WO2023275715A1 - Metabolites of selective androgen receptor modulators - Google Patents

Metabolites of selective androgen receptor modulators Download PDF

Info

Publication number
WO2023275715A1
WO2023275715A1 PCT/IB2022/055952 IB2022055952W WO2023275715A1 WO 2023275715 A1 WO2023275715 A1 WO 2023275715A1 IB 2022055952 W IB2022055952 W IB 2022055952W WO 2023275715 A1 WO2023275715 A1 WO 2023275715A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
pharmaceutically acceptable
acceptable salt
mmol
methyl
Prior art date
Application number
PCT/IB2022/055952
Other languages
French (fr)
Inventor
Anne Elizabeth Hagen
Gregory Scott Walker
Original Assignee
Pfizer Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pfizer Inc. filed Critical Pfizer Inc.
Publication of WO2023275715A1 publication Critical patent/WO2023275715A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/22—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the nitrogen-containing ring
    • C07D217/26—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/24—Heterocyclic radicals containing oxygen or sulfur as ring hetero atom

Definitions

  • the present invention provides metabolites of certain selective androgen receptor modulators (SARMs), including salts and compositions thereof, which are useful in the prevention and/or treatment of diseases and disorders that are related to the androgen receptors as well as analytical methods related to the administration of these selective androgen receptor modulators.
  • SARMs selective androgen receptor modulators
  • BACKGROUND OF THE INVENTION The androgen receptor (“AR”) is a ligand-activated transcriptional regulatory protein that mediates induction of male sexual development and function through its activity with endogenous androgens.
  • Androgenic steroids play an important role in many physiologic processes, including the development and maintenance of male sexual characteristics such as muscle and bone mass, prostate growth, spermatogenesis, and the male hair pattern.
  • the endogenous steroidal androgens include testosterone and dihydrotestosterone ("DHT").
  • Steroidal ligands which bind the AR and act as androgens (e.g. testosterone enanthate) or as antiandrogens (e.g. cyproterone acetate) have been known for many years and are used clinically.
  • Patent Nos.9,328,104, 9,920,043, and 10,328,082 disclose certain SARMs and their uses in treating and/or preventing a variety of hormone-related conditions, for example, a disease or disorder or condition that is selected from anemia; anorexia; arthritis; bone disease; benign prostate hyperplasia; musculoskeletal impairment; cachexia; cachexia associated with cancer; cancer; frailty; age-related functional decline in the elderly; growth hormone deficiency; hematopoietic disorders; hormone replacement; hypergonadism; loss of muscle strength and/or function; muscular dystrophies; muscle loss following surgery; muscular atrophy; neurodegenerative diseases; neuromuscular disease; obesity; osteoporosis; sarcopenia, including sarcopenia in chronic obstructive pulmonary disease; a method of improving dexterity and movement in a subject; atherosclerosis and its associated diseases; dysmenorrhea; dysspermtogenic sterility; muscle wasting; respiratory tract disease; o
  • Compound 1 is a selective androgen receptor modulator (SARM).
  • SARM selective androgen receptor modulator
  • Compound 1 in its free base form, has the chemical formula C 14 H 14 N 4 SO 2 and the following structural formula:
  • the present invention provides a compound of Formula X-1, X-2, X-3, or X-4: wherein: A 1 is N or CR 0 ; R 0 is hydrogen, C 1 -C 6 linear or branched chain alkyl, C 1 -C 6 linear or branched chain perfluoroalkyl, aryl, perfluoroaryl, alkylaryl, heteroaryl or alkylheteroaryl; R 1 is glucuronidation; and R 3 and R 4 are each independently hydrogen, C 1 -C 6 linear or branched chain alkyl, C 1 -C 6 linear or branched chain perfluoroalkyl, C 1 -C 6 linear or branched chain perfluoroalkoxy, halogen, cyano, hydroxyl, amino, carboxy, hydroxyl, aryl, heteroaryl, C 1 -C 6 linear or branched chain alkoxylcarbonyl, C 1 -C
  • the substituent -R 1 , -OH, or -OR 1 is substituted at the part of the 4-methyl-1,1-dioxido-1,2,6-thiadiazinan-2-yl moiety within the dotted oval shape ⁇ i.e., one of the hydrogen atoms (including the hydrogen bonded to the N atom as shown or any hydrogen bonded to a ring-forming C atom or the C atom of the methyl group) on the part of the 4-methyl-1,1-dioxido- 1,2,6-thiadiazinan-2-yl moiety is replaced by the substituent.
  • the compound of Formula X-1, X-2, X-3, or X-4 or pharmaceutically acceptable salt thereof of the present invention is substantially isolated.
  • the present invention provides a compound of Formula Y-1, Y-2, Y- 3, or Y-4,
  • R 1A is and 3 R and R 4 are each independently hydrogen, C 1 -C 6 linear or branched chain alkyl, C 1 -C 6 linear or branched chain perfluoroalkyl, C 1 -C 6 linear or branched chain perfluoroalkoxy, halogen, cyano, hydroxyl, amino, carboxy, hydroxyl, aryl, heteroaryl, C 1 -C 6 linear or branched chain alkoxylcarbonyl, C 1 -C 6 linear or branched chain alkylamino-carbonylamino, or C 1 -C 6 linear or branched chain alkylaminocarbonyl, or a pharmaceutically acceptable salt thereof.
  • the compound of Formula Y-1, Y-2, Y-3, or Y-4, or pharmaceutically acceptable salt thereof of the present invention is substantially isolated.
  • the present invention further provides compositions comprising a compound of the invention, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.
  • the present invention further provides preparations comprising a compound of the invention, or a pharmaceutically acceptable salt thereof.
  • the present invention further provides methods of treating or preventing a disease or disorder or condition in a human by administering to the human a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, wherein the disease or disorder or condition is selected from selected from anemia; anorexia; arthritis; bone disease; benign prostate hyperplasia; musculoskeletal impairment; cachexia; cachexia associated with cancer; cancer; frailty; age-related functional decline in the elderly; growth hormone deficiency; hematopoietic disorders; hormone replacement; hypergonadism; loss of muscle strength and/or function; muscular dystrophies; muscle loss following surgery; muscular atrophy; neurodegenerative diseases; neuromuscular disease; obesity; osteoporosis; sarcopenia, including sarcopenia in chronic obstructive pulmonary disease; a method of improving dexterity and movement in a subject; atherosclerosis and its associated diseases; dysmenorrhea; dysspermtogenic sterility; muscle wasting; respiratory
  • the present invention further provides a compound of the invention, or pharmaceutically acceptable salt thereof, for use as a medicament.
  • the present invention further provides a compound of the invention, or pharmaceutically acceptable salt thereof, for use in a method of treating or preventing a disease or disorder or condition, wherein the disease or disorder or condition is selected from selected from anemia; anorexia; arthritis; bone disease; benign prostate hyperplasia; musculoskeletal impairment; cachexia; cachexia associated with cancer; cancer; frailty; age-related functional decline in the elderly; growth hormone deficiency; hematopoietic disorders; hormone replacement; hypergonadism; loss of muscle strength and/or function; muscular dystrophies; muscle loss following surgery; muscular atrophy; neurodegenerative diseases; neuromuscular disease; obesity; osteoporosis; sarcopenia, including sarcopenia in chronic obstructive pulmonary disease; a method of improving dexterity and movement in a subject; atherosclerosis and its associated diseases; dysmenorrhea
  • the present invention further provides methods of detecting or confirming the administration of Compound 1 to a human, comprising identifying a metabolite of Compound 1 (e.g. a compound of the invention), or a salt thereof, in a biological sample obtained from the human.
  • the present invention further provides methods of measuring the rate of metabolism of Compound 1 in a patient comprising measuring the amount of a metabolite of Compound 1 (e.g. a compound of the invention), or a salt thereof, in the patient at one or more time points after administration of Compound 1.
  • the present invention further provides methods of determining the therapeutic or prophylactic response of a patient to Compound 1 in the treatment of a disease or disorder or condition, comprising measuring the amount of a metabolite of Compound 1 (e.g.
  • the present invention further provides methods of optimizing the dose of Compound 1 for a patient in need of treatment with Compound 1, comprising measuring the amount of a metabolite of Compound 1 (e.g. a compound of the invention), or a salt thereof, in the patient at one or more time points after administration of Compound 1.
  • a metabolite of Compound 1 e.g. a compound of the invention
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows HPLC-UV chromatogram of pre-dose (Top) and pooled (Bottom) extracted human plasma samples from a multi dose study (100 mg of Compound 1 BID; 14 Days).
  • Figure 2 shows HPLC-UV chromatogram of pre-dose (Top) and pooled (Bottom) extracted human urine samples from a multi dose study (100 mg of Compound 1 BID; 14 Days).
  • Figure 3 shows product ion scan of Compound 1.
  • Figure 4 shows product ion spectrum of Compound/Metabolite M1 (m/z 479).
  • Figure 5 shows full 1 H NMR Spectra of Compound/Metabolite M1.
  • Figure 6 shows 1 H- 1 H TOCSY of Compound/Metabolite M1.
  • Figure 7 shows 1 H- 13 C HSQC Spectrum of Compound/Metabolite M1.
  • Figure 8 shows product ion scan of Compound/Metabolite M2 (m/z 256).
  • Figure 9 shows full 1 H NMR Spectra of Compound/Metabolite M2.
  • Figure 10 shows 1 H- 1 H TOCSY Spectrum of Compound/Metabolite M2.
  • Figure 11 shows product ion scan of Metabolite 495 (m/z 495) in human urine pool of patients dosed with Compound 1 (Day 14100 mg oral BID).
  • DETAILED DESCRIPTION In a first aspect, the present invention provides a compound of Formula X-1, X-2, X-3, or X- 4:
  • a 1 is N or CR 0 ;
  • R 0 is hydrogen, C 1 -C 6 linear or branched chain alkyl, C 1 -C 6 linear or branched chain perfluoroalkyl, aryl, perfluoroaryl, alkylaryl, heteroaryl or alkylheteroaryl;
  • R 1 is glucuronidation;
  • R 3 and R 4 are each independently hydrogen, C 1 -C 6 linear or branched chain alkyl, C 1 -C 6 linear or branched chain perfluoroalkyl, C 1 -C 6 linear or branched chain perfluoroalkoxy, halogen, cyano, hydroxyl, amino, carboxy, hydroxyl, aryl, heteroaryl, C 1 -C 6 linear or branched chain alkoxylcarbonyl, C 1 -C 6 linear or branched chain alkylamino-carbonylamino, or C 1 -C 6 linear or branched branched chain al
  • the present invention provides a compound of Formula X-1, or a pharmaceutically acceptable salt thereof.
  • the compound of Formula X-1 or a pharmaceutically acceptable salt thereof is a compound of Formula Y-1 or a pharmaceutically acceptable salt thereof, wherein R 1A is e.g. .
  • the present invention provides a compound of Formula X-2, or a pharmaceutically acceptable salt thereof.
  • the compound of Formula X-2 or a pharmaceutically acceptable salt thereof is a compound of Formula Y-2 or a pharmaceutically acceptable salt thereof.
  • the present invention provides a compound of Formula X-3, or a pharmaceutically acceptable salt thereof.
  • the substituents R 1 and OH are substituted on the part of the structure of Formula X-3 within the dotted oval shape (i.e., each of R 1 and OH replaces a hydrogen atom with the dotted oval shape).
  • the compound of Formula X-3 or a pharmaceutically acceptable salt thereof is a compound of Formula Y-3 or a pharmaceutically acceptable salt thereof, wherein R 1A is e.g.
  • a compound Formula X-3 or a pharmaceutically acceptable salt thereof is a compound of Formula X-3A or X-3B: X-3A ⁇ wherein the OH is substituted on the part of the structure of Formula X-3A within the dotted oval shape (i.e., the OH replaces a hydrogen atom with the dotted oval shape) ⁇ 1 X-3B ⁇ wherein R is substituted on the part of the structure of Formula X-3B within the dotted oval shape (i.e., R 1 replaces a hydrogen atom with the dotted oval shape) ⁇ or a pharmaceutically acceptable salt thereof.
  • the present invention provides a compound of Formula X-4, or a pharmaceutically acceptable salt thereof.
  • the substituent OR 1 is substituted on the part of the structure of Formula X-4 within the dotted oval shape (i.e., OR 1 replaces a hydrogen atom with the dotted oval shape).
  • the compound of Formula X-4 or a pharmaceutically acceptable salt thereof is a compound of Formula Y-4 or a pharmaceutically acceptable salt thereof, wherein R 1A is e.g.
  • the present invention is directed to metabolites of Compound 1 or a pharmaceutically acceptable salt thereof and uses thereof.
  • the metabolite results from Compound 1 (or a pharmaceutically acceptable salt thereof) which has undergone (1) glucuronidation (see e.g.
  • the metabolite is selected from Compounds M1; M2; a compound of Formula Y-3; and a compound of Formula Y- 4.
  • the present invention provides Compound M1.
  • the present invention provides Compound M1-A. or a pharmaceutically acceptable salt thereof, which is substantially isolated/purified. In some embodiments, the present invention provides Compound M2, or a pharmaceutically acceptable salt thereof, which is substantially isolated. In some embodiments, the present invention provides Compound M2-A, or a pharmaceutically acceptable salt thereof, which is substantially isolated. In some embodiments, the present invention provides Compound M2-B, or a pharmaceutically acceptable salt thereof, which is substantially isolated. In some embodiments, the compound of Formula Y-3 or pharmaceutically acceptable salt thereof is a compound of Formula Y-3A or a pharmaceutically acceptable salt thereof wherein R 1A is e.g. (i.e.
  • a compound of Formula Y-3A or a pharmaceutically acceptable salt thereof is a compound of Formula Y-3A-1
  • the compound of Formula Y-3A or pharmaceutically acceptable salt thereof is a compound of Formula Y-3A-2 OH or a pharmaceutically acceptable salt thereof, wherein R 1A is or (i.e.
  • the present invention provides a compound of Formula Y-4A or a pharmaceutically acceptable salt thereof, wherein R 1A is or (i.e. the metabolite is the glucuronic acid conjugation of a hydroxylated Compound 1); and wherein OR 1A is substituted on the part of the structure of Formula Y-4 within the dotted oval shape (i.e., OR 1A replaces a hydrogen atom with the dotted oval shape).
  • a compound of Formula Y-4A or a pharmaceutically acceptable salt thereof is a compound of Formula Y-4A-1
  • a salt generally refers to a derivative of a disclosed compound wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
  • a pharmaceutically acceptable salt is one that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • the salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid.
  • Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17 ed., Mack Publishing Company, Easton, Pa., 1985, p.1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
  • the pharmaceutically acceptable salt is a sodium salt.
  • the metabolite compounds (or the compounds of invention), or salts thereof are substantially isolated. By “substantially isolated” is meant that the metabolite compound, or salt thereof, is at least partially or substantially separated from the environment in which it was formed or detected.
  • Partial separation can include, for example, a composition enriched in the compound of the invention.
  • Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the metabolite, or salt thereof.
  • each of compounds Formula X-1 or X-2 (including compounds of Formula Y-1 or Y-2, such as Compound M1 or M2), compounds of Formula X-3 or X4 (including e.g. Compounds of Formula Y-3A or Y-4A) or their salts is substantially isolated.
  • each of compounds Formula X-1 or X-2 (including compounds of Y-1 or Y-2, such as Compounds M1 and M2) and compounds of Formula X-3 or X4 (including e.g. Compounds of Formula Y-3A or Y-4A) or their salts is substantially isolated.
  • one or more of the metabolite compounds, or salts thereof are prepared by metabolism of Compound 1 or a pharmaceutically salt thereof (for example, in a mammal or a mammalian cell environment); and the metabolite compounds thus prepared are substantially isolated.
  • one or more of the metabolite compounds, or salts thereof are prepared by chemical synthesis other than metabolism of Compound 1 or a pharmaceutically salt thereof (for example, in a mammal or a mammalian cell environment) and the synthesized metabolite compounds are substantially isolated.
  • a metabolite of the invention, or its salt can be present in a composition where the composition includes at least one compound other than the metabolite.
  • the composition includes more than one metabolite of the invention.
  • the composition comprises one or more metabolites of the invention, or salts thereof, and Compound 1, or a salt thereof.
  • compositions can be mixtures containing a metabolite of the invention, or salt thereof, and one or more solvents, substrates, carriers, etc.
  • the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 25% by weight.
  • the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 50% by weight.
  • the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 75% by weight.
  • the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 80% by weight.
  • the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 85% by weight. In some embodiments, the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 90% by weight. In some embodiments, the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 95% by weight.
  • a preparation of a metabolite of the invention, or salt thereof can be prepared by chemical synthesis or by isolation of the metabolite from a biological sample. Preparations can have a purity of greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% purity.
  • the metabolites of the invention are asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Metabolites of the invention also include all isotopes of atoms occurring in the metabolites. Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium.
  • the metabolite includes at least one deuterium.
  • compound or “metabolite,” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
  • metabolite as used herein is meant to include any and all metabolic derivatives of a parent drug molecule (e.g.
  • Compound 1 or a pharmaceutically acceptable salt thereof including derivatives that have undergone one or more transformative processes selected from (1) glucuronidation, (2) hydrolysis of the 1,1-dioxido-1,2,6-thiadiazinane ring followed by oxidation, (3) glucuronidation and hydroxylation, (4) glucuronic acid conjugation of a hydroxylated-Compound 1, (5) hydroxylation, or a combination thereof (including a pharmaceutically acceptable salt thereof).
  • the present invention provides a metabolite of Compound 1 or a pharmaceutically acceptable salt thereof.
  • a glucuronide conjugation (or a glucuronide adduct, or glucuronidation) of a parent compound refers to replacing a hydrogen atom of the parent compound with a chemical moiety that is glucuronic acid without one of its four alcohol hydroxyl groups, e.g., a moiety having the structure of: O H H or wherein indicates the point of contact of the moiety to the parent compound.
  • -O-glucuronidation or -O-glucuronide refers to a moiety of the structure of
  • Compound 1 can also be considered a prodrug of the metabolites of the invention (e.g., a prodrug of metabolites M1, M2, a compound of Formula Y-3A or Y-4A and the like) because Compound 1 metabolically transforms upon administration to provide the metabolites of the invention. Accordingly, Compound 1 can be administered to a human as a means of providing a metabolite of the invention to the human, for example, for preventing or treating a disease or disorder or condition in the human as described herein.
  • a prodrug of the metabolites of the invention e.g., a prodrug of metabolites M1, M2, a compound of Formula Y-3A or Y-4A and the like
  • the present of invention provides a metabolite of a compound of Formula 3-A: wherein A 1 is N or CR 0 ; R 0 is hydrogen, C 1 -C 6 linear or branched chain alkyl, C 1 -C 6 linear or branched chain perfluoroalkyl, aryl, perfluoroaryl, alkylaryl, heteroaryl or alkylheteroaryl; and R 3 and R 4 are each independently hydrogen, C 1 -C 6 linear or branched chain alkyl, C 1 -C 6 linear or branched chain perfluoroalkyl, C 1 -C 6 linear or branched chain perfluoroalkoxy, halogen, cyano, hydroxyl, amino, carboxy, hydroxyl, aryl, heteroaryl, C 1 -C 6 linear or branched chain alkoxylcarbonyl, C 1 -C 6 linear or branched chain alkylamino-carbonylamino, or C 1 -C 6
  • the compound of Formula 3-A or pharmaceutically acceptable salt thereof including any of the derivatives that have undergone one or more transformative processes selected from (1) glucuronidation, (2) hydrolysis of the 1,1-dioxido-1,2,6-thiadiazinane ring followed by oxidation, (3) glucuronidation and hydroxylation, (4) glucuronic acid conjugation of a hydroxylated-Compound 1, (5) hydroxylation, or a combination thereof (including a pharmaceutically acceptable salt thereof).
  • the present of invention provides a metabolite of a compound of Formula 3-B: wherein R 3 and R 4 are each independently hydrogen, C 1 -C 6 linear or branched chain alkyl, C 1 -C 6 linear or branched chain perfluoroalkyl, C 1 -C 6 linear or branched chain perfluoroalkoxy, halogen, cyano, amino, carboxy, hydroxyl, aryl, heteroaryl, C 1 -C 6 linear or branched chain alkoxy-carbonyl-, C 1 -C 6 linear or branched chain alkylamino-carbonylamino, or C 1 -C 6 linear or branched chain alkylaminocarbonyl, or a pharmaceutically acceptable salt thereof, and wherein the metabolite is a derivative of the parent drug molecule (i.e.
  • the compound of Formula 3-A or pharmaceutically acceptable salt thereof including any of the derivatives that have undergone one or more transformative processes selected from (1) glucuronidation, (2) hydrolysis of the 1,1-dioxido-1,2,6-thiadiazinane ring followed by oxidation, (3) glucuronidation and hydroxylation, (4) glucuronic acid conjugation of a hydroxylated-Compound 1, (5) hydroxylation, or a combination thereof (including a pharmaceutically acceptable salt thereof).
  • one or more of the metabolite compounds, or salts thereof are prepared by metabolism of its parent compound, e.g., a compound of Formula 3-A or 3-B or a pharmaceutically salt thereof (for example, in a mammal or a mammalian cell environment); and the metabolite compounds thus prepared are substantially isolated.
  • one or more of the metabolite compounds, or salts thereof are prepared by chemical synthesis other than metabolism of a compound of Formula 3-A or 3-B or a pharmaceutically salt thereof (for example, in a mammal or a mammalian cell environment) and the synthesized metabolite compounds are substantially isolated.
  • a compound of Formula 3-A or 3-B or its salt can be prepared, for example, by the methods described in U.S. Patent No.9328104.
  • alkyl alone or in combination, means an acyclic, saturated hydrocarbon group of the formula C n H 2n+1 which may be linear or branched. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl and hexyl. Unless otherwise specified, an alkyl group comprises from 1 to 6 carbon atoms.
  • C i -C j indicates a moiety of the integer "i" to the integer "j" carbon atoms, inclusive.
  • C 1 -C 6 alkyl refers to alkyl of one to six carbon atoms, inclusive.
  • aryl alone or in combination, means phenyl or naphthyl.
  • -alkylaryl means an -alkyl-aryl moiety that is attached through the alkyl part.
  • heteroaryl refers to an aromatic heterocycle which may be attached via a ring carbon atom (in all cases) or a ring nitrogen atom with an appropriate valency (when the heterocycle is attached to a carbon atom). Equally, when substituted, the substituent may be located on a ring carbon atom (in all cases) or a ring nitrogen atom with an appropriate valency (if the substituent is joined through a carbon atom).
  • thienyl furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl and pyrazinyl.
  • -alkylheteroaryl means an -alkyl-heteroaryl moiety that is attached through the alkyl part.
  • perfluoroalkyl means an alkyl radical wherein each of the hydrogen on the alkyl is replaced by a fluorine atom.
  • perfluoroaryl means an aryl radical wherein each of the hydrogen on the aryl is replaced by a fluorine atom.
  • hydroxy means an OH radical.
  • alkoxy means a radical comprising an alkyl radical that is bonded to an oxygen atom, such as a methoxy radical. Examples of such radicals include methoxy, ethoxy, propoxy, isopropoxy, butoxy and tert-butoxy.
  • halogen means, fluoro, chloro, bromo or iodo.
  • amino means -NH 2 .
  • a wavy line,“ ” denotes a point of attachment of a substituent to another group.
  • R 3 may be bonded to any ring-forming carbon atom of the left ring of the bicyclic ring that is substitutable (i.e., any one of the carbon atoms of a -CH- group of the left ring).
  • R 4 may be bonded to any ring- forming carbon atom of the right ring of the bicyclic ring that is substitutable (i.e., any one of the carbon atoms of a -CH- group).
  • the present invention further includes a pharmaceutical composition comprising a compound (or a metabolite) of the invention, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.
  • the compound (or the metabolite) of the invention or pharmaceutically acceptable salt thereof is present in the composition in an amount greater than about 0.001%, 0.01%, 0.05%, 0.08%, 0.1%, 0.5%, or 1.0% by weight
  • pharmaceutically acceptable carrier is meant to refer to any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • the present invention further relates to a method of treating or preventing a disease or disorder or condition in a human by administering to the human a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, wherein the disease or disorder or condition is selected from selected from anemia; anorexia; arthritis; bone disease; benign prostate hyperplasia; musculoskeletal impairment; cachexia; cachexia associated with cancer; cancer; frailty; age-related functional decline in the elderly; growth hormone deficiency; hematopoietic disorders; hormone replacement; hypergonadism; loss of muscle strength and/or function; muscular dystrophies; muscle loss following surgery; muscular atrophy; neurodegenerative diseases; neuromuscular disease; obesity; osteoporosis; sarcopenia, including sarcopenia in chronic obstructive pulmonary disease; a method of improving dexterity and movement in a subject; atherosclerosis and its associated diseases; dysmenorrhea; dysspermtogenic sterility;
  • the human may have or be at risk of having the disease or disorder.
  • treating in connection with a disease or disorder as used herein embraces palliative treatment, including reversing, relieving, alleviating, eliminating, or slowing the progression of the disease or disorder , or one or more symptoms of the disease or disorder, or any tissue damage associated with one or more symptoms of the disease or disorder.
  • prevention or “preventing” in connection with a disease or disorder refers to delaying or forestalling the onset or development of the disease or disorder a period of time from minutes to indefinitely. The term also includes prevention of the appearance of symptoms of the disease or disorder. The term further includes reducing risk of developing the disease or disorder.
  • an effective amount refers to an amount of a metabolite according to the invention, which when administered to a patient in need thereof, is sufficient to effect treatment for disease-states, conditions, or disorders for which the compounds have utility. Such an amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is sought by a researcher or clinician.
  • the amount of a metabolite according to the invention which constitutes a therapeutically effective amount will vary depending on such factors as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of the treatment, the type of disease-state or disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds of the invention, and the age, body weight, general health, sex and diet of the patient.
  • a therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure.
  • Administration of the metabolites of the invention, or their pharmaceutically acceptable salts can be carried out via any of the accepted modes of administration of agents for serving similar utilities.
  • compositions of the invention can be prepared by combining a metabolite of the invention, or a pharmaceutically acceptable salt thereof, with an appropriate pharmaceutically acceptable carrier and, in specific embodiments, are formulated into preparations in solid, semi solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
  • pharmaceutical compositions of the invention are tablets.
  • compositions of the invention are injection (intramuscular (IM) or intraperitoneal (IP)).
  • Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
  • composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease or disorder of interest in accordance with the teachings described herein.
  • the present invention further relates to a method of detecting or confirming the administration of Compound 1 to a patient comprising identifying a metabolite of Compound 1 (e.g. a metabolite of the invention), or salt thereof, in a biological sample obtained from the patient.
  • the biological sample is derived from plasma, urine, or feces.
  • the present invention further relates to a method of measuring the rate of metabolism of Compound 1 in a patient comprising measuring the amount of a metabolite, or salt thereof, in the patient at one or more time points after administration of Compound 1.
  • the present invention further relates to a method of determining the prophylactic or therapeutic response of a patient to Compound 1 in the treatment of a disease or disorder comprising measuring the amount of a metabolite of Compound 1 (e.g. a metabolite of the invention), or salt thereof, in the patient at one or more time points after administration of Compound 1.
  • the present invention further relates to a method of optimizing the dose of Compound 1 for a patient in need of treatment with Compound 1 comprising measuring the amount of a metabolite of Compound 1 (including, e.g. a metabolite of the invention) or salt thereof, in the patient at one or more time points after administration of Compound 1.
  • the amount of metabolite may be indicative of the rate at which the patient metabolizes Compound 1.
  • Patients who metabolize Compound 1 more quickly or more effectively than other patients may form higher amounts of metabolite and potentially require higher doses of Compound 1, or additional doses, compared with patients who metabolize Compound 1 more slowly.
  • the method of optimizing the dose of Compound 1 may further include determining whether the measured amounts of metabolite are higher or lower than average, and adjusting the dosage of Compound 1 accordingly.
  • Measuring the amount of metabolite, or salt thereof, in a patient can be carried out by obtaining a biological sample from the patient and measuring the amount of metabolite, or salt thereof, in the sample.
  • the sample is blood.
  • the sample is plasma.
  • the sample is urine.
  • the sample is feces.
  • patient is meant to refer to a human or another mammal such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as non-human primates, mammalian wildlife, and the like, that are in need of therapeutic or preventative treatment for a disease or disorder described herein.
  • Combination Therapies One or more additional pharmaceutical agents can be used in combination with the compounds, salts, and compositions of the present invention for preventing or treating a disease or disorder described herein, e.g., in a human patient.
  • the composition of the invention further comprises one or more additional therapeutic agents.
  • the composition of the invention further comprises one to three additional therapeutic agents.
  • additional therapeutic agents include, but are not limited to: (i) estrogen and estrogen derivatives (such as conjugated estrogens and synthetic estrogens) including, but not limited to, steroidal compounds having estrogenic activity such as, for example, 17.beta.-estradiol, estrone, conjugated estrogen (PREMARIN.RTM.), equine estrogen, 17.beta.-ethynyl estradiol, and the like.
  • the estrogen or estrogen derivative can be employed alone or in combination with a progestin or progestin derivative.
  • progestin derivatives are norethindrone and medroxy-progesterone acetate; (ii) a bisphosphonate compound, including, but not limited to: (a) alendronate (also known as alendronic acid, 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid, alendronate sodium, alendronate monosodium trihydrate or 4-amino-1-hydroxybutylidene-1,1- bisp- hosphonic acid monosodium trihydrate. Alendronate is described in U.S. Pat. No.4,922,007, to Kieczykowski et al., issued May 1, 1990; U.S. Pat.
  • a selective estrogen receptor modulator including, but not limited to tamoxifen, 4- hydroxytamoxifen, raloxifene (see, e.g., U.S. Pat.
  • PSK- 3471 PSK- 3471; (iv) calcitonin and analogue thereof, including, but not limited to, salmon, Elcatonin, SUN-8577 or TJN-135, wherein if the calcitonin analogue is salmon it is optionally dosed as a nasal spray (for example as disclosed in Azra et al., Calcitonin.1996. In: J. P.
  • cathepsin K formerly known as cathepsin O.sub.2, for example as described in PCT International Application Publication No. WO 96/13523; U.S. Pat. Nos.5,501,969 and 5,736,357, and which include those which at an acidic pH degrade type-I collagen.
  • cathepsin K include, but are not limited to, those disclosed in WO 01/49288, and WO 01/77073.
  • cathepsin K inhibitors include, but are not limited to AAE581 and Odanacatib; (vi) alpha.v.beta.3 Integrin receptor antagonists peptidyl as well as peptidomimetic antagonists of the .alpha.v.beta.3 integrin receptor which indluce, but are not limited to those disclosed in the following publications W. J. Hoekstra and B. L. Poulter, Curr. Med.
  • HMG-CoA reductase inhibitors also known as the "statins", including, but not limited to, statins in their lactonized or dihydroxy open acid forms and pharmaceutically acceptable salts and esters thereof, including but not limited to lovastatin (see U.S. Pat. No.4,342,767); simvastatin (see U.S. Pat. No.4,444,784); dihydroxy open-acid simvastatin, particularly the ammonium or calcium salts thereof; pravastatin, particularly the sodium salt thereof (see U.S. Pat.
  • NK-104 pitavastatin
  • itavastatin lovastatin
  • pravastatin sodium nisvastatin
  • osteoanabolic agents including, but not limited to, parathyroid hormone (PTH) and fragments thereof, such as naturally occurring PTH (1-84), PTH (1-34), analogs thereof, native or with substitutions and particularly parathyroid hormone subcutaneous injection, for example Forteo (teriparatide);
  • protein kinase inhibitors including, but not limited to, those disclosed in WO 01/17562 and which are in one embodiment selected from inhibitors of p38, non-limiting example of which include SB 203580 [Badger et al., J.
  • agonists including, but not liited to, bezafibrate, clofibrate, fenofibrate including micronized fenofibrate, and gemiibrozil; (xiv) dual acting peroxisome proliferator-activated alpha./.gamma.
  • agonists including, but not limited to, muraglitazar, naveglitazar, farglitazar, tesaglitazar, ragaglitazar, oxeglitazar, PN-2034, PPAR.delta, such as for example, GW-501516;
  • calcium receptor antagonists which induce the secretion of PTH as described by Gowen et al., J. Clin.
  • growth hormone and its analogs including, but not limited to, human growth hormone, such as, for example, somatotropin or analogues, nutropin A; growth promoting agents such as, for example, TRH, diethylstilbesterol, theophylline, enkephalins, E series prostaglandins, such as, for example, Ep1, EP2, EP4, FP, IP and derivatives thereof, prostanoids, compounds disclosed in U.S. Pat. No.3,239,345, e.g., zeranol, and compounds disclosed in U.S. Pat.
  • human growth hormone such as, for example, somatotropin or analogues, nutropin A
  • growth promoting agents such as, for example, TRH, diethylstilbesterol, theophylline, enkephalins, E series prostaglandins, such as, for example, Ep1, EP2, EP4, FP, IP and derivatives thereof, prostanoids, compounds disclosed in U.S. Pat. No.3,
  • No.4,036,979 e.g., sulbenox or peptides disclosed in U.S. Pat. No.4,411,890
  • growth hormone secretagogues such as, for example, anamorelin, pralmorelin, examorelin, tabimorelin, capimorelin, capromorelin, ipamorelin, EP-01572, EP-1572, or JMV-1843, GHRP-6, GHRP-1 (as described in U.S. Pat. No.
  • growth hormone releasing factor and its analogues such as, for example (a) epidermal growth factor (EGF); (b) transforming growth factor-.alpha.
  • TGF-.alpha. platelet derived growth factor
  • PDGF platelet derived growth factor
  • FGFs fibroblast growth factors
  • FGFs including acidic fibroblast growth factor (.alpha.- FGF) and basic fibroblast growth factor (.beta.-FGF), including, but not limited to aFGF, bFGF and related peptides with FGF activity [Hurley Florkiewicz, "Fibroblast growth factor and vascular endothelial growth factor families," 1996. In: J. P. Bilezikian, et al., Ed. Principles of Bone Biology, San Diego: Academic Press]; (e) transforming growth factor-.beta.
  • TGF-.beta. insulin like growth factors
  • IGF-1 and IGF-2 insulin like growth factors selected from, but not limited to, Insulin-like Growth Factor I, alone or in combination with IGF binding protein 3 and IGF II [See Johannson and Rosen, "The IGFs as potential therapy for metabolic bone diseases," 1996, In: Bilezikian, et al., Ed., Principles of Bone Biology, San Diego: Academic Press; and Ghiron et al., J. Bone Miner.
  • IGF-1 IGF-1 analogues and secretagogue IGF-1 (xviii) a bone morphogenetic protein (BMP), including, but not limited to, chordin, fetuin, BMP 2, 3, 5, 6, 7, as well as related molecules TGF beta and GDF 5 [Rosen et al., "Bone morphogenetic proteins," 1996. In: J. P.
  • BMP bone morphogenetic protein
  • Bilezikian et al., Ed., Principles of Bone Biology, San Diego: Academic Press; and Wang E A, Trends Biotechnol., 11: 379-383 (1993)]; (xix) an inhibitor of BMP antagonism including, but not limited to, sclerostin, SOST, noggin, chordin, gremlin, and dan [see Massague and Chen, "Controlling TGF-beta signaling," Genes Dev., 14: 627-644, 2000; Aspenberg et al., J. Bone Miner. Res.16: 497-500, 2001; and Brunkow et al., Am. J. Hum.
  • Vitamin D vitamin D derivatives, vitamin D analogs, including, but not limited to, D.sub.3 (cholecaciferol), D.sub.2 (ergocalciferol), 25-OH-vitamin D.sub.3, 1.alpha.,25(OH).sub.2 vitamin D.sub.3, 1.alpha.-OH-vitamin D.sub.3, 1.alpha.-OH-vitamin D.sub.2, dihydrotachysterol, 26,27-F6- 1.alpha.,25(OH).sub.2 vitamin D.sub.3, 19-nor-1.alpha.,25(OH).sub.2 vitamin D.sub.3, 22- oxacalcitriol, calcipotriol, 1.alpha.,25(OH).sub.2-16-ene-23-yne-vitamin D.sub.3 (Ro 23-7553), EB1089, 20-epi-1.alpha.,25(OH).sub.2 vitamin D analogs, including, but not limited to, D.sub.3 (
  • Vitamin K and Vitamin K derivatives including, but not limited to, menatetrenone (vitamin K2) [see Shiraki et al., J. Bone Miner.
  • a steroidal or nonsteroidal androgen receptor antagonist including, but not limited to, enzalutamide, ARN-509, flutamide, hydroxyflutamide, bicalutamide, nilutamide, or hydroxysteroid dehydrogenase inhibitor or abiraterone; a reversible antiandrogen; or a SARM agent, including, but not limited to those disclosed herein, RU-58642, RU-56279, WS9761 A and B, RU-59063, RU-58841, bexlosteride, LG-2293, L-245976, LG-121071, LG-121091, LG-121104, LGD-2226, LGD-2941, LGD-3303, LGD-4033, YM-92088, YM-175735, LGD-1331, BMS-3575
  • Pat. No.3,865,801 or recombinantly produced protein and analogs thereof, for example, as described in U.S. Pat. Nos.5,441,868, 5,547,933, 5,618,698 and 5,621,080 as well as human erythropoietin analogs with increased glycosylation and/or changes in the amino acid sequence as those described in European Patent Publication No. EP 668351 and the hyperglycosylated analogs having 1-14 sialic acid groups and changes in the amino acid sequence described in PCT Publication No.
  • erythropoietin-like polypeptides comprise darbepoietin (from Amgen; also known as Aranesp and novel erthyropoiesis stimulating protein (NESP)); (xxviii) an immunomodulating agent, including, but not limited to, immunosuppressive cytotoxic drugs, such as, for example, mechlorethamine, chlorambucil; immunosuppressive agent such as, for example, mycophenolate motefil or 6-thioguanine, including those which can optionally be administered topically such as tacrolimus, pimecrolimus, imiquimod, 5-fluorouracil, or mechlorethamin; immunostimulatory agents such as, for example, a non-specificimmunostimulator for example Freund's complete adjuvant, Freund's incomplete adjuvant, a montanide ISA adjuvant, a Ribi's adjuvant, a Hunter's TiterMax, an aluminum salt adjuvant,
  • the adrenomimetic drug is a catecholamine.
  • adrenomimetic drugs include but are not limited to isoproterenol, norepinephrine, epinephrine, ephedrine, or dopamine.
  • the adrenomimetic drug is a directly acting adrenomimetic drug.
  • directly acting adrenomimetic drugs include but are not limited to phenylephrine, metaraminol, or methoxamine; (xliii) an appetite stimulants such as megestrol acetate, cyproheptadine; (xliv) a luteinizing hormone releasing hormone (LHRH), a LHRH analog or derivative, a luteinizing hormone agonists or antagonists including, but not limited to, letrozole, anastrazole, atamestane, fadrozole, minamestane, exemestane, plomestane, liarozole, NKS-01, vorozole, YM-511, finrozole, 4-hydroxyandrostenedione, aminogluethimide, or rogletimide; (xlv) a vitronectin receptor antagonist; (xlv) a vitr
  • Dual ET/AII antagonist e.g., compounds disclosed in WO 00/01389, neutral endopeptidase (NEP) inhibitors, vasopepsidase inhibitors (dual NEP-ACE inhibitors) (e.g., omapatrilat and gemopatrilat), or nitrates; (xcx) a melanocortin 4 receptor antagonist.
  • NEP neutral endopeptidase
  • dual NEP-ACE inhibitors e.g., omapatrilat and gemopatrilat
  • xcx melanocortin 4 receptor antagonists
  • melanocortin 4 receptor (MC4R) antagonists include, for example, those in U.S. Provisional Application No.63/036,798 filed June 09, 2020, and U.S. Provisional Application No.63/167,271 filed March 29, 2021.
  • melanocortin 4 receptor antagonist is selected from: (2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1; 2-(6-methoxy-2-methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1; 2-[6-(difluoromethoxy)pyridin-3-yl]-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4- di
  • an anticancer agent including, but not limited to, (a) a monoclonal antibody, which antibody may be optionally used for diagnosis, monitoring, or treatment of cancer, including monoclonal antibodies which react against specific antigens on cancer cells such as the monoclonal antibody acts as a cancer cell receptor antagonist, those which monoclonal antibodies enhance the patient's immune response, those which act against cell growth factors, thus blocking cancer cell growth, those which are conjugated or linked to anti-cancer drugs, radioisotopes, other biologic response modifiers, other toxins, or a combination thereof; (b) a selective tyrosine kinase inhibitor including those embodiments where the selective tyrosine kinase inhibitor inhibits catalytic sites of cancer promoting receptors thereby inhibiting tumor growth; the selective tyrosine kinase inhibitor modulates growth factor signaling; the selective tyrosine kinase inhibitor targets EGFR (ERB B/HER) family members; the selective tyrosine kinase
  • the cancer vaccine comprises viral vectors and/or DNA vaccines, including those embodiments where the cancer vaccine is an idiotype vaccine; (ci) a cholesterol acyltransferase (ACAT) inhibitors including selective inhibitors of ACAT-1 or ACAT-2 as well as dual inhibitors of ACAT-1 and -2; (cii) an amylin analogue such as pramlintide; (ciii) a cholesteryl ester transfer protein or CETP Inhibitor, including, but not limited to, JTT-705, CETi-1; (civ) a vasodilator; (cv) an anti-anginal agent including, but not limited to, nifedipine; (cvi) a glucagon-like peptide-1 (GLP-1) and analogues, including, but not limited to, exenatide or liraglutide; (cvii) a H.sub.2-receptor antagonist, including, but not limited to, cimetidine and ran
  • a metabolite disclosed herein, or a pharmaceutically acceptable salt thereof is combined with two, three, four or more additional therapeutic agents.
  • the two, three four or more additional therapeutic agents can be different therapeutic agents selected from the same class of therapeutic agents, or they can be selected from different classes of therapeutic agents.
  • different components/APIs active pharmaceutical ingredients
  • simultaneous administration of drug combinations is used.
  • each component/API may be administered in any order and each of them can be administered in an independent frequency or dose regimen.
  • such administration be oral.
  • such administration can be oral and simultaneous.
  • the administration of each may be by the same or by different methods.
  • administration of one component/API is oral but administration of another component/API is not oral (for example, is injectable).
  • a metabolite disclosed herein is combined with one or more additional therapeutic agents as described above, the components of the composition are administered as a simultaneous or separate (e.g. sequential) regimen.
  • the combination may be administered in two or more administrations.
  • a metabolite disclosed herein is combined with one or more additional therapeutic agents in a unitary dosage form for simultaneous administration to a patient, for example as a solid dosage form for oral administration (e.g., a fixed dose combination tablet).
  • a metabolite disclosed herein is administered with one or more additional therapeutic agents.
  • Co-administration of a metabolite disclosed herein, or a pharmaceutically acceptable salt thereof, with one or more additional therapeutic agents generally refers to simultaneous or separate (e.g. sequential) administration of a compound disclosed herein and one or more additional therapeutic agents, such that therapeutically effective amounts of the metabolite and one or more additional therapeutic agents are both present in the body of the patient.
  • Co-administration includes administration of unit dosages of the metabolites disclosed herein before or after administration of unit dosages of one or more additional therapeutic agents, for example, administration of the metabolites disclosed herein within seconds, minutes, or hours of the administration of one or more additional therapeutic agents.
  • a unit dose of a metabolite disclosed herein is administered first, followed within seconds or minutes by administration of a unit dose of one or more additional therapeutic agents.
  • a unit dose of one or more additional therapeutic agents is administered first, followed by administration of a unit dose of a metabolite disclosed herein within seconds or minutes.
  • a unit dose of a metabolite disclosed herein is administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of one or more additional therapeutic agents.
  • a unit dose of one or more additional therapeutic agents is administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of a metabolite disclosed herein.
  • Pharmaceutical Formulations and Dosage Forms The pharmaceutical compositions disclosed herein can be prepared by methodologies well known in the pharmaceutical art. For example, in certain embodiments, a pharmaceutical composition intended to be administered by injection can prepared by combining a metabolite of the invention with sterile, distilled water so as to form a solution.
  • a surfactant is added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
  • the metabolites of the invention, or their pharmaceutically acceptable salts can be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • Example X-1 Investigation of Metabolism of Compound 1 (in the form of its tris salt) in a Clinical Study Study Design and Objectives The metabolism of Compound 1 (dosed in its free form) was investigated in humans. A primary objective of this study was to gain a preliminary assessment of the human metabolites of Compound 1 using plasma and urine sample after repeat dosing (multi-dose).
  • Plasma and urine samples from five human subjects following oral administration of 100 mg BID for 14 days in a clinical study were examined for metabolites.
  • MATERIALS AND METHODS Multiple Dose Metabolite Identification in Human Plasma And in Human Urine Human plasma and urine samples were acquired from five individual subjects in a multiple dose study. The five subjects were dosed every 12 hours (BID oral dose) for 14 days at 100 mg. Both plasma and urine were from Day 14 collected 0 to 12 hours postdose. Plasma from individual subjects were pooled in proportion to the time period represented by each sampling interval to yield a composite sample for profiling that is representative of C average (AUC 0-12h) according to the method of Hamilton et al. [Hamilton RA, Garnett WR, Kline BJ.
  • the residue was reconstituted in 0.2 mL of mobile phase (90% 1% formic acid/10% acetonitrile).
  • the reconstituted samples were transferred to micro-centrifuge tubes and centrifuged at 16,000 x g for 3 minutes.
  • the supernatants were transferred to limited volume insert and injected (50 ⁇ L) on HPLC-UV-MS for analysis.
  • Urine from individual subjects were pooled in proportion to the volume excreted for the time interval for each subject to yield a pool for profiling that is representative of 0-12 h. An equivalent aliquot from each subject’s Day 1, 0 h sample was pooled to act as a blank control sample.
  • HPLC/UV/MS Sample Analysis The multi-dose human plasma and urine samples were analyzed by positive ion HPLC-UV- MS using a Thermo Fisher Scientific LTQ-Obitrap mass spectrometer.
  • the HPLC system consisted of an Accela quaternary solvent delivery pump, an Accela autosampler, and an Accela PDA Plus photodiode array detector.
  • a Polaris C18 column (250 x 4.6 mm, 5 ⁇ m) was used with a flow rate of 0.8 mL/min.
  • the mobile phase was comprised of 0.1% formic acid (A) and acetonitrile (B).
  • the gradient system used was as follows: initially, 95% A held for 5 minutes followed by a linear gradient to 90% B at 35 minutes, held for 5 minutes, and then re-equilibration at 95/5 A/B for 15 minutes.
  • UV spectra were collected from 200-400 nm.
  • the mass spectrometer was operated in the positive ion mode with an ESI (Electron Spray Ion) source. Capillary temperature was set at 275°C and the source potential was 5000V. Other potentials were optimized to get optimal ionization and fragmentation of the parent. Sheath, auxiliary, and sweep gas flows were set to 20, 10, and 5 arbitrary units, respectively.
  • Table E1-2 summarizes the metabolites observed in the multi-dose human urine samples
  • parent Compound 1
  • M1 N-glucuronide of the parent
  • M2 thiadiazinane ring opened methyl propanoic acid
  • Urine was found to contain both M1 and M2 and a hydroxy glucuronide metabolite.
  • a total of 8 metabolites were observed in plasma (see table below).
  • O-debenzylation to Metabolite/Compound M2 was the major circulating metabolite.
  • Table E1-1 Metabolite Summary of Compound 1 in Human Day 14 Pooled Extracted Plasma Samples in Multi-dose Subjects (100 mg BID for 14 Days)
  • Table E1-2 Metabolite Summary of Compound 1 in Human Day 14 Pooled Extracted Urine Samples in Multi-dose Subjects (100 mg BID for 14 Days)
  • a CID mass spectrum generated for Compound 1 in the LTQ-Orbitrap is shown in Figure 3 with some of the diagnostic fragments assigned.
  • the major fragment ion at m/z 232 (radical cation) corresponds to the loss of the butyl-amine portion of the molecule.
  • the ion at m/z 239 resulted from a neutral loss of sulfur dioxide. Fragmentation through the thiadiazinane ring yields m/z 210 (cation) and 197 (radical cation).
  • the Collision-Induced Dissociation (CID) mass spectrum generated for M1 in the LTQ- Orbitrap is shown in Figure 4 with some of the diagnostic fragments assigned.
  • the N-glucuronide metabolite M1 was detected as a major metabolite in human plasma and urine.
  • An isolated Metabolite M1 sample was prepared and analyzed by NMR.
  • the 1 H spectrum of the isolated Compound 1-glucuronide (dissolved in dimethyl sulfoxide-d 6 ) is contained in Figure 5. The presence of the glucuronic acid is confirmed both in the TOCSY data ( Figure 6) and the HSQC data ( Figure 7).
  • Metabolite M2 has a protonated molecular ion of m/z 256.1081 (0.2327 ppm) which is consistent with the empirical formula of C 14 H 14 O 2 N 3 ( Figure 8).
  • the MS/MS spectrum yields fragment ions of 238, 196, and 182.
  • the fragments at m/z 238 and 182 corresponds to the neutral loss of water and propionic acid, respectively. It is proposed to arise via hydrolysis of the thiadiazinane ring followed by subsequent oxidations.
  • a sample of isolated metabolite M2 (dissolved in acetonitrile–d 3 ) was submitted for 1 H NMR analysis for definitive structure elucidation.
  • the 1 H spectrum of the isolated Metabolite M2 (thiadiazinane ring open methyl propanoic acid) is contained in Figure 9.
  • a resonance (broad singlet, Integration 1 H) at 5.60 ppm is assigned as the NH of the ring open acid. This assignment is further supported by the TOCSY data ( Figure 10Error! Reference source not found.).
  • Step 1 Synthesis of methyl (5-chloro-2-methoxypyridin-4-yl)acetate (C1).
  • a solution of lithium diisopropylamide in tetrahydrofuran (2 M; 1.9 L, 3.8 mol) was added in a drop-wise manner to a ⁇ 30 °C solution of 5-chloro-2-methoxy-4-methylpyridine (197 g, 1.25 mol) in tetrahydrofuran (1.4 L).
  • dimethyl carbonate (338 g, 3.75 mol) was added drop-wise; at the end of the addition, the reaction mixture was warmed to 25 °C and stirred for 1 hour.
  • reaction mixture was stirred at ⁇ 78 °C for 1 hour, whereupon a solution of iodomethane (172.6 g, 1.216 mol) in tetrahydrofuran (100 mL) was added drop-wise at ⁇ 78 °C, and stirring was continued at this temperature for 2 hours.
  • the reaction mixture was then poured into saturated aqueous ammonium chloride solution (500 mL) and extracted with ethyl acetate (2 x 100 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo to provide C2 as a brown oil.
  • aqueous layer was then adjusted to pH 4 by addition of 3 M hydrochloric acid and extracted with ethyl acetate (2 x 500 mL); the combined ethyl acetate layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide P1 as a white solid. Yield: 122 g, 0.566 mol, 77%.
  • the first-eluting enantiomer an oil which solidified on standing, was designated as P2, and the second-eluting enantiomer as P3.
  • the indicated absolute stereochemistry was assigned via X-ray crystal structure determination of 15, which was synthesized using this lot of P2 (see below, Example 15, Alternate Step 3).
  • Retention time 3.98 minutes (Analytical conditions.
  • Step 1 Synthesis of dibenzyl (5-fluoro-2-methoxypyridin-4-yl)propanedioate (C8). This reaction was carried out in three parallel batches. To a 25 °C solution of dibenzyl propanedioate (607 g, 2.13 mol) in tetrahydrofuran (1.5 L) was added pyridine-2-carboxylic acid (35.0 g, 284 mmol), followed by copper(I) iodide (27.1 g, 142 mmol), and then freshly ground cesium carbonate (1.39 kg, 4.27 mol).
  • reaction mixture After the reaction mixture had been heated to 70 °C, it was treated in a drop-wise manner with a solution of 5-fluoro-4-iodo-2-methoxypyridine (360 g, 1.42 mol) in tetrahydrofuran (800 mL), whereupon stirring was continued for 16 hours at 70 °C.
  • the three reaction mixtures were combined at this point, cooled to 25 °C, and filtered through diatomaceous earth.
  • the filter pad was rinsed with ethyl acetate (3 x 500 mL), and the combined filtrates were concentrated in vacuo, while keeping the internal temperature below 40 °C.
  • the combined filtrates were concentrated at 40 °C and the residue was partitioned between ethyl acetate (2 L) and water (500 mL).
  • the aqueous layer was extracted with ethyl acetate (2 x 1 L), and the combined organic layers were washed with saturated aqueous sodium chloride solution (1 L), dried over sodium sulfate, filtered, and concentrated under reduced pressure at 40 °C.
  • the resulting crude product was dissolved in petroleum ether (1.5 L) and stirred at 0 °C for 2 hours; a solid was collected via filtration.
  • the filtrate was concentrated in vacuo, and the residue was taken up in petroleum ether (500 mL), then cooled to 0 °C to provide additional solid, which was isolated via filtration.
  • the first-eluting enantiomer was designated as P7, and the second-eluting enantiomer as P8; both were isolated as solids.
  • P7 - Yield 260 g, 1.30 mol, 37%.
  • Retention time 3.17 minutes (Analytical conditions. Column: Chiral Technologies Chiralpak AD-H, 4.6 x 250 mm, 5 ⁇ m; Mobile phase A: carbon dioxide; Mobile phase B: 2-propanol; Gradient: 5% B for 1.00 minute, then 5% to 60% B over 8 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar).
  • P8 - Yield 400 g, 2.01 mol, 57%.
  • Retention time 3.36 minutes (Analytical conditions identical to those used for P7). The indicated absolute stereochemistries for P7 and P8 were assigned on the basis of comparison to the sample of P7 synthesized in Alternate Preparation (#1) of P7; the configuration of that material was established via X-ray crystallographic study of the derived compound 14 (see below). Retention time for P7 from Preparations P7 and P8: 2.86 minutes. Retention time for P7 from Alternate Preparation (#1) of P7: 2.86 minutes. Retention times for a racemic mixture of P7 and P8: 2.87 and 3.16 minutes.
  • a 1.0 M, pH 8.0 buffer solution was prepared in the following manner: a solution of 2-amino- 2-(hydroxymethyl)propane-1,3-diol (Tris; 121 g, 1.00 mol) in water (900 mL) was adjusted to pH 8.0 by addition of hydrochloric acid (37.5 weight%, approximately 40 mL), and then brought to a volume of 1 L by addition of water.
  • a hydrogenation reactor was charged with palladium hydroxide on carbon (10%; 5.00 g).
  • Step 1 Synthesis of dibenzyl (5-fluoro-2-methoxypyridin-4-yl)propanedioate (C8).
  • reaction mixture After the reaction mixture had been heated at 60 °C to 70 °C for approximately 3 to 6 hours, it was allowed to cool to 15 °C to 30 °C and filtered through diatomaceous earth (250 g). The filter cake was washed with tetrahydrofuran (500 mL, 2 volumes) and the combined filtrates, containing C8, were used directly in the following step.
  • the resulting mixture was diluted with propan-2-yl acetate (1.25 L, 3.1 volumes), washed sequentially with water (750 mL, 1.8 volumes), aqueous ammonium chloride solution (20%; 750 mL), and aqueous sodium chloride solution (20%; 750 mL), and concentrated in vacuo.
  • the remaining solvent was exchanged with heptane, and precipitation was allowed to proceed from heptane (2 to 3 volumes) at 15 °C to 25 °C.
  • the resulting solid was collected via filtration and triturated with a mixture of heptane (450 mL) and propan-2-yl acetate (50 mL) to afford C9 as a solid.
  • a buffer solution [pH 8.0; 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris; 121 g, 1.00 mol), and concentrated hydrochloric acid (46 mL, 0.23 volumes) in water (1 L, 5 volumes)]
  • palladium hydroxide on carbon 10%, 20 g
  • a solution of sodium hydroxide (38.8 g, 0.970 mol) in water (1 L, 5 volumes) was added, whereupon the mixture was stirred for approximately 10 to 20 minutes.
  • Step 4 Synthesis of (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)propanoic acid (P7).
  • reaction mixture was warmed to ⁇ 30 °C and allowed to stir at that temperature for 3 hours, whereupon it was diluted with aqueous ammonium chloride solution and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo while keeping the temperature below 45 °C. Purification via silica gel chromatography (Eluent: 1:3 ethyl acetate / petroleum ether) provided C11 as a colorless oil. Yield: 376 mg, 1.37 mmol, 86%.
  • Step 1 Synthesis of dimethyl (2-methoxypyridin-4-yl)propanedioate (C15).
  • 2-methoxy-4-methylpyridine 5.00 g, 40.6 mmol
  • lithium diisopropylamide 2 M solution in tetrahydrofuran; 81.2 mL, 162 mmol
  • dimethyl carbonate (14.6 g, 162 mmol) was added and stirring was continued at ⁇ 10 °C for 1.5 hours.
  • Step 4 Synthesis of methyl 2-(2-methoxypyridin-4-yl)propanoate (C18).
  • the reaction mixture was then concentrated in vacuo, washed with aqueous sodium bicarbonate solution, and extracted with ethyl acetate (2 x 20 mL).
  • Step 1 Synthesis of 1-(difluoromethoxy)-3-methoxy-5-methylbenzene (C20).
  • Aqueous potassium hydroxide solution (20% solution; 60.9 g, 217 mmol) and [bromo(difluoro)methyl](trimethyl)silane (11.3 mL, 72.7 mmol) were sequentially added to a 0 °C solution of 3-methoxy-5-methylphenol (5.00 g, 36.2 mmol) in dichloromethane (50 mL). After the reaction mixture had been stirred at 0 °C for 4.5 hours, it was diluted with water (50 mL) and extracted with dichloromethane (3 x 100 mL).
  • reaction mixture was poured into aqueous sodium bicarbonate solution (100 mL), and the resulting mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with aqueous sodium dithionite solution (200 mL), filtered, concentrated in vacuo, and purified by silica gel chromatography (Gradient: 0% to 15% methanol in dichloromethane), providing C27 as a white solid. Yield: 685 mg, 2.40 mmol, 32%.
  • reaction mixture After the reaction mixture had been stirred at ⁇ 50 °C for 1 hour, it was cooled to ⁇ 78 °C, and a solution of di-tert-butyl dicarbonate (8.51 mL, 37.0 mmol) in tetrahydrofuran (30 mL) was added. The reaction mixture was then warmed to ⁇ 30 °C, stirred for 2 hours, and diluted with water (100 mL). The resulting mixture was extracted with ethyl acetate (3 x 50 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo.
  • the reaction mixture was stirred at 0 °C for 8 hours, whereupon it was diluted with diethyl ether (25 mL) and washed sequentially with 10% aqueous citric acid solution (5 mL), saturated aqueous sodium bicarbonate solution (15 mL), and saturated aqueous sodium chloride solution (25 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide the crude diazoketone. This material was suspended in methanol (10 mL) in an ultrasonic bath; a solution of silver benzoate (512 mg, 2.24 mmol) in triethylamine (1.86 mL, 13.3 mmol) was gradually added at room temperature while the reaction mixture was sonicated.
  • Step 1 Synthesis of 2-chloro-3-iodo-6-methylpyridine (C40).
  • 2-chloro-6-methylpyridin-3-amine 400 g, 2.80 mol
  • hydrochloric acid 5.0 M; 3.3 L, 16.5 mol
  • a solution of sodium nitrite 290 g, 4.20 mol
  • water 800 mL
  • reaction mixture was stirred under ice-cooling for 30 minutes, then cooled to ⁇ 5 °C, whereupon tert-butyl methyl ether (3.0 L) was added, followed by drop-wise addition of a solution of potassium iodide (929 g, 5.60 mol) in water (800 mL), while the internal reaction temperature was maintained below 10 °C. The reaction mixture was then allowed to slowly warm to 25 °C and stirring was continued at 25 °C for 16 hours.
  • reaction mixture was stirred at ⁇ 78 °C for 1 hour, whereupon a solution of 1- benzylpyrrolidin-3-one (1.50 kg, 8.56 mol) in tetrahydrofuran (1.5 L) was added drop-wise. After completion of the addition, the reaction mixture was warmed to 20 °C, stirred at 20 °C for 16 hours, and subsequently poured into aqueous ammonium chloride solution. The resulting mixture was extracted with ethyl acetate (2 x 2.0 L), and the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo to provide C41 as a yellow oil.
  • Step 7 Synthesis of 1-benzyl-3-[2-(2-chloro-6-methylpyridin-3-yl)ethyl]-N-[(4- methoxyphenyl)methyl]pyrrolidin-3-amine (C46).
  • a mixture of C45 (40.0 g, 89.7 mmol) and platinum(IV) oxide (4.09 g, 18.0 mmol) in methanol (400 mL) was hydrogenated (60 psi) at 25 °C for 3 hours.
  • the reaction mixture was then filtered, and the filtrate was concentrated in vacuo to provide C46 as a black oil. Yield: 40.5 g, assumed quantitative.
  • Step 9 Synthesis of 1'-benzyl-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine] (C48).
  • C47 190 g, 0.459 mol
  • dichloromethane 1.5 L
  • trifluoroacetic acid 523 g, 4.59 mol
  • the reaction mixture was stirred at 25 °C for 3 hours. It was then concentrated in vacuo; the residue was diluted with ethyl acetate (1.5 L) and washed with saturated aqueous sodium carbonate solution (1.0 L), and this aqueous layer was extracted with ethyl acetate (2 x 300 mL).
  • the first-eluting enantiomer was designated as P19 and the second-eluting enantiomer as P20. Both were isolated as solids.
  • Retention time 3.96 minutes (Analytical conditions identical to those used for P19). The indicated absolute stereochemistries were assigned based on the conversion of this batch of P19 to P23 in Alternate Preparation (#1) of P23 below. The absolute configuration of P23 was established via its use in the synthesis of 14, which was analyzed via single-crystal X-ray crystallography (see below).
  • 1,3-Dibromo-5,5-dimethylimidazolidine-2,4-dione (2.47 g, 8.64 mmol) was added in portions over 20 minutes to a 0 °C solution of P17 (5.25 g, 17.3 mmol) in dichloromethane (69 mL).
  • LCMS analysis indicated conversion to P22: LCMS m/z 384.3 (bromine isotope pattern observed) [M+H] + .
  • the reaction mixture was treated with saturated aqueous sodium sulfite solution (100 mL), and the mixture was extracted with dichloromethane.
  • 1,3-Dibromo-5,5-dimethylimidazolidine-2,4-dione (5.65 g, 19.8 mmol) was added in portions to a 0 °C solution of P17 (10.0 g, 32.9 mmol) in dichloromethane (150 mL), and the reaction mixture was stirred at 0 °C to 5 °C for 1 hour, at which time LCMS analysis indicated that bromination had occurred: LCMS m/z 382.3 [M+H] + .
  • Saturated aqueous sodium sulfite solution (20 mL) was added, followed by water (50 mL); the resulting aqueous layer was extracted with dichloromethane (2 x 50 mL).
  • Retention time of P23 from Preparations P23 and P24 4.01 minutes Retention time of P24 from Preparations P23 and P24: 4.32 minutes
  • Mobile phase A carbon dioxide
  • Mobile phase B methanol containing 0.2% (7 M ammonia in methanol)
  • Gradient 5% B for 1 minute, then 5% to 60% B over 8 minutes
  • Flow rate 3.0 mL/minute
  • Step 1 Synthesis of (2-chloro-6-methylpyridin-3-yl)methanol (C49).
  • Sodium bis(2-methoxyethoxy)aluminum hydride solution (70%; 1.05 kg, 2.5 eq) was added to a ⁇ 5 °C to 5 °C solution of 2-chloro-6-methylpyridine-3-carboxylic acid (250 g, 1.46 mol) in toluene (2.5 L).
  • the reaction mixture had been stirred at ⁇ 5 °C to 5 °C for 19 hours, it was treated with a solution of sodium hydroxide (145.7 g, 3.642 mol, 2.50 eq) in water (1.25 L), while the internal temperature was maintained below 0 °C to 10 °C.
  • the aqueous layer was extracted with 2- methyltetrahydrofuran (7.0 L), and the combined organic layers were washed with a solution of acetic acid (288 g, 4.80 mol) in water (4.2 L) and then with an aqueous solution of sodium sulfate (10%; 2 x 3.50 kg).
  • the organic layers were concentrated in vacuo to 2 to 3 volumes, while keeping the temperature below 50 °C.
  • Ethanol (4.90 L, 7 volumes) was added, and the solution was again concentrated in vacuo to 2 to 3 volumes, while keeping the temperature below 50 °C.
  • reaction vessel After addition of wet palladium on carbon (10%; 12 g), the reaction vessel was evacuated and charged with argon three times, and then evacuated and charged with hydrogen three times. Hydrogenation was then carried out at 40 to 50 psi and 40 °C to 50 °C for 24 hours.
  • the resulting mixture was filtered through diatomaceous earth (50 g); the filter cake was washed with ethanol (240 mL, 2 volumes), and the combined filtrates were concentrated in vacuo to 2.5 to 3.5 volumes while keeping the temperature at or below 45 °C.
  • Di-tert-butyl dicarbonate (19.7 g, 90.3 mmol) was added in a drop-wise manner to a 20 °C to 30 °C mixture of C54 (88.12 g, 0.2412 mol) and triethylamine (73.33 g, 0.7247 mol) in dichloromethane (881 mL, 10 volumes). Additional di-tert-butyl dicarbonate (19.2 g, 88.0 mmol and 19.3 g, 88.4 mmol) was added drop-wise after periodic HPLC analysis.
  • the initial reaction pH was 7.08; after stirring at 20 °C to 30 °C for 1 hour, the pH decreased to 6.58.
  • a pH autotitrator was used to maintain the pH at 7.5 by addition of aqueous sodium hydroxide solution (2 M; 121 mL, 0.242 mol) over 24 hours.
  • Hydrochloric acid (6 M; 52 mL, 0.312 mol) was added, bringing the pH to 2.39; ethyl acetate (435 mL, 6.0 volumes) was then added, and the mixture was stirred for 30 minutes at 20 °C to 30 °C.
  • Filtration through diatomaceous earth (18.0 g) provided a filter cake, which was rinsed with ethyl acetate (2 x 75 mL).
  • Toluene (170 mL, 1.2 volumes) was added to a solution of C56 in toluene (3.8 volumes, containing 28.9% by weight of C56, 146.4 g, 0.5096 mol); the solution was heated to 80 °C to 90 °C. To this was slowly added, over 2 hours, a mixture of triethylamine (77.4 g, 0.765 mol) and diphenyl phosphorazidate (140.3 g, 0.5098 mol) in toluene (732 mL, 5 volumes).
  • reaction mixture was stirred at 80 °C to 90 °C for 3 hours, whereupon it was cooled to 50 °C and treated drop-wise, over 2 hours, with a solution of benzyl alcohol (55.12 g, 0.5097 mol) in toluene (290 mL, 2 volumes).
  • a solution of benzyl alcohol 55.12 g, 0.5097 mol
  • toluene 290 mL, 2 volumes
  • the reaction mixture had been stirred at 100 °C for 16 hours, it was cooled to 15 °C to 25 °C and partitioned between toluene (1.46 L, 10 volumes) and water (2.20 L, 15 volumes) by stirring for 30 minutes.
  • the organic layer was washed sequentially with aqueous potassium carbonate solution (10%; 3 x 1.46 L) and with water (2 x 750 mL).
  • the mixture was then warmed to 20 °C to 30 °C, diluted with ethyl acetate (1.60 L, 10 volumes) and stirred for 10 minutes, whereupon the organic layer was concentrated in vacuo to 2 to 3 volumes while maintaining the temperature at or below 50 °C.
  • the resulting mixture was diluted with acetonitrile (880 mL) and concentrated in vacuo to 2 to 3 volumes while maintaining the temperature at or below 50 °C; this dilution / concentration procedure was carried out a total of three times.
  • the mixture was then heated to 40 °C to 50 °C and stirred for 1 hour, whereupon it was cooled over 4 hours to 15 °C to 25 °C.
  • Step 13 Synthesis of tert-butyl (3S)-3- ⁇ [(benzyloxy)carbonyl]amino ⁇ -3-[2-(2-chloro-6-methylpyridin- 3-yl)ethyl]pyrrolidine-1-carboxylate (C62).
  • a reaction vessel containing a mixture of C60 and C61 (283.0 g, 0.5996 mol) and rhodium on alumina (5%; 14.15 g) in methanol (1.98 L) was evacuated and charged with argon three times, then evacuated and charged with hydrogen three times. Hydrogenation was then carried out for 40 hours at 30 to 40 psi and 20 °C to 25 °C.
  • the resulting mixture was purged three times with nitrogen, whereupon tris(dibenzylideneacetone)dipalladium(0) (34.78 g, 37.98 mmol) was added, and three additional rounds of purging with nitrogen were carried out.
  • the reaction mixture was stirred for 24 hours at 75 °C to 85 °C.
  • Potassium phosphate (16.2 g, 0.117 mol) was added, and stirring was continued at 75 °C to 85 °C for an additional 16 hours.
  • potassium tert-butoxide (76.7 g, 0.684 mol) was added, and the reaction mixture was stirred for 2 hours at 75 °C to 85 °C.
  • the combined citric acid layers were washed with ethyl acetate (2 x 1.07 L), then adjusted to pH 7 by addition of aqueous sodium hydroxide solution (30%; 596 g) at 20 °C to 30 °C. Extraction of the aqueous layer with ethyl acetate (3 x 1.07 L), followed by combination of these three organic layers, provided P19 as a solution in ethyl acetate (3.218 kg, 2.7% P19 by weight); The bulk of this material was progressed to the following step. Estimated yield: 86.9 g, 0.286 mol, 75%. HPLC purity: 98.9%.
  • reaction mixture After the reaction mixture had been stirred for 1 hour at 0 °C to 5 °C, it was quenched by addition of aqueous sodium sulfite solution (10%; 203 g) and water (456 mL), and the resulting mixture was stirred at 10 °C to 20 °C for 20 minutes.
  • the aqueous layer was extracted twice with ethyl acetate (415 mL, 5.1 volumes) by stirring at 10 °C to 20 °C for 20 minutes; the combined organic layers were then stirred for 20 minutes with aqueous sodium sulfate solution (10%; 456 g).
  • reaction vial was then sealed and heated at 100 °C in an aluminum block for 2 hours, whereupon it was allowed to cool to room temperature.5-Bromo-2-methyl-2H-tetrazole (134 mg, 0.822 mmol), dichlorobis(triphenylphosphine)palladium(II) (27.5 mg, 39.2 ⁇ mol), and a degassed aqueous solution of sodium carbonate (2.0 M; 0.981 mL, 1.96 mmol) were added, and the reaction mixture was again degassed with bubbling nitrogen for 5 minutes. It was then stirred at 90 °C for 18 hours, cooled to room temperature, diluted with ethyl acetate, and filtered through diatomaceous earth.
  • reaction mixture was then heated in a 105 °C aluminum block for 2 hours, whereupon it was allowed to cool to room temperature and then treated with 2-bromopyrimidine (840 mg, 5.28 mmol), additional [1,1’- bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex (216 mg, 0.264 mmol), and aqueous sodium carbonate solution (2.0 M; 7.93 mL, 15.9 mmol). After this reaction mixture had been sparged with nitrogen, it was heated to 100 °C for 18 hours, at which time LCMS analysis indicated conversion to C69: LCMS m/z 382.4 [M+H] + .
  • the reaction mixture was cooled, partitioned between aqueous ammonium chloride solution and ethyl acetate, and then the entire mixture was filtered through a pad of diatomaceous earth.
  • the filter pad was rinsed with both water and ethyl acetate, and the aqueous layer of the combined filtrate was extracted with ethyl acetate (2 x 30 mL). After all the organic layers had been combined, they were washed sequentially with water (100 mL) and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo.
  • a solution of hydrogen chloride was prepared by slow addition of acetyl chloride (1.50 mL, 21.1 mmol) to 2-propanol (4 mL).
  • C69 from the previous step; 2 g; ⁇ 5.28 mmol
  • the hydrogen chloride solution was slowly added to it, and the reaction mixture was heated at 50 °C for 2 hours. It was then allowed to cool slowly to room temperature while being stirred; stirring was continued at room temperature for 18 hours.
  • Step 2 Synthesis of tert-butyl 3-[(3-chloro-6-methylpyridin-2-yl)amino]-3-(prop-2-en-1- yl)pyrrolidine-1-carboxylate (C71).
  • Step 3 Synthesis of tert-butyl 4,7-dimethyl-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'- carboxylate (P29).
  • reaction mixture was diluted with dichloromethane (20 mL), washed sequentially with saturated aqueous sodium sulfite solution, saturated aqueous sodium bicarbonate solution, and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo.
  • Silica gel chromatography (Gradient: 0% to 50% ethyl acetate in heptane) afforded C73 as an oil. Yield: 980 mg, 2.12 mmol, 68%.
  • a reaction vial was charged with 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi-1,3,2-dioxaborolane (148 mg, 0.583 mmol), C73 (180 mg, 0.389 mmol), [1,1’- bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex (31.8 mg, 38.9 ⁇ mol), and oven-dried potassium acetate (153 mg, 1.56 mmol) in 1,4-dioxane (5 mL). Nitrogen was bubbled through the reaction mixture for 5 minutes, whereupon the vial was sealed and heated at 100 °C in an aluminum block for 2 hours.
  • Step 4 Synthesis of 6-[5-(difluoromethyl)-1-methyl-1H-1,2,4-triazol-3-yl]-7-methyl-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidine] (P31).
  • Palladium on carbon (10%, wet with water; 20 mg) was added to a solution of C75 (105 mg, 0.204 mmol) in methanol (5 mL) containing a drop of formic acid, and the resulting mixture was hydrogenated overnight at room temperature and 70 psi. After filtration, the filtrate was concentrated in vacuo to provide P31 as a light-yellow solid.
  • Step 1 Synthesis of 7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidine], dihydrochloride salt (C77).
  • a solution of hydrogen chloride in 1,4-dioxane (4.0 M; 0.587 mL, 2.35 mmol) was added to a solution of P25 (285 mg, 0.587 mmol) in a mixture of dichloromethane (1 mL) and 1,1,1,3,3,3- hexafluoropropan-2-ol (1 mL).
  • Retention time 2.32 minutes [Analytical conditions. Column: Chiral Technologies Chiralpak IB, 4.6 x 100 mm, 5 ⁇ m; Mobile phase 3:2 carbon dioxide / (0.2% ammonium hydroxide in methanol); Flow rate: 1.5 mL/minute; Back pressure: 120 bar]. 2 – Yield: 7.9 mg, 16 ⁇ mol, 6%. LCMS m/z 483.2 [M+H] + . Retention time: 2.53 minutes (Analytical conditions identical to those used for 1).
  • Trifluoroacetic acid (2 mL) was added to a solution of C68 (280 mg, 0.726 mmol) in dichloromethane (10 mL), and the reaction mixture was stirred at room temperature for 2 hours. It was then concentrated in vacuo and evaporated twice with ethyl acetate to afford the deprotected material as a dark brown oil (200 mg), LCMS m/z 286.3 [M+H] + .
  • Retention time 2.47 minutes [Analytical conditions. Column: Chiral Technologies Chiralpak IA, 4.6 x 100 mm, 5 ⁇ m; Mobile phase: 65:35 carbon dioxide / (methanol containing 0.5% ammonium hydroxide); Flow rate: 1.5 mL/minute; Back pressure: 120 bar]. 4 – Yield: 3.6 mg, 7.8 ⁇ mol, 6%. LCMS m/z 486.3 [M+Na + ]. Retention time: 2.92 minutes (Analytical conditions identical to those used for 3).
  • the first-eluting diastereomer was designated as 3 ⁇ 2-(6- methoxy-2-methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1 ⁇
  • the second-eluting diastereomer as 4 ⁇ 2-(6-methoxy-2-methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol- 5-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2 ⁇ .
  • Retention time 4.92 minutes (Analytical conditions. Column: Chiral Technologies Chiralcel OJ, 4.6 x 250 mm, 5 ⁇ m; Mobile phase A: carbon dioxide; Mobile phase B: 2-propanol containing 0.2% propan-2-amine; Gradient: 5% B for 1.00 minute, then 5% to 60% B over 8.00 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar). 4 – Yield: 30 mg, 65 ⁇ mol, 22%. LCMS m/z 464.2 [M+H] + .
  • Examples 5 and 6 2-[6-(Difluoromethoxy)pyridin-3-yl]-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1 (5) and 2-[6-(Difluoromethoxy)pyridin-3- yl]-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[
  • the first-eluting diastereomer was designated as 5 ⁇ 2-[6-(difluoromethoxy)pyridin-3-yl]- 1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'- yl]propan-1-one, DIAST-1 ⁇ and the second-eluting diastereomer was designated as 6 ⁇ 2-[6- (difluoromethoxy)pyridin-3-yl]-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2 ⁇ ; both were isolated as white solids.
  • Retention time 10.66 minutes (Analytical conditions identical to those used for 5).
  • 1,1’-Carbonyldiimidazole (240 mg, 1.48 mmol) was added portion-wise to a solution of 2-[4- (trifluoromethyl)phenyl]propanoic acid (323 mg, 1.48 mmol) in acetonitrile (5 mL).
  • acetonitrile 5 mL
  • P28 500 mg, 1.41 mmol
  • N,N-diisopropylethylamine 0.504 mL, 2.89 mmol
  • the first-eluting diastereomer was designated as 7 ⁇ 1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]-2-[4-(trifluoromethyl)phenyl]propan-1-one, DIAST-1 ⁇ and the second-eluting diastereomer was designated as 8 ⁇ 1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]-2-[4-(trifluoromethyl)phenyl]propan-1-one, DIAST- 2 ⁇ ; both were isolated as solids.
  • Retention time 4.28 minutes [Column: Chiral Technologies Chiralcel OJ, 4.6 x 250 mm, 5 ⁇ m; Mobile phase A: carbon dioxide; Mobile phase B: methanol containing 0.2% (7 M ammonia in methanol); Gradient: 5% B for 1.0 minute, then 5% to 60% B over 8.0 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar]. 8 – Yield: 260 mg, 0.540 mmol, 38%. LCMS m/z 482.4 [M+H] + .
  • the first-eluting diastereomer was designated as 9 ⁇ 1-(4,7-dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4- fluorophenyl)ethan-1-one, DIAST-1 ⁇ , the second-eluting as 10 ⁇ 1-(4,7-dimethyl-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4-fluorophenyl)ethan-1-one, DIAST-2 ⁇ , the third- eluting as 11 ⁇ 1-(4,7-dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4- fluorophenyl)ethan-1-
  • Retention time 2.77 minutes (Analytical conditions. Column: Chiral Technologies Chiralcel OJ-H, 4.6 x 100 mm, 5 ⁇ m; Mobile phase: 85:15 carbon dioxide / (methanol containing 0.2% ammonium hydroxide); Flow rate: 1.5 mL/minute; Back pressure: 120 bar). 10 – Yield: 1.3 mg, 3.7 ⁇ mol, 6%. LCMS m/z 354.3 [M+H] + .
  • Recrystallization from a 3:2 mixture of ethyl acetate and heptane provided material with a diastereomeric excess of 99.1%; further recrystallization from acetonitrile afforded the single crystal that was used for X-ray structural determination.
  • Single-crystal X-ray structural determination of 14 Single Crystal X-Ray Analysis Data collection was performed on a Bruker D8 Quest diffractometer at room temperature. Data collection consisted of omega and phi scans. The structure was solved by intrinsic phasing using SHELX software suite in the triclinic class group P1. The structure was subsequently refined by the full-matrix least squares method. All non- hydrogen atoms were found and refined using anisotropic displacement parameters.
  • the hydrogen atoms located on nitrogen were found from the Fourier difference map and refined with distances restrained. The remaining hydrogen atoms were placed in calculated positions and were allowed to ride on their carrier atoms. The final refinement included isotropic displacement parameters for all hydrogen atoms.
  • Analysis of the absolute structure using likelihood methods (Hooft, 2008) was performed using PLATON (Spek). The results indicate that the absolute structure has been correctly assigned. The method calculates that the probability that the structure is correctly assigned is 100%.
  • the Hooft parameter is reported as 0.05 with an esd (estimated standard deviation) of (10) and the Parson’s parameter is reported as 0.04 with an esd of (10).
  • the final R-index was 4.5%.
  • a final difference Fourier revealed no missing or misplaced electron density.
  • the present invention provides a crystalline form of (2R)-2-(5-fluoro- 2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidin]-1'-yl]propan-1-one.
  • the crystalline form of (2R)-2-(5- Fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one is the one described (or as prepared) in Example 14.
  • Step 1 Synthesis of tert-butyl (2S)-6-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-7-methyl-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (C79).
  • Di(1-adamantyl)-n-butylphosphine (cataCXium® A; 2.21 g, 6.16 mmol), followed by palladium(II) acetate (0.461 mg, 2.05 mmol), was added to 2-methyltetrahydrofuran (170 mL); the catalyst mixture was sparged with argon for 10 to 20 minutes between each manipulation.
  • the catalyst mixture was then added via cannula, over less than 2 minutes, and the reaction mixture was heated to reflux at a rate of 1 °C / minute. After 4 hours at reflux, it was cooled to 10 °C, held at that temperature overnight, and rapidly treated drop-wise, over 15 minutes, with aqueous sodium hydroxide solution (1.0 M; 410 mL, 410 mmol). The internal temperature was maintained below 17 °C during the addition. The resulting mixture was warmed to 20 °C, diluted with tert-butyl methyl ether (180 mL) and mixed well for 5 minutes, whereupon the aqueous layer was confirmed to be at pH 10.
  • aqueous sodium hydroxide solution 1.0 M; 480 mL, 480 mmol
  • aqueous sodium hydroxide solution 1.0 M; 480 mL, 480 mmol
  • the combined sodium hydroxide extracts were mixed with toluene (240 mL), and treated portion-wise with hydrochloric acid (12.2 M; 62.3 mL, 760 mmol), at a rate that maintained the temperature below 30 °C.
  • the pH of the resulting mixture was 14; additional hydrochloric acid (12.2 M; 34 mL, 415 mmol) was added to adjust the pH to 10.
  • reaction mixture After the reaction mixture had been heated at 50 °C for 3.5 hours, it was cooled to 20 °C, allowed to stir overnight, and filtered. The filter cake was rinsed with toluene (150 mL), and the organic layer of the combined filtrates was washed with water by stirring for 5 minutes and then allowing the mixture to stand for 30 minutes; solids in the mixture were kept with the organic layer, which was subjected to short-path distillation at 100 mbar and 60 °C. The mixture was distilled until approximately 275 mL remained, whereupon it was cooled to 20 °C at a rate of 1 °C/minute.
  • the mixture was treated drop-wise with additional 6 M aqueous sodium hydroxide solution (approximately 20 drops) to a pH of 9.6 to 9.7, at which point haziness persisted. Stirring was continued for 45 minutes, whereupon additional 6 M aqueous sodium hydroxide solution (to a total of approximately 80 mL, 480 mmol) was added, and stirring was continued at 20 °C for 30 minutes. The mixture was then heated to 50 °C at a rate of 1 °C/minute, stirred for 1.5 hours, and cooled to 20 °C at a rate of 1 °C/minute.
  • aqueous sodium bicarbonate solution (1.14 M; 250 mL, 285 mmol) was added ⁇ Caution: gas evolution ⁇ and stirring was continued for 10 minutes at 20 °C.
  • the resulting mixture was heated to 40 °C, stirred for 30 minutes, and again treated with aqueous sodium bicarbonate solution (1.14 M; 125 mL, 142 mmol). After this mixture had been stirred for 80 minutes, water (75 mL) was added and stirring was continued for 10 minutes.
  • the organic layer was subjected to distillation at 60 °C and 500 mbar, until the mixture had been reduced to 5 volumes.
  • 2- Methyltetrahydrofuran 125 mL was added, the temperature was adjusted to 45 °C to 50 °C, and the mixture was filtered through diatomaceous earth. Additional 2-methyltetrahydrofuran (50 mL) was used to rinse the filter pad, and the combined filtrates were distilled at 60 °C and 500 mbar to approximately 3 volumes. The heat was increased to 80 °C until solids at the bottom of the reactor were released, then decreased to 50 °C.
  • the resulting material was treated at 50 °C, over 15 minutes, with heptane (250 mL), and allowed to stir at 50 °C for 90 minutes. It was then cooled to 20 °C at a rate of 1 °C/minute and allowed to stir for 3 days, whereupon it was diluted to a volume of 600 mL by addition of 10 mol% 2-methyltetrahydrofuran in heptane.
  • reaction vial After the reaction vial had been sealed, it was heated to 100 °C in an aluminum block for 2 hours, then allowed to cool to room temperature.2-Bromopyrimidine (109 mg, 0.686 mmol), dichlorobis(triphenylphosphine)palladium(II) (22.9 mg, 32.6 ⁇ mol), and a degassed solution of aqueous sodium carbonate (2.0 M; 0.817 mL, 1.63 mmol) were then added to the reaction mixture, and it was heated at 90 °C for 18 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and filtered through diatomaceous earth.
  • reaction mixture was then partitioned between ethyl acetate and aqueous sodium bicarbonate solution; the organic layer was washed sequentially with water and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo.
  • the hydrogen atoms located on nitrogen were found from the Fourier difference map and refined with distances restrained. The remaining hydrogen atoms were placed in calculated positions and were allowed to ride on their carrier atoms. The final refinement included isotropic displacement parameters for all hydrogen atoms.
  • Analysis of the absolute structure using likelihood methods (Hooft, 2008) was performed using PLATON (Spek). The results indicate that the absolute structure has been correctly assigned. The method calculates that the probability that the structure is correctly assigned is 100.0%.
  • the Hooft parameter is reported as 0.04 with an esd (estimated standard deviation) of (3) and the Parson’s parameter is reported as 0.05 with an esd of (3).
  • the final R-index was 6.9%.
  • a final difference Fourier revealed no missing or misplaced electron density.
  • the present invention provides a crystalline form of (2R)-2-(5-chloro- 2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidin]-1'-yl]propan-1-one.
  • the crystalline form of (2R)-2-(5- chloro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one is the one described (or as prepared) in Example 15.
  • the first-eluting diastereomer was designated as 16 ⁇ (2R)-2- (5-fluoro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1 ⁇
  • the second-eluting diastereomer as 17 ⁇ (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2 ⁇ .
  • Retention time 3.71 minutes [Analytical conditions. Column: Phenomenex Lux Cellulose-1, 4.6 x 100 mm, 5 ⁇ m; Mobile phase: 3:1 carbon dioxide / (methanol containing 0.2% ammonium hydroxide); Flow rate: 1.5 mL/minute; Back pressure: 200 bar]. 17 – Yield: 6.2 mg, 13.3 ⁇ mol, 20%. LCMS m/z 466.6 [M+H] + . Retention time: 4.64 minutes (Analytical conditions identical to those used for 16).
  • Step 1 Synthesis of (4,6- 2 H 2 )pyrimidin-2-amine (C82).
  • methanol-d 4 10 mL
  • palladium on carbon 100 mg
  • triethylamine 1.3 mL, 9.3 mmol
  • reaction mixture had been poured into aqueous sodium bicarbonate solution (10 mL), it was extracted with ethyl acetate (2 x 20 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo.
  • Example AA In Vitro Binding Affinity Assay Using hMC4R The binding affinity of test compounds at the ⁇ -melanocyte-stimulating hormone receptor (hMC4R) was assessed using a radioligand competition binding assay.
  • hMC4R membranes were grown in Dulbecco's Modified Essential Medium and Ham's F-12 Medium (DMEM/F12), 10% heat inactivated fetal bovine serum (FBS), 0.4 mg/mL Geneticin and 2 mM L-glutamine. Cell membranes were bulked and frozen until the assay was performed.
  • DMSO dimethyl sulfoxide
  • Control wells containing 1 ⁇ L of 2 mM (2 ⁇ M final) alpha- melanocyte stimulating hormone ( ⁇ -MSH-Tocris # 2584) was added to the non-specific binding wells and 1 ⁇ L 100% DMSO for the total binding control wells. This was followed by the addition of 80 ⁇ L of assay buffer [25 mM HEPES, 5 mM MgCl 2 , 2.5 mM CaCl 2 , 150 mM NaCl, Complete EDTA-free Protease Inhibitor Tablet (Thermo Scientific #11873580001) and 0.25% BSA].
  • assay buffer [25 mM HEPES, 5 mM MgCl 2 , 2.5 mM CaCl 2 , 150 mM NaCl, Complete EDTA-free Protease Inhibitor Tablet (Thermo Scientific #11873580001) and 0.25% BSA].
  • the competition binding reaction was initiated by the addition of 10 ⁇ L MC4R membrane solution to the assay-ready plates containing test compound and [ 125 I]-(Nle4, D-Phe7)- ⁇ -MSH. The plates were incubated for 2 hours at room temperature. Assay samples were then rapidly filtered through Unifilter-96 GF/B PEI coated filter plates using a filter plate harvester (PerkinElmer) and rinsed with ice-cold wash buffer [25 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM MgCl 2 , 2.5 mM CaCl 2 , and 500 mM NaCl]. Filter plates were dried overnight at room temperature.
  • K i IC 50 / (1+ ([L]/ K d )), where [L] is the concentration of the radioligand used in the experiment and K d is the affinity of the radioligand (determined in separate saturation experiments).
  • K i IC 50 / (1+ ([L]/ K d )
  • the functional in vitro antagonist potency for test compounds was determined by monitoring intracellular cyclic adenosine monophosphate (cAMP) levels in Chinese hamster ovary (CHO-) cells stably expressing the human Melanocortin-4 receptor (MC4R). Following agonist activation, human MC4R associates with the G-protein complex causing the G ⁇ subunit to exchange bound GDP for GTP, followed by dissociation of the G ⁇ -GTP complex. The activated G ⁇ subunit can couple to downstream effectors to regulate the levels of second messengers or cAMP within the cell. Thereby, determination of intracellular cAMP levels allows for pharmacological characterization.
  • cAMP cyclic adenosine monophosphate
  • Intracellular cAMP levels are quantitated using a homogenous assay utilizing the Homogeneous Time-Resolved Fluorescence (HTRF) technology from CisBio.
  • the method is a competitive immunoassay between native cAMP produced by cells and the cAMP labelled with the acceptor dye, d2. The two entities then compete for binding to a monoclonal anti-cAMP antibody labeled with cryptate. The specific signal is inversely proportional to the concentration of cAMP in the cells.
  • Test compounds were solubilized to 30 mM in 100% dimethyl sulfoxide (DMSO) and stored.
  • DMSO dimethyl sulfoxide
  • CHO- cells stably expressing the Gs-coupled human MC4R receptor were plated in 384-well assay plates (Corning, Cat No.3570) in 50 ⁇ L/well of Ham’s F-12 containing 10% heat inactivated FBS, 1x penicillin/streptomycin, 1 mM Glutamax (Invitrogen) at a density of 2,500 cells per well and incubated at 37 o C (95% O 2 : 5% CO 2 ) overnight, with micro- clime lids (Labcyte, Cat No. LLS-0310).
  • Intracellular cAMP levels were quantified as per Cisbio’s protocol (5 uL of D2 and then 5 uL Cryptate, incubated for 1-2 hours at room temperature). Samples were measured on an Envision plate reader (PerkinElmer Life and Analytical Sciences; excitation, 320 nm; emission, 665 nm/620 nm). Data were analyzed using the ratio of fluorescence intensity at 620 and 665 nm for each well, extrapolated from the cAMP standard curve to express data as nM cAMP for each well. Data expressed as nM cAMP were then normalized to control wells using Activity Base (IDBS).
  • IDBS Activity Base
  • Zero percent effect was defined as nM of cAMP generated from EC 80 agonist stimulation (200 nM ⁇ MSH).
  • HPE one hundred percent effect
  • concentration and % effect values for each compound were plotted by Activity Base using a four-parameter logistic dose response equation, and the concentration required for 50% inhibition (IC 50 ) was determined.
  • Table MC4R-1 lists biological activities (K i values, see Example AA; and K b values, see Example BB) and compound names for Examples 1 – 18.
  • Table MC4R-1 Biological activity and Compound name for Examples 1 – 18 of MC4R antagonists. All references, including publications, patents, and patent documents are hereby incorporated by reference herein, as though individually incorporated by reference. The present disclosure provides reference to various embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the scope of the present disclosure.

Abstract

The present invention provides compounds of Formula X-1, X-2, X-3, or X-4, or metabolites of Compound 1 or metabolites of a compound of Formula 3-A or 3-B, including compositions and salts thereof, which are useful in the prevention and/or treatment of a disease or disorder or condition such as anorexia or cachexia, as well as analytical methods related to the administration of Compound 1 or a compound of Formula 3-A or 3-B.

Description

METABOLITES OF SELECTIVE ANDROGEN RECEPTOR MODULATORS FIELD OF THE INVENTION The present invention provides metabolites of certain selective androgen receptor modulators (SARMs), including salts and compositions thereof, which are useful in the prevention and/or treatment of diseases and disorders that are related to the androgen receptors as well as analytical methods related to the administration of these selective androgen receptor modulators. BACKGROUND OF THE INVENTION The androgen receptor ("AR") is a ligand-activated transcriptional regulatory protein that mediates induction of male sexual development and function through its activity with endogenous androgens. Androgenic steroids play an important role in many physiologic processes, including the development and maintenance of male sexual characteristics such as muscle and bone mass, prostate growth, spermatogenesis, and the male hair pattern. The endogenous steroidal androgens include testosterone and dihydrotestosterone ("DHT"). Steroidal ligands which bind the AR and act as androgens (e.g. testosterone enanthate) or as antiandrogens (e.g. cyproterone acetate) have been known for many years and are used clinically. U.S. Patent Nos.9,328,104, 9,920,043, and 10,328,082 disclose certain SARMs and their uses in treating and/or preventing a variety of hormone-related conditions, for example, a disease or disorder or condition that is selected from anemia; anorexia; arthritis; bone disease; benign prostate hyperplasia; musculoskeletal impairment; cachexia; cachexia associated with cancer; cancer; frailty; age-related functional decline in the elderly; growth hormone deficiency; hematopoietic disorders; hormone replacement; hypergonadism; loss of muscle strength and/or function; muscular dystrophies; muscle loss following surgery; muscular atrophy; neurodegenerative diseases; neuromuscular disease; obesity; osteoporosis; sarcopenia, including sarcopenia in chronic obstructive pulmonary disease; a method of improving dexterity and movement in a subject; atherosclerosis and its associated diseases; dysmenorrhea; dysspermtogenic sterility; muscle wasting; respiratory tract disease; otorhinolaryngologic disease; hormonal disorder/ disruption or imbalance; androgen deprivation therapy; injuries of the central nervous system; hair loss; an infection; digestive system disease; urologic or male genital disease; dermatological disorder; endocrine disorder; hemic or lymphatic disorder; congenital/hereditary or neonatal disease; connective tissue disease; metabolic disease; disorder of environmental origin; a behavior mechanism; a mental disorder; a cognitive disorder; liver disease; kidney disease and diabetic nephropathy, and stress urinary incontinence. For example, 6-[(4R)-4-Methyl-1,1-dioxido- 1,2,6-thiadiazinan-2-yl]isoquinoline-1-carbonitrile (Compound 1) is a selective androgen receptor modulator (SARM). Compound 1, in its free base form, has the chemical formula C14H14N4SO2 and the following structural formula:
Figure imgf000004_0001
There is a continuing need for new and improved SARMs and for analytical methods related to the administration of SARMs. The metabolites of Compound 1 (including the salts thereof), as well as their compositions and methods of use described herein, are directed toward fulfilling this need. SUMMARY OF THE INVENTION In one embodiment, the present invention provides a compound of Formula X-1, X-2, X-3, or X-4:
Figure imgf000004_0002
wherein: A1 is N or CR0; R0 is hydrogen, C1-C6 linear or branched chain alkyl, C1-C6 linear or branched chain perfluoroalkyl, aryl, perfluoroaryl, alkylaryl, heteroaryl or alkylheteroaryl; R1 is glucuronidation; and R3 and R4 are each independently hydrogen, C1-C6 linear or branched chain alkyl, C1-C6 linear or branched chain perfluoroalkyl, C1-C6 linear or branched chain perfluoroalkoxy, halogen, cyano, hydroxyl, amino, carboxy, hydroxyl, aryl, heteroaryl, C1-C6 linear or branched chain alkoxylcarbonyl, C1-C6 linear or branched chain alkylamino-carbonylamino, or C1-C6 linear or branched chain alkylaminocarbonyl, or a pharmaceutically acceptable salt thereof. In Formulas X-3 and X-4, the substituent -R1 , -OH, or -OR1 is substituted at the part of the 4-methyl-1,1-dioxido-1,2,6-thiadiazinan-2-yl moiety within the dotted oval shape {i.e., one of the hydrogen atoms (including the hydrogen bonded to the N atom as shown or any hydrogen bonded to a ring-forming C atom or the C atom of the methyl group) on the part of the 4-methyl-1,1-dioxido- 1,2,6-thiadiazinan-2-yl moiety is replaced by the substituent. In some embodiments, the compound of Formula X-1, X-2, X-3, or X-4 or pharmaceutically acceptable salt thereof of the present invention is substantially isolated. In another embodiment, the present invention provides a compound of Formula Y-1, Y-2, Y- 3, or Y-4,
Figure imgf000005_0001
Figure imgf000006_0001
wherein R1A is and 3
Figure imgf000006_0002
R and R4 are each independently hydrogen, C1-C6 linear or branched chain alkyl, C1-C6 linear or branched chain perfluoroalkyl, C1-C6 linear or branched chain perfluoroalkoxy, halogen, cyano, hydroxyl, amino, carboxy, hydroxyl, aryl, heteroaryl, C1-C6 linear or branched chain alkoxylcarbonyl, C1-C6 linear or branched chain alkylamino-carbonylamino, or C1-C6 linear or branched chain alkylaminocarbonyl, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula Y-1, Y-2, Y-3, or Y-4, or pharmaceutically acceptable salt thereof of the present invention is substantially isolated. The present invention further provides compositions comprising a compound of the invention, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. The present invention further provides preparations comprising a compound of the invention, or a pharmaceutically acceptable salt thereof. The present invention further provides methods of treating or preventing a disease or disorder or condition in a human by administering to the human a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, wherein the disease or disorder or condition is selected from selected from anemia; anorexia; arthritis; bone disease; benign prostate hyperplasia; musculoskeletal impairment; cachexia; cachexia associated with cancer; cancer; frailty; age-related functional decline in the elderly; growth hormone deficiency; hematopoietic disorders; hormone replacement; hypergonadism; loss of muscle strength and/or function; muscular dystrophies; muscle loss following surgery; muscular atrophy; neurodegenerative diseases; neuromuscular disease; obesity; osteoporosis; sarcopenia, including sarcopenia in chronic obstructive pulmonary disease; a method of improving dexterity and movement in a subject; atherosclerosis and its associated diseases; dysmenorrhea; dysspermtogenic sterility; muscle wasting; respiratory tract disease; otorhinolaryngologic disease; hormonal disorder/ disruption or imbalance; androgen deprivation therapy; injuries of the central nervous system; hair loss; an infection; digestive system disease; urologic or male genital disease; dermatological disorder; endocrine disorder; hemic or lymphatic disorder; congenital/hereditary or neonatal disease; connective tissue disease; metabolic disease; disorder of environmental origin; a behavior mechanism; a mental disorder; a cognitive disorder; liver disease; kidney disease and diabetic nephropathy, and stress urinary incontinence. The present invention further provides a compound of the invention, or pharmaceutically acceptable salt thereof, for use as a medicament. The present invention further provides a compound of the invention, or pharmaceutically acceptable salt thereof, for use in a method of treating or preventing a disease or disorder or condition, wherein the disease or disorder or condition is selected from selected from anemia; anorexia; arthritis; bone disease; benign prostate hyperplasia; musculoskeletal impairment; cachexia; cachexia associated with cancer; cancer; frailty; age-related functional decline in the elderly; growth hormone deficiency; hematopoietic disorders; hormone replacement; hypergonadism; loss of muscle strength and/or function; muscular dystrophies; muscle loss following surgery; muscular atrophy; neurodegenerative diseases; neuromuscular disease; obesity; osteoporosis; sarcopenia, including sarcopenia in chronic obstructive pulmonary disease; a method of improving dexterity and movement in a subject; atherosclerosis and its associated diseases; dysmenorrhea; dysspermtogenic sterility; muscle wasting; respiratory tract disease; otorhinolaryngologic disease; hormonal disorder/ disruption or imbalance; androgen deprivation therapy; injuries of the central nervous system; hair loss; an infection; digestive system disease; urologic or male genital disease; dermatological disorder; endocrine disorder; hemic or lymphatic disorder; congenital/hereditary or neonatal disease; connective tissue disease; metabolic disease; disorder of environmental origin; a behavior mechanism; a mental disorder; a cognitive disorder; liver disease; kidney disease and diabetic nephropathy, and stress urinary incontinence. The present invention further provides methods of detecting or confirming the administration of Compound 1 to a human, comprising identifying a metabolite of Compound 1 (e.g. a compound of the invention), or a salt thereof, in a biological sample obtained from the human. The present invention further provides methods of measuring the rate of metabolism of Compound 1 in a patient comprising measuring the amount of a metabolite of Compound 1 (e.g. a compound of the invention), or a salt thereof, in the patient at one or more time points after administration of Compound 1. The present invention further provides methods of determining the therapeutic or prophylactic response of a patient to Compound 1 in the treatment of a disease or disorder or condition, comprising measuring the amount of a metabolite of Compound 1 (e.g. a compound of the invention), or a salt thereof, in the patient at one or more time points after administration of Compound 1. The present invention further provides methods of optimizing the dose of Compound 1 for a patient in need of treatment with Compound 1, comprising measuring the amount of a metabolite of Compound 1 (e.g. a compound of the invention), or a salt thereof, in the patient at one or more time points after administration of Compound 1. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows HPLC-UV chromatogram of pre-dose (Top) and pooled (Bottom) extracted human plasma samples from a multi dose study (100 mg of Compound 1 BID; 14 Days). Figure 2 shows HPLC-UV chromatogram of pre-dose (Top) and pooled (Bottom) extracted human urine samples from a multi dose study (100 mg of Compound 1 BID; 14 Days). Figure 3 shows product ion scan of Compound 1. Figure 4 shows product ion spectrum of Compound/Metabolite M1 (m/z 479). Figure 5 shows full 1H NMR Spectra of Compound/Metabolite M1. Figure 6 shows 1H-1H TOCSY of Compound/Metabolite M1. Figure 7 shows 1H-13C HSQC Spectrum of Compound/Metabolite M1. Figure 8 shows product ion scan of Compound/Metabolite M2 (m/z 256). Figure 9 shows full 1H NMR Spectra of Compound/Metabolite M2. Figure 10 shows 1H-1H TOCSY Spectrum of Compound/Metabolite M2. Figure 11 shows product ion scan of Metabolite 495 (m/z 495) in human urine pool of patients dosed with Compound 1 (Day 14100 mg oral BID). DETAILED DESCRIPTION In a first aspect, the present invention provides a compound of Formula X-1, X-2, X-3, or X- 4:
Figure imgf000008_0001
Figure imgf000009_0001
or a pharmaceutically acceptable salt thereof, wherein: A1 is N or CR0; R0 is hydrogen, C1-C6 linear or branched chain alkyl, C1-C6 linear or branched chain perfluoroalkyl, aryl, perfluoroaryl, alkylaryl, heteroaryl or alkylheteroaryl; R1 is glucuronidation; and R3 and R4 are each independently hydrogen, C1-C6 linear or branched chain alkyl, C1-C6 linear or branched chain perfluoroalkyl, C1-C6 linear or branched chain perfluoroalkoxy, halogen, cyano, hydroxyl, amino, carboxy, hydroxyl, aryl, heteroaryl, C1-C6 linear or branched chain alkoxylcarbonyl, C1-C6 linear or branched chain alkylamino-carbonylamino, or C1-C6 linear or branched chain alkylaminocarbonyl, or a pharmaceutically acceptable salt thereof, which is substantially isolated. In some embodiments, the present invention provides a compound of Formula X-1, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula X-1 or a pharmaceutically acceptable salt thereof is a compound of Formula Y-1
Figure imgf000009_0002
or a pharmaceutically acceptable salt thereof, wherein R1A is
Figure imgf000010_0001
e.g.
Figure imgf000010_0002
. In some embodiments, the present invention provides a compound of Formula X-2, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula X-2 or a pharmaceutically acceptable salt thereof is a compound of Formula Y-2
Figure imgf000010_0003
or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a compound of Formula X-3, or a pharmaceutically acceptable salt thereof. The substituents R1 and OH are substituted on the part of the structure of Formula X-3 within the dotted oval shape (i.e., each of R1 and OH replaces a hydrogen atom with the dotted oval shape). In some embodiments, the compound of Formula X-3 or a pharmaceutically acceptable salt thereof is a compound of Formula Y-3
Figure imgf000010_0004
or a pharmaceutically acceptable salt thereof, wherein R1A is e.g.
Figure imgf000011_0001
Figure imgf000011_0002
In some embodiments, a compound Formula X-3 or a pharmaceutically acceptable salt thereof is a compound of Formula X-3A or X-3B:
Figure imgf000011_0003
X-3A {wherein the OH is substituted on the part of the structure of Formula X-3A within the dotted oval shape (i.e., the OH replaces a hydrogen atom with the dotted oval shape)} 1
Figure imgf000011_0004
X-3B {wherein R is substituted on the part of the structure of Formula X-3B within the dotted oval shape (i.e., R1 replaces a hydrogen atom with the dotted oval shape)} or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a compound of Formula X-4, or a pharmaceutically acceptable salt thereof. The substituent OR1 is substituted on the part of the structure of Formula X-4 within the dotted oval shape (i.e., OR1 replaces a hydrogen atom with the dotted oval shape). In some embodiments, the compound of Formula X-4 or a pharmaceutically acceptable salt thereof is a compound of Formula Y-4
Figure imgf000012_0001
or a pharmaceutically acceptable salt thereof, wherein R1A is e.g.
Figure imgf000012_0002
Figure imgf000012_0003
In a second aspect, the present invention is directed to metabolites of Compound 1 or a pharmaceutically acceptable salt thereof and uses thereof. In some embodiments, the metabolite results from Compound 1 (or a pharmaceutically acceptable salt thereof) which has undergone (1) glucuronidation (see e.g. Compound M1), (2) hydrolysis of the 1,1-dioxido-1,2,6-thiadiazinane ring followed by oxidation (see Compound M2), (3) glucuronidation and hydroxylation (see e.g. a compound of Formula Y-3), (4) glucuronic acid conjugation of a hydroxylated-Compound 1 (see e.g. a compound of Formula Y-4), or a combination thereof. In some embodiments, the metabolite is selected from Compounds M1; M2; a compound of Formula Y-3; and a compound of Formula Y- 4. In some embodiments, the present invention provides Compound M1.
Figure imgf000013_0001
or a pharmaceutically acceptable salt thereof, which is substantially isolated/purified. In some embodiments, the present invention provides Compound M1-A.
Figure imgf000013_0002
or a pharmaceutically acceptable salt thereof, which is substantially isolated/purified. In some embodiments, the present invention provides Compound M2, or a pharmaceutically acceptable salt thereof, which is substantially isolated.
Figure imgf000013_0003
In some embodiments, the present invention provides Compound M2-A, or a pharmaceutically acceptable salt thereof, which is substantially isolated.
Figure imgf000013_0004
In some embodiments, the present invention provides Compound M2-B, or a pharmaceutically acceptable salt thereof, which is substantially isolated.
Figure imgf000014_0001
In some embodiments, the compound of Formula Y-3 or pharmaceutically acceptable salt thereof is a compound of Formula Y-3A
Figure imgf000014_0002
or a pharmaceutically acceptable salt thereof wherein R1A is e.g.
Figure imgf000014_0003
(i.e. the metabolite results from Compound 1 that has undergone both
Figure imgf000014_0004
glucuronidation and hydroxylation); and wherein both R1A and the OH are substituted on the part of the structure of Formula Y-3A within the dotted oval shape (i.e., each of R1A and the OH replaces a hydrogen atom with the dotted oval shape). In some further embodiments, a compound of Formula Y-3A or a pharmaceutically acceptable salt thereof is a compound of Formula Y-3A-1
Figure imgf000015_0001
or a pharmaceutically acceptable salt thereof, wherein R1A is e.g.
Figure imgf000015_0002
Figure imgf000015_0003
(i.e. the metabolite results from Compound 1 that has undergone both N- glucuronidation and hydroxylation); and wherein the OH is substituted on the part of the structure of Formula Y-3A-1 within the dotted oval shape (i.e., the OH replaces a hydrogen atom with the dotted oval shape). In some further embodiments, the compound of Formula Y-3A or pharmaceutically acceptable salt thereof is a compound of Formula Y-3A-2 OH
Figure imgf000015_0004
or a pharmaceutically acceptable salt thereof, wherein R1A is or
Figure imgf000016_0001
(i.e. the metabolite results from Compound 1 that has undergone both
Figure imgf000016_0002
glucuronidation and N-hydroxylation); and wherein R1A is substituted on the part of the structure of Formula Y-3B within the dotted oval shape (i.e., R1A replaces a hydrogen atom with the dotted oval shape). In some embodiments, the present invention provides a compound of Formula Y-4A
Figure imgf000016_0003
or a pharmaceutically acceptable salt thereof, wherein R1A is or
Figure imgf000016_0004
(i.e. the metabolite is the glucuronic acid conjugation of a hydroxylated
Figure imgf000016_0005
Compound 1); and wherein OR1A is substituted on the part of the structure of Formula Y-4 within the dotted oval shape (i.e., OR1A replaces a hydrogen atom with the dotted oval shape). In some further embodiments, a compound of Formula Y-4A or a pharmaceutically acceptable salt thereof is a compound of Formula Y-4A-1
Figure imgf000017_0001
or a pharmaceutically acceptable salt thereof, wherein R1A is or
Figure imgf000017_0002
(i.e. the metabolite is the glucuronic acid conjugation of a hydroxylated
Figure imgf000017_0003
Compound 1); and wherein OR1A is substituted on the part of the structure of Formula Y-4A-1 within the dotted oval shape (i.e., OR1A replaces a hydrogen atom with the dotted oval shape). The present invention further includes salts of the metabolites of the invention, such as pharmaceutically acceptable salts. A salt generally refers to a derivative of a disclosed compound wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. A pharmaceutically acceptable salt is one that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17 ed., Mack Publishing Company, Easton, Pa., 1985, p.1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety. In one embodiment, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the metabolite compounds (or the compounds of invention), or salts thereof, are substantially isolated. By “substantially isolated” is meant that the metabolite compound, or salt thereof, is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the metabolite, or salt thereof. In some embodiments, each of compounds Formula X-1 or X-2 (including compounds of Formula Y-1 or Y-2, such as Compound M1 or M2), compounds of Formula X-3 or X4 (including e.g. Compounds of Formula Y-3A or Y-4A) or their salts is substantially isolated. In some embodiments, each of compounds Formula X-1 or X-2 (including compounds of Y-1 or Y-2, such as Compounds M1 and M2) and compounds of Formula X-3 or X4 (including e.g. Compounds of Formula Y-3A or Y-4A) or their salts is substantially isolated. In some embodiments, one or more of the metabolite compounds, or salts thereof, are prepared by metabolism of Compound 1 or a pharmaceutically salt thereof (for example, in a mammal or a mammalian cell environment); and the metabolite compounds thus prepared are substantially isolated. In some other embodiments, one or more of the metabolite compounds, or salts thereof, are prepared by chemical synthesis other than metabolism of Compound 1 or a pharmaceutically salt thereof (for example, in a mammal or a mammalian cell environment) and the synthesized metabolite compounds are substantially isolated. A metabolite of the invention, or its salt, can be present in a composition where the composition includes at least one compound other than the metabolite. In some embodiments, the composition includes more than one metabolite of the invention. In some embodiments, the composition comprises one or more metabolites of the invention, or salts thereof, and Compound 1, or a salt thereof. Compositions can be mixtures containing a metabolite of the invention, or salt thereof, and one or more solvents, substrates, carriers, etc. In some embodiments, the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 25% by weight. In some embodiments, the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 50% by weight. In some embodiments, the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 75% by weight. In some embodiments, the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 80% by weight. In some embodiments, the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 85% by weight. In some embodiments, the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 90% by weight. In some embodiments, the composition comprises a metabolite of the invention, or salt thereof, in an amount greater than about 95% by weight. A preparation of a metabolite of the invention, or salt thereof, can be prepared by chemical synthesis or by isolation of the metabolite from a biological sample. Preparations can have a purity of greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% purity. Purity can be measured by any of conventional means, such as by chromatographic methods or spectroscopic methods like NMR, MS, LC-MS, etc. The metabolites of the invention are asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Metabolites of the invention also include all isotopes of atoms occurring in the metabolites. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. In some embodiments, the metabolite includes at least one deuterium. The term, “compound” or “metabolite,” as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. The term, “metabolite” as used herein is meant to include any and all metabolic derivatives of a parent drug molecule (e.g. Compound 1 or a pharmaceutically acceptable salt thereof), including derivatives that have undergone one or more transformative processes selected from (1) glucuronidation, (2) hydrolysis of the 1,1-dioxido-1,2,6-thiadiazinane ring followed by oxidation, (3) glucuronidation and hydroxylation, (4) glucuronic acid conjugation of a hydroxylated-Compound 1, (5) hydroxylation, or a combination thereof (including a pharmaceutically acceptable salt thereof). In some embodiments, the present invention provides a metabolite of Compound 1 or a pharmaceutically acceptable salt thereof. As used herein, a glucuronide conjugation (or a glucuronide adduct, or glucuronidation) of a parent compound refers to replacing a hydrogen atom of the parent compound with a chemical moiety that is glucuronic acid without one of its four alcohol hydroxyl groups, e.g., a moiety having the structure of:
Figure imgf000020_0001
O H H or wherein
Figure imgf000020_0002
Figure imgf000020_0003
indicates the point of contact of the moiety to the parent compound. As used herein, -O-glucuronidation or -O-glucuronide refers to a moiety of the structure of
Figure imgf000020_0004
Figure imgf000020_0005
Figure imgf000021_0001
Compound 1 can also be considered a prodrug of the metabolites of the invention (e.g., a prodrug of metabolites M1, M2, a compound of Formula Y-3A or Y-4A and the like) because Compound 1 metabolically transforms upon administration to provide the metabolites of the invention. Accordingly, Compound 1 can be administered to a human as a means of providing a metabolite of the invention to the human, for example, for preventing or treating a disease or disorder or condition in the human as described herein. In another aspect, the present of invention provides a metabolite of a compound of Formula 3-A: wherein
Figure imgf000021_0002
A1 is N or CR0; R0 is hydrogen, C1-C6 linear or branched chain alkyl, C1-C6 linear or branched chain perfluoroalkyl, aryl, perfluoroaryl, alkylaryl, heteroaryl or alkylheteroaryl; and R3 and R4 are each independently hydrogen, C1-C6 linear or branched chain alkyl, C1-C6 linear or branched chain perfluoroalkyl, C1-C6 linear or branched chain perfluoroalkoxy, halogen, cyano, hydroxyl, amino, carboxy, hydroxyl, aryl, heteroaryl, C1-C6 linear or branched chain alkoxylcarbonyl, C1-C6 linear or branched chain alkylamino-carbonylamino, or C1-C6 linear or branched chain alkylaminocarbonyl, or a pharmaceutically acceptable salt thereof, and wherein the metabolite is a derivative of the parent drug molecule (i.e. the compound of Formula 3-A or pharmaceutically acceptable salt thereof), including any of the derivatives that have undergone one or more transformative processes selected from (1) glucuronidation, (2) hydrolysis of the 1,1-dioxido-1,2,6-thiadiazinane ring followed by oxidation, (3) glucuronidation and hydroxylation, (4) glucuronic acid conjugation of a hydroxylated-Compound 1, (5) hydroxylation, or a combination thereof (including a pharmaceutically acceptable salt thereof). In some embodiments, the present of invention provides a metabolite of a compound of Formula 3-B:
Figure imgf000022_0001
wherein R3 and R4 are each independently hydrogen, C1-C6 linear or branched chain alkyl, C1-C6 linear or branched chain perfluoroalkyl, C1-C6 linear or branched chain perfluoroalkoxy, halogen, cyano, amino, carboxy, hydroxyl, aryl, heteroaryl, C1-C6 linear or branched chain alkoxy-carbonyl-, C1-C6 linear or branched chain alkylamino-carbonylamino, or C1-C6 linear or branched chain alkylaminocarbonyl, or a pharmaceutically acceptable salt thereof, and wherein the metabolite is a derivative of the parent drug molecule (i.e. the compound of Formula 3-A or pharmaceutically acceptable salt thereof), including any of the derivatives that have undergone one or more transformative processes selected from (1) glucuronidation, (2) hydrolysis of the 1,1-dioxido-1,2,6-thiadiazinane ring followed by oxidation, (3) glucuronidation and hydroxylation, (4) glucuronic acid conjugation of a hydroxylated-Compound 1, (5) hydroxylation, or a combination thereof (including a pharmaceutically acceptable salt thereof). In some embodiments, one or more of the metabolite compounds, or salts thereof, are prepared by metabolism of its parent compound, e.g., a compound of Formula 3-A or 3-B or a pharmaceutically salt thereof (for example, in a mammal or a mammalian cell environment); and the metabolite compounds thus prepared are substantially isolated. In some other embodiments, one or more of the metabolite compounds, or salts thereof, are prepared by chemical synthesis other than metabolism of a compound of Formula 3-A or 3-B or a pharmaceutically salt thereof (for example, in a mammal or a mammalian cell environment) and the synthesized metabolite compounds are substantially isolated. A compound of Formula 3-A or 3-B or its salt can be prepared, for example, by the methods described in U.S. Patent No.9328104. The term “alkyl”, alone or in combination, means an acyclic, saturated hydrocarbon group of the formula CnH2n+1 which may be linear or branched. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl and hexyl. Unless otherwise specified, an alkyl group comprises from 1 to 6 carbon atoms. The carbon atom content of alkyl and various other hydrocarbon-containing moieties is indicated by a prefix designating a lower and upper number of carbon atoms in the moiety, that is, the prefix Ci-Cj indicates a moiety of the integer "i" to the integer "j" carbon atoms, inclusive. Thus, for example, C1-C6 alkyl refers to alkyl of one to six carbon atoms, inclusive. The term “aryl”, alone or in combination, means phenyl or naphthyl. The term “-alkylaryl” means an -alkyl-aryl moiety that is attached through the alkyl part. The term “heteroaryl” refers to an aromatic heterocycle which may be attached via a ring carbon atom (in all cases) or a ring nitrogen atom with an appropriate valency (when the heterocycle is attached to a carbon atom). Equally, when substituted, the substituent may be located on a ring carbon atom (in all cases) or a ring nitrogen atom with an appropriate valency (if the substituent is joined through a carbon atom). Specific examples include thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl and pyrazinyl. The term “-alkylheteroaryl” means an -alkyl-heteroaryl moiety that is attached through the alkyl part. The term “perfluoroalkyl” means an alkyl radical wherein each of the hydrogen on the alkyl is replaced by a fluorine atom. The term “perfluoroaryl” means an aryl radical wherein each of the hydrogen on the aryl is replaced by a fluorine atom. The term “hydroxy,” as used herein, means an OH radical. The term “oxo” means a double-bonded oxygen (=O). The term “alkoxy” means a radical comprising an alkyl radical that is bonded to an oxygen atom, such as a methoxy radical. Examples of such radicals include methoxy, ethoxy, propoxy, isopropoxy, butoxy and tert-butoxy. The term “halogen” means, fluoro, chloro, bromo or iodo. The term “carboy” means -C(=O)OH. The term “amino” means -NH2. The term “alkylamino-carbonylamino” means (alkyl)HN-C(=O)NH-. The term “alkoxylcarbonyl” means alkyl-O-C(=O)-. The term “alkylaminocarbonyl” means (alkyl)HN-C(=O)-. As used herein, a wavy line,“ ” denotes a point of attachment of a substituent to another group. As used herein, when a bond to a substituent is shown to cross a ring (or a bond connecting two atoms in a ring), then such substituent may be bonded to any of the ring-forming atoms in that ring that are substitutable (i.e., any ring forming atom that is bonded to one or more hydrogen atoms), unless otherwise specified or otherwise implicit from the context. For example, as shown in Formula 3-A below, R3 may be bonded to any ring-forming carbon atom of the left ring of the bicyclic ring that is substitutable (i.e., any one of the carbon atoms of a -CH- group of the left ring). For another example, as shown in Formula 3-A below, R4 may be bonded to any ring- forming carbon atom of the right ring of the bicyclic ring that is substitutable (i.e., any one of the carbon atoms of a -CH- group).
Figure imgf000024_0001
The present invention further includes a pharmaceutical composition comprising a compound (or a metabolite) of the invention, or pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In some further embodiments, the compound (or the metabolite) of the invention or pharmaceutically acceptable salt thereof is present in the composition in an amount greater than about 0.001%, 0.01%, 0.05%, 0.08%, 0.1%, 0.5%, or 1.0% by weight As used herein, “pharmaceutically acceptable carrier” is meant to refer to any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Methods The present invention further relates to a method of treating or preventing a disease or disorder or condition in a human by administering to the human a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, wherein the disease or disorder or condition is selected from selected from anemia; anorexia; arthritis; bone disease; benign prostate hyperplasia; musculoskeletal impairment; cachexia; cachexia associated with cancer; cancer; frailty; age-related functional decline in the elderly; growth hormone deficiency; hematopoietic disorders; hormone replacement; hypergonadism; loss of muscle strength and/or function; muscular dystrophies; muscle loss following surgery; muscular atrophy; neurodegenerative diseases; neuromuscular disease; obesity; osteoporosis; sarcopenia, including sarcopenia in chronic obstructive pulmonary disease; a method of improving dexterity and movement in a subject; atherosclerosis and its associated diseases; dysmenorrhea; dysspermtogenic sterility; muscle wasting; respiratory tract disease; otorhinolaryngologic disease; hormonal disorder/ disruption or imbalance; androgen deprivation therapy; injuries of the central nervous system; hair loss; an infection; digestive system disease; urologic or male genital disease; dermatological disorder; endocrine disorder; hemic or lymphatic disorder; congenital/hereditary or neonatal disease; connective tissue disease; metabolic disease; disorder of environmental origin; a behavior mechanism; a mental disorder; a cognitive disorder; liver disease; kidney disease and diabetic nephropathy, and stress urinary incontinence. The human may have or be at risk of having the disease or disorder. The term "treating", "treat", or "treatment" in connection with a disease or disorder as used herein embraces palliative treatment, including reversing, relieving, alleviating, eliminating, or slowing the progression of the disease or disorder , or one or more symptoms of the disease or disorder, or any tissue damage associated with one or more symptoms of the disease or disorder. The term “prevention” or “preventing” in connection with a disease or disorder refers to delaying or forestalling the onset or development of the disease or disorder a period of time from minutes to indefinitely. The term also includes prevention of the appearance of symptoms of the disease or disorder. The term further includes reducing risk of developing the disease or disorder. The terms “effective amount” or “therapeutically effective amount” refer to an amount of a metabolite according to the invention, which when administered to a patient in need thereof, is sufficient to effect treatment for disease-states, conditions, or disorders for which the compounds have utility. Such an amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is sought by a researcher or clinician. The amount of a metabolite according to the invention which constitutes a therapeutically effective amount will vary depending on such factors as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of the treatment, the type of disease-state or disorder being treated and its severity, drugs used in combination with or coincidentally with the compounds of the invention, and the age, body weight, general health, sex and diet of the patient. Such a therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure. Administration of the metabolites of the invention, or their pharmaceutically acceptable salts, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions of the invention can be prepared by combining a metabolite of the invention, or a pharmaceutically acceptable salt thereof, with an appropriate pharmaceutically acceptable carrier and, in specific embodiments, are formulated into preparations in solid, semi solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Exemplary routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. In one embodiment, pharmaceutical compositions of the invention are tablets. In another embodiment, pharmaceutical compositions of the invention are injection (intramuscular (IM) or intraperitoneal (IP)). Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disease or disorder of interest in accordance with the teachings described herein. The present invention further relates to a method of detecting or confirming the administration of Compound 1 to a patient comprising identifying a metabolite of Compound 1 (e.g. a metabolite of the invention), or salt thereof, in a biological sample obtained from the patient. In some embodiments, the biological sample is derived from plasma, urine, or feces. The present invention further relates to a method of measuring the rate of metabolism of Compound 1 in a patient comprising measuring the amount of a metabolite, or salt thereof, in the patient at one or more time points after administration of Compound 1. The present invention further relates to a method of determining the prophylactic or therapeutic response of a patient to Compound 1 in the treatment of a disease or disorder comprising measuring the amount of a metabolite of Compound 1 (e.g. a metabolite of the invention), or salt thereof, in the patient at one or more time points after administration of Compound 1. The present invention further relates to a method of optimizing the dose of Compound 1 for a patient in need of treatment with Compound 1 comprising measuring the amount of a metabolite of Compound 1 (including, e.g. a metabolite of the invention) or salt thereof, in the patient at one or more time points after administration of Compound 1. The amount of metabolite may be indicative of the rate at which the patient metabolizes Compound 1. Patients who metabolize Compound 1 more quickly or more effectively than other patients may form higher amounts of metabolite and potentially require higher doses of Compound 1, or additional doses, compared with patients who metabolize Compound 1 more slowly. Patients who metabolize Compound 1 less quickly or less effectively than other patients may form lower amounts of metabolite and potentially require lower doses of Compound 1, or fewer doses, compared with patients who metabolize Compound 1 more quickly. Accordingly, the method of optimizing the dose of Compound 1 may further include determining whether the measured amounts of metabolite are higher or lower than average, and adjusting the dosage of Compound 1 accordingly. Measuring the amount of metabolite, or salt thereof, in a patient can be carried out by obtaining a biological sample from the patient and measuring the amount of metabolite, or salt thereof, in the sample. In some embodiments, the sample is blood. In other embodiments, the sample is plasma. In other embodiments, the sample is urine. In other embodiments, the sample is feces. The term “patient” is meant to refer to a human or another mammal such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as non-human primates, mammalian wildlife, and the like, that are in need of therapeutic or preventative treatment for a disease or disorder described herein. Combination Therapies One or more additional pharmaceutical agents can be used in combination with the compounds, salts, and compositions of the present invention for preventing or treating a disease or disorder described herein, e.g., in a human patient. In some embodiments, the composition of the invention further comprises one or more additional therapeutic agents. In some embodiments, the composition of the invention further comprises one to three additional therapeutic agents. Examples of additional therapeutic agents include, but are not limited to: (i) estrogen and estrogen derivatives (such as conjugated estrogens and synthetic estrogens) including, but not limited to, steroidal compounds having estrogenic activity such as, for example, 17.beta.-estradiol, estrone, conjugated estrogen (PREMARIN.RTM.), equine estrogen, 17.beta.-ethynyl estradiol, and the like. The estrogen or estrogen derivative can be employed alone or in combination with a progestin or progestin derivative. Nonlimiting examples of progestin derivatives are norethindrone and medroxy-progesterone acetate; (ii) a bisphosphonate compound, including, but not limited to: (a) alendronate (also known as alendronic acid, 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid, alendronate sodium, alendronate monosodium trihydrate or 4-amino-1-hydroxybutylidene-1,1- bisp- hosphonic acid monosodium trihydrate. Alendronate is described in U.S. Pat. No.4,922,007, to Kieczykowski et al., issued May 1, 1990; U.S. Pat. No.5,019,651, to Kieczykowski, issued May 28, 1991; U.S. Pat. No.5,510,517, to Dauer et al., issued Apr.23, 1996; U.S. Pat. No.5,648,491, to Dauer et al., issued Jul.15, 1997; (b) [(cycloheptylamino)-methylene]-bis-phosphonate (incadronate), which is described in U.S. Pat. No.4,970,335, to Isomura et al., issued Nov.13, 1990; (c) (dichloromethylene)-bis-phosphonic acid (clodronic acid) and the disodium salt (clodronate), which are described in Belgium Patent 672,205 (1966) and J. Org. Chem 32, 4111 (1967); (d) [1-hydroxy-3-(1-pyrrolidinyl)-propylidene]-bis-phosphonate (EB-1053); (e) (1-hydroxyethylidene)-bis-phosphonate (etidronate); (f) [1-hydroxy-3-(methylpentylamino)propylidene]-bis-phosphonate (ibandronate), which is described in U.S. Pat. No.4,927,814, issued May 22, 1990; (g) (6-amino-1-hydroxyhexylidene)-bis-phosphonate (neridronate); (h) [3-(dimethylamino)-1-hydroxypropylidene]-bis-phosphonate (olpadronate); (i) (3-amino-1-hydroxypropylidene)-bis-phosphonate (pamidronate); (j) [2-(2-pyridinyl)ethylidene]-bis-phosphonate (piridronate), which is described in U.S. Pat. No. 4,761,406; (k) [1-hydroxy-2-(3-pyridinyl)-ethylidene]-bis-phosphonate (risedronate); (l) {[(4-chlorophenyl)thio]methylene}-bis-phosphonate (tiludronate), which is described in U.S. Pat. No.4,876,248, to Breliere et al., Oct.24, 1989; (m) [1-hydroxy-2-(1H-imidazol-1-yl)ethylidene]-bis-phosphonate (zoledronate); and (n) [1-hydroxy-2-imidazopyridin-(1,2-a)-3-ylethylidene]-bis-phospho- nate (minodronate). (iii) a selective estrogen receptor modulator (SERM), including, but not limited to tamoxifen, 4- hydroxytamoxifen, raloxifene (see, e.g., U.S. Pat. No.5,393,763), lasofoxifene, ospemifene, tesmilifene, toremifene, azorxifene, EM-800, EM-652, TSE 424, pipendoxifene, clomiphene, zuclomiphene, enclomiphene, droloxifene, idoxifene, levormeloxifene, nafoxidene, zindoxifene, RU 58,688, EM 139, ICI-164,384, ICI-182,780, CI-680, CI-628, CN-55,945-27, Mer-25, U-11,555A, U- 100A, bazedoxifene, miproxifene phosphate, PPT (1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H- pyrazole), diarylpropionitrile (DPN), diethylstibestrol, coumestrol, genistein, GW5638, LY353581, delmadinone acetate, tibolone, DPPE, (N,N-diethyl-2-{4-(phenylmethyl)-phenoxy}ethanamine), TSE-424, WAY-070, WAY-292, WAY-818, cyclocommunol, prinaberel, ERB-041, WAY-397, WAY- 244, ERB-196, WAY-169122, MF-101, ERb-002, ERB-037, ERB-017, BE-1060, BE-380, BE-381, WAY-358, [18F]FEDNP, LSN-500307, AA-102, CT-101, CT-102, or VG-101and salts thereof, and the like (see, e.g., U.S. Pat. Nos.4,729,999 and 4,894,373) [Goldstein, et al., "A pharmacological review of selective estrogen receptor modulators," Human Reproduction Update, 6: 212-224 (2000); Lufkin, et al., Rheumatic Disease Clinics of North America, 27: 163-185 (2001), and "Targeting the Estrogen Receptor with SERMs," Ann. Rep. Med. Chem.36: 149-158 (2001)]. PSK- 3471; (iv) calcitonin and analogue thereof, including, but not limited to, salmon, Elcatonin, SUN-8577 or TJN-135, wherein if the calcitonin analogue is salmon it is optionally dosed as a nasal spray (for example as disclosed in Azra et al., Calcitonin.1996. In: J. P. Bilezikian, et al., Ed., Principles of Bone Biology, San Diego: Academic Press; and Silverman, "Calcitonin," Rheumatic Disease Clinics of North America.27: 187-196, 2001); (v) a cysteine protease cathepsin K, formerly known as cathepsin O.sub.2, for example as described in PCT International Application Publication No. WO 96/13523; U.S. Pat. Nos.5,501,969 and 5,736,357, and which include those which at an acidic pH degrade type-I collagen. Examples of cathepsin K include, but are not limited to, those disclosed in WO 01/49288, and WO 01/77073. Examples of cathepsin K inhibitors include, but are not limited to AAE581 and Odanacatib; (vi) alpha.v.beta.3 Integrin receptor antagonists peptidyl as well as peptidomimetic antagonists of the .alpha.v.beta.3 integrin receptor which indluce, but are not limited to those disclosed in the following publications W. J. Hoekstra and B. L. Poulter, Curr. Med. Chem.5: 195-204 (1998) and references cited therein; WO 95/32710; WO 95/37655; WO 97/01540; WO 97/37655; WO 98/08840; WO 98/18460; WO 98/18461; WO 98/25892; WO 98/31359; WO 98/30542; WO 99/15506; WO 99/15507; WO 00/03973; EP 853084; EP 854140; EP 854145; U.S. Pat. Nos. 5,204,350; 5,217,994; 5,639,754; 5,741,796; 5,780,426; 5,929,120; 5,952,341; 6,017,925; and 6,048,861. Other .alpha.v.beta.3 antagonists are described in R. M. Keenan et al., J. Med. Chem. 40: 2289-2292 (1997); R. M. Keenan et al., Bioorg. Med. Chem. Lett.8: 3165-3170 (1998); and R. M. Keenan et al., Bioorg. Med. Chem. Lett.8: 3171-3176 (1998). Other non-limiting representative examples of published patent and patent applications that describe various .alpha.v.beta.3 integrin receptor antagonists include: those comprising benzazepine and benzocycloheptene-PCT Patent Application Nos. WO 96/00574, WO 96/00730, WO 96/06087, WO 96/26190, WO 97/24119, WO 97/24122, WO 97/24124, WO 98/14192, WO 98/15278, WO 99/05107, WO 99/06049, WO 99/15170, WO 99/15178, WO 97/34865, WO 99/15506, and U.S. Pat. No.6,159,964; those comprising dibenzpcyclopheptene, and dibenzoxapine--PCT Patent Application Nos. WO 97/01540, WO 98/30542, WO 99/11626, WO 99/15508, and U.S. Pat. Nos.6,008,213 and 6,069,158; those having a phenol constraint--PCT Patent Application Nos. WO 98/00395, WO 99/32457, WO 99/37621, WO 99/44994, WO 99/45927, WO 99/52872, WO 99/52879, WO 99/52896, WO 00/06169, European Patent Nos. EP 0820,988, EP 0820,991, and U.S. Pat. Nos. 5,741,796, 5,773,644, 5,773,646, 5,843,906, 5,852,210, 5,929,120, 5,952,281, 6,028,223 and 6,040,311; those having a monocyclic ring constraint--PCT Patent Application Nos. WO 99/26945, WO 99/30709, WO 99/30713, WO 99/31099, WO 99/59992, WO 00/00486, WO 00/09503, European Patent Nos. EP 0796,855, EP 0928,790, EP 0928,793, and U.S. Pat. Nos.5,710,159, 5,723,480, 5,981,546, 6,017,926, and 6,066,648; and those having a bicyclic ring constraint--PCT Patent Application Nos. WO 98/23608, WO 98/35949, and WO 99/33798, European Patent No. EP 0853,084, and U.S. Pat. Nos.5,760,028, 5,919,792, and 5,925,655 (vii) osteoclast vacuolar ATPase inhibitors, also called proton pump inhibitors, due to the role they play in the bone resportive process [see C. Farina et al., DDT, 4: 163-172 (1999)],including, but not limited to, omeprazole, lansoprazole, pantoprazole, rebeprazole, or esomeprazole; (viii) angiogenic factor VEGF, due to the role they play in stimulating bone-resorbing activity of isolated mature rabbit osteoclasts via binding to its receptors on osteoclasts [see M. Nakagawa et al., FEBS Letters, 473: 161-164 (2000)] including, but not limited to KDR/Flk-1 and Flt-1; (ix) HMG-CoA reductase inhibitors, also known as the "statins", including, but not limited to, statins in their lactonized or dihydroxy open acid forms and pharmaceutically acceptable salts and esters thereof, including but not limited to lovastatin (see U.S. Pat. No.4,342,767); simvastatin (see U.S. Pat. No.4,444,784); dihydroxy open-acid simvastatin, particularly the ammonium or calcium salts thereof; pravastatin, particularly the sodium salt thereof (see U.S. Pat. No.4,346,227); fluvastatin, particularly the sodium salt thereof (see U.S. Pat. No.5,354,772); atorvastatin, particularly the calcium salt thereof (see U.S. Pat. No.5,273,995); cerivastatin, particularly the sodium salt thereof (see U.S. Pat. No.5,177,080), rosuvastatin, also known as ZD4522 (see U.S. Pat. No.5,260,440) and pitavastatin, also referred to as NK-104, itavastatin, lovastatin, pravastatin sodium, or nisvastatin (see PCT international application publication number WO 97/23200); (x) osteoanabolic agents including, but not limited to, parathyroid hormone (PTH) and fragments thereof, such as naturally occurring PTH (1-84), PTH (1-34), analogs thereof, native or with substitutions and particularly parathyroid hormone subcutaneous injection, for example Forteo (teriparatide); (xi) protein kinase inhibitors including, but not limited to, those disclosed in WO 01/17562 and which are in one embodiment selected from inhibitors of p38, non-limiting example of which include SB 203580 [Badger et al., J. Pharmacol. Exp. Ther., 279: 1453-1461 (1996)]; (xii) activators of peroxisome proliferator-activated receptor-.gamma. (PPAR.gamma.), inlcuidng, but not limited to, those compounds included within the structural class known as thiazolidinediones, those compounds outside the thiazolidinedione structural class, and glitazones, such as, for example, darglitazone, isaglitazone, rivoglitazone, netoglitazone, troglitazone, pioglitazone, rosiglitazone, and BRL 49653; (xiii) activators of peroxisome proliferator-activated receptor-.alpha (PPAR.alpha. agonists), including, but not liited to, bezafibrate, clofibrate, fenofibrate including micronized fenofibrate, and gemiibrozil; (xiv) dual acting peroxisome proliferator-activated alpha./.gamma. agonists including, but not limited to, muraglitazar, naveglitazar, farglitazar, tesaglitazar, ragaglitazar, oxeglitazar, PN-2034, PPAR.delta, such as for example, GW-501516; (xv) the polypeptide osteoprotegerin, and derivatives or analogues thereof, including, but not limited to mammalian osteoprotegerin and human osteoprotegerin; (xvi) calcium receptor antagonists which induce the secretion of PTH as described by Gowen et al., J. Clin. Invest.105: 1595-604 (2000); (xvii) growth hormone and its analogs, including, but not limited to, human growth hormone, such as, for example, somatotropin or analogues, nutropin A; growth promoting agents such as, for example, TRH, diethylstilbesterol, theophylline, enkephalins, E series prostaglandins, such as, for example, Ep1, EP2, EP4, FP, IP and derivatives thereof, prostanoids, compounds disclosed in U.S. Pat. No.3,239,345, e.g., zeranol, and compounds disclosed in U.S. Pat. No.4,036,979, e.g., sulbenox or peptides disclosed in U.S. Pat. No.4,411,890; growth hormone secretagogues such as, for example, anamorelin, pralmorelin, examorelin, tabimorelin, capimorelin, capromorelin, ipamorelin, EP-01572, EP-1572, or JMV-1843, GHRP-6, GHRP-1 (as described in U.S. Pat. No. 4,411,890 and publications WO 89/07110 and WO 89/07111), GHRP-2 (as described in WO 93/04081), NN.sub.7O.sub.3 (Novo Nordisk), LY444711 (Lilly), MK-677 (Merck), CP424391 (Pfizer) and B-HT920 and other representative examples disclosed in U.S. Pat. Nos.3,239,345, 4,036,979, 4,411,890, 5,206,235, 5,283,241, 5,284,841, 5,310,737, 5,317,017, 5,374,721, 5,430,144, 5,434,261, 5,438,136, 5,494,919, 5,494,920, 5,492,916 and 5,536,716; European Patent Pub. Nos.0,144,230 and 0,513,974; PCT Patent Pub. Nos. WO 94/07486, WO 94/08583, WO 94/11012; WO 94/13696, WO 94/19367, WO 95/03289, WO 95/03290, WO 95/09633, WO 95/11029, WO 95/12598, WO 95/13069, WO 95/14666, WO 95/16675, WO 95/16692, WO 95/17422, WO 95/17423, WO 95/34311, and WO 96/02530; articles, Science, 2601640-1643 (Jun. 11, 1993); Ann. Rep. Med. Chem., 28: 177-186 (1993); Bioorg. Med. Chem. Lett., 4: 2709-2714 (1994); and Proc. Natl. Acad. Sci. USA, 92: 7001-7005 (1995); and growth hormone releasing factor and its analogues such as, for example (a) epidermal growth factor (EGF); (b) transforming growth factor-.alpha. (TGF-.alpha.); (c) platelet derived growth factor (PDGF); (d) fibroblast growth factors (FGFs) including acidic fibroblast growth factor (.alpha.- FGF) and basic fibroblast growth factor (.beta.-FGF), including, but not limited to aFGF, bFGF and related peptides with FGF activity [Hurley Florkiewicz, "Fibroblast growth factor and vascular endothelial growth factor families," 1996. In: J. P. Bilezikian, et al., Ed. Principles of Bone Biology, San Diego: Academic Press]; (e) transforming growth factor-.beta. (TGF-.beta.) (f) insulin like growth factors (IGF-1 and IGF-2) selected from, but not limited to, Insulin-like Growth Factor I, alone or in combination with IGF binding protein 3 and IGF II [See Johannson and Rosen, "The IGFs as potential therapy for metabolic bone diseases," 1996, In: Bilezikian, et al., Ed., Principles of Bone Biology, San Diego: Academic Press; and Ghiron et al., J. Bone Miner. Res.10: 1844-1852 (1995)] IGF-1, IGF-1 analogues and secretagogue IGF-1 (xviii) a bone morphogenetic protein (BMP), including, but not limited to, chordin, fetuin, BMP 2, 3, 5, 6, 7, as well as related molecules TGF beta and GDF 5 [Rosen et al., "Bone morphogenetic proteins," 1996. In: J. P. Bilezikian, et al., Ed., Principles of Bone Biology, San Diego: Academic Press; and Wang E A, Trends Biotechnol., 11: 379-383 (1993)]; (xix) an inhibitor of BMP antagonism including, but not limited to, sclerostin, SOST, noggin, chordin, gremlin, and dan [see Massague and Chen, "Controlling TGF-beta signaling," Genes Dev., 14: 627-644, 2000; Aspenberg et al., J. Bone Miner. Res.16: 497-500, 2001; and Brunkow et al., Am. J. Hum. Genet.68: 577-89 (2001)]; (xx) Vitamin D, vitamin D derivatives, vitamin D analogs, including, but not limited to, D.sub.3 (cholecaciferol), D.sub.2 (ergocalciferol), 25-OH-vitamin D.sub.3, 1.alpha.,25(OH).sub.2 vitamin D.sub.3, 1.alpha.-OH-vitamin D.sub.3, 1.alpha.-OH-vitamin D.sub.2, dihydrotachysterol, 26,27-F6- 1.alpha.,25(OH).sub.2 vitamin D.sub.3, 19-nor-1.alpha.,25(OH).sub.2 vitamin D.sub.3, 22- oxacalcitriol, calcipotriol, 1.alpha.,25(OH).sub.2-16-ene-23-yne-vitamin D.sub.3 (Ro 23-7553), EB1089, 20-epi-1.alpha.,25(OH).sub.2 vitamin D.sub.3, KH1060, ED71, 1.alpha.,24(S)--(OH).sub.2 vitamin D.sub.3, 1.alpha.,24(R)--(OH).su- b.2 vitamin D.sub.3 [See, Jones G., "Pharmacological mechanisms of therapeutics: vitamin D and analogs," 1996. In: J. P. Bilezikian, et al. Ed. Principles of Bone Biology, San Diego: Academic Press] and vitamin D receptor ligand and analogues such as calcitriol, topitriol, ZK-150123, TEI-9647, BXL-628, Ro-26-9228, BAL-2299, Ro-65-2299 or DP- 035; (xxi) Vitamin K and Vitamin K derivatives, including, but not limited to, menatetrenone (vitamin K2) [see Shiraki et al., J. Bone Miner. Res., 15: 515-521 (2000)]; (xxii) soy isoflavones, including ipriflavone; (xxiii) dietary calcium supplements including, but not limited to, calcium carbonate, calcium citrate, and natural calcium salts (Heaney. Calcium.1996. In: J. P. Bilezikian, et al., Ed., Principles of Bone Biology, San Diego: Academic Press); (xxiv) fluoride salts, including, but not limited to, sodium fluoride (NaF) and monosodium fluorophosphate (MFP); (xxv) androgen receptor modulators, such as those disclosed in Edwards, J. P. et. al., Bio. Med. Chem. Let., 9, 1003-1008 (1999) and Hamann, L. G. et. al., J. Med. Chem., 42, 210-212 (1999); a steroidal or nonsteroidal androgen receptor antagonist, including, but not limited to, enzalutamide, ARN-509, flutamide, hydroxyflutamide, bicalutamide, nilutamide, or hydroxysteroid dehydrogenase inhibitor or abiraterone; a reversible antiandrogen; or a SARM agent, including, but not limited to those disclosed herein, RU-58642, RU-56279, WS9761 A and B, RU-59063, RU-58841, bexlosteride, LG-2293, L-245976, LG-121071, LG-121091, LG-121104, LGD-2226, LGD-2941, LGD-3303, LGD-4033, YM-92088, YM-175735, LGD-1331, BMS-357597, BMS-391197, S-40542, S-40503, BMS-482404, EM-4283, EM-4977, BMS-564929, BMS-391197, BMS-434588, BMS- 487745, BMS-501949, SA-766, YM-92088, YM-580, LG-123303, LG-123129, PMCol, YM-175735, BMS-591305, BMS-591309, BMS-665139, BMS-665539, CE-590, 116BG33, 154BG31, arcarine, or ACP-105; (xxvi) an antiemetic drug including, but not limited to, a dopamine antagonist such as, for example, domperidone droperidol, chlorpromazine, promethazine, or metoclopramide; or an antihistamine such as, for example, cyclizine, diphenhydramine, dimenhydrinate, or meclizine; or tropisetron; (xxvii) erythropoietin, including obtained by natural sources (e.g., urinary erythropoietin; See U.S. Pat. No.3,865,801), or recombinantly produced protein and analogs thereof, for example, as described in U.S. Pat. Nos.5,441,868, 5,547,933, 5,618,698 and 5,621,080 as well as human erythropoietin analogs with increased glycosylation and/or changes in the amino acid sequence as those described in European Patent Publication No. EP 668351 and the hyperglycosylated analogs having 1-14 sialic acid groups and changes in the amino acid sequence described in PCT Publication No. WO 91/05867, including erythropoietin-like polypeptides comprise darbepoietin (from Amgen; also known as Aranesp and novel erthyropoiesis stimulating protein (NESP)); (xxviii) an immunomodulating agent, including, but not limited to, immunosuppressive cytotoxic drugs, such as, for example, mechlorethamine, chlorambucil; immunosuppressive agent such as, for example, mycophenolate motefil or 6-thioguanine, including those which can optionally be administered topically such as tacrolimus, pimecrolimus, imiquimod, 5-fluorouracil, or mechlorethamin; immunostimulatory agents such as, for example, a non-specificimmunostimulator for example Freund's complete adjuvant, Freund's incomplete adjuvant, a montanide ISA adjuvant, a Ribi's adjuvant, a Hunter's TiterMax, an aluminum salt adjuvant, a nitrocellulose-adsorbed protein, a Gerbu Adjuvant; (xxix) a retinoid, including, but not limited to, isotretinoin, acitretin, tretinoin, adapalene, tazarotene, bexarotene, alitretinoin, or beta-carotene; (xxx) an antacid agent; (xxxi) a 17-beta hydroxysteroid dehydrogenase inhibitor; (xxxii) an anti-rheumatic drug, including, but not limited to, chloroquine, hydroxychloroquine, , sulfasalazine, cyclosporine, sulfasalazine, aurothioglucose, gold sodium thiomalate, or auranofin; (xxxiii) a gene therapy agent, including but not limited to, an antisense agent such as, for example, anti-sense oligonucleotides; or a replacement gene; (xxxiv) a PDE5 inhibitor, for example sildenafil, tadalafil or vardenafil; (xxxv) strontium ranelate (xxxvi) a chemotherapeutic agent and/or therapy, including but not limited to, ifosfamide, adriamycin, doxorubicin, cyclosporine; (xxxvii) an MMP inhibitor; (xxxviii) an anti-thyroid agent, including, but not limited to, thyroid hormone supplement thyroxine, L-thyroxine; (xxxix) an angiotensin converting enzyme (ACE) inhibitor, including, but not limited to, benazepril, captopril, cilazapril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril, or enalaprilat; or angiotensin II antagonists such as, for example, losartan; (xl) a neurodegenerative disorder medication including, but not limited to, acetylcholinesterase inhibitor such as, for example, tacrine, donepezil, galanthamine, or rivastigmine; N-methyl-D-aspartate (NMDA) antagonist such as, for example, memantine; dopaminergic agonist; AMPA regulator; cholinesterase inhibitor; dopaminergic drugs such as, for example, amantadine, biperiden, bromocriptine, entacapone, selegiline/deprenyl, iphenhydramine, pergolide, procyclidine, selegiline, or trihexyphenidyl; gamma secretase inhibitor; or A beta lowering drug; riluzole; an agent which silences the gene that causes the progression of the disease; or a cholinesterase inhibitor, including but not limited to a quaternary ammonium agent, such as, for example, edrophonium or ambenonium; (xli) an anti-hypercholesterolemic agent including, but not limited to, a cholesterol absorption inhibitors, such as, for example, SCH-58235, also known as ezetimibe; 1-(4- fluorophenyl)-3(R)-[3(S)-(4-fluorophenyl- )-3-hydroxypropyl)]-4(S)-(4-hydroxyphenyl)-2-azetidinone, described in U.S. Pat. Nos.5,767,115 and 5,846,966; niacin-lovastatin; colestipol HCl; sodium, gemfibrozil; cholestyramine; cholestyramine light; colesevelam HCl; (xlii) an adrenomimetic drug, such as a beta-adrenoceptor agonist, alpha- adrenoceptor agonist, In one embodiment, the adrenomimetic drug is a catecholamine. In one embodiment, adrenomimetic drugs include but are not limited to isoproterenol, norepinephrine, epinephrine, ephedrine, or dopamine. In one embodiment, the adrenomimetic drug is a directly acting adrenomimetic drug. In some embodiments, directly acting adrenomimetic drugs include but are not limited to phenylephrine, metaraminol, or methoxamine; (xliii) an appetite stimulants such as megestrol acetate, cyproheptadine; (xliv) a luteinizing hormone releasing hormone (LHRH), a LHRH analog or derivative, a luteinizing hormone agonists or antagonists including, but not limited to, letrozole, anastrazole, atamestane, fadrozole, minamestane, exemestane, plomestane, liarozole, NKS-01, vorozole, YM-511, finrozole, 4-hydroxyandrostenedione, aminogluethimide, or rogletimide; (xlv) a vitronectin receptor antagonist; (xlvi) a Src SH2 antagonists or a Src kinase inhibitors; (xlvii) a protein synthesis inhibitor including, but not limited to, abrin, aurintricarboxylic acid, colicin E3, cycloheximide, diphtheria toxin, edeine A, emetine, erythromycin, ethionine, 5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate and guanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, and O-methyl threonine, modeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin, ricin, .alpha.-sarcin, shiga toxin, showdomycin, sparsomycin, spectinomycin, streptomycin, thiostrepton; (xlviii) an inhibitor of an enzyme involved in the androgen biosynthetic pathway, including, but not limited to, 17-ketoreductase inhibitor, a 17-aldoketoreductase inhibitor, a3- .DELTA.H4,6-isomerase inhibitor, a 3-.DELTA.H4,5-isomerase inhibitor, a 17,20 desmolase inhibitor, a p450c17 inhibitor, a p450ssc inhibitor, a 17.beta.-hydroxysteroid dehydrogenase inhibitor, or a 17,20-lyase inhibitor such as abiraterone; (xlix) an anti-inflammatory agent, including, but not limited to, non-steroidal anti- inflammatory agents such as salsalate, diflunisal, ibuprofen, fenoprofen, flubiprofen, fenamate, ketoprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, sulindac, tolmetin, etodolac, ketorolac, oxaprozin, or celecoxib, cyclooxygenase-2 inhibitors, such as rofecoxib and celecoxib; 5-amino-salicylate, corticosteroid, metronidazole, ciprofloxacin, infiximab, budesonide, or anti-TNF alpha antibody; (l) an anti- diabetic agent, including, but not limited to, a sulfonylurea, such as, for example tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glyburide, glimepiride, or gliclazide; a meglitinide, for example prandin or nateglinide; a biguanide, such as, for example metformin; a thiazolidinedione such as, for example rosiglitazone, pioglitazone, or troglitazone; (lii) an analgesic agent, including, but not limited to, paracetamol; (liii) an expectorant, including, but not limited to a mucolytic agent; (liv) an anti-estrogen; (lv) an antiviral agent, including, but not limited to, abacavir, acyclovir, amantadine, didanosine, emtricitabine, enfuvirtide, entecavir, lamivudine, nevirapine, oseltamivir, ribavirin, rimantadine, stavudine, valaciclovir, vidarabine, zalcitabine, or zidovudine;nucleotide analog reverse transcriptase inhibitor such as, for example, otenofovir or adefovir; or interferon alpha; a protease inhibitor include but are not limited to saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, fosamprenavir, or tipranavir; (lvi) a cortisone, cortisol, icortisone, corticosterone, corticosteroid, glucocorticosteroid including, but not limited to, including glucocorticoid or analogues thereof, corticotrophin,cyclosporine, cyclophosphamide, tacrolimus--FK-506, anti-thymocyte globulin, mycophenylate prednisone or dexamethasone moeftil, betamethasone dipropionate, clobetasol, diflorasone, amcinonide, desoximetasone, fluocinonide, aclometasone, desonide triamcinolone, fluticasone, halobetasol, mometasone, or hydrocortisone, prednisone; or steroidal or nonsteroidal glucocorticoid receptor ligands, such as, ZK-216348, ZK-243149, ZK-243185, LGD-5552, mifepristone, RPR-106541, ORG-34517, GW-215864.times., Sesquicillin, CP-472555, CP-394531, A-222977, AL-438, A-216054, A-276575, CP-394531, CP-409069, UGR-07; (lvii) somatostatin analogue or agents which inhibit somatostatin or its release, including, but not limited to, physostigmine and pyridostigmine; (lviii) a Bax activity modulator such as alisol B acetate; (lix) a cytokine, including, but not limited to, IL-3, IL-7, GM-CSF, anticytokine antibodies, cytokine inhibitors; (lx) an insulin, including but not limited to, short-, intermediate-, and long acting formulations; (lxi) insulin-sensitizers, including but not limited to, biguanides such as, for example, metformin; (lxii) gonadotropin; gonadotropin-releasing hormone or analogue or derivatives thereof; gonadotropin-releasing hormone agonists or antagonists, including, but not limited to, leuprolide, goserelin, triptorelin, alfaprostol, histrelin, detirelix, ganirelix, antide iturelix, cetrorelix, ramorelix, ganirelix, antarelix, teverelix, abarelix, ozarelix, sufugolix, prazarelix, degarelix, NBI- 56418, TAK-810, acyline; (lxiii) a ghrelin, a ghrelin receptor ligand or analogs thereof, including, but not limited to, human ghrelin, CYT-009-GhrQb, L-692429, GHRP-6, SK&F-110679, or U-75799E, leptin, metreleptin, pegylated leptin; a leptin receptor agonist, such as LEP(116-130), OB3, [D- Leu4]-0B3, rAAV-leptin, AAV-hOB, rAAVhOB; or a steroidal or nonsteroidal GR ligand; (lxiv) a 5a-Reductase Inhibitor, including, but not limited to, finasteride, dutasteride, izonsteride; (lxv) an aromatase inhibitor, including, but not limited to, letrozole, anastrazole, atamestane, fadrozole, minamestane, exemestane, plomestane, liarozole, NKS-01, vorozole, YM- 511, finrozole, 4-hydroxyandrostenedione, aminogluethimide, rogletimide; (lxvi) an agent for treating an ophthalmic disease, including, but not limited to, betagan, betimol, timoptic, betoptic, betoptic, ocupress, optipranolol, xalatan, alphagan, azopt, trusopt, cospot, pilocar, pilagan, propine, opticrom, acular, livostin, alomide, emadine, patanol, alrex, poly-pred, pred-g, dexacidin, erythromycin, maxitrol, FML, ocufen, voltaren, profenal, pred forte, betadine, gramicidin, prednisolone, betaxolol, humorsol, proparacaine, betoptic, hylartin, flurbiprofen, methazolamide, timolol, terramycin, ciprofloxacin, miostat, triamcinolone, miconazole, tobramycin, physostimine, gentamicin, pilocarpine, goniosol, oxytetracycline, viroptic, suprofen, celluvisc, ciloxan, ocuflox, brinzolamide, cefazolin, tobrex, latanoprost, indocycanine, trifluridine, phenylephrine, demecarium, neomycin, tropicamide, dexamethasone, neptazane, dipivefrin, vidarabine, dorzolamide, ofloxacin, epinephrine, acyclovir, carbonic anhydrase inhibitor, vitamin A, zinc, copper, atropine, flarex, eflone, illotycin, or garamycin; (lxvii) an adrenoceptor antagonist including, but not limited to, a haloalkylamine, such as, for example, phenoxybenzamine; imidazoline, such as, for example, phentolamine or tolazoline; quinazoline such as, for example, prazosin, terazosin, doxazosin, or trimazosin; or an agent with combined alpha and blocking activity, such as, for example, labetalol, bucindolol, carvedilol, or medroxalol; (lxviii) a progestin, a progestin deriviative or analog, a synthetic progestin, progesterone, progesterone receptor agonists ("PRA"), such as levonorgestrel, medroxyprogesterone acetate (MPA)prostaglandins (for osteo) or steroidal or nonsterodial progesterone receptor ligands; (lxix) an alpha glucosidase inhibitor such as acarbose, miglitol; (lxx) an anti-arrhythmic agent including, but not limited to, a sodium channel blocker such as, for example, quinidine, procainamide, disopyramide, lidocaine, tocamide, mexiletine, encamide, or flecamide; a beta-adrenergic blocker, such as, for example acebutolol, esmolol, or sotalol; or an agent that prolong repolarization, such as, for example, amiodarone; adenosine or digoxin; (lxxi) an agent wich interferes with tumor necrosis factore, including, but not limited to, etanercept; (lxxii) a beta-blocker, including, but not limited to, acebutolol, atenolol, betaxolol hydrochloride, bisoprolol fumarate, carteolol hydrochloride, carvedilol, celiprolol hydrochloride, esmolol hydrochloride, labetalol hydrochloride, levobunolol, metoprolol tartrate, metipranolol, nadolol, nebivolol, oxprenolol hydrochloride, pindolol, sotalol hydrochloride, or timolol maleate; (lxxiii) a photochemotherapy agent including, but not limited to, PUVA or psoralen such as oxsoralen; (lxxiv) a photodynamic agent, including, but not limited to, porphyrin; (lxxv) an anti-diuretic hormone or antidiuretic hormone analogue; (lxxxvi) a steroidal or nonsteroidal AR antagonists such as flutamide, hydroxyflutamide, bicalutamide, nilutamide, enzalutamide, ARN-509; (lxxxvii) a myostatin antibody or a myostatin analog; (lxxxviii) a RANK ligand monoclonal antibody (mAb), including, but not limited to, denosumab (Prolia.TM.) formerly AMG162 (Amgen) (lxxxix) a diuretic, including, but not limited to thiazide diuretic, such as, for example, bendrofluazide, bendroflumethiazide, benzthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, Diucardin.RTM., Diuril.RTM., Enduron.RTM., Esidrix.RTM., Exna.RTM., HCTZ, Hydrochlorothiazide, HydroDIURIL.RTM., hydroflumethiazide, Hydromox.RTM., Hygroton.RTM., indapamide, Lozol.RTM., methyclothiazide, metolazone, Mykrox.RTM., Naqua.RTM., Naturetin.RTM., Oretic.RTM., polythiazide, quinethazone, Renese.RTM., trichlormethiazide, xipamide, or Zaroxolyn.RTM; a loop diuretic such as, for example, furosemide, bumetanide, or torsemide; a potassium-sparing diuretic such as, for example, amiloride, triamterene, aldosterone antagonists, or spironolactone; organomercurial, ethacrynic acid, furosemide, bumetanide, piretanide, muzolimine, chlorothiazide and thiazide, phthalimidine, chlorthalidone, clorexolone, quinazolinone, quinethazone, metolazone ilenzenesulphonamide, mefruside, chlorobenzamide, clopamidesalicylamide, xipamide, xanthine, aminophylline, carbonic anhydrase inhibitor, acetazolamide mannitol, potassium-sparing compound, aldosterone antagonist, spironolactone; (xc) a steroid, including, but not limited to, an androgenic/anabolic steroid such as testosterone/oxandrolone; (xci) a proteasome inhibitor; (xcii) a melanocortin 4 receptor agonist, including, but not limited to, bremelanotide; (xciii) a squalene epoxidase inhibitor or a squalene synthetase inhibitors (also known as squalene synthase inhibitors); (xciv) a calcium channel blocker, including but not limited to, verapamil, diltiazem, or mebefradil; (xcv) a mineral, including, but not limited to, selenium, magnesium, zinc, chromium, calcium, potassium, platinum or derivatives or salts thereof; (xcvi) a calcium receptor antagonist; (xcvii) a beta-2 agonist; (xcviii) an anti-cholinergic bronchodilator, including, but not limited to, theophylline, aminophylline; (xcix) a vasoactive agent or an inotrope including, but not limited to, digoxin, dopamine, dobutamine, hydralazine, prazosin, carvedilol, nitroprusside, nitroglycerin, lisinopril, diltiazem, hydrochlorothiazide, furosemide, spironolactone, AT-1 receptor antagonists (e.g., losartan, irbesartan, valsartan), ET receptor antagonists (e.g., sitaxsentan, atrsentan and compounds disclosed in U.S. Pat. Nos.5,612,359 and 6,043,265), Dual ET/AII antagonist (e.g., compounds disclosed in WO 00/01389), neutral endopeptidase (NEP) inhibitors, vasopepsidase inhibitors (dual NEP-ACE inhibitors) (e.g., omapatrilat and gemopatrilat), or nitrates; (xcx) a melanocortin 4 receptor antagonist. Examples of melanocortin 4 receptor (MC4R) antagonists include, for example, those in U.S. Provisional Application No.63/036,798 filed June 09, 2020, and U.S. Provisional Application No.63/167,271 filed March 29, 2021. An example of melanocortin 4 receptor antagonist is selected from: (2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1; 2-(6-methoxy-2-methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1; 2-[6-(difluoromethoxy)pyridin-3-yl]-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2; 1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'- yl]-2-[4-(trifluoromethyl)phenyl]propan-1-one, DIAST-1; 1-(4,7-dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4- fluorophenyl)ethan-1-one, DIAST-1; (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one; (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one; (2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one; (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1; and (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-{(2S)-7-methyl-6-[(4,6-2H2)pyrimidin-2-yl]-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl}propan-1-one,or pharmaceutically acceptable salt thereof. (c) an anticancer agent including, but not limited to, (a) a monoclonal antibody, which antibody may be optionally used for diagnosis, monitoring, or treatment of cancer, including monoclonal antibodies which react against specific antigens on cancer cells such as the monoclonal antibody acts as a cancer cell receptor antagonist, those which monoclonal antibodies enhance the patient's immune response, those which act against cell growth factors, thus blocking cancer cell growth, those which are conjugated or linked to anti-cancer drugs, radioisotopes, other biologic response modifiers, other toxins, or a combination thereof; (b) a selective tyrosine kinase inhibitor including those embodiments where the selective tyrosine kinase inhibitor inhibits catalytic sites of cancer promoting receptors thereby inhibiting tumor growth; the selective tyrosine kinase inhibitor modulates growth factor signaling; the selective tyrosine kinase inhibitor targets EGFR (ERB B/HER) family members; the selective tyrosine kinase inhibitor is a BCR-ABL tyrosine kinase inhibitor; the selective tyrosine kinase inhibitor is an epidermal growth factor receptor tyrosine kinase inhibitor; the selective tyrosine kinase inhibitor is a vascular endothelial growth factor tyrosine kinase inhibitor; the selective tyrosine kinase inhibitor is a Platelet Derived Growth Factor (PDGF) inhibitor; (c) an alkylating agent (d) a vinca alkaloid, including, but not limited to, vindesine (e) platinum compounds, including, but not limited to, carboplatin (f) taxanes, including, but not limited to, docetaxel (g) antineoplastic agents, including, but not limited to, alkylating agents such as, for example, alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, carboquone, meturedepa and uredepa; ethylenimines and methylmelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylol melamine; nitrogen mustards such as chlorambucil, chlomaphazine, estramustine, iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitroso ureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol and pipobroman, hormonal antineoplastics and antimetabolites; (h) inhibitors of DNA synthesis, including alkylating agents such as dimethyl sulfate, mitomycin C, nitrogen and sulfur mustards, MNNG and NMS; intercalating agents such as acridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene, ethidium bromide, propidium diiodide-intertwining, and agents such as distamycin and netropsin; (i) DNA base analogs such as acyclovir, adenine, .beta.-1-D-arabinoside, amethopterin, aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine, 6-azauracil, 2'-azido- 2'-deoxynucliosides, 5-bromodeoxycytidine, cytosine, .beta.-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides, 5-fluorodeoxycytidine, 5-fluorodeoxyuridine, 5- fluorouracil, hydroxyurea and 6-mercaptopurine; (j) topoisomerase inhibitors, such as coumermycin, nalidixic acid, novobiocin and oxolinic acid, inhibitors of cell division, including colcemide, vinblastine and vincristine; and RNA synthesis inhibitors including actinomycin D, .alpha.-amanitine and other fungal amatoxins, cordycepin (3'-deoxyadenosine), dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin and streptolydigin; (k) an ER antagonist, including, but not limited to, fulvestrant; (l) a cancer vaccine, including a therapeutic vaccine thus, treating an existing cancer; a a prophylactic vaccine thus, preventing the development of cancer, which vaccine may be a antigen/adjuvant vaccine, or a whole cell tumor vaccine, or a dendritic cell vaccine. In one embodiment, the cancer vaccine comprises viral vectors and/or DNA vaccines, including those embodiments where the cancer vaccine is an idiotype vaccine; (ci) a cholesterol acyltransferase (ACAT) inhibitors including selective inhibitors of ACAT-1 or ACAT-2 as well as dual inhibitors of ACAT-1 and -2; (cii) an amylin analogue such as pramlintide; (ciii) a cholesteryl ester transfer protein or CETP Inhibitor, including, but not limited to, JTT-705, CETi-1; (civ) a vasodilator; (cv) an anti-anginal agent including, but not limited to, nifedipine; (cvi) a glucagon-like peptide-1 (GLP-1) and analogues, including, but not limited to, exenatide or liraglutide; (cvii) a H.sub.2-receptor antagonist, including, but not limited to, cimetidine and ranitidine, famotidine, or nizatidine (cviii) a hypocholesterolemic agent; (cix) an anti-hypertensive including, but not limited to, methyldopa, reserpine, clonidine, and verapamil; (cx) a AR partial antagonists, including, but not limited to, spironolactone, eplerenone; (cxi) an endothelin antagonist; (cxii) a vacuolar-H.sup.+-ATPase inhibitor; (cxiii) a alpha.nu.beta.3 Integrin receptor antagonist; (cxiv) an agent to decrease prostate (benign or malignant) hypertrophy; (cxv) a microsomal triglyceride transfer protein (MTP) inhibitor; (cxvi) a FSH agonist/antagonist; (cxvii) a colchicine; (cxviii) a LDL (low density lipoprotein) receptor inducer; (cxix) an agent such as a LXR ligand that enhances ABC1 gene expression; (cxx) a steroidal or nonsterodial PR ligand; (cxxi) a cytotoxic antibiotic; (cxxii) an antimetabolite; (cxxiii) an analgesic agent; (cxxiv) a cholinomimetic agent, including, but not limited to, a direct-acting parasympathomimetic drug such as, for example, methacholine, pilocarpine, carbachol, or bethanechol (cxxv) a selective serotonin receptor inhibitor; (cxxvi) a serotonin norepinephrine receptor inhibitor; (cxxvii) an anti-infective agent; (cxxviii) a AT-II receptor antagonist, including, but not limited to, valsartan or telmisartan; (cxxix) an agent treating neuromuscular transmission, a nervous system stimulant; (cxxx) androgen deprevation therapy; (cxxxi) a muscarinic blocking agent , including, but not limited to, belladonna alkaloid such as, for example, atropine or scopolamine; (cxxxii) a 5-HT.sub.3 receptor antagonist including, but not limited to, dolasetron, granisetron, ondansetron; (cxxxiii) a beta-3 adrenergic agonist; (cxxxiv) a DPP-IV inhibitor, including, but not limited to, vildagliptin or sitagliptin; (cxxxv) a pancreatic lipase inhibitor, including, but not limited to, orlistat, cetilistat; (cxxxvi) a muscle relaxant including but not limited to methocarbamol, baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, dantrolene, metaxalone, orphenadrine, amyl nitrite, pancuronium, tizanidine, clonidine, or gabapentin; (cxxxvii) a vasoconstrictor agent including, but not limited to, adrenalin dimethylarginine, caffeine, cannabis, catecholamines, decongestants, pseudoephedrinse, norepinephrines, tetrahydrozoline, or thromboxane; (cxxxviii) a fusion inhibitor such as enfuvirtide; (cxxxix) a SGLT (sodium-dependent glucose transporter 1) inhibitor; (cxl) a FBPase (fructose 1,6-bisphosphatase) inhibitor; (cxli) a dipeptidyl peptidase IV (DPP4) inhibitors such as those disclosed in WO 01/68603; (cxlii) a fibrinogen receptor antagonist; (cxliii) coenzyme Q10; (cxliv) folic antioxidants; (cxlv) one or more nucleic acids which encode bone-stimulating compounds; (cxlvi) acyl-coenzyme A; or (cxlvii) an HDL-elevating agent including, but not limited to, 1-hydroxyalkyl-3- phenylthiourea, and analogs thereof; (cxlviii) an antimuscarinic agent including, but not limited to, tolterodine or fesoteridine, or (cxlix) an alpha 2 delta agent including, but not limited to, gabapentin or pregablin; or pharmaceutically acceptable salts or derivatives thereof. In certain embodiments, a metabolite disclosed herein, or a pharmaceutically acceptable salt thereof, is combined with two, three, four or more additional therapeutic agents. The two, three four or more additional therapeutic agents can be different therapeutic agents selected from the same class of therapeutic agents, or they can be selected from different classes of therapeutic agents. When different components/APIs (active pharmaceutical ingredients) in a combination of the present invention are administered together, such administration is simultaneous. In some embodiments, simultaneous administration of drug combinations is used. For a separate administration, each component/API may be administered in any order and each of them can be administered in an independent frequency or dose regimen. In some embodiments, such administration be oral. In some embodiments, such administration can be oral and simultaneous. When different components/APIs are administered separately (including, for example, sequentially), the administration of each may be by the same or by different methods. In some embodiments, administration of one component/API is oral but administration of another component/API is not oral (for example, is injectable). In certain embodiments, when a metabolite disclosed herein is combined with one or more additional therapeutic agents as described above, the components of the composition are administered as a simultaneous or separate (e.g. sequential) regimen. When administered sequentially, the combination may be administered in two or more administrations. In certain embodiments, a metabolite disclosed herein is combined with one or more additional therapeutic agents in a unitary dosage form for simultaneous administration to a patient, for example as a solid dosage form for oral administration (e.g., a fixed dose combination tablet). In certain embodiments, a metabolite disclosed herein is administered with one or more additional therapeutic agents. Co-administration of a metabolite disclosed herein, or a pharmaceutically acceptable salt thereof, with one or more additional therapeutic agents generally refers to simultaneous or separate (e.g. sequential) administration of a compound disclosed herein and one or more additional therapeutic agents, such that therapeutically effective amounts of the metabolite and one or more additional therapeutic agents are both present in the body of the patient. Co-administration includes administration of unit dosages of the metabolites disclosed herein before or after administration of unit dosages of one or more additional therapeutic agents, for example, administration of the metabolites disclosed herein within seconds, minutes, or hours of the administration of one or more additional therapeutic agents. For example, in some embodiments, a unit dose of a metabolite disclosed herein is administered first, followed within seconds or minutes by administration of a unit dose of one or more additional therapeutic agents. Alternatively, in other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed by administration of a unit dose of a metabolite disclosed herein within seconds or minutes. In some embodiments, a unit dose of a metabolite disclosed herein is administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of one or more additional therapeutic agents. In other embodiments, a unit dose of one or more additional therapeutic agents is administered first, followed, after a period of hours (e.g., 1-12 hours), by administration of a unit dose of a metabolite disclosed herein. Pharmaceutical Formulations and Dosage Forms The pharmaceutical compositions disclosed herein can be prepared by methodologies well known in the pharmaceutical art. For example, in certain embodiments, a pharmaceutical composition intended to be administered by injection can prepared by combining a metabolite of the invention with sterile, distilled water so as to form a solution. In some embodiments, a surfactant is added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system. The metabolites of the invention, or their pharmaceutically acceptable salts, can be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. EXAMPLES Example X-1: Investigation of Metabolism of Compound 1 (in the form of its tris salt) in a Clinical Study Study Design and Objectives The metabolism of Compound 1 (dosed in its free form) was investigated in humans. A primary objective of this study was to gain a preliminary assessment of the human metabolites of Compound 1 using plasma and urine sample after repeat dosing (multi-dose). Plasma and urine samples from five human subjects following oral administration of 100 mg BID for 14 days in a clinical study were examined for metabolites. MATERIALS AND METHODS Multiple Dose Metabolite Identification in Human Plasma And in Human Urine Human plasma and urine samples were acquired from five individual subjects in a multiple dose study. The five subjects were dosed every 12 hours (BID oral dose) for 14 days at 100 mg. Both plasma and urine were from Day 14 collected 0 to 12 hours postdose. Plasma from individual subjects were pooled in proportion to the time period represented by each sampling interval to yield a composite sample for profiling that is representative of Caverage (AUC 0-12h) according to the method of Hamilton et al. [Hamilton RA, Garnett WR, Kline BJ. Determination of a mean valproic acid serum level by assay of a single pooled sample. Clin Pharmacol Ther 1981; 29(3):408-13]. A 1 mL aliquot from each subject’s composite sample was combined to make a multi-subject composite sample. An equivalent aliquot from each subject’s Day 1, 0 h sample was pooled to act as a blank control sample. To these pooled samples was added 1 mL of water followed by 20 mL of acetonitrile. The samples were vortexed, centrifuged at 1850 x g for 5 minutes; the supernatants were transferred to clean test tubes, and evaporated in vacuo to near dryness (Genevac®). The residue was reconstituted in 0.2 mL of mobile phase (90% 1% formic acid/10% acetonitrile). The reconstituted samples were transferred to micro-centrifuge tubes and centrifuged at 16,000 x g for 3 minutes. The supernatants were transferred to limited volume insert and injected (50 µL) on HPLC-UV-MS for analysis. Urine from individual subjects were pooled in proportion to the volume excreted for the time interval for each subject to yield a pool for profiling that is representative of 0-12 h. An equivalent aliquot from each subject’s Day 1, 0 h sample was pooled to act as a blank control sample. The samples were vortexed, centrifuged at 16,870 x g for 3 minutes; the supernatants were transferred to limited volume inserts and injected (50 µL) on HPLC-UV-MS for analysis. HPLC/UV/MS Sample Analysis The multi-dose human plasma and urine samples were analyzed by positive ion HPLC-UV- MS using a Thermo Fisher Scientific LTQ-Obitrap mass spectrometer. The HPLC system consisted of an Accela quaternary solvent delivery pump, an Accela autosampler, and an Accela PDA Plus photodiode array detector. A Polaris C18 column (250 x 4.6 mm, 5 µm) was used with a flow rate of 0.8 mL/min. The mobile phase was comprised of 0.1% formic acid (A) and acetonitrile (B). The gradient system used was as follows: initially, 95% A held for 5 minutes followed by a linear gradient to 90% B at 35 minutes, held for 5 minutes, and then re-equilibration at 95/5 A/B for 15 minutes. UV spectra were collected from 200-400 nm. The mass spectrometer was operated in the positive ion mode with an ESI (Electron Spray Ion) source. Capillary temperature was set at 275°C and the source potential was 5000V. Other potentials were optimized to get optimal ionization and fragmentation of the parent. Sheath, auxiliary, and sweep gas flows were set to 20, 10, and 5 arbitrary units, respectively. Full scan mass spectra were acquired over the range of 100– 800 m/z at 15,000 resolving power (specified at m/z 400). Data-dependent scanning was used to trigger MS2 and MS3 analysis of molecular ions over a threshold intensity at a resolving power of 7,500 using CID fragmentation with an isolation width of 2 Da and a normalized collision energy of 35%. RESULTS AND ANALYSES The biotransformation of Compound 1 was studied in the human plasma and urine samples from the Day 14100 mg BID multiple dose group. An examination of the HPLC-UV chromatograms and extracted ion chromatograms for both the predose and the pooled plasma and urine samples from the multi-dose human study are in Figures 1 and 2, respectively. A summary listing of metabolites observed in the multi-dose human plasma samples is in Table 0-1. Table E1-2 summarizes the metabolites observed in the multi--dose human urine samples In plasma, parent (Compound 1) and two metabolites were observed by UV detection: M1 (N-glucuronide of the parent) and M2 (thiadiazinane ring opened methyl propanoic acid). Urine was found to contain both M1 and M2 and a hydroxy glucuronide metabolite. A total of 8 metabolites were observed in plasma (see table below). O-debenzylation to Metabolite/Compound M2 was the major circulating metabolite. Seven additional minor metabolites observed in circulation were a single hydroxylation (Compound M4), oxidation and ring opening of the piperidine ring (Compound 574a), oxidation and ring opening of the oxetane ring (Compound 574b), N-dealkylation (Compound M3), acyl glucuronide (Compound M1), and two O- glucuronides (Compounds 748a and 748b). Each of the minor metabolites individually represented less than 10% of the total circulating drug related material based on UV response.
Table E1-1. Metabolite Summary of Compound 1 in Human Day 14 Pooled Extracted Plasma Samples in Multi-dose Subjects (100 mg BID for 14 Days)
Figure imgf000047_0001
Table E1-2. Metabolite Summary of Compound 1 in Human Day 14 Pooled Extracted Urine Samples in Multi-dose Subjects (100 mg BID for 14 Days)
Figure imgf000047_0002
A. Compound 1 (m/z 303). A CID mass spectrum generated for Compound 1 in the LTQ-Orbitrap is shown in Figure 3 with some of the diagnostic fragments assigned. The major fragment ion at m/z 232 (radical cation) corresponds to the loss of the butyl-amine portion of the molecule. The ion at m/z 239 resulted from a neutral loss of sulfur dioxide. Fragmentation through the thiadiazinane ring yields m/z 210 (cation) and 197 (radical cation).
Figure imgf000048_0001
Compound 1 B. Metabolite/Compound M1 (m/z 479). The Collision-Induced Dissociation (CID) mass spectrum generated for M1 in the LTQ- Orbitrap is shown in Figure 4 with some of the diagnostic fragments assigned. The N-glucuronide metabolite M1 was detected as a major metabolite in human plasma and urine. An isolated Metabolite M1 sample was prepared and analyzed by NMR. The 1H spectrum of the isolated Compound 1-glucuronide (dissolved in dimethyl sulfoxide-d6) is contained in Figure 5. The presence of the glucuronic acid is confirmed both in the TOCSY data (Figure 6) and the HSQC data (Figure 7). Within the TOCSY data, there is a resonance at 4.71 (d, j=8.7 Hz, Integration 1H) that has cross peaks to resonances at 3.23, 3.50 and 5.23. In the HSQC data the resonance at 4.71 correlates to a carbon chemical shift of 87.2. These resonances (1H 4.71 and 13C 87.2) are attributed to the anomeric CH of the Glucuronide. Additionally, the TOCSY data contain cross peaks from a doublet at 0.9 ppm to resonances at 2.61, 3.40 and 3.81 ppm. These are assigned as H4, H3 and H5 respectively. All of the two-dimensional NMR data is consistent with M1 being a glucuronide of Compound 1. The specific site of attachment is assigned at the 2 position of the thiadiazinane. This is based on the absence of the NH resonance (7.76 ppm) in the 1H spectrum of the metabolite. All collected NMR data is consistent with the proposed structure. C. Metabolite/Compound M2 (m/z 256). The CID mass spectrum generated for M2 in the LTQ-Orbitrap is shown in Figure 8Error! Reference source not found. with some of the diagnostic fragments assigned. M2 was detected as a minor metabolite in human plasma and urine. Metabolite M2 has a protonated molecular ion of m/z 256.1081 (0.2327 ppm) which is consistent with the empirical formula of C14H14O2N3 (Figure 8). The MS/MS spectrum yields fragment ions of 238, 196, and 182. The fragments at m/z 238 and 182 corresponds to the neutral loss of water and propionic acid, respectively. It is proposed to arise via hydrolysis of the thiadiazinane ring followed by subsequent oxidations. A sample of isolated metabolite M2 (dissolved in acetonitrile–d3) was submitted for 1H NMR analysis for definitive structure elucidation. The 1H spectrum of the isolated Metabolite M2 ( thiadiazinane ring open methyl propanoic acid) is contained in Figure 9. A resonance (broad singlet, Integration 1H) at 5.60 ppm is assigned as the NH of the ring open acid. This assignment is further supported by the TOCSY data (Figure 10Error! Reference source not found.). The TOCSY data set contains cross peaks that indicate, the 5.60 ppm resonance is coupled to a set of resonances at 3.51 and 3.32, which are strongly coupled to each other. These resonances, in turn are coupled to a resonances at 2.86 ppm which is finally coupled to a resonance that is a doublet f(J=7.5 Hz) at 1.25 ppm. All of this data is consistent with the thiadiazinane ring open methyl propanoic acid structure proposed for M2. D. Metabolite/Compound m/z 495. A metabolite possessing a protonated molecular ion at m/z 495.1180 (0.0079 ppm) consistent with the empirical formula C20H23O9N4S is proposed to be the addition of a hydroxy and a glucuronide to the parent/Compound 1 (or addition of a hydroxy and then glucuronidation through the added hydroxy). Fragmentation of m/z 495, yielded m/z 319 and subsequent fragmentation yielded 255, 239, 210, 182, and 170 (Figure Error! Reference source not found.11). The site of hydroxylation is proposed to be on the thiadiazinane ring; however, no definitive conclusions could be drawn from the fragmentation as to the exact location of the glucuronide (not wishing to be bound by any particular theory, it is believed that glucuronide is likely on the N atom of thiadiazinane ring). The hydroxy N-glucuronide metabolite was only detected in human urine. PREPARATIONS Preparations P1 – P33 describe preparations of some starting materials or intermediates that may be used for preparation of certain MC4R antagonists that can be used in the invention. Preparation P1 2-(5-Chloro-2-methoxypyridin-4-yl)propanoic acid (P1)
Figure imgf000050_0001
Step 1. Synthesis of methyl (5-chloro-2-methoxypyridin-4-yl)acetate (C1). A solution of lithium diisopropylamide in tetrahydrofuran (2 M; 1.9 L, 3.8 mol) was added in a drop-wise manner to a −30 °C solution of 5-chloro-2-methoxy-4-methylpyridine (197 g, 1.25 mol) in tetrahydrofuran (1.4 L). After the reaction mixture had been stirred at −30 °C for 1 hour, dimethyl carbonate (338 g, 3.75 mol) was added drop-wise; at the end of the addition, the reaction mixture was warmed to 25 °C and stirred for 1 hour. It was then poured into hydrochloric acid (0.5 M, 7 L, 3.5 mol) and extracted with ethyl acetate (2 x 1.5 L); the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification via silica gel chromatography (Gradient: 0% to 20% ethyl acetate in petroleum ether) provided C1 as a yellow oil. Yield: 203 g, 0.941 mol, 75%. LCMS m/z 216.1 (chlorine isotope pattern observed) [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.10 (s, 1H), 6.82 (s, 1H), 3.90 (s, 3H), 3.79 (s, 2H), 3.71 (s, 3H). Step 2. Synthesis of methyl 2-(5-chloro-2-methoxypyridin-4-yl)propanoate (C2). To a −78 °C solution of C1 (175 g, 0.812 mol) in tetrahydrofuran (1.2 L) was added a solution of sodium bis(trimethylsilyl)amide in tetrahydrofuran (2 M; 455 mL, 0.910 mol) in a drop- wise manner. The reaction mixture was stirred at −78 °C for 1 hour, whereupon a solution of iodomethane (172.6 g, 1.216 mol) in tetrahydrofuran (100 mL) was added drop-wise at −78 °C, and stirring was continued at this temperature for 2 hours. The reaction mixture was then poured into saturated aqueous ammonium chloride solution (500 mL) and extracted with ethyl acetate (2 x 100 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo to provide C2 as a brown oil. By NMR and LCMS analysis, this material was contaminated with some of the dimethylated side product methyl 2-(5-chloro-2-methoxypyridin-4-yl)-2-methylpropanoate. Yield: 136 g, ≤0.592 mol, ≤73%. LCMS m/z 230.1 (chlorine isotope pattern observed) [M+H]+.1H NMR (400 MHz, methanol- d4), product peak only: δ 8.10 (s, 1H), 6.76 (s, 1H), 4.10 (q, J = 7.2 Hz, 1H), 3.89 (s, 3H), 3.69 (s, 3H), 1.48 (d, J = 7.2 Hz, 3H). Step 3. Synthesis of 2-(5-chloro-2-methoxypyridin-4-yl)propanoic acid (P1). To a 25 °C solution of C2 (168 g, 0.732 mol) in tetrahydrofuran (1 L) was added, in a drop- wise manner, a solution of lithium hydroxide monohydrate (61.4 g, 0.146 mol) in water (300 mL) at 25 °C. The mixture was stirred for 2 hours, whereupon it was concentrated in vacuo. The aqueous residue was poured into water (500 mL) and washed with tert-butyl methyl ether (2 x 500 mL). The aqueous layer was then adjusted to pH 4 by addition of 3 M hydrochloric acid and extracted with ethyl acetate (2 x 500 mL); the combined ethyl acetate layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide P1 as a white solid. Yield: 122 g, 0.566 mol, 77%. LCMS m/z 216.1 (chlorine isotope pattern observed) [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.10 (s, 1H), 6.79 (s, 1H), 4.06 (q, J = 7.1 Hz, 1H), 3.89 (s, 3H), 1.48 (d, J = 7.2 Hz, 3H). Preparations P2 and P3 (2R)-2-(5-Chloro-2-methoxypyridin-4-yl)propanoic acid (P2) and (2S)-2-(5-Chloro-2- methoxypyridin-4-yl)propanoic acid (P3)
Figure imgf000051_0001
Separation of P1 (5.00 g, 23.2 mmol) into its component enantiomers was carried out via supercritical fluid chromatography (Column: Chiral Technologies Chiralpak IG, 30 x 250 mm, 5 µm; Mobile phase: 95:5 carbon dioxide / methanol; Flow rate: 80 mL/minute; Back pressure: 120 bar). The first-eluting enantiomer, an oil which solidified on standing, was designated as P2, and the second-eluting enantiomer as P3. The indicated absolute stereochemistry was assigned via X-ray crystal structure determination of 15, which was synthesized using this lot of P2 (see below, Example 15, Alternate Step 3). P2 – Yield: 2.4 g, 11.1 mmol, 48%.1H NMR (400 MHz, chloroform-d) δ 8.13 (s, 1H), 6.75 (s, 1H), 4.12 (q, J = 7.2 Hz, 1H), 3.91 (s, 3H), 1.53 (d, J = 7.2 Hz, 3H). Retention time: 3.98 minutes (Analytical conditions. Column: Chiral Technologies Chiralpak IG, 4.6 x 250 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: methanol; Gradient: 5% B for 1.00 minute, then 5% to 60% B over 8 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar). P3 – Yield: 2.4 g, 11.1 mmol, 48%. Retention time: 4.22 minutes (Analytical conditions identical to those used for P2). Preparation P4 Lithium 2-(6-methoxy-2-methylpyrimidin-4-yl)propanoate (P4)
Figure imgf000052_0001
Step 1. Synthesis of dimethyl (6-methoxy-2-methylpyrimidin-4-yl)(methyl)propanedioate (C3) and methyl 2-(6-methoxy-2-methylpyrimidin-4-yl)propanoate (C4). Sodium hydride (60% in mineral oil; 1.14 g, 28.5 mmol) was added to a solution of dimethyl methylpropanedioate (5.53 g, 37.8 mmol) in N,N-dimethylformamide (25 mL). After 30 minutes, 4- chloro-6-methoxy-2-methylpyrimidine (3.00 g, 18.9 mmol) was added, whereupon the reaction mixture was heated at 100 °C for 16 hours. It was then diluted with water (150 mL) and extracted with ethyl acetate (3 x 50 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, concentrated in vacuo, and purified via silica gel chromatography (Gradient: 0% to 10% ethyl acetate in petroleum ether), affording the product (2.60 g) as a yellow oil. On the basis of NMR and LCMS analysis, this was judged to be an impure mixture of C3 and C4, which was taken directly into the following step. LCMS m/z 211.1 and 269.2 [M+H]+.1H NMR (400 MHz, methanol-d4), characteristic peaks: δ 6.68 (s), 6.60 (s), 3.96 (s), 3.94 (s), 3.81 (q, J = 7.2 Hz), 3.75 (s), 3.68 (s), 2.54 (s), 2.52 (s), 1.79 (s), 1.47 (d, J = 7.3 Hz). Step 2. Synthesis of lithium 2-(6-methoxy-2-methylpyrimidin-4-yl)propanoate (P4). A solution of C3 and C4 (from the previous step; 2.60 g, ≤18.9 mmol) and lithium hydroxide monohydrate (1.22 g, 29.1 mmol) in a mixture of tetrahydrofuran (45 mL) and water (15 mL) was stirred at 45 °C for 3 hours. After the reaction mixture had been concentrated in vacuo, the residue was subjected to lyophilization, providing P4 as a white solid. Yield: 2.3 g, 11 mmol, 58% over 2 steps. LCMS m/z 197.1 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 6.66 (s, 1H), 3.94 (s, 3H), 3.61 (q, J = 7.3 Hz, 1H), 2.53 (s, 3H), 1.44 (d, J = 7.2 Hz, 3H).
Figure imgf000053_0001
Step 1. Synthesis of 2-(difluoromethoxy)-5-iodopyridine (C5). Sodium chloro(difluoro)acetate (4.62 g, 30.3 mmol) and potassium carbonate (5.58 g, 40.4 mmol) were added to a 25 °C solution of 5-iodopyridin-2-ol (4.46 g, 20.2 mmol) in N,N- dimethylformamide (100 mL), and the reaction mixture was stirred at 50 °C for 16 hours. It was then diluted with water (500 mL) and extracted with ethyl acetate (3 x 100 mL), and the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (Gradient: 0% to 8% ethyl acetate in petroleum ether) provided C5 as an oil. Yield: 2.10 g, 7.75 mmol, 38%.1H NMR (400 MHz, chloroform-d) δ 8.39 (br d, J = 2.2 Hz, 1H), 7.97 (dd, J = 8.6, 2.3 Hz, 1H), 7.40 (t, JHF = 72.6 Hz, 1H), 6.74 (br d, J = 8.6 Hz, 1H). Step 2. Synthesis of diethyl [6-(difluoromethoxy)pyridin-3-yl]propanedioate (C6). A mixture of C5 (1.9 g, 7.0 mmol), diethyl propanedioate (1.68 g, 10.5 mmol), copper(I) iodide (133 mg, 0.698 mmol), pyridine-2-carboxylic acid (172 mg, 1.40 mmol), and cesium carbonate (7.42 g, 22.8 mmol) in tetrahydrofuran (50 mL) was stirred at 80 °C for 16 hours, whereupon the reaction mixture was diluted with ethyl acetate (100 mL) and washed with aqueous ammonium chloride solution (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 50 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification using silica gel chromatography (Gradient: 0% to 15% ethyl acetate in petroleum ether) afforded C6 as a colorless oil (2.4 g). By 1H NMR analysis, this material contained residual diethyl propanedioate; a portion of this sample was taken directly to the following step. LCMS m/z 304.0 [M+H]+.1H NMR (400 MHz, chloroform-d), product peaks only: δ 8.14 (br s, 1H), 7.90 (br d, J = 8.4 Hz, 1H), 7.45 (t, JHF = 72.9 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.59 (s, 1H), 4.26 – 4.17 (m, 4H, assumed; partially obscured by residual diethyl propanedioate), 1.31 – 1.24 (m, 6H, assumed; partially obscured by residual diethyl propanedioate). Step 3. Synthesis of diethyl [6-(difluoromethoxy)pyridin-3-yl](methyl)propanedioate (C7). To a solution of C6 (from the previous step; 750 mg, ≤2.2 mmol) in N,N-dimethylformamide (15 mL) was added potassium carbonate (1.03 g, 7.45 mmol). Iodomethane (527 mg, 3.71 mmol) was added drop-wise, and the reaction mixture was stirred at 25 °C for 4 hours. It was then combined with a similar reaction carried out using C6 (250 mg, ≤0.73 mmol), poured into water (200 mL), and extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, and concentrated in vacuo; chromatography on silica gel (Gradient: 0% to 15% ethyl acetate in petroleum ether) provided C7 as an oil. Combined yield: 738 mg, 2.33 mmol, 80% over 2 steps. LCMS m/z 318.2 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.20 (br s, 1H), 7.81 (br d, J = 8.7 Hz, 1H), 7.45 (t, JHF = 72.9 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 4.30 – 4.18 (m, 4H), 1.87 (s, 3H), 1.27 (t, J = 7.1 Hz, 6H). Step 4. Synthesis of 2-[6-(difluoromethoxy)pyridin-3-yl]propanoic acid (P5). To a 25 °C solution of C7 (738 mg, 2.33 mmol) in tetrahydrofuran (10 mL) was added a solution of lithium hydroxide (279 mg, 11.6 mmol) in water (3 mL). The reaction mixture was stirred at 25 °C for 16 hours, whereupon it was diluted with water (100 mL) and washed with dichloromethane (3 x 50 mL). These organic layers were discarded. After the aqueous layer had been adjusted to pH 5 by addition of 5 M hydrochloric acid, it was extracted with dichloromethane (3 x 50 mL); the combined organic layers were concentrated in vacuo to afford P5 as a solid. Yield: 337 mg, 1.55 mmol, 67%. LCMS m/z 218.1 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.15 (d, J = 2.5 Hz, 1H), 7.83 (dd, J = 8.5, 2.5 Hz, 1H), 7.51 (t, JHF = 73.2 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H), 3.78 (q, J = 7.2 Hz, 1H), 1.49 (d, J = 7.2 Hz, 3H).
Figure imgf000055_0001
Step 1. Synthesis of dibenzyl (5-fluoro-2-methoxypyridin-4-yl)propanedioate (C8). This reaction was carried out in three parallel batches. To a 25 °C solution of dibenzyl propanedioate (607 g, 2.13 mol) in tetrahydrofuran (1.5 L) was added pyridine-2-carboxylic acid (35.0 g, 284 mmol), followed by copper(I) iodide (27.1 g, 142 mmol), and then freshly ground cesium carbonate (1.39 kg, 4.27 mol). After the reaction mixture had been heated to 70 °C, it was treated in a drop-wise manner with a solution of 5-fluoro-4-iodo-2-methoxypyridine (360 g, 1.42 mol) in tetrahydrofuran (800 mL), whereupon stirring was continued for 16 hours at 70 °C. The three reaction mixtures were combined at this point, cooled to 25 °C, and filtered through diatomaceous earth. The filter pad was rinsed with ethyl acetate (3 x 500 mL), and the combined filtrates were concentrated in vacuo, while keeping the internal temperature below 40 °C. The residue was dissolved in ethyl acetate (2 L), washed sequentially with saturated aqueous ammonium chloride solution (500 mL) and saturated aqueous sodium chloride solution (500 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure at 40 °C. Chromatography on silica gel (Gradient: 1% to 8% ethyl acetate in petroleum ether) afforded C8 (1.87 kg) as a yellow oil. By 1H NMR analysis, this material was contaminated with dibenzyl propanedioate; a portion of it was used in the following step. LCMS m/z 410.1 [M+H]+.1H NMR (400 MHz, chloroform-d), product peaks only: δ 8.01 (d, J = 1.3 Hz, 1H), 7.40 – 7.25 (m, 10H, assumed; partially obscured by residual dibenzyl propanedioate), 6.83 (d, J = 4.8 Hz, 1H), 5.20 (AB quartet, JAB = 12.2 Hz, ΔνAB = 11.9 Hz, 4H), 5.00 (s, 1H), 3.89 (s, 3H). Step 2. Synthesis of dibenzyl (5-fluoro-2-methoxypyridin-4-yl)(methyl)propanedioate (C9). This reaction was carried out in two parallel batches. A solution of C8 (from the previous step; 575 g, ≤1.31 mol) in acetonitrile (1.5 L) was stirred in an ice-water bath for 20 minutes, whereupon potassium carbonate (582 g, 4.21 mol) was added. Stirring was continued for an additional 10 minutes. Iodomethane (302 g, 2.13 mol) was then added to the reaction mixture at 0 °C, and the reaction was allowed to proceed until LCMS analysis indicated conversion to C9. After the two reaction mixtures had been combined, they were filtered through diatomaceous earth, and the filter cake was washed with acetonitrile (2 x 1 L). The combined filtrates were concentrated at 40 °C and the residue was partitioned between ethyl acetate (2 L) and water (500 mL). The aqueous layer was extracted with ethyl acetate (2 x 1 L), and the combined organic layers were washed with saturated aqueous sodium chloride solution (1 L), dried over sodium sulfate, filtered, and concentrated under reduced pressure at 40 °C. The resulting crude product was dissolved in petroleum ether (1.5 L) and stirred at 0 °C for 2 hours; a solid was collected via filtration. The filtrate was concentrated in vacuo, and the residue was taken up in petroleum ether (500 mL), then cooled to 0 °C to provide additional solid, which was isolated via filtration. The two solids were combined, suspended in petroleum ether (800 mL), and stirred at 20 °C for 16 hours. Subsequent collection via filtration afforded C9 as a yellow solid. Yield: 670 g, 1.58 mol, 60% over 2 steps. LCMS m/z 423.8 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.94 (d, J = 2.6 Hz, 1H), 7.36 – 7.20 (m, 10H), 6.54 (d, J = 5.1 Hz, 1H), 5.18 (s, 4H), 3.87 (s, 3H), 1.85 (s, 3H). Step 3. Synthesis of 2-(5-fluoro-2-methoxypyridin-4-yl)propanoic acid (P6). This reaction was carried out in four parallel batches. To a 25 °C solution of C9 (200 g, 472 mmol) in ethyl acetate (1 L) was added 10% palladium on carbon (wet; 40 g). The mixture was degassed under vacuum and then purged with nitrogen; this evacuation-purge cycle was carried out a total of three times. The mixture was again degassed under vacuum and then purged with hydrogen; this evacuation-purge cycle was also carried out a total of three times. The mixture was hydrogenated (30 psi) at 50 °C for 16 hours. The four reaction mixtures were combined and filtered through a pad of diatomaceous earth, and the filtrate was concentrated in vacuo at 45 °C. Chromatography on silica gel (Gradient: 10% to 20% ethyl acetate in petroleum ether) provided P6 as a white solid. Combined yield: 270 g, 1.36 mmol, 72%. LCMS m/z 199.7 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.98 (d, J = 1.7 Hz, 1H), 6.70 (d, J = 4.9 Hz, 1H), 3.97 (q, J = 7.3 Hz, 1H), 3.89 (s, 3H), 1.53 (d, J = 7.3 Hz, 3H). Preparations P7 and P8 (2R)-2-(5-Fluoro-2-methoxypyridin-4-yl)propanoic acid (P7) and (2S)-2-(5-Fluoro-2-methoxypyridin- 4-yl)propanoic acid (P8)
Figure imgf000057_0001
Separation of P6 (700 g, 3.51 mol) into its component enantiomers was carried out by supercritical fluid chromatography (Column: Chiral Technologies Chiralpak AD-H, 50 x 250 mm, 5 µm; Mobile phase: 9:1 carbon dioxide / 2-propanol; Flow rate: 250 mL/minute; Back pressure: 120 bar). The first-eluting enantiomer was designated as P7, and the second-eluting enantiomer as P8; both were isolated as solids. P7 - Yield: 260 g, 1.30 mol, 37%. Retention time: 3.17 minutes (Analytical conditions. Column: Chiral Technologies Chiralpak AD-H, 4.6 x 250 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: 2-propanol; Gradient: 5% B for 1.00 minute, then 5% to 60% B over 8 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar). P8 - Yield: 400 g, 2.01 mol, 57%. Retention time: 3.36 minutes (Analytical conditions identical to those used for P7). The indicated absolute stereochemistries for P7 and P8 were assigned on the basis of comparison to the sample of P7 synthesized in Alternate Preparation (#1) of P7; the configuration of that material was established via X-ray crystallographic study of the derived compound 14 (see below). Retention time for P7 from Preparations P7 and P8: 2.86 minutes. Retention time for P7 from Alternate Preparation (#1) of P7: 2.86 minutes. Retention times for a racemic mixture of P7 and P8: 2.87 and 3.16 minutes. These three analyses were run using the same analytical method: [Column: Chiral Technologies Chiralpak IG, 4.6 x 250 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: methanol; Gradient: 5% B for 1 minute, then 5% to 60% B over 7 minutes; Flow rate: 3 mL/minute; Back pressure: 120 bar]. Alternate Preparation (#1) of P7 (2R)-2-(5-Fluoro-2-methoxypyridin-4-yl)propanoic acid (P7)
Figure imgf000058_0001
Step 1. Synthesis of disodium (5-fluoro-2-methoxypyridin-4-yl)(methyl)propanedioate (C10). A 1.0 M, pH 8.0 buffer solution was prepared in the following manner: a solution of 2-amino- 2-(hydroxymethyl)propane-1,3-diol (Tris; 121 g, 1.00 mol) in water (900 mL) was adjusted to pH 8.0 by addition of hydrochloric acid (37.5 weight%, approximately 40 mL), and then brought to a volume of 1 L by addition of water. A hydrogenation reactor was charged with palladium hydroxide on carbon (10%; 5.00 g). To this was added a solution of C9 (50.0 g, 118 mmol) in toluene (50 mL, 1 volume); additional toluene (50 mL) was used to rinse the flask, and this was also added to the reaction mixture. A mixture of aqueous sodium hydroxide solution (2.0 M, 118 mL, 236 mmol), the pH 8.0 buffer solution described above (1.0 M; 250 mL, 250 mmol), and water (132 mL) was added, and the resulting mixture was purged with nitrogen (3.5 bar) followed by hydrogen (3.5 bar); this purging process was carried out a total of three times. After the mixture had been brought to 20 °C, stirring at 100 rpm, it was pressurized with hydrogen to 3.45 bar, whereupon the rate of stirring was increased to 750 rpm. After the hydrogenation had proceeded for 4 hours at 20 °C, the stirring rate was decreased to 250 rpm and the reaction was purged three times with nitrogen (3.5 bar). The catalyst was removed via filtration, and the reactor was rinsed with water (100 mL), which was then used to wash the filter cake. The aqueous phase of the combined filtrates (590 mL, pH 8.2), containing C10, was progressed directly to the following step. LCMS m/z 244.2 [M+H]+. Step 2. Synthesis of (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)propanoic acid (P7). A 2 L jacketed vessel (set to a 20 °C jacket temperature) with overhead stirrer was charged with C10 (aqueous solution from the previous step; ≤118 mmol), and the stirring rate was set at 200 rpm. A solution of Bordetella bronchiseptica AMDase lyophilized cell-free extract powder (1.75 gm) [This aryl malonate decarboxylase (AMDase) from Bordetella bronchiseptica is a wild-type enzyme described in the literature with accession number Q05115, which was recombinantly expressed in E. coli and charged as a lyophilized cell-free extract powder. Literature references: S. K. Gaßmeyer et al., ChemCatChem, 2016, 8, 916 – 921; K. Okrasa et al., Angew. Chem. Int. Ed. 2009, 48, 7691 –7694] in water (17.5 mL) was then charged to the reactor, along with a water rinse of the enzyme vessel (5 mL). After 15 hours, the stirring speed was lowered to 100 rpm, and the pH of the reaction mixture was adjusted to pH 6.0 by sequential additions of hydrochloric acid (4.0 M, 5 mL portions, 38 mL). At this point, the mixture was stirred for 1.5 hours to allow off-gassing to subside, whereupon it was acidified to a pH of ≤2.0 via further addition of hydrochloric acid (4.0 M, total of 85 mL). tert-Butyl methyl ether (300 mL) was added and stirring was continued at 200 rpm for 15 minutes. The mixture was then filtered through diatomaceous earth (25 g), using a Büchner funnel and filter paper; the reactor was rinsed with tert-butyl methyl ether (100 mL), which was then used to wash the filter cake. The aqueous layer of the combined filtrates was extracted in the same manner with tert-butyl methyl ether (300 mL). The combined organic layers were dried over sodium sulfate (50 g) and filtered; the filter cake was washed with tert-butyl methyl ether (25 mL). The combined filtrates were concentrated in vacuo at 30 °C to provide an oil, which solidified under vacuum drying overnight to afford P7 as an off-white solid. Yield: 18.88 g, 94.8 mmol, 80% over 2 steps.1H NMR (400 MHz, chloroform-d) δ 11.4 – 10.3 (br s, 1H), 7.98 (d, J = 1.6 Hz, 1H), 6.70 (d, J = 4.9 Hz, 1H), 3.97 (q, J = 7.2 Hz, 1H), 3.89 (s, 3H), 1.53 (d, J = 7.2 Hz, 3H). Combination of P7 from the previous step (18.6 g, 93.4 mmol) and P7 (24.9 g, 125 mmol) from a similar reaction of C10 with AMDase afforded a slightly pink solid, with an enantiomeric excess of 98.5%.1H NMR (400 MHz, methanol-d4) δ 7.94 (d, J = 1.9 Hz, 1H), 6.74 (d, J = 5.0 Hz, 1H), 3.93 (q, J = 7.3 Hz, 1H), 3.87 (s, 3H), 1.48 (d, J = 7.3 Hz, 3H). Retention time: 2.86 minutes [Column: Chiral Technologies Chiralpak IG, 4.6 x 250 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: methanol; Gradient: 5% B for 1 minute, then 5% to 60% B over 7 minutes; Flow rate: 3 mL/minute; Back pressure: 120 bar]. The indicated absolute stereochemistry of P7 was assigned on the basis of conversion of this lot of P7 to Example 14; the absolute stereochemistry of 14 was established via single-crystal X-ray analysis (see below).
Figure imgf000060_0001
Step 1. Synthesis of dibenzyl (5-fluoro-2-methoxypyridin-4-yl)propanedioate (C8). A mixture of pyridine-2-carboxylic acid (24.6 g, 0.200 mol), copper(I) iodide (19.1 g, 0.100 mol), and cesium carbonate (977 g, 3.00 mol) in tetrahydrofuran (1.26 L; 5 volumes), was heated to an internal temperature of 60 °C to 70 °C, whereupon a solution of 5-fluoro-4-iodo-2- methoxypyridine (253 g, 1.00 mol) and dibenzyl propanedioate (426 g, 1.50 mol) in tetrahydrofuran (250 mL, 1 volume) was added. After the reaction mixture had been heated at 60 °C to 70 °C for approximately 3 to 6 hours, it was allowed to cool to 15 °C to 30 °C and filtered through diatomaceous earth (250 g). The filter cake was washed with tetrahydrofuran (500 mL, 2 volumes) and the combined filtrates, containing C8, were used directly in the following step. Representative 1H NMR (500 MHz, chloroform-d) δ 8.00 (d, J = 1.3 Hz, 1H), 7.40 – 7.24 (m, 10H, assumed; partially obscured by residual dibenzyl propanedioate), 6.82 (d, J = 4.8 Hz, 1H), 5.20 (AB quartet, JAB = 12.3 Hz, ΔνAB = 14.9 Hz, 4H), 4.99 (s, 1H), 3.88 (s, 3H). Step 2. Synthesis of dibenzyl (5-fluoro-2-methoxypyridin-4-yl)(methyl)propanedioate (C9). Iodomethane (284 g, 2.00 mol) was slowly added to a 10 °C to 20 °C mixture of cesium carbonate (977 g, 3.00 mol) and a solution of C8 (from the previous step, solution in tetrahydrofuran; ≤1.00 mol). After the reaction mixture had been stirred at 10 °C to 20 °C for approximately 10 to 12 hours, it was filtered through diatomaceous earth (250 g). The filter cake was washed with tetrahydrofuran (500 mL, 1.2 volumes), and the combined filtrates were concentrated to 1 to 2 volumes. The resulting mixture was diluted with propan-2-yl acetate (1.25 L, 3.1 volumes), washed sequentially with water (750 mL, 1.8 volumes), aqueous ammonium chloride solution (20%; 750 mL), and aqueous sodium chloride solution (20%; 750 mL), and concentrated in vacuo. The remaining solvent was exchanged with heptane, and precipitation was allowed to proceed from heptane (2 to 3 volumes) at 15 °C to 25 °C. The resulting solid was collected via filtration and triturated with a mixture of heptane (450 mL) and propan-2-yl acetate (50 mL) to afford C9 as a solid. Three batches of the chemistry in steps 1 and 2 were carried out, and the final lots of C9 were combined. Yield: 675 g, 1.59 mol, approximately 53% over 2 steps. Representative 1H NMR (500 MHz, DMSO-d6) δ 8.15 (d, J = 2.0 Hz, 1H), 7.39 – 7.21 (m, 10H), 6.75 (d, J = 5.0 Hz, 1H), 5.21 (s, 4H), 3.81 (s, 3H), 1.81 (s, 3H). Step 3. Synthesis of disodium (5-fluoro-2-methoxypyridin-4-yl)(methyl)propanedioate (C10). A buffer solution [pH 8.0; 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris; 121 g, 1.00 mol), and concentrated hydrochloric acid (46 mL, 0.23 volumes) in water (1 L, 5 volumes)], and palladium hydroxide on carbon (10%, 20 g) were added to a 15 °C to 25 °C mixture of C9 (200 g, 0.472 mol) in toluene (400 mL, 2 volumes). A solution of sodium hydroxide (38.8 g, 0.970 mol) in water (1 L, 5 volumes), was added, whereupon the mixture was stirred for approximately 10 to 20 minutes. After the reactor had been purged with nitrogen, then purged with hydrogen, the reaction mixture was stirred at 15 °C to 30 °C under a bag of hydrogen (approximately 10 L), until HPLC analysis indicated ≤0.5% of C9 was present (approximately 22 hours) (Retention time: 11.44 minutes. HPLC conditions. Column: Agilent Technologies ZORBAX Eclipse Plus C18, 4.6 x 100 mm, 3.5 µm; Mobile phase A: 0.1% phosphoric acid in water; Mobile phase B: acetonitrile; Gradient: 5% B for 3 minutes, then 5% to 100% B over 9 minutes, then 100% B for 3 minutes; Flow rate: 1.5 mL/minute). The reaction mixture was filtered, and the filter cake was washed with water (2.6 volumes); the aqueous layer of the filtrate, containing C10, was taken directly to the following step. Step 4. Synthesis of (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)propanoic acid (P7). A mixture of AMDase (7 g) in water (70 mL, 0.35 volumes) and C10 (from the previous step, as a solution in water, ≤0.472 mol) was stirred at 15 °C to 30 °C until HPLC analysis indicated that ≤0.5% of C10 was present (approximately 16 hours) [Retention time: 5.80 minutes. HPLC conditions identical to those described in Step 3, Synthesis of disodium (5-fluoro-2-methoxypyridin- 4-yl)(methyl)propanedioate (C10)]. Hydrochloric acid (4.0 M) was then slowly added until the pH of the reaction mixture reached 6.0, whereupon stirring was continued for 1.5 hours. The pH was then adjusted to ≤2.0 (range, 1.5 to 2.0) by further addition of hydrochloric acid (4.0 M). After addition of tert-butyl methyl ether (1.2 L, 6 volumes), the mixture was filtered through diatomaceous earth (100 g), and the aqueous phase of the filtrate was extracted with tert-butyl methyl ether (800 mL, 4 volumes). The combined organic layers were washed with aqueous sodium chloride solution (15%; 600 mL, 3 volumes), and concentrated to 2 to 2.5 volumes at a temperature of ≤45 °C and a pressure of ≤ −0.08 MPa. n-Heptane (600 mL, 3 volumes) was added, and the mixture was concentrated to 3 to 5 volumes at a temperature of ≤45 °C and a pressure of ≤ −0.08 MPa; this heptane dilution / concentration was carried out a total of 3 times. After the resulting mixture had been stirred at 0 °C to 10 °C for approximately 1 to 2 hours, the precipitate was collected via filtration, providing P7 as an off-white solid with an enantiomeric excess of 99.8%. Yield: 80.0 g, 0.402 mol, 85% over 2 steps. Representative 1H NMR (500 MHz, chloroform-d) δ 11.68 (v br s, 1H), 7.99 (br s, 1H), 6.70 (d, J = 4.9 Hz, 1H), 3.97 (q, J = 7.2 Hz, 1H), 3.88 (s, 3H), 1.52 (d, J = 7.3 Hz, 3H).
Figure imgf000062_0001
Step 1. Synthesis of methyl 2-(5-bromo-2-methoxypyridin-4-yl)propanoate (C11). A solution of sodium bis(trimethylsilyl)amide in tetrahydrofuran (2 M; 1 mL, 2 mmol) was added drop-wise to a −78 °C solution of methyl (5-bromo-2-methoxypyridin-4-yl)acetate (415 mg, 1.60 mmol) in tetrahydrofuran (50 mL). After the reaction mixture had been stirred at −78 °C for 1 hour, a solution of iodomethane (0.5 mL, 8 mmol) was added drop-wise. At the completion of the addition, the reaction mixture was warmed to −30 °C and allowed to stir at that temperature for 3 hours, whereupon it was diluted with aqueous ammonium chloride solution and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo while keeping the temperature below 45 °C. Purification via silica gel chromatography (Eluent: 1:3 ethyl acetate / petroleum ether) provided C11 as a colorless oil. Yield: 376 mg, 1.37 mmol, 86%. LCMS m/z 276.0 (bromine isotope pattern observed) [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.23 (s, 1H), 6.76 (s, 1H), 4.10 (q, J = 7.1 Hz, 1H), 3.89 (s, 3H), 3.69 (s, 3H), 1.48 (d, J = 7.2 Hz, 3H). Step 2. Synthesis of methyl 2-(5-ethenyl-2-methoxypyridin-4-yl)propanoate (C12). A mixture of C11 (376 mg, 1.37 mmol), potassium vinyltrifluoroborate (460 mg, 3.43 mmol), [1,1’-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (201 mg, 0.275 mmol), and potassium phosphate (872 mg, 4.11 mmol) in N,N-dimethylformamide (20 mL) was stirred at 100 °C for 16 hours. The reaction mixture was then filtered; the filtrate was poured into water and extracted with ethyl acetate (2 x 30 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, concentrated in vacuo, and purified via chromatography on silica gel (Gradient: 0% to 30% ethyl acetate in petroleum ether) to afford C12 as a colorless oil. Yield: 188 mg, 0.850 mmol, 62%. LCMS m/z 222.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.21 (s, 1H), 6.81 (dd, J = 17.3, 10.9 Hz, 1H), 6.63 (s, 1H), 5.56 (br d, J = 17.3 Hz, 1H), 5.32 (br d, J = 10.8 Hz, 1H), 3.95 – 3.87 (m, 1H), 3.93 (s, 3H), 3.67 (s, 3H), 1.46 (d, J = 7.1 Hz, 3H). Step 3. Synthesis of methyl 2-(5-formyl-2-methoxypyridin-4-yl)propanoate (C13). A solution of C12 (195 mg, 0.881 mmol) in dichloromethane (10 mL) was cooled to −78 °C, and then treated with a stream of ozone-enriched oxygen until a blue color persisted. After 5 minutes, a stream of dry nitrogen was bubbled through the reaction mixture until the blue color had disappeared, whereupon triphenylphosphine (439 mg, 1.67 mmol) was added. The resulting mixture was warmed to 25 °C and stirred for 2 hours, at which point it was combined with a similar reaction carried out using C12 (63 mg, 0.28 mmol) and concentrated in vacuo. The residue was purified using silica gel chromatography (Gradient: 0% to 30% ethyl acetate in petroleum ether) to provide C13 as a colorless oil. Combined yield: 124 mg, 0.555 mmol, 48%. LCMS m/z 224.0 [M+H]+. Step 4. Synthesis of methyl 2-[5-(difluoromethyl)-2-methoxypyridin-4-yl]propanoate (C14). To a solution of C13 (124 mg, 0.555 mmol) in dichloromethane (5 mL) was added [bis(2- methoxyethyl)amino]sulfur trifluoride (614 mg, 2.78 mmol). After the reaction mixture had been stirred at 25 °C for 16 hours, it was poured into saturated aqueous sodium bicarbonate solution (50 mL) and extracted with dichloromethane (50 mL). The organic layer was washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 20% ethyl acetate in petroleum ether) provided C14 as a colorless oil. Yield: 110 mg, 0.449 mmol, 81%. LCMS m/z 246.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.28 (s, 1H), 6.87 (s, 1H), 6.76 (t, JHF = 54.5 Hz, 1H), 4.11 (q, J = 6.9 Hz, 1H), 4.03 (s, 3H), 3.69 (s, 3H), 1.52 (d, J = 7.0 Hz, 3H). Step 5. Synthesis of 2-[5-(difluoromethyl)-2-methoxypyridin-4-yl]propanoic acid (P9). To a solution of C14 (145 mg, 0.591 mmol) in methanol (10 mL) was added a solution of lithium hydroxide (43 mg, 1.8 mmol) in water (4 mL), and the reaction mixture was stirred at 20 °C for 4 hours, whereupon it was concentrated in vacuo and washed with tert-butyl methyl ether (2 x 5 mL). The aqueous layer was adjusted to pH 5 by addition of 2 M hydrochloric acid and then extracted with ethyl acetate (3 x 10 mL). The combined ethyl acetate layers were washed with water (3 x 10 mL) and with saturated aqueous sodium chloride solution (20 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide P9 as a yellow oil. Yield: 132 mg, 0.571 mmol, 97%. LCMS m/z 232.1 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.26 (s, 1H), 6.96 (t, JHF = 54.4 Hz, 1H), 6.84 (s, 1H), 4.12 (q, J = 7.2 Hz, 1H), 3.94 (s, 3H), 1.48 (d, J = 7.2 Hz, 3H).
Figure imgf000064_0001
Step 1. Synthesis of dimethyl (2-methoxypyridin-4-yl)propanedioate (C15). To a −10 °C solution of 2-methoxy-4-methylpyridine (5.00 g, 40.6 mmol) in tetrahydrofuran (30 mL) was added lithium diisopropylamide (2 M solution in tetrahydrofuran; 81.2 mL, 162 mmol). After the reaction mixture had been stirred at −10 °C for 1.5 hours, dimethyl carbonate (14.6 g, 162 mmol) was added and stirring was continued at −10 °C for 1.5 hours. The reaction mixture was then warmed to 25 °C and allowed to stir for 16 hours, whereupon it was quenched by addition of aqueous ammonium chloride solution. The resulting mixture was extracted with ethyl acetate (3 x 30 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Purification via chromatography on silica gel (Gradient: 0% to 20% ethyl acetate in petroleum ether) provided C15 as a yellow oil. Yield: 4.92 g, 20.6 mmol, 51%. LCMS m/z 240.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.17 (d, J = 5.0 Hz, 1H), 6.95 (d, J = 4.8 Hz, 1H), 6.80 (s, 1H), 4.59 (s, 1H), 3.96 (s, 3H), 3.77 (s, 6H). Also obtained from the chromatographic purification was the product of mono-acylation, methyl (2-methoxypyridin-4-yl)acetate. Yield: 1.29 g, 7.12 mmol, 18%. LCMS m/z 182.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.11 (br d, J = 5.3 Hz, 1H), 6.81 (dd, J = 5.4, 1.5 Hz, 1H), 6.68 – 6.66 (m, 1H), 3.93 (s, 3H), 3.71 (s, 3H), 3.57 (s, 2H). Step 2. Synthesis of dimethyl (2-methoxypyridin-4-yl)(methyl)propanedioate (C16). Sodium bis(trimethylsilyl)amide (2 M solution in tetrahydrofuran; 14.0 mL, 28.0 mmol) was added to a −78 °C solution of C15 (4.47 g, 18.7 mmol) in tetrahydrofuran (30 mL). After the reaction mixture had been stirred at −78 °C for 1 hour, iodomethane (1.40 mL, 22.5 mmol) was added. The reaction mixture was then warmed to −40 °C, stirred for 2 hours, warmed to 25 °C, and stirred for a further 16 hours, whereupon it was quenched with aqueous ammonium chloride solution. The resulting mixture was extracted with ethyl acetate (2 x 30 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% ethyl acetate in petroleum ether) afforded C16 as a yellow oil. Yield: 3.29 g, 13.0 mmol, 70%. LCMS m/z 254.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.15 (d, J = 5.5 Hz, 1H), 6.88 (br d, J = 5.5 Hz, 1H), 6.74 (br s, 1H), 3.95 (s, 3H), 3.78 (s, 6H), 1.83 (s, 3H). Step 3. Synthesis of 2-(2-methoxypyridin-4-yl)propanoic acid (C17). A solution of C16 (3.28 g, 13.0 mmol) and lithium hydroxide (1.24 g, 51.8 mmol) in a mixture of tetrahydrofuran (20 mL) and water (10 mL) was stirred at 45 °C for 5 hours. LCMS analysis indicated conversion to C17: LCMS m/z 182.1 [M+H]+, and the reaction mixture was concentrated in vacuo, providing C17 as a white solid (2.40 g). This material was used directly in the following step. Step 4. Synthesis of methyl 2-(2-methoxypyridin-4-yl)propanoate (C18). A mixture of C17 (from the previous step; 2.40 g, ≤13.0 mmol) and sulfuric acid (2.5 mL) in methanol (25 mL) was stirred at 60 °C for 16 hours. The reaction mixture was then concentrated in vacuo, washed with aqueous sodium bicarbonate solution, and extracted with ethyl acetate (2 x 20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford C18 as a colorless oil. Yield: 1.56 g, 7.99 mmol, 61% over 2 steps. LCMS m/z 196.2 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.10 (d, J = 5.4 Hz, 1H), 6.81 (dd, J = 5.4, 1.5 Hz, 1H), 6.67 (br s, 1H), 3.93 (s, 3H), 3.67 (s, 3H), 3.66 (q, J = 7.1 Hz, 1H), 1.47 (d, J = 7.2 Hz, 3H). Step 5. Synthesis of methyl 2-fluoro-2-(2-methoxypyridin-4-yl)propanoate (C19). To a −78 °C solution of C18 (500 mg, 2.56 mmol) in tetrahydrofuran (13 mL) was added lithium bis(trimethylsilyl)amide (1 M solution in tetrahydrofuran 3.33 mL, 3.33 mmol). After the reaction mixture had been stirred at −78 °C for 30 minutes, a solution of N-(benzenesulfonyl)-N- fluorobenzenesulfonamide (969 mg, 3.07 mmol) in tetrahydrofuran (2 mL) was added. The reaction mixture was stirred at −10 °C for 3 hours, whereupon it was quenched with aqueous ammonium chloride solution and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo; silica gel chromatography (Gradient: 0% to 10% ethyl acetate in petroleum ether) afforded C19 as a yellow oil. Yield: 400 mg, 1.88 mmol, 73%. LCMS m/z 214.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.18 (d, J = 5.4 Hz, 1H), 7.00 (dd, J = 5.5, 1.6 Hz, 1H), 6.88 (br d, J = 1.5 Hz, 1H), 3.96 (s, 3H), 3.78 (s, 3H), 1.89 (d, JHF = 22.3 Hz, 3H). Step 6. Synthesis of 2-fluoro-2-(2-methoxypyridin-4-yl)propanoic acid (P10). A solution of C19 (400 mg, 1.88 mmol) and lithium hydroxide (89.9 mg, 3.75 mmol) in a mixture of tetrahydrofuran (10 mL) and water (2 mL) was stirred at 45 °C for 4 hours. The reaction mixture was then concentrated in vacuo, diluted with water (12 mL), and adjusted to pH 6 by addition of 3 M hydrochloric acid. The resulting mixture was extracted with ethyl acetate (2 x 20 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide P10 as a yellow oil. Yield: 300 mg, 1.51 mmol, 80%. LCMS m/z 200.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 9.9 – 9.4 (br s, 1H), 8.21 (d, J = 5.6 Hz, 1H), 7.08 (dd, J = 5.6, 1.6 Hz, 1H), 6.95 (br s, 1H), 3.95 (s, 3H), 1.92 (d, JHF = 22.2 Hz, 3H).
Figure imgf000067_0001
Step 1. Synthesis of 1-(difluoromethoxy)-3-methoxy-5-methylbenzene (C20). Aqueous potassium hydroxide solution (20% solution; 60.9 g, 217 mmol) and [bromo(difluoro)methyl](trimethyl)silane (11.3 mL, 72.7 mmol) were sequentially added to a 0 °C solution of 3-methoxy-5-methylphenol (5.00 g, 36.2 mmol) in dichloromethane (50 mL). After the reaction mixture had been stirred at 0 °C for 4.5 hours, it was diluted with water (50 mL) and extracted with dichloromethane (3 x 100 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, concentrated in vacuo, and purified via chromatography on silica gel (Gradient: 0% to 5% ethyl acetate in petroleum ether), affording C20 as a colorless oil. Yield: 6.27 g, 33.3 mmol, 92%.1H NMR (400 MHz, methanol-d4) δ 6.76 (t, JHF = 74.4 Hz, 1H), 6.60 (br s, 1H), 6.53 (br s, 1H), 6.49 – 6.46 (m, 1H), 3.76 (s, 3H), 2.30 (s, 3H). Step 2. Synthesis of 1-(bromomethyl)-3-(difluoromethoxy)-5-methoxybenzene (C21). A mixture of C20 (3.00 g, 15.9 mmol), 2,2’-azobisisobutyronitrile (262 mg, 1.60 mmol), and N-bromosuccinimide (2.84 g, 15.9 mmol) in tetrachloromethane (90 mL) was stirred at 80 °C for 8 hours. Concentration in vacuo provided C21 as a yellow oil. Yield: 4.0 g, 15 mmol, 94%.1H NMR (400 MHz, methanol-d4), product peaks only, characteristic peaks: δ 6.84 (s, 1H), 6.77 (s, 1H), 6.63 – 6.60 (m, 1H), 4.50 (s, 2H), 3.81 (s, 3H). Step 3. Synthesis of [3-(difluoromethoxy)-5-methoxyphenyl]acetonitrile (C22). To a solution of C21 (4.0 g, 15 mmol) in acetonitrile (150 mL) were sequentially added potassium carbonate (3.11 g, 22.5 mmol) and trimethylsilyl cyanide (2.2 g, 22 mmol). The resulting mixture was stirred at 80 °C for 16 hours, at which time LCMS analysis indicated the presence of C22: LCMS m/z 214.1 [M+H]+. The reaction mixture was concentrated under reduced pressure, diluted with water (50 mL), and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, concentrated in vacuo, and purified using silica gel chromatography (Gradient: 0% to 30% ethyl acetate in petroleum ether) to afford C22 as a yellow oil. Yield: 1.20 g, 5.63 mmol, 38%.1H NMR (400 MHz, methanol-d4) δ 6.84 (t, JHF = 73.9 Hz, 1H), 6.81 (br s, 1H), 6.73 (br s, 1H), 6.68 – 6.66 (m, 1H), 3.89 (s, 2H), 3.82 (s, 3H). Step 4. Synthesis of 2-[3-(difluoromethoxy)-5-methoxyphenyl]propanenitrile (C23). Conversion of C22 (3.00 g, 14.1 mmol) to C23 was carried out using the procedure described for synthesis of C16 from C15 in Preparation P10. Silica gel chromatography (Gradient: 0% to 5% ethyl acetate in petroleum ether) provided C23 as a yellow oil. Yield: 1.00 g, 4.40 mmol, 31%. LCMS m/z 228.1 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 6.85 (t, JHF = 73.9 Hz, 1H), 6.84 – 6.82 (m, 1H), 6.77 – 6.74 (m, 1H), 6.69 – 6.66 (m, 1H), 4.11 (q, J = 7.2 Hz, 1H), 3.82 (s, 3H), 1.60 (d, J = 7.3 Hz, 3H). Step 5. Synthesis of ethyl 2-[3-(difluoromethoxy)-5-methoxyphenyl]propanoate (C24). Thionyl chloride (5.3 mL, 73 mmol) was added in a drop-wise manner to a 0 °C solution of C23 (900 mg, 3.96 mmol) in ethanol (40 mL). The reaction mixture was stirred at 85 °C for 16 hours, whereupon it was diluted with water (50 mL) and extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 6% ethyl acetate in petroleum ether) provided C24 (700 mg, 2.55 mmol, 64%) as a yellow oil. Yield: 700 mg, 2.55 mmol, 64%. LCMS m/z 275.1 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 6.80 (t, JHF = 74.2 Hz, 1H), 6.74 – 6.71 (m, 1H), 6.65 (br s, 1H), 6.59 (dd, J = 2.2, 2.2 Hz, 1H), 4.19 – 4.05 (m, 2H), 3.79 (s, 3H), 3.72 (q, J = 7.2 Hz, 1H), 1.44 (d, J = 7.2 Hz, 3H), 1.20 (t, J = 7.1 Hz, 3H). Step 6. Synthesis of 2-[3-(difluoromethoxy)-5-methoxyphenyl]propanoic acid (P11). To a solution of C24 (700 mg, 2.55 mmol) in tetrahydrofuran (30 mL) was added a solution of lithium hydroxide monohydrate (535 mg, 12.8 mmol) in water (10 mL). After the reaction mixture had been stirred at 25 °C for 16 hours, it was concentrated in vacuo, diluted with water (20 mL), and washed with dichloromethane (3 x 25 mL). These organic layers were discarded. The aqueous layer was adjusted to a pH of approximately 2 using 2 M hydrochloric acid; it was then extracted with dichloromethane (3 x 25 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution (10 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure, affording P11 as a yellow oil. Yield: 628 mg, 2.55 mmol, quantitative. LCMS m/z 247.1 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 6.79 (t, JHF = 74.2 Hz, 1H), 6.77 – 6.73 (m, 1H), 6.69 – 6.66 (m, 1H), 6.59 (dd, J = 2.2, 2.2 Hz, 1H), 3.79 (s, 3H), 3.69 (q, J = 7.2 Hz, 1H), 1.44 (d, J = 7.2 Hz, 3H). Preparation P12 2-[5-Fluoro-2-(trifluoromethoxy)pyridin-4-yl]propanoic acid (P12)
Figure imgf000069_0001
Step 1. Synthesis of diethyl (5-fluoro-2-methoxypyridin-4-yl)propanedioate (C25). Reaction of 5-fluoro-4-iodo-2-methoxypyridine (3.45 g, 13.6 mmol) with diethyl propanedioate (3.28 g, 20.5 mmol) was carried using the method described for synthesis of C6 from C5 in Preparation P5. Purification using silica gel chromatography (Gradient: 0% to 15% ethyl acetate in petroleum ether) afforded C25 as a colorless oil. Yield: 2.80 g, 9.82 mmol, 72%. LCMS m/z 286.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.00 (br s, 1H), 6.84 (br d, J = 4.8 Hz, 1H), 4.87 (s, 1H), 4.30 – 4.21 (m, 4H), 3.90 (s, 3H), 1.28 (t, J = 7.1 Hz, 6H). Step 2. Synthesis of diethyl (5-fluoro-2-methoxypyridin-4-yl)(methyl)propanedioate (C26). To a solution of C25 (2.80 g, 9.82 mmol) in acetonitrile (100 mL) was added potassium carbonate (4.07 g, 29.4 mmol), followed by drop-wise addition of iodomethane (2.09 g, 14.7 mmol). The reaction mixture was stirred at 25 °C for 2 days, whereupon LCMS analysis indicated conversion to C26: LCMS m/z 300.1 [M+H]+. The reaction mixture was poured into water (1 L) and extracted with ethyl acetate (2 x 100 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo to provide C26 as a yellow oil. Yield: 2.25 g, 7.52 mmol, 77%.1H NMR (400 MHz, chloroform-d) δ 7.95 (d, J = 2.7 Hz, 1H), 6.58 (d, J = 5.2 Hz, 1H), 4.30 – 4.22 (m, 4H), 3.90 (s, 3H), 1.81 (s, 3H), 1.27 (t, J = 7.1 Hz, 6H). Step 3. Synthesis of diethyl (5-fluoro-2-hydroxypyridin-4-yl)(methyl)propanedioate (C27). Trimethylsilyl iodide (7.52 g, 37.6 mmol) was added in a drop-wise manner to a solution of C26 (2.25 g, 7.52 mmol) in acetonitrile (100 mL), and the reaction mixture was stirred at 100 °C for 4 hours, at which time LCMS analysis indicated conversion to C27: LCMS m/z 286.1 [M+H]+. The reaction mixture was poured into aqueous sodium bicarbonate solution (100 mL), and the resulting mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed with aqueous sodium dithionite solution (200 mL), filtered, concentrated in vacuo, and purified by silica gel chromatography (Gradient: 0% to 15% methanol in dichloromethane), providing C27 as a white solid. Yield: 685 mg, 2.40 mmol, 32%.1H NMR (400 MHz, chloroform-d) δ 7.29 – 7.26 (m, 1H, assumed; partially obscured by solvent peak), 6.43 (d, J = 6.4 Hz, 1H), 4.35 – 4.19 (m, 4H), 1.80 (s, 3H), 1.27 (t, J = 7.1 Hz, 6H). Step 4. Synthesis of diethyl [5-fluoro-2-(trifluoromethoxy)pyridin-4-yl](methyl)propanedioate (C28). A solution of 1-trifluoromethyl-1,2-benziodoxol-3-(1H)-one (759 mg, 2.40 mmol) and C27 (685 mg, 2.40 mmol) in nitromethane (20 mL) was stirred at 100 °C for 16 hours. After removal of solvent in vacuo, the residue was purified via chromatography on silica gel (Gradient: 0% to 20% ethyl acetate in petroleum ether) to afford C28 as a colorless oil. Yield: 283 mg, 0.801 mmol, 33%. LCMS m/z 354.0 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.13 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 5.0 Hz, 1H), 4.34 – 4.22 (m, 4H), 1.85 (s, 3H), 1.28 (t, J = 7.1 Hz, 6H). Step 5. Synthesis of 2-[5-fluoro-2-(trifluoromethoxy)pyridin-4-yl]propanoic acid (P12). To a solution of C28 (300 mg, 0.849 mmol) in tetrahydrofuran (10 mL) was added a solution of lithium hydroxide (102 mg, 4.26 mmol) in water (3 mL) at 25 °C. After the reaction mixture had been stirred at 25 °C for 16 hours, it was combined with a similar reaction carried out using C28 (50 mg, 0.14 mmol), diluted with water (100 mL), and washed with dichloromethane (3 x 50 mL). These organic layers were discarded. The aqueous layer was adjusted to pH 5 by addition of 5 M hydrochloric acid and extracted with dichloromethane (3 x 50 mL); the combined dichloromethane layers were concentrated in vacuo to provide P12 as a white solid. Combined yield: 230 mg, 0.909 mmol, 92%. LCMS m/z 254.0 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.17 (d, J = 1.5 Hz, 1H), 7.20 (d, J = 4.8 Hz, 1H), 4.05 (q, J = 7.3 Hz, 1H), 1.53 (d, J = 7.3 Hz, 3H).
Figure imgf000071_0001
Step 1. Synthesis of methyl 2-(5-fluoro-2-methoxypyridin-4-yl)propanoate (C29). Sulfuric acid (0.2 mL) was added to a solution of a solution of P6 (1.80 g, 9.04 mmol) in methanol (20 mL), and the reaction mixture was stirred at 70 °C for 12 hours, whereupon it was concentrated under reduced pressure. The residue was treated with saturated aqueous sodium bicarbonate solution (30 mL) until the pH reached 8, and it was then extracted with ethyl acetate (3 x 30 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo to provide C29 as a colorless oil. Yield: 1.85 g, 8.68 mmol, 96%. LCMS m/z 214.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.94 (br s, 1H), 6.65 (d, J = 5.0 Hz, 1H), 3.93 (q, J = 7.3 Hz, 1H), 3.89 (s, 3H), 3.69 (s, 3H), 1.49 (d, J = 7.3 Hz, 3H). Step 2. Synthesis of methyl 2-(5-fluoro-2-hydroxypyridin-4-yl)propanoate (C30). A solution of C29 (700 mg, 3.28 mmol) and trimethylsilyl iodide (1.97 g, 9.85 mmol) in acetonitrile (10 mL) was stirred at 80 °C for 4 hours. After the reaction mixture had been concentrated in vacuo, the residue was purified using silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane), affording C30 as a pale brown oil. Yield: 550 mg, 2.76 mmol, 84%. LCMS m/z 200.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.99 (d, J = 3.5 Hz, 1H), 6.93 (d, J = 5.8 Hz, 1H), 3.99 (q, J = 7.2 Hz, 1H), 3.75 (s, 3H), 1.58 (d, J = 7.2 Hz, 3H). Step 3. Synthesis of methyl 2-[2-(difluoromethoxy)-5-fluoropyridin-4-yl]propanoate (C31). A mixture of C30 (580 mg, 2.91 mmol) and sodium chloro(difluoro)acetate (888 mg, 5.82 mmol) in acetonitrile (10.0 mL) was stirred at 100 °C for 12 hours. The reaction mixture was then concentrated in vacuo and subjected to silica gel chromatography (Gradient: 0% to 30% ethyl acetate in petroleum ether), providing C31 as a colorless oil. Yield: 550 mg, 2.21 mmol, 76%. LCMS m/z 250.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.99 (d, J = 1.3 Hz, 1H), 7.36 (t, JHF = 72.9 Hz, 1H), 6.86 (d, J = 4.8 Hz, 1H), 3.99 (q, J = 7.3 Hz, 1H), 3.72 (s, 3H), 1.53 (d, J = 7.3 Hz, 3H). Step 4. Synthesis of 2-[2-(difluoromethoxy)-5-fluoropyridin-4-yl]propanoic acid (P13). A solution of lithium hydroxide monohydrate (455 mg, 10.8 mmol) in water (5 mL) was added to a solution of C31 (1.00 g, 4.01 mmol) in tetrahydrofuran (10 mL). The reaction mixture was stirred at 25 °C for 10 hours, whereupon it was concentrated under reduced pressure, and the aqueous residue was washed with dichloromethane (3 x 10 mL). The aqueous layer was then adjusted to pH 7 by addition of 1 M hydrochloride acid, and the resulting mixture was extracted with ethyl acetate (3 x 30 mL). The combined ethyl acetate layers were concentrated in vacuo, providing P13 as a colorless oil. Yield: 830 mg, 3.53 mmol, 88%. LCMS m/z 236.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.99 (br s, 1H), 7.35 (t, JHF = 72.8 Hz, 1H), 6.86 (d, J = 4.8 Hz, 1H), 3.98 (q, J = 7.2 Hz, 1H), 1.52 (d, J = 7.3 Hz, 3H). Preparation P14 2-[2-(Dimethylamino)-5-fluoropyridin-4-yl]propanoic acid (P14)
Figure imgf000072_0001
Figure imgf000073_0001
Step 1. Synthesis of tert-butyl (2-chloro-5-fluoropyridin-4-yl)acetate (C32). Lithium diisopropylamide (2 M solution in tetrahydrofuran; 50.5 mL, 101 mmol) was added to a −78 °C solution of 2-chloro-5-fluoro-4-methylpyridine (4.90 g, 33.7 mmol) in tetrahydrofuran (200 mL). After the reaction mixture had been stirred at −50 °C for 1 hour, it was cooled to −78 °C, and a solution of di-tert-butyl dicarbonate (8.51 mL, 37.0 mmol) in tetrahydrofuran (30 mL) was added. The reaction mixture was then warmed to −30 °C, stirred for 2 hours, and diluted with water (100 mL). The resulting mixture was extracted with ethyl acetate (3 x 50 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% ethyl acetate in petroleum ether) provided C32 as an oil. Yield: 4.90 g, 19.9 mmol, 59%. LCMS m/z 246.1 (chlorine isotope pattern observed) [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.21 (br s, 1H), 7.29 (d, J = 5.2 Hz, 1H), 3.59 (s, 2H), 1.46 (s, 9H). Step 2. Synthesis of tert-butyl 2-(2-chloro-5-fluoropyridin-4-yl)propanoate (C33). Conversion of C32 (4.60 g, 18.7 mmol) to C33 was carried out using the method described for synthesis of C16 from C15 in Preparation P10. Silica gel chromatography (Gradient 0% to 20% ethyl acetate in petroleum ether) provided C33 as an oil. Yield: 4.40 g, 16.9 mmol, 90%. LCMS m/z 262.1 (chlorine isotope pattern observed) [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.19 (br s, 1H), 7.28 (d, J = 5.1 Hz, 1H), 3.87 (q, J = 7.3 Hz, 1H), 1.48 (d, J = 7.2 Hz, 3H), 1.42 (s, 9H). Step 3. Synthesis of 2-[2-(dimethylamino)-5-fluoropyridin-4-yl]propanoic acid (P14). A mixture of C33 (3.00 g, 11.6 mmol), dimethylamine (2 M solution in tetrahydrofuran; 8.66 mL, 17.3 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.06 g, 1.16 mmol), 2- dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (RuPhos; 1.08 g, 2.31 mmol), and sodium tert- butoxide (3.33 g, 34.7 mmol) in toluene (100 mL) was stirred at 100 °C for 16 hours. After the reaction mixture had been concentrated in vacuo, it was diluted with water and washed with dichloromethane (3 x 30 mL). The aqueous layer was then adjusted to pH 5 by addition of 5 M hydrochloric acid, and extracted with ethyl acetate (2 x 50 mL). The combined ethyl acetate layers were dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane) afforded P14 as a gray solid. Yield: 700 mg, 3.30 mmol, 28%. LCMS m/z 213.1 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.87 (d, J = 2.1 Hz, 1H), 6.57 (d, J = 4.9 Hz, 1H), 3.90 (q, J = 7.2 Hz, 1H), 3.04 (s, 6H), 1.48 (d, J = 7.2 Hz, 3H).
Figure imgf000074_0001
Step 1. Synthesis of dimethyl (2,5-dichloropyrimidin-4-yl)(methyl)propanedioate (C34). Sodium hydride (60% dispersion in mineral oil; 1.31 g, 33 mmol) was slowly added to a 0 °C solution of dimethyl methylpropanedioate (4.78 g, 32.7 mmol) in tetrahydrofuran (40 mL). The reaction mixture was stirred at 0 °C for 30 minutes, whereupon a solution of 2,4,5- trichloropyrimidine (5.00 g, 27.3 mmol) in tetrahydrofuran (10 mL) was added drop-wise at 0 °C. Stirring was continued at 0 °C for 30 minutes, at which point the reaction mixture was slowly warmed to 25 °C and allowed to stir at that temperature for 30 minutes. After addition of saturated aqueous ammonium chloride solution (100 mL), the mixture was extracted with ethyl acetate (3 x 100 mL). The combined organic layers were washed sequentially with water and with saturated aqueous sodium chloride solution, then combined with the organic layer from a similar reaction carried out using 2,4,5-trichloropyrimidine (500 mg, 2.73 mmol), dried over sodium sulfate, filtered, and concentrated in vacuo while keeping the temperature below 40 °C. Silica gel chromatography (Gradient: 10% to 13% ethyl acetate in petroleum ether) provided C34 as a colorless oil. Combined yield: 6.82 g, 23.3 mmol, 78%. LCMS m/z 293.0 (dichloro isotope pattern observed) [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.74 (s, 1H), 3.79 (s, 6H), 1.90 (s, 3H). Step 2. Synthesis of dimethyl (5-chloro-2-methoxypyrimidin-4-yl)(methyl)propanedioate (C35). A solution of sodium methoxide in methanol (30% solution; 4.66 g, 26 mmol) was added drop-wise to a solution of C34 (6.32 g, 21.6 mmol) in methanol (120 mL). After the reaction mixture had been stirred at 25 °C for 2 hours, it was concentrated in vacuo while keeping the temperature below 40 °C, diluted with water (50 mL), and extracted with ethyl acetate (2 x 100 mL). The organic layers were combined with those from a similar reaction carried out using C34 (500 mg, 1.71 mmol), washed sequentially with water and with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 11% to 15% ethyl acetate in petroleum ether) afforded C35 as a colorless oil. Combined yield: 4.00 g, 13.9 mmol, 60%. LCMS m/z 289.0 (chlorine isotope pattern observed) [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.53 (s, 1H), 3.95 (s, 3H), 3.79 (s, 6H), 1.88 (s, 3H). Step 3. Synthesis of lithium 2-(5-chloro-2-methoxypyrimidin-4-yl)propanoate (P15). A solution of lithium hydroxide monohydrate (1.65 g, 39.3 mmol) in water (20 mL) was added drop-wise to a solution of C35 (3.78 g, 13.1 mmol) in tetrahydrofuran (60 mL). The reaction mixture was stirred at 35 °C for 3 hours, whereupon it was concentrated in vacuo. The resulting aqueous mixture was washed with dichloromethane and then purified via reversed-phase chromatography (Column: C18; Gradient: 0% to 10% acetonitrile in water), providing P15 as a white solid. Yield: 1.87 g, 8.40 mmol, 64%. LCMS m/z 217.1 [M+H]+.1H NMR (400 MHz, methanol- d4) δ 8.37 (s, 1H), 4.05 (q, J = 7.2 Hz, 1H), 4.00 (s, 3H), 1.55 (d, J = 7.2 Hz, 3H).
Figure imgf000075_0001
Figure imgf000076_0001
Step 1. Synthesis of methyl 2-(difluoromethoxy)-6-methoxypyridine-4-carboxylate (C36). Methyl 2-hydroxy-6-methoxypyridine-4-carboxylate (900 mg, 4.91 mmol) was converted to C36 using the method described for synthesis of C5 from 5-iodopyridin-2-ol in Preparation P5. Chromatography on silica gel (Gradient: 0% to 8% ethyl acetate in petroleum ether) provided C36 as a colorless oil. Yield: 720 mg, 3.09 mmol, 63%. LCMS m/z 234.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.39 (t, JHF = 73.0 Hz, 1H), 7.10 (br s, 1H), 7.00 (br s, 1H), 3.94 (s, 3H), 3.93 (s, 3H). Step 2. Synthesis of 2-(difluoromethoxy)-6-methoxypyridine-4-carboxylic acid (C37). Using the method described for synthesis of P11 from C24 in Preparation P11, C36 (1.10 g, 4.72 mmol) was hydrolyzed, affording C37 as a white solid. Yield: 980 mg, 4.47 mmol, 95%. LCMS m/z 220.1 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.41 (t, JHF = 72.8 Hz, 1H), 7.15 (d, J = 1.1 Hz, 1H), 7.05 (d, J = 1.0 Hz, 1H), 3.95 (s, 3H). Step 3. Synthesis of methyl [2-(difluoromethoxy)-6-methoxypyridin-4-yl]acetate (C38). A solution of C37 (980 mg, 4.47 mmol) in thionyl chloride (6.49 mL, 89.0 mmol) was stirred at 70 °C for 2.5 hours, whereupon it was concentrated under reduced pressure. After the resulting acyl chloride had been dissolved in a mixture of tetrahydrofuran (8 mL) and acetonitrile (8 mL), it was cooled to 0 °C and treated with freshly distilled triethylamine (0.87 mL, 6.2 mmol), followed by (diazomethyl)(trimethyl)silane (2 M solution in diethyl ether; 3.35 mL, 6.70 mmol). The reaction mixture was stirred at 0 °C for 8 hours, whereupon it was diluted with diethyl ether (25 mL) and washed sequentially with 10% aqueous citric acid solution (5 mL), saturated aqueous sodium bicarbonate solution (15 mL), and saturated aqueous sodium chloride solution (25 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide the crude diazoketone. This material was suspended in methanol (10 mL) in an ultrasonic bath; a solution of silver benzoate (512 mg, 2.24 mmol) in triethylamine (1.86 mL, 13.3 mmol) was gradually added at room temperature while the reaction mixture was sonicated. After 30 minutes, volatiles were removed in vacuo, and the residue was purified using chromatography on silica gel (Gradient: 0% to 10% ethyl acetate in petroleum ether) to provide C38 as a colorless oil. Yield: 340 mg, 1.38 mmol, 31%. LCMS m/z 248.0 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.40 (t, JHF = 73.4 Hz, 1H), 6.45 (br s, 1H), 6.40 (br s, 1H), 3.88 (s, 3H), 3.71 (s, 3H), 3.57 (s, 2H). Step 4. Synthesis of methyl 2-[2-(difluoromethoxy)-6-methoxypyridin-4-yl]propanoate (C39). To a −78 °C solution of C38 (230 mg, 0.930 mmol) in tetrahydrofuran (20 mL) was added sodium bis(trimethylsilyl)amide (2 M solution in tetrahydrofuran; 0.56 mL, 1.1 mmol), and the reaction mixture was stirred at −78 °C for 1 hour. Iodomethane (57.9 µL, 0.93 mmol) was then added and stirring was continued for 2 hours at −78 °C. After addition of saturated aqueous ammonium chloride solution (10 mL), the mixture was combined with a similar reaction carried out using C38 (100 mg, 0.405 mmol) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo; silica gel chromatography (Gradient: 0% to 4% ethyl acetate in petroleum ether) afforded C39 as a colorless oil. Combined yield: 150 mg, 0.574 mmol, 43%. LCMS m/z 262.1 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.51 (t, JHF = 73.3 Hz, 1H), 6.50 (br d, J = 1 Hz, 1H), 6.43 (br d, J = 1 Hz, 1H), 3.88 (s, 3H), 3.78 (q, J = 7.2 Hz, 1H), 3.68 (s, 3H), 1.44 (d, J = 7.2 Hz, 3H). Step 5. Synthesis of 2-[2-(difluoromethoxy)-6-methoxypyridin-4-yl]propanoic acid (P16). Hydrolysis of C39 (130 mg, 0.498 mmol) was carried out using the method described for synthesis of P12 from C28 in Preparation P12, providing P16 as a colorless oil. Yield: 101 mg, 0.409 mmol, 82%. LCMS m/z 248.0 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.51 (t, JHF = 73.3 Hz, 1H), 6.53 (br d, J = 1.1 Hz, 1H), 6.46 (br d, J = 1.1 Hz, 1H), 3.88 (s, 3H), 3.72 (q, J = 7.1 Hz, 1H), 1.44 (d, J = 7.2 Hz, 3H). Preparations P17 and P18 tert-Butyl 7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (P17) and Di-tert-butyl 7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1,1'-dicarboxylate (P18)
Figure imgf000077_0001
Figure imgf000078_0001
Step 1. Synthesis of 2-chloro-3-iodo-6-methylpyridine (C40). To a 0 °C mixture of 2-chloro-6-methylpyridin-3-amine (400 g, 2.80 mol) in water (5.0 L) and hydrochloric acid (5.0 M; 3.3 L, 16.5 mol) was added a solution of sodium nitrite (290 g, 4.20 mol) in water (800 mL) in a drop-wise manner, at a rate that maintained the internal reaction temperature below 5 °C. The reaction mixture was stirred under ice-cooling for 30 minutes, then cooled to −5 °C, whereupon tert-butyl methyl ether (3.0 L) was added, followed by drop-wise addition of a solution of potassium iodide (929 g, 5.60 mol) in water (800 mL), while the internal reaction temperature was maintained below 10 °C. The reaction mixture was then allowed to slowly warm to 25 °C and stirring was continued at 25 °C for 16 hours. After the pH had been adjusted to 9 by addition of 2 M aqueous sodium hydroxide solution, the mixture was extracted with ethyl acetate (3 x 2.0 L); the combined organic layers were washed twice with aqueous sodium sulfite solution and once with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification via silica gel chromatography (Gradient: 0% to 5% ethyl acetate in petroleum ether) afforded C40 as a white solid. Yield: 610 g, 2.41 mol, 86%. LCMS m/z 253.9 (chlorine isotope pattern observed) [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.13 (d, J = 7.9 Hz, 1H), 6.96 (d, J = 7.9 Hz, 1H), 2.44 (s, 3H). Step 2. Synthesis of 1-benzyl-3-[(trimethylsilyl)ethynyl]pyrrolidin-3-ol (C41). A solution of n-butyllithium in tetrahydrofuran (2.5 M; 3.75 L, 9.4 mol) was added in a drop- wise manner to a −78 °C solution of ethynyl(trimethyl)silane (1.01 kg, 10.3 mol) in tetrahydrofuran (4.0 L). The reaction mixture was stirred at −78 °C for 1 hour, whereupon a solution of 1- benzylpyrrolidin-3-one (1.50 kg, 8.56 mol) in tetrahydrofuran (1.5 L) was added drop-wise. After completion of the addition, the reaction mixture was warmed to 20 °C, stirred at 20 °C for 16 hours, and subsequently poured into aqueous ammonium chloride solution. The resulting mixture was extracted with ethyl acetate (2 x 2.0 L), and the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo to provide C41 as a yellow oil. Yield: 2.25 kg, 8.23 mol, 96%. LCMS m/z 274.2 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.37 – 7.28 (m, 4H), 7.28 – 7.22 (m, 1H), 3.66 (AB quartet, JAB = 12.7 Hz, ΔνAB = 12.2 Hz, 2H), 2.89 – 2.77 (m, 3H), 2.65 (ddd, J = 9.4, 7.9, 5.5 Hz, 1H), 2.30 – 2.21 (m, 1H), 2.12 – 2.03 (m, 1H), 0.14 (s, 9H). Step 3. Synthesis of 1-benzyl-3-ethynylpyrrolidin-3-ol (C42). A mixture of C41 (2.77 kg, 10.1 mol) and potassium carbonate (2.80 kg, 20.3 mol) in methanol (10 L) was stirred at 25 °C for 3 hours, whereupon the reaction mixture was filtered, and the filtrate was concentrated in vacuo. After the residue had been diluted with ethyl acetate (10 L), it was filtered. Concentration of this filtrate under reduced pressure afforded C42 as a black oil (2.30 kg). This material was taken directly to the following step. LCMS m/z 202.2 [M+H]+.1H NMR (400 MHz, methanol-d4), characteristic peaks: δ 7.37 – 7.28 (m, 4H), 7.28 – 7.22 (m, 1H), 3.66 (AB quartet, JAB = 12.7 Hz, ΔνAB = 12.7 Hz, 2H), 2.89 – 2.78 (m, 3H), 2.65 (ddd, J = 9.4, 7.9, 5.7 Hz, 1H), 2.27 (ddd, J = 13.3, 7.9, 6.8 Hz, 1H), 2.14 – 2.04 (m, 1H). Step 4. Synthesis of 1-benzyl-3-ethynylpyrrolidin-3-yl acetate (C43). To a 0 °C solution of C42 (from the previous step; 2.30 kg, ≤10.1 mol) and triethylamine (3.17 L, 22.7 mol) in dichloromethane (10 L) was added acetyl chloride (1.35 kg, 17.2 mol) in a drop-wise manner. The reaction mixture was then stirred at 25 °C for 30 minutes, whereupon water (10 L) was added. The resulting mixture was extracted with dichloromethane (2 x 3.0 L), and the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo to provide C43 as a brown oil (2.82 kg). A portion of this material was used in the following step. LCMS m/z 244.2 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.38 – 7.23 (m, 5H), 3.65 (s, 2H), 3.05 (s, 2H), 3.03 (s, 1H), 2.77 (ddd, J = 9.5, 7.4, 6.1 Hz, 1H), 2.66 (ddd, J = 9.5, 7.4, 6.5 Hz, 1H), 2.46 (ddd, J = 13.6, 7.4, 6.1 Hz, 1H), 2.40 – 2.31 (m, 1H), 2.02 (s, 3H). Step 5. Synthesis of 1-benzyl-3-ethynyl-N-[(4-methoxyphenyl)methyl]pyrrolidin-3-amine (C44). A mixture of C43 (from the previous step; 1.20 kg, ≤4.30 mol), 1-(4- methoxyphenyl)methanamine (1.35 kg, 9.84 mmol), and copper(I) chloride (48.8 g, 0.493 mol) in tetrahydrofuran (6.0 L) was degassed under vacuum and then purged with nitrogen; this evacuation-purge cycle was carried out a total of three times. The reaction mixture was then stirred at reflux for 45 minutes, whereupon it was concentrated in vacuo. This material was combined with that from three similar reactions carried out using C43 (from the previous step; 900 g of C43 employed in the three reactions, ≤3.2 mol) and purified by chromatography on silica gel (Gradient: 0% to 50% ethyl acetate in petroleum ether) to provide C44 as a brown oil. Combined yield: 620 g, 1.93 mol, 26% over 3 steps. LCMS m/z 321.3 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.36 – 7.21 (m, 7H), 6.85 (br d, J = 8.7 Hz, 2H), 3.81 – 3.69 (m, 2H), 3.77 (s, 3H), 3.65 (AB quartet, JAB = 12.7 Hz, ΔνAB = 9.9 Hz, 2H), 2.88 (s, 1H), 2.82 – 2.67 (m, 2H), 2.79 (AB quartet, JAB = 9.8 Hz, ΔνAB = 37.8 Hz, 2H), 2.27 (ddd, J = 13.4, 7.7, 6.0 Hz, 1H), 2.09 – 2.01 (m, 1H). Step 6. Synthesis of 1-benzyl-3-[(2-chloro-6-methylpyridin-3-yl)ethynyl]-N-[(4- methoxyphenyl)methyl]pyrrolidin-3-amine (C45). A mixture of C44 (426 g, 1.33 mol), C40 (303 g, 1.20 mmol), dichlorobis(triphenylphosphine)palladium(II) (46.6 g, 66.4 mmol), and copper(I) iodide (12.6 g, 66.2 mmol) in triethylamine (2.0 L) was degassed under vacuum and then purged with nitrogen; this evacuation-purge cycle was carried out a total of three times. The reaction mixture was stirred at reflux for 16 hours, whereupon it was filtered; the filtrate was concentrated in vacuo and combined with material from two similar reactions carried out using C40 (12.17 g, 48.0 mmol; 146 g, 0.576 mol). The resulting mixture was purified via silica gel chromatography (Gradient: 20% to 50% ethyl acetate in petroleum ether), affording C45 as a black oil. Combined yield: 420 g, 0.942 mol, 52%. LCMS m/z 446.2 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.80 (d, J = 7.8 Hz, 1H), 7.39 – 7.20 (m, 8H), 6.86 (d, J = 8.6 Hz, 2H), 3.87 (AB quartet, JAB = 12.0 Hz, ΔνAB = 29.6 Hz, 2H), 3.76 (s, 3H), 3.70 (AB quartet, JAB = 13.0 Hz, ΔνAB = 9.3 Hz, 2H), 3.00 (d, component of AB quartet, J = 9.9 Hz, 1H), 2.87 – 2.77 (m, 3H), 2.51 (s, 3H), 2.44 – 2.33 (m, 1H), 2.21 – 2.10 (m, 1H). Step 7. Synthesis of 1-benzyl-3-[2-(2-chloro-6-methylpyridin-3-yl)ethyl]-N-[(4- methoxyphenyl)methyl]pyrrolidin-3-amine (C46). A mixture of C45 (40.0 g, 89.7 mmol) and platinum(IV) oxide (4.09 g, 18.0 mmol) in methanol (400 mL) was hydrogenated (60 psi) at 25 °C for 3 hours. The reaction mixture was then filtered, and the filtrate was concentrated in vacuo to provide C46 as a black oil. Yield: 40.5 g, assumed quantitative. LCMS m/z 450.3 [M+H]+.1H NMR (400 MHz, methanol-d4), characteristic peaks: δ 7.61 (d, J = 7.7 Hz, 1H), 7.38 – 7.23 (m, 7H), 7.17 (d, J = 7.7 Hz, 1H), 6.88 (br d, J = 8.7 Hz, 2H), 3.78 (s, 3H), 3.64 (AB quartet, JAB = 12.0 Hz, ΔνAB = 21.6 Hz, 2H), 3.64 (s, 2H), 2.46 (s, 3H). Step 8. Synthesis of 1'-benzyl-1-[(4-methoxyphenyl)methyl]-7-methyl-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine] (C47). A mixture of C46 (400 g, 0.89 mol), palladium(II) acetate (9.97 g, 44.4 mmol), 2- dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (RuPhos; 41.5 g, 88.9 mmol) and sodium tert- butoxide (170 g, 1.77 mol) in 1,4-dioxane (4.0 L) was stirred at 90 °C for 10 hours, whereupon the reaction mixture was filtered, and the filtrate was concentrated in vacuo. After the residue had been partitioned between ethyl acetate (2 L) and water (2 L), the aqueous layer was extracted with ethyl acetate (1 L). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, concentrated in vacuo, and subjected to silica gel chromatography (Gradient: 0% to 10% ethyl acetate in petroleum ether), affording C47 as a white solid. Yield: 195 g, 0.472 mol, 53%. LCMS m/z 414.3 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.38 – 7.21 (m, 5H, assumed; partially obscured by solvent peak), 7.17 (d, J = 8.2 Hz, 2H), 7.03 (d, J = 7.2 Hz, 1H), 6.77 (d, J = 8.2 Hz, 2H), 6.32 (d, J = 7.3 Hz, 1H), 5.07 – 4.92 (m, 2H), 3.77 (s, 3H), 3.54 (br AB quartet, JAB = 13 Hz, ΔνAB = 40 Hz, 2H), 2.95 (d, J = 10.2 Hz, 1H), 2.92 – 2.83 (m, 1H), 2.83 – 2.73 (m, 1H), 2.73 – 2.63 (m, 1H), 2.43 – 2.31 (m, 1H), 2.29 – 2.08 (m, 2H), 2.23 (s, 3H), 2.03 – 1.73 (m, 3H). Step 9. Synthesis of 1'-benzyl-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine] (C48). To a 0 °C solution of C47 (190 g, 0.459 mol) in dichloromethane (1.5 L) was added trifluoroacetic acid (523 g, 4.59 mol), and the reaction mixture was stirred at 25 °C for 3 hours. It was then concentrated in vacuo; the residue was diluted with ethyl acetate (1.5 L) and washed with saturated aqueous sodium carbonate solution (1.0 L), and this aqueous layer was extracted with ethyl acetate (2 x 300 mL). The combined organic layers were concentrated in vacuo and purified via silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to afford C48 as a brown oil (179 g). This material was progressed directly to the following step. LCMS m/z 294.3 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 9.1 – 8.3 (br s, 1H), 7.41 – 7.35 (m, 2H), 7.35 – 7.28 (m, 2H), 7.28 – 7.22 (m, 2H, assumed; partially obscured by solvent peak), 6.35 (d, J = 7.3 Hz, 1H), 3.72 (s, 2H), 2.96 – 2.85 (m, 1H), 2.80 – 2.62 (m, 5H), 2.42 (s, 3H), 2.05 (ddd, J = 13.1, 8.1, 5.0 Hz, 1H), 1.98 – 1.81 (m, 3H). Step 10. Synthesis of tert-butyl 7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'- carboxylate (P17) and di-tert-butyl 7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine]-1,1'-dicarboxylate (P18). A mixture of C48 (from the previous step; 179 g, ≤0.459 mol), di-tert-butyl dicarbonate (199.7 g, 915 mmol), and palladium hydroxide (17.9 g, 127 mmol) in methanol (2.0 L) and ethyl acetate (2.0 L) was hydrogenated at 55 psi and 25 °C for 18 hours. The reaction mixture was then filtered through a pad of diatomaceous earth and the filtrate was concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 50% dichloromethane in ethyl acetate) provided P17 and P18, both as white solids. P17 – Yield: 101 g, 0.333 mol, 73% over 2 steps. LCMS m/z 304.3 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.20 (d, J = 7.3 Hz, 1H), 6.44 (d, J = 7.4 Hz, 1H), 3.62 – 3.44 (m, 2H), 3.37 – 3.3 (m, 2H, assumed; partially obscured by solvent peak), 2.84 – 2.66 (m, 2H), 2.27 (s, 3H), 2.05 – 1.92 (m, 2H), 1.92 – 1.76 (m, 2H), [1.48 (s) and 1.46 (s), total 9H]. P18 – Yield: 21.3 g, 52.8 mmol, 12% over 2 steps. LCMS m/z 404.3 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.49 (d, J = 7.7 Hz, 1H), 7.02 (br d, J = 7.7 Hz, 1H), [3.85 (d, J = 11.3 Hz) and 3.75 (d, J = 11.2 Hz), total 1H], 3.62 – 3.47 (m, 2H), 3.40 – 3.24 (m, 1H, assumed; partially obscured by solvent peak), 2.89 – 2.73 (m, 2H), 2.54 – 2.27 (m, 1H), 2.44 (s, 3H), 2.13 – 1.82 (m, 3H), 1.46 (s, 9H), [1.43 (s) and 1.43 (s), total 9H].
Figure imgf000082_0001
Di-tert-butyl dicarbonate (3.97 g, 18.2 mmol) was added to a solution of C48 (4.45 g, 15.2 mmol) in a mixture of methanol (20 mL) and ethyl acetate (25 mL). After addition of palladium hydroxide on carbon (900 mg), the reaction mixture was hydrogenated at 80 psi for 18 hours, at which time LCMS analysis indicated complete conversion to P19 / P20: LCMS m/z 304.2 [M+H]+. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure; the residue was dissolved in ethyl acetate, washed sequentially with saturated sodium bicarbonate solution and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. Separation of the component enantiomers was carried out via supercritical fluid chromatography {Column: Chiral Technologies Chiralpak IB, 30 x 250 mm, 5 µm; Mobile phase 9:1 carbon dioxide / [ethanol containing 0.2% (7 M ammonia in methanol)]; Flow rate: 80 mL/minute; Back pressure: 100 bar}. The first-eluting enantiomer was designated as P19 and the second-eluting enantiomer as P20. Both were isolated as solids. P19 – Yield: 1.60 g, 5.27 mmol, 35%. Retention time: 3.75 minutes [Analytical conditions. Column: Chiral Technologies Chiralpak IB, 4.6 x 250 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: ethanol containing 0.2% (7 M ammonia in methanol); Gradient: 5% B for 1 minute, then 5% to 60% B over 8 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar]. P20 – Yield: 1.50 g, 4.94 mmol, 32%. Retention time: 3.96 minutes (Analytical conditions identical to those used for P19). The indicated absolute stereochemistries were assigned based on the conversion of this batch of P19 to P23 in Alternate Preparation (#1) of P23 below. The absolute configuration of P23 was established via its use in the synthesis of 14, which was analyzed via single-crystal X-ray crystallography (see below). Preparation P21 Di-tert-butyl 6-bromo-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1,1'- dicarboxylate (P21)
Figure imgf000083_0001
To a 0 °C solution of P18 (20 g, 50 mmol) in dichloromethane (200 mL) was added 1,3- dibromo-5,5-dimethylimidazolidine-2,4-dione (7.09 g, 24.8 mmol) in six portions over 30 minutes. The reaction mixture was stirred at 0 °C for 1 hour, whereupon it was treated with saturated aqueous sodium sulfite solution (200 mL) and extracted with dichloromethane (3 x 100 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 40% ethyl acetate in petroleum ether) provided P21 as a white solid. Yield: 22.8 g, 47.2 mmol, 94%. LCMS m/z 384.1 (bromine isotope pattern observed) {[M – (2-methylprop-1-ene and CO2)]+H}+.1H NMR (400 MHz, chloroform-d) δ 7.51 (br s, 1H), [3.89 (d, J = 11.0 Hz) and 3.73 (d, J = 11.0 Hz), total 1H], 3.65 – 3.51 (m, 1H), 3.46 (d, J = 11.0 Hz, 1H), 3.38 – 3.26 (m, 1H), [2.87 – 2.56 (m) and 2.15 – 1.70 (m), total 6H], 2.57 (s, 3H), [1.46 (s) and 1.45 (s), total 18H].
Figure imgf000084_0001
1,3-Dibromo-5,5-dimethylimidazolidine-2,4-dione (2.47 g, 8.64 mmol) was added in portions over 20 minutes to a 0 °C solution of P17 (5.25 g, 17.3 mmol) in dichloromethane (69 mL). After the reaction mixture had been stirred at 0 °C for 45 minutes, LCMS analysis indicated conversion to P22: LCMS m/z 384.3 (bromine isotope pattern observed) [M+H]+. After 1 hour at 0 °C, the reaction mixture was treated with saturated aqueous sodium sulfite solution (100 mL), and the mixture was extracted with dichloromethane. The organic layer was washed sequentially with saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo to provide P22 as a solid. Yield: 6.60 g, 17.3 mmol, quantitative.1H NMR (400 MHz, methanol-d4) δ 7.40 (br s, 1H), 3.61 – 3.43 (m, 2H), 3.37 – 3.3 (m, 2H, assumed; largely obscured by water peak), 2.85 – 2.67 (m, 2H), 2.37 (s, 3H), 2.06 – 1.92 (m, 2H), 1.92 – 1.75 (m, 2H), [1.47 (s) and 1.46 (s), total 9H]. Preparations P23 and P24 tert-Butyl (2S)-6-bromo-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'- carboxylate (P23) and tert-Butyl (2R)-6-bromo-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidine]-1'-carboxylate (P24)
Figure imgf000085_0001
1,3-Dibromo-5,5-dimethylimidazolidine-2,4-dione (5.65 g, 19.8 mmol) was added in portions to a 0 °C solution of P17 (10.0 g, 32.9 mmol) in dichloromethane (150 mL), and the reaction mixture was stirred at 0 °C to 5 °C for 1 hour, at which time LCMS analysis indicated that bromination had occurred: LCMS m/z 382.3 [M+H]+. Saturated aqueous sodium sulfite solution (20 mL) was added, followed by water (50 mL); the resulting aqueous layer was extracted with dichloromethane (2 x 50 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo; silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) afforded a racemic mixture of P23 and P24 as a light-yellow foam (11.8 g). This was combined with the product of a similar reaction carried out using P17 (7.40 g, 24.4 mmol) to provide a light-yellow foam (20.9 g, 54.6 mmol, combined yield 95%), and separated into its component enantiomers via supercritical fluid chromatography [Column: Chiral Technologies Chiralcel OJ, 50 x 250 mm, 5 µm; Mobile phase 4:1 carbon dioxide / (1:1 methanol / acetonitrile); Flow rate: 250 mL/minute; Back pressure: 120 bar]. The first-eluting enantiomer was designated as P23, and the second-eluting enantiomer was designated as P24. The indicated absolute stereochemistry was assigned on the basis of conversion of this batch of P23 to P28 (see Preparation P28) and then to Example 14; the absolute stereochemistry of 14 was established via single-crystal X-ray analysis (see below). P23, isolated as a yellow oil that solidified on standing – Combined yield: 9.37 g, 24.5 mmol, 43%. 1H NMR (400 MHz, DMSO-d6) δ 7.37 (s, 1H), 7.02 – 6.96 (m, 1H), [3.55 – 3.40 (m), 3.36 – 3.26 (m, assumed; partially obscured by water peak), and 3.24 – 3.13 (m), total 4H], 2.75 – 2.55 (m, 2H), 2.31 (s, 3H), 1.95 – 1.78 (m, 2H), 1.76 – 1.60 (m, 2H), [1.40 (s) and 1.38 (s), total 9H]. Retention time: 4.01 minutes [Analytical conditions. Column: Chiral Technologies Chiralcel OJ-H, 4.6 x 250 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: methanol containing 0.2% (7 M ammonia in methanol); Gradient: 5% B for 1 minute, then 5% to 60% B over 8 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar]. P24 - Combined yield: 11.8 g, which contained ethanol; corrected estimate: 28.4 mmol, 50%.1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ 7.37 (s, 1H), 7.01 – 6.96 (m, 1H), 2.75 – 2.55 (m, 2H), 2.31 (s, 3H), 1.95 – 1.78 (m, 2H), 1.76 – 1.60 (m, 2H), [1.40 (s) and 1.38 (s), total 9H]. Retention time: 4.32 minutes (Analytical conditions identical to those used for P23). Alternate Preparation (#1) of P23 tert-Butyl (2S)-6-bromo-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'- carboxylate (P23)
Figure imgf000086_0001
1,3-Dibromo-5,5-dimethylimidazolidine-2,4-dione (625 mg, 2.19 mmol) was added in portions to a 0 °C solution of P19 (material from Preparations P19 and P20; 1.10 g, 3.63 mmol) in dichloromethane (20 mL). After the reaction mixture had been stirred at room temperature for 1 hour, LCMS analysis indicated conversion to P23: LCMS m/z 384.2 (bromine isotope pattern observed) [M+H]+. Saturated aqueous sodium sulfite solution was then added, and the resulting mixture was extracted with dichloromethane. The organic layer was washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, concentrated in vacuo, and purified using silica gel chromatography (Gradient: 10% to 40% ethyl acetate in heptane) to afford P23 as a white solid. Yield: 1.25 g, 3.27 mmol, 90%.1H NMR (400 MHz, chloroform-d) δ 7.33 (s, 1H), 5.15 – 5.01 (br s, 1H), 3.59 – 3.45 (m, 2H), 3.43 – 3.25 (m, 2H), 2.81 – 2.66 (m, 2H), 2.43 (s, 3H), 2.01 – 1.74 (m, 4H), 1.48 – 1.43 (br s, 9H). The absolute stereochemistry of this sample of P23 was assigned as indicated, via comparison with samples from Preparations P23 and P24: Retention time of P23 from Alternate Preparation (#1) of P23: 4.08 minutes Retention times of a racemic mixture of P23 and P24: 4.07 and 4.36 minutes. Retention time of P23 from Preparations P23 and P24: 4.01 minutes Retention time of P24 from Preparations P23 and P24: 4.32 minutes These four analyses were run using the same analytical method: [Column: Chiral Technologies Chiralcel OJ-H, 4.6 x 100 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: methanol containing 0.2% (7 M ammonia in methanol); Gradient: 5% B for 1 minute, then 5% to 60% B over 8 minutes; Flow rate: 3.0 mL/minute].
Figure imgf000087_0001
Figure imgf000088_0001
Step 1. Synthesis of (2-chloro-6-methylpyridin-3-yl)methanol (C49). Sodium bis(2-methoxyethoxy)aluminum hydride solution (70%; 1.05 kg, 2.5 eq) was added to a −5 °C to 5 °C solution of 2-chloro-6-methylpyridine-3-carboxylic acid (250 g, 1.46 mol) in toluene (2.5 L). After the reaction mixture had been stirred at −5 °C to 5 °C for 19 hours, it was treated with a solution of sodium hydroxide (145.7 g, 3.642 mol, 2.50 eq) in water (1.25 L), while the internal temperature was maintained below 0 °C to 10 °C. The resulting mixture was then warmed to 25 °C; after 15 minutes, the aqueous layer was extracted with propan-2-yl acetate (2 x 1.25 L). These two extracts were combined with the toluene layer and filtered through silica gel (125 g). The filter cake was rinsed with propan-2-yl acetate (125 mL), and the combined filtrates were concentrated to 8 volumes at a temperature of 40 °C to 45 °C, affording C49 as a solution in toluene (1.602 kg, 11.2% C49 by weight); the bulk of this solution was used in the following step. Estimated yield: 179.4 g, 1.138 mol, 78%.1H NMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 5.48 (t, J = 5.6 Hz, 1H), 4.50 (d, J = 5.6 Hz, 2H), 2.43 (s, 3H). Step 2. Synthesis of (2-chloro-6-methylpyridin-3-yl)methyl methanesulfonate (C50). Triethylamine (134.2 g, 1.326 mol) was added to a solution of C49 in toluene (from the previous step; 1.537 kg, containing 11.2% C49, 172.1 g, 1.09 mol). The solution was cooled to −5 °C to 5 °C, and then treated in a drop-wise manner with methanesulfonyl chloride (128.5 g, 1.122 mol), while maintaining the internal temperature at −5 °C to 5 °C. After the reaction mixture had been stirred at this temperature for 2 hours, triethylamine (22.7 g, 0.224 mol) was again added, followed by drop-wise addition of methanesulfonyl chloride (25.7 g, 0.224 mol). Stirring was continued at −5 °C to 5 °C for 1 hour, whereupon the reaction mixture was treated with water (805 mL) while the internal temperature was maintained below 25 °C, and then stirred for 15 minutes at 25 °C. The organic layer was washed with water (805 mL) and concentrated to provide C50 as a solution in toluene (861 g). This solution was used directly in the following step.1H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 7.7 Hz, 1H), 7.36 (d, J = 7.7 Hz, 1H), 5.30 (s, 2H), 3.29 (s, 3H), 2.48 (s, 3H). Step 3. Synthesis of 2-chloro-3-(iodomethyl)-6-methylpyridine (C51). Sodium iodide (230 g, 1.53 mol) was dissolved in acetone (1.13 kg) at 25 °C; to this solution was added a solution of C50 in toluene (from the previous step; 861 g, ≤1.09 mol of C50). After the reaction mixture had been stirred at 25 °C for 1 hour, a solution of sodium metabisulfite (57.86 g, 0.3044 mol) in water (1.45 L) was added and stirring was continued for 30 minutes. The organic layer was separated, diluted with toluene (417 mL), and concentrated to 5 volumes, providing C51 as a solution in toluene (1.110 kg, 22.93% C51 by weight). This solution was used directly in the following step. Estimated yield: 254.5 g, 0.9514 mol, 87% over 2 steps.1H NMR (400 MHz, DMSO- d6) δ 7.90 (d, J = 7.7 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 4.55 (s, 2H), 2.41 (s, 3H). Step 4. Synthesis of [(2-chloro-6-methylpyridin-3-yl)methyl](triphenyl)phosphonium iodide (C52). A solution of C51 in toluene (from the previous step; 1.110 kg, 22.93% C51 by weight, 254.5 g, 0.9514 mol) was diluted with acetonitrile (1.29 L) and treated with triphenylphosphine (262 g, 0.999 mol). After the reaction mixture had been stirred for 4 hours at 25 °C, it was cooled to 10 °C, stirred at that temperature for 16 hours, and filtered. The filter cake was washed with toluene (255 mL) and dried at 45 °C for 4 hours, affording C52 as a solid. Yield: 412.6 g, 0.7788 mol, 56% over 4 steps. Purity: 99.7% by HPLC.1H NMR (400 MHz, DMSO-d6) δ 7.97 – 7.90 (m, 3H), 7.80 – 7.71 (m, 8H), 7.71 – 7.66 (m, 4H), 7.44 (dd, J = 7.8, 2.4 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 5.15 (d, JHP = 15.0 Hz, 2H), 2.40 (d, J = 2.4 Hz, 3H). Step 5. Synthesis of diethyl 1-benzylpyrrolidine-3,3-dicarboxylate (C53). A solution of ethyl 1-benzylpyrrolidine-3-carboxylate (700 g, 3.00 mol) in tetrahydrofuran (4.20 L) was added in a drop-wise manner over 5 hours to a −80 °C to −70 °C solution of lithium diisopropylamide (2.0 M, 2.40 L, 4.80 mol). Stirring was continued at −80 °C to −70 °C for 2 hours, whereupon ethyl chloroformate (423.5 g, 3.90 mol) was added over 3 hours, while the reaction temperature was maintained at −80 °C to −70 °C. After the reaction mixture had been stirred for 2 hours at −80 °C to −70 °C, the temperature was adjusted to −50 °C to −40 °C, and the reaction was quenched by addition of a solution of acetic acid (288 g, 4.80 mol) in tetrahydrofuran (1.40 L), while the temperature was kept at −50 °C to −40 °C. The resulting mixture was warmed to 15 °C to 25 °C and partitioned between water (3.50 L) and 2-methyltetrahydrofuran (7.0 L). After this mixture had been stirred for 30 minutes at 15 °C to 25 °C, the aqueous layer was extracted with 2- methyltetrahydrofuran (7.0 L), and the combined organic layers were washed with a solution of acetic acid (288 g, 4.80 mol) in water (4.2 L) and then with an aqueous solution of sodium sulfate (10%; 2 x 3.50 kg). The organic layers were concentrated in vacuo to 2 to 3 volumes, while keeping the temperature below 50 °C. Ethanol (4.90 L, 7 volumes) was added, and the solution was again concentrated in vacuo to 2 to 3 volumes, while keeping the temperature below 50 °C. This ethanol addition / concentration was carried out a total of three times, with the final round employing 2.80 L of ethanol, followed by concentration to 4 to 5 volumes. This provided C53 as a solution in ethanol (3.148 kg, 24.23% C53 by weight). A portion of this solution was used in the following step. Estimated yield: 762.8 g, 2.498 mol, 83%.1H NMR (400 MHz, DMSO-d6) δ 7.35 – 7.20 (m, 5H), 4.12 (q, J = 7.1 Hz, 4H), 3.57 (s, 2H), 2.90 (s, 2H), 2.55 (t, J = 6.9 Hz, 2H), 2.29 (t, J = 6.8 Hz, 2H), 1.14 (t, J = 7.1 Hz, 6H). Step 6. Synthesis of diethyl pyrrolidine-3,3-dicarboxylate, L-tartrate salt (C54). Ethanol (720 mL, 6 volumes) was added to a solution of C53 (120 g, 0.393 mol) in ethanol (from the previous step; approximately 500 mL). After addition of wet palladium on carbon (10%; 12 g), the reaction vessel was evacuated and charged with argon three times, and then evacuated and charged with hydrogen three times. Hydrogenation was then carried out at 40 to 50 psi and 40 °C to 50 °C for 24 hours. The resulting mixture was filtered through diatomaceous earth (50 g); the filter cake was washed with ethanol (240 mL, 2 volumes), and the combined filtrates were concentrated in vacuo to 2.5 to 3.5 volumes while keeping the temperature at or below 45 °C. This solution was added, over 2 hours, to a 40 °C to 50 °C solution of L-tartaric acid (76.7 g, 0.511 mol) in water (85 mL, 0.7 volumes) and ethanol (465 mL). After the mixture had been stirred at 40 °C to 50 °C for 1 hour, a seed of C54 (0.4 g; see below) was added at 45 °C. The mixture was cooled to 10 °C over 6 hours, and then stirred at 10 °C for 4 hours; filtration provided a filter cake, which was washed with ethanol (2 volumes) and dried at 40 °C for 20 hours to afford C54 as a solid. Yield: 127.4 g, 0.3487 mol, 89%. HPLC purity: 99.1%.1H NMR (400 MHz, DMSO-d6) δ 4.16 (q, J = 7.1 Hz, 4H), 4.03 (s, 2H), 3.49 (s, 2H), 3.08 (t, J = 7.1 Hz, 2H), 2.32 (t, J = 7.1 Hz, 2H), 1.18 (t, J = 7.1 Hz, 6H). The seed material used above was obtained from another run of the same synthesis of C54, in which solid C54 formed directly upon cooling. Step 7. Synthesis of 1-tert-butyl 3,3-diethyl pyrrolidine-1,3,3-tricarboxylate (C55). Di-tert-butyl dicarbonate (19.7 g, 90.3 mmol) was added in a drop-wise manner to a 20 °C to 30 °C mixture of C54 (88.12 g, 0.2412 mol) and triethylamine (73.33 g, 0.7247 mol) in dichloromethane (881 mL, 10 volumes). Additional di-tert-butyl dicarbonate (19.2 g, 88.0 mmol and 19.3 g, 88.4 mmol) was added drop-wise after periodic HPLC analysis. After the reaction mixture had been stirred at 20 °C to 30 °C for 18 hours, the pH was adjusted to 7 by addition of hydrochloric acid (1 M; 309 g), and stirring was continued for 15 minutes. The organic layer was stirred with aqueous sodium sulfate solution (10%; 485.30 g) at 20 °C to 30 °C for 15 minutes, and then the organic layer was concentrated in vacuo to 1 to 2 volumes while the temperature was maintained below 40 °C. Dimethyl sulfoxide (71.7 g) was added to afford C55 as a solution in dimethyl sulfoxide (154.2 g, 48.9% C55 by weight). The bulk of this material was progressed to the following step. Estimated yield: 75.4 g, 0.239 mol, 99%.1H NMR (400 MHz, DMSO-d6) δ 4.16 (q, J = 7.1 Hz, 4H), 3.67 (br s, 2H), 3.34 – 3.26 (m, 2H), 2.37 – 2.28 (m, 2H), 1.39 (s, 9H), 1.17 (br t, J = 7.1 Hz, 6H). Step 8. Synthesis of (3R)-1-(tert-butoxycarbonyl)-3-(ethoxycarbonyl)pyrrolidine-3-carboxylic acid (C56). ECS-Esterase 03 enzyme [Bacillus stearothermophilus, recombinant from Escherichia coli, (EC 3.1.1.1); 0.540 g] was added to phosphate buffer (0.1 M; pH = 6.92, 580 mL, 8.2 volumes) at 20 °C to 30 °C. A solution of C55 (72.2 g, 0.229 mol) in dimethyl sulfoxide (from the previous step; approximately 148 g) was added; additional dimethyl sulfoxide (9 mL) was used to rinse the initial vessel, and this was also added to the reaction mixture. The initial reaction pH was 7.08; after stirring at 20 °C to 30 °C for 1 hour, the pH decreased to 6.58. A pH autotitrator was used to maintain the pH at 7.5 by addition of aqueous sodium hydroxide solution (2 M; 121 mL, 0.242 mol) over 24 hours. Hydrochloric acid (6 M; 52 mL, 0.312 mol) was added, bringing the pH to 2.39; ethyl acetate (435 mL, 6.0 volumes) was then added, and the mixture was stirred for 30 minutes at 20 °C to 30 °C. Filtration through diatomaceous earth (18.0 g) provided a filter cake, which was rinsed with ethyl acetate (2 x 75 mL). The combined filtrates were stirred at 20 °C to 30 °C for 30 minutes, and then the aqueous layer was stirred with ethyl acetate (217 mL, 3.0 volumes) for 30 minutes. The combined organic layers were washed twice with water (360 mL, 5.0 volumes) by stirring for 30 minutes. The resulting solution was concentrated in vacuo to 1 to 2 volumes, while maintaining the temperature below 40 °C, then diluted with toluene (360 mL); this concentration / dilution procedure was carried out a total of three times, providing C56 as a solution in toluene (418.3 g, 15.67% C56 by weight). Estimated yield: 65.6 g, 0.228 mol, quantitative.1H NMR (400 MHz, chloroform-d) δ 7.99 (v br s, 1H), 4.22 (q, J = 7.1 Hz, 2H), [3.88 (br s) and 3.83 (br s), total 2H], 3.51 – 3.38 (m, 2H), 2.41 (t, J = 7.1 Hz, 2H), 1.44 (s, 9H), 1.26 (br t, J = 7 Hz, 3H). Step 9. Synthesis of 1-tert-butyl 3-ethyl (3S)-3-{[(benzyloxy)carbonyl]amino}pyrrolidine-1,3- dicarboxylate (C57). Toluene (170 mL, 1.2 volumes) was added to a solution of C56 in toluene (3.8 volumes, containing 28.9% by weight of C56, 146.4 g, 0.5096 mol); the solution was heated to 80 °C to 90 °C. To this was slowly added, over 2 hours, a mixture of triethylamine (77.4 g, 0.765 mol) and diphenyl phosphorazidate (140.3 g, 0.5098 mol) in toluene (732 mL, 5 volumes). The reaction mixture was stirred at 80 °C to 90 °C for 3 hours, whereupon it was cooled to 50 °C and treated drop-wise, over 2 hours, with a solution of benzyl alcohol (55.12 g, 0.5097 mol) in toluene (290 mL, 2 volumes). After the reaction mixture had been stirred at 100 °C for 16 hours, it was cooled to 15 °C to 25 °C and partitioned between toluene (1.46 L, 10 volumes) and water (2.20 L, 15 volumes) by stirring for 30 minutes. The organic layer was washed sequentially with aqueous potassium carbonate solution (10%; 3 x 1.46 L) and with water (2 x 750 mL). It was then concentrated in vacuo to 1 to 2 volumes, while the temperature was maintained below 50 °C, and diluted with tetrahydrofuran (1.0 L); this concentration / dilution procedure was carried out a total of three times, whereupon the mixture was concentrated in vacuo to 4 to 5 volumes while maintaining the temperature below 50 °C. This afforded C57 as a solution in tetrahydrofuran (595.8 g, 19.14% C57 by weight). Estimated yield: 114 g, 0.290 mol, 57%.1H NMR (400 MHz, chloroform-d) δ 7.40 – 7.28 (m, 5H), 5.25 (v br s, 1H), 5.10 (br s, 2H), 4.28 – 4.12 (m, 2H), 3.91 – 3.76 (m, 1H), 3.71 – 3.38 (m, 3H), 2.54 – 2.15 (m, 2H), 1.45 (s, 9H), 1.27 – 1.16 (m, 3H). Step 10. Synthesis of tert-butyl (3S)-3-{[(benzyloxy)carbonyl]amino}-3-(hydroxymethyl)pyrrolidine- 1-carboxylate (C58). A solution of lithium borohydride in tetrahydrofuran (2 M; 511 mL, 1.02 mol) was added over 2 hours to a 0 °C to 10 °C solution of C57 in tetrahydrofuran (835.6 g, containing 19.20% C57 by weight, 160.4 g, 0.4087 mol). After the reaction mixture had been stirred at 0 °C to 10 °C for 15 hours, it was cooled to −5 °C to 5 °C and treated in a drop-wise manner with hydrochloric acid (0.5 M; 2.08 L, 1.04 mol, 13 volumes), to a pH of 7. The mixture was then warmed to 20 °C to 30 °C, diluted with ethyl acetate (1.60 L, 10 volumes) and stirred for 10 minutes, whereupon the organic layer was concentrated in vacuo to 2 to 3 volumes while maintaining the temperature at or below 50 °C. The resulting mixture was diluted with acetonitrile (880 mL) and concentrated in vacuo to 2 to 3 volumes while maintaining the temperature at or below 50 °C; this dilution / concentration procedure was carried out a total of three times. The mixture was then heated to 40 °C to 50 °C and stirred for 1 hour, whereupon it was cooled over 4 hours to 15 °C to 25 °C. Water (164 mL) was added drop-wise over 2 hours at 15 °C to 25 °C, and the mixture was stirred at 15 °C to 25 °C for 12 hours. The resulting solid was collected via filtration and dried in vacuo for 40 hours, at a temperature at or below 50 °C, to afford C58 as a solid. Yield: 123.2 g, 0.3516 mol, 86%. HPLC purity: 99.8%.1H NMR (400 MHz, DMSO-d6) δ 7.40 – 7.27 (m, 5H), 4.99 (s, 2H), 4.93 (t, J = 5.8 Hz, 1H), 3.58 – 3.45 (m, 3H), 3.31 – 3.21 (m, 3H), 2.10 – 1.85 (m, 2H), 1.38 (s, 9H). Step 11. Synthesis of tert-butyl (3S)-3-{[(benzyloxy)carbonyl]amino}-3-formylpyrrolidine-1- carboxylate (C59). A solution of C58 (125 g, 0.357 mol) and dimethyl sulfoxide (144.5 g, 1.849 mol) in dichloromethane (2.02 L) was stirred for 2 hours at 35 °C to 45 °C; Karl Fischer analysis indicated a water content of 0.029%. The solution was concentrated in vacuo to 3 to 4 volumes at 35 °C to 45 °C, and then diluted with dichloromethane (1.80 L). Another Karl Fischer analysis revealed a water content of 0.034%. The solution was concentrated in vacuo at 35 °C to 45 °C to 6 to 7 volumes, whereupon triethylamine (112.3 g, 1.110 mol) was added at 20 °C to 30 °C, and the reaction mixture was cooled to −5 °C to 0 °C and stirred at that temperature for 15 minutes. Sulfur trioxide pyridine complex (141.3 g, 0.8878 mol) was added in portions over 2 hours; stirring was then continued at −5 °C to 0 °C for 16 hours, at which time the reaction mixture was warmed to 35 °C to 45 °C and concentrated to 2 to 3 volumes. After the mixture had cooled to 20 °C to 30 °C, it was partitioned between ethyl acetate (945 mL) and water (675 mL), and the aqueous layer was extracted with ethyl acetate (675 mL). The combined organic layers were washed sequentially with hydrochloric acid (1 M; 675 mL), water (675 mL), and saturated aqueous sodium bicarbonate solution (675 mL), then concentrated to dryness at 30 °C to 40 °C, providing C59 as an oil. Yield: 118.7 g, 0.3407 mol, 95%. HPLC purity: 91.2%.1H NMR (400 MHz, chloroform-d) δ 9.59 (s, 1H), 7.42 – 7.29 (m, 5H), 5.39 (br s, 1H), 5.12 (s, 2H), 3.85 – 3.70 (m, 1H), 3.63 – 3.43 (m, 3H), 2.44 – 2.04 (m, 2H), 1.45 (s, 9H). Step 12. Synthesis of tert-butyl (3R)-3-{[(benzyloxy)carbonyl]amino}-3-[(E)-2-(2-chloro-6- methylpyridin-3-yl)ethenyl]pyrrolidine-1-carboxylate (C60) and tert-butyl (3R)-3- {[(benzyloxy)carbonyl]amino}-3-[(Z)-2-(2-chloro-6-methylpyridin-3-yl)ethenyl]pyrrolidine-1- carboxylate (C61). A mixture of C59 (237.1 g, 0.6805 mol) and C52 (393.6 g, 0.7429 mol) in dimethyl sulfoxide (2.40 L, 10 volumes) was treated with potassium carbonate (188.7 g, 1.365 mol) and heated at 60 °C for 2 hours. Propan-2-yl acetate (1.54 L, 6.5 volumes), water (6.40 L, 27 volumes), and aqueous sodium sulfate solution (10%; 710 mL, 3.0 volumes) were then added, and the mixture was stirred for 20 minutes at 25 °C. The organic layer was washed three times with aqueous sodium sulfate solution (10%; 1.30 L, 5.5 volumes) by stirring each mixture for 20 minutes before separation. It was then washed with aqueous sodium bicarbonate solution (7%; 1.30 L, 5.5 volumes) in the same manner, and concentrated in vacuo to 1 to 2 volumes at a temperature at or below 50 °C. Propan- 2-yl acetate (1.06 L) was added, and the mixture was concentrated in vacuo to 1 to 2 volumes at a temperature at or below 50 °C. Propan-2-yl acetate (480 mL) was added, followed by drop-wise addition of methylcyclohexane (1.66 L) at 20 °C to 30 °C. After the resulting mixture had been stirred at 20 °C to 30 °C for 1 hour, it was cooled to −15 °C to −5 °C and stirred at that temperature for 16 hours. Filtration of the slurry was carried out at −15 °C to −5 °C, and the filter cake was washed with a mixture of propan-2-yl acetate and methylcyclohexane (3:7 ratio, 710 mL) at −15 °C to −5 °C. The combined filtrates were concentrated in vacuo, diluted with methylcyclohexane (20 volumes) and subjected to silica gel chromatography (Gradient: 14% to 25% ethyl acetate in methylcyclohexane) to afford a mixture of C60 and C61 as an oil. This material was judged by 1H NMR analysis to consist of 3 to 4 isomers / rotamers. Yield: 268.1 g, 0.5680 mol, 83%. HPLC purity: >99.7%.1H NMR (400 MHz, DMSO-d6), characteristic peaks: δ [8.00 (d, J = 7.9 Hz) and 7.59 (d, J = 7.6 Hz), total 1H], [7.85 (s) and 7.41 (s), total 1H], [7.38 – 7.25 (m), 7.22 (br d, J = 7.2 Hz), and 7.16 (d, J = 7.7 Hz), total 6H], [6.62 (d, component of AB quartet, J = 16.1 Hz) and 6.34 (d, J = 12.3 Hz), total 1H], [6.49 (br d, component of AB quartet, J = 16.0 Hz), 5.88 (d, J = 12.3 Hz), and 5.87 (d, J = 12.3 Hz), total 1H], [5.04 (AB quartet, JAB = 12.7 Hz, ΔνAB = 16.4 Hz), 4.74 (d, component of AB quartet, J = 12.4 Hz), and 4.70 – 4.62 (m), total 2H], 3.83 – 3.68 (m, 1H), 3.32 – 3.16 (m, 3H), [2.43 (s) and 2.36 (s), total 3H], 2.27 – 2.12 (m, 1H), 2.00 – 1.85 (m, 1H), [1.40 (s), 1.39 (s), 1.37 (s), and 1.34 (s), total 9H]. Step 13. Synthesis of tert-butyl (3S)-3-{[(benzyloxy)carbonyl]amino}-3-[2-(2-chloro-6-methylpyridin- 3-yl)ethyl]pyrrolidine-1-carboxylate (C62). A reaction vessel containing a mixture of C60 and C61 (283.0 g, 0.5996 mol) and rhodium on alumina (5%; 14.15 g) in methanol (1.98 L) was evacuated and charged with argon three times, then evacuated and charged with hydrogen three times. Hydrogenation was then carried out for 40 hours at 30 to 40 psi and 20 °C to 25 °C. After the reaction mixture had been filtered through diatomaceous earth (424 g), the filter cake was washed with methanol (5 volumes); the combined filtrates were concentrated in vacuo at 35 °C to 45 °C. The resulting material was treated with toluene (5 volumes) and concentrated in vacuo at 50 °C to 60 °C; this toluene addition / concentration procedure was carried out a total of three times, providing C62. Yield: 254.4 g, 0.5367 mol, 90%. HPLC purity: 97.1%.1H NMR (400 MHz, chloroform-d) δ 7.41 – 7.28 (m, 6H), 6.99 (br d, J = 7.6 Hz, 1H), 5.06 (s, 2H), 4.91 – 4.79 (m, 1H), 3.62 (d, J = 11.7 Hz, 1H), 3.57 – 3.36 (m, 2H), 3.36 – 3.26 (m, 1H), 2.74 – 2.55 (m, 2H), 2.48 (s, 3H), [2.48 – 2.40 (m), 2.39 – 2.07 (m) and 2.05 – 1.82 (m), total 4H], 1.45 (br s, 9H). Step 14. Synthesis of tert-butyl (2S)-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine]-1'-carboxylate (P19). A solution of C62 in toluene (947.73 g, containing 19% C62 by weight, 180 g, 0.380 mol) was diluted with toluene (1.17 L, 6.5 volumes) and treated sequentially with 2- dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (RuPhos; 35.44 g, 75.95 mmol) and potassium phosphate (145.1 g, 0.6836 mol). The resulting mixture was purged three times with nitrogen, whereupon tris(dibenzylideneacetone)dipalladium(0) (34.78 g, 37.98 mmol) was added, and three additional rounds of purging with nitrogen were carried out. The reaction mixture was stirred for 24 hours at 75 °C to 85 °C. Potassium phosphate (16.2 g, 0.117 mol) was added, and stirring was continued at 75 °C to 85 °C for an additional 16 hours. After the reaction mixture had been cooled to 20 °C to 30 °C, potassium tert-butoxide (76.7 g, 0.684 mol) was added, and the reaction mixture was stirred for 2 hours at 75 °C to 85 °C. It was then cooled and partitioned between water (2.25 L) and ethyl acetate (2.25 L); after being stirred for 30 minutes at 20 °C to 30 °C, the mixture was filtered through diatomaceous earth (180 g) and the filter cake was washed with ethyl acetate (1.80 L). The organic layer of the combined filtrates was washed sequentially with water (2 x 2.25 L) and aqueous sodium sulfate solution (10%; 2.25 L), then extracted three times with aqueous citric acid solution (0.5 M; 1.072 kg, 1.4 eq.). The combined citric acid layers were washed with ethyl acetate (2 x 1.07 L), then adjusted to pH 7 by addition of aqueous sodium hydroxide solution (30%; 596 g) at 20 °C to 30 °C. Extraction of the aqueous layer with ethyl acetate (3 x 1.07 L), followed by combination of these three organic layers, provided P19 as a solution in ethyl acetate (3.218 kg, 2.7% P19 by weight); The bulk of this material was progressed to the following step. Estimated yield: 86.9 g, 0.286 mol, 75%. HPLC purity: 98.9%.1H NMR (400 MHz, chloroform-d) δ 7.11 (d, J = 7.3 Hz, 1H), 6.41 (d, J = 7.4 Hz, 1H), 4.90 (br s, 1H), 3.59 – 3.43 (m, 2H), [3.40 (d, component of AB quartet, J = 11.1 Hz) and 3.36 – 3.25 (m), total 2H], 2.80 – 2.65 (m, 2H), 2.31 (s, 3H), 2.00 – 1.75 (m, 4H), [1.45 (s) and 1.44 (s), total 9H]. Step 15. Synthesis of tert-butyl (2S)-6-bromo-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine]-1'-carboxylate (P23). 1,3-Dibromo-5,5-dimethylimidazolidine-2,4-dione (45.60 g, 0.1595 mol) was added to a 0 °C to 5 °C solution of P19 in ethyl acetate (from the previous step; 2986 g, containing 2.7% P19, 80.6 g, 0.266 mol). After the reaction mixture had been stirred for 1 hour at 0 °C to 5 °C, it was quenched by addition of aqueous sodium sulfite solution (10%; 203 g) and water (456 mL), and the resulting mixture was stirred at 10 °C to 20 °C for 20 minutes. The aqueous layer was extracted twice with ethyl acetate (415 mL, 5.1 volumes) by stirring at 10 °C to 20 °C for 20 minutes; the combined organic layers were then stirred for 20 minutes with aqueous sodium sulfate solution (10%; 456 g). Concentration of the organic layer to 1 to 2 volumes in vacuo below 50 °C was followed by dilution with methanol (480 mL, 6 volumes). This concentration / dilution procedure was carried out a total of three times, and the final solution was concentrated in vacuo, below 50 °C, to 5 to 6 volumes. The resulting solution was cooled to 15 °C to 25 °C and water (415 mL) was slowly added, over 2 hours at 15 °C to 25 °C, and then stirring was carried out for 14 hours at 15 °C to 25 °C. Filtration provided a filter cake, which was washed with a mixture of methanol and water (1:1, 2 x 200 mL) and then dried under vacuum at 45 °C for 48 hours to afford P23 as a solid. Yield: 99.50 g, 0.2603 mol, 98%. HPLC purity: 99.7%. LCMS m/z 384.1 (bromine isotope pattern observed) [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 7.37 (s, 1H), 7.03 – 6.97 (m, 1H), 3.55 – 3.43 (m, 1H), 3.3 – 3.25 (m, 1H, assumed; partially obscured by water peak), 3.24 – 3.13 (m, 2H), 2.75 – 2.55 (m, 2H), 2.30 (s, 3H), 1.95 – 1.77 (m, 2H), 1.76 – 1.59 (m, 2H), [1.40 (s) and 1.38 (s), total 9H].
Figure imgf000096_0001
Step 1. Synthesis of di-tert-butyl 6-ethenyl-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine]-1,1'-dicarboxylate (C63). A mixture of P21 (15.0 g, 31.1 mmol), potassium vinyltrifluoroborate (10.4 g, 77.6 mmol), [1,1’-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (2.27 g, 3.10 mmol), and potassium phosphate (19.8 g, 93.3 mmol) in N,N-dimethylformamide (500 mL) was stirred at 95 °C for 16 hours, whereupon the reaction mixture was filtered; the filtrate was poured into water (4 L) and extracted with ethyl acetate (2 x 800 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. After the residue had been combined with the product of a similar reaction carried out using P21 (5.00 g, 10.4 mmol), it was purified via chromatography on silica gel (Gradient: 0% to 20% ethyl acetate in petroleum ether), affording C63 as a white solid. Combined yield: 17.1 g, 38.9 mmol, 94%. LCMS m/z 430.3 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.46 (br s, 1H), 6.83 (dd, J = 17.4, 11.1 Hz, 1H), 5.59 (br d, J = 17.4 Hz, 1H), 5.37 – 5.24 (m, 1H), [3.90 (d, J = 11.0 Hz) and 3.72 (d, J = 11.0 Hz), total 1H], 3.64 – 3.41 (m, 2H), 3.38 – 3.24 (m, 1H), [2.86 – 2.64 (m), 2.62 – 2.39 (m), and 2.16 – 1.72 (m), total 9H], 1.45 (s, 18H). Step 2. Synthesis of di-tert-butyl 6-formyl-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine]-1,1'-dicarboxylate (C64). A solution of C63 (17.0 g, 39.6 mmol) in dichloromethane (200 mL) was cooled to −78 °C, and a stream of ozone-enriched oxygen was introduced until a blue color persisted. After an additional 5 minutes, a stream of dry nitrogen was bubbled through the reaction mixture until the blue color disappeared, whereupon triphenylphosphine (20.7 g, 78.9 mmol) was added. The resulting mixture was warmed to 25 °C and stirred for 2 hours, at which point LCMS analysis indicated the presence of C64: LCMS m/z 454.3 [M+Na+]. After the reaction mixture had been concentrated in vacuo, the residue was purified using silica gel chromatography (Gradient: 0% to 50% ethyl acetate in petroleum ether) to provide C64 as a colorless gum. Yield: 9.98 g, 23.1 mmol, 58%. Step 3. Synthesis of di-tert-butyl 7-methyl-6-[(2-methylhydrazinylidene)methyl]-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1,1'-dicarboxylate (C65). A solution of methylhydrazine sulfate (3.20 g, 22.2 mmol) and triethylamine (7.78 mL, 55.8 mmol) in methanol (50 mL) was stirred at 25 °C for 5 minutes, whereupon a solution of C64 (7.98 g, 18.5 mmol) in methanol (20 mL) was added. After the reaction mixture had been stirred at 25 °C for 1 hour, collection of the precipitate via filtration afforded C65 as a white solid. Yield: 7.60 g, 16.5 mmol, 89%. LCMS m/z 460.3 [M+H]+. Step 4. Synthesis of di-tert-butyl 7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine]-1,1'-dicarboxylate (P25). To a solution of C65 (6.70 g, 14.6 mmol) in a mixture of 2,2,2-trifluoroethanol (35 mL) and dichloromethane (35 mL) was added di-tert-butyl azodicarboxylate (4.36 g, 18.9 mmol), followed by [bis(trifluoroacetoxy)iodo]benzene (33.2 g, 77.2 mmol). The reaction mixture was stirred at 25 °C for 30 minutes, whereupon it was poured into saturated aqueous sodium sulfite solution (300 mL) and extracted with dichloromethane (2 x 100 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo; silica gel chromatography (Gradient: 0% to 20% ethanol in dichloromethane) provided P25 as a white solid. Yield: 2.10 g, 4.32 mmol, 30%. LCMS m/z 508.3 [M+Na+].1H NMR (400 MHz, methanol-d4) δ 8.11 (s, 1H), 4.44 (s, 3H), [3.93 (d, J = 11.3 Hz) and 3.86 (d, J = 11.1 Hz), total 1H], 3.68 – 3.56 (m, 1H), 3.56 – 3.46 (m, 1H), 3.46 – 3.3 (m, 1H, assumed; partially obscured by solvent peak), 2.92 – 2.81 (m, 2H), 2.73 (s, 3H), [2.69 – 2.58 (m) and 2.58 – 2.47 (m), total 1H], 2.15 – 1.88 (m, 3H), 1.48 (br s, 18H).
Figure imgf000098_0001
Step 1. Separation of di-tert-butyl (2R)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1,1'-dicarboxylate (C66) and di-tert-butyl (2S)-7-methyl-6- (2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1,1'- dicarboxylate (C67). Separation of P25 (2.37 g, 4.88 mmol) into its component diastereomers was carried out using supercritical fluid chromatography {Column: Chiral Technologies Chiralcel OJ-H, 21.2 x 250 mm, 5 µm; Mobile phase 9:1 carbon dioxide / [methanol containing 0.2% (7 M ammonia in methanol)]; Flow rate: 80 mL/minute; Back pressure: 120 bar}. The first-eluting diastereomer was designated as C66, and the second-eluting diastereomer was designated as C67. C66 was isolated as a solid. Yield: 1.01 g, 2.08 mmol, 43%. Retention time: 2.68 minutes [Analytical conditions. Column: Chiral Technologies Chiralcel OJ-H, 4.6 x 250 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: methanol containing 0.2% (7 M ammonia in methanol); Gradient: 5% B for 1.0 minute, then 5% to 60% B over 8.0 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar]. C67 was isolated as an oil. Yield: 1.00 g, 2.06 mmol, 42%. Retention time: 3.33 minutes (Analytical conditions identical to those used for C66). See below for assignment of absolute stereochemistry. Step 2. Synthesis of (2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine], dihydrochloride salt (P26). A solution of C67 (150 mg, 0.309 mmol) in a mixture of dichloromethane (1.0 mL) and 1,1,1,3,3,3-hexafluoropropan-2-ol (1.0 mL) was treated with a solution of hydrogen chloride in 1,4- dioxane (4 M; 0.309 mL, 1.24 mmol). After the reaction mixture had been stirred at room temperature for 2 hours, LCMS analysis indicated conversion to P26: LCMS m/z 286.3 [M+H]+. Concentration of the reaction mixture in vacuo afforded P26 as a solid. Yield: 105 mg, 0.293 mmol, 95%. The indicated absolute stereochemistry was established in the following manner. This batch of P26 was used to prepare 3 and 4 in Alternate Synthesis of Examples 3 and 4. Correlation between those batches of 3 and 4 with the same compounds prepared from a precursor of known absolute stereochemistry (see Examples 3 and 4) is provided in Alternate Synthesis of Examples 3 and 4. Alternate Preparation of P26 (2S)-7-Methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine], dihydrochloride salt (P26)
Figure imgf000100_0001
Step 1. Synthesis of tert-butyl (2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (C68). A mixture of 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi-1,3,2-dioxaborolane (299 mg, 1.18 mmol), P23 (300 mg, 0.785 mmol), [1,1’-bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex (32.0 mg, 39.2 µmol), and oven-dried potassium acetate (308 mg, 3.14 mmol) in 1,4-dioxane (10 mL) was degassed by bubbling nitrogen through it for 5 minutes. The reaction vial was then sealed and heated at 100 °C in an aluminum block for 2 hours, whereupon it was allowed to cool to room temperature.5-Bromo-2-methyl-2H-tetrazole (134 mg, 0.822 mmol), dichlorobis(triphenylphosphine)palladium(II) (27.5 mg, 39.2 µmol), and a degassed aqueous solution of sodium carbonate (2.0 M; 0.981 mL, 1.96 mmol) were added, and the reaction mixture was again degassed with bubbling nitrogen for 5 minutes. It was then stirred at 90 °C for 18 hours, cooled to room temperature, diluted with ethyl acetate, and filtered through diatomaceous earth. The organic layer of the filtrate was washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo; LCMS analysis indicated the presence of C68: LCMS m/z 386.3 [M+H]+. Silica gel chromatography (Gradient: 20% to 50% ethyl acetate in heptane) provided C68 as a light-yellow oil. Yield: 280 mg, 0.726 mmol, 92%. This batch of C68 was used in Examples 3 and 4 below. Step 2. Synthesis of (2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine], dihydrochloride salt (P26). A mixture of C68 (185 mg, 0.480 mmol) and a solution of hydrogen chloride in 2-propanol (1.25 M; 1.9 mL, 2.4 mmol) was heated to 50 °C for 1 hour. Concentration of the reaction mixture in vacuo provided P26 as a solid, which was used without additional purification. Yield: 170 mg, 0.47 mmol, 98%.
Figure imgf000101_0001
A reaction vessel containing a mixture of P23 (19.5 g, 51.0 mmol), potassium acetate (12.5 g, 127 mmol), sodium tert-butoxide (49.0 mg, 0.510 mmol), chloro(2-dicyclohexylphosphino-2′,4′,6′- triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) [XPhos Pd G2; 401 mg, 0.510 mmol), and 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos; 729 mg, 1.53 mmol) was purged with nitrogen. Methanol (200 mL), ethane-1,2-diol (20 mL), and tetrahydroxydiboron (11.4 g, 127 mmol) were then added, whereupon nitrogen was bubbled through the reaction mixture for 10 minutes. The reaction mixture was heated to an internal temperature of 50 °C for 2 hours, cooled to room temperature and then to 0 °C, and adjusted to pH 14 by addition of aqueous sodium hydroxide solution (4 M; 80 mL) {Caution: gas evolution}. The resulting mixture was stirred at room temperature for 30 minutes and filtered; the filtrate was concentrated in vacuo and extracted twice with tert-butyl methyl ether. The combined organic layers were then extracted with aqueous sodium hydroxide solution (2 M; 2 x 100 mL). All the aqueous layers were combined, and the stirring mixture was treated drop-wise with hydrochloric acid (4 M; approximately 20 mL) until solids precipitated (this occurred at a pH of approximately 9). After the mixture had been stirred at room temperature for an additional 30 minutes, it was extracted four times with ethyl acetate. The combined ethyl acetate layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo to provide P27 as a light-yellow powder. Yield: 12.5 g, 36.0 mmol, 71%. LCMS m/z 348.4 [M+H]+.1H NMR (400 MHz, methanol-d4), characteristic peaks: δ 7.74 (br s, 1H), 3.46 – 3.35 (m, 2H), 2.92 – 2.72 (m, 2H), 2.48 (s, 3H), 2.12 – 1.83 (m, 4H), [1.47 (s) and 1.46 (s), total 9H]. Preparation P28 (2S)-7-Methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine], dihydrochloride salt (P28)
Figure imgf000102_0001
Step 1. Synthesis of tert-butyl (2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (C69). Nitrogen was bubbled through a mixture of oven-dried potassium acetate (2.07 g, 21.1 mmol), P23 (material from Preparations P23 and P24; 2.02 g, 5.28 mmol), 5,5,5′,5′-tetramethyl- 2,2′-bi-1,3,2-dioxaborinane (1.79 g, 7.92 mmol), and [1,1’- bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex (216 mg, 0.264 mmol) in 1,4-dioxane (20 mL) for 5 minutes. The reaction mixture was then heated in a 105 °C aluminum block for 2 hours, whereupon it was allowed to cool to room temperature and then treated with 2-bromopyrimidine (840 mg, 5.28 mmol), additional [1,1’- bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex (216 mg, 0.264 mmol), and aqueous sodium carbonate solution (2.0 M; 7.93 mL, 15.9 mmol). After this reaction mixture had been sparged with nitrogen, it was heated to 100 °C for 18 hours, at which time LCMS analysis indicated conversion to C69: LCMS m/z 382.4 [M+H]+. The reaction mixture was cooled, partitioned between aqueous ammonium chloride solution and ethyl acetate, and then the entire mixture was filtered through a pad of diatomaceous earth. The filter pad was rinsed with both water and ethyl acetate, and the aqueous layer of the combined filtrate was extracted with ethyl acetate (2 x 30 mL). After all the organic layers had been combined, they were washed sequentially with water (100 mL) and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue (2.9 g) was dissolved in ethyl acetate (10 mL) and treated with SiliaMetS Thiol (SiliCycle, R51030B; 2 g); the resulting mixture was heated at reflux for 10 minutes and then cooled to room temperature. Filtration through a pad of diatomaceous earth provided a filtrate, which was concentrated under reduced pressure to afford C69 as a brown gum (2 g). This material was employed in the following step without additional purification. Step 2. Synthesis of (2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine], dihydrochloride salt (P28). A solution of hydrogen chloride was prepared by slow addition of acetyl chloride (1.50 mL, 21.1 mmol) to 2-propanol (4 mL). In a separate flask, C69 (from the previous step; 2 g; ≤5.28 mmol) was dissolved in a mixture of propan-2-yl acetate (15 mL) and 2-propanol (15 mL); this required heating at 50 °C. Once a solution had been attained, the hydrogen chloride solution was slowly added to it, and the reaction mixture was heated at 50 °C for 2 hours. It was then allowed to cool slowly to room temperature while being stirred; stirring was continued at room temperature for 18 hours. The resulting solid was collected via vacuum filtration under nitrogen, providing P28 as a hygroscopic solid. Yield: 750 mg, 2.12 mmol, 40% over 2 steps. LCMS m/z 282.3 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 8.91 (d, J = 4.9 Hz, 2H), 8.58 (s, 1H), 7.43 (t, J = 4.9 Hz, 1H), 3.76 – 3.66 (m, 1H), 3.66 – 3.52 (m, 2H), 3.46 (d, component of AB quartet, J = 12.5 Hz, 1H), 3.12 – 2.95 (m, 2H), 2.90 (s, 3H), 2.49 – 2.38 (m, 1H), 2.37 – 2.25 (m, 1H), 2.24 – 2.06 (m, 2H). The indicated absolute stereochemistry was assigned on the basis of conversion of this lot of P28 to Example 14; the absolute stereochemistry of 14 was established via single-crystal X-ray analysis (see Example 14 below). Preparation P29 tert-Butyl 4,7-dimethyl-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (P29)
Figure imgf000103_0001
Figure imgf000104_0001
Step 1. Synthesis of tert-butyl 3-amino-3-(prop-2-en-1-yl)pyrrolidine-1-carboxylate (C70). A mixture of tert-butyl 3-oxopyrrolidine-1-carboxylate (500 mg, 2.70 mmol) and a solution of ammonia in methanol (7 M; 3.9 mL, 27 mmol) was stirred at room temperature for 30 minutes. To this was then added, in a drop-wise manner, a solution of 4,4,5,5-tetramethyl-2-(prop-2-en-1-yl)- 1,3,2-dioxaborolane (907 mg, 5.40 mmol) in methanol, and the reaction mixture was stirred at room temperature for 18 hours. Volatiles were removed in vacuo, and the residue was subjected to silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) to provide C70. Yield: 200 mg, 0.884 mmol, 33%.1H NMR (400 MHz, chloroform-d) δ 5.91 – 5.76 (m, 1H), 5.20 (m, 1H), 5.15 (br d, J = 11 Hz, 1H), 3.53 – 3.38 (m, 2H), 3.32 – 3.08 (m, 2H), 2.28 (d, J = 7.5 Hz, 2H), 1.89 – 1.79 (m, 1H), 1.73 – 1.63 (m, 1H), 1.46 (s, 9H). Step 2. Synthesis of tert-butyl 3-[(3-chloro-6-methylpyridin-2-yl)amino]-3-(prop-2-en-1- yl)pyrrolidine-1-carboxylate (C71). A vial containing a mixture of 2,3-dichloro-6-methylpyridine (100 mg, 0.617 mmol), C70 (168 mg, 0.742 mmol), palladium(II) acetate (6.93 mg, 30.9 µmol), 2-dicyclohexylphosphino-2′,6′- diisopropoxybiphenyl (RuPhos; 28.8 mg, 61.7 µmol), and sodium tert-butoxide (119 mg, 1.24 mmol) in 1,4-dioxane (8 mL) was sparged with nitrogen, sealed, and heated at 80 °C overnight. LCMS analysis indicated conversion to C71: LCMS m/z 352.3 (chlorine isotope pattern observed) [M+H]+, whereupon the reaction mixture was cooled to room temperature and partitioned between water and dichloromethane. The aqueous layer was extracted with dichloromethane, and the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, concentrated in vacuo, and subjected to silica gel chromatography (Gradient: 0% to 50% ethyl acetate in heptane) to provide C71 as an oil that solidified upon standing. Yield: 121 mg, 0.344 mmol, 56%.1H NMR (400 MHz, chloroform-d) δ 7.29 (d, J = 8.1 Hz, 1H), 6.42 – 6.34 (m, 1H), 5.82 – 5.68 (m, 1H), 5.11 – 5.03 (m, 1H), 5.03 – 4.93 (m, 1H), 3.79 – 3.69 (m, 1H), [3.62 (d, component of AB quartet, J = 11.6 Hz), 3.56 (d, component of AB quartet, J = 11.4 Hz), and 3.54 – 3.36 (m), total 3H], 2.95 – 2.83 (m, 1H), 2.76 – 2.63 (m, 1H), 2.45 – 2.28 (m, 1H), 2.34 (s, 3H), 2.08 – 1.96 (m, 1H), 1.50 – 1.41 (br s, 9H). Step 3. Synthesis of tert-butyl 4,7-dimethyl-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'- carboxylate (P29). A mixture of C71 (40 mg, 0.11 mmol), tris(dibenzylideneacetone)dipalladium(0) (5.20 mg, 5.68 µmol), N-cyclohexyl-N-methylcyclohexanamine (111 mg, 0.568 mmol), and tri-tert- butylphosphine (1.15 mg, 5.68 µmol) in N,N-dimethylformamide (1.0 mL) was degassed and then heated at 80 °C for 2 hours. The heat was increased to 120 °C, and the reaction mixture was maintained at that temperature for 3 days. LCMS analysis indicated conversion to P29: LCMS m/z 316.3 [M+H]+. After the reaction mixture had cooled to room temperature, it was partitioned between ethyl acetate and water, and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo; silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) afforded P29. Yield: 30 mg, 95 µmol, 86%.1H NMR (400 MHz, chloroform-d), characteristic peaks: δ 7.13 (d, J = 7.5 Hz, 1H), 6.40 (d, J = 7.5 Hz, 1H), 5.27 (br s, 1H), 5.06 – 4.99 (br s, 1H), 3.58 – 3.40 (m, 3H), 3.31 – 3.21 (m, 1H), 2.31 (s, 3H), [1.96 (s) and 1.96 (s), total 3H], [1.46 (s) and 1.44 (s), total 9H].
Figure imgf000105_0001
Step 1. Synthesis of 1,1'-dibenzyl-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine] (C72). Conversion of C43 to C72 was carried out using the method described for synthesis of C47 from C43 in Preparations P17 and P18, by utilizing 1-phenylmethanamine in place of 1-(4- methoxyphenyl)methanamine. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) provided C72. Yield for cyclization step to provide C72: 580 mg, 1.51 mmol, 69%. Step 2. Synthesis of tert-butyl 1-benzyl-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine]-1'-carboxylate (P30). A mixture of C72 (550 mg, 1.43 mmol), palladium on carbon (50 mg, 0.143 mmol), and di- tert-butyl dicarbonate (376 mg, 1.72 mmol) in methanol (20 mL) was hydrogenated overnight at 75 psi. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated in vacuo; silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) afforded P30 as a white semi-solid. Yield: 482 mg, 1.22 mmol, 85%.1H NMR (400 MHz, chloroform-d) δ 7.29 – 7.07 (m, 6H, assumed; partially obscured by solvent peak), 6.39 (d, J = 7.2 Hz, 1H), 5.15 – 4.99 (m, 1H), 4.97 – 4.78 (m, 1H), 3.58 – 3.19 (m, 4H), 2.87 – 2.71 (m, 2H), 2.31 – 2.16 (m, 1H), 2.24 (s, 3H), 2.07 – 1.95 (m, 1H), 1.92 – 1.79 (m, 1H), 1.75 – 1.63 (m, 1H), [1.45 (s) and 1.43 (s), total 9H].
Figure imgf000106_0001
Figure imgf000107_0001
Step 1. Synthesis of 1,1'-dibenzyl-6-bromo-7-methyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine] (C73). 1,3-Dibromo-5,5-dimethylimidazolidine-2,4-dione (532 mg, 1.86 mmol) was added in portions to a 0 °C solution of C72 (1.19 g, 3.10 mmol) in dichloromethane (16 mL). LCMS analysis after 1 hour indicated conversion to C73: LCMS m/z 462.2 (bromine isotope pattern observed) [M+H]+. The reaction mixture was diluted with dichloromethane (20 mL), washed sequentially with saturated aqueous sodium sulfite solution, saturated aqueous sodium bicarbonate solution, and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 50% ethyl acetate in heptane) afforded C73 as an oil. Yield: 980 mg, 2.12 mmol, 68%.1H NMR (400 MHz, chloroform-d) δ 7.32 – 7.12 (m, 11H, assumed; partially obscured by solvent peak), 5.03 (AB quartet, JAB = 16.3 Hz, ΔνAB = 26.6 Hz, 2H), 3.54 (AB quartet, JAB = 13.1 Hz, ΔνAB = 41.8 Hz, 2H), 2.93 (d, J = 10.2 Hz, 1H), 2.88 (ddd, J = 8.5, 8.5, 3.4 Hz, 1H), 2.84 – 2.75 (m, 1H), 2.74 – 2.65 (m, 1H), 2.40 – 2.32 (m, 1H), 2.29 (s, 3H), 2.19 (d, J = 10.2 Hz, 1H), 2.12 (ddd, J = 13.4, 8.3, 8.3 Hz, 1H), 1.99 (ddd, J = 13.7, 8.8, 5.2 Hz, 1H), 1.93 – 1.85 (m, 1H), 1.81 (ddd, J = 13.4, 7.3, 3.5 Hz, 1H). Step 2. Synthesis of 1,1'-dibenzyl-7-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine] (C74). A reaction vial was charged with 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi-1,3,2-dioxaborolane (148 mg, 0.583 mmol), C73 (180 mg, 0.389 mmol), [1,1’- bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex (31.8 mg, 38.9 µmol), and oven-dried potassium acetate (153 mg, 1.56 mmol) in 1,4-dioxane (5 mL). Nitrogen was bubbled through the reaction mixture for 5 minutes, whereupon the vial was sealed and heated at 100 °C in an aluminum block for 2 hours. LCMS analysis indicated the presence of C74: LCMS m/z 510.4 [M+H]+. After the reaction mixture had cooled to room temperature, it was diluted with ethyl acetate, and filtered through a pad of diatomaceous earth. The filtrate was concentrated in vacuo to provide C74, which was used directly in the following step. Step 3. Synthesis of 1,1'-dibenzyl-6-[5-(difluoromethyl)-1-methyl-1H-1,2,4-triazol-3-yl]-7-methyl-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine] (C75). Dichlorobis(triphenylphosphine)palladium(II) (5.24 mg, 7.46 µmol), an aqueous solution of potassium phosphate (2.0 M; 0.466 mL, 0.932 mmol), and 3-bromo-5-(difluoromethyl)-1-methyl-1H- 1,2,4-triazole (P33; 79.1 mg, 0.373 mmol) were added to a solution of C74 (from the previous step; ≤0.389 mmol) in tetrahydrofuran (5 mL). After the reaction mixture had been sparged with nitrogen, the reaction vessel was sealed and heated at 70 °C in an aluminum block for 1 hour. The temperature was then increased to 100 °C, and heating was continued overnight.3-Bromo-5- (difluoromethyl)-1-methyl-1H-1,2,4-triazole (P33; 79.1 mg, 0.373 mmol) was again added, and heating was carried out for an additional 6 hours, whereupon the reaction mixture was cooled and partitioned between ethyl acetate and water. The aqueous layer was extracted twice with ethyl acetate, and the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) provided C75. Yield: 105 mg, 0.204 mmol, 52% over 2 steps. LCMS m/z 515.3 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.69 (s, 1H), 7.33 – 7.19 (m, 9H, assumed; partially obscured by solvent peak), 7.18 – 7.12 (m, 1H), 6.85 (t, JHF = 52.6 Hz, 1H), 5.14 (AB quartet, JAB = 16.3 Hz, ΔνAB = 17.6 Hz, 2H), 4.05 (s, 3H), 3.54 (AB quartet, JAB = 13.0 Hz, ΔνAB = 38.8 Hz, 2H), 2.97 (d, J = 10.2 Hz, 1H), 2.93 – 2.81 (m, 2H), 2.81 – 2.72 (m, 1H), 2.53 (s, 3H), 2.41 – 2.32 (m, 1H), 2.22 (d, J = 10.2 Hz, 1H), 2.15 (ddd, J = 13.5, 8.3, 8.2 Hz, 1H), 2.06 – 1.97 (m, 1H), 1.96 – 1.88 (m, 1H), 1.84 (ddd, J = 13.4, 7.3, 3.4 Hz, 1H). Step 4. Synthesis of 6-[5-(difluoromethyl)-1-methyl-1H-1,2,4-triazol-3-yl]-7-methyl-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidine] (P31). Palladium on carbon (10%, wet with water; 20 mg) was added to a solution of C75 (105 mg, 0.204 mmol) in methanol (5 mL) containing a drop of formic acid, and the resulting mixture was hydrogenated overnight at room temperature and 70 psi. After filtration, the filtrate was concentrated in vacuo to provide P31 as a light-yellow solid. Yield: 63 mg, 0.19 mmol, 93%. LCMS m/z 335.2 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.20 (s, 1H), 6.86 (t, JHF = 52.4 Hz, 1H), 4.10 (s, 3H), 3.78 – 3.51 (m, 3H), 3.41 (d, J = 12.3 Hz, 1H), 2.99 – 2.85 (m, 2H), 2.83 (s, 3H), 2.29 – 2.19 (m, 2H), 2.19 – 2.01 (m, 2H).
Figure imgf000109_0001
Step 1. Synthesis of tert-butyl (2S)-6-[5-(difluoromethyl)-1-methyl-1H-1,2,4-triazol-3-yl]-7-methyl- 3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (C76). Using the method described for synthesis of C68 from P23 in Alternate Preparation of P26, P23 (220 mg, 0.575 mmol) and 3-bromo-5-(difluoromethyl)-1-methyl-1H-1,2,4-triazole (P33;128 mg, 0.604 mmol) were used to prepare C76. Silica gel chromatography (Gradient: 20% to 50% ethyl acetate in heptane) afforded C76 as a white solid. Yield: 110 mg, 0.253 mmol, 44%. LCMS m/z 435.4 [M+H]+. Step 2. Synthesis of (2S)-6-[5-(difluoromethyl)-1-methyl-1H-1,2,4-triazol-3-yl]-7-methyl-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine], dihydrochloride salt (P32). A mixture of C76 (110 mg, 0.253 mmol) in a solution of hydrogen chloride in 2-propanol (1.25 M, 1.0 mL, 1.2 mmol) was heated at 50 °C for 1 hour. LCMS analysis indicated formation of P32: LCMS m/z 335.3 [M+H]+. Concentration in vacuo afforded P32 as a solid. Yield: 74 mg, 0.182 mmol, 72%.
Figure imgf000110_0001
[Bis(2-methoxyethyl)amino]sulfur trifluoride (47.0 mL, 255 mmol) was added in a drop-wise manner to a 0 °C mixture of 3-bromo-1-methyl-1H-1,2,4-triazole-5-carbaldehyde (24.2 g, 127 mmol) in dichloromethane (400 mL); the reaction mixture was allowed to warm to 20 °C and stir at 20 °C for 16 hours. After drop-wise addition of aqueous sodium bicarbonate solution, the resulting mixture was extracted with dichloromethane (3 x 300 mL). The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (Gradient: 50% to 70% dichloromethane in petroleum ether) afforded 3-bromo-5-(difluoromethyl)-1-methyl-1H-1,2,4-triazole (P33) as a light- yellow oil (17.7 g). This material was combined with the product of a similar reaction carried out using 3-bromo-1-methyl-1H-1,2,4-triazole-5-carbaldehyde (12.0 g, 63.2 mmol); concentration under reduced pressure provided P33 as a white solid. Combined yield: 25.2 g, 119 mmol, 63%. LCMS m/z 212 (bromine isotope pattern observed) [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.06 (t, JHF = 52.2 Hz, 1H), 4.01 (s, 3H). Examples 1 and 2 (2R)-2-(5-Chloro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1 (1) and (2R)-2-(5-Chloro-2- methoxypyridin-4-yl)-1-[7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2 (2)
Figure imgf000110_0002
Figure imgf000111_0001
Step 1. Synthesis of 7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidine], dihydrochloride salt (C77). A solution of hydrogen chloride in 1,4-dioxane (4.0 M; 0.587 mL, 2.35 mmol) was added to a solution of P25 (285 mg, 0.587 mmol) in a mixture of dichloromethane (1 mL) and 1,1,1,3,3,3- hexafluoropropan-2-ol (1 mL). After the reaction mixture had been stirred at room temperature for 2 hours, LCMS analysis indicated the presence of C77: LCMS m/z 286.3 [M+H]+. Removal of volatiles in vacuo afforded C77 as a white solid. Yield: 210 mg, 0.586 mmol, quantitative. Step 2. Synthesis of (2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(2-methyl-2H-tetrazol-5- yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1 (1) and (2R)- 2-(5-chloro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2 (2). To a solution of P2 (65.7 mg, 0.305 mmol) in acetonitrile (1 mL) was added pyridinium trifluoromethanesulfonate (140 mg, 0.611 mmol), and the mixture was stirred until it was a solution. 1,1’-Carbonyldiimidazole (49.4 mg, 0.305 mmol) was added in one portion, and the reaction mixture was stirred at room temperature for 45 minutes, whereupon a solution of C77 (104 mg, 0.290 mmol) in acetonitrile (2 mL) was introduced. After the reaction mixture had been stirred at room temperature for 3 hours, it was diluted with aqueous ammonium chloride solution, and the resulting mixture was extracted three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 20% to 100% ethyl acetate in heptane) afforded a mixture of 1 and 2 as a white solid (105 mg), LCMS m/z 483.3 (chlorine isotope pattern observed) [M+H]+. Separation of the diastereomers was carried out via supercritical fluid chromatography [Column: Chiral Technologies Chiralpak IB, 21 x 250 mm, 5 µm; Mobile phase 85:15 carbon dioxide / (0.2% ammonium hydroxide in methanol); Flow rate: 75 mL/minute; Back pressure: 200 bar]; the first-eluting diastereomer was designated as 1 {(2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(2- methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1}, and the second-eluting diastereomer as 2 {(2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[7- methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'- yl]propan-1-one, DIAST-2}. 1 – Yield: 7.2 mg, 15 µmol, 5%. LCMS m/z 483.2 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [8.15 (s) and 8.14 (s), total 1H], [7.87 (s) and 7.83 (s), total 1H], [6.81 (s) and 6.75 (s), total 1H], [4.39 (s) and 4.39 (s), total 3H], [4.31 (q, J = 6.8 Hz) and 4.22 (q, J = 6.9 Hz), total 1H], 3.90 (s, 3H), [3.9 – 3.81 (m) and 3.76 – 3.52 (m), total 3H], [3.48 (d, component of AB quartet, J = 12.3 Hz) and 3.35 (d, J = 10.7 Hz), total 1H], [2.93 – 2.72 (m) and 2.6 – 2.46 (m), total 2H], [2.60 (s) and 2.58 (s), total 3H], 2.16 – 1.84 (m, 3H), 1.80 – 1.72 (m, 1H), [1.43 (d, J = 6.8 Hz) and 1.42 (d, J = 6.9 Hz), total 3H]. Retention time: 2.32 minutes [Analytical conditions. Column: Chiral Technologies Chiralpak IB, 4.6 x 100 mm, 5 µm; Mobile phase 3:2 carbon dioxide / (0.2% ammonium hydroxide in methanol); Flow rate: 1.5 mL/minute; Back pressure: 120 bar]. 2 – Yield: 7.9 mg, 16 µmol, 6%. LCMS m/z 483.2 [M+H]+. Retention time: 2.53 minutes (Analytical conditions identical to those used for 1). Examples 3 and 4 2-(6-Methoxy-2-methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1 (3) and 2-(6-Methoxy-2- methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2 (4)
Figure imgf000112_0001
Figure imgf000113_0001
Trifluoroacetic acid (2 mL) was added to a solution of C68 (280 mg, 0.726 mmol) in dichloromethane (10 mL), and the reaction mixture was stirred at room temperature for 2 hours. It was then concentrated in vacuo and evaporated twice with ethyl acetate to afford the deprotected material as a dark brown oil (200 mg), LCMS m/z 286.3 [M+H]+. A portion of this oil (35 mg) and P4 (24.9 mg, 0.123 mmol) were dissolved in dichloromethane (3 mL) and treated with O-(7- azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU; 70.0 mg, 0.184 mmol) and triethylamine (51.3 µL, 0.368 mmol), followed by N,N-dimethylformamide (2 drops) to aid solubility. After the reaction mixture had been stirred at room temperature overnight, it was diluted with dichloromethane, washed sequentially with aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution, filtered, dried, and concentrated under reduced pressure. Separation of the component diastereomers was carried out using supercritical fluid chromatography [Column: Chiral Technologies Chiralpak IA, 21 x 250 mm, 5 µm; Mobile phase: 7:3 carbon dioxide / (0.5% ammonium hydroxide in methanol); Flow rate: 75 mL/minute; Back pressure: 120 bar]; the first-eluting diastereomer was designated as 3 {2-(6-methoxy-2- methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1} and the second-eluting diastereomer as 4 {2-(6-methoxy-2-methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2}. 3 – Yield: 3.1 mg, 6.7 µmol, 5%. LCMS m/z 464.3 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [7.86 (s) and 7.85 (s), total 1H], [6.65 (s) and 6.61 (s), total 1H], [4.39 (s) and 4.39 (s), total 3H], [4.05 (q, J = 7.0 Hz), 4.01 – 3.89 (m), 3.88 – 3.55 (m), 3.59 (s), and 3.53 (s), total 5H], [3.98 (s) and 3.96 (s), total 3H], 2.95 – 2.75 (m, 2H), [2.60 (s), 2.58 (s), and 2.55 (s), total 6H], 2.19 – 1.71 (m, 4H), [1.46 (d, J = 7.1 Hz) and 1.44 (d, J = 7.1 Hz), total 3H]. Retention time: 2.47 minutes [Analytical conditions. Column: Chiral Technologies Chiralpak IA, 4.6 x 100 mm, 5 µm; Mobile phase: 65:35 carbon dioxide / (methanol containing 0.5% ammonium hydroxide); Flow rate: 1.5 mL/minute; Back pressure: 120 bar]. 4 – Yield: 3.6 mg, 7.8 µmol, 6%. LCMS m/z 486.3 [M+Na+]. Retention time: 2.92 minutes (Analytical conditions identical to those used for 3).
Figure imgf000114_0001
A solution of P26 (material from Preparation P26; 105 mg, 0.293 mmol), P4 (69.0 mg, 0.352 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI; 169 mg, 0.882 mmol), 1H-benzotriazol-1-ol (119 mg, 0.881 mmol) and N,N-diisopropylethylamine (0.255 mL, 1.46 mmol) in N,N-dimethylformamide (3 mL) was stirred at 25 °C for 16 hours. The reaction mixture was then diluted with water (40 mL) and extracted with ethyl acetate (3 x 30 mL), and the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) was followed by separation of the two diastereomers using supercritical fluid chromatography [Column: Chiral Technologies Chiralcel OJ, 30 x 250 mm, 5 µm; Mobile phase: 85:15 carbon dioxide / (2-propanol containing 0.2% propan-2-amine); Flow rate: 80 mL/minute; Back pressure: 100 bar]. The first-eluting diastereomer was designated as 3 {2-(6- methoxy-2-methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1}, and the second-eluting diastereomer as 4 {2-(6-methoxy-2-methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol- 5-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2}. 3 – Yield: 30 mg, 65 µmol, 22%. LCMS m/z 464.2 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [7.86 (s) and 7.84 (s), total 1H], [6.65 (s) and 6.61 (s), total 1H], [4.39 (s) and 4.39 (s), total 3H], [4.04 (q, J = 7.0 Hz), 4.00 – 3.89 (m), 3.88 – 3.60 (m), 3.59 (s), and 3.53 (s), total 5H], [3.97 (s) and 3.96 (s), total 3H], [2.94 – 2.74 (m) and 2.67 – 2.59 (m), total 2H], [2.60 (s), 2.58 (s), and 2.55 (s), total 6H], [2.16 – 2.06 (m) and 2.06 – 1.71 (m), total 4H], [1.46 (d, J = 7.1 Hz) and 1.44 (d, J = 7.1 Hz), total 3H]. Retention time: 4.92 minutes (Analytical conditions. Column: Chiral Technologies Chiralcel OJ, 4.6 x 250 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: 2-propanol containing 0.2% propan-2-amine; Gradient: 5% B for 1.00 minute, then 5% to 60% B over 8.00 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar). 4 – Yield: 30 mg, 65 µmol, 22%. LCMS m/z 464.2 [M+H]+.1H NMR (400 MHz, methanol-d4) δ 7.85 (s, 1H), [6.62 (s) and 6.59 (s), total 1H], [4.40 (s) and 4.39 (s), total 3H], [4.04 (q, J = 7.1 Hz), 3.98 – 3.85 (m), 3.77 – 3.60 (m), 3.58 (d, component of AB quartet, J = 10.6 Hz), and 3.55 – 3.48 (m), total 5H], [3.96 (s) and 3.91 (s), total 3H], 2.92 – 2.76 (m, 2H), [2.59 (s), 2.57 (s), 2.56 (s), and 2.37 (s), total 6H], [2.21 – 2.09 (m), 2.08 – 2.01 (m), and 2.01 – 1.78 (m), total 4H], [1.47 (d, J = 6.9 Hz) and 1.42 (d, J = 7.0 Hz), total 3H]. Retention time: 5.05 minutes (Analytical conditions identical to those used for 3). Assignment of the two diastereomers as 3 and 4 was carried out on the basis of the similarity of the 1H NMR spectra of this first-eluting enantiomer (3) with the sample of 3 from Examples 3 and 4 above. Further support was provided by comparison of the chromatographic retention time for this batch of 3 with the products from Examples 3 and 4 above: Retention time of 3 from Alternate Synthesis of Examples 3 and 4: 2.28 minutes Retention time of 3 from Examples 3 and 4: 2.46 minutes Retention time of 4 from Examples 3 and 4: 2.91 minutes These analyses were run using the same analytical method: [Column: Chiral Technologies Chiralpak IA, 4.6 x 100 mm, 5 µm; Mobile phase: 65:35 carbon dioxide / (methanol containing 0.5% ammonium hydroxide); Flow rate: 1.5 mL/minute; Back pressure: 120 bar]. The biological activity (Ki) of the respective examples from these two experiments was also consistent with the given assignments (data from individual batches that are summarized in Table 2): Example 3 from Examples 3 and 4: 0.36 nM Example 3 from Alternate Synthesis of Examples 3 and 4: 1.2 nM Example 4 from Examples 3 and 4: 25 nM Example 4 from Alternate Synthesis of Examples 3 and 4: 34 nM Examples 5 and 6 2-[6-(Difluoromethoxy)pyridin-3-yl]-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1 (5) and 2-[6-(Difluoromethoxy)pyridin-3- yl]-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'- yl]propan-1-one, DIAST-2 (6)
Figure imgf000116_0001
A mixture of P28 (50 mg, 0.14 mmol), P5 (30.6 mg, 0.141 mmol), N,N- diisopropylethylamine (0.12 mL, 0.69 mmol), and 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution in ethyl acetate; 0.25 mL, 0.42 mmol) in dichloromethane (10 mL) was stirred at 25 °C for 16 hours, whereupon it was diluted with water (20 mL) and extracted with dichloromethane (2 x 20 mL). The combined organic layers were sequentially washed with aqueous sodium bicarbonate solution (30 mL) and saturated aqueous sodium chloride solution (30 mL), dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane) afforded a mixture of 5 and 6; these diastereomers were separated using reversed-phase HPLC (Column: Chiral Technologies Chiralpak IE; 50 x 250 mm; 10 µm; Mobile phase: 95:5 ethanol / acetonitrile; Flow rate: 60 mL/minute). The first-eluting diastereomer was designated as 5 {2-[6-(difluoromethoxy)pyridin-3-yl]- 1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'- yl]propan-1-one, DIAST-1} and the second-eluting diastereomer was designated as 6 {2-[6- (difluoromethoxy)pyridin-3-yl]-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2}; both were isolated as white solids. 5 – Yield: 10 mg, 21 µmol, 15%. LCMS m/z 481.3 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [8.82 (d, J = 4.9 Hz) and 8.81 (d, J = 4.9 Hz), total 2H], [8.19 (d, J = 2.5 Hz) and 8.12 (d, J = 2.5 Hz), total 1H], 7.88 – 7.76 (m, 2H), [7.52 (t, JHF = 73.2 Hz) and 7.43 (t, JHF = 73.1 Hz), total 1H], [7.31 (t, J = 4.9 Hz) and 7.31 (t, J = 4.9 Hz), total 1H], [6.96 (d, J = 8.5 Hz) and 6.89 (d, J = 8.5 Hz), total 1H], [4.07 (q, J = 6.9 Hz), 4.03 – 3.91 (m), 3.74 – 3.63 (m), 3.60 (d, component of AB quartet, J = 12.1 Hz), 3.58 – 3.51 (m), 3.44 (d, J = 12.4 Hz) and 3.40 (d, J = 10.6 Hz), total 5H], 2.92 – 2.77 (m, 2H), [2.58 (s) and 2.54 (s), total 3H], [2.22 – 2.10 (m), 2.08 – 1.93 (m) and 1.93 – 1.77 (m), total 4H], [1.46 (d, J = 6.9 Hz) and 1.42 (d, J = 6.9 Hz), total 3H]. Retention time: 7.12 minutes (Analytical conditions. Column: Chiral Technologies Chiralpak AY-H; 4.6 x 250 mm; Mobile phase: 95:5:0.1 ethanol / acetonitrile / diethylamine; Flow rate: 0.6 mL/minute). 6 – Yield: 9.8 mg, 20 µmol, 14%. LCMS m/z 481.3 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [8.81 (d, J = 4.9 Hz) and 8.80 (d, J = 4.9 Hz), total 2H], [8.21 (d, J = 2.5 Hz) and 8.16 (d, J = 2.5 Hz), total 1H], 7.90 – 7.78 (m, 2H), [7.54 (t, JHF = 73.2 Hz) and 7.53 (t, JHF = 73.2 Hz), total 1H], [7.31 (t, J = 4.9 Hz) and 7.30 (t, J = 4.9 Hz), total 1H], [6.98 (d, J = 8.5 Hz) and 6.96 (d, J = 8.5 Hz), total 1H], [4.08 (q, J = 6.9 Hz) and 4.00 (q, J = 6.9 Hz), total 1H], [3.95 – 3.87 (m), 3.78 – 3.54 (m), 3.51 (AB quartet, JAB = 12.3 Hz, ΔνAB = 33.2 Hz), and 3.39 (d, J = 10.7 Hz), total 4H], [2.94 – 2.71 (m) and 2.62 – 2.49 (m), total 2H], [2.57 (s) and 2.54 (s), total 3H], [2.16 – 2.04 (m) and 2.02 – 1.84 (m), total 3H], 1.78 – 1.70 (m, 1H), [1.45 (d, J = 7.0 Hz) and 1.42 (d, J = 7.0 Hz), total 3H]. Retention time: 10.66 minutes (Analytical conditions identical to those used for 5).
Figure imgf000117_0001
1,1’-Carbonyldiimidazole (240 mg, 1.48 mmol) was added portion-wise to a solution of 2-[4- (trifluoromethyl)phenyl]propanoic acid (323 mg, 1.48 mmol) in acetonitrile (5 mL). After the reaction mixture had been stirred at room temperature for 45 minutes, a mixture of P28 (500 mg, 1.41 mmol) and N,N-diisopropylethylamine (0.504 mL, 2.89 mmol) in acetonitrile (2 mL) was added. Stirring was continued at room temperature for 18 hours, whereupon the reaction mixture was extracted with ethyl acetate. The combined organic layers were washed sequentially with saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue was separated into its component diastereomers via supercritical fluid chromatography {Column: Chiral Technologies Chiralcel OJ, 30 x 250 mm, 5 µm; Mobile phase 85:15 carbon dioxide / [methanol containing 0.2% (7 M ammonia in methanol)]; Flow rate: 80 mL/minute; Back pressure: 100 bar}. The first-eluting diastereomer was designated as 7 {1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]-2-[4-(trifluoromethyl)phenyl]propan-1-one, DIAST-1} and the second-eluting diastereomer was designated as 8 {1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]-2-[4-(trifluoromethyl)phenyl]propan-1-one, DIAST- 2}; both were isolated as solids. 7 – Yield: 250 mg, 0.519 mmol, 37%. LCMS m/z 482.4 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [8.80 (d, J = 4.9 Hz) and 8.79 (d, J = 4.9 Hz), total 2H], [7.83 (s) and 7.75 (s), total 1H], 7.68 – 7.62 (m, 2H), [7.54 (d, component of AB quartet, J = 8.1 Hz) and 7.49 (d, component of AB quartet, J = 8.1 Hz), total 2H], [7.28 (t, J = 4.9 Hz) and 7.28 (t, J = 4.9 Hz), total 1H], [4.10 (q, J = 6.9 Hz) and 4.00 (q, J = 6.9 Hz), total 1H], [3.92 – 3.83 (m) and 3.71 (ddd, J = 12.5, 8.5, 6.2 Hz), total 1H], [3.62 – 3.46 (m), 3.46 (d, component of AB quartet, J = 12.3 Hz), and 3.26 (d, J = 10.7 Hz), total 3H], [2.91 – 2.75 (m), 2.68 – 2.58 (m), and 2.35 – 2.25 (m), total 2H], [2.56 (s) and 2.53 (s), total 3H], [2.13 – 1.99 (m) and 1.99 – 1.81 (m), total 3H], 1.66 – 1.58 (m, 1H), [1.45 (d, J = 6.9 Hz) and 1.42 (d, J = 6.9 Hz), total 3H]. Retention time: 4.28 minutes [Column: Chiral Technologies Chiralcel OJ, 4.6 x 250 mm, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: methanol containing 0.2% (7 M ammonia in methanol); Gradient: 5% B for 1.0 minute, then 5% to 60% B over 8.0 minutes; Flow rate: 3.0 mL/minute; Back pressure: 120 bar]. 8 – Yield: 260 mg, 0.540 mmol, 38%. LCMS m/z 482.4 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [8.80 (d, J = 4.9 Hz) and 8.79 (d, J = 4.9 Hz), total 2H], [7.82 (s) and 7.81 (s), total 1H], [7.64 (d, component of AB quartet, J = 8.1 Hz) and 7.57 (d, component of AB quartet, J = 8.2 Hz), total 2H], [7.52 (d, component of AB quartet, J = 8.1 Hz) and 7.47 (d, component of AB quartet, J = 8.2 Hz), total 2H], [7.29 (t, J = 4.9 Hz) and 7.28 (t, J = 4.9 Hz), total 1H], [4.09 (q, J = 6.9 Hz) and 4.03 (q, J = 6.9 Hz), total 1H], [3.96 – 3.87 (m) and 3.46 – 3.37 (m), total 1H], [3.73 – 3.63 (m), 3.52 (AB quartet, JAB = 12.3 Hz, ΔνAB = 62.6 Hz), and 3.27 (d, J = 10.6 Hz), total 3H].2.90 – 2.71 (m, 2H), [2.57 (s) and 2.53 (s), total 3H], [2.15 – 2.05 (m), 2.04 – 1.90 (m), and 1.89 – 1.70 (m), total 4H], [1.45 (d, J = 6.9 Hz) and 1.43 (d, J = 6.9 Hz), total 3H]. Retention time: 4.74 minutes (Analytical conditions identical to those used for 7). Examples 9, 10, 11, and 12 1-(4,7-Dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4- fluorophenyl)ethan-1-one, DIAST-1 (9), 1-(4,7-Dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidin]-1'-yl)-2-(4-fluorophenyl)ethan-1-one, DIAST-2 (10), 1-(4,7-Dimethyl-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4-fluorophenyl)ethan-1-one, DIAST-3 (11), and 1- (4,7-Dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4-fluorophenyl)ethan- 1-one, DIAST-4 (12)
Figure imgf000119_0001
Step 1. Synthesis of 1-(4,7-dimethyl-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4- fluorophenyl)ethan-1-one (C78). Trifluoroacetic acid (0.5 mL) was added to a solution of P29 (30 mg, 95 µmol) in dichloromethane (3 mL), and the reaction mixture was stirred at room temperature for 1 hour. After removal of volatiles via concentration in vacuo, the residue was coevaporated twice with ethyl acetate and heptane, then dissolved in dichloromethane (5 mL). To this solution were added triethylamine (13.3 µL, 95.4 µmol), (4-fluorophenyl)acetic acid (14.7 mg, 95.4 µmol), and O-(7- azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU; 36.2 mg, 95.2 µmol). After the reaction mixture had been stirred at room temperature for 1 hour, it was concentrated in vacuo and purified via chromatography on silica gel (Gradient: 0% to 10% methanol in dichloromethane), affording C78 as an off-white powder. Yield: 34 mg, quantitative. LCMS m/z 352.2 [M+H]+. Step 2. Synthesis of 1-(4,7-dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2- (4-fluorophenyl)ethan-1-one, DIAST-1 (9), 1-(4,7-dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidin]-1'-yl)-2-(4-fluorophenyl)ethan-1-one, DIAST-2 (10), 1-(4,7-dimethyl-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4-fluorophenyl)ethan-1-one, DIAST-3 (11), and 1- (4,7-dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4-fluorophenyl)ethan- 1-one, DIAST-4 (12). A solution of C78 (22 mg, 63 µmol) in methanol (3 mL) was treated with palladium on carbon (10%; 5 mg) and hydrogenated overnight at 50 psi. The reaction mixture was then filtered, concentrated in vacuo, and subjected to supercritical fluid chromatography (Column: Chiral Technologies Chiralcel OJ-H, 5 µm; Mobile phase A: carbon dioxide; Mobile phase B: methanol containing 0.2% ammonium hydroxide; Gradient: 3% to 5% B; Flow rate: 75 mL/minute; Back pressure: 200 bar) to separate the four diastereomers. The first-eluting diastereomer was designated as 9 {1-(4,7-dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4- fluorophenyl)ethan-1-one, DIAST-1}, the second-eluting as 10 {1-(4,7-dimethyl-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4-fluorophenyl)ethan-1-one, DIAST-2}, the third- eluting as 11 {1-(4,7-dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4- fluorophenyl)ethan-1-one, DIAST-3}, and the fourth-eluting as 12 {1-(4,7-dimethyl-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4-fluorophenyl)ethan-1-one, DIAST-4}. 9 – Yield: 1.2 mg, 3.4 µmol, 5%. LCMS m/z 354.3 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 7.31 – 7.21 (m, 2H, assumed; partially obscured by solvent peak), 7.21 – 7.16 (m, 1H), 7.05 – 6.94 (m, 2H), [6.48 (d, J = 7.5 Hz) and 6.47 (d, J = 7.5 Hz), total 1H], [3.77 – 3.52 (m) and 3.44 (d, component of AB quartet, J = 12.1 Hz), total 4H], [3.62 (s) and 3.39 (s), total 2H], [2.90 – 2.77 (m) and 2.61 – 2.48 (m), total 1H], 2.33 (s, 3H), 2.13 – 2.03 (m, 1H), 2.02 – 1.94 (m, 1H), 1.89 – 1.74 (m, 1H), [1.33 (d, J = 6.7 Hz) and 1.28 (d, J = 6.7 Hz), total 3H]. Retention time: 2.77 minutes (Analytical conditions. Column: Chiral Technologies Chiralcel OJ-H, 4.6 x 100 mm, 5 µm; Mobile phase: 85:15 carbon dioxide / (methanol containing 0.2% ammonium hydroxide); Flow rate: 1.5 mL/minute; Back pressure: 120 bar). 10 – Yield: 1.3 mg, 3.7 µmol, 6%. LCMS m/z 354.3 [M+H]+.1H NMR (400 MHz, chloroform- d) δ 7.30 – 7.19 (m, 3H, assumed; partially obscured by solvent peak), 7.06 – 6.98 (m, 2H), [6.48 (d, J = 7.4 Hz) and 6.47 (d, J = 7.4 Hz), total 1H], [3.75 – 3.55 (m) and 3.50 – 3.40 (m), total 6H], 2.95 – 2.82 (m, 1H), 2.33 (s, 3H), [2.13 – 1.79 (m) and 1.74 – 1.66 (m, assumed; partially obscured by water peak), total 4H], 1.36 – 1.30 (m, 3H). Retention time: 2.92 minutes (Analytical conditions identical to those used for 9). 11 – Yield: 1.3 mg, 3.7 µmol, 6%.LCMS m/z 354.3 [M+H]+.1H NMR (400 MHz, chloroform- d) δ 7.29 – 7.21 (m, 2H, assumed; partially obscured by solvent peak), 7.21 – 7.15 (m, 1H), 7.05 – 6.93 (m, 2H), [6.48 (d, J = 7.5 Hz) and 6.47 (d, J = 7.5 Hz), total 1H], [3.74 – 3.52 (m) and 3.45 (d, component of AB quartet, J = 12.0 Hz), total 4H], [3.62 (s) and 3.39 (s), total 2H], [2.90 – 2.78 (m) and 2.61 – 2.49 (m), total 1H], [2.33 (s) and 2.32 (s), total 3H], 2.10 – 2.04 (m, 1H), 2.00 – 1.94 (m, 1H), 1.88 – 1.74 (m, 1H), [1.32 (d, J = 6.7 Hz) and 1.28 (d, J = 6.7 Hz), total 3H]. Retention time: 3.48 minutes (Analytical conditions identical to those used for 9). 12 – Yield: 2.1 mg, 5.9 µmol, 9%. LCMS m/z 354.3 [M+H]+.1H NMR (400 MHz, chloroform- d) δ 7.29 – 7.20 (m, 3H, assumed; partially obscured by solvent peak), 7.06 – 6.98 (m, 2H), [6.48 (d, J = 7.4 Hz) and 6.46 (d, J = 7.4 Hz), total 1H], [3.74 – 3.55 (m) and 3.50 – 3.40 (m), total 6H], 2.95 – 2.82 (m, 1H), 2.32 (s, 3H), [2.12 – 1.78 (m) and 1.74 – 1.66 (m, assumed; partially obscured by water peak), total 4H], 1.36 – 1.30 (m, 3H). Retention time: 4.14 minutes (Analytical conditions identical to those used for 9). By comparison of the 1H NMR data, 9 and 11 are enantiomers of one another. Similarly, 10 and 12 comprise a pair of enantiomers. Example 13 (2R)-2-(5-Fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (13)
Figure imgf000121_0001
A solution of C68 (280 mg, 0.726 mmol) in dichloromethane (10 mL) was treated with trifluoroacetic acid (2 mL) and the reaction mixture was stirred at room temperature for 2 hours. It was then concentrated in vacuo and evaporated twice from ethyl acetate, providing the deprotected substrate as a dark brown oil (200 mg); a portion of this material was used in the subsequent coupling. To a solution of P7 (36.4 mg, 0.183 mmol) in acetonitrile (3 mL) was added pyridinium trifluoromethanesulfonate (88.0 mg, 0.384 mmol), followed by 1,1’-carbonyldiimidazole (31.1 mg, 0.192 mmol). After this mixture had been stirred at room temperature for 45 minutes, a portion of the deprotected material from above (73 mg, ≤0.18 mmol), as a solution in acetonitrile (3 mL), was added, and the reaction mixture was stirred at room temperature overnight. It was then partitioned between dichloromethane and dilute aqueous ammonium chloride solution; the organic layer was washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane), followed by supercritical fluid chromatography [Column: Chiral Technologies Chiralpak IA, 21 x 250 mm, 5 µm; Mobile phase: 7:3 carbon dioxide / (0.5% ammonium hydroxide in methanol); Flow rate: 75 mL/minute; Back pressure: 120 bar] provided (2R)-2-(5-fluoro-2- methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (13). Yield: 13.6 mg, 29.1 µmol, approximately 16%. LCMS m/z 489.3 [M+Na+]. Retention time: 2.6 minutes [Analytical conditions. Column: Chiral Technologies Chiralpak IA, 4.6 x 100 mm, 5 µm; Mobile phase: 65:35 carbon dioxide / (methanol containing 0.5% ammonium hydroxide); Flow rate: 1.5 mL/minute; Back pressure: 120 bar]. Example 14 (2R)-2-(5-Fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (14)
Figure imgf000122_0001
Pyridinium trifluoromethanesulfonate (1.02 g, 4.45 mmol) was added to a solution of P7 (material from Step 2 of Alternate Preparation (#1) of P7; 422 mg, 2.12 mmol) in acetonitrile (10 mL). To the resulting solution was added 1,1’-carbonyldiimidazole (360 mg, 2.22 mmol) in one portion, and the reaction mixture was allowed to stir at room temperature for 45 minutes, whereupon a solution of P28 (material from Step 2 of Preparation P28; 750 mg, 2.12 mmol) in acetonitrile (5 mL) was added in one portion. After the reaction had been stirred at room temperature for an additional 3 hours, it was diluted with saturated aqueous ammonium chloride solution and extracted three times with ethyl acetate. The combined organic layers were washed sequentially with saturated aqueous sodium bicarbonate solution and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo; silica gel chromatography (Gradient: 30% to 100% ethyl acetate in heptane) afforded (2R)-2-(5-fluoro-2- methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidin]-1'-yl]propan-1-one (14) as a white solid. The indicated absolute stereochemistry was assigned on the basis of a single crystal X-ray structure analysis carried out on 14 derived from crystallization of this lot (see below). Yield: 670 mg, 1.45 mmol, 68%. LCMS m/z 463.4 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [8.81 (d, J = 4.9 Hz) and 8.80 (d, J = 4.9 Hz), total 2H], [7.99 (d, J = 1.6 Hz) and 7.98 (d, J = 1.7 Hz), total 1H], [7.84 (s) and 7.81 (s), total 1H], [7.30 (t, J = 4.9 Hz) and 7.29 (t, J = 4.9 Hz), total 1H], [6.78 (d, J = 4.9 Hz) and 6.73 (d, J = 4.9 Hz), total 1H], [4.27 (q, J = 6.9 Hz) and 4.19 (q, J = 6.9 Hz), total 1H], [3.93 – 3.83 (m) and 3.76 – 3.67 (m), total 1H], [3.88 (s) and 3.88 (s), total 3H], [3.67 – 3.57 (m), 3.53 (AB quartet, JAB = 12.3 Hz, ΔνAB = 34.7 Hz), and 3.39 (d, component of AB quartet, J = 10.6 Hz), total 3H], [2.94 – 2.72 (m) and 2.63 – 2.54 (m), total 2H], [2.57 (s) and 2.55 (s), total 3H], 2.15 – 1.83 (m, 3H), 1.83 – 1.74 (m, 1H), [1.45 (d, J = 6.8 Hz) and 1.43 (d, J = 6.8 Hz), total 3H]. Recrystallization from a 3:2 mixture of ethyl acetate and heptane provided material with a diastereomeric excess of 99.1%; further recrystallization from acetonitrile afforded the single crystal that was used for X-ray structural determination. Single-crystal X-ray structural determination of 14 Single Crystal X-Ray Analysis Data collection was performed on a Bruker D8 Quest diffractometer at room temperature. Data collection consisted of omega and phi scans. The structure was solved by intrinsic phasing using SHELX software suite in the triclinic class group P1. The structure was subsequently refined by the full-matrix least squares method. All non- hydrogen atoms were found and refined using anisotropic displacement parameters. The hydrogen atoms located on nitrogen were found from the Fourier difference map and refined with distances restrained. The remaining hydrogen atoms were placed in calculated positions and were allowed to ride on their carrier atoms. The final refinement included isotropic displacement parameters for all hydrogen atoms. Analysis of the absolute structure using likelihood methods (Hooft, 2008) was performed using PLATON (Spek). The results indicate that the absolute structure has been correctly assigned. The method calculates that the probability that the structure is correctly assigned is 100%. The Hooft parameter is reported as 0.05 with an esd (estimated standard deviation) of (10) and the Parson’s parameter is reported as 0.04 with an esd of (10). The final R-index was 4.5%. A final difference Fourier revealed no missing or misplaced electron density. Pertinent crystal, data collection, and refinement information is summarized in Table A. Atomic coordinates, bond lengths, bond angles, and displacement parameters are listed in Tables B – D. Software and References SHELXTL, Version 5.1, Bruker AXS, 1997. PLATON, A. L. Spek, J. Appl. Cryst.2003, 36, 7-13. MERCURY, C. F. Macrae, P. R. Edington, P. McCabe, E. Pidcock, G. P. Shields, R. Taylor, M. Towler, and J. van de Streek, J. Appl. Cryst.2006, 39, 453-457. OLEX2, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, and H. Puschmann, J. Appl. Cryst.2009, 42, 339-341. R. W. W. Hooft, L. H. Straver, and A. L. Spek, J. Appl. Cryst.2008, 41, 96-103. H. D. Flack, Acta Cryst.1983, A39, 867-881. Table A. Crystal data and structure refinement for 14.
Figure imgf000124_0001
Z 2
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Table C. Bond lengths [Å] and angles [°] for 14. ____________________________________
Figure imgf000127_0002
Figure imgf000128_0001
Figure imgf000129_0001
C(26) C(31) 1345(5) C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Symmetry transformations used to generate equivalent atoms. Table D. Anisotropic displacement parameters (Å2 x 103) for 14. The anisotropic displacement factor exponent takes the form: −2π2[h2 a*2U11 + ... + 2 h k a* b* U12 ].
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0002
Thus, the absolute stereochemistry of compound Example 14 was determined by single crystal X-ray crystallography Figure 1 is the obtained single crystal X-ray structure (ORTEP drawing) of the crystalline compound Example 14: (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[(2S)- 7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1- one. In some embodiments, the present invention provides a crystalline form of (2R)-2-(5-fluoro- 2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidin]-1'-yl]propan-1-one. In some further embodiments, the crystalline form of (2R)-2-(5- Fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one is the one described (or as prepared) in Example 14. Alternate Synthesis of Example 14 (2R)-2-(5-Fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (14)
Figure imgf000140_0001
Figure imgf000141_0001
Step 1. Synthesis of tert-butyl (2S)-6-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-7-methyl-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (C79). Di(1-adamantyl)-n-butylphosphine (cataCXium® A; 2.21 g, 6.16 mmol), followed by palladium(II) acetate (0.461 mg, 2.05 mmol), was added to 2-methyltetrahydrofuran (170 mL); the catalyst mixture was sparged with argon for 10 to 20 minutes between each manipulation. The mixture was heated at reflux for 1 hour, then cooled to ≤50 °C. In a separate reactor, P23 (98.2% by mass; 80.0 g, 205 mmol), 5,5,5′,5′-tetramethyl-2,2′-bi-1,3,2- dioxaborinane (60.3 g, 267 mmol), potassium acetate (97% by mass; 62.4 g, 617 mmol), and water (2.37 mL, 132 mmol) were added to 2-methyltetrahydrofuran (220 mL). The sides of the reactor were rinsed with 2-methyltetrahydrofuran (100 mL), and the resulting mixture was sparged with argon for approximately 1 hour. The catalyst mixture was then added via cannula, over less than 2 minutes, and the reaction mixture was heated to reflux at a rate of 1 °C / minute. After 4 hours at reflux, it was cooled to 10 °C, held at that temperature overnight, and rapidly treated drop-wise, over 15 minutes, with aqueous sodium hydroxide solution (1.0 M; 410 mL, 410 mmol). The internal temperature was maintained below 17 °C during the addition. The resulting mixture was warmed to 20 °C, diluted with tert-butyl methyl ether (180 mL) and mixed well for 5 minutes, whereupon the aqueous layer was confirmed to be at pH 10. To the organic layer was added aqueous sodium hydroxide solution (1.0 M; 480 mL, 480 mmol) in four portions over 4 minutes; after stirring for 5 minutes, the organic layer was separated and similarly extracted with aqueous sodium hydroxide solution (1.0 M; 480 mL, 480 mmol). The combined sodium hydroxide extracts were mixed with toluene (240 mL), and treated portion-wise with hydrochloric acid (12.2 M; 62.3 mL, 760 mmol), at a rate that maintained the temperature below 30 °C. The pH of the resulting mixture was 14; additional hydrochloric acid (12.2 M; 34 mL, 415 mmol) was added to adjust the pH to 10. After the mixture had been stirred for 5 minutes, the aqueous layer was extracted with toluene (2 x 240 mL), and the toluene layers were combined, affording C79 as a solution in toluene. The bulk of this material was used in the following step. Estimated yield: 73.2 g (by quantitative NMR), 176 mmol, 86% yield, as a solution in toluene. Step 2. Synthesis of tert-butyl (2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (C69). To a solution of C79 in toluene (from the previous step; 509 mL, containing 72.7 g, 175 mmol, of C79) was added aqueous sodium hydroxide solution (1 M; 530 mL, 530 mmol) followed by 2- bromopyrimidine (39.0 g, 245 mmol). The resulting mixture was sparged with argon for 30 minutes, whereupon 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos; 1.27 g, 2.19 mmol) and palladium(II) acetate (394 mg, 1.76 mmol) were added. After the reaction mixture had been heated at 50 °C for 3.5 hours, it was cooled to 20 °C, allowed to stir overnight, and filtered. The filter cake was rinsed with toluene (150 mL), and the organic layer of the combined filtrates was washed with water by stirring for 5 minutes and then allowing the mixture to stand for 30 minutes; solids in the mixture were kept with the organic layer, which was subjected to short-path distillation at 100 mbar and 60 °C. The mixture was distilled until approximately 275 mL remained, whereupon it was cooled to 20 °C at a rate of 1 °C/minute. After the mixture had stirred for 30 minutes, during which time solids were noted, heptane (727 mL) was slowly added drop-wise, over 30 minutes. The resulting solution was stirred for 10 minutes, heated to 60 °C at a rate of 1 °C/minute, and stirred at 60 °C for 90 minutes, whereupon it was cooled to 20 °C at a rate of 1 °C/minute and allowed to stir for 3 days. Filtration, followed by rinsing of the solid cake twice with the filtrate and once with heptane (220 mL), provided C69 as a solid. Yield: 63.85 g, 167.4 mmol, 96%. HPLC purity: 99.4%. 1H NMR (600 MHz, DMSO-d6) δ 8.80 (d, J = 4.8 Hz, 2H), 7.90 (s, 1H), 7.27 (t, J = 4.8 Hz, 1H), [7.25 (br s) and 7.24 (br s), total 1H], 3.56 – 3.49 (m, 1H), 3.37 – 3.30 (m, 1H), 3.28 – 3.21 (m, 2H), 2.80 – 2.73 (m, 1H), 2.73 – 2.65 (m, 1H), 2.59 (s, 3H), 1.99 – 1.84 (m, 2H), 1.82 – 1.69 (m, 2H), [1.41 (s) and 1.39 (s), total 9H]. Step 3. Synthesis of (2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine] (P28, free base). A solution of C69 (96% by mass; 50.0 g, 126 mmol) in water (100 mL) and 2-propanol (150 mL) was added over 10 minutes to an 80 °C mixture of water (150 mL) and concentrated sulfuric acid (14.5 mL, 272 mmol). After the reaction mixture had been stirred at 80 °C for 4 hours, it was cooled to 25 °C and was then subjected to short-path distillation at 120 °C and atmospheric pressure. When the mixture had been distilled to a volume of approximately 200 mL, the temperature was lowered to 50 °C, activated carbon (Darco G-60; 10 g) was added, and stirring was continued for 1.5 hours at 50 °C. The mixture was then cooled to 25 °C and filtered using a 10 μm filter. The filter cake was rinsed with water (100 mL), and the combined filtrates were diluted with 2-propanol (20 mL); the resulting mixture, of pH 0.86, was basified to the point of haziness that then cleared up, by addition of 6 M aqueous sodium hydroxide solution (approximately 75 mL). The resulting pH was 9.32. The mixture was treated drop-wise with additional 6 M aqueous sodium hydroxide solution (approximately 20 drops) to a pH of 9.6 to 9.7, at which point haziness persisted. Stirring was continued for 45 minutes, whereupon additional 6 M aqueous sodium hydroxide solution (to a total of approximately 80 mL, 480 mmol) was added, and stirring was continued at 20 °C for 30 minutes. The mixture was then heated to 50 °C at a rate of 1 °C/minute, stirred for 1.5 hours, and cooled to 20 °C at a rate of 1 °C/minute. After stirring for 1.5 hours, the mixture was filtered; the filter cake was rinsed with aqueous sodium hydroxide solution (1 M; 100 mL, 100 mmol), and dried overnight in vacuo at 50 °C to provide P28, free base. Yield: 30.87 g, 98.1% P28 via quantitative NMR, 108 mmol, 86%.1H NMR (600 MHz, DMSO-d6) δ 8.79 (d, J = 4.8 Hz, 2H), 7.88 (s, 1H), 7.25 (t, J = 4.8 Hz, 1H), 7.01 (s, 1H), 2.99 (ddd, J = 11.0, 8.4, 6.4 Hz, 1H), 2.79 (ddd, J = 10.9, 8.6, 5.6 Hz, 1H), 2.75 – 2.68 (m, 3H), 2.61 (d, J = 11.3 Hz, 1H), 2.58 (s, 3H), 1.80 – 1.68 (m, 3H), 1.65 (ddd, J = 12.7, 8.6, 6.4 Hz, 1H). Step 4. Synthesis of (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (14). A slurry of P7 (19.1 g, 95.9 mmol) in 2-methyltetrahydrofuran (200 mL) was treated with P28, free base (98.1% by mass, 25 g, 87.2 mmol) followed by N,N-diisopropylethylamine (19 mL, 110 mmol).2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphinane 2,4,6-trioxide (50% solution by weight in ethyl acetate; 65 mL, 110 mmol) was added over 15 minutes, at a rate that maintained the internal reaction temperature below 30 °C. After the reaction mixture had been stirred for 100 minutes, aqueous sodium bicarbonate solution (1.14 M; 250 mL, 285 mmol) was added {Caution: gas evolution} and stirring was continued for 10 minutes at 20 °C. The resulting mixture was heated to 40 °C, stirred for 30 minutes, and again treated with aqueous sodium bicarbonate solution (1.14 M; 125 mL, 142 mmol). After this mixture had been stirred for 80 minutes, water (75 mL) was added and stirring was continued for 10 minutes. The organic layer was subjected to distillation at 60 °C and 500 mbar, until the mixture had been reduced to 5 volumes.2- Methyltetrahydrofuran (125 mL) was added, the temperature was adjusted to 45 °C to 50 °C, and the mixture was filtered through diatomaceous earth. Additional 2-methyltetrahydrofuran (50 mL) was used to rinse the filter pad, and the combined filtrates were distilled at 60 °C and 500 mbar to approximately 3 volumes. The heat was increased to 80 °C until solids at the bottom of the reactor were released, then decreased to 50 °C. The resulting material was treated at 50 °C, over 15 minutes, with heptane (250 mL), and allowed to stir at 50 °C for 90 minutes. It was then cooled to 20 °C at a rate of 1 °C/minute and allowed to stir for 3 days, whereupon it was diluted to a volume of 600 mL by addition of 10 mol% 2-methyltetrahydrofuran in heptane. Filtration provided a filter cake, which was rinsed with heptane (75 mL) and dried overnight at 50 °C in vacuo, affording (2R)- 2-(5-fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (14) as a solid. Yield: 29.63 g, 64.06 mmol, 73%. HPLC purity: 100%. LCMS m/z 463.3 [M+H]+.1H NMR (600 MHz, DMSO-d6) δ [8.81 (d, J = 4.8 Hz) and 8.80 (d, J = 4.7 Hz), total 2H], [8.12 (s) and 8.10 (s), total 1H], [7.90 (s) and 7.87 (s), total 1H], [7.33 (s) and 7.23 (s), total 1H], 7.30 – 7.26 (m, 1H), [6.75 (d, J = 4.8 Hz) and 6.69 (d, J = 4.8 Hz), total 1H], [4.15 (q, J = 6.9 Hz) and 4.10 (q, J = 6.9 Hz), total 1H], [3.83 (s) and 3.82 (s), total 3H], [3.78 – 3.71 (m), 3.61 – 3.49 (m), 3.47 – 3.41 (m), 3.42 (d, J = 11.2 Hz), 3.32 – 3.28 (m, assumed; partially obscured by water peak), and 3.25 (d, J = 10.4 Hz), total 4H], [2.80 – 2.65 (m) and 2.5 – 2.43 (m, assumed; partially obscured by solvent peak), total 2H], [2.59 (s) and 2.57 (s), total 3H], [2.03 – 1.94 (m) and 1.87 – 1.72 (m), total 3H], 1.67 – 1.60 (m, 1H), 1.36 – 1.30 (m, 3H). Example 15 (2R)-2-(5-Chloro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (15)
Figure imgf000144_0001
Step 1. Synthesis of tert-butyl (2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (C69). A mixture of 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi-1,3,2-dioxaborolane (249 mg, 0.981 mmol), P23 (250 mg, 0.654 mmol), bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex (26.7 mg, 32.7 µmol), and oven-dried potassium acetate (257 mg, 2.62 mmol) in 1,4-dioxane (12 mL) was degassed by bubbling nitrogen through it for 5 minutes. After the reaction vial had been sealed, it was heated to 100 °C in an aluminum block for 2 hours, then allowed to cool to room temperature.2-Bromopyrimidine (109 mg, 0.686 mmol), dichlorobis(triphenylphosphine)palladium(II) (22.9 mg, 32.6 µmol), and a degassed solution of aqueous sodium carbonate (2.0 M; 0.817 mL, 1.63 mmol) were then added to the reaction mixture, and it was heated at 90 °C for 18 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and filtered through diatomaceous earth. The organic layer of the filtrate was washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo; chromatography on silica gel (Eluents: 20%, then 50%, then 100% ethyl acetate in heptane) provided C69 as a white solid. Yield: 55.0 mg, 0.144 mmol, 22%. LCMS m/z 382.3 [M+H]+.1H NMR (400 MHz, chloroform-d) δ 8.76 (d, J = 4.8 Hz, 2H), 7.92 (s, 1H), 7.11 (t, J = 4.9 Hz, 1H), 5.37 (br s, 1H), 3.62 – 3.26 (m, 4H), 2.88 – 2.76 (m, 2H), 2.68 (s, 3H), 2.06 – 1.77 (m, 4H), 1.46 (br s, 9H). Step 2. Synthesis of (2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidine], dihydrochloride salt (P28). A solution of hydrogen chloride in 1,4-dioxane (4.0 M; 0.144 mL, 0.576 mmol) was added to a solution of C69 (55.0 mg, 0.144 mmol) in a mixture of dichloromethane (0.5 mL) and 1,1,1,3,3,3- hexafluoropropan-2-ol (0.5 mL), and the reaction mixture was stirred at room temperature for 2 hours, whereupon LCMS analysis indicated conversion to P28: LCMS m/z 282.3 [M+H]+. The reaction mixture was concentrated in vacuo, providing P28 as a yellow gum. Yield: 50 mg, 0.141 mmol, 98%. Step 3. Synthesis of (2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)- 3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (15). Pyridinium trifluoromethanesulfonate (35.6 mg, 0.155 mmol) was added to a solution of P2 (16.7 mg, 77.4 µmol) in acetonitrile (1 mL). The resulting solution was treated with 1,1’- carbonyldiimidazole (12.6 mg, 77.7 µmol) in one portion, and the reaction mixture was stirred at room temperature for 45 minutes. A solution of P28 (25.0 mg, 70.6 µmol) in acetonitrile (2 mL) was then added in one portion, and stirring was continued at room temperature for 3 hours, whereupon the reaction mixture was diluted with aqueous ammonium chloride solution and extracted three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification via silica gel chromatography (Gradient: 20% to 100% ethyl acetate in heptane) followed by supercritical fluid chromatography (Column: Chiral Technologies Chiralcel OJ-H, 21 x 250 mm, 5 µm; Mobile phase 9:1 carbon dioxide / (methanol containing 0.2% ammonium hydroxide); Flow rate: 75 mL/minute; Back pressure: 150 bar) afforded (2R)-2-(5- chloro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (15). Yield: 5.9 mg, 12 µmol, 17%. LCMS m/z 479.3 (chlorine isotope pattern observed) [M+H]+.1H NMR (400 MHz, methanol-d4) δ [8.81 (d, J = 5.0 Hz) and 8.81 (d, J = 5.0 Hz), total 2H], [8.15 (s) and 8.14 (s), total 1H], [7.85 (s) and 7.81 (s), total 1H], [7.31 (t, J = 4.9 Hz) and 7.30 (t, J = 4.9 Hz), total 1H], [6.81 (s) and 6.76 (s), total 1H], [4.32 (q, J = 7.0 Hz) and 4.23 (q, J = 6.9 Hz), total 1H], 3.91 (br s, 3H), [3.9 – 3.83 (m) and 3.76 – 3.52 (m), total 3H], [3.49 (d, J = 12.2 Hz) and 3.38 – 3.3 (m, assumed; partially obscured by solvent peak), total 1H], [2.93 – 2.72 (m) and 2.56 – 2.47 (m), total 2H], [2.57 (s) and 2.56 (s), total 3H], [2.16 – 2.07 (m) and 2.05 – 1.84 (m), total 3H], 1.80 – 1.73 (m, 1H), [1.43 (d, J = 6.9 Hz) and 1.42 (d, J = 6.9 Hz), total 3H]. Alternate Step 3. Synthesis of (2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6- (pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one (15) for X- ray crystal structure determination. Pyridinium trifluoromethanesulfonate (112 mg, 0.487 mmol) was added to a solution of P2 (material from Preparations P2 and P3; 50.0 mg, 0.232 mmol) in acetonitrile (3 mL). The resulting solution was treated with 1,1’-carbonyldiimidazole (39.5 mg, 0.244 mmol) in one portion, and the reaction mixture was stirred at room temperature for 30 minutes. A solution of P28 (82.1 mg, 0.232 mmol) was then added in one portion; after 1 hour, a drop of water was added to provide a solution. After the reaction mixture had been stirred at room temperature for 2 hours, LCMS analysis indicated conversion to 15: LCMS m/z 479.3 (chlorine isotope pattern observed) [M+H]+. The reaction mixture was then partitioned between ethyl acetate and aqueous sodium bicarbonate solution; the organic layer was washed sequentially with water and saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) afforded (2R)-2-(5-chloro-2- methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidin]-1'-yl]propan-1-one (15) as a solid. Yield: 102 mg, 0.213 mmol, 92%. This material was dissolved in a mixture (approximately 12 mL) of 10% ethyl acetate in heptane by application of heat. The solution was allowed to cool and then stand, partially capped, at room temperature for 3 days. The resulting solid provided a crystal for X-ray structure determination (see below). Single-crystal X-ray structural determination of 15 Single Crystal X-Ray Analysis Data collection was performed on a Bruker D8 Venture diffractometer at room temperature. Data collection consisted of omega and phi scans. The micro-sized and multi-domain type of crystalline material used produced Theta diffraction above 0.90-0.94 Å resolution region. The structure was solved by intrinsic phasing using SHELX software suite in the triclinic class space group P1. The structure was subsequently refined by the full-matrix least squares method. All non-hydrogen atoms were found and refined using anisotropic displacement parameters. The hydrogen atoms located on nitrogen were found from the Fourier difference map and refined with distances restrained. The remaining hydrogen atoms were placed in calculated positions and were allowed to ride on their carrier atoms. The final refinement included isotropic displacement parameters for all hydrogen atoms. Analysis of the absolute structure using likelihood methods (Hooft, 2008) was performed using PLATON (Spek). The results indicate that the absolute structure has been correctly assigned. The method calculates that the probability that the structure is correctly assigned is 100.0%. The Hooft parameter is reported as 0.04 with an esd (estimated standard deviation) of (3) and the Parson’s parameter is reported as 0.05 with an esd of (3). The final R-index was 6.9%. A final difference Fourier revealed no missing or misplaced electron density. Pertinent crystal, data collection, and refinement information is summarized in Table E. Atomic coordinates, bond lengths, and displacement parameters are listed in Tables F – H. Software and References See list provided above for Single-crystal X-ray structural determination of 14. Table E. Crystal data and structure refinement for 15. _______________________________________________________________
Figure imgf000147_0001
Figure imgf000148_0001
Table F. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2 x 103) for 15. U(eq) is defined as one-third of the trace of the orthogonalized Uij tensor. _____________________________________________________________
Figure imgf000148_0002
Figure imgf000149_0001
Figure imgf000150_0001
Table G. Bond lengths [Å] for 15.
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Symmetry transformations used to generate equivalent atoms. Table H. Anisotropic displacement parameters (Å2 x 103) for 15. The anisotropic displacement factor exponent takes the form: −2π2[h2 a*2U11 + ... + 2 h k a* b* U12 ]. ____________________________________________________________________
Figure imgf000153_0002
Figure imgf000154_0001
Figure imgf000155_0001
Thus, the absolute stereochemistry of compound Example 15 was determined by single crystal X-ray crystallography Figure 2 is the obtained single crystal X-ray structure (ORTEP drawing) of the crystalline compound Example 15: (2R)-2-(5-Chloro-2-methoxypyridin-4-yl)-1-[(2S)- 7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1- one. In some embodiments, the present invention provides a crystalline form of (2R)-2-(5-chloro- 2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidin]-1'-yl]propan-1-one. In some further embodiments, the crystalline form of (2R)-2-(5- chloro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one is the one described (or as prepared) in Example 15. Examples 16 and 17 (2R)-2-(5-Fluoro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1 (16) and (2R)-2-(5-Fluoro-2- methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2 (17)
Figure imgf000156_0001
Step 1. Synthesis of tert-butyl 7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (C80). A mixture of P22 (100 mg, 0.262 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-pyrazole (109 mg, 0.524 mmol), bis(diphenylphosphino)ferrocene]dichloropalladium(II), dichloromethane complex (10.7 mg, 13.1 µmol), and aqueous sodium carbonate solution (2.0 M; 0.33 mL, 0.66 mmol) in 1,4-dioxane (3 mL) was sparged with nitrogen. The reaction vial was sealed and heated to 80 °C overnight, whereupon LCMS analysis indicated conversion to C80: LCMS m/z 384.3 [M+H]+. After the reaction mixture had cooled to room temperature, it was partitioned between ethyl acetate and water, and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered, and purified via silica gel chromatography (Gradient: 0% to 100% ethyl acetate in heptane) to afford C80 as a solid. Yield: 93 mg, 0.24 mmol, 92%.1H NMR (400 MHz, chloroform-d), characteristic peaks: δ 7.52 (s, 1H), 7.36 (s, 1H), 7.19 (s, 1H), 3.95 (s, 3H), 3.62 – 3.26 (m, 4H), 2.85 – 2.68 (m, 2H), 2.41 (s, 3H), [1.47 (s) and 1.45 (s), total 9H]. Step 2. Synthesis of 7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine- 2,3'-pyrrolidine], bis(trifluoroacetate) salt (C81). Trifluoroacetic acid (1.0 mL) was added to a solution of C80 (92 mg, 0.24 mmol) in dichloromethane (3 mL), and the reaction mixture was stirred at room temperature for 2 hours. It was then concentrated in vacuo, and the residue was coevaporated twice with ethyl acetate/heptane to afford C81 as a gum. Yield: 128 mg, assumed quantitative. LCMS m/z 284.2 [M+H]+. Step 3. Synthesis of (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4- yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1 (16) and (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2 (17). To a solution of P7 (23.9 mg, 0.120 mmol) in acetonitrile (1 mL) was added pyridinium trifluoromethanesulfonate (57.8 mg, 0.252 mmol), followed by 1,1’-carbonyldiimidazole (20.4 mg, 0.126 mmol). After the reaction mixture had stirred at room temperature for 45 minutes, a solution of C81 (34.0 mg, 66.5 µmol) in acetonitrile was added, and stirring was continued overnight at room temperature. LCMS analysis at this point indicated the presence of the coupling product: LCMS m/z 465.3 [M+H]+. The reaction mixture was then partitioned between dichloromethane and dilute aqueous ammonium chloride solution; the organic layer was washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification via silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) was followed by supercritical fluid chromatography (Column: Phenomenex Lux Cellulose-1, 21 x 250 mm, 5 µm; Mobile phase: 4:1 carbon dioxide / (methanol containing 0.2% ammonium hydroxide); Flow rate: 75 mL/minute; Back pressure: 120 bar]. The first-eluting diastereomer was designated as 16 {(2R)-2- (5-fluoro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1}, and the second-eluting diastereomer as 17 {(2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2}. 16 – Yield: 7.3 mg, 15.7 µmol, 24%. LCMS m/z 465.5 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [7.99 (d, J = 1.6 Hz) and 7.97 (d, J = 1.7 Hz), total 1H], [7.67 (s) and 7.67 (s), total 1H], 7.55 – 7.52 (m, 1H), [7.29 (s) and 7.27 (s), total 1H], [6.78 (d, J = 4.9 Hz) and 6.72 (d, J = 4.9 Hz), total 1H], [4.27 (q, J = 6.9 Hz) and 4.18 (q, J = 6.9 Hz), total 1H], [3.92 (s) and 3.92 (s), total 3H], [3.88 (s) and 3.88 (s), total 3H], [3.88 – 3.83 (m), 3.75 – 3.56 (m), and 3.54 (d, component of AB quartet, J = 12.1 Hz), total 3H], [3.45 (d, component of AB quartet, J = 12.3 Hz) and 3.36 (d, J = 10.6 Hz), total 1H], [2.89 – 2.70 (m) and 2.59 – 2.49 (m), total 2H], [2.37 (s) and 2.34 (s), total 3H], 2.13 – 1.81 (m, 3H), 1.80 – 1.71 (m, 1H), 1.47 – 1.40 (m, 3H). Retention time: 3.71 minutes [Analytical conditions. Column: Phenomenex Lux Cellulose-1, 4.6 x 100 mm, 5 µm; Mobile phase: 3:1 carbon dioxide / (methanol containing 0.2% ammonium hydroxide); Flow rate: 1.5 mL/minute; Back pressure: 200 bar]. 17 – Yield: 6.2 mg, 13.3 µmol, 20%. LCMS m/z 466.6 [M+H]+. Retention time: 4.64 minutes (Analytical conditions identical to those used for 16). Example 18 (2R)-2-(5-Fluoro-2-methoxypyridin-4-yl)-1-{(2S)-7-methyl-6-[(4,6-2H2)pyrimidin-2-yl]-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl}propan-1-one (18)
Figure imgf000158_0001
Figure imgf000159_0001
Step 1. Synthesis of (4,6-2H2)pyrimidin-2-amine (C82). To a solution of 4,6-dichloropyrimidin-2-amine (500 mg, 3.05 mmol) in methanol-d4 (10 mL) were added palladium on carbon (100 mg) and triethylamine (1.3 mL, 9.3 mmol). The reaction mixture was stirred under deuterium gas at 20 °C for 6 hours, whereupon it was filtered to remove the catalyst. After the collected catalyst had been washed with methanol (2 x 10 mL), the combined filtrates were concentrated in vacuo, then subjected to silica gel chromatography (Gradient: 0% to 80% ethyl acetate in petroleum ether), affording C82 as a white solid. Yield: 210 mg, 2.16 mmol, 71%. LCMS m/z 98.2 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 6.60 – 6.52 (br s, 2H), 6.53 (s, 1H). Step 2. Synthesis of 2-chloro(4,6-2H2)pyrimidine (C83). Intermediate C82 (210 mg, 2.16 mmol) was added portion-wise to concentrated hydrochloric acid (0.7 mL) at 0 °C, and the resulting mixture was stirred until it became homogeneous. The solution was then cooled to about −15 °C, whereupon a cold solution of sodium nitrite (298 mg, 4.32 mmol) in water (0.5 mL) was added drop-wise over 1 hour, while the reaction temperature was maintained between −15 °C and −10 °C. The reaction mixture was stirred for 1 hour, and the temperature was allowed to rise to about −5 °C; it was then carefully neutralized to a pH of 7 by addition of 30% aqueous sodium hydroxide solution, while the reaction temperature was maintained below 0 °C. The resulting mixture was extracted with diethyl ether (3 x 5 mL), and the combined organic layers were washed with saturated aqueous sodium chloride solution (10 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to afford C83 as a white solid. Yield: 115 mg, 0.987 mmol, 46%.1H NMR (400 MHz, DMSO-d6) δ 7.60 (s, 1H). Step 3. Synthesis of tert-butyl (2S)-7-methyl-6-[(4,6-2H2)pyrimidin-2-yl]-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine]-1'-carboxylate (C84). A mixture of C83 (40 mg, 0.34 mmol), P27 (119 mg, 0.34 mmol), 2-dicyclohexylphosphino- 2′,6′-dimethoxybiphenyl (SPhos; 5.6 mg, 14 µmol), chloro(2-dicyclohexylphosphino-2′,6′-dimethoxy- 1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (SPhos Pd G2; 4.9 mg, 6.8 µmol), and aqueous lithium hydroxide solution (2 M; 0.4 mL, 0.8 mmol) in tetrahydrofuran (5 mL) was purged with nitrogen for 3 minutes, whereupon the reaction mixture was stirred at 60 °C for 4 hours. It was then concentrated in vacuo; the residue was purified using chromatography on silica gel (Gradient: 0% to 50% ethyl acetate in petroleum ether) to provide C84 as a yellow solid. Yield: 116 mg, 0.302 mmol, 89%. LCMS m/z 384.3 [M+H]+.1H NMR (400 MHz, chloroform-d) δ [8.04 (s) and 8.01 (s), total 1H], 7.14 (s, 1H), 3.67 – 3.30 (m, 4H), 2.92 – 2.76 (m, 2H), [2.74 (s) and 2.73 (s), total 3H], 2.12 – 1.79 (m, 4H), [1.47 (s) and 1.46 (s), total 9H]. Step 4. Synthesis of (2S)-7-methyl-6-[(4,6-2H2)pyrimidin-2-yl]-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidine], dihydrochloride salt (C85). A solution of hydrogen chloride in 1,4-dioxane (4 M; 3 mL) was added to a solution of C84 (116 mg, 0.302 mmol) in dichloromethane (3 mL), and the reaction mixture was stirred at 20 °C for 2 hours. Concentration in vacuo afforded C85 as a yellow solid. Yield: 108 mg, 0.303 mmol, quantitative. LCMS m/z 284.2 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 10.09 – 9.93 (br s, 1H), 9.82 – 9.67 (br s, 1H), 9.01 (s, 1H), 8.45 (s, 1H), 7.50 (s, 1H), 3.50 – 3.34 (m, 2H), 3.34 – 3.27 (m, 2H), 3.01 – 2.84 (m, 2H), 2.82 (s, 3H), 2.26 – 2.07 (m, 3H), 1.99 – 1.87 (m, 1H). Step 5. Synthesis of (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-{(2S)-7-methyl-6-[(4,6-2H2)pyrimidin- 2-yl]-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl}propan-1-one (18). A solution of C85 (80 mg, 0.22 mmol), P7 (45 mg, 0.23 mmol), fluoro-N,N,N′,N′- bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH; 85 mg, 0.27 mmol), and pyridine (71 mg, 0.890 mmol) in dichloromethane (10 mL) was stirred at 25 °C for 16 hours. After the reaction mixture had been poured into aqueous sodium bicarbonate solution (10 mL), it was extracted with ethyl acetate (2 x 20 mL); the combined organic layers were washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated in vacuo. Silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane) afforded (2R)-2-(5- fluoro-2-methoxypyridin-4-yl)-1-{(2S)-7-methyl-6-[(4,6-2H2)pyrimidin-2-yl]-3,4-dihydro-1H-spiro[1,8- naphthyridine-2,3'-pyrrolidin]-1'-yl}propan-1-one (18) as a white solid. Yield: 27 mg, 58 µmol, 26%. LCMS m/z 465.3 [M+H]+.1H NMR (400 MHz, methanol-d4) δ [8.00 (d, J = 1.7 Hz) and 7.98 (d, J = 1.7 Hz), total 1H], [7.85 (s) and 7.82 (s), total 1H], [7.31 (s) and 7.30 (s), total 1H], [6.78 (d, J = 4.9 Hz) and 6.73 (d, J = 4.9 Hz), total 1H], [4.28 (q, J = 6.9 Hz) and 4.20 (q, J = 6.9 Hz), total 1H], [3.93 – 3.85 (m), 3.77 – 3.67 (m), 3.67 – 3.57 (m), 3.53 (AB quartet, JAB = 12.2 Hz, ΔνAB = 35.5 Hz), and 3.39 (d, J = 10.6 Hz), total 4H], [3.89 (s) and 3.88 (s), total 3H], [2.95 – 2.75 (m) and 2.64 – 2.55 (m), total 2H], [2.58 (s) and 2.55 (s), total 3H], [2.16 – 2.06 (m) and 2.05 – 1.85 (m), total 3H], 1.84 – 1.75 (m, 1H), [1.45 (d, J = 6.9 Hz) and 1.44 (d, J = 6.9 Hz), total 3H]. Example AA. In Vitro Binding Affinity Assay Using hMC4R The binding affinity of test compounds at the α-melanocyte-stimulating hormone receptor (hMC4R) was assessed using a radioligand competition binding assay. Recombinant Chinese hamster ovaries (CHO) cells stably expressing hMC4R (PerkinElmer # ES-191-C) were used for competitive binding. hMC4R membranes were grown in Dulbecco's Modified Essential Medium and Ham's F-12 Medium (DMEM/F12), 10% heat inactivated fetal bovine serum (FBS), 0.4 mg/mL Geneticin and 2 mM L-glutamine. Cell membranes were bulked and frozen until the assay was performed. Compounds were solubilized in 100% dimethyl sulfoxide (DMSO) to a concentration of 30 mM. A 10-point intermediate dilution series using half log dilutions was created in 100% DMSO with a top concentration of 0.03 mM. The serially diluted compounds were spotted as 1 µL/well, in 96-well Costar 3363 plates. The final compound range in the assay was 300 nM to 0.01 nM with a final DMSO concentration of 1%. Control wells, containing 1 µL of 2 mM (2 µM final) alpha- melanocyte stimulating hormone (α-MSH-Tocris # 2584) was added to the non-specific binding wells and 1 µL 100% DMSO for the total binding control wells. This was followed by the addition of 80 µL of assay buffer [25 mM HEPES, 5 mM MgCl2, 2.5 mM CaCl2, 150 mM NaCl, Complete EDTA-free Protease Inhibitor Tablet (Thermo Scientific #11873580001) and 0.25% BSA]. 10 µL of [125I]-(Nle4, D-Phe7)-α-MSH (PerkinElmer #NEX3520) was added to all wells at 10-fold the final concentration of 0.5 nM. The radioligand concentration used was below the equilibrium dissociation constant (Kd) of 2.59 nM. The exact concentration of radioligand used for each experiment was determined by liquid scintillation counting and adjusted if necessary. Frozen hMC4R cell membranes were thawed and Dounce homogenized. Homogenates were resuspended in assay buffer to a concentration of 2 µg per well. The competition binding reaction was initiated by the addition of 10 µL MC4R membrane solution to the assay-ready plates containing test compound and [125I]-(Nle4, D-Phe7)-α-MSH. The plates were incubated for 2 hours at room temperature. Assay samples were then rapidly filtered through Unifilter-96 GF/B PEI coated filter plates using a filter plate harvester (PerkinElmer) and rinsed with ice-cold wash buffer [25 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM MgCl2, 2.5 mM CaCl2, and 500 mM NaCl]. Filter plates were dried overnight at room temperature. Plates were then bottom-sealed prior to the addition of 50 µL/well Ultima Gold XR scintillation fluid (PerkinElmer 6013111). Plates were then top-sealed, incubated for 60 minutes at room temperature and then the amount of radioactivity present was determined by liquid scintillation counting on a Microbeta Trilux (PerkinElmer # 2450-0060). The raw data (expressed as counts per minute) were analyzed using ActivityBase (IDBS Data Management Software). The percent effect at each concentration of compound was calculated by ActivityBase based on the values for the uninhibited wells (total binding controls) and fully inhibited wells (non-specific binding controls) on each assay plate. A concentration required for 50% inhibition (IC50) value was determined from these data using a 4-parameter logistic model. Equilibrium dissociation constant for inhibitor of ligand and receptor interaction (Ki) values were then calculated from the IC50 values using the Cheng-Prusoff equation: Ki = IC50 / (1+ ([L]/ Kd)), where [L] is the concentration of the radioligand used in the experiment and Kd is the affinity of the radioligand (determined in separate saturation experiments). Example BB. Functional In Vitro MC4R Antagonist Potency Assay The functional in vitro antagonist potency for test compounds was determined by monitoring intracellular cyclic adenosine monophosphate (cAMP) levels in Chinese hamster ovary (CHO-) cells stably expressing the human Melanocortin-4 receptor (MC4R). Following agonist activation, human MC4R associates with the G-protein complex causing the Gα subunit to exchange bound GDP for GTP, followed by dissociation of the Gα-GTP complex. The activated Gα subunit can couple to downstream effectors to regulate the levels of second messengers or cAMP within the cell. Thereby, determination of intracellular cAMP levels allows for pharmacological characterization. Intracellular cAMP levels are quantitated using a homogenous assay utilizing the Homogeneous Time-Resolved Fluorescence (HTRF) technology from CisBio. The method is a competitive immunoassay between native cAMP produced by cells and the cAMP labelled with the acceptor dye, d2. The two entities then compete for binding to a monoclonal anti-cAMP antibody labeled with cryptate. The specific signal is inversely proportional to the concentration of cAMP in the cells. Test compounds were solubilized to 30 mM in 100% dimethyl sulfoxide (DMSO) and stored. An 11-point dilution series using 1 in 3162-fold serial dilutions was created in 100% DMSO with a top concentration of 800 μM. The serially diluted compound was spotted into a 384-well plate (Greiner, Cat No.781280) at 40 nL/well with duplicate points at each concentration, and then diluted 1 in 1000 with 40 µL assay buffer containing HBSS, 20 mM HEPES (Invitrogen), 0.1% BSA, and 250 μM IBMX (Sigma Aldrich) to create an intermediate plate at 2x final assay concentration (FAC). The final compound concentration range in the assay was 400 nM to 4 pM, with a final DMSO concentration of 0.1%. In-house generated CHO- cells stably expressing the Gs-coupled human MC4R receptor were plated in 384-well assay plates (Corning, Cat No.3570) in 50 μL/well of Ham’s F-12 containing 10% heat inactivated FBS, 1x penicillin/streptomycin, 1 mM Glutamax (Invitrogen) at a density of 2,500 cells per well and incubated at 37 oC (95% O2: 5% CO2) overnight, with micro- clime lids (Labcyte, Cat No. LLS-0310). On day of assay, media was removed from the assay plate through gentle flicking and blotting plate on a paper towel and replaced with 5 μL of 2x antagonist compound in assay buffer (HBSS, 20 mM HEPES, 0.1% BSA, 250 µM IBMX) and 0.1% DMSO. Cells were incubated with compound for 30 minutes at 37 oC (95% O2: 5% CO2) before addition of 5 μL EC80 agonist stimulation (200 nM α-melanocyte stimulating hormone, αMSH, Bachem) and another 30-minute incubation at 37 oC (95% O2: 5% CO2). Intracellular cAMP levels were quantified as per Cisbio’s protocol (5 uL of D2 and then 5 uL Cryptate, incubated for 1-2 hours at room temperature). Samples were measured on an Envision plate reader (PerkinElmer Life and Analytical Sciences; excitation, 320 nm; emission, 665 nm/620 nm). Data were analyzed using the ratio of fluorescence intensity at 620 and 665 nm for each well, extrapolated from the cAMP standard curve to express data as nM cAMP for each well. Data expressed as nM cAMP were then normalized to control wells using Activity Base (IDBS). Zero percent effect (ZPE) was defined as nM of cAMP generated from EC80 agonist stimulation (200 nM αMSH). In the absence of an antagonist control compound, one hundred percent effect (HPE) was defined as nM of cAMP generated from assay buffer/vehicle only. The concentration and % effect values for each compound were plotted by Activity Base using a four-parameter logistic dose response equation, and the concentration required for 50% inhibition (IC50) was determined. Equilibrium dissociation constant (Kb) values were then calculated according to the Leff-Dougall equation: Kb = [IC50] / ((2+ ([A]/[EC50])n)1/n -1 ), wherein A is the concentration of the agonist challenged used in the experiment (200 nM) and n= the slope. Table MC4R-1 lists biological activities (Ki values, see Example AA; and Kb values, see Example BB) and compound names for Examples 1 – 18. Table MC4R-1. Biological activity and Compound name for Examples 1 – 18 of MC4R antagonists.
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
All references, including publications, patents, and patent documents are hereby incorporated by reference herein, as though individually incorporated by reference. The present disclosure provides reference to various embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the scope of the present disclosure.

Claims

What is claimed is: 1. A compound of Formula M1, M2, Y-3A, or Y-4:
Figure imgf000167_0001
1A
Figure imgf000168_0001
wherein R is , or a pharmaceutically acceptable salt thereof, and wherein the compound or pharmaceutically acceptable salt thereof is substantially isolated or purified.
2. The compound of claim 1, wherein the compound is a compound of Formula M1 or a pharmaceutically acceptable salt thereof.
3. The compound of claim 2, wherein the compound is a compound of Formula M1-A
Figure imgf000168_0002
or a pharmaceutically acceptable salt thereof.
4. The compound of claim 1, wherein the compound is a compound of Formula M2 or a pharmaceutically acceptable salt thereof.
5. The compound of claim 1, wherein the compound is a compound of Formula M2-A or M2-B:
Figure imgf000168_0003
Figure imgf000168_0004
or a pharmaceutically acceptable salt thereof.
6. The compound of claim 1, wherein the compound is a compound of Formula Y-3A or a pharmaceutically acceptable salt thereof.
7. The compound of claim 1, wherein the compound is a compound of Formula Y-4A or a pharmaceutically acceptable salt thereof.
8. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 7, which has greater than about 50% purity by weight.
9. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 7, which has greater than about 70% purity by weight.
10. The compound or pharmaceutically acceptable salt thereof of any one of claims 1 to 7, which has greater than about 90% purity by weight.
11. A pharmaceutical composition comprising (1) a compound of Formula M1, M2, Y-3A, or Y-4:
Figure imgf000169_0001
Figure imgf000170_0001
wherein R1A , or a pharmaceutically acceptable salt thereof, and (2) a
Figure imgf000170_0002
pharmaceutically acceptable carrier.
12. The pharmaceutical composition of claim 11, wherein the composition comprises a compound of Formula M1 or a pharmaceutically acceptable salt thereof.
13. The pharmaceutical composition of claim 11, wherein the composition comprises a compound of Formula M2 or a pharmaceutically acceptable salt thereof.
14. The pharmaceutical composition of claim 11, wherein the composition comprises a compound of Formula Y-3A or a pharmaceutically acceptable salt thereof.
15. The pharmaceutical composition of claim 11, wherein the composition comprises a compound of Formula Y-4A or a pharmaceutically acceptable salt thereof.
16. The pharmaceutical composition of any one of claims 1 to 15, wherein the compound or pharmaceutically acceptable salt thereof is present in the composition in an amount greater than about 0.001% by weight.
17. A preparation of a compound of Formula M1, M2, M2-A, M2-B, Y-3A, or Y-4A, or a pharmaceutically acceptable salt thereof, which has greater than about 90% or 95% purity by weight.
18. A pharmaceutical combination comprising (1) a compound of Formula M1, M2, Y-3A, or Y-4A, or a pharmaceutically acceptable salt thereof, and (2) an additional therapeutic agent.
19. A pharmaceutical composition comprising (1) a compound of Formula M1, M2, Y-3A, or Y-4A, or a pharmaceutically acceptable salt thereof, (2) an additional therapeutic agent, and a pharmaceutically acceptable carrier.
20. The pharmaceutical combination of claim 18 or the pharmaceutical composition of claim 19, wherein the additional therapeutic agent is a melanocortin 4 receptor antagonist.
21. The pharmaceutical combination of claim 20 or the pharmaceutical composition of claim 20, wherein the additional therapeutic agent is selected from: (2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1; 2-(6-methoxy-2-methylpyrimidin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1; 2-[6-(difluoromethoxy)pyridin-3-yl]-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H- spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-2; 1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'- pyrrolidin]-1'-yl]-2-[4-(trifluoromethyl)phenyl]propan-1-one, DIAST-1; 1-(4,7-dimethyl-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl)-2-(4- fluorophenyl)ethan-1-one, DIAST-1; (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(2-methyl-2H-tetrazol-5-yl)- 3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one; (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one; (2R)-2-(5-chloro-2-methoxypyridin-4-yl)-1-[(2S)-7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro- 1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one; (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-[7-methyl-6-(1-methyl-1H-pyrazol-4-yl)-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1-one, DIAST-1; and (2R)-2-(5-fluoro-2-methoxypyridin-4-yl)-1-{(2S)-7-methyl-6-[(4,6-2H2)pyrimidin-2-yl]-3,4- dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl}propan-1-one,or pharmaceutically acceptable salt thereof.
22. The pharmaceutical combination of claim 20 or the pharmaceutical composition of claim 20, wherein the additional therapeutic agent is (2R)-2-(5-Fluoro-2-methoxypyridin-4-yl)-1-[(2S)- 7-methyl-6-(pyrimidin-2-yl)-3,4-dihydro-1H-spiro[1,8-naphthyridine-2,3'-pyrrolidin]-1'-yl]propan-1- one, or a pharmaceutically acceptable salt thereof.
23. The pharmaceutical combination of claim 18 or the pharmaceutical composition of claim 19, wherein the additional therapeutic agent is a SARM agent.
24. The pharmaceutical combination of claim 23 or the pharmaceutical composition of claim 23, wherein the additional therapeutic agent is Compound 1 or a pharmaceutically acceptable salt thereof.
25. A method for treating or preventing a disease or disorder or condition in a human, which method comprises administering to the human in need thereof a therapeutically effective amount of a compound or pharmaceutically acceptable salt of any one of claims 1 to 10, or a pharmaceutical composition or combination of any one of claims 11 to 24, wherein the disease or disorder or condition is selected from anemia; anorexia; arthritis; bone disease; benign prostate hyperplasia; musculoskeletal impairment; cachexia; cachexia associated with cancer; cancer; frailty; age-related functional decline in the elderly; growth hormone deficiency; hematopoietic disorders; hormone replacement; hypergonadism; loss of muscle strength and/or function; muscular dystrophies; muscle loss following surgery; muscular atrophy; neurodegenerative diseases; neuromuscular disease; obesity; osteoporosis; sarcopenia, including sarcopenia in chronic obstructive pulmonary disease; a method of improving dexterity and movement in a subject; atherosclerosis and its associated diseases; dysmenorrhea; dysspermtogenic sterility; muscle wasting; respiratory tract disease; otorhinolaryngologic disease; hormonal disorder/ disruption or imbalance; androgen deprivation therapy; injuries of the central nervous system; hair loss; an infection; digestive system disease; urologic or male genital disease; dermatological disorder; endocrine disorder; hemic or lymphatic disorder; congenital/hereditary or neonatal disease; connective tissue disease; metabolic disease; disorder of environmental origin; a behavior mechanism; a mental disorder; a cognitive disorder; liver disease; kidney disease and diabetic nephropathy, and stress urinary incontinence.
26. The method of claim 25, wherein the disease or disorder or condition is selected from the group consisting of anorexia; cachexia; cachexia associated with cancer; and frailty.
27. A method for detecting or confirming the administration of Compound 1 or a pharmaceutically acceptable salt thereof to a patient comprising identifying a compound or pharmaceutically acceptable salt of any one of claims 1 to 7, in a biological sample obtained from the patient.
28. The method of claim 27 wherein the biological sample is derived from plasma.
29. The method of claim 39 wherein the biological sample is derived from urine.
30. A method of measuring the rate of metabolism of Compound 1 or a pharmaceutically acceptable salt thereof in a patient comprising measuring the amount of a compound or pharmaceutically acceptable salt of any one of claims 1 to 7, in the patient at one or more time points after administration of Compound 1 or pharmaceutically acceptable salt thereof.
31. The method of claim 30 wherein the amount of compound is measured from a blood sample or plasma.
32. A method for determining the prophylactic or therapeutic response of a patient treated with Compound 1 or a pharmaceutically acceptable salt thereof comprising measuring the amount of a compound or pharmaceutically acceptable salt of any one of claims 1 to 7, in the patient at one or more time points after administration of Compound 1 or pharmaceutically acceptable salt thereof.
33. A method for optimizing the dose of Compound 1 or a pharmaceutically acceptable salt thereof for a patient in need of treatment with Compound 1 or pharmaceutically acceptable salt thereof comprising measuring the amount of a compound or pharmaceutically acceptable salt of any one of claims 1 to 7, in the patient at one or more time points after administration of Compound 1 or pharmaceutically acceptable salt thereof.
PCT/IB2022/055952 2021-06-30 2022-06-27 Metabolites of selective androgen receptor modulators WO2023275715A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163216849P 2021-06-30 2021-06-30
US63/216,849 2021-06-30

Publications (1)

Publication Number Publication Date
WO2023275715A1 true WO2023275715A1 (en) 2023-01-05

Family

ID=82547133

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2022/055952 WO2023275715A1 (en) 2021-06-30 2022-06-27 Metabolites of selective androgen receptor modulators

Country Status (1)

Country Link
WO (1) WO2023275715A1 (en)

Citations (147)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3239345A (en) 1965-02-15 1966-03-08 Estrogenic compounds and animal growth promoters
BE672205A (en) 1963-03-18 1966-05-10
US3865801A (en) 1973-06-15 1975-02-11 Atomic Energy Commission Stabilization of urinary erythropoietin using sodium p-aminosalicylate and extracting into phenol
US4036979A (en) 1974-01-25 1977-07-19 American Cyanamid Company Compositions containing 4,5,6,7-tetrahydrobenz[b]thien-4-yl-ureas or derivatives and methods of enhancing growth rate
US4342767A (en) 1980-01-23 1982-08-03 Merck & Co., Inc. Hypocholesteremic fermentation products
US4346227A (en) 1980-06-06 1982-08-24 Sankyo Company, Limited ML-236B Derivatives and their preparation
US4411890A (en) 1981-04-14 1983-10-25 Beckman Instruments, Inc. Synthetic peptides having pituitary growth hormone releasing activity
US4444784A (en) 1980-08-05 1984-04-24 Merck & Co., Inc. Antihypercholesterolemic compounds
EP0144230A2 (en) 1983-12-07 1985-06-12 Pfizer Limited Growth promotants for animals
US4729999A (en) 1984-10-12 1988-03-08 Bcm Technologies Antiestrogen therapy for symptoms of estrogen deficiency
US4761406A (en) 1985-06-06 1988-08-02 The Procter & Gamble Company Regimen for treating osteoporosis
WO1989007110A1 (en) 1988-01-28 1989-08-10 Eastman Kodak Company Polypeptide compounds having growth hormone releasing activity
WO1989007111A1 (en) 1988-01-28 1989-08-10 Eastman Kodak Company Polypeptide compounds having growth hormone releasing activity
US4876248A (en) 1982-07-29 1989-10-24 Sanofi Anti-inflammatory products derived from methylene-diphosphonic acid, and process for their preparation
US4922007A (en) 1989-06-09 1990-05-01 Merck & Co., Inc. Process for preparing 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid or salts thereof
US4927814A (en) 1986-07-11 1990-05-22 Boehringer Mannheim Gmbh Diphosphonate derivatives, pharmaceutical compositions and methods of use
US4970335A (en) 1988-01-20 1990-11-13 Yamanouchi Pharmaceutical Co., Ltd. (Cycloalkylamino)methylenebis(phosphonic acid)
WO1991005867A1 (en) 1989-10-13 1991-05-02 Amgen Inc. Erythropoietin isoforms
US5019651A (en) 1990-06-20 1991-05-28 Merck & Co., Inc. Process for preparing 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid (ABP) or salts thereof
EP0513974A1 (en) 1991-03-20 1992-11-19 Merck & Co. Inc. Novel benzo-fused lactams that promote the release of growth hormone
US5177080A (en) 1990-12-14 1993-01-05 Bayer Aktiengesellschaft Substituted pyridyl-dihydroxy-heptenoic acid and its salts
WO1993004081A1 (en) 1991-08-22 1993-03-04 Administrators Of The Tulane Educational Fund Peptides having growth hormone releasing activity
US5204350A (en) 1991-08-09 1993-04-20 Merck & Co., Inc. Method of inhibiting osteoclast-mediated bone resorption by administration of n-heterocyclicalkyl-substituted phenyl derivatives
US5217994A (en) 1991-08-09 1993-06-08 Merck & Co., Inc. Method of inhibiting osteoclast-mediated bone resorption by administration of aminoalkyl-substituted phenyl derivatives
US5260440A (en) 1991-07-01 1993-11-09 Shionogi Seiyaku Kabushiki Kaisha Pyrimidine derivatives
US5273995A (en) 1989-07-21 1993-12-28 Warner-Lambert Company [R-(R*R*)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl-3-phenyl-4-[(phenylamino) carbonyl]- 1H-pyrrole-1-heptanoic acid, its lactone form and salts thereof
US5283241A (en) 1992-08-28 1994-02-01 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
US5284841A (en) 1993-02-04 1994-02-08 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1994007486A1 (en) 1992-09-25 1994-04-14 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1994008583A1 (en) 1992-10-14 1994-04-28 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1994011012A1 (en) 1992-11-06 1994-05-26 Merck & Co., Inc. Substituted dipeptide analogs promote release of growth hormone
US5317017A (en) 1992-09-30 1994-05-31 Merck & Co., Inc. N-biphenyl-3-amido substituted benzolactams stimulate growth hormone release
WO1994013696A1 (en) 1992-12-11 1994-06-23 Merck & Co., Inc. Spiro piperidines and homologs which promote release of growth hormone
WO1994019367A1 (en) 1992-12-11 1994-09-01 Merck & Co., Inc. Spiro piperidines and homologs promote release of growth hormone
US5354772A (en) 1982-11-22 1994-10-11 Sandoz Pharm. Corp. Indole analogs of mevalonolactone and derivatives thereof
WO1995003289A1 (en) 1993-07-26 1995-02-02 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1995003290A1 (en) 1993-07-26 1995-02-02 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
US5393763A (en) 1992-07-28 1995-02-28 Eli Lilly And Company Methods for inhibiting bone loss
WO1995009633A1 (en) 1993-10-04 1995-04-13 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1995011029A1 (en) 1993-10-19 1995-04-27 Merck & Co., Inc. Combination of bisphosphonates and growth hormone secretagogues
WO1995012598A1 (en) 1993-11-02 1995-05-11 Merck & Co., Inc. Benzo-fused macrocycles promote release of growth hormone
WO1995013069A1 (en) 1993-11-09 1995-05-18 Merck & Co., Inc. Piperidines, pyrrolidines and hexahydro-1h-azepines promote release of growth hormone
WO1995014666A1 (en) 1993-11-24 1995-06-01 Merck & Co., Inc. Indolyl group containing compounds and the use thereof to promote the release of growth hormone(s)
WO1995016692A1 (en) 1993-12-14 1995-06-22 Merck & Co., Inc. Heterocyclic-fused lactams promote release of growth hormone
WO1995016675A1 (en) 1993-12-13 1995-06-22 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1995017423A1 (en) 1993-12-23 1995-06-29 Novo Nordisk A/S Compounds with growth hormone releasing properties
WO1995017422A1 (en) 1993-12-23 1995-06-29 Novo Nordisk A/S Compounds with growth hormone releasing properties
US5441868A (en) 1983-12-13 1995-08-15 Kirin-Amgen, Inc. Production of recombinant erythropoietin
WO1995032710A1 (en) 1994-05-27 1995-12-07 Merck & Co., Inc. Compounds for inhibiting osteoclast-mediated bone resorption
WO1995034311A1 (en) 1994-06-13 1995-12-21 Merck & Co., Inc. Piperazine compounds promote release of growth hormone
WO1996000574A1 (en) 1994-06-29 1996-01-11 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1996000730A1 (en) 1994-06-29 1996-01-11 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1996002530A1 (en) 1994-07-20 1996-02-01 Merck & Co., Inc. Piperidines and hexahydro-1h-azepines spiro substituted at the 4-position promote release of growth hormone
US5492916A (en) 1993-12-23 1996-02-20 Merck & Co., Inc. Di- and tri-substituted piperidines, pyrrolidines and hexahydro-1H-azepines promote release of growth hormone
US5494919A (en) 1993-11-09 1996-02-27 Merck & Co., Inc. 2-substituted piperidines, pyrrolidines and hexahydro-1H-azepines promote release of growth hormone
US5494920A (en) 1994-08-22 1996-02-27 Eli Lilly And Company Methods of inhibiting viral replication
WO1996006087A1 (en) 1994-08-22 1996-02-29 Smithkline Beecham Corporation Bicyclic compounds
US5501969A (en) 1994-03-08 1996-03-26 Human Genome Sciences, Inc. Human osteoclast-derived cathepsin
US5510517A (en) 1993-08-25 1996-04-23 Merck & Co., Inc. Process for producing N-amino-1-hydroxy-alkylidene-1,1-bisphosphonic acids
WO1996013523A1 (en) 1994-10-27 1996-05-09 Khepri Pharmaceuticals, Inc. Cathepsin o2 protease
US5547933A (en) 1983-12-13 1996-08-20 Kirin-Amgen, Inc. Production of erythropoietin
WO1996026190A1 (en) 1995-02-22 1996-08-29 Smithkline Beecham Corporation Integrin receptor antagonists
WO1997001540A1 (en) 1995-06-29 1997-01-16 Smithkline Beecham Corporation Integrin receptor antagonists
US5612359A (en) 1994-08-26 1997-03-18 Bristol-Myers Squibb Company Substituted biphenyl isoxazole sulfonamides
US5639754A (en) 1994-07-12 1997-06-17 Janssen Pharmaceutica N.V. Urea and thiourea derivatives of azolones
WO1997023200A1 (en) 1995-12-22 1997-07-03 Kowa Company, Ltd. Pharmaceutical composition stabilized with a basic agent
WO1997024124A1 (en) 1995-12-29 1997-07-10 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1997024119A1 (en) 1995-12-29 1997-07-10 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1997024122A1 (en) 1995-12-29 1997-07-10 Smithkline Beecham Corporation Vitronectin receptor antagonists
US5648491A (en) 1993-08-25 1997-07-15 Merck & Co., Inc. Process for producing n-amino-1-hydroxy-alkyl-idene-1,1-bisphosphonic acids
EP0796855A1 (en) 1996-03-20 1997-09-24 Hoechst Aktiengesellschaft Inhibitors of bone resorption and vitronectin receptor antagonists
WO1997034865A1 (en) 1996-03-20 1997-09-25 Hoechst Marion Roussel TRICYCLIC COMPOUNDS HAVING ACTIVITY SPECIFIC FOR INTEGRINS, PARTICULARLY αvβ3 INTEGRINS, METHOD FOR PREPARING SAME, INTERMEDIATES THEREFOR, USE OF SAID COMPOUNDS AS DRUGS, AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAME
WO1997037655A1 (en) 1996-04-10 1997-10-16 Merck & Co., Inc. αvβ3 ANTAGONISTS
WO1998000395A1 (en) 1996-06-28 1998-01-08 Merck Patent Gmbh Phenylalamine derivatives as integrin inhibitors
US5710159A (en) 1996-05-09 1998-01-20 The Dupont Merck Pharmaceutical Company Integrin receptor antagonists
EP0820988A2 (en) 1996-07-24 1998-01-28 Hoechst Aktiengesellschaft Imino derivatives as bone resorption inhibitors and vitronectin receptor antagonists
EP0820991A2 (en) 1996-07-24 1998-01-28 Hoechst Aktiengesellschaft Cycloalkyl derivatives as bone resorption inhibitors and vitronectin receptor antagonists
US5723480A (en) 1993-09-23 1998-03-03 Merck Patent Gesellschaft Mit Beschrankter Haftung Adhesion receptor antagonists III
WO1998008840A1 (en) 1996-08-29 1998-03-05 Merck & Co., Inc. Integrin antagonists
US5736357A (en) 1994-10-27 1998-04-07 Arris Pharmaceutical Cathespin O protease
WO1998014192A1 (en) 1996-10-02 1998-04-09 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1998015278A1 (en) 1996-10-07 1998-04-16 Smithkline Beecham Corporation Method for stimulating bone formation
WO1998018460A1 (en) 1996-10-30 1998-05-07 Merck & Co., Inc. Integrin antagonists
WO1998018461A1 (en) 1996-10-30 1998-05-07 Merck & Co., Inc. Integrin antagonists
US5760028A (en) 1995-12-22 1998-06-02 The Dupont Merck Pharmaceutical Company Integrin receptor antagonists
WO1998023608A1 (en) 1996-11-27 1998-06-04 Dupont Pharmaceuticals Company Novel integrin receptor antagonists
US5767115A (en) 1993-09-21 1998-06-16 Schering-Plough Corporation Hydroxy-substituted azetidinone compounds useful as hypocholesterolemic agents
WO1998025892A1 (en) 1996-12-09 1998-06-18 Eli Lilly And Company Integrin antagonists
US5773646A (en) 1996-03-29 1998-06-30 G. D. Searle & Co. Meta-substituted phenylene derivatives
US5773644A (en) 1996-03-29 1998-06-30 G. D. Searle & Co. Cyclopropyl alkanoic acid derivatives
US5780426A (en) 1995-06-07 1998-07-14 Ixsys, Incorporated Fivemer cyclic peptide inhibitors of diseases involving αv β3
EP0853084A2 (en) 1996-12-20 1998-07-15 Hoechst Aktiengesellschaft Substituted purine derivatives as vitronectin receptor antagonists
WO1998030542A1 (en) 1997-01-08 1998-07-16 Smithkline Beecham Corporation Vitronectin receptor antagonists
EP0854145A2 (en) 1996-12-20 1998-07-22 Hoechst Aktiengesellschaft Vitronectin receptor antagonists, their production and their use
EP0854140A2 (en) 1996-12-20 1998-07-22 Hoechst Aktiengesellschaft Vitronectin receptor antagonists, their production and their use
WO1998031359A1 (en) 1997-01-17 1998-07-23 Merck & Co., Inc. Integrin antagonists
WO1998035949A1 (en) 1997-02-13 1998-08-20 Merck Patent Gmbh Bicyclic amino acids
US5843906A (en) 1996-03-29 1998-12-01 G. D. Searle & Co. Meta-substituted phenylene sulphonamide derivatives
US5852210A (en) 1996-03-29 1998-12-22 G. D. Searle & Co. Cinnamic acid derivatives
WO1999005107A1 (en) 1997-07-25 1999-02-04 Smithkline Beecham Corporation Vitronectin receptor antagonist
WO1999006049A1 (en) 1997-08-04 1999-02-11 Smithkline Beecham Corporation Integrin receptor antagonists
WO1999011626A1 (en) 1997-09-04 1999-03-11 Smithkline Beecham Corporation Integrin receptor antagonists
WO1999015507A1 (en) 1997-09-24 1999-04-01 Hoechst Marion Roussel Hydrazono-benzazulene derivatives, pharmaceutical compositions and intermediates
WO1999015170A1 (en) 1997-09-24 1999-04-01 Smithkline Beecham Corporation Vitronectin receptor antagonist
WO1999015178A1 (en) 1997-09-24 1999-04-01 Smithkline Beecham Corporation Vitronectin receptor antagonist
WO1999015508A1 (en) 1997-09-19 1999-04-01 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1999015506A1 (en) 1997-09-24 1999-04-01 Hoechst Marion Roussel Tricyclic compounds, preparation method and said method intermediates, application as medicines and pharmaceutical compositions containing same
WO1999026945A1 (en) 1997-11-26 1999-06-03 Du Pont Pharmaceuticals Company 1,3,4-THIADIAZOLES AND 1,3,4-OXADIAZOLES AS αvβ3 ANTAGONISTS
WO1999030709A1 (en) 1997-12-17 1999-06-24 Merck & Co., Inc. Integrin receptor antagonists
WO1999030713A1 (en) 1997-12-17 1999-06-24 Merck & Co., Inc. Integrin receptor antagonists
WO1999031099A1 (en) 1997-12-17 1999-06-24 Merck & Co., Inc. Integrin receptor antagonists
WO1999032457A1 (en) 1997-12-19 1999-07-01 Aventis Pharma Deutschland Gmbh Novel acylguanidine derivatives as inhibitors of bone resorption and as vitronectin receptor antagonists
US5919792A (en) 1996-10-30 1999-07-06 Merck & Co., Inc. Integrin antagonists
WO1999033798A1 (en) 1997-12-25 1999-07-08 Yamanouchi Pharmaceutical Co., Ltd. Nitrogenous heterocyclic derivatives
EP0928793A1 (en) 1998-01-02 1999-07-14 F. Hoffmann-La Roche Ag Thiazole derivatives
EP0928790A1 (en) 1998-01-02 1999-07-14 F. Hoffmann-La Roche Ag Thiazole derivatives
US5925655A (en) 1996-04-10 1999-07-20 Merck & Co., Inc. αv β3 antagonists
WO1999037621A1 (en) 1998-01-23 1999-07-29 Aventis Pharma Deutschland Gmbh Novel sulfonamide derivatives as inhibitors of bone resorption and as inhibitors of cell adhesion
WO1999044994A1 (en) 1998-03-04 1999-09-10 G.D. Searle & Co. Meta-azacyclic amino benzoic acid compounds and derivatives thereof being integrin antagonists
US5952341A (en) 1996-10-30 1999-09-14 Merck & Co., Inc. Integrin antagonists
US5952281A (en) 1993-08-04 1999-09-14 Colgate Palmolive Company Aqueous cleaning composition which may be in microemulsion form containing a silicone antifoam agent
WO1999045927A1 (en) 1998-03-10 1999-09-16 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1999052872A1 (en) 1998-04-09 1999-10-21 Meiji Seika Kaisha, Ltd. AMINOPIPERIDINE DERIVATIVES AS INTEGRIN αvβ3 ANTAGONISTS
WO1999052896A1 (en) 1998-04-10 1999-10-21 G.D. Searle & Co. Heterocyclic glycyl beta-alanine derivatives as vitronectin antagonists
WO1999052879A1 (en) 1998-04-14 1999-10-21 American Home Products Corporation Acylresorcinol derivatives as selective vitronectin receptor inhibitors
US5981546A (en) 1996-08-29 1999-11-09 Merck & Co., Inc. Integrin antagonists
WO1999059992A1 (en) 1998-05-19 1999-11-25 Aventis Pharma Deutschland Gmbh Thienyl substituted acylguanidines as inhibitors of bone resorption and vitronectin receptor antagonists
US6008213A (en) 1995-06-29 1999-12-28 Smithkline Beecham Corporation Integrin receptor antagonists
WO2000000486A1 (en) 1998-06-29 2000-01-06 Biochem Pharma Inc. Thiophene and furan 2,5-dicarboxamides useful in the treatment of cancer
WO2000001389A1 (en) 1998-07-06 2000-01-13 Bristol-Myers Squibb Co. Biphenyl sulfonamides as dual angiotensin endothelin receptor antagonists
US6017925A (en) 1997-01-17 2000-01-25 Merck & Co., Inc. Integrin antagonists
US6017926A (en) 1997-12-17 2000-01-25 Merck & Co., Inc. Integrin receptor antagonists
WO2000003973A1 (en) 1998-07-15 2000-01-27 Merck Patent Gmbh Diacylhydrazine derivatives as integrin inhibitors
WO2000006169A1 (en) 1998-07-29 2000-02-10 Merck & Co., Inc. Integrin receptor antagonists
US6028223A (en) 1995-08-30 2000-02-22 G. D. Searle & Co. Meta-guanidine, urea, thiourea or azacyclic amino benzoic acid compounds and derivatives thereof
WO2000009503A1 (en) 1998-08-13 2000-02-24 Merck & Co., Inc. Integrin receptor antagonists
US6043265A (en) 1997-01-30 2000-03-28 Bristol-Myers Squibb Co. Isoxazolyl endothelin antagonists
US6048861A (en) 1997-12-17 2000-04-11 Merck & Co., Inc. Integrin receptor antagonists
US6066648A (en) 1997-12-17 2000-05-23 Merck & Co., Inc. Integrin receptor antagonists
WO2001017562A1 (en) 1999-09-02 2001-03-15 Yamanouchi Pharmaceutical Co., Ltd. Osteogenesis promoting agents
WO2001049288A1 (en) 2000-01-06 2001-07-12 Merck Frosst Canada & Co. Novel compounds and compositions as protease inhibitors
WO2001068603A2 (en) 2000-03-10 2001-09-20 Bristol-Myers Squibb Co. Cyclopropyl-fused pyrrolidine-based inhibitors of dipeptidyl iv, processes for their preparation, and their use
WO2001077073A1 (en) 2000-04-06 2001-10-18 Merck Frosst Canada & Co. Cathepsin cysteine protease inhibitors
RU58688U1 (en) 2005-11-28 2006-11-27 Закрытое акционерное общество "Предприятие с иностранными инвестициями "Интервыбухпром" (Iнтервибухпром) UNIVERSAL WELL LOADING MACHINE
WO2014087298A1 (en) * 2012-12-03 2014-06-12 Pfizer Inc. Novel selective androgen receptor modulators
WO2015173684A1 (en) * 2014-05-15 2015-11-19 Pfizer Inc. Crystalline form of 6-[(4r)-4-methyl-1,2-dioxido-1,2,6-thiadiazinan-2-yl]isoquinoline-1-carbonitrile
WO2015181676A1 (en) * 2014-05-30 2015-12-03 Pfizer Inc. Carbonitrile derivatives as selective androgen receptor modulators

Patent Citations (167)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE672205A (en) 1963-03-18 1966-05-10
US3239345A (en) 1965-02-15 1966-03-08 Estrogenic compounds and animal growth promoters
US3865801A (en) 1973-06-15 1975-02-11 Atomic Energy Commission Stabilization of urinary erythropoietin using sodium p-aminosalicylate and extracting into phenol
US4036979A (en) 1974-01-25 1977-07-19 American Cyanamid Company Compositions containing 4,5,6,7-tetrahydrobenz[b]thien-4-yl-ureas or derivatives and methods of enhancing growth rate
US4342767A (en) 1980-01-23 1982-08-03 Merck & Co., Inc. Hypocholesteremic fermentation products
US4346227A (en) 1980-06-06 1982-08-24 Sankyo Company, Limited ML-236B Derivatives and their preparation
US4444784A (en) 1980-08-05 1984-04-24 Merck & Co., Inc. Antihypercholesterolemic compounds
US4411890A (en) 1981-04-14 1983-10-25 Beckman Instruments, Inc. Synthetic peptides having pituitary growth hormone releasing activity
US4876248A (en) 1982-07-29 1989-10-24 Sanofi Anti-inflammatory products derived from methylene-diphosphonic acid, and process for their preparation
US5354772A (en) 1982-11-22 1994-10-11 Sandoz Pharm. Corp. Indole analogs of mevalonolactone and derivatives thereof
EP0144230A2 (en) 1983-12-07 1985-06-12 Pfizer Limited Growth promotants for animals
US5621080A (en) 1983-12-13 1997-04-15 Kirin-Amgen, Inc. Production of erythropoietin
US5618698A (en) 1983-12-13 1997-04-08 Kirin-Amgen, Inc. Production of erythropoietin
US5547933A (en) 1983-12-13 1996-08-20 Kirin-Amgen, Inc. Production of erythropoietin
US5441868A (en) 1983-12-13 1995-08-15 Kirin-Amgen, Inc. Production of recombinant erythropoietin
US4894373A (en) 1984-10-12 1990-01-16 Bcm Technologies, Inc. Antiestrogens and their use in treatment of menopause and osteoporosis
US4729999A (en) 1984-10-12 1988-03-08 Bcm Technologies Antiestrogen therapy for symptoms of estrogen deficiency
US4761406A (en) 1985-06-06 1988-08-02 The Procter & Gamble Company Regimen for treating osteoporosis
US4927814A (en) 1986-07-11 1990-05-22 Boehringer Mannheim Gmbh Diphosphonate derivatives, pharmaceutical compositions and methods of use
US4970335A (en) 1988-01-20 1990-11-13 Yamanouchi Pharmaceutical Co., Ltd. (Cycloalkylamino)methylenebis(phosphonic acid)
WO1989007111A1 (en) 1988-01-28 1989-08-10 Eastman Kodak Company Polypeptide compounds having growth hormone releasing activity
WO1989007110A1 (en) 1988-01-28 1989-08-10 Eastman Kodak Company Polypeptide compounds having growth hormone releasing activity
US4922007A (en) 1989-06-09 1990-05-01 Merck & Co., Inc. Process for preparing 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid or salts thereof
US5273995A (en) 1989-07-21 1993-12-28 Warner-Lambert Company [R-(R*R*)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl-3-phenyl-4-[(phenylamino) carbonyl]- 1H-pyrrole-1-heptanoic acid, its lactone form and salts thereof
WO1991005867A1 (en) 1989-10-13 1991-05-02 Amgen Inc. Erythropoietin isoforms
EP0668351A1 (en) 1989-10-13 1995-08-23 Amgen Inc. Erythropoietin isoforms
US5019651A (en) 1990-06-20 1991-05-28 Merck & Co., Inc. Process for preparing 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid (ABP) or salts thereof
US5177080A (en) 1990-12-14 1993-01-05 Bayer Aktiengesellschaft Substituted pyridyl-dihydroxy-heptenoic acid and its salts
EP0513974A1 (en) 1991-03-20 1992-11-19 Merck & Co. Inc. Novel benzo-fused lactams that promote the release of growth hormone
US5310737A (en) 1991-03-20 1994-05-10 Merck & Co., Inc. Benzo-fused lactams that promote the release of growth hormone
US5206235A (en) 1991-03-20 1993-04-27 Merck & Co., Inc. Benzo-fused lactams that promote the release of growth hormone
US5260440A (en) 1991-07-01 1993-11-09 Shionogi Seiyaku Kabushiki Kaisha Pyrimidine derivatives
US5217994A (en) 1991-08-09 1993-06-08 Merck & Co., Inc. Method of inhibiting osteoclast-mediated bone resorption by administration of aminoalkyl-substituted phenyl derivatives
US5204350A (en) 1991-08-09 1993-04-20 Merck & Co., Inc. Method of inhibiting osteoclast-mediated bone resorption by administration of n-heterocyclicalkyl-substituted phenyl derivatives
WO1993004081A1 (en) 1991-08-22 1993-03-04 Administrators Of The Tulane Educational Fund Peptides having growth hormone releasing activity
US5393763A (en) 1992-07-28 1995-02-28 Eli Lilly And Company Methods for inhibiting bone loss
US5283241A (en) 1992-08-28 1994-02-01 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1994007486A1 (en) 1992-09-25 1994-04-14 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
US5317017A (en) 1992-09-30 1994-05-31 Merck & Co., Inc. N-biphenyl-3-amido substituted benzolactams stimulate growth hormone release
WO1994008583A1 (en) 1992-10-14 1994-04-28 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
US5374721A (en) 1992-10-14 1994-12-20 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1994011012A1 (en) 1992-11-06 1994-05-26 Merck & Co., Inc. Substituted dipeptide analogs promote release of growth hormone
US5536716A (en) 1992-12-11 1996-07-16 Merck & Co., Inc. Spiro piperidines and homologs which promote release of growth hormone
WO1994019367A1 (en) 1992-12-11 1994-09-01 Merck & Co., Inc. Spiro piperidines and homologs promote release of growth hormone
WO1994013696A1 (en) 1992-12-11 1994-06-23 Merck & Co., Inc. Spiro piperidines and homologs which promote release of growth hormone
US5284841A (en) 1993-02-04 1994-02-08 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1995003289A1 (en) 1993-07-26 1995-02-02 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
US5430144A (en) 1993-07-26 1995-07-04 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
US5434261A (en) 1993-07-26 1995-07-18 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1995003290A1 (en) 1993-07-26 1995-02-02 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
US5952281A (en) 1993-08-04 1999-09-14 Colgate Palmolive Company Aqueous cleaning composition which may be in microemulsion form containing a silicone antifoam agent
US5648491A (en) 1993-08-25 1997-07-15 Merck & Co., Inc. Process for producing n-amino-1-hydroxy-alkyl-idene-1,1-bisphosphonic acids
US5510517A (en) 1993-08-25 1996-04-23 Merck & Co., Inc. Process for producing N-amino-1-hydroxy-alkylidene-1,1-bisphosphonic acids
US5846966A (en) 1993-09-21 1998-12-08 Schering Corporation Combinations of hydroxy-substituted azetidinone compounds and HMG CoA Reductase Inhibitors
US5767115A (en) 1993-09-21 1998-06-16 Schering-Plough Corporation Hydroxy-substituted azetidinone compounds useful as hypocholesterolemic agents
US5723480A (en) 1993-09-23 1998-03-03 Merck Patent Gesellschaft Mit Beschrankter Haftung Adhesion receptor antagonists III
WO1995009633A1 (en) 1993-10-04 1995-04-13 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1995011029A1 (en) 1993-10-19 1995-04-27 Merck & Co., Inc. Combination of bisphosphonates and growth hormone secretagogues
US5438136A (en) 1993-11-02 1995-08-01 Merck & Co., Inc. Benzo-fused macrocycles promote release of growth hormone
WO1995012598A1 (en) 1993-11-02 1995-05-11 Merck & Co., Inc. Benzo-fused macrocycles promote release of growth hormone
US5494919A (en) 1993-11-09 1996-02-27 Merck & Co., Inc. 2-substituted piperidines, pyrrolidines and hexahydro-1H-azepines promote release of growth hormone
WO1995013069A1 (en) 1993-11-09 1995-05-18 Merck & Co., Inc. Piperidines, pyrrolidines and hexahydro-1h-azepines promote release of growth hormone
WO1995014666A1 (en) 1993-11-24 1995-06-01 Merck & Co., Inc. Indolyl group containing compounds and the use thereof to promote the release of growth hormone(s)
WO1995016675A1 (en) 1993-12-13 1995-06-22 Merck & Co., Inc. Benzo-fused lactams promote release of growth hormone
WO1995016692A1 (en) 1993-12-14 1995-06-22 Merck & Co., Inc. Heterocyclic-fused lactams promote release of growth hormone
WO1995017423A1 (en) 1993-12-23 1995-06-29 Novo Nordisk A/S Compounds with growth hormone releasing properties
US5492916A (en) 1993-12-23 1996-02-20 Merck & Co., Inc. Di- and tri-substituted piperidines, pyrrolidines and hexahydro-1H-azepines promote release of growth hormone
WO1995017422A1 (en) 1993-12-23 1995-06-29 Novo Nordisk A/S Compounds with growth hormone releasing properties
US5501969A (en) 1994-03-08 1996-03-26 Human Genome Sciences, Inc. Human osteoclast-derived cathepsin
US5741796A (en) 1994-05-27 1998-04-21 Merck & Co., Inc. Pyridyl and naphthyridyl compounds for inhibiting osteoclast-mediated bone resorption
WO1995032710A1 (en) 1994-05-27 1995-12-07 Merck & Co., Inc. Compounds for inhibiting osteoclast-mediated bone resorption
US5929120A (en) 1994-05-27 1999-07-27 Merck & Co., Inc. Guainidino, formamidino, amino and related compounds for inhibiting osteoclast-mediated bone resorption
WO1995034311A1 (en) 1994-06-13 1995-12-21 Merck & Co., Inc. Piperazine compounds promote release of growth hormone
WO1996000730A1 (en) 1994-06-29 1996-01-11 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1996000574A1 (en) 1994-06-29 1996-01-11 Smithkline Beecham Corporation Vitronectin receptor antagonists
US5639754A (en) 1994-07-12 1997-06-17 Janssen Pharmaceutica N.V. Urea and thiourea derivatives of azolones
WO1996002530A1 (en) 1994-07-20 1996-02-01 Merck & Co., Inc. Piperidines and hexahydro-1h-azepines spiro substituted at the 4-position promote release of growth hormone
US5494920A (en) 1994-08-22 1996-02-27 Eli Lilly And Company Methods of inhibiting viral replication
WO1996006087A1 (en) 1994-08-22 1996-02-29 Smithkline Beecham Corporation Bicyclic compounds
US5612359A (en) 1994-08-26 1997-03-18 Bristol-Myers Squibb Company Substituted biphenyl isoxazole sulfonamides
WO1996013523A1 (en) 1994-10-27 1996-05-09 Khepri Pharmaceuticals, Inc. Cathepsin o2 protease
US5736357A (en) 1994-10-27 1998-04-07 Arris Pharmaceutical Cathespin O protease
WO1996026190A1 (en) 1995-02-22 1996-08-29 Smithkline Beecham Corporation Integrin receptor antagonists
US5780426A (en) 1995-06-07 1998-07-14 Ixsys, Incorporated Fivemer cyclic peptide inhibitors of diseases involving αv β3
WO1997001540A1 (en) 1995-06-29 1997-01-16 Smithkline Beecham Corporation Integrin receptor antagonists
US6008213A (en) 1995-06-29 1999-12-28 Smithkline Beecham Corporation Integrin receptor antagonists
US6028223A (en) 1995-08-30 2000-02-22 G. D. Searle & Co. Meta-guanidine, urea, thiourea or azacyclic amino benzoic acid compounds and derivatives thereof
WO1997023200A1 (en) 1995-12-22 1997-07-03 Kowa Company, Ltd. Pharmaceutical composition stabilized with a basic agent
US5760028A (en) 1995-12-22 1998-06-02 The Dupont Merck Pharmaceutical Company Integrin receptor antagonists
WO1997024124A1 (en) 1995-12-29 1997-07-10 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1997024122A1 (en) 1995-12-29 1997-07-10 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1997024119A1 (en) 1995-12-29 1997-07-10 Smithkline Beecham Corporation Vitronectin receptor antagonists
US6159964A (en) 1995-12-29 2000-12-12 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1997034865A1 (en) 1996-03-20 1997-09-25 Hoechst Marion Roussel TRICYCLIC COMPOUNDS HAVING ACTIVITY SPECIFIC FOR INTEGRINS, PARTICULARLY αvβ3 INTEGRINS, METHOD FOR PREPARING SAME, INTERMEDIATES THEREFOR, USE OF SAID COMPOUNDS AS DRUGS, AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAME
EP0796855A1 (en) 1996-03-20 1997-09-24 Hoechst Aktiengesellschaft Inhibitors of bone resorption and vitronectin receptor antagonists
US5773646A (en) 1996-03-29 1998-06-30 G. D. Searle & Co. Meta-substituted phenylene derivatives
US5852210A (en) 1996-03-29 1998-12-22 G. D. Searle & Co. Cinnamic acid derivatives
US5843906A (en) 1996-03-29 1998-12-01 G. D. Searle & Co. Meta-substituted phenylene sulphonamide derivatives
US5773644A (en) 1996-03-29 1998-06-30 G. D. Searle & Co. Cyclopropyl alkanoic acid derivatives
US5925655A (en) 1996-04-10 1999-07-20 Merck & Co., Inc. αv β3 antagonists
WO1997037655A1 (en) 1996-04-10 1997-10-16 Merck & Co., Inc. αvβ3 ANTAGONISTS
US5710159A (en) 1996-05-09 1998-01-20 The Dupont Merck Pharmaceutical Company Integrin receptor antagonists
WO1998000395A1 (en) 1996-06-28 1998-01-08 Merck Patent Gmbh Phenylalamine derivatives as integrin inhibitors
EP0820991A2 (en) 1996-07-24 1998-01-28 Hoechst Aktiengesellschaft Cycloalkyl derivatives as bone resorption inhibitors and vitronectin receptor antagonists
EP0820988A2 (en) 1996-07-24 1998-01-28 Hoechst Aktiengesellschaft Imino derivatives as bone resorption inhibitors and vitronectin receptor antagonists
US5981546A (en) 1996-08-29 1999-11-09 Merck & Co., Inc. Integrin antagonists
WO1998008840A1 (en) 1996-08-29 1998-03-05 Merck & Co., Inc. Integrin antagonists
WO1998014192A1 (en) 1996-10-02 1998-04-09 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1998015278A1 (en) 1996-10-07 1998-04-16 Smithkline Beecham Corporation Method for stimulating bone formation
US5919792A (en) 1996-10-30 1999-07-06 Merck & Co., Inc. Integrin antagonists
WO1998018460A1 (en) 1996-10-30 1998-05-07 Merck & Co., Inc. Integrin antagonists
WO1998018461A1 (en) 1996-10-30 1998-05-07 Merck & Co., Inc. Integrin antagonists
US5952341A (en) 1996-10-30 1999-09-14 Merck & Co., Inc. Integrin antagonists
WO1998023608A1 (en) 1996-11-27 1998-06-04 Dupont Pharmaceuticals Company Novel integrin receptor antagonists
WO1998025892A1 (en) 1996-12-09 1998-06-18 Eli Lilly And Company Integrin antagonists
EP0854140A2 (en) 1996-12-20 1998-07-22 Hoechst Aktiengesellschaft Vitronectin receptor antagonists, their production and their use
EP0854145A2 (en) 1996-12-20 1998-07-22 Hoechst Aktiengesellschaft Vitronectin receptor antagonists, their production and their use
EP0853084A2 (en) 1996-12-20 1998-07-15 Hoechst Aktiengesellschaft Substituted purine derivatives as vitronectin receptor antagonists
WO1998030542A1 (en) 1997-01-08 1998-07-16 Smithkline Beecham Corporation Vitronectin receptor antagonists
US6069158A (en) 1997-01-08 2000-05-30 Smithkline Beecham Corporation Vitronectin receptor antagonists
US6017925A (en) 1997-01-17 2000-01-25 Merck & Co., Inc. Integrin antagonists
WO1998031359A1 (en) 1997-01-17 1998-07-23 Merck & Co., Inc. Integrin antagonists
US6043265A (en) 1997-01-30 2000-03-28 Bristol-Myers Squibb Co. Isoxazolyl endothelin antagonists
WO1998035949A1 (en) 1997-02-13 1998-08-20 Merck Patent Gmbh Bicyclic amino acids
WO1999005107A1 (en) 1997-07-25 1999-02-04 Smithkline Beecham Corporation Vitronectin receptor antagonist
WO1999006049A1 (en) 1997-08-04 1999-02-11 Smithkline Beecham Corporation Integrin receptor antagonists
WO1999011626A1 (en) 1997-09-04 1999-03-11 Smithkline Beecham Corporation Integrin receptor antagonists
WO1999015508A1 (en) 1997-09-19 1999-04-01 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1999015507A1 (en) 1997-09-24 1999-04-01 Hoechst Marion Roussel Hydrazono-benzazulene derivatives, pharmaceutical compositions and intermediates
WO1999015170A1 (en) 1997-09-24 1999-04-01 Smithkline Beecham Corporation Vitronectin receptor antagonist
WO1999015178A1 (en) 1997-09-24 1999-04-01 Smithkline Beecham Corporation Vitronectin receptor antagonist
WO1999015506A1 (en) 1997-09-24 1999-04-01 Hoechst Marion Roussel Tricyclic compounds, preparation method and said method intermediates, application as medicines and pharmaceutical compositions containing same
WO1999026945A1 (en) 1997-11-26 1999-06-03 Du Pont Pharmaceuticals Company 1,3,4-THIADIAZOLES AND 1,3,4-OXADIAZOLES AS αvβ3 ANTAGONISTS
US6017926A (en) 1997-12-17 2000-01-25 Merck & Co., Inc. Integrin receptor antagonists
US6066648A (en) 1997-12-17 2000-05-23 Merck & Co., Inc. Integrin receptor antagonists
WO1999031099A1 (en) 1997-12-17 1999-06-24 Merck & Co., Inc. Integrin receptor antagonists
WO1999030709A1 (en) 1997-12-17 1999-06-24 Merck & Co., Inc. Integrin receptor antagonists
WO1999030713A1 (en) 1997-12-17 1999-06-24 Merck & Co., Inc. Integrin receptor antagonists
US6048861A (en) 1997-12-17 2000-04-11 Merck & Co., Inc. Integrin receptor antagonists
WO1999032457A1 (en) 1997-12-19 1999-07-01 Aventis Pharma Deutschland Gmbh Novel acylguanidine derivatives as inhibitors of bone resorption and as vitronectin receptor antagonists
WO1999033798A1 (en) 1997-12-25 1999-07-08 Yamanouchi Pharmaceutical Co., Ltd. Nitrogenous heterocyclic derivatives
EP0928790A1 (en) 1998-01-02 1999-07-14 F. Hoffmann-La Roche Ag Thiazole derivatives
EP0928793A1 (en) 1998-01-02 1999-07-14 F. Hoffmann-La Roche Ag Thiazole derivatives
WO1999037621A1 (en) 1998-01-23 1999-07-29 Aventis Pharma Deutschland Gmbh Novel sulfonamide derivatives as inhibitors of bone resorption and as inhibitors of cell adhesion
WO1999044994A1 (en) 1998-03-04 1999-09-10 G.D. Searle & Co. Meta-azacyclic amino benzoic acid compounds and derivatives thereof being integrin antagonists
WO1999045927A1 (en) 1998-03-10 1999-09-16 Smithkline Beecham Corporation Vitronectin receptor antagonists
WO1999052872A1 (en) 1998-04-09 1999-10-21 Meiji Seika Kaisha, Ltd. AMINOPIPERIDINE DERIVATIVES AS INTEGRIN αvβ3 ANTAGONISTS
WO1999052896A1 (en) 1998-04-10 1999-10-21 G.D. Searle & Co. Heterocyclic glycyl beta-alanine derivatives as vitronectin antagonists
WO1999052879A1 (en) 1998-04-14 1999-10-21 American Home Products Corporation Acylresorcinol derivatives as selective vitronectin receptor inhibitors
WO1999059992A1 (en) 1998-05-19 1999-11-25 Aventis Pharma Deutschland Gmbh Thienyl substituted acylguanidines as inhibitors of bone resorption and vitronectin receptor antagonists
WO2000000486A1 (en) 1998-06-29 2000-01-06 Biochem Pharma Inc. Thiophene and furan 2,5-dicarboxamides useful in the treatment of cancer
WO2000001389A1 (en) 1998-07-06 2000-01-13 Bristol-Myers Squibb Co. Biphenyl sulfonamides as dual angiotensin endothelin receptor antagonists
WO2000003973A1 (en) 1998-07-15 2000-01-27 Merck Patent Gmbh Diacylhydrazine derivatives as integrin inhibitors
US6040311A (en) 1998-07-29 2000-03-21 Merck & Co., Inc. Integrin receptor antagonists
WO2000006169A1 (en) 1998-07-29 2000-02-10 Merck & Co., Inc. Integrin receptor antagonists
WO2000009503A1 (en) 1998-08-13 2000-02-24 Merck & Co., Inc. Integrin receptor antagonists
WO2001017562A1 (en) 1999-09-02 2001-03-15 Yamanouchi Pharmaceutical Co., Ltd. Osteogenesis promoting agents
WO2001049288A1 (en) 2000-01-06 2001-07-12 Merck Frosst Canada & Co. Novel compounds and compositions as protease inhibitors
WO2001068603A2 (en) 2000-03-10 2001-09-20 Bristol-Myers Squibb Co. Cyclopropyl-fused pyrrolidine-based inhibitors of dipeptidyl iv, processes for their preparation, and their use
WO2001077073A1 (en) 2000-04-06 2001-10-18 Merck Frosst Canada & Co. Cathepsin cysteine protease inhibitors
RU58688U1 (en) 2005-11-28 2006-11-27 Закрытое акционерное общество "Предприятие с иностранными инвестициями "Интервыбухпром" (Iнтервибухпром) UNIVERSAL WELL LOADING MACHINE
WO2014087298A1 (en) * 2012-12-03 2014-06-12 Pfizer Inc. Novel selective androgen receptor modulators
US9328104B2 (en) 2012-12-03 2016-05-03 Pfizer Inc. Selective androgen receptor modulators
WO2015173684A1 (en) * 2014-05-15 2015-11-19 Pfizer Inc. Crystalline form of 6-[(4r)-4-methyl-1,2-dioxido-1,2,6-thiadiazinan-2-yl]isoquinoline-1-carbonitrile
US9920043B2 (en) 2014-05-15 2018-03-20 Pfizer Inc. Crystalline form of 6-[(4R)-4-methyl-1,2-dioxido-1,2,6-thiadiazinan-2-yl]iosoquinoline-1-carbonitrile
WO2015181676A1 (en) * 2014-05-30 2015-12-03 Pfizer Inc. Carbonitrile derivatives as selective androgen receptor modulators
US10328082B2 (en) 2014-05-30 2019-06-25 Pfizer Inc. Methods of use and combinations

Non-Patent Citations (35)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING COMPANY, pages: 1418
"Targeting the Estrogen Receptor with SERMs", ANN. REP. MED. CHEM., vol. 36, 2001, pages 149 - 158
A. L. SPEK, J. APPL. CRYST., vol. 36, 2003, pages 7 - 13
ANN. REP. MED. CHEM., vol. 28, 1993, pages 177 - 186
ASPENBERG ET AL., J. BONE MINER. RES., vol. 16, 2001, pages 497 - 500
BADGER ET AL., J. PHARMACOL. EXP. THER., vol. 279, 1996, pages 1453 - 1461
BIOORG. MED. CHEM. LETT., vol. 4, 1994, pages 2709 - 2714
BRUNKOW ET AL., AM. J. HUM. GENET., vol. 68, 2001, pages 577 - 89
C. F. MACRAEP. R. EDINGTONP. MCCABEE. PIDCOCKG. P. SHIELDSR. TAYLORM. TOWLERJ. VAN DE STREEK, J. APPL. CRYST., vol. 39, 2006, pages 453 - 457
C. FARINA ET AL., DDT, vol. 4, 1999, pages 163 - 172
EDWARDS, J. P., BIO. MED. CHEM. LET., vol. 9, 1999, pages 1003 - 1008
GHIRON ET AL., J. BONE MINER. RES., vol. 10, 1995, pages 1844 - 1852
GOLDSTEIN ET AL.: "A pharmacological review of selective estrogen receptor modulators", HUMAN REPRODUCTION UPDATE, vol. 6, 2000, pages 212 - 224, XP002936407, DOI: 10.1093/humupd/6.3.212
GOWEN ET AL., J. CLIN. INVEST., vol. 105, 2000, pages 1595 - 604
H. D. FLACK, ACTA CRYST., vol. 39, 1983, pages 867 - 881
HAMANN, L. G., J. MED. CHEM., vol. 42, 1999, pages 210 - 212
HAMILTON RAGARNETT WRKLINE BJ: "Determination of a mean valproic acid serum level by assay of a single pooled sample", CLIN PHARMACOL THER, vol. 29, no. 3, 1981, pages 408 - 13
HURLEY FLORKIEWICZ, FIBROBLAST GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR FAMILIES, 1996
J. ORG. CHEM, vol. 32, 1967, pages 4111
JOHANNSONROSEN ET AL.: "Principles of Bone Biology", 1996, ACADEMIC PRESS, article "The IGFs as potential therapy for metabolic bone diseases"
JONES G., PHARMACOLOGICAL MECHANISMS OF THERAPEUTICS: VITAMIN D AND ANALOGS, 1996
JOURNAL OF PHARMACEUTICAL SCIENCE, vol. 66, no. 2, 1977
LUFKIN ET AL.: "Calcitonin", RHEUMATIC DISEASE CLINICS OF NORTH AMERICA, vol. 27, 2001, pages 187 - 196
M. NAKAGAWA ET AL., FEBS LETTERS, vol. 473, 2000, pages 161 - 164
MASSAGUECHEN: "Controlling TGF-beta signaling", GENES DEV., vol. 14, 2000, pages 627 - 644, XP055165529
O. V. DOLOMANOVL. J. BOURHISR. J. GILDEAJ. A. K. HOWARDH. PUSCHMANN, J. APPL. CRYST., vol. 42, 2009, pages 7691 - 7694
PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 7001 - 7005
R. M. KEENAN ET AL., BIOORG. MED. CHEM. LETT., vol. 8, 1998, pages 3171 - 3176
R. M. KEENAN ET AL., J. MED. CHEM., vol. 40, 1997, pages 2289 - 2292
R. W. W. HOOFTL. H. STRAVERA. L. SPEK, J. APPL. CRYST., vol. 41, 2008, pages 96 - 103
S. K. GALLMEYER ET AL., CHEMCATCHEM, vol. 8, 2016, pages 916 - 921
SCIENCE, vol. 260, 11 June 1993 (1993-06-11), pages 1640 - 1643
SHIRAKI ET AL., J. BONE MINER. RES., vol. 15, 2000, pages 515 - 521
W. J. HOEKSTRAB. L. POULTER, CURR. MED. CHEM., vol. 5, 1998, pages 195 - 204
WANG E A, TRENDS BIOTECHNOL., vol. 11, 1993, pages 379 - 383

Similar Documents

Publication Publication Date Title
EP3774791B1 (en) Heterocyclic compounds as immunomodulators
KR101972719B1 (en) Androgen receptor modulators and uses thereof
EP1755572B1 (en) N- (2-benzyl) -2-phenylbutanamides as androgen receptor modulators
EP3148587B1 (en) Carbonitrile derivatives as selective androgen receptor modulators
CN114008033A (en) Glucagon-like peptide 1 receptor agonists
US7534796B2 (en) Imidazo[4,5-b]pyridine antagonists of gonadotropin releasing hormone receptor
EP1960417B1 (en) Polymorphs of androgen receptor modulator - (n-3h-imidazo [4 , 5-b] pyridin-2-yl-methyl) -2-fluoro-4-methyl-3-oxo-4-aza-androst- 1-en-17 . beta . -carboxamide
TWI402073B (en) A nitrogen-containing condensed ring derivative, a pharmaceutical composition containing the same, and a pharmaceutical use thereof
JP2023505100A (en) Covalent RAS inhibitors and uses thereof
KR20210061329A (en) Quinazoline derivatives as antitumor agents
CA3120530A1 (en) Novel compounds having estrogen receptor alpha degradation activity and uses thereof
US20060189618A1 (en) 4-Substituted imidazo[4,5-c]pyridine antagonists of gonadotropin releasing hormone receptor
UA111739C2 (en) IMIDAZOPYRIDASINE AS ACT-KINASE INHIBITORS
JP2016522231A (en) Diaminoheteroaryl substituted pyrazoles
US11939328B2 (en) Quinoline compounds as inhibitors of KRAS
CN114007697A (en) Antiproliferative agents for the treatment of PAH
US20210353630A1 (en) 1,2,4-triazin-3(2h)-one compounds for the treatment of hyperproliferative diseases
WO2023275715A1 (en) Metabolites of selective androgen receptor modulators
US11952377B2 (en) Quinolines and azaquinolines as inhibitors of CD38
WO2020157236A1 (en) Pyridyl substituted dihydrooxadiazinones

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22741574

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE