103 mmol) followed by TFA (7.6 μLo 0.103 mmol) and the reaction mixture was stirred for 2 h at rt. The mixture was diluted with saturated aqueous NaHCO3 and the aqueous layer was extracted with DCM (2×10 mL). The combined organic layers were washed with brine (10 mL)o then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 1-5% MeOH in DCM to afford title compound (12 mgo 76%) as a solid. 1H NMR (500 MHzo CD3OD) δ 7.92 (ddo J=5.0o 1.2 Hzo 1H)o 7.51 (dddo J=8.6o 7.1o 1.7 Hzo 1H)o 6.74 (do J=1.4 Hzo 1H)o 6.67 (do J=7.9 Hzo 2H)o 6.56 (do J=8.5 Hzo 1H)o 3.25-3.21 (mo 2H)o 2.76 (to J=6.4 Hzo 2H)2.46-2.40 (mo 2H)o 1.92-1.86 (mo 2H)o 1.58-1.49 (mo 2H)o 1.35-1.24 (mo 4H)o 0.87 (to J=7.0 Hzo 3H). LCMS m/z: ES+ [M+H]+=296.3 QC tR: 3.83 min.
To a solution of 1-(1-benzyl-8-bromo-3o4-dihydro-2H-quinolin-6-yl)pentan-1-one (350 mgo 0.906 mmol) in methanol (5 mL) at 0° C.o was added NaBH4 (68.6 mgo 1.81 mmol) and the reaction mixture was stirred for 30 min at 0° C. then 1 h at rt. The mixture was diluted with water and the aqueous layer was extracted with EtOAc (2×10 mL). The combined organic layers were washed with brine (10 mL)o then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a mixture of 30% EtOAc in hexane to afford title compound (300 mgo 86%) as a solid. LCMS m/z: ES+ [M+H]+=388.1o tR: 3.01 min.
To a solution of 1-(1-benzyl-8-bromo-3o4-dihydro-2H-quinolin-6-yl) pentan-1-ol (500 mgo 1.29 mmol) in anhydrous THF (20 mL) at 0° C.o was added NaH (60% oil dispersiono 44 mgo 1.93 mmol) and the mixture was warmed to rt and stirred for 20 min. MeI (96 μLo 1.55 mmol) was then added at 0° C. and the reaction mixture warmed to rt and stirred for 12 h. The mixture was diluted with saturated aqueous NH4Cl (10.0 mL) and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (500 mgo 85%) as a solid. LCMS (ES+): m/z [M+H]+ 402.1o tR=2.46 min.
To a solution of 1-benzyl-8-bromo-6-(1-methoxypentyl)-3o4-dihydro-2H-quinoline (200 mgo 0.497 mmol) in anhydrous DMF (3 mL)o was added 2-aminopyridine (46.9 mgo 0.498 mmol) followed by Cs2CO3 (324 mgo 0.994 mmol)o and then the mixture was degassed for 5 min by bubbling argon. Xantphos (35 mgo 0.06 mmol) and Pd2dba3 (57 mgo 0.01 mmol) were added and the mixture was degassed for another 5 min and then the reaction mixture was stirred at 100° C. for 12 h. The mixture was cooled to rt then diluted with water (10 mL) and EtOAc (50 mL). The separated organic layer was washed with brineo then dried (Na2SO4) filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient 0-100% EtOAc in hexane to afford title compound (90 mgo 44%) as a solid. LCMS m/z: ES+ [M+H]+=416.3o tR=2.25 min.
A mixture of 1-benzyl-6-(1-methoxypentyI)-N-(2-pyridyl)-3o4-dihydro-2H-quinolin-8-amine (50.0 mgo 0.120 mmol) and Pd/C (10% on carbono 2.0 mgo 0.012 mmol) in EtOAc (5 mL) was hydrogenated under hydrogen atmosphere for 6 h at rt. The mixture was filtered on Celiteo washed and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-50% EtOAc in hexane to afford title compound (25 mgo 64%) as a solid. 1H NMR (500 MHzo) δ 8.02-7.94 (mo 1H)o 7.53-7.42 (mo 1H)o 6.86 (do J=1.4 Hzo 1H)o 6.77 (so 1H)o 6.67 (ddo J=6.5o 5.7 Hzo 1H)o 6.49 (do J=8.5 Hzo 1H)o 3.94 (to J=6.9 Hzo 1H)o 3.30-3.26 (mo 2H)o 3.16 (so 3H)o 2.80 (to J=6.3 Hzo 2H)o 1.99-1.85 (mo 2H)o 1.77 (tddo J=12.1o 6.9o 4.9 Hzo 1H)o 1.65-1.52 (mo 1H)o 1.39-1.24 (mo 3H)o 1.19 (tto J=11.4o 4.2 Hzo 1H)o 0.87 (to J=7.1 Hzo 3H). LCMS m/z: ES+ [M+H]+=326.3 QC tR=3.56 min.
A mixture of HNO3 (3.80 go 60.3 mmol) and concentrated H2SO4 (9 mL) was added dropwise to a solution of 3-methyl-4-(propanoylamino)benzoic acid (2.50 go 12.1 mmol) in H2SO4 (3 mL) at 0° C. and the mixture was stirred for 3 h at rt. The mixture was poured into ice-water and the resulting precipitate was collected by filtration and washed with water. The material was recrystallized from MeOH to afford title compound (810 mgo 29%) as a solid. 1H NMR (500 MHzo CD3OD) δ 8.68 (do J=1.5 Hzo 1H)o 8.16 (so 1H)o 3.23-3.08 (mo 2H)o 2.79-2.58 (mo 2H). LCMS m/z: ES+ [M+H]+=237.1o QC tR=3.37 min.
To a solution of 8-nitro-2-oxo-3o4-dihydro-1H-quinoline-6-carboxylic acid (500 mgo 2.12 mmol) in DMF (15 mL)o were successively added NoO-dimethylhydroxylamineo HCl (227 mgo 2.33 mmol)o HATU (966 mgo 2.54 mmol) and DIPEA (410 mgo 3.18 mmol) and the reaction mixture was stirred for 8 h. The mixture was diluted with water and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with 0.1 N aqueous HClo and brineo then dried (Na2SO4)o filteredo and concentrated under reduced. The material was purified by column chromatography silica gel using a gradient 0-40% EtOAc in hexane to afford title compound (810 mgo 99%) as a solid. LCMS m/z: ES+ [M+H]+=280.1o LCMS; tR=1.91 min.
Sulfuric acid (193 mgo 1.97 mmol) was added to a solution of N-methoxy-N-methyl-8-nitro-2-oxo-3o4-dihydro-1H-quinoline-6-carboxamide (550 mgo 1.97 mmol) in absolute ethanol (15 ml) at room temperature. After refluxing for 3 ho the reaction mixture was concentrated in vacuo and purified by silica-gel column chromatography using a gradient 0-100% EtOAc in hexane to afford title compound (200 mgo 35%) as a solid. LC-MS m/z: ES+ [M+H]+:265.1o LCMS; tR=2.30 min.
To a solution of ethyl 8-nitro-2-oxo-3o4-dihydro-1H-quinoline-6-carboxylate (400 mgo 1.51 mmol) in acetone (5 mL) at rto was added saturated aqueous NH4Cl (5.0 mL) followed by zinc (297 mgo 4.54 mmol)o and the resulting mixture was stirred vigorously for 30 min. The mixture was diluted with EtOAc (25 mL) and then filtered on Celite. The organic layer was washed with saturated aqueous NaHCO3 (10 mL) and brine (15 mL)o then dried (Na2SO4)o filteredo concentrated under reduced pressure to afford title compound (250 mgo 64%) as a solid which was used in the next step without further purification. LCMS m/z: ES+ [M+H]+=235.1o LCMS; tR=1.94 min.
To a mixture of ethyl 8-amino-2-oxo-3o4-dihydro-1H-quinoline-6-carboxylate (50 mgo 0.213 mmol) and cyclopentanone (18 mgo 0.213 mmol) in DCM (5 mL) at rto was added NaBH(OAc)3 (90 mgo 0.427 mmol) and the reaction mixture was stirred for 16 h at rt. The mixture was diluted with saturated aqueous NaHCO3 (10 mL) and the mixture was gently stirred for 5 min. The layers were separatedo and the aqueous layer was extracted with DCM (3×10 mL). The combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-60% EtOAc in hexane to afford title compound (15 mgo 22%) as a solid. 1H NMR (500 MHzo CDCl3) δ 9.21 (so 1H)o 7.33 (do J=6.2 Hzo 2H)o 4.39-4.32 (mo 2H)o 3.01-2.94 (mo 2H)o 2.66-2.59 (mo 2H)o 2.03 (dto J=13.5o 6.6 Hzo 2H)o 1.81-1.73 (mo 2H)o 1.69-1.55 (mo 6H)o 1.43-1.35 (mo 3H); LCMS m/z: ES+ [M+H]+=303.2 tR=4.91 min.
To a solution of 1-(8-amino-1o2o3o4-tetrahydroquinolin-6-yl)pentan-1-one (25.0 mgo 0.108 mmol) in DCM (5.0 mL) at rto was added triethylamine (0.2 mLo 0.143 mmol) followed by DMAP (2.00 mgo 0.0164 mmol) and trifluoroacetic anhydride (24.9 mgo 0.118 mmol) and the reaction mixture was stirred at rt for 4 h and then stirred at 40° C. for 1 h. The mixture was poured onto saturated aqueous NaHCO3 and the layers were separated. The aqueous layer was extracted with EtOAc (2×)o and the combined organic layers were dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica using a gradient of 0-50% EtOAc in hexane to afford title compound (20 mgo 60%) as a solid. 1H NMR (500 MHzo CD3OD) δ 8.26 (so 1H)o 7.85 (so 1H)o 4.46-4.41 (mo 2H)o 3.12-3.05 (mo 4H)o 2.37-2.27 (mo 2H)o 1.74-1.66 (mo 2H)o 1.41 (dto J=14.7o 7.4 Hzo 2H)o 0.96 (to J=7.4 Hzo 3H); LCMS m/z: ES+ [M+H]+=311.2o tR=2.60 min.
To a solution of 1-(8-amino-1-benzyl-3o4-dihydro-2H-quinolin-6-yl)pentan-1-one (25 mgo 0.078 mmol) in DCM (3 mL) at 0° C.o were successively added DMAP (2.0 mgo 0.016 mmol) triethylamine (6.2 μLo 0.085 mmol) then a solution of isobutanesulfonyl chloride (24 mgo 0.16 mmol) in DCM (0.5 mL) and the reaction mixture was stirred at rt for 12 h. The mixture was diluted with saturated aqueous NaHCO3 and the aqueous layer was extracted with DCM. The combined organic layers were washed with brineo then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a mixture of 5% EtOAc in hexane to afford title compound (7 mgo 16%) as an oil. LCMS m/z: ES+ [M+H]+=443.2o tR=3.07 min.
A mixture of N-(1-benzyl-6-pentanoyl-3o4-dihydro-2H-quinolin-8-yl)-N-isobutylsulfonyl-2-methyl-propane-1-sulfonamide (17 mgo 0.030 mmol) and Pd/C (10% on carbono 32 mgo 0.302 mmol) in anhydrous MeOH (5 mL)o was hydrogenated under hydrogen atmosphere for 6 h at rt. The mixture was filtered on Celiteo rinsed with MeOH and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-50% EtOAc in hexane to afford title compound (7 mgo 49%) as a solid. 1H NMR (500 MHzo CDCl3+CD3OD) δ 7.24 (so 1H)o 7.21 (so 1H)o 3.22 (ddo J=13.6o 6.8 Hzo 2H)o 3.11-3.00 (mo 4H)o 2.45 (dto J=13.2o 6.8 Hzo 4H)o 2.00 (dpo J=13.4o 6.7 Hzo 2H)o 1.59-1.50 (mo 2H)o 1.32-1.22 (mo 2H)o 1.04-0.95 (mo 2H)o 0.73 (ddo J=6.6o 4.3 Hzo 12H)o 0.55 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=473.2o QC tR: 6.29 min.
To a solution of 2-chloro-N-cyclopentyl-pyrido[3o2-d]pyrimidin-4-amine (20 mgo 0.080 mmol) in anhydrous ethanol (10 mL)o was added PtO2 (1.83 mgo 0.008 mmol) followed by TFA (0.6 μLo 0.008 mmol) and the resulting mixture was hydrogenated under hydrogen atmosphere for 6 h. The mixture was filtered on Celiteo washed and the filtrate was concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-50% EtOAc in hexane to afford title compound (8 mgo 39%) as a solid. 1H NMR (500 MHzo CD3OD) δ 4.42 (qo J=6.9 Hzo 1H)o 2.69 (to J=6.4 Hzo 2H)o 2.07 (tdo J=12.1o 6.5 Hzo 2H)o 1.97-1.89 (mo 2H)o 1.83-1.72 (mo 2H)o 1.67 (dddo J=10.8o 10.1o 6.0 Hzo 2H)o 1.54 (tdo J=13.6o 6.9 Hzo 2H). LCMS m/z: ES+ [M+H]+=253.1; QC tR=3.67 min.
A mixture composed of 2-chloro-N-cyclopentyl-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (60 mgo 0.237 mmol)o 1-pentenylboronic acid (35 mgo 0.309 mmol)o and K2CO3 (98 mgo 0.71 mmol) in toluene (1.5 mL)o ethanol (0.4 mL)o and water (0.4 mL) was degassed for 10 min by bubbling argon. Pd(dppf)2Cl2 (35 mgo 0.048 mmol) and triphenylphosphine (25 mgo 0.095 mmol) were then addedo the resulting mixture was heated at 100° C. overnight. The mixture was cooled to rt and diluted with saturated aqueous NaHCO3 and EtOAc. The layers were separatedo and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brineo then dried (Na2SO4)o filteredo and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (4 g) using a gradient of 0-70% EtOAc in hexane to afford title compound (35 mgo 52%) as a solid. 1H NMR (500 MHzo CDCl3) δ 8.72 (so 1H)o 7.04-6.85 (mo 1H)o 6.38 (do J=15.1 Hzo 1H)o 4.54-4.38 (mo 2H)o 3.22-3.12 (mo 2H)o 2.73-2.62 (mo 2H)o 2.22 (ddo J=14.2o 7.0 Hzo 2H)o 2.14-2.01 (mo 2H)o 1.90-1.79 (mo 2H)o 1.78-1.69 (mo 2H)o 1.67-1.58 (mo 2H)o 1.57-1.46 (mo 4H)o 0.94 (to J=7.3 Hzo 3H). LCMS m/z: ES+ [M+H]+=287.2; tR=2.05 min.
To a solution of 1-(8-fluoro-6-quinolyl)pentan-1-one (92 mgo 399 μmol) and tetrahydrofuran-3-amine (343 μLo 3.99 mmol) in dry DMSO (1 mL) at re was added DIPEA (139 μLo 797 μmol) and the reaction mixture was stirred at 150° C. for 40 h. The mixture was cooled to rt and diluted with water (25 mL) and DCM (10 mL). The layers were separatedo and the aqueous layer was extracted with DCM (3×10 mL). The combined organic layers were washed with brine (30 mL)o then dried (Na2SO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (12 g cartridge) using a gradient of 0-30% EtOAc and hexane and was further purified by reversed chromatography on C18 (12 g) using 50-100% MeCN and water (contains 0.1% formic acid) to afford title compound (65 mgo 55%) as an oil. 1H NMR (500 MHzo CDCl3) δ 8.80 (ddo J=4.2o 1.7 Hzo 1H)o 8.18 (ddo J=8.3o 1.7 Hzo 1H)o 7.70 (do J=1.7 Hzo 1H)o 7.45 (ddo J=8.2o 4.2 Hzo 1H)o 7.20 (do J=1.7 Hzo 1H)o 6.34 (do J=6.9 Hzo 1H)o 4.41-4.33 (mo 1H)o 4.14 (ddo J=9.2o 5.6 Hzo 1H)o 4.10-4.00 (mo 1Hr 3.94 (tdo J=8.4o 5.2 Hzo 1H)o 3.88 (ddo J=9.2o 3.3 Hzo 1H)o 3.13-3.03 (mo 2H)o 2.48-2.32 (mo 1H)o 2.13-2.00 (mo 1H)o 1.78 (dto J=15.0o 7.5 Hzo 2H)o 1.50-1.40 (mo 2H)o 0.98 (to J=7.3 Hzo 3H). LCMS m/z: ES+[M+H]+=299.92; (A05) tR=1.89 min.
To a solution of 1-[8-(cyclopentylamino)-6-quinolyl]pentan-1-one (65 mgo 218 μmol) and Hantzsch ester (276 mgo 1.09 mmol) in CHCl3 (2 mL)o was added Fe(ClO4)2 (11.1 mgo 44 μmol) at rto and the reaction mixture was stirred at rt for 60 h. The mixture was concentrated under reduced pressure and the material was purified by column chromatography on silica gel (12 g) using a gradient 0-60% of EtOAc in hexane and was further purified by preparative HPLC (BEH 5 μm C18 30×100 mm; using 42-62% MeCN and 10 mM ammonium formate pH 3.8) to afford title compound (12.0 mgo 18%) as a solid. 1H NMR (500 MHzo CD3OD) δ 7.23 (so 1H)o 7.04 (do J=1.6 Hzo 1H)o 4.15-4.06 (mo 1H)o 4.03-3.93 (mo 2H)o 3.84 (tdo J=8.3o 5.4 Hzo 1H)o 3.71 (ddo J=9.0o 3.2 Hzo 1H)o 3.43-3.36 (mo 2H)o 2.87 (to J=7.5 Hzo 2H)o 2.78 (to J=6.2 Hzo 2H)o 2.34-2.26 (mo 1H)o 1.96-1.86 (mo 3H)o 1.69-1.61 (mo 2H)o 1.46-1.35 (mo 2H)o 0.95 (to 3H). LCMS m/z: ES+ [M+H]+=302.70; (A05) tR=1.73 m. LCMS m/z: ES+ [M+H]+=302.62; (B05) tR=1.88 min.
To a solution of 4-fluoro-3-nitro-benzonitrile (10.0 go 60.2 mmol) and 2-methylbut-3-yn-2-amine (6.3 mLo 60.2 mmol) in DMF (60 mL)o was added Et3N (9.2 mLo 66.2 mmol) and the reaction was stirred at rt for 2 h. The volatiles were evaporated under reduced pressure and the residue was diluted with DCM. Water was added (20 mL) and the aqueous layer was extracted with DCM (3×60 mL). The combined organic layers were dried (MgSO4)o filtered and concentrated under reduced pressure. The resulting solid was triturated with Et2O and filtered to afford title compound (12.5 go 91%) as solido which was used in the nest step without further purification. 1H NMR (500 MHzo DMSO) δ 8.57 (do J=2.0 Hzo 1H)o 8.28 (so 1H)o 7.95 (ddo J=9.1o 2.0 Hzo 1H)o 7.64 (do J=9.1 Hzo 1H)o 3.65 (so 1H)o 1.70 (so 6H).
To a suspension of 4-(1o1-dimethylprop-2-ynylamino)-3-nitro-benzonitrile (5.00 go 21.8 mmol) in EtOH (220.0 mL)o were added AcOH (6.2 mLo 0.109 mmol) and Zn (7.13 go 0.109 mmol) and the resulting mixture was stirred at rt for 4 h. The mixture was then filtered on Celiteo washed and the filtrate was concentrated under reduced pressure. The residue was diluted with water (60 mL) and the aqueous layer was extracted with DCM (4×100 mL). The combined organic layers were dried (MgSO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel using a gradient of 0-50% EtOAc in hexane to afford title compound (1.70 go 39%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.29 (do J=8.4 Hzo 1H)o 7.16 (ddo J=8.4o 1.9 Hzo 1H)o 6.97 (do J=1.9 Hzo 1H)o 4.01 (so 1H)o 3.34 (so 2H)o 2.42 (so 1H)o 1.66 (so 6H). LCMS m/z: ES+ [M+H]+=200.06; (B05) tR=1.68 min.
To a solution of 3-amino-4-(1o1-dimethylprop-2-ynylamino)benzonitrile (345 mgo 1.73 mmol) in toluene (3.5 mL)o was added CuCl (86 mgo 0.87 mmol) and the reaction mixture was degassed with nitrogen for 5 min and then refluxed for 6 h. The mixture was cooled at rt and diluted with water (3.5 mL). The layers were separatedo and the aqueous layer was extracted with EtOAc (3×10 mL). The combined organic layers were dried (MgSO4)o filtered and concentrated under reduced pressure. The material was purified by column chromatography on silica gel (24 g) using a gradient of 0-100% EtOAc in hexane to afford title compound (135 mgo 39%) as a solid. 1H NMR (500 MHzo CDCl3) δ 7.44 (do J=1.7 Hzo 1H)o 7.24 (ddo J=8.2o 1.9 Hzo 1H)o 6.49 (do J=8.2 Hzo 1H)o 4.04 (so 1H)o 2.17 (so 3H)o 1.37 (so 6H). LCMS m/z: ES+ [M+H]+=200.05; (B05) tR=1.54 min.
To a solution of N-tert-butyl-6-chloro-3-(trifluoromethyl)-1o7-naphthyridin-8-amine (100 mgo 0.296 mmol) in NoN-Dimethylformamide (1.27 mL) was successively added cesium carbonate (290 mgo 0.889 mmol) and BrettPhos (32 mgo 0.059 mmol). The resulting mixture was degassed by bubbling argon for 5 mins under stirring then 2o2o2-Trifluoroethanol (0.043 mLo 0.59 mmol) and Pd2(dba)3 (14 mgo 0.015 mmol) were added. The vial was sealed then stirred for 1 h at 160° C. in the microwave oven. The mixture was diluted with sat. aq. NaHCO3 and extracted with EtOAc (3×5 mL). The combined organic layers were washed with brineo dried over Na2SO4o filteredo then concentrated. The residue obtained was purified by silica-gel column chromatography (0-100% DCM in hexanes) to afford the title compound (35 mgo 33%) as an oil. 1H NMR (500 MHzo CDCl3) ∂ 8.62 (do J=1.9 Hzo 1H)o 8.05 (so 1H)o 7.10 (so 1H)o 6.27 (so 1H)o 4.80 (qo J=8.6 Hzo 2H)o 1.59 (so 9H). LC-MS m/z: ES+ [M+H]+=368.2o LCMS; tR=3.06 min.
To a solution of N-tert-butyl-6-(2o2o2-trifluoroethoxy)-3-(trifluoromethyl)-1o7-naphthyridin-8-amine (33 mgo 0.09 mmol) in EtOH (1.65 mL) under argon at rt was added TFA (6 μLo 0.09 mmol) followed by PtO2 (13 mgo 0.108 mmol). The mixture was hydrogenated under hydrogen atmosphere for 10 h. The mixture was degassed with nitrogeno then filtered on celiteo rinsed with EtOH and the filtrate was concentrated under reduced pressure. The residue was purified by reversed phase gel column chromatography C18 (5.5 g) using a gradient of 10-100% acetonitrile in water (contains 0.1% formic acid) to afford the title compound (22 mgo 66%) as a solid. 1H NMR (500 MHzo CDCl3) δ 5.89 (so 1H)o 4.71-4.57 (mo 2H)o 3.55 (do J=11.9 Hzo 1H)o 3.06 (ddo J=13.0o 10.4 Hzo 1H)o 2.93-2.72 (mo 2H)o 2.59-2.39 (mo 1H)o 1.46 (so 9H). LC-MS m/z: ES+ [M+H]+=372.1o LCMS; tR=3.16 min.
Structures of the following synthesized compounds are shown in Table 1 above.
A solution of N2-(3-methyltetrahydrofuran-3-yl)-6-(3-pyridyl)pyridine-2o3-diamine (0.220 go 0.81 mmol) in methanol (3 mL) was successively treated with tetrahydrofuran-3-one (0.14 go 1.6 mmolo 2 eq) and then glacial acetic acid (93 uLo 1.6 mmolo 2eq). After 20 mino the reaction mixture was treated with sodium cyanoborohydride (77 mgo 1.2 mmolo 1.5 eq). After stirring overnighto LC/MS analysis showed clean conversion to the desired product. The reaction mixture was dried and purified by flash chromatography (4 g silicao 0-10% methanol/methylene chloride) to afford N2-(3-methyltetrahydrofuran-3-yl)-6-(3-pyridyl)-N3-tetrahydrofuran-3-yl-pyridine-2o3-diamine (0.26 go 0.277 g theoro 93%) as a brown viscous oil.
A solution of N2-(3-methyltetrahydrofuran-3-yl)-6-(4-pyridyl)pyridine-2o3-diamine (0.155 go 0.57 mmol) in methanol (3 mL) was successively treated with tetrahydrofuran-3-one (0.10 go 1.15 mmolo 2 eq) and then acetic acid (66 uLo 1.15 mmolo 2 eq). After 10 mino the reaction mixture was then treated with sodium cyanoborohydride (55 mgo 0.86 mmolo 1.5 eq). After stirring overnighto LC/MS analysis showed clean conversion to the desired product. The reaction mixture was adsorbed onto silica (4 g) and then purified by flash chromatography (12 g silicao 0-10% methanol/methylene chloride) to afford N2-(3-methyltetrahydrofuran-3-yl)-6-(4-pyridyl)-N3-tetrahydrofuran-3-yl-pyridine-2o3-diamine (0.115 go 0.195 g theoro 58%) as a reddish-brown solid.
A solution of N-(3-methyltetrahydrofuran-3-yl)-2-(2-pyridyl)pyrido[3o2-d]pyrimidin-4-amine (0.15 go 0.49 mmol) in ethanol (2 mL) was treated with TFA (36 uLo 0.49 mmolo 1 eq) and then degassed with nitrogen by bubbling through the solution. The reaction mixture was then treated with Pt (IV) oxide (23 mgo 98 umolo 0.2 eq) and the solution was bubbled with hydrogen gas via balloon for 10 min. The needle was removed from the solution and the reaction mixture was stirred overnight under a balloon pressure of hydrogen gas. LC/MS analysis showed partial complete consumption of the starting material. The reaction mixture was filtered through Celite and the solvent was removed in vacuo. The residue was purified by flash chromatography (12 g silicao 0-10% methanol/methylene chloride) to afford N-(3-methyltetrahydrofuran-3-yl)-2-(2-pyridyl)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (0.15 go 0.152 g theoro 99%) as a reddish-brown solid.
In a 40-mL vialo 2-(4-fluorophenyl)-N-(3-methyltetrahydrofuran-3-yl)pyrido[3o2-d]pyrimidin-4-amine (M-13o presumed to contain 0.245 g desired material) was stirred in ethanol (5 mL). To this was added 0.056 mL TFA. The solution was stirred and degassed by bubbling N2 gas through the mixture. After 10 mino PtO2 (0.0343 go 0.2 eq) was added. The reaction mixture was again purged with nitrogen. A balloon of hydrogen was then addedo and the reaction stirred at room temperature No reaction seen after 1 hr by LCMS. Minimal reaction after 4.5 hours. LCMS shows complete reaction after weekend. Reaction mix filtered and loaded onto silica for purification. Initial purification in hexanes/EtOAc left most of desired product stuck on column. Re-ran purification in DCM/methanol to elute desired product. Fractions 12-14 were dried down separately from fractions 15-17. Fractions 12-14: orange solid 0.0979 g; fractions 15-17: yellow glassy solid 0.1568 g. 1H-NMR (400 MHzo DMSO-d6): δ 8.15 (mo 2H)o 7.41 (mo 2H)o 4.08 (do 1H)o 3.85 (mo 3H)o 3.30 (mo 2H)o 2.83 (mo 2H)o 2.50 (mo 1H)o 2.09 (mo 1H)o 1.89 (mo 2H)o 1.61 (so 3H).
A vial was charged with 6-(4-fluorophenyl)-N2-(3-methyltetrahydrofuran-3-yl)pyridine-2o3-diamine (N-01o 0.06 go 0.209 mmol) and methanol (2 mL). A stir baro tetrahydrofuran-3-one (2 eqo 0.036 go 0.418 mmol) and acetic acid (2 eqo 0.024 mLo 0.418 mmol) were added. After 20 mino sodium cyanoborohydride (1.5 eq.o 0.0197 go 0.313 mmol) was added. The reaction was stirred at room temperature overnight. LCMS at this time suggests predominant peak is desired producto with minor impurity peaks present. The reaction mixture was loaded directly onto a plug of silicao driedo and purified by column chromatography (0-100% Hex/EtOAc). Two dominant peakso each containing desired product with trace impurity. Dried fractions 22-25 (42 mg) and 26-28 (32 mg) for total 74 mg. 1H-NMR (400 MHzo DMSO-d6): δ 7.90 (mo 2H)o 7.19 (mo 2H)o 7.04 (do 1H)o 6.63 (do 1H)o 5.80 (mo 1H (NH))o 5.24 (mo 1H (NH))o 4.00 (mo 2H)o 3.90 (mo 2H)o 3.82 (mo 3H)o 3.72 (mo 1H)o 3.61 (mo 1H)o 2.42 (mo 1H)o 2.22 (mo 1H)o 2.02 (mo 1H)o 1.82 (mo 1H)o 1.58 (so 3H).
A 40 mL vial was charged with 6-(4-fluorophenyl)-N2-(3-methyltetrahydrofuran-3-yl)pyridine-2o3-diamine (300 mgo 1.04 mmol) and a stir baro tetrahydropyran-4-one (1.25 eqo 131 mgo 1.60 mmol)o TFA (2.5 eqo 0.194 mLo 2.61 mmol)o and isopropyl acetate(3 mLo 0.3 M) were added. To this was added sodium triacetoxyborohydride (2.5 eqo 553 mgo 2.61 mmol). The reaction was then allowed to stir at room temperature. After 20 minuteso the reaction mixture was made basic with the careful addition of sat. NaHCO3 and then partitioned between 25 mL of water and 25 mL of EtOAc. The water layer was extracted twice with 15 mL EtOAco dried over Na2SO4o filtered and concentrated under reduced pressure. The organic layer was concentrated to provide a grey solid that was recrystallized from MeOH to provide 60 mg of white solid. The remaining MeOH was concentrated. The residue was purified on silica gel (24 go 0-100% EtOAc/hexanes) to provide a total of 220 mg of 6-(4-fluorophenyl)-N2-(3-methyl-tetrahydrofuran-3-yl)-N3-tetrahydropyran-4-yl-pyridine-2o3-diamine (388 mg theo.o 58%) as a white solid. LCMS: 372.1 M+H+. 1H NMR: δ 7.90 (to 2H)o 7.08 (mo 3H)o 6.90 (to 1H)o 4.36 (bso 1H)o 4.14 (do 1H)o 3.98 (mo 5H)o 3.52 (mo 2H)o 3.45(bso 1H)o 2.88 (bso 1H)o 2.13 (mo 1H)o 2.03 (mo 2H)o 1.72 (so 3H)o 1.56 (mo 2H).
N2-(3o3-difluoro-1-methyl-cyclobutyI)-6-(4-fluorophenyl)pyridine-2o3-diamine (163 mg) was dissolved in 10 ml of isopropyl acetate. 48 mg butan-2-one was added followed by 82 uL of TFA. The mixture was stirred at RT for 10-15 min and then sodium triacetoxyborohydride (147 mg) was added in 2 portions. The mixture was stirred at RT for 2 hrs. LC-MS indicated the reaction is complete. The reaction mixture was diluted with EtOAc and washed with water. The EtOAc was evaporated and the residue was run through a 24 g silica column with a gradient of DCM in hexane. LC-MS showed clean producto but the material is a dark blue tar. The material was dissolved in a small amount of dioxane and 0.5 ml of 4N HCl in dioxane was added and no precipitate percieved. The mixture was evaporated down to give a gray solid. NMR and LC-MS indicate the desired product in good purity.
100 mg N-03 was dissolved in 10 ml of isopropylacetate. 25 mg of cyclobutanone was added and the mixture was stirred at RT for 10 to 15 min. 44 uL of TFA was added and stirred was continued for an additional 10 to 15 min. 81 mgs of sodium triacetoxyborohydride was added in 2 portions. The reaction mixture was stirred at RT for 1.5 hrs. LC-MS indicated the reaction was complete. The reaction was diluted with EtOAc and washed with water. The EtOAc layer was evaporated down and run through a 12 g silica column. The product was eluted with a gradient of EtOAc in hexane to give 69 mg (60%) pale yellow solid.
100 mg of N-01 was dissolved in 10 ml of isopropylacetate. 31 uL of cycolbutanone was added and the mixture was stirred at RT for 10 to 15 min. 52 uL of TFA was added and stirred was continued for an additional 10 to 15 min. 96 mgs of sodium triacetoxyborohydride was added in 2 portions and the mixture was stirred at RT for 1.5 hrs. LC-MS indicated the reaction was complete. The reaction mixture was diluted with EtOAc and washed with water. The EtOAc was evaporated down and the residue was run through a 24 g silica column. The product was eluted with a gradient of EtOAc in hexane to give 70 mg (59%) white solid.
2-chloro-N-(3-methyltetrahydrofuran-3-yl)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (120 mg) and 4-pyridylboronic acid (82 mg) were dissolved in a mixture of 10 ml of dioxane and 2 ml of water. The mixture was de-aerated by bubbling nitrogen through the solution for 15 min. 142 mg sodium carbonate was addedo followed by 33 mg of Pd(dppf)Cl2-DCM. The mixture was heated in a microwave for 1 h at 100° C. LC-MS indicated the reaction was about 50% complete. The reaction was worked up by evaporating the solvent. The residue was run through a 24 g silica column the product was eluted with a MeOH in DCM gradient to give 32 mg final product.
2-chloro-N-(3-methyltetrahydrofuran-3-yl)-5o6o7o8-tetrahydropyrido[3o2-d]pyrimidin-4-amine (120 mg) and 3-pyridylboronic acid (89 mg) were dissolved in a mixture of 10 ml of dioxane and 2 ml of water. The mixture was de-aerated by bubbling nitrogen through the solution for 15 min. 154 mg sodium carbonate was addedo followed by 40 mgs of Pd(dppf)Cl2-DCM. The mixture was heated in a microwave for 1 h at 100° C. LC-MS indicated the reaction to be about 50% complete. After heating for an additional 30 mino LC-MS showed the reaction to be about 60% complete. The reaction mixture was worked up by evaporating the solvents and running the residue through a 24 g silica column. The product was eluted with a MeOH/DCM gradient to give 37 mg final product.
Wild type (strain N2)o the temperature sensitive-sterile strain TJ1060: spe-9(hc88); fer-15(b26) and the DAF-16 reporter strain TJ356: zls356 [Pdaf-16::daf-16a/b::gfp+rol-6(su1006)] were obtained from the Caenorhabditis Genetics Center. The wild type strain was maintained at 20° C. on standard nematode growth media (NGM) and aged at 20° C. or 25° C. as required. TJ1060 was maintained at 16° C. and also aged at 20° C. or 25° C. as required. TJ1060 was predominately used to remove the inconvenience of progeny production and can be regarded as a proxy for wild type.
Compounds used in this study include:
Diethyl maleate (DEM; Sigma-Aldrich) was added to neat DMSO and added to molten NGM at 55° C. to a final concentration of 5o 10o 15 or 20 mM DEM and 0.5% v/v DMSO. Plates were seeded with OP50 and used within 24 hours. As aboveo data was collected at 25 (±1) ° C. using the temperature sensitive-sterile strain TJ1060. A synchronous population was obtained by transferring egg-laying adults to fresh plates at 16° C. for 2-3 hours. The adults were removed and the plates with eggs then transferred to 25° C. to ensure sterility. After 48 hours at 25° C.o when worms were at the late L4/young adult stageo 25-35 nematodes were transferred to fresh plates containing either vehicle controlo 250 μM SIHo or 200 μM Lip-1. Worms were aged at 25° C. for a further 4 days and then transferred to DEM plates. Survivalo determined by touch-provoked movemento was scored at 24 and 48 hours after exposure to DEM.
Aging studies were also undertaken to determine changes with age of both survival after DEM exposure and basal glutathione levels. Initial populations were obtained as describe aboveo with worms aged on standard NGA plates. Note that here we refer to the age of adults as determined by the number of days following the last larval molt and therefore reflects the number of days of adulthoodo not the time since egg.
Quantification of Total Glutathione
Measurement of total glutathione was based on established protocols and is based on a kinetic spectrophotometric assay using the reaction between GSH and 5o5′-dithio-bis (2-nitrobenzoic acid) (DTNB) measured at 412 nm (Caito and Aschnero 2015; Rahman et al.o 2006). All reagents were freshly prepared prior to the assay and for each estimate 50 adults were collected in 200 μL of S-basal (Brennero 1974) in 1.7 ml microfuge tubes. Animals were washed twice in S-basalo pelleted via centrifugation and total volume reduced to 20 μL. A 50 μL aliquot of Extraction Buffer was added then the samples were frozen in Liquid N2 and store at −80° C. until required. Extraction buffer consisted of 6 mg/mL 5-sulfosalicylic acid dehydrateo 0.1% v/v Triton X-100 and Completeo EDTA-free Proteinase inhibitor cocktail (Roche) in KPE buffer (0.1 M potassium phosphate buffer and 5 mM EDTA at pH 7.5).
Samples were homogenized with a Bioruptor Next Gen (Diagenode) bath sonicatoro set on HIGH and cooled to 4° C.o using 10 cycles of 10 seconds ON and 10 seconds OFF. Supernatant was collected following a 14K×g spin at 4° C. Assays were performed in 96 well microplates (clear polystyreneo flat-bottomedo Greiner bio-one)o in a total volume of 200 μL per well. To each well was added 50 μL of lysate supernatanto 50 μL of milli-Q H2O and then 100 μL of GA buffer (NADPH 400 μMo glutathione reductase 1 U/mL and 0.3 mM DTNB in KPE buffer diluent). Reactions were incubated for 1-2 min at room temperature and then absorbance measured at 412 nm for 10 min with 1 min interval using a Powerwave plate spectrophotometer (BioTek). The rate of change in absorbance per minute is linearly proportional to the total concentration of GSH. Total GSH in the samples was interpolated from using linear regression from a standard curve of known GSH concentrations (0 to 1 μM) run in tandem. In parallelo the concentration of total protein per sample was also determined by a Bicinchoninic acid (BCA) assay (Pierce) using the manufacturers protocol. Total GSH estimates were then normalized for protein load thous accounting for any size differences between populations. Within experiment results are presented as relative glutathione levelso where results are normalized to the mean of the starting population.
Measurement of malondialdehyde (MDA) was performed using a Thiobarbituric acid reactive substances (TBARS) assay kit (10009055o Caymen Chemical) as per manufacturer instructions using reduced reaction volumes of 1 mL. For C. elegans samples with acute glutathione depletiono Day 1 adults were treated with and without 20 mM DEM for 6 h at 25° C. prior to collection. For agingo animals were aged at 25° C. and treated with Lip-1 or SIH as previously described. Replicate samples were collectedo washed twice in S-basalo pelleted by centrifugation. Following removal of excess buffer samples (˜40 μL) were frozen in liquid-N2 and stored at −80° C. until needed. Samples were then homogenized via a Bioruptor bath sonicator (Diagenodeo set on ‘high power’ with 10 cycles of 10 s pulses with a 10 s pause between pulseso at 4° C.)o then centrifuged at 21o500×g at 4° C. for 30 min and the supernatant retained. The concentration of protein was determined using a BCA assay kit (Pierce) and equivalent aliquots of 20-25 μg total protein used for subsequent measurements.
Analysis of Hydroxynonenal (4-HNE) protein adducts was also used as a proxy for lipid peroxidation. Duplicate samples of 50 and 200 worms were collected and washed twice in S-basalo pelleted by centrifugation and the supernatant discarded. These samples (˜30 μL) were frozen in liquid-N2 and stored at −80° C. until needed. To each sample an 10 μL 4× Bolt LDS sample buffer (Invitrogen) and 3 μL TCEP (Invitrogen) was added and the sample heated to 95° C. for 10 min. Lysates were loaded onto NuPAGE™ 4-12% Bis-Tris acrylamide gels (1.0 mmo 10-wello Invitrogen)o electrophoresed with MES running buffer and then transferred onto 0.45 μm PVDF membrane by electroblot using a Mini Blot module (Invitrogen). 4-HNE protein adducts were detected by an anti 4-HNE protein adduct antibody (1:2000o AB5605o Millipore) in Tris-buffer saline with 5% skim milko and ECL (GE Healthcare). The membranes were stripped using a 1× ReBlot Strong Antibody Stripping Solution (Merck) for 15 mino reprobed for tubulin using an anti-Tubulin antibody (1:10o000oT6074o Sigma-Aldrich).
The red-fluorescent propidium iodide (PI)o was used to visualize dead cells within live C. elegans after DEM treatment and during aging. Populations were incubated for 24 h at 25° C. with PI (a 10 μL volume of 0.25 mg/mL solution added to the bacterial lawn on 50 mm NGM plates) prior to the described age or with concurrent exposure to 10 mM DEM (as described above) and PI. For aging experimentso animals were visualized at Day 6 and Day 8. Cohorts of live animals (i.e. showing spontaneous or touch-provoked movement) were isolated and mounted under glass coverslips on 2% agarose pads without anesthetic. Imaging were captures with on a Leica DMI3000B inverted microscopeo DsRed filter set and a DFC 3000G digital.
Liquid chromatography was performed using established protocols. Brieflyo samples of aged C. elegans were lysed using a Bioruptor Next Gen (Diagenode) bath sonicator set on HIGH and cooled to 4° C. using 10 cycles of 10 sec ON and 10 sec OFFo in a 1:1 volume ratio of Tris-buffered saline (pH 8.0) with added proteinase inhibitors (EDTA-free; Roche). Sample homogenization was confirmed by microscopic inspection. Lysates were then centrifuged for 15 min at 175o000 g at 4° C. The supernatant was removed and total protein concentration in the soluble fraction was determined using a NanoDrop UV spectrometer (Thermo Fisher Scientific) before being transferred to standard chromatography vials with polypropylene inserts (Agilent Technologies) and kept at 4° C. on a Peltier cooler for analysis. Size exclusion chromatography-inductively coupled plasma-mass spectrometry was performed using an Agilent Technologies 1100 Series liquid chromatography system with a BioSEC 5 SEC column (5 μm particle sizeo 300 Å pore sizeo I.D. 4.6 mmo Agilent Technologies) and 7700× Series ICP-MS as previously described (Hare et al.o 2016b). A buffer of 200 mM NH4NO3 was used for all separations at a flow rate of 0.4 mL min−1. A total of 50 μg of soluble protein was loaded onto the column by manually adjusting the injection volume for each sample. Mass-to-charge ratios (m/z) for phosphorus (31) and iron (56) were monitored in time resolved analysis mode.
Plots of the mean (±standard deviation) of three independent biological replicates are shown. Integration of the three major peaks was performed using Prism (ver. 7 for Mac OS X, Graphpad).
Specimens were prepared for XFM using previously described protocols. Briefly, adult C. elegans were removed from NGM, washed four times in excess S-basal (0.1 M NaCl; 0.05 M KHPO4 at pH 6.0), briefly in ice-cold 18 MΩ resistant de-ionized H2O (Millipore) and twice in ice-cold CH3COONH4 (1.5% w/v). Samples were transferred onto 0.5 μm-thick silicon nitride (Si3N4) window (Silson), excess buffer wicked away and then the slide was frozen in liquid nitrogen (N2)-chilled liquid propane using a KF-80 plunge freezer (Leica Microsystems). The samples were lyophilised overnight at −40° C. and stored under low vacuum until required.
The distribution of metals was mapped at the X-ray Fluorescence Microscopy beamline at the Australian Synchrotron using the Maia detector system. The distribution of elements with atomic number<37 were mapped using an incident beam of 15.6 keV X-rays. This incident energy allowed clear separation of X-ray fluorescence (XRF) peaks from the relatively intense elastic and inelastic scatter. The incident beam (˜1.71×109 photons s−1) was focussed to approximately 2×2 μm2(H×V, FWHM) in the sample plane and the specimen was continuously scanned through focus (1 mm sec−1). The resulting XRF was binned in 0.8 jim intervals in both the horizontal and vertical giving virtual pixels spanning 0.64 jim2 of the specimen probed with a dwell time of 8 jisec. XRF intensity was normalized to the incident beam flux monitored with a nitrogen filled ionization chamber with a 27 cm path length placed upstream of the focusing optics. Three single-element thin metal foils of known areal density (Mn 18.9 jig cm−2, Fe 50.1 jig cm−2 and Pt 42.2 jig cm−2, Micromatter, Canada) were used to calibrate the relationship between fluorescence flux at the detector and elemental abundance. Dynamic Analysis, as implemented in GeoPIXE 7.3 (CSIRO), was used to deconvolve the full XRF spectra at each pixel in the scan region to produce quantitative elemental maps. This procedure includes a correction for an assumed specimen composition and thickness, in this case 30 jim of cellulose. Though unlikely to exactly match the actual sample characteristics, deviations from these assumptions are not significant for the results presented in this study as the effects of beam attenuation and self-absorption on calcium and iron XRF are negligible for a dried specimen of this type and size.
Analysis of elemental XRF maps was performed using a combination of tools native to GeoPIXE and ImageJ. Incident photons inelastically scattered (Compton scatter) from the sample detail the extent and internal structure of individual C. elegans. The differential scattering power of the specimens and substrate allowed individual animals (or parts thereof) to be identified as regions of interest (ROI) facilitating analysis of elemental content on a ‘per worm’ basis. This segmentation of each elemental map was achieved using the histogram of pixel intensities from Compton maps to locate the clusters within the image. ROIs composed of <10,000 pixels were deemed to be so small that their elemental content was not reflective of the elemental content of whole animals and so these were excluded from the analysis. The ‘non-worm’ region of each scan was used to calculate the value specimen elemental content was distinguishable from background noise, i.e. the critical value. The background corrected elemental maps were used to establish the areal densities and the total mass of each element associated with individual ROIs.
Sample Preparation—jXANES Imaging
Adult C. elegans were removed from NGM, washed four times in excess ice-cold S-basal (0.1 M NaCl; 0.05 M KHPO4 at pH 6.0). Samples were transferred onto 0.5 μm-thick silicon nitride (Si3N4) window (Silson), excess buffer wicked away and then the slide was frozen in situ under a laminar stream of 100° K. dry nitrogen (N2) gas.
jXANES Imaging
The beam energy was selected using a Si(311) double-crystal monochromator with a resolution of ˜0.5 eV. !XANES imaging was achieved by recording Fe XRF at 106 incident energies spanning the Fe Kedge (7112 eV). Measurement energy interval was commensurate with anticipated structure in the XANES:
As for XFM, !XANES measurements used a beam spot ˜2×2 jim but data was recorded using continuous scanning at 0.2 mm sec−1 (binned at 2 jim intervals). Transit time through each virtual pixel was 10 ms and the incident X-ray intensity at 7455 eV was ˜1.67×1010 photons s−1. These imaging parameters gave a total dose associated with the qXANES measurement estimated at ˜5 MGy. This value is commensurate with doses typically delivered during bulk X-ray absorption spectroscopy.
qXANES Analysis
The XANES spectra from an iron foil (50.1 jig cm−2, Micromatter Canada) was measured to monitor the energy calibration of the beamline. The maxima of the first peak in the derivative spectra of the iron foil was subsequently defined as 7112.0 eV. The energy stability of beamline has been determined at <0.25 eV over 24 hrs making energy drift over the course of a scan negligible. Consistency of the measured edge positions in conjunction with stability of beam position and flux recorded in ion chambers upstream the specimen position provide confidence that energy stability was high through the duration of the experiment. Small position drifts were aligned by cross-correlation of the calcium map which remains essentially constant throughout the energy series.
XANES probes the density of states on the absorbing atom and reveals electronic and structural details of coordination environment. The aligned qXANES image series is stack of images, one per incident energy allowing the XANES of individual cells to be assessed. Previous work has shown that the distribution of calcium is a useful marker for the position of C. elegans intestinal cells, and we used this information to identify regions of interest in the qXANES stack corresponding to anterior intestinal cells. Anterior intestinal cells were chosen due to their consistent and robust iron content.
As all points on the specimen represent a heterogenous mixture of iron binding species the resulting XANES spectra are admixtures with contributions from all of these components. The technical particulars of the XFM beamline (being primarily designed for elemental mapping) are not optimized for high resolution spectroscopy and our XANES spectra are relatively sparse. For iron K-edge XANES the abrupt increase in absorption coefficient at the critical threshold obscures the presence of 1s→4s and 1s→4p electronic transitions. It has been shown that the relative intensity of these transitions provides the proportional contribution of Fe2+ and Fe3+ to the XANES and can be assessed by interrogating the first derivative of the XANES spectra.
Lifespan was measured using established protocols. SIH was dissolved in neat dimethyl sulfoxide (DMSO; Sigma-Aldrich) then added to the molten NGM at 55° C. (to a final concentration of 250 μM SIH in 0.5% v/v DMSO). Lip-1 was dissolved in neat DMSO then added to the molten NGM at 55° C. (to a final concentration of 200 μM Lip-1 in 0.5% v/v DMSO). Media containing equivalent vehicle alone (0.5% v/v DMSO) was used for comparison. Standard overnight culture of the Escherichia coli (E. coli) strain OP50 was used as the food source.
Lifespan data was collected at 25 (±1) ° C. using the temperature sensitive-sterile strain TJ1060 [spe-9(hc88); fer-15(b26)]. A synchronous population was obtained by transferring egg-laying adults to fresh plates at 16° C. for 2-3 hours. The adults were removed and the plates with eggs then transferred to 25° C. to ensure sterility. After 48 hours at 25° C., when worms were at the late L4/young adult stage, 25-35 nematodes were transferred to fresh plates containing either vehicle control, 250 μM SIH, or 200 μM Lip-1. All plates were coded to allowing blinding of the experimenter to the treatment regime during scoring. Nematodes were scored for survival at one to three-day intervals and transferred to freshly prepared plates as needed (2-5 days).
To determine whether the increased lifespan seen with SIH treatment could be explained solely by an antibiotic effect of iron reduction, nematodes were treated with ampicillin, with and without SIH co-administration. Even in the presence of ampicillin, SIH increased median lifespan by 6 days, similar to its benefits in the absence of ampicillin (median increase of 7 days).
The effects of test compounds on growth of the OP50 E. coli feed was assayed using optical density at 600 nm (OD600). Using standard microtitre plates, replicate wells of 200 μL of sterile Luria broth were inoculated with 2 μL of an overnight OP50 culture in addition to the stated final concentrations of ampicillin (Amp), Lip-1 and SIH. OD600 measures were taken after 12 hours in an EnSpire (PerkinElmer) spectrophotometric plate reader preset to 37° C., with 30 sec of 200 rpm orbital shaking every 10 minutes.
Data was averaged across duplicate experiments, each with eight replicate wells per treatment where a baseline of Luria broth without an inoculate was subtracted. Results indicated that ampicillin at either 50 or 100 μg/mL completely suppressed bacterial growth. In contrast, neither Lip-1 nor SIH suppressed bacterial growth.
C. elegans are bacteriophores and the E. coli (OP50) monoxenic diet can colonize the pharynx and intestine, resulting in death. Consequently, antibiotics are known to extend C. elegans lifespan. In addition, iron chelating compounds, such as EDTA have been reported to have antibiotic properties. We performed a disk diffusion test on both Lip-1 and SIH and observed no evidence for inhibition of E. coli (strain OP50) growth). Furthermore, an additive effect on media lifespan extension was seen when SIH and the antibiotic ampicillin were co-administered to C. elegans, consistent with independent effects on lifespan.
It is well documented that differences are observed between independent measures of lifespan, with micro-environmental factors such as minor temperature fluctuations potentially resulting differences in median and maximum lifespan between replicates. After determining the optimal doses of 250 μM SIH and 200 μM Lip-1, respectively, cohorts of nematodes were compared in 8 independent replicates. As the number of worms measured is known to influence the likelihood of accurately observing differences in lifespan, the starting populations for all treatments within experiments were in excess of 70 individuals. The median and maximum lifespans observed of control and treated populations for these 8 replicates are shown in Table 2. As can be seen in this table, the median lifespan of treated populations was always greater than that of control populations, however the magnitude of the difference varied between experiments, with the median lifespan of control populations ranging from 7 to 9 days.
A developmentally synchronous population, derived from eggs laid over a 2-hour window, were cultured on NGA media at 25° C. for 48 h, and then as young adult worms were transferred onto three treatment plates for an additional 24 h. The treatment plates included NGA with 0.5% (v/v) DMSO (vehicle control, Ctl), 250 μM SIH, or 200 μM Lip-1 (as described above).
Cohorts of approximately 100 animals were transferred into a 1.5 ml centrifuge tube containing 400 μL S-basal. Following a brief centrifugation excess S-basal was removed leaving the animals suspended in 50 μL. Animals were euthanized and straightened by a 15 second exposure to 60° C. (using a heated water bath). Samples were then mounted between glass slides and a cover slip and immediately imaged. Micrographs were collected using a Leica M80 stereomicroscope and Leica DFC290 HD 3 MP) digital camera. Pixel sizes were defined using a calibrated 25 μm grid slide (Microbrightfield, Inc). Size and shape metrics were extracted from brightfield images were analyzed using the WormSizer plugin for ImageJ.
Wild type (N2) adults (4-day post egg lay) were transferred to fresh plates for 30 minutes at 20° C. to establish a developmentally synchronous population. Adult nematodes were then removed, and eggs were then transferred to 25° C. As with the survival analyses, after 48 hours at 25° C., when worms were at the late L4/young adult stage individual nematodes were transferred to plates containing vehicle control, 250 μM SIH, or 200 μM Lip-1. After 24 hours, adult worms were transferred to fresh plates and transferred daily until the end of the fertile period. After allowing progeny to develop for 2 days at 20° C., they were then counted to determine daily and total fertility. Early fertility is determined by the number of progeny laid in the first 24-hour period.
A developmentally synchronous population, derived from eggs laid over a 2-hour window, were cultured on NGA media at 25° C. for 48 h, and then as young adult worms were transferred onto three treatment plates for an additional 24 h. The treatment plates included NGM+0.5% (v/v) DMSO (vehicle control, Ctl), NGM+250 μM SIH, and NGM+200 μM Lip-1 (as described above).
Single worms were transferred to a 55 mm NGA assay plate devoid of a bacterial lawn, without a lid, and left to recover from the transfer for 2 minutes. Movement of the adults was then recorded using a stereomicroscope (Leica M80) with transmitted illumination from below. A 30 second video recording was captured using a 3 MP DFC290 HD digital camera (Leica Microsystems) at a rate of 30 frames per second. Pixel length was calibrated using a 25 μm grid slide (Microbrightfield, Inc). Recorded series were analysed using the wrMTrck plugin for ImageJ (www.phage.dk/plugins) and Fiji (a distribution of ImageJ).
The maximum velocity achieved was expressed as mm per second (as derived from the distance between displaced centroids per second). Additional metrics of movement were determined including mean velocity (mm s−1) and (total) distance travelled (mm). These variables were collated in Prism (v7.0a GraphPad Software) and presented as a scatter plot with medians and interquartile range.
Glutathione Depletion Vulnerability
Glutathione is suggested to be the dominant coordinating ligand for cytosolic ferrous iron and is also the substrate used by glutathione peroxidase-4 (GPX4) to clear the lipid peroxides that induce ferroptotic cell death. Deletion of four C. elegans homologs of GPX4 decreases lifespan, but whether ferroptosis mediates this is unknown. We tested whether acute depletion of glutathione can initiate ferroptosis in adult C. elegans using diethyl maleate (DEM), which conjugates glutathione. DEM has been reported to produce a nonlinear response to glutathione depletion, with a minor glutathione loss induced by DEM at 10-100 μM increasing lifespan via hormesis, but a major glutathione loss induced by DEM≥1 mM shortening lifespan. We found that DEM≥1 mM induced death in 4-day old adult worms (at the end of their reproductive phase) in a dose- and time-dependent manner, with ≈50% lethality occurring after 24-hour exposure to 10 mM DEM associated with ≈50% depletion of glutathione. We also found that total glutathione levels steadily decrease with normal aging, approaching ≈50% on Day 10 of the levels on Day 1. This may contribute to C. elegans becoming disproportionately more vulnerable to DEM lethality as they enter the midlife stage.
We tested whether lethality associated with glutathione depletion was caused by ferroptosis. We examined the treatment of C. elegans with the selective ferroptosis inhibitor, liproxstatin (Lip-1, 200 μM). We also targeted the accumulation of late life iron, that catalyses (phospho)lipid hydroperoxide propagation, using salicylaldehyde isonicotinoyl hydrazone (SIH, 250 μM), a lipophilic acylhydrazone that scavenges intracellular iron and mobilizes it for extracellular clearance. Importantly, unlike chelators such as CaEDTA, iron bound by SIH does not redox cycle (Chen et al., 2018). For both interventions, C. elegans were treated from early adulthood (late L4) onwards to eliminate any potential developmental effects.
DEM toxicity in 4-day old worms was rescued by both Lip-1 and SIH, with more marked protection by SIH. This is consistent with ferroptosis contributing to the death mechanism. Therefore, the fall in glutathione with aging would be expected to interact synergistically with the concomitant rise in labile iron to increase the risk of ferroptosis. We found that this age-dependent rise in iron itself may contribute to the fall in glutathione, since pretreatment of the worms with SIH from L4 prevented the age-dependent decrease in glutathione when assayed on Day 4 of adult life. Furthermore, SIH mitigated the glutathione depletion induced by DEM in Day 4 animals, demonstrating that cytosolic iron synergizes the depletion of glutathione initiated by DEM. While Lip-1 alleviated the lethality of DEM, it did not prevent the fall in glutathione that was induced by aging (as assayed on Day 4) or by DEM. Thus, Lip-1 inhibition of ferroptosis in C. elegans occurs downstream of glutathione depletion, consistent with its effect in rescuing ferroptosis in cultured cells.
Testing for Departure from Temporal Rescaling
We determined whether the results observed with both the SIH and Lip-1 interventions were due to temporal scaling of aging. A modified Kolmogorov-Smirnov (K-S) test was applied to the residuals from a replicate-specific accelerated failure time (AFT) model fitted according to the Buckley-James method that uses a nonparametric baseline hazard function. The function bj in R package rms was used to fit the replicate-specific model with interventions as categorical independent variables. We used the same approach for testing whether the temperature difference results in simple temporal rescaling, with the only difference being using temperature rather than intervention as categorical independent variable in the AFT model.
Characterizing Departure from Temporal Rescaling
Parametric survival models with Weibull baseline hazards and Gamma frailty were fitted to replicate-specific data using the R package flexsurv. A likelihood ratio test was used to compare models that assume simple temporal rescaling to models that allow varying degrees of departure from temporal rescaling. The best model for each replicate was selected using a likelihood ratio test and the goodness of fit (GOF) of the best model is evaluated using a chi-square GOF test. To combine data across different replicates, we performed fixed-effect and random-effect meta-analyses for each parameter in the best model. Briefly, the fixed-effect meta-analysis estimates were derived using Inverse Variance Weighting (IVW) in which the estimates from each replicate were weighted by the inverse of their variance estimates. The meta-analysis estimates were then calculated simply as the weighted average of estimates from all replicates. The fixed-effect meta-analysis assumes that there is insignificant variation between the estimates of the same parameter across different replicates. The random-effect meta-analysis also derives the estimates by assigning weights to estimates from each replicate, but in this case the weights take into account the variation of estimates across replicates.
The fixed-effects and random-effects meta-analysis estimates are quite similar; the meta-analysis estimates provide the best fit to SIH data and worst for Lip-1 data. Since there is significant between-replicate variation for the majority of the parameters, it is not surprising that the when the meta-analysis estimates are applied to the real data, a chi-square goodness of fit reveals significant lack of fit (X2(3)=237.0 for control worms, X2(5)=258.0 for Lip-1 and X2(3)=49.7 for SIH, all p-values<0.001).
One notable pattern from these data is that for nearly all replicates, there is more heterogeneity due to unobserved factors among the control worms, as indicated by the negative Alog(a2) parameter estimates for Lip-1 and SIH data. This heterogeneity is also reflected in a de-acceleration of the hazard function for control worms beyond 7-8 days. This de-acceleration of the hazard function is the main contributor to the crossing behavior we observe when comparing the survival functions, and it is what causes a violation of the simple temporal rescaling assumption.
For survival with increasing DEM dose response and protection by compounds (Lip-1 and SIH), data was plotted as fraction of animal alive with upper and lower 95% confidence interval, using the Wilson ‘score’ method using asymptotic variance and fitted with a sigmoidal curve (Prism). Pairwise comparisons of treated groups versus control at each concentration of DEM was determined using the N-1 chi-squared test.
Differences in fertility (i.e. early and total reproductive output) were assessed using an ordinary one-way analysis of variance (ANOVA), followed by a Tukey's multiple comparison test (as implemented by Prism v7.0a, GraphPad Software).
Data of estimated adult body length and volume were initially assessed for normality using a D'Agostino & Pearson test. Based on this analysis a nonparametric Kruskal-Wallis Analysis of Variance (ANOVA) was performed followed by a Dunn-Šidák test for multiple comparisons (as implemented by Prism v7.0a, GraphPad Software). There was a significant difference between body length (H(10)=432.6, p<0.0001) amongst the groups measured. The results of the pairwise comparisons, corrected for multiple comparisons, are shown in Table 3
There was a significant difference between body volume (H(10)=489, p<0.0001) amongst the groups measured. Comparisons between age and treatment groups a Kruskal-Wallace ANOVA was performed, followed by Dunn's multiple comparisons Post-hoc tests. The results of the pairwise comparisons, corrected for multiple comparisons, are shown in Table 4.
Data of estimated maximum velocity were initially assessed for normality (see Table 5).
There was a significant difference between maximum velocity (H(7)=298.5, p<0.0001) amongst the groups measured. The results of the pairwise comparisons, corrected for multiple comparisons, are shown in Table 6.
Mean velocity and total distance travelled were also determined. Results summaries and comparisons between treatments are shown in Tables 7-10. The data for the three movement parameters were combined across treatments and ages to determine the relationship between the estimated parameters, all were found to be positively correlated. Movement parameters measured included maximum velocity, mean velocity and total distance travelled. Treatment with either Lip-1 or SIH attenuates the age-related decline in mean velocity (Kruskal-Wallis ANOVA: H(7)=339.2, p<0.0001).
Summary statistics for normality of mean velocity (mm s-1) are included in Table 7. Not all data sets were normally distributed, as indicated below.
To compare between age and treatment groups a Kruskal-Wallace ANOVA was performed, followed by Dunn's multiple comparisons Post-hoc tests. There was a significant difference between mean velocity (H(7)=339.2, p<0.0001) amongst the groups measured. The results of the pairwise comparisons, corrected for multiple comparisons, are shown in Table 8.
Summary statistics and tests for normality of total distance traveled (mm) are included in Table 9. Not all data were normally distributed, as indicated below.
To compare between age and treatment groups a Kruskal-Wallace ANOVA was performed, followed by Dunn's multiple comparisons Post-hoc tests. There was a significant difference between total distance travelled (H(7)=340.6, p<0.0001) amongst the groups measured. The results of the pairwise comparisons, corrected for multiple comparisons, are shown in Table 10.
Pooling all groups and ages reveals that all movement parameters (maximum velocity, mean velocity and distance travelled in 30s) are all positively correlated.
Differences between the proportion of live animals with fluorescently labelled nuclei in control versus Lip-1 and SIH treatment, either aged or exposed to DEM, were compared using a z-test.
Unless otherwise stated, all statistical tests are conducted with type I error set at 0.05.
A feature of ferroptosis is the propagation of cell death in a paracrine manner mediated by uncertain signals that might include the toxic lipid peroxidation end-products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). Compared to strong oxidants like the hydroxyl radical, 4-HNE and MDA are relatively stable and able react with macromolecules, such as proteins distal to the site of origin. To determine whether individual cell death precedes organismal death in our model of aging, we used propidium iodide to visualize moribund cells in vivo after DEM treatment and during aging. Propidium iodide (PI) is a fluorescent intercalating agent that binds to DNA, but cannot cross the membrane of live cells, making it possible to identify the nuclei of recently dead or dying cells.
Examination of aged cohorts, or young animals treated with DEM, indicated that cell death (particularly death of intestinal cells) preceded organismal death in both 4-day old and 6 and 8 day old adults, and was significantly attenuated by both Lip-1 or SIH. Hence, the animal dies cell by cell, rather than in a single event, and this progressive degeneration is likely to contribute to the frailty phenotype.
The PI-positive dying cells did not accumulate with aging, perhaps because the dead cells are cleared during the remaining lifespan of the animal. It is known that as C. elegans ages, intestinal nuclei are lost and the propidium iodide cannot stain nuclei if they are absent. Additionally, we would not expect a linear increase proportional to age in the prevalence of animals with stained cells during longitudinal studies of our cohorts, because dead animals are removed from the population and the rate of death changed over time for the cohorts (see below). Thus, the prevalence of PI-positive cells per animal would be a complex product of the rate of PI emergence, the rate of PI disappearance, the rate of nuclear disappearance and the rate of organismal death. However, we were able to survey the prevalence of animals with any dead cells on particular days in the adult life span. This determined that cell death begins to be detected after 4 days of age, and that our interventions with SIH and Lip-1 completely suppressed this cell death at 6 and 8 days of age.
To estimate changes in lipid peroxidation, we assayed MDA via the thiobarbituric acid reactive substance assay. As expected, acute glutathione depletion by DEM exposure caused a marked increase in the relative amounts of MDA. We also observed an aged-related increase in MDA, consistent with an age-related increase in ferroptotic signaling in C. elegans, that was ameliorated by both Lip-1 and SIH treatment. Consistent with the MDA results, we also found a concomitant qualitative increase in 4-HNE protein adducts with age that was suppressed by both Lip-1 and SIH treatments.
We considered whether the higher levels of glutathione in animals treated with SIH ( ) could reflect a hormetic response to sublethal oxidative stress, which has been described for SIH at low concentrations (10 μM) combining with the cellular labile iron pool within hepatocellular carcinoma cells in culture. The decrease we observed in our oxidation markers, MDA and 4-HNE, by SIH treatment at 250 μM in C. elegans suggests that this higher dose of SIH was sufficient to debulk reservoirs of total iron. To further discount possible off-target stress responses elicited by our interventions, we interrogated DAF-16 localization. Nuclear localization of the DAF-16 transcription factor is known to be an indicator of insulin-like signalling, which occurs under stress conditions. Neither 250 μM SIH nor 200 μM Lip-1 induced DAF-16 nuclear translocation. As a positive control, treatment with 10 mM DEM did induce nuclear localization of DAF-16, consistent with this challenge inducing acute stress. Taken together, these findings argue against hormesis mediating the benefits of SIH or Lip-1 under these conditions in C. elegans.
Lowering cellular iron suppresses ferroptosis, but the peroxyl radical trapping ferroptosis inhibitors, such as Lip-1, are not expected to change iron levels. We examined the impact of SIH and Lip-1 interventions on iron levels over lifespan using synchrotron-based X-ray fluorescence microscopy to measure both iron concentration (presented as areal density, pg μm−2) and total (pg inhibitors, such as Lip-1, are not expected to change iron levels. We examined the impact of SIH and Lip-1 interventions on iron levels over lifespan using synchrotron-based X-ray fluorescence microscopy to measure both iron concentration (presented as areal density, pg μm−2) and total (per worm) iron. Both total iron and areal density increased with age in control animals (Tables 11 and 12), as expected.
There was a significant difference between mean areal density of iron (F (6, 148)=171.3, p<0.0001) amongst the groups measured. Comparisons between age and treatment groups an Ordinary one-way ANOVA was performed, followed by Sidak's multiple comparisons test. The results of the pairwise comparisons, corrected for multiple comparisons, are shown in Table 12.
SIH dramatically reduced the areal density of iron (and reduced variance) with aging (Tables 11 and 12), but Lip-1 did not alter iron density. Notably, by Day 8, animals treated with SIH contained total iron load on par with the untreated control group (Tables 13 & 14), as the lower areal density was offset by an increase in body size of SIH-treated worms compared to age matched controls.
There was a significant difference between total body iron (F(6,148)=97.3, p<0.0001) amongst the groups measured. Comparisons between age and treatment groups an Ordinary one-way ANOVA was performed, followed by Sidak's multiple comparisons test. The results of the pairwise comparisons, corrected for multiple comparisons, are shown in Table 14.
These results highlight how bulk measures of total iron or measurements by inference can be confounded by changes in the animal morphology when exploring aging interventions.
We had previously determined age-related changes to the C. elegans iron-proteome, characterized on size exclusion chromatography by three major peaks: a high molecular weight peak (HMW, >1 MDa), ferritin, and a low MW peak (LMW, 600 Da) that may contain labile iron. With aging, iron redistributes in C. elegans out of the ferritin peak (where it is sequestered in redox-silent storage reserves) and accumulates in the HMW and LMW peaks. The chromatographic profile of aged C. elegans (10 days post adulthood) treated with SIH revealed decreased iron associated with the LMW peak (normalized peak area approximately 40%). Ferritin-bound iron was also similarly decreased by SIH (normalized peak area approximately 50%), but iron bound within HMW species was unaffected. The age-related changes in LMW iron are consistent with increased labile iron, which is withdrawn as the substrate for ferroptosis by SIH treatment.
X-ray absorption near edge structure (XANES) spectroscopy, using fluorescence detection for visualization, directly assesses the in vivo coordination environments of metal ions in biological specimens (ϕXANES). The centroid of the XANES pre-edge feature reflects the relative abundance of ferrous [Fe2+] and ferric [Fe3+] species. Since Fe2+ in the labile iron pool is the specific substrate for ferroptosis, and rises with aging in C. elegans, we investigated the impact of our interventions using jXANES. This synchrotron-based spectroscopy allowed us to evaluate steady state iron speciation (Fe2+/Fe3+) in a specific region (anterior intestinal) of intact, cryogenically-stabilized control, Lip-1 and SIH—treated worms. We found that the age-related increase in the Fe2+ fraction was normalized to that of a young animal by both Lip-1 and SIH treatments (Table 15). There was a significant difference between the fractional Fe2+/Fe(total) estimates, determined by non-overlapping 95% Cl, between aged TJ1060 animals (Table S7). Treatment with Lip-1 or SIH restored the Fe2+/Fe(total) estimate. Similarly, treatment of wild type (N2) animals with DEM markedly increased Fe2+/Fe(total).
There was a significant difference be
Higher levels of pro-ferroptotic Fe2+ might be compounded by a loss of glutathione. So, we also assessed changes in fractional Fe2+ induced by lethal glutathione depletion by DEM. jXANES of 4 day old wild type worms treated with DEM identified a marked increase in the Fe2+ fraction (Table 15), revealing the upper limit for tolerable Fe2+ fraction being about 0.3 of the total iron. These results help to contextualize the observed increase in Fe2+ during normal aging also being about 0.3 of the total iron, which was normalized to ≈0.2 by Lip-1 or SIH intervention.
Since Fe2+ accumulates with aging and contributes to C. elegans frailty by executing cells before organismal death, we hypothesized that ferroptosis directly impacts on lifespan and may represent an underlying process that contributes to organismal aging. We found that treatment of C. elegans with Lip-1 markedly extended lifespan (average ˜70% increase in median lifespan (8 independent replicates; p<0.002)). An alternative ferroptosis inhibitor, ferrostatin, was also examined, producing a significant but more modest median lifespan extension. Targeting the accumulation of late life iron using SIH also resulted in a marked increase in median lifespan (average ˜100% median increase (8 independent replicates; p<0.0001)). Exposing C. elegans to 250 μM SIH as an iron complex (Fe(SIH)2NO3) neutralized the benefits of SIH on lifespan, confirming that the rescue mechanism required SIH being free to ligate iron.
Lip-1 and SIH had distinct effects on aging. Treatment with Lip-1 primarily altered late life survival, while SIH extended mid-life with a squaring of the survival curve. Interventions that increase lifespan in C. elegans are not uncommon, but it has recently been demonstrated that the great majority of longevity interventions e.g. dietary and temperature alteration, oxidative stress, and genetic disruptions of the insulin/IGF-1 pathway (e.g. daf-2 and daf-16), heat shock factor hsf-1, or hypoxia-inducible factor hif-1, each alter lifespan by temporal scaling—an apparent stretching or shrinking of time. For an intervention to extend lifespan by temporal scaling it must alter, to the same extent throughout adult life, all physiological determinants of the risk of death. In effect temporal scaling arises when the risk of death is modulated by an intervention acting solely on the rate constant associated with a single stochastic process. It is important to note that temporal scaling is determined by statistical analysis rather than subjective assessment, and also that reproducibility of results depends upon adequate sample size.
Combining the replicate data from 8 independent experiments, we assessed whether Lip-1 and SIH treatment effects can be explained by the temporal scaling model of accelerated failure time (AFT). We found that the lifespan increases were not consistent with the temporal scaling model (p<0.01; Tables 16-), so the interventions may target previously unrecognized aging mechanisms. For SIH treatment, the risk of death (hazard) in early adulthood was greatly reduced compared to control populations but rose precipitously in late life. In contrast, Lip-1 markedly reduced the rate of mortality in the post-reproductive period (late-life) with early life mortality closer to that seen in untreated populations. These findings are consistent with ferroptotic cell death limiting lifespan in late life rather than being a global regulator (e.g. insulin/IGF-1 pathway) of aging. This raises the possibility of targeted intervention with minimal or no metabolic cost.
Using the modified Kolmogorov-Smirnov (K-S) test (Fleming et al., 1980) we examined whether the treatment effects can be reasonably modelled using the Accelerated Failure Time (AFT) model to determine whether we can reasonably assume that the treatment effect manifests in temporal rescaling. To control for inter-replicate differences, the test is conducted on the residuals a replicate-specific AFT model with the Buckley-James method (Buckley and James, 1979) using the nonparametric baseline hazards form. The function bj in R package rms was used to fit the models. The null hypothesis for the two-sample K-S test is that the simple temporal rescaling holds and the residuals for the two treatment groups under comparison come from the same distribution.
Since the R function can only take right-censored data and our lifespan data are interval-censored, we use the mid-point of the interval to assign the time of event. Treating interval-censored data as right-censored is expected to underestimate the variability in the statistical estimates (Lindsey and Ryan, 1998) which in turn will produce an optimistic (smaller than it should be) p-value. To reduce the likelihood of false rejection of the null hypothesis merely because of the optimistic p-value, we chose a more stringent Type I error (0.01) than the usual 0.05 when conducting the K-S test.
1 × 10−15
4 × 10−11
4 × 10−13
3 × 10−15
2 × 10−31
7 × 10−28
4 × 10−14
As can be seen from Table 16, the effect of Lip-1 and SIH treatment relative to control always deviates away from simple temporal rescaling (all p-values<10-2) with the exception of Lip-1 in replicate 2. However, the AFT assumption is not reasonable since the survival curves of residuals from the AFT models show ‘crossing’ behavior. If the simple temporal rescaling assumption is reasonable, we would expect the survival curves for the different treatments to be very similar to each other. The observed crossing of the curves is primarily caused by the de-acceleration in the survival function for control worms.
When all the replicates are combined, and the modified KS test were performed on the residuals of the AFT models with the best parametric form, we found that the p-value for comparing Control vs Lip-1, Control vs SIH and Lip-1 vs SIH are 2×10-24, 1×10-24 and 3×10-37 respectively. These results indicate that failure to control for inter-replicate differences would lead to even stronger evidence of departure from simple temporal rescaling.
Determining AFT Models with the Best Baseline Hazard Form
In order to investigate the possible reasons for departure from simple temporal rescaling, we used parametric survival models which require specification of a parametric baseline hazard form. To minimize the risk of model misspecification, we identified the most appropriate baseline hazard form for each replicate using the Bayesian Information Criterion (BIC), with the best parametric form chosen as the model that minimizes the BIC.
The following parametric baseline hazards were fitted:Gompertz, Gompertz with Frailty, Weibull, Weibull with Frailty, Log-normal and Log-logistic. The mathematical formulae for each parametric form are detailed below:
Gompertz: h(t|a,b)=(a/b)exp(t/b)
Gompertz with frailty: h(t|a,b,σ)=(a/b)exp(t/b)/[1+σ2a exp((t/b)−1)] Weibull: h(t|α,f3)=(α/f3)(t/f3)α−1
Weibull with frailty: h(t|α,f3,σ)=(α/)(t/f3)α−1/[1+σ2(t/f3)α]
Log-normal: h(t|μ,σ)=φ((log t−μ)/σ)/σt[1−CD ((log t−μ)/σ)] Log-logistic: h(t|α,f3)=(α/f3)(t/f3)α−1[1+(t/f3)α]
Here φ and CD denote the probability density function (PDF) and cumulative distribution function (CDF), respectively, of the standard normal distribution; p and o denote the mean and standard deviation (in the case of the log-normal, the mean and standard deviation of the logarithm of x); λ, α, and a are shape parameters; β and b are scale parameters. In the case of frailty, individual hazards hi(t) are related to a baseline hazard by a random factor Z that follows a Gamma distribution with mean 1 and variance o2.
Bayesian Information Criterion (BIC) is used to determine the best parametric form of the hazards; with better fit indicated by lower BIC value. All computations are done using flexsury R package, taking into account that events are interval censored to account for the fact that we do not observe the exact event time and only know that events occurred within an interval (a,b).
However, Gompertz baseline hazard form does not fit the data well, except when frailty is used. Weibull baseline hazard fits some replicates quite well and the fit is further improved when frailty is assumed. In fact, Weibull with frailty provides the best parametric baseline hazards form for nearly all the replicates, followed closely by the log-normal models.
Possible Causes of Departure from Temporal Rescaling
Unobserved heterogeneity (e.g. due to heterogeneity in the temperature the worms were exposed to) could cause de-acceleration and further, when the degree of heterogeneity is different between treatments, this could give rise to apparent departure from temporal rescaling. We investigated whether there is significant difference in the degree of heterogeneity by comparing two models for each replicate: (M1) model with Weibull frailty (Weibull hazard, Gamma frailty) where the degree of heterogeneity (represented by parameter a2 and a) is assumed to be the same for all three treatments, (M2) where the parameter a2 is allowed to be different but parameter a fixed across treatments and (M3) where the parameter a2 and a are allowed to be different across treatments. We compared the three models based on their BIC values and also performed likelihood ratio tests, comparing M1 vs M2 and M1 vs M3.
Note that only M1 can be classified as an AFT model while M2 and M3 are not AFT models, as the treatment effects also manifest in the other parameters apart from the location (shift) parameter. Table S10 shows that both M3 and M2 provide better fit than M1 for all replicates as indicated by small likelihood ratio test (LRT) p-values, with M3 providing more convincing p-values.
8 × 10−11
To investigate whether M3 provides an adequate fit to the data, for each replicate, we performed a chi-square goodness of fit test, comparing the observed survival curve to the fitted curve for each treatment group. The results are presented in Table 18. While the controls and SIH are always well-fitted by the Weibull frailty models (all p-values>0.01), the Lip-1 data from replicates 1, 4, 5 and 7 are not adequately fitted by the Weibull frailty model. The lack of fit for replicate 4 in particular is mainly caused by the estimated survival underestimating the observed counterparts in the middle-section between 7 and 15 days and overestimation on the tails.
We tried to identify the most parsimonious model that can best fit the combined data from all replicates. If some of the parameters are quite similar across replicates, we can fit a simpler model than the saturated model where all parameters are allowed to be different across replicates. A range of models are fitted and the best simple model for the combined data is Model 6 (M6) with the same treatment-dependent shapes and heterogeneity parameters across replicates but replicate-specific parameters for scale parameter of the control worms and temporal rescaling parameters. The BIC value for this model is smaller than that for the saturated model (15079 vs 15347) and the LR test statistic is 79.2 (df=90) with p-value=0.78, indicating that based on LR test the combined model (M6) does not provide worse fit to the data. The need for replicate-specific scale parameters and temporal rescaling parameters corroborates the evidence showing these parameters as having considerable variations across replicates.
Goodness-of-fit (GOF) test at replicate-level based on model M6 (Table 19) shows that this model provides more or less the same level of fit to the replicate-specific model (Table 18), with replicates showing good fit before still showing good fit now.
4 × 10−11
To investigate if changing the temperature showed evidence of temporal scaling, we compared worms aged at 20° C. and 25° C. during the same time frame. The effect of temperature is evaluated separately for control and SIH treated worms, population sizes for each replicate are shown in Table 20.
For each of two replicates and treatment factors, the AFT model with Weibull baseline hazard was fitted with temperature as the covariate. The residuals from this model were then subjected to the K-S test. The p-values from the K-S test are given in Table 21. As can be seen, the p-values are generally not very small (only one p-value<0.01), indicating that the evidence of departure from simple temporal rescaling is not strong.
We also performed meta-analysis for each worm population and the results are given below. The log a2 provides an indication of the level of heterogeneity, and it is interesting to note that the control worms exhibit greater heterogeneity than the SIH worms, consistent with that observed at 25° C.
For both populations, being exposed to the higher temperature of 25° C. accelerates life as expected, by approximately 30% for control worms (Otemp=0.72 (95% Cl: 0.68; 0.77) and 20% for SIH worms (Otemp=0.81 (95% Cl: 0.78; 0.84).
To minimize the likelihood that our findings are due to either intrinsic bias in our experiment or inflation of effect size (the Winner's curse phenomenon) we also examined the effect of temperature on lifespan intervention. It has been reported that changing temperature results in simple temporal rescaling of lifespans; our data corroborated this result and showed that SIH still extended lifespan by a similar dimension at both 20° C. and 25° C. (Tables 20-22). Our results indicate that while iron accumulation may impact many processes that influence aging rate, ferroptosis inhibition predominantly reduces frailty rather than slows a global rate of aging.
Preventing ferroptosis improves fitness and healthspan. Interventions that increase lifespan in C. elegans often do so at the detriment of fitness and healthspan. Adult body size can inform on fitness; reduced size may reflect a trade-off between longevity and fitness, as typically seen under dietary restriction where the cost of increased longevity can be lowered size, fertility and movement. Distinctly, SIH-treated animals grew substantially larger. Following one day of treatment all animals were of similar body length. After 4 days and 8 days of intervention, adult SIH-treated animals were significantly longer compared to similarly aged controls (e.g. control 1440±123 μm versus SIH 1696±64 μm, means±SD on Day 8, p<0.001). In addition, SIH induced an increase in body volume between Days 1 and 4, but not thereafter. SIH-treated worms grew to greater volume than both control and Lip-1 treated worms at Day 4, indicating that preventing iron accumulation can improve animal robustness (for all comparisons see Tables 3-4). Lip-1 had no effect on length or volume.
We also examined whether the interventions altered early and total reproductive output when worms were treated from early adulthood/late L4 (as used in the lifespan experiments). Early fertility (first 24 hours) was not altered by either SIH or Lip-1 treatment (p>0.4). Lip-1 treatment resulted in a small decrease in lifetime reproductive output (p<0.05), but SIH had no effect. Early fertility in C. elegans is paramount with respect to Darwinian fitness, so the reduction in lifetime fertility with Lip-1 treatment is consistent with a mild deleterious effect in early adulthood.
The effects of both interventions on movement parameters were assessed, since peak motile velocity has been previously demonstrated to correlate strongly with C. elegans healthspan and longevity and may be considered the best estimate of healthspan. As expected, control animals showed a steady decline in maximum velocity as they aged. Treatment with SIH or Lip-1 markedly improved the maximum velocity of aging animals, with increases also in distance traveled and mean velocity (Tables 5-10).
| Number | Date | Country | |
|---|---|---|---|
| 63054515 | Jul 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/041569 | Jul 2021 | US |
| Child | 18155281 | US |
