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AU2013267570A1 - Novel compounds and compositions for treatment of breathing control disorders or diseases - Google Patents

Novel compounds and compositions for treatment of breathing control disorders or diseases Download PDF

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Publication number
AU2013267570A1
AU2013267570A1 AU2013267570A AU2013267570A AU2013267570A1 AU 2013267570 A1 AU2013267570 A1 AU 2013267570A1 AU 2013267570 A AU2013267570 A AU 2013267570A AU 2013267570 A AU2013267570 A AU 2013267570A AU 2013267570 A1 AU2013267570 A1 AU 2013267570A1
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AU
Australia
Prior art keywords
propylamino
bis
hydroxylamine
triazin
methyl
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
AU2013267570A
Inventor
Scott L. Dax
Francis John Golder
Duncan Euan Macintyre
James C. Mannion
James Francis Mcleod
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Galleon Pharmaceuticals Inc
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Galleon Pharmaceuticals Inc
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Filing date
Publication date
Priority claimed from US13/482,837 external-priority patent/US20120295911A1/en
Application filed by Galleon Pharmaceuticals Inc filed Critical Galleon Pharmaceuticals Inc
Publication of AU2013267570A1 publication Critical patent/AU2013267570A1/en
Abandoned legal-status Critical Current

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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • 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
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5355—Non-condensed oxazines and containing further heterocyclic rings
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • 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
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/16—Central respiratory analeptics
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/50—Three nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
    • C07D251/40—Nitrogen atoms
    • C07D251/54—Three nitrogen atoms
    • 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
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/08—Bridged systems
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    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
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    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

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  • Bioinformatics & Cheminformatics (AREA)
  • Pulmonology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

The present invention includes compositions that are useful in the treatment of breathing control diseases or disorders in a subject in need thereof. The present invention also includes a method of treating a respiratory disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation of the invention. The present invention further includes a method of preventing destabilization or stabilizing breathing rhythm in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation of the invention.

Description

WO 2013/181217 PCT/US2013/043052 TITLE OF THE INVENTION Novel Compounds and Compositions for Treatment of Breathing Control Disorders or Diseases 5 CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority to U.S. Patent Application No. 13/482,837, filed May 29, 2012, which is a continuation-in-part of and claims priority to U.S. Patent Application No. 13/306,349, filed November 29, 2011, which is 10 entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Applications No. 61/417,777, filed November 29, 2010, and No. 61/494,268, filed June 7, 2011, all of which are hereby incorporated by reference in their entireties herein. BACKGROUND OF THE INVENTION 15 Normal control of breathing is a complex process that involves, in part, the body's interpretation and response to chemical stimuli such as carbon dioxide, pH and oxygen levels in blood, tissues and the brain. Breathing control is also affected by other factors such as wakefulness (i.e., whether the patient is awake or sleeping), emotion, posture and vocalization. Within the brain medulla, there are respiratory 20 control centers that interpret various feedforward and feedback signals that affect respiration and issues commands to the muscles that perform the work of breathing. Key muscle groups are located in the abdomen, diaphragm, pharynx and thorax. Sensors located centrally and peripherally then provide input to the brain's central respiration control areas that enables response to changing metabolic requirements. 25 For example, ventilation sufficient to meet the body's metabolic needs is maintained primarily by the body's rapid response to changes in carbon dioxide levels (C0 2 ). Increased CO 2 levels signal the body to increase breathing rate and depth, resulting in higher blood oxygen levels and subsequent lower blood CO 2 levels. Conversely, low CO 2 levels can result in periods of hyponea (decreased breathing) or, 30 in the extreme case, apnea (no breathing) since the stimulation to breathe is diminished. This is what happens when a person hyperventilates. There are many diseases in which loss of normal breathing control is a primary or secondary feature of the disease. Examples of diseases with a primary loss of breathing control are apneas (central, mixed or obstructive; where the breathing - 1 - WO 2013/181217 PCT/US2013/043052 repeatedly stops for 10 to 60 seconds) and congenital central hypoventilation syndrome. Secondary loss of breathing control may be due to chronic cardio pulmonary diseases (e.g., heart failure, chronic bronchitis, emphysema, and impending respiratory failure), excessive weight (e.g., obesity-hypoventilation 5 syndrome), certain drugs (e.g., anesthetics, sedatives, anxiolytics, hypnotics, alcohol, and narcotic analgesics and/or factors that affect the neurological system (e.g., stroke, tumor, trauma, radiation damage, and ALS). In chronic obstructive pulmonary diseases where the body is exposed to chronically high levels of carbon dioxide, the body adapts to the lower pH by a kidney mediated retention of bicarbonate, which has 10 the effect of partially neutralizing the C0 2 /pH respiratory stimulation. Thus, the patient is unable to mount a normal ventilatory response to changes in metabolic demand. Sleep disordered breathing is an example of where abnormalities in the control of breathing lead to a serious and prevalent disease in humans. Sleep apnea is 15 characterized by frequent periods of no or partial breathing. Key factors that contribute to these apneas include anatomical factors (such as obesity), decreased hypercapnic and hypoxic ventilatory responses (e.g., decreased response to high carbon dioxide and low oxygen levels, respectively) and loss of "wakefulness" (i.e., drive to pharyngeal dilator muscles). Apneic events result in hypoxia (and the 20 associated oxidative stress) and eventually severe cardiovascular consequences (high blood pressure, stroke, heart attack). Estimates for U.S. individuals afflicted with conditions wherein there is compromised respiratory control include sleep apneas (15-20 millions); obesity hypoventilation syndrome (5-10 millions); chronic heart disease (5 millions); chronic 25 obstructive pulmonary disease (COPD)/chronic bronchitis (10 millions); drug-induced hypoventilation (2-5 millions); and mechanical ventilation weaning (0.5 million). There is a need in the art for novel chemical compounds that can be used to restore all or part of the body's normal breathing control system in response to changes in CO 2 and/or oxygen, with minimal side effects. Such compounds would be 30 of benefit in decreasing the incidence and severity of breathing control disturbances. The present invention addresses and meets these needs. -2- WO 2013/181217 PCT/US2013/043052 BRIEF SUMMARY OF THE INVENTION The invention includes a composition comprising at least one compound selected from the group consisting of: N-(2,4-bis-n-propylamino)-N-(6 methylamino)-[1,3,5]triazine (CLXXII), N-(2,4,6-tris-n-propylamino)-[1,3,5]triazine 5 (CLXXIII), N-(2,4,6-tris-n-propylamino)-1,3-pyrimidine (CLXXIV), N-(2,4-bis-n propylamino)-N-(6-i-propylamino)-1,3-pyrimidine (CLXXV), a salt thereof, and any combinations thereof. In one embodiment, the salt is hydrogen sulfate, hydrochloride, phosphate, hydrogen phosphate or dihydrogen phosphate. In another embodiment, 10 the composition further comprises at least one pharmaceutically acceptable carrier. The invention also includes a method of preventing or treating a breathing control disorder or disease in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least 15 one compound selected from the group consisting of: N-(2,4-bis-n-propylamino)-N (6-methylamino)-[1,3,5]triazine (CLXXII), N-(2,4,6-tris-n-propylamino) [1,3,5]triazine (CLXXIII), N-(2,4,6-tris-n-propylamino)-1,3-pyrimidine (CLXXIV), N-(2,4-bis-n-propylamino)-N-(6-i-propylamino)-1,3-pyrimidine (CLXXV), a salt thereof, and any combinations thereof. 20 In one embodiment, the breathing control disorder or disease is selected from the group consisting of respiratory depression, sleep apnea, apnea of prematurity, obesity-hypoventilation syndrome, primary alveolar hypoventilation syndrome, dyspnea, hypoxia, and hypercapnia. In another embodiment, the respiratory depression is caused by an anesthetic, a sedative, an anxiolytic agent, a 25 hypnotic agent, alcohol or a narcotic. In yet another embodiment, the subject is further administered a composition comprising at least one additional compound useful for treating the breathing control disorder or disease. In yet another embodiment, the at least one additional compound is selected from the group consisting of acetazolamide, almitrine, theophylline, caffeine, methyl progesterone, a 30 serotinergic modulator, a cannabinoid and an ampakine. In yet another embodiment, the formulation is administered in conjunction with the use of a mechanical ventilation device or positive airway pressure device on the subject. In yet another embodiment, the subject is a mammal. In yet another embodiment, the mammal is a human. In yet another embodiment, the formulation is administered to the subject by -3 - WO 2013/181217 PCT/US2013/043052 an inhalational, topical, oral, buccal, rectal, vaginal, intramuscular, subcutaneous, transdermal, intrathecal, intraperitoneal, intrathoracic, intrapleural or intravenous route. The invention also includes a method of preventing destabilization or 5 stabilizing breathing rhythm in a subject in need thereof. The method comprising administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound selected from the group consisting of: N-(2,4-bis-n-propylamino)-N-(6 methylamino)-[1,3,5]triazine (CLXXII), N-(2,4,6-tris-n-propylamino)-[1,3,5]triazine 10 (CLXXIII), N-(2,4,6-tris-n-propylamino)-1,3-pyrimidine (CLXXIV), N-(2,4-bis-n propylamino)-N-(6-i-propylamino)-1,3-pyrimidine (CLXXV), a salt thereof, and any combinations thereof. In one embodiment, the destabilization is associated with a breathing control disorder or disease selected from the group consisting of respiratory 15 depression, sleep apnea, apnea of prematurity, obesity-hypoventilation syndrome, primary alveolar hypoventilation syndrome, dyspnea, hypoxia, and hypercapnia. In another embodiment, the respiratory depression is caused by an anesthetic, a sedative, an anxiolytic agent, a hypnotic agent, alcohol or a narcotic. In yet another embodiment, the subject is further administered a composition comprising at least one 20 additional compound useful for treating the breathing control disorder or disease. In yet another embodiment, the at least one additional compound is selected from the group consisting of acetazolamide, almitrine, theophylline, caffeine, methyl progesterone, a serotinergic modulator, a cannabinoid and an ampakine. In yet another embodiment, the formulation is administered in conjunction with the use of a 25 mechanical ventilation device or positive airway pressure device on the subject. In yet another embodiment, the subject is a mammal. In yet another embodiment, the mammal is a human. In yet another embodiment, the formulation is administered to the subject by an inhalational, topical, oral, buccal, rectal, vaginal, intramuscular, subcutaneous, transdermal, intrathecal, intraperitoneal, intrathoracic, intrapleural or 30 intravenous route. The invention also includes a composition comprising N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) or a salt thereof, wherein 4,6-bis-(n-propylamino)-1,3,5-triazin-2-ol (CLXXVIII) is present at -4- WO 2013/181217 PCT/US2013/043052 a level equal to or lower than about 1% (w/w) with respect to (XXXV) or the salt thereof. In one embodiment, 4,6-bis-(n-propylamino)-1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 0.5% (w/w) with respect 5 to (XXXV) or the salt thereof. In another embodiment, 4,6-bis-(n-propylamino) 1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 0.3% (w/w) with respect to (XXXV) or the salt thereof. In yet another embodiment, 4,6 bis-(n-propylamino)-1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 0.2% (w/w) with respect to (XXXV) or the salt thereof. In yet 10 another embodiment, 4,6-bis-(n-propylamino)-1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 0.15% (w/w) with respect to (XXXV) or the salt thereof. In yet another embodiment, 4,6-bis-(n-propylamino)-1,3,5-triazin 2-ol (CLXXVIII) is present at a level equal to or lower than about 0.l1% (w/w) with respect to (XXXV) or the salt thereof. In yet another embodiment, 4,6-bis-(n 15 propylamino)-1,3,5-triazin-2-ol (CLXXVIII) is essentially absent from the composition. The invention also includes a composition comprising N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) or a salt thereof, further comprising a buffer and a liquid carrier. 20 In one embodiment, the salt of (XXXV) is the hydrogen sulfate salt (XXXVI). In another embodiment, the composition further comprises citric acid. In yet another embodiment, the pH of the composition is adjusted with a base. In yet another embodiment, the base comprises sodium hydroxide. In yet another embodiment, the liquid carrier comprises water. In yet another embodiment, the pH 25 of the composition ranges from about 2.5 to about 6. In yet another embodiment, the pH of the composition ranges from about 2.5 to about 5. In yet another embodiment, the pH of the composition ranges from about 3 to about 4. In yet another embodiment, the concentration of (XXXV) or the salt thereof in the composition is about 1-10 mg/mL. In yet another embodiment, the concentration of (XXXV) or the 30 salt thereof in the composition is about 5-10 mg/mL. In yet another embodiment, the concentration of (XXXV) or the salt thereof in the composition is about 10 mg/mL. In yet another embodiment, the composition comprises less than 0.5% (w/w) (CLXXVIII) with respect to (XXXV). In yet another embodiment, less than 0.5% (w/w) of N-(4,6-Bis-n-propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) with respect to -5- WO 2013/181217 PCT/US2013/043052 (XXXV) forms over a six-month period of storage of the composition at 2-8 0 C. In yet another embodiment, wherein less than 0.5% (w/w) of N-(4,6-Bis-n propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) forms over a twelve-month period of storage of the composition at 2-8 0 C. In yet another 5 embodiment, less than 0.5% (w/w) of N-(4,6-Bis-n-propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) forms over an eighteen-month period of storage of the composition at 2-8 0 C. In yet another embodiment, less than 0.5% (w/w) of N (4,6-Bis-n-propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) forms over a twenty-four-month period of storage of the composition at 2-8 0 C. 10 The invention also includes a crystalline free base of N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV), Form A, with a XRPD spectrum as per Figure 22. The invention also includes at least one crystalline salt of N-(4,6-Bis n-propylamino-[1,3,5]triazin-2-yl)-N.0-dimethyl-hydroxylamine (XXXV) selected 15 from the group consisting of: crystalline hydrogen sulfate salt (XXXVI) Form A, with a XRPD spectrum as per Figure 14; crystalline hydrogen sulfate salt (XXXVI) comprising a mixture of Form A and Form B, with a XRPD spectrum as per Figure 23; crystalline hydrogen sulfate salt (XXXVI) Form C, with a XRPD spectrum as per Figure 24; crystalline hydrogen sulfate salt (XXXVI) Form D, with a XRPD spectrum 20 as per Figure 25; crystalline hydrogen sulfate salt (XXXVI) comprising a mixture of Form A and Form D, with a XRPD spectrum as per Figure 26; crystalline phosphoric acid salt (CLXXX) Form A, with a XRPD spectrum as per Figure 27; crystalline phosphoric acid salt (CLXXX) Form C, with a XRPD spectrum as per Figure 28; crystalline phosphoric acid salt (CLXXX) comprising a mixture of Form A and Form 25 B, with a XRPD spectrum as per Figure 29; crystalline phosphoric acid salt (CLXXX) comprising a mixture of Form C and Form D, with a XRPD spectrum as per Figure 30; crystalline phosphoric acid salt (CLXXX) comprising a mixture of Form C and Form E, with a XRPD spectrum as per Figure 31; and any mixtures thereof. The invention also includes a salt of N-(4,6-Bis-n-propylamino 30 [1,3,5]triazin-2-yl)-hydroxylamine (XXXV), comprising approximately one mole equivalent of sulfuric acid. In one embodiment, the salt further comprises one mole equivalent of water. The invention also includes a salt of N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-N.0-dimethyl-hydroxylamine (XXXV), comprising at least one -6- WO 2013/181217 PCT/US2013/043052 mole equivalent of phosphoric acid. In one embodiment, the salt comprises about one mole equivalent of phosphoric acid. The invention also includes a method of reducing or minimizing the open channel fraction of the potassium maxi-K or BK channel in a subject in need 5 thereof. The method comprises administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound of formula (I): R X-R 2 Zbl b2 Y N A-KA R 5 R3 N R5 R4 H (I), wherein:
R
1 and R 2 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted 10 cycloalkyl, alkenyl, substituted alkenyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, heteroaryl or substituted heteroaryl; or R and R2 combine as to form a biradical selected from the group consisting of 3 hydroxy-pentane-1,5-diyl, 6-hydroxy-cycloheptane-1,4-diyl, propane- 1,3-diyl, 15 butane-1,4-diyl and pentane-1,5-diyl; R 3 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -NR 1
R
2 , -C(O)OR 1 , acyl, or aryl;
R
4 is H, alkyl, or substituted alkyl; R 5 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -OR', -NR 1
R
2 , -C(O)OR 1 , acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, or substituted 20 heterocyclic; or R3 and R combine as to form a biradical selected from the group consisting of 3,6,9-trioxa-undecane- 1,11 -diyl and 3,6-dioxa-octane- 1,8-diyl; R6 is H, alkyl, substituted alkyl or alkenyl; X is a bond, 0 or NR 4 ; and, Y is N, CR 6 or C; wherein: if Y is N or CR 6 , then bond bl is nil and: (i) Z is H, bond b2 is a single bond, and A is 25 CH; or, (ii) Z is nil, bond b2 is nil, and A is a single bond; and, if Y is C, then bond bl is a single bond, and: (i) Z is CH 2 , bond b2 is a single bond, and A is CH; or, (ii) Z is CH, bond b2 is a double bond, and A is C; or a salt thereof. The invention also includes a method of inhibiting the TASK-I (KCNK3) channel in a subject in need thereof. The method comprises administering -7- WO 2013/181217 PCT/US2013/043052 to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound of formula (I): R X-R 2 N b2 Z-bY "N R3 "I N N RN I I R4 H (I), wherein: R and R2 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted 5 cycloalkyl, alkenyl, substituted alkenyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, heteroaryl or substituted heteroaryl; or R and R 2 combine as to form a biradical selected from the group consisting of 3 hydroxy-pentane-1,5-diyl, 6-hydroxy-cycloheptane-1,4-diyl, propane- 1,3-diyl, 10 butane-1,4-diyl and pentane-1,5-diyl; R 3 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -NR 1
R
2 , -C(O)OR 1 , acyl, or aryl;
R
4 is H, alkyl, or substituted alkyl; R 5 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -OR', -NR 1
R
2 , -C(O)OR 1 , acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, or substituted 15 heterocyclic; or R 3 and R 5 combine as to form a biradical selected from the group consisting of 3,6,9-trioxa-undecane- 1,11 -diyl and 3,6-dioxa-octane- 1,8-diyl; R 6 is H, alkyl, substituted alkyl or alkenyl; X is a bond, 0 or NR 4 ; and, Y is N, CR 6 or C; wherein: if Y is N or CR 6 , then bond bl is nil and: (i) Z is H, bond b 2 is a single bond, 20 and A is CH; or, (ii) Z is nil, bond b2 is nil, and A is a single bond; and, if Y is C, then bond bl is a single bond, and: (i) Z is CH 2 , bond b2 is a single bond, and A is CH; or, (ii) Z is CH, bond b 2 is a double bond, and A is C; or a salt thereof. The invention also includes a method of increasing minute ventilation 25 in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound of formula (I): -8- WO 2013/181217 PCT/US2013/043052 R X-R 2 b 2 -Y N R N N R5 R4 H (I), wherein: R and R2 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, 5 heteroarylalkyl, substituted heteroarylalkyl, heteroaryl or substituted heteroaryl; or R and R2 combine as to form a biradical selected from the group consisting of 3 hydroxy-pentane-1,5-diyl, 6-hydroxy-cycloheptane-1,4-diyl, propane- 1,3-diyl, butane-1,4-diyl and pentane-1,5-diyl; R 3 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -NRR 2 , -C(O)OR 1 , acyl, or aryl; 10 R 4 is H, alkyl, or substituted alkyl; R 5 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -OR', -NR 1
R
2 , -C(O)OR 1 , acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic; or R3 and R combine as to form a biradical selected from the group consisting of 3,6,9-trioxa-undecane- 1,11 -diyl and 3,6-dioxa-octane- 1,8-diyl; R6 is H, 15 alkyl, substituted alkyl or alkenyl; X is a bond, 0 or NR 4 ; and, Y is N, CR 6 or C; wherein: if Y is N or CR 6 , then bond bl is nil and: (i) Z is H, bond b 2 is a single bond, and A is CH; or, (ii) Z is nil, bond b2 is nil, and A is a single bond; and, if Y is C, then bond bl is a single bond, and: (i) Z is CH 2 , bond b2 is a single 20 bond, and A is CH; or, (ii) Z is CH, bond b2 is a double bond, and A is C; or a salt thereof. In one embodiment, the compound of formula (I) is selected from the group consisting of: N-(4,6-Bis-methylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, N-(4,6-Bis-ethylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl 25 hydroxylamine, N-(4-Cyclopropylmethyl)-N-(6-n-propylamino) [1,3,5]triazin-2-yl) N,O-dimethyl-hydroxylamine, N-(4-Ethylamino)-N-(6-n-propylamino)-[1,3,5]triazin 2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2-methylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2,2 dimethylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6 30 (2,2-dimethylpropylamino))[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4,6 -9- WO 2013/181217 PCT/US2013/043052 Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4 (Methoxy(methyl)amino)-6-(n-propylamino)-1,3,5-triazin-2-yl)propionamide, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-methyl-hydroxylamine, O-Allyl-N-(4,6-bis n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine, N-(4,6-Bis-n-propylamino 5 [1,3,5]triazin-2-yl)-hydroxylamine, 6-(Methoxy(methyl)amino)-N 2 -propyl-1,3,5 triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-isopropyl hydroxylamine, 6-[1,2]Oxazinan-2-yl-N,N'-dipropyl-[1,3,5]triazine-2,4-diamine, N 10 (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-N-methyl-hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-hydroxylamine, N (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-hydroxylamine, 6 ((Benzyloxy)(isopropyl) amino)-N 2
,N
4 -dipropyl-1,3,5-triazine-2,4-diamine, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-O-isopropyl-hydroxylamine, N-(4,6 15 Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl-N-methyl-hydroxylamine, 6 (Methyl(thiophen-2-ylmethoxy)amino)-N 2
,N
4 -dipropyl-1,3,5-triazine-2,4-diamine, N (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-cyclopropylmethyl-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-ethyl-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl) 20 hydroxylamine, 4-N-(2-Dimethylaminoethyl)amino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4-N-(3-(1-N-Methylimidazol-2-yl) propyl)-amino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, 4-N-(1-N-Methylimidazol-2-yl)-methylamino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-(N-(2 25 dimethylaminoethyl)amino)-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6 Bis-(N-(pyridin-4-yl-methyl)amino)-[1,3,5]triazin-2-yl)-N,0-dimethyl hydroxylamine, 4,6-Bis-[N-(3-methoxy-n-propyl)amino]-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine, 4,6-Bis-[N-(tetrahydropyran-4-yl-methyl)amino] [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(5,8,11 -Trioxa-2,14,16,18,19 30 pentaazabicyclo[13.3.1 ]nonadeca-1(18),15(19),16(17)-trien-17-yl)-N,O dimethylhydroxylamine, 2,6-Bis-(N-n-propylamino)-[1,3]pyrimidin-4-yl)-N,O dimethyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N',N' dimethylhydrazine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl-N' methylhydrazine, 2-(n-Propyl)amino-4-(i-propylamino-7-methyl-pyrrolidino[2,3 - 10 - WO 2013/181217 PCT/US2013/043052 d]pyrimidine, 2-(n-Propyl)amino-4-dimethylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine, 2-(n-Propyl)amino-4-methylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine, 2-(n-Propyl)amino-4-(i-propyl)amino-7-i-propyl-pyrrolidino[2,3 d]pyrimidine, 2,4-Bis-(n-propyl)amino-7H-pyrrolidino[2,3-d]pyrimidine, 2-(n 5 Propyl)amino-4-(4-hydroxypiperidin-1-yl)-7-methyl-pyrrolidino[2,3-d]pyrimidine, 8 (7-Methyl-2-(n-propylamino)-pyrrolidino[2,3-d]pyrimidin-4-yl)-8 azabicyclo[3.2.1 ]octan-3-ol, N-(2-Propylamino-7H-pyrrolo[2,3d]pyrimidin-4-yl) N,O-dimethyl-hydroxylamine, N-(2-(Propen-2-yl)amino-7-methyl pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-(Propen-2 10 yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine, N-(2-n Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) 15 hydrazine, N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4 yl)-hydrazine, N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine, N (2,4,6-Tris-n-propylamino)-[1,3,5]triazine, N-(2,4,6-Tris-n-propylamino)-1,3 pyrimidine, N-(2,4-Bis-n-propylamino)-N-(6-i-propylamino)-1,3-pyrimidine, a salt thereof and mixtures thereof. 20 In one embodiment, the compound of formula (I) is selected from the group consisting of: N-(4,6-Bis-n-propylamino- 1,3,5]triazin-2-yl)-O-methyl hydroxylamine; N-(4,6-Bis-n-propylamino-1,3,5]triazin-2-yl)-N',N' dimethylhydrazine; N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine; N-[(2,6-Bis-n-propylamino)-pyrimidin-4-yl]-N,O-dimethyl-hydroxylamine; N,N 25 Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine; a salt thereof and mixtures thereof. In one embodiment, the compound of formula (I) is N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, or a salt thereof. In one embodiment, the pharmaceutical formulation is administered 30 via intravenous infusion. In another embodiment, the infusion dose of the pharmaceutical formulation is at least about 0.016 mg/kg/min. In yet another embodiment, the infusion dose of the pharmaceutical formulation is about 0.016 mg/kg/min. In yet another embodiment, the infusion dose of the pharmaceutical composition produce plasma concentrations of at least about 726 ng/mL in the - 11 - WO 2013/181217 PCT/US2013/043052 subject. In yet another embodiment, the subject is a mammal. In yet another embodiment, the mammal is human. BRIEF DESCRIPTION OF THE DRAWINGS 5 For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings. Figure 1 is a graph illustrating results of plethysmography experiments 10 which monitored minute ventilation in the opioid-treated rat upon administration of Compound (XXXVI) [labeled as cmpd (A)]. Figure 2, comprising Figures 2A-B, illustrates arterial blood gas analysis results for administration of Compound (XXXVI) [labeled as cmpd (A)] in the opioid-treated rat. Figure 2A - PaCO 2 (mmHg); Figure 2B - SaO 2 (%). 15 Figure 3 is a graph illustrating results of plethysmography experiments monitoring minute ventilation in the rat upon administration of Compound (XXXVI) [labeled as cmpd (A)] under conditions of hypoxia. Figure 4 is a graph illustrating end-tidal CO 2 and minute ventilation in the opioid-treated monkey upon administration of Compound (XXXVI) [labeled as 20 cmpd (A)]. Figure 5 is a graph illustrating the dose-dependent effect of Compound (XXXVI) [labeled as cmpd (A)] and Compound (L) [labeled as cmpd (B)] on minute ventilation, in terms of maximum peak response, in the rat. Figure 6 is a graph illustrating the dose-dependent effect of Compound 25 (XXXVI) [labeled as cmpd (A)] and Compound (CXXI) [labeled as cmpd (C)] on minute ventilation, in terms of maximum peak response, in the rat. Figure 7, comprising Figures 7A-D, illustrates the dose-dependent effect of Compound (L) [labeled as cmpd (B)] on blood gases and pH in the opioid treated rat. Figure 7A - pH; Figure 7B - SaO 2 ; Figure 7C - pO2; Figure 7D 30 pCO 2 . Figure 8, comprising Figures 8A-D, illustrates the dose-dependent effect of Compound (CXLII) [labeled as cmpd (D)] on blood gases and pH in the opioid-treated rat. Figure 8A - PO2; Figure 8B - SaO 2 ; Figure 8C - pCO 2 ; Figure 8D - pH. - 12 - WO 2013/181217 PCT/US2013/043052 Figure 9 is a graph illustrating the effect of Compound (CXLII) [labeled as cmpd (D)] in the minute ventilation of the opioid-treated rat. Figure 10 illustrates the 1H -NMR spectrum for Compound (XXXVI) in DMSO-d 6 at 25'C. 5 Figure 11 illustrates the 13 C-NMR spectrum for Compound (XXXVI) in DMSO-d 6 at 25'C. Figure 12 illustrates the FTIR spectrum for Compound (XXXVI). Figure 13 illustrates the thermal analysis by DSC of N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate 10 (XXXVI). Figure 14 illustrates the X-ray diffraction spectrum of Compound (XXXVI) Form A (hydrogen sulfate salt Form A). Figure 15 is a graph illustrating the effect of Compound (XXXVI) on reversing the effects of midazolam on minute ventilation (MV) in the rat. 15 Figure 16 is a graph illustrating the effect of Compound (XXXVI) on reversing the effects of midazolam on tidal volume (TV) in the rat. Figure 17 is a graph illustrating the effect of Compound (XXXVI) on reversing the effects of midazolam on respiratory frequency (f) in the rat. Figure 18 is a graph illustrating the effect of Compound (XXXVI) 20 infusion on the minute volume response to acute hypercapnia (3% C0 2 ). Figure 19A is a graph illustrating the effect of pH on aqueous solubility of Compound (XXXV) at ambient temperature. Figure 19B is a graph illustrating the effect of pH on aqueous solubility of Compound (XXXV) at ambient temperature 25 Figure 20A is a graph illustrating the effect of pH on degradation of Compound (XXXV) at pH 2-6. Figure 20B is a graph illustrating the effect of pH on degradation of Compound (XXXV) at pH 2-3.6. Figure 21 is a graph illustrating the formation rate of Compound (CLXXVIII) from Compound (XXXV) in solution at 2-8'C, pH 3-4. 30 Figure 22 is a graph illustrating the X-ray powder diffraction pattern of the free base of Compound (XXXV) Figure 23 is a graph illustrating the X-ray powder diffraction spectrum of a mixture of Compound (XXXVI) Form A and Compound (XXXVI) Form B. - 13 - WO 2013/181217 PCT/US2013/043052 Figure 24 is a graph illustrating the X-ray powder diffraction spectrum of Compound (XXXVI) Form C. Figure 25 is a graph illustrating the X-ray powder diffraction spectrum of Compound (XXXVI) Form D. 5 Figure 26 is a graph illustrating the X-ray powder diffraction spectrum of a mixture of Compound (XXXVI) Form A and Compound (XXXVI) Form D. Figure 27 is a graph illustrating the X-ray powder diffraction spectrum of Compound (CLXXX) Form A. Figure 28 is a graph illustrating the X-ray powder diffraction spectrum 10 of Compound (CLXXX) Form C. Figure 29 is a graph illustrating the X-ray powder diffraction spectrum of a mixture of Compound (CLXXX) Form A and Compound (CLXXX) Form B. Figure 30 is a graph illustrating the X-ray powder diffraction spectrum of a mixture of Compound (CLXXX) Form C and Compound (CLXXX) Form D. 15 Figure 31 is a graph illustrating the X-ray powder diffraction spectrum of a mixture of Compound (CLXXX) Form C and Compound (CLXXX) Form E. Figure 32, comprising Figures 32A-D, are graphs illustrating the effect of Compound (XLII) on respiratory rate (Figure 32A), tidal volume (Figure 32B), minute volume (Figure 32C) and 5 min average change in minute volume (Figure 20 32D) respectively, in rat. Figure 33, comprising Figures 33A-D, are graphs illustrating the effect of Compound (CLXXII) on respiratory rate (Figure 33A), tidal volume (Figure 33B), minute volume (Figure 33C) and 5 min average change in minute volume (Figure 33D) respectively, in rat. 25 Figure 34, comprising Figures 34A-C, illustrates the effect of BK channel activity in response to Compound (XXXV) at a concentration of 1 pM. Figure 34A: sample recordings of channel activity in control conditions and with 1 pM (XXXV). Arrow, closed state. Figure 34B: all points histogram obtained from 1 min of recording in control condition. Figure 34C: all points histogram obtained 30 from 1 min of ecording with 1 pM (XXXV). Patch ID: C 05 SP 101008_0000. Figure 35 illustrates the design of study GAL-021-101: infusion rates and duration by study period and group (cohort). The dose levels that were pooled for PD analyses are delineated on the left side. - 14 - WO 2013/181217 PCT/US2013/043052 Figure 36 is an illustrative design diagram of respiratory depression Proof-of-Concept study (GAL-021-104). Figure 37 is a graph illustrating tidal volume during first 2 hour after starting the infusion. 5 Figure 38, comprising Figures 38A-D, is a set of graphs illustrating the integrated percent change from baseline during the first hour (0-lh) and first 2 hours (0-2h) after starting the infusion for minute ventilation (Figure 38A), end tidal CO 2 (Figure 38B), respiratory rate (Figure 38C), and tidal volume (Figure 38D) in the initial clinical study (GAL-021-101). Data from the first hour of period 9 (0.96 10 mg/kg/h) was pooled with data from similar infusion rate subjects in groups 6 and 7 for the 0-lh assessments. Figure 39, comprising Figures 39A-D, is a set of graphs illustrating integrated percent change from baseline during the first hour (0-1h) and during first 2 hours (0-2h) after starting the infusion for minute ventilation (Figure 39A), end tidal 15 CO 2 (Figure 39B), respiratory rate (Figure 39C), and tidal volume (Figure 39D) in the second clinical study (GAL-021-102). Figure 40 is a graph illustrating transcutaneous hemoglobin oxygen saturation (GAL-021-102). Figure 41, comprising Figures 4 1A-C, illustrates results for Part I of 20 the GAL-021-104 clinical study: integrated percent change from baseline during the last ten minutes of each segment for minute ventilation (Figure 41A), tidal volume (Figure 41B) and respiratory rate (Figure 41C). Segments were thirty minutes in durantion, except for the segment labeled (XXXVI)-high, which had duration of 40 minutes due to a twenty-minute loading dose infusion. The end-tidal was clamped 25 during the study and was largely invariant. Figure 42 is a graph illustrating Part 2 of the GAL-021-104 study: Percent change in current at onset of the pain from a TENS device placed over anterior tibial area (n=8). Figure 43, comprising Figures 43A-D, illustrates results for Part 2 of 30 the GAL-021-104 study: Integrated percent change from baseline during the last ten minutes of each segment for minute ventilation (Figure 43A), end-tidal CO 2 (Figure 43B), respiratory rate (Figure 43C), and tidal volume (Figure 43D). Figure 44, comprising Figures 44A-B, illustrate mean plasma concentration-time profiles of N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O - 15 - WO 2013/181217 PCT/US2013/043052 dimethyl-hydroxylamine hydrogen sulfate (XXXVI) following IV infusion to healthy volunteers during the First-in-Human study (Figure 44A) and the second study (Figure 44B). 5 DETAILED DESCRIPTION OF THE INVENTION The present invention relates in one aspect to the unexpected discovery that the compounds of the invention are respiratory stimulants and useful in the treatment of breathing control disorders or diseases. 10 Definitions As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one skilled in the art to 15 which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, separation science and organic chemistry are those known and commonly employed in the art. As used herein, the articles "a" and "an" refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, 20 "an element" means one element or more than one element. As used herein, the term "about" is understood by persons of ordinary skill in the art and varies to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term "about" is meant to encompass variations of ±20% or ±10%, 25 more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. As used herein, a "subject" may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, 30 porcine, canine, feline and murine mammals. Preferably, the subject is human. In a non-limiting embodiment, the following terminology used to report blood gas measurements is well known to those skilled in the art and may be defined as such: minute ventilation (MV) is a measure of breathing volume per unit time and is given herein as mL/min; pCO 2 is partial pressure of carbon dioxide (gas) - 16 - WO 2013/181217 PCT/US2013/043052 in (arterial) blood measured in mm Hg (millimeters of Hg); P 0 2 is partial pressure of oxygen (gas) in (arterial) blood measured in mmHg (millimeters of Hg); SaO 2 is the percentage of hemoglobin in the form of oxy-hemoglobin in blood; end-tidal CO 2 is the measurement of exhaled carbon dioxide gas as detected using colorimetry, or 5 capnometry. As used herein, the term ED 50 refers to the effective dose of a formulation that produces a given effect in 50 % of the subjects that are administered that formulation. As used herein, a "disease" is a state of health of an animal wherein the 10 animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. As used herein, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a 15 disorder does not necessarily cause a further decrease in the animal's state of health. As used herein, an "effective amount", "therapeutically effective amount" or pharmaceuticallyy effective amount" of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered. The term to "treat," as used herein, means reducing the 20 frequency with which symptoms are experienced by a patient or subject or administering an agent or compound to reduce the severity with which symptoms are experienced. As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity 25 or properties of the compound useful within the invention, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the term "salt" refers to an addition salt of free acid or 30 base that is useful within the methods of the invention. The term "salt" also refers to a mixture of a salt with its corresponding free acid or base, either in isolated form (e.g., as a solid) or in solution. As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compound prepared from pharmaceutically acceptable - 17 - WO 2013/181217 PCT/US2013/043052 non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof. As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound useful within the invention 5 with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject. As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening 10 agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other 15 ingredients of the formulation, including the compound useful within the invention, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; 20 powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and 25 aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that 30 are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical - 18- WO 2013/181217 PCT/US2013/043052 compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference. As used herein, the phrase "process-related impurities" refers to 5 impurities formed during the process of making the active pharmaceutical ingredient, as caused by exposure of any starting material, intermediate, reactant, solvent, and the active pharmaceutical ingredient itself to the conditions of the synthetic process. As used herein, "treating a disease or disorder" means reducing the frequency with which a symptom of the disease or disorder is experienced by a 10 subject. Disease and disorder are used interchangeably herein. By the term "specifically bind" or "specifically binds," as used herein, is meant that a first molecule preferentially binds to a second molecule (e.g., a particular receptor or enzyme), but does not necessarily bind only to that second molecule. 15 As used herein, the term "alkyl," by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e. C 1
-C
10 means one to ten carbon atoms) and includes straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, 20 hexyl, and cyclopropylmethyl. Most preferred is (CI-C 6 )alkyl, such as, but not limited to, ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl. As used herein, the term "cycloalkyl," by itself or as part of another substituent means, unless otherwise stated, a cyclic chain hydrocarbon having the 25 number of carbon atoms designated (i.e. C 3
-C
6 means a cyclic group comprising a ring group consisting of three to six carbon atoms) and includes straight, branched chain or cyclic substituent groups. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Most preferred is
(C
3
-C
6 )cycloalkyl, such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl 30 and cyclohexyl. As used herein, the term "alkenyl," employed alone or in combination with other terms, means, unless otherwise stated, a stable mono-unsaturated or di-unsaturated straight chain or branched chain hydrocarbon group having the stated number of carbon atoms. Examples include vinyl, propenyl (or allyl), crotyl, - 19 - WO 2013/181217 PCT/US2013/043052 isopentenyl, butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, and the higher homologs and isomers. A functional group representing an alkene is exemplified by -CH 2 CH=CH 2 . As used herein, the term "alkynyl," employed alone or in combination 5 with other terms, means, unless otherwise stated, a stable straight chain or branched chain hydrocarbon group with a triple carbon-carbon bond, having the stated number of carbon atoms. Examples include ethynyl and propynyl, and the higher homologs and isomers. As used herein, the term "substituted alkyl," "substituted cycloalkyl," 10 "substituted alkenyl" or "substituted alkynyl" means alkyl, cycloalkyl, alkenyl or alkynyl, as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, tetrahydro-2-H-pyranyl, -NH 2 , N(CH 3
)
2 , (1-methyl-imidazol-2-yl), pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, C(=O)OH, trifluoromethyl, -C-N, -C(=O)O(C 1
-C
4 )alkyl, -C(=O)NH 2 , -C(=O)NH(C 1 15 C 4 )alkyl, -C(=O)N((CI-C 4 )alkyl) 2 , -SO 2
NH
2 , -C(=NH)NH 2 , and -NO 2 , preferably containing one or two substituents selected from halogen, -OH, alkoxy, -NH 2 , trifluoromethyl, -N(CH 3
)
2 , and -C(=O)OH, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl. 20 As used herein, the term "alkoxy" employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. Preferred are (CI-C 3 )alkoxy, 25 such as, but not limited to, ethoxy and methoxy. As used herein, the term "halo" or "halogen" alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine. 30 As used herein, the term "heteroalkyl" by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of 0, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally - 20 - WO 2013/181217 PCT/US2013/043052 quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -O-CH 2
-CH
2
-CH
3 , -CH 2
-CH
2
-CH
2 -OH, -CH 2
-CH
2
-NH
5 CH 3 , -CH 2
-S-CH
2
-CH
3 , and -CH 2
CH
2
-S(=O)-CH
3 . Up to two heteroatoms may be consecutive, such as, for example, -CH 2
-NH-OCH
3 , or -CH 2
-CH
2
-S-S-CH
3 As used herein, the term "heteroalkenyl" by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain monounsaturated or di-unsaturated hydrocarbon group consisting of the stated number 10 of carbon atoms and one or two heteroatoms selected from the group consisting of 0, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Up to two heteroatoms may be placed consecutively. Examples include -CH=CH-0-CH 3 , -CH=CH-CH 2 -OH,
-CH
2
-CH=N-OCH
3 , -CH=CH-N(CH 3
)-CH
3 , and -CH 2
-CH=CH-CH
2 -SH. 15 As used herein, the term "aromatic" refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n+2) delocalized iT (pi) electrons, where n is an integer. As used herein, the term "aryl," employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system 20 containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl, anthracyl, and naphthyl. Preferred are phenyl and naphthyl, most preferred is phenyl. As used herein, the term "aryl-(C1-C 3 )alkyl" means a functional group 25 wherein a one to three carbon alkylene chain is attached to an aryl group, e.g., -CH 2
CH
2 -phenyl or -CH 2 -phenyl (benzyl). Preferred is aryl-CH 2 - and aryl
CH(CH
3 )-. The term "substituted aryl-(CI-C 3 )alkyl" means an aryl-(CI-C 3 )alkyl functional group in which the aryl group is substituted. Preferred is substituted aryl(CH 2 )-. Similarly, the term "heteroaryl-(CI-C 3 )alkyl" means a functional group 30 wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e.g., -CH 2
CH
2 -pyridyl. Preferred is heteroaryl-(CH 2 )-. The term "substituted heteroaryl-(CI-C 3 )alkyl" means a heteroaryl-(CI-C 3 )alkyl functional group in which the heteroaryl group is substituted. Preferred is substituted heteroaryl-(CH 2 )-. -21- WO 2013/181217 PCT/US2013/043052 As used herein, the term "heterocycle" or "heterocyclyl" or "heterocyclic" by itself or as part of another substituent means, unless otherwise stated, an unsubstituted or substituted, stable, mono- or multi-cyclic heterocyclic ring system that consists of carbon atoms and at least one heteroatom selected from the 5 group consisting of N, 0, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or non aromatic in nature. In one embodiment, the heterocycle is a heteroaryl. 10 As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. A polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include tetrahydroquinoline and 2,3-dihydrobenzofuryl. Examples of non-aromatic heterocycles include monocyclic groups 15 such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4 dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 20 1,3-dioxepane, 4,7-dihydro-1,3-dioxepin and hexamethyleneoxide. Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (such as, but not limited to, 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 25 1,3,4-oxadiazolyl. Examples of polycyclic heterocycles include indolyl (such as, but not limited to, 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (such as, but not limited to, 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (such as, but not limited to, 30 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (such as, but not limited to, 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (such as, but not limited to, 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (such as, but not limited to, - 22 - WO 2013/181217 PCT/US2013/043052 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl. The aforementioned listing of heterocyclyl and heteroaryl moieties is intended to be representative and not limiting. 5 As used herein, the term "substituted" means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. For aryl, aryl-(CI-C 3 )alkyl and heterocyclyl groups, the term "substituted" as applied to the rings of these groups refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is 10 permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In one embodiment, the substituents vary in number between one and four. In another embodiment, the substituents vary in number between one and three. In yet another embodiment, the substituents vary in number between one and two. In yet another embodiment, the substituents are 15 independently selected from the group consisting of C1- 6 alkyl, -OH, C1_ 6 alkoxy, halo, amino, acetamido and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic, with straight being preferred. As used herein, the term "AcOH" refers to acetic acid; the term 20 "nBuOH" refers to n-butanol; the term "CH 2 Cl 2 " refers to dichloromethane (also known as methylene dichloride); the term "DMSO" refers to dimethylsulfoxide; the term "EtOAc" refers to ethyl acetate; the term "EtOH" refers to ethanol; the term "HCl" refers to hydrochloric acid or a hydrochloride salt; the term "HPLC" refers to high pressure liquid chromatography; the term "H 2
SO
4 " refers to sulfuric acid; the 25 term "LCMS" refers to liquid chromatography-mass spectrometry; the term "MS" refers to mass spectrometry; the term "MeOH" refers to methanol; the term "NaCl" refers to sodium chloride; the term "NaHCO 3 " refers to sodium bicarbonate; the term "NaOH" refers to sodium hydroxide; the term "Na 2
SO
4 " refers to sodium sulfate; the term "mpk" refers to mg/kg; the term "NMR" refers to nuclear magnetic resonance; 30 the term "PE" or "pet ether" refers to petroleum ether; the term "POCl 3 " refers to phosphorous oxychloride; the term "ppm" refers to part per million; the term "xphos" refers to 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl; the term "dba" refers to trans, trans-dibenzylideneacetone. -23 - WO 2013/181217 PCT/US2013/043052 "Instructional material," as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the composition and/or compound of the invention in a kit. The instructional material of the kit may, for example, be affixed to 5 a container that contains the compound and/or composition of the invention or be shipped together with a container that contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical 10 delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website. 15 Compounds and Compositions of the Invention The invention includes compounds of the invention, as well as any compositions comprising such compounds. The invention includes at least one compound selected from the group consisting of: N-(2,4-bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine 20 (CLXXII), N-(2,4,6-tris-n-propylamino)-[1,3,5]triazine (CLXXIII), N-(2,4,6-tris-n propylamino)-1,3-pyrimidine (CLXXIV), N-(2,4-bis-n-propylamino)-N-(6-i propylamino)-1,3-pyrimidine (CLXXV), a salt thereof, and any combinations thereof. In one embodiment, the salt is hydrogen sulfate, hydrochloride, phosphate, hydrogen phosphate or dihydrogen phosphate. In another embodiment, 25 the at least one compound is formulated with at least one pharmaceutically acceptable carrier. The invention also includes a composition comprising N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) or a salt thereof, wherein 4,6-bis-(n-propylamino)-1,3,5-triazin-2-ol is present at a level equal 30 to or lower than about 1% (w/w) with respect to (XXXV) or the salt thereof. In one embodiment, 4,6-bis-(n-propylamino)-1,3,5-triazin-2-ol is present at a level equal to or lower than about 0.5% (w/w) with respect to (XXXV) or the salt thereof. In another embodiment, 4,6-bis-(n-propylamino)-1,3,5-triazin-2-ol is present at a level equal to or lower than about 0.3% (w/w) with respect to (XXXV) or - 24 - WO 2013/181217 PCT/US2013/043052 the salt thereof. In yet another embodiment, 4,6-bis-(n-propylamino)-1,3,5-triazin-2 ol is present at a level equal to or lower than about 0.2% (w/w) with respect to (XXXV) or the salt thereof. In yet another embodiment, 4,6-bis-(n-propylamino) 1,3,5-triazin-2-ol is present at a level equal to or lower than about 0.15% (w/w) with 5 respect to (XXXV) or the salt thereof. In yet another embodiment, 4,6-bis-(n propylamino)-1,3,5-triazin-2-ol is present at a level equal to or lower than about 0. 1% (w/w) with respect to (XXXV) or the salt thereof. In yet another embodiment, 4,6 bis-(n-propylamino)-1,3,5-triazin-2-ol is essentially absent from the composition. The invention also includes a composition comprising N-(4,6-bis-n 10 propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) or a salt thereof, further comprising a buffer and a liquid carrier. In one embodiment, the salt of (XXXV) is the hydrogen sulfate salt (XXXVI). In another embodiment, the composition further comprises citric acid. In yet another embodiment, the pH of the composition is adjusted with a base. In yet 15 another embodiment, the base comprises sodium hydroxide. In yet another embodiment, the liquid carrier comprises water. In yet another embodiment, the pH of the composition ranges from about 2.5 to about 6. In yet another embodiment, the pH of the composition ranges from about 2.5 to about 5. In yet another embodiment, the pH of the composition ranges from about 3 to about 4. In yet another 20 embodiment, the concentration of (XXXV) or the salt thereof in the composition is about 1-10 mg/mL. In yet another embodiment, the concentration of (XXXV) or the salt thereof in the composition is about 5-10 mg/mL. In yet another embodiment, the concentration of (XXXV) or the salt thereof in the composition is about 10 mg/mL. In yet another embodiment, the composition comprises less than about 0.5% (w/w) 25 4,6-bis-(n-propylamino)-1,3,5-triazin-2-ol (CLXXVIII) with respect to (XXXV). In yet another embodiment, less than about 0.5% (w/w) of N-(4,6-Bis-n-propylamino) [1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) forms over at least a 6 month period of storage of the composition at 2-8'C. In yet another embodiment, less than about 0.5% (w/w) of N-(4,6-Bis-n-propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) 30 with respect to (XXXV) forms over at least a 12-month period of storage of the composition at 2-8'C. In yet another embodiment, less than about 0.5% (w/w) of N (4,6-Bis-n-propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) forms over at least an 18-month period of storage of the composition at 2-8'C. In yet another embodiment, less than about 0.5% (w/w) of N-(4,6-Bis-n-propylamino) - 25 - WO 2013/181217 PCT/US2013/043052 [1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) forms over at least a 24 month period of storage of the composition at 2-8'C. The invention also includes a crystalline free base of N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV), Form A, 5 with a XRPD spectrum as per Figure 22. The invention also includes at least one crystalline salt of N-(4,6-Bis n-propylamino-[1,3,5]triazin-2-yl)-N.0-dimethyl-hydroxylamine (XXXV) selected from the group consisting of: crystalline hydrogen sulfate salt (XXXVI) Form A, with a XRPD spectrum as per Figure 14; crystalline hydrogen sulfate salt (XXXVI) 10 comprising a mixture of Form A and Form B, with a XRPD spectrum as per Figure 23; crystalline hydrogen sulfate salt (XXXVI) Form C, with a XRPD spectrum as per Figure 24; crystalline hydrogen sulfate salt (XXXVI) Form D, with a XRPD spectrum as per Figure 25; crystalline hydrogen sulfate salt (XXXVI) comprising a mixture of Form A and Form D, with a XRPD spectrum as per Figure 26; crystalline phosphoric 15 acid salt (CLXXX) Form A, with a XRPD spectrum as per Figure 27; crystalline phosphoric acid salt (CLXXX) Form C, with a XRPD spectrum as per Figure 28; crystalline phosphoric acid salt (CLXXX) comprising a mixture of Form A and Form B, with a XRPD spectrum as per Figure 29; crystalline phosphoric acid salt (CLXXX) comprising a mixture of Form C and Form D, with a XRPD spectrum as per Figure 20 30; crystalline phosphoric acid salt (CLXXX) comprising a mixture of Form C and Form E, with a XRPD spectrum as per Figure 31; and any mixtures thereof. The invention also includes a salt of N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-hydroxylamine (XXXV), comprising approximately one mole equivalent of sulfuric acid. In one embodiment, the salt further comprises one mole 25 equivalent of water. The invention also includes a salt of N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-N.0-dimethyl-hydroxylamine (XXXV), comprising at least one mole equivalent of phosphoric acid. In one embodiment, the salt comprises about one mole equivalent of phosphoric acid. 30 The invention also includes a compound of formula (I) or a salt thereof: -26- WO 2013/181217 PCT/US2013/043052
R
1
X-R
2 N b 2 ZbY N N N R5 I I R4 H (I), wherein R and R2 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, 5 heteroarylalkyl, substituted heteroarylalkyl, heteroaryl or substituted heteroaryl; or R and R2 combine as to form a biradical selected from the group consisting of 3 hydroxy-pentane-1,5-diyl, 6-hydroxy-cycloheptane-1,4-diyl, propane- 1,3-diyl, butane-1,4-diyl and pentane-1,5-diyl; R 3 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -NRR 2 , -C(O)OR 1 , acyl, or aryl; 10 R 4 is H, alkyl, or substituted alkyl; R 5 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -OR', -NR 1
R
2 , -C(O)OR 1 , acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic; or R3 and R combine as to form a biradical selected from the group consisting of 3,6,9-trioxa-undecane- 1,11 -diyl and 3,6-dioxa-octane- 1,8-diyl; R6 is H, 15 alkyl, substituted alkyl or alkenyl; X is a bond, 0 or NR 4 ; and, Y is N, CR 6 or C; wherein: if Y is N or CR 6 , then bond bl is nil and: (i) Z is H, bond b 2 is a single bond, and A is CH; or, (ii) Z is nil, bond b2 is nil, and A is a single bond; and, if Y is C, then bond bl is a single bond, and: (i) Z is CH 2 , bond b2 is a single 20 bond, and A is CH; or, (ii) Z is CH, bond b2 is a double bond, and A is C. In one embodiment, R 3 is H, alkyl, substituted alkyl, alkenyl, cycloalkyl, substituted cycloalkyl, or substituted alkenyl. In another embodiment, R 5 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, acyl, cycloalkyl or substituted cycloalkyl. 25 In one embodiment, Y is N, bond bl is nil, Z is H, bond b 2 is a single bond, A is CH, and the compound of the invention is a 1,3,5-triazine of formula (II-a) or a salt thereof: - 27 - WO 2013/181217 PCT/US2013/043052 R N X-R 2 N N R3 N N N R5 R4 H (II-a). In one embodiment, Y is N, bond bl is nil, Z is nil, bond b 2 is nil, and A is a bond, and the compound of the invention is a 1,3,5-triazine of formula (II-b) or a salt thereof: R N X-R 2 N N N N N' R ' I 5 R H (IIb). In one embodiment, Y is CR 6 , bond b 1 is nil, Z is H, bond b2 is a single bond, A is CH, and the compound of the invention is a pyrimidine of formula (Ill-a) or a salt thereof:
R
1 N X-R 2 R 6 NN
R
3 N NAN-R5 I I R4 H (III-a). 10 In one embodiment, Y is CR 6 , bond b 1 is nil, Z is nil, bond b 2 is nil, and A is a bond, and the compound of the invention is a pyrimidine of formula (III-b) or a salt thereof: R N X-R 2 N RN N N'R5 In one embodiment, Y is C, bond b is a single bond, Z is CH 2 , bond b2 15 is a single bond, A is CH, and the compound of the invention is a pyrrolidinopyrimidine of formula (IV) or a salt thereof: -28- WO 2013/181217 PCT/US2013/043052 R N X-R 2
R
3 N N-NR5 R4 H (IV). In one embodiment, Y is C, bond b is a single bond, Z is CH, bond b2 is a double bond, A is C, and the compound of the invention is a pyrrolopyrimidine of formula (V) or a salt thereof: R N X-R 2 R3[N N1 NR 5 RH (V). In one embodiment, the compound of formula (I) is selected from the group consisting of: N-(4,6-Bis-methylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, N-(4,6-Bis-ethylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, N-(4-Cyclopropylmethyl)-N-(6-n-propylamino) [1,3,5]triazin-2-yl) 10 N,O-dimethyl-hydroxylamine, N-(4-Ethylamino)-N-(6-n-propylamino)-[1,3,5]triazin 2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2-methylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2,2 dimethylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6 (2,2-dimethylpropylamino))[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4,6 15 Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4 (Methoxy(methyl)amino)-6-(n-propylamino)-1,3,5-triazin-2-yl)propionamide, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-methyl-hydroxylamine, O-Allyl-N-(4,6-bis n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine, N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-hydroxylamine, 6-(Methoxy(methyl)amino)-N 2 -propyl-1,3,5 20 triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-isopropyl hydroxylamine, 6-[1,2]Oxazinan-2-yl-N,N'-dipropyl-[1,3,5]triazine-2,4-diamine, N (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-N-methyl-hydroxylamine, 25 O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-hydroxylamine, N (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-hydroxylamine, 6 -29- WO 2013/181217 PCT/US2013/043052 ((Benzyloxy)(isopropyl) amino)-N 2
,N
4 -dipropyl-1,3,5-triazine-2,4-diamine, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-O-isopropyl-hydroxylamine, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl-N-methyl-hydroxylamine, 6 (Methyl(thiophen-2-ylmethoxy)amino)-N 2
,N
4 -dipropyl-1,3,5-triazine-2,4-diamine, N 5 (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-cyclopropylmethyl-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-ethyl-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl) hydroxylamine, 4-N-(2-Dimethylaminoethyl)amino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4-N-(3-(1-N-Methylimidazol-2-yl) 10 propyl)-amino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, 4-N-(1-N-Methylimidazol-2-yl)-methylamino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-(N-(2 dimethylaminoethyl)amino)-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6 Bis-(N-(pyridin-4-yl-methyl)amino)-[1,3,5]triazin-2-yl)-N,O-dimethyl 15 hydroxylamine, 4,6-Bis-[N-(3-methoxy-n-propyl)amino]-[1,3,5]triazin-2-yl)-N,0 dimethyl-hydroxylamine, 4,6-Bis-[N-(tetrahydropyran-4-yl-methyl)amino] [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(5,8,11 -Trioxa-2,14,16,18,19 pentaazabicyclo[13.3.1 ]nonadeca-1(18),15(19),16(17)-trien-17-yl)-N,O dimethylhydroxylamine, 2,6-Bis-(N-n-propylamino)-[1,3]pyrimidin-4-yl)-N,O 20 dimethyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N',N' dimethylhydrazine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl-N' methylhydrazine, 2-(n-Propyl)amino-4-(i-propylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine, 2-(n-Propyl)amino-4-dimethylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine, 2-(n-Propyl)amino-4-methylamino-7-methyl-pyrrolidino[2,3 25 d]pyrimidine, 2-(n-Propyl)amino-4-(i-propyl)amino-7-i-propyl-pyrrolidino[2,3 d]pyrimidine, 2,4-Bis-(n-propyl)amino-7H-pyrrolidino[2,3-d]pyrimidine, 2-(n Propyl)amino-4-(4-hydroxypiperidin-1-yl)-7-methyl-pyrrolidino[2,3-d]pyrimidine, 8 (7-Methyl-2-(n-propylamino)-pyrrolidino[2,3-d]pyrimidin-4-yl)-8 azabicyclo[3.2.1 ]octan-3-ol, N-(2-Propylamino-7H-pyrrolo[2,3d]pyrimidin-4-yl) 30 N,O-dimethyl-hydroxylamine, N-(2-(Propen-2-yl)amino-7-methyl pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-(Propen-2 yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine, N-(2-n Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl - 30 - WO 2013/181217 PCT/US2013/043052 hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4 yl)-hydrazine, N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine, N 5 (2,4,6-Tris-n-propylamino)-[1,3,5]triazine, N-(2,4,6-Tris-n-propylamino)-1,3 pyrimidine, N-(2,4-Bis-n-propylamino)-N-(6-i-propylamino)-1,3-pyrimidine, a salt thereof and mixtures thereof. In one embodiment, the at least one compound is 2,6-bis-(N-n propylamino)-[1,3]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine or a salt thereof. In 10 another embodiment, the salt is hydrogen sulfate, hydrochloride, phosphate, hydrogen phosphate or dihydrogen phosphate. In one embodiment, the at least one compound is selected from the group consisting of: 2-(n-Propyl)amino-4-(i-propylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine (CXXVI), 2-(n-Propyl)amino-4-dimethylamino-7-methyl 15 pyrrolidino[2,3-d]pyrimidine (CXXVIII), 2-(n-Propyl)amino-4-methylamino-7 methyl-pyrrolidino[2,3-d]pyrimidine (CXXXI), 2-(n-Propyl)amino-4-(i propyl)amino-7-i-propyl-pyrrolidino[2,3-d]pyrimidine (CXXXVI), 2,4-Bis-(n propyl)amino-7H-pyrrolidino[2,3-d]pyrimidine (CXLIX), 2-(n-Propyl)amino-4-(4 hydroxypiperidin-1-yl)-7-methyl-pyrrolidino[2,3-d]pyrimidine (CLII), 8-(7-Methyl-2 20 (propylamino)-pyrrolidino[2,3-d]pyrimidin-4-yl)-8-azabicyclo[3.2.11 octan-3-ol (CLV), a salt thereof and mixtures thereof. In another embodiment, the salt is hydrogen sulfate, hydrochloride, phosphate, hydrogen phosphate or dihydrogen phosphate. In one embodiment, the at least one compound is selected from the 25 group consisting of: N-(2-Propylamino-7H-pyrrolo[2,3d]pyrimidin-4-yl)-O,N dimethyl-hydroxylamine (CXLI), N-(2-(Propen-2-yl)amino-7-methyl pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine (CLVIII), N-(2-(Propen 2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine (CLX), N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O,N-dimethyl 30 hydroxylamine (CLXII), N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) 0-methyl-hydroxylamine (CLXIV), N-(2-n-Propylamino-7-methyl pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (CLXVI), N-Methyl-N-(2-n-propylamino-7 methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (CLXVIII), N,N-dimethyl-N'-(2-n propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (CLXX), a salt thereof -31- WO 2013/181217 PCT/US2013/043052 and mixtures thereof. In another embodiment, the salt is hydrogen sulfate, hydrochloride, phosphate, hydrogen phosphate or dihydrogen phosphate. In one embodiment, the compound is formulated with at least one pharmaceutically acceptable carrier. 5 Preparation of the Compounds of the Invention The compounds of the invention may be prepared according to the general methodology illustrated in the synthetic schemes described below. The reagents and conditions described herein may be modified to allow the preparation of 10 the compounds of the invention, and such modifications are known to those skilled in the art. The scheme included herein are intended to illustrate but not limit the chemistry and methodologies that one skilled in the art may use to make compounds of the invention. In one aspect, compounds of formula (I) may be prepared by the 15 successive additions of (i) primary amines, (ii) a N-alkoxy-N-alkylamine or (iii) an appropriately substituted hydrazine (H 2
N-NHR
2 or R 1
HN-NHR
2 ) to suitably chlorinated intermediate (VI), as illustrated below in Scheme 1. CI
R
1 N X-R 2 Z )- ~H 2
N-R
5 H Z ---- Y ' N
R
1 -N-X-R2 Z----Y N R3-- NNCI R3- N N N'R I' R R 41 R4 H (VI) (I) Scheme 1 20 In another aspect, a compound of formula (IV) or (V) may be prepared by reductive alkylation of a suitably chlorinated amino-pyrrolidino-pyrimidine or amino-pyrrolo-pyrimidine, respectively (Scheme 2). - 32 - WO 2013/181217 PCT/US2013/043052 CI O R N X-R 2 N H R H
R
3 N N NH 2 R-N-X-R2' R 3 N 4 R4 (IV) CI O R N X-R 2 H R 5 H_ N I . R 1
-N-X-R
2 R
R
3 N N NH 2
R
3 N N N 4 4 R(V) Scheme 2 In yet another aspect, a triazine compound of formula (II) may be 5 prepared by the successive additions of primary amines and (i) a N-alkoxy-N alkylamine, (ii) a hydrazine H 2
N-NHR
2 , or (iii) a hydrazine R HN-NHR2 to a suitably chlorinated triazine. Under appropriate conditions, the reaction may allow the addition of either one or two amine substituents to the triazine ring. Alternatively, first the N-alkoxy-N-alkylamine, the hydrazine H 2
N-NHR
2 , or the hydrazine R 1
HN
10 NHR2 may be added to the triazine, followed by the addition of the amines. In a non-limiting example, to a solution of 2,4,6-trichlorotriazine in an appropriate aprotic or protic solvent containing an inorganic or organic base, is added a solution of a primary amine (VII) and the reaction is allowed to proceed at ambient temperature or heated, to isolate mono-amine adduct (VIII) or bis-amine adduct (IX). 15 In a subsequent reaction, mono-amine adduct (VIII) is reacted with another primary amine or a secondary amine (X) to yield the unsymmetrical monochloro-bis-amino-triazine adduct (XI). In a subsequent reaction, monochloro bis-amino-triazine adduct (XI) is reacted with (i) a N-alkoxy-N-alkylamine, (ii) a hydrazine H 2
N-NHR
2 or (iii) a hydrazine R HN-NHR2 in an appropriate aprotic or 20 protic solvent containing an inorganic or organic base to produce desired compounds of formula (II) (Scheme 3). Alternatively, in a subsequent reaction, bis-amine adduct (IX) is reacted with (i) a N-alkoxy-N-alkylamine, (ii) a hydrazine H 2
N-NHR
2 or (iii) a hydrazine R' HN-NHR2 in an appropriate aprotic or protic solvent containing an 25 inorganic or organic base to produce desired compounds of formula (II), wherein RiCH 2 is R5 (Scheme 4). - 33 - WO 2013/181217 PCT/US2013/043052 CI H 2
N-R
5 CI R 3 AN-H N (VII) N N (X) R 4 CI N >CI CI N N'R5 H CI (VIII) H R 1
,X-R
2 N N H 2 5 R-N-X-R 2 N N
R
3 IN N N R 3 N N R 4 H 4 (XI) R14 H II Scheme 3 CI H 2
N-R
5 CI R NX-R 2 N N (VII) N N R-N-X-R 2 CI N I RN N R5 R5 NL ~ R 5 H H N N N (IX) (II), wherein R 3
CH
2 is R 5 5 Scheme 4 In yet another aspect, the pyrimidine compound of the formula (III) may be prepared by the successive additions of primary amines and (i) a N-alkoxy-N H 2 10 alkylamine, (ii) a hydrazine H 2
N-NHR
2 or (iii) a hydrazine R HwN-NHR to a suitably chlorinated pyrimidine. In a non-limiting example, to a solution of 2,4,6-trichloropyrimidine (XII) in an appropriate aprotic or protic solvent containing an inorganic or organic base is added a solution of a primary amine (VII) and the reaction is allowed to 15 proceed at ambient temperature or heated, yielding bis-amine adduct (XIII). In a subsequent reaction, bis-amine adduct (XIII) is reacted with (i) a N-alkoxy-N alkylamine, (ii) a hydrazine H 2
N-NHR
2 , or (iii) a hydrazine R 1
HN-NHR
2 in an appropriate aprotic or protic solvent containing an inorganic or organic base to produce desired compounds of formula (III) (Scheme 5). - 34 - WO 2013/181217 PCT/US2013/043052 CI H2N-R5 CI H R< NX-R 2 (Vll)
NR
1
-N-X-R
2 N , NN N CR I l - R 5 511 CI N CI N N' N' R-N N R 5 H H (Xll) (Xlll) (III), R 3
CH
2 is R' Scheme 5 In yet another aspect, a pyrrolidino-pyrimidine of formula (IV) or a 5 pyrrolo-pyrimidine compounds of formula (V) may be prepared from an appropriately chlorinated aminopyrrolidinopyrimidine or aminopyrrolopyrimidine intermediate, respectively. In a non-limiting example, 2-chloroacetaldehyde may be added to a solution of 2,6-diamino-4-hydroxy-1,3-pyrimidine (XIV) in a polar protic solvent, at 10 ambient temperature or under heating, to yield cyclized adduct (XV). Subsequent treatment with a chlorinating agent, such as, but not limited to, phosphorous oxychloride produces the chloro intermediate (XVI). Intermediate (XVI) may be submitted to reductive alkylation with an aldehyde in the presence of a reducing agent, such as a borohydride (in a non-limiting example, cyanoborohydride) in a 15 protic solvent, at ambient temperature or elevated temperature, to produce the amino substituted adduct (XVII). In a subsequent reaction, amino substituted adduct (XVII) is reacted with (i) a N-alkoxy-N-alkylamine, (ii) a hydrazine H 2
N-NHR
2 , or (iii) a hydrazine R HN-NHR2 in an appropriate aprotic or protic solvent containing an inorganic or organic base to produce desired compounds of formula (V), wherein R 3 20 and R 4 are H (Scheme 6). In a non-limiting example, a pyrrolidinopyrimidine compound of the formula (IV) may be prepared from the corresponding pyrrolopyrimidine analog via reduction (Scheme 7). The manufacture of active pharmaceutical ingredients (APIs) may 25 introduce or generate impurities in an intermediate or final active pharmaceutical compound. These impurities may include trace metals, processing aide residuals (e.g., silica, cellulose fibers), or solvents derived from purchased starting materials. Other impurities may form as a consequence of the conditions to which the starting materials, process intermediates, or active ingredient itself are exposed. These 30 impurities may be problematic, affecting the manufacturing process as a whole and - 35 - WO 2013/181217 PCT/US2013/043052 the active ingredient in particular (e.g., its purity and crystallinity, and conditions for its isolation or crystallization). Such impurities often carry forward into the isolated API. HO N 0' i 0O O1 CI HO O N CI~ NH Ol -NNH H , N NH 2 N N NH
H
2 N (XIV) (XV) (XVI) CI H
N'X-R
2 H R 5 / N R 1
-N-X-R
2 / R 5 R 5~ reduction N N RN N RN H HN H H (XV l) (V )
R
3 and R 4 are H 5 Scheme 6 R 1NX-R 2 R N X-R 2 NN N R5 [RED] N N R 5 I I R4 K4 H (IV)H Scheme 7 10 Process-related impurities derived from degradation or side reactions involving the starting materials, intermediates, reactants, solvents or APIs are often related to the structure of the API itself. Regulatory guidances (e.g., IQC Q3b R3) provide accepted limits to which such potentially structurally related species may be present in the active ingredient, and provide threshold levels above which such 15 impurities should be identified, or both identified and qualified with toxicology data. In some instances, a given impurity may be present within acceptable levels and still affect the behavior of the active ingredient as a drug product formulated for exploratory or approved medical use. For example, a process impurity present within acceptable levels in the active ingredient from a regulatory perspective may form an 20 unacceptable precipitate in preparations for parenteral use. - 36 - WO 2013/181217 PCT/US2013/043052 Under certain conditions where the compounds of the present invention and their synthetic intermediates are exposed to acidic or alkaline aqueous environments, the bond between the aromatic heterocycle and the ring substituents may be susceptible to hydrolytic cleavage. For example, in the intermediates to 5 compounds of formula (11-a), a halo substituent in the 2 position (Schemes 3 and 4) may be hydrolytically replaced with a hydroxyl group to yield N-(4,6-Bis(n propylamino)-1,3,5-triazin-2-ol (CLXXVIII). Similarly, the amine substituents of compounds of formula (II-a) may be displaced by a hydroxyl group. Without wishing to be bound by theory, a series of chemical changes may occur to enable this 10 substitution, such as a prior oxidation of the appended amine side chain, with hydrolytic replacement by hydroxyl as the final transformation. As a specific non limiting example, the N,O-dimethylhydroxylamino substituent of N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) may be partially displaced by hydroxyl during processing in the presence of water, forming 15 (CLXXVIII) as a process impurity. (CLXXVIII) is unreported, has very low solubility in most organic solvents and is sparingly soluble in aqueous environments. It was unexpectedly observed that even at elevated temperatures (CLXXVIII) is very insoluble relative to the active ingredient. Thus, trace quantities of this compound in the API can be problematic in liquid formulations. In one embodiment, (CLXXVIII) 20 formed during processing is removed from the system by filtering a solution of the active ingredient as the free base at an elevated temperature. Remaining levels of the impurity present after such filtration may be purged during solid product isolation in the mother liquor and final product washes. In one embodiment, the level of (CLXXVIII) in the isolated (XXXVI) is less that 1% (w/w) with respect to (XXXVI). 25 In yet another embodiment, the level of (CLXXVIII) in the isolated (XXXVI) is less that 0.5% (w/w) with respect to (XXXVI). In yet another embodiment, the level of (CLXXVIII) in the isolated (XXXVI) is less that 0.3% (w/w) with respect to (XXXVI). In yet another embodiment, the level of (CLXXVIII) in the isolated (XXXVI) is less that 0.2% (w/w) with respect to (XXXVI). In yet another 30 embodiment, the level of (CLXXVIII) in the isolated (XXXVI) is less that 0.15% (w/w) with respect to (XXXVI). In yet another embodiment, the level of (CLXXVIII) in the isolated (XXXVI) is less that 0.1% (w/w) with respect to (XXXVI). In yet another embodiment, the isolated (XXXVI) is essentially free of 4,6-bis(n propylamino)-1,3,5-triazin-2-ol (CLXXVIII). - 37 - WO 2013/181217 PCT/US2013/043052 Formulation and Drug Product Stability Extensive experimentation was conducted to devise a suitable liquid formulation for (XXXVI). Experimentation had shown the pKa of (XXXVI) to be ca. 5 5.6. A pH solubility study was performed to assess the feasibility of formulating (XXXVI) as a simple buffer solution at a reasonable pH for an IV formulation. Solubility was assessed for (XXXV) alone (Figure 19A) or when admixed with citric acid and adjusted to pH 3-5.5 with 6N sodium hydroxide (Figure 19B). From the resulting data (Table 12A, Figure 19B), solubility of (XXXV) was calculated to be 10 10 mg/mL at ca. pH 3.86, 25 mg/mL at ca. pH 3.46, and 50 mg/mL at ca. pH 3.16. The solubility studies in water suggested solubility might be inadequate above a pH of ca. 3.6 to enable useful concentrations. Formulation at pH <3.6 may be tolerable when the formulated drug is diluted prior to administration, however the drug stability could be an issue at such a low pH. 15 To further evaluate the possibility for an all-aqueous formulation, stability studies of (XXXV) as a function of pH were undertaken in the presence of a buffer, with special attention to drug and buffer concentration to minimize drug precipitation and avoid buffer-catalyzed drug degradation (Figure 20A,Table 12B). (XXXVI) in water (1 mg/mL) was admixed with a solution of citric acid and glycine 20 (10 mM each), the mix adjusted to pH 2-5, and exposed to 40'C, 60'C, 25'C (control) and -70'C (control) and evaluated for physical appearance, pH, assay and related substances after 1 week, 2 weeks and 4 weeks of storage. At 40'C and 60'C, degradant levels at pH 2 were ca. 10-fold higher vs.pH 3-6, however at pH 3-6 growth in impurities to levels > 0.2 was generally still observed. Due to the limited solubility 25 of (XXXV) above pH 3.6, the stability was evaluated in the pH range 2-3.6 using methodology similar to that used for pH 2-5 (Figure 20B, Table 12C). Assay and sample pH showed little change, however impurities growth occurred across the range, with impurity levels at pH 2 >10 fold higher than impurity levels at pH 3.6. As another option, formulations including a co-solvent matrix could be 30 useful in providing good solubility for the salt and free base forms at a suitable pH. The solubilities of (XXXV) and (XXXVI) were evaluated in various solvents and standard IV solutions, including 0.9% NaCl, 5% dextrose, ethanol, methanol, acetonitrile, 30% PEG 400, 70% PEG 400 and 100% PEG 400, 5% Tween 80, and propylene glycol (Table 12D). The solubility of (XXXVI) exceeded 90 mg/mL in - 38 - WO 2013/181217 PCT/US2013/043052 water (>400 mg/mL), IV solutions (0.9% saline and 5% dextrose), ethanol and methanol, and in 30%, 70% and 100% propylene glycol. Its solubility in 100% PEG 400 was ca. 36mg/mL but > 90 mg/mL in 30 % and 70% PEG-400. The saturation solubility of (XXXV) in water was ca. 0.21 mg/mL, which agreed with the predicted 5 value from the pH solubility study. The solubility in ethanol, methanol and acetonitrile were > 90 mg/mL. The solubility in PEG-400 and propylene glycol systems improved with increasing levels of the cosolvent. The impact of buffer strength on stability was studied using admixtures of (XXXV) with 10-250 mmole citric acid at pH 3 (Table 12F). Impurity levels 10 increased noticeably at the highest buffer concentration, but there was no effect of buffer strength at 40 'C within the range of buffer concentration relevant to the formulation (ca. 10-50 mM). A total of nine (XXXVI) formulation prototypes were prepared, including a simple buffered solution, co-solvent systems as well as non-aqueous 15 solutions (Table 12G). The solutions were prepared at 25 mg/mL (XXXV) as free base, corresponding to 35 mg/mL (XXXVI). Prototypes 6, 7 and 8 resulted in significant precipitation during preparation and hence were not evaluated further. The remaining prototypes were stored at 2-8'C, room temperature, 40'C and 60'C in serum vials. Samples stored at 2-8'C and RT were analyzed at selected time points 20 for stability (Tables 12G.1-12G.6). The study suggested that a buffered aqueous formulation of (XXXV) near pH 3 would be stable under refrigerated or 25'C conditions (Table 12G.1). Based on the data used to prepare Figure 19B, reducing the (XXXV) concentration by half would allow the formulation pH to be increased by about 0.3 units, and as can be 25 seen in Table 12C, a 0.3 unit pH increase substantially improved stability of the formulation. Accordingly, the concentration of (XXXV) in the formulation as per Table 12G, line 1, was reduced from 25 mg/mL to 10 mg/mL, the pH increased from 3.0 to 3.2, and the buffer concentration was proportionately decreased from 50 mM to 20 mM. The final formulation is provided in Table 12H. 30 A batch of (XXXVI) formulated for clinical use was placed under formal GMP stability (Tables 201.1 and 121.2). The data obtained indicate that a single degradant was formed over time. The degradant was identified as N-(4,6 Bis(propylamino)-1,3,5-triazin-2-ol (CLXXVIII). The result of the GMP stability - 39 - WO 2013/181217 PCT/US2013/043052 study indicated the degradant can be expected to remain below 0.5% for at least two years (Figure 21, Table 12J). In one aspect, when compounds of the invention are partly or entirely dissolved in water or aqueous mixtures comprising suitable organic solvents, 5 hydrolytic degradants may be observed over time. For example, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) may degrade to 4,6-Bis(propylamino)-1,3,5-triazin-2-ol (CLXXVIII) in water-based formulations. Degradation was increasingly more pronounced as the pH was reduced below a value of 3 (Figures 20A and 20B). For (XXXV) solubility was high at pH 3.5 or lower, and 10 decreased rapidly at ca. pH 3.5 to ca. 4.4 (Figures 19A and 19B). In another aspect, the pH of the mixture containing (XXXV) is adjusted to a specific range to balance solubility and hydrolytic stability at any temperature selected. The rate of degradation for (XXXV) was highest in the pH range where (XXXV) is soluble to clinically useful levels. In one embodiment, the 15 clinically useful concentration of (XXXV) in a liquid carrier is about 1-10 mg/mL. In another embodiment, the clinically useful concentration of (XXXV) in a liquid carrier is about 5-10 mg/mL. Degradation of (XXXV) in an aqueous environment was also highly temperature-dependent, wherein cold temperatures slowed degradation. In one embodiment, (XXXV) as its hydrogen sulfate salt (XXXVI) in a 20 liquid carrier is admixed with agents to adjust pH to optimize solubility and minimize degradation. In another embodiment, (XXXV) as its hydrogen sulfate salt (XXXVI) in a liquid carrier is admixed with a buffering aid. Suitable buffering aids include, but are not limited to citric acid, ascorbic acid, monobasic phosphoric acid, and lactic acid. In an embodiment, the buffering aid is citric acid. In another embodiment, 25 (XXXV) as its hydrogen sulfate salt (XXXVI) is admixed with citric acid and a base to adjust pH as necessary to optimize solubility and minimize hydrolytic degradation. Suitable bases include, but are not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, and dibasic potassium hydrogen phosphate. In one embodiment, the pH is adjusted with sodium hydroxide. In another embodiment, the 30 liquid carrier is a mixture of water and a suitable organic co-solvent. Suitable organic co-solvents include, but are not limited to, glycerin, propylene glycol, polyethylene glycol 400, ethanol, 1,4-butanediol, and dimethylsulfoxide. In another embodiment, the liquid carrier is water. - 40 - WO 2013/181217 PCT/US2013/043052 In one embodiment, the mixture comprising (XXXV) as its sulfate salt (XXXVI) with citric acid and sodium hydroxide in water is adjusted to a pH of about 2.5 to 6. In another embodiment, the mixture is adjusted to a pH of about 3 to 5. In yet another embodiment, the mixture is adjusted to a pH of about 3 to 4. 5 In one embodiment, the mixture comprising (XXXV) as it hydrogen sulfate salt may be stored at low temperatures to retard the rate of degradation of the compound. In another embodiment, the temperature may be in the range of about 0 15'C. In yet another embodiment, the cold temperature may be in the range of 2-8'C. In yet another embodiment, the mixture comprising (XXXV) as its hydrogen sulfate 10 salt (XXXVI) and citric acid and sodium hydroxide is adjusted to pH of about 3-4 and stored at about 2-8 C. The rate of degradant growth was slow at 2-8 C and much faster at 25'C or above. As illustrated in Figure 21, the level of (CLXXVIII) formed when (XXXVI) is formulated for parenteral administration (non-limiting conditions: aqueous media at pH 3-4) may be maintained below 0.5% for over 2 years by storing 15 the preparation at 2-8'C (Figure 21). The present invention includes a formulation comprising an active ingredient, salt former, admixed buffering aid, admixed base pH and liquid carrier, along with storage conditions necessary to optimize drug solubility and maintain hydrolytic degradants within acceptable levels. 20 Salts The compounds described herein may form salts with acids, and such salts are included in the present invention. In one embodiment, the salts are pharmaceutically acceptable salts. The term "salts" embraces addition salts of free 25 acids that are useful within the methods of the invention. The term "pharmaceutically acceptable salt" refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, 30 purification or formulation of compounds useful within the methods of the invention. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and -41 - WO 2013/181217 PCT/US2013/043052 dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, 5 aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, p-hydroxybutyric, salicylic, galactaric and galacturonic acid. 10 Suitable pharmaceutically acceptable base addition salts of compounds of the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N' 15 dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound. 20 Combination Therapies In one embodiment, the compounds of the invention are useful in the methods of present invention in combination with at least one additional compound useful for treating breathing control disorders. These additional compounds may comprise compounds of the present invention or other compounds, such as 25 commercially available compounds, known to treat, prevent, or reduce the symptoms of breathing disorders. In embodiment, the combination of at least one compound of the invention or a salt thereof and at least one additional compound useful for treating breathing disorders has additive, complementary or synergistic effects in the treatment of disordered breathing, and in the treatment of sleep-related breathing disorders. 30 In a non-limiting example, the compounds of the invention or a salt thereof may be used in combination with one or more of the following drugs: acetazolamide, almitrine, theophylline, caffeine, methylprogesterone and related compounds, serotinergic modulators, cannabinoids (such as but not limited to dronabinol), and compounds known as ampakines. Non-limiting examples of - 42 - WO 2013/181217 PCT/US2013/043052 ampakines are the pyrrolidine derivative racetam drugs such as piracetam and aniracetam; the "CX" series of drugs which encompass a range of benzoylpiperidine and benzoylpyrrolidine structures, such as CX-516 (6-(piperidin-1-yl carbonyl)quinoxaline), CX-546 (2,3-dihydro-1,4-benzodioxin-7-yl-(1-piperidyl) 5 methanone), CX-614 (2H,3H,6aH-pyrrolidino(2,1-3',2')-1,3-oxazino (6',5'-5,4)benzo(e)1,4-dioxan-10-one), CX-691 (2,1,3-benzoxadiazol-6-yl-piperidin-1 yl-methanone), CX-717, CX-701, CX-1739, CX-1763, and CX-1837; benzothiazide derivatives such as cyclothiazide and IDRA-21 (7-chloro-3-methyl-3,4-dihydro-2H 1,2,4-benzothiadiazine 1,1-dioxide); biarylpropylsulfonamides such as LY-3 92,098, 10 LY-404,187 (N-[2-(4'-cyanobiphenyl-4-yl)propyl]propane-2-sulfonamide), LY-451,646 and LY-503,430 (4'-{( iS)-1-fluoro-2-[(isopropylsulfonyl)amino]-1 methylethyl} -N-methylbiphenyl-4-carboxamide). A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 15 19981, Clin. Pharmacokinet. 6: 429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22: 27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding 20 graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively. Methods of the Invention The invention includes a method of preventing or treating a breathing 25 control disorder or disease in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound selected from the group consisting of: N-(2,4-bis-n-propylamino)-N-(6 methylamino)-[1,3,5]triazine (CLXXII), N-(2,4,6-tris-n-propylamino)-[1,3,5]triazine 30 (CLXXIII), N-(2,4,6-tris-n-propylamino)-1,3-pyrimidine (CLXXIV), N-(2,4-bis-n propylamino)-N-(6-i-propylamino)-1,3-pyrimidine (CLXXV), a salt thereof, and any combinations thereof. The invention also includes a method of preventing destabilization or stabilizing breathing rhythm in a subject in need thereof. The method comprises - 43 - WO 2013/181217 PCT/US2013/043052 administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound selected from the group consisting of: N-(2,4-bis-n-propylamino)-N-(6 methylamino)-[1,3,5]triazine (CLXXII), N-(2,4,6-tris-n-propylamino)-[1,3,5]triazine 5 (CLXXIII), N-(2,4,6-tris-n-propylamino)-1,3-pyrimidine (CLXXIV), N-(2,4-bis-n propylamino)-N-(6-i-propylamino)-1,3-pyrimidine (CLXXV), a salt thereof, and any combinations thereof. In one embodiment, the breathing control disorder or disease is selected from the group consisting of respiratory depression, sleep apnea, apnea of 10 prematurity, obesity-hypoventilation syndrome, primary alveolar hypoventilation syndrome, dyspnea, hypoxia, and hypercapnia. In another embodiment, the respiratory depression is caused by an anesthetic, a sedative, an anxiolytic agent, a hypnotic agent, alcohol or a narcotic. In yet another embodiment, the subject is further administered a composition comprising at least one additional compound 15 useful for treating the breathing control disorder or disease. In yet another embodiment, the at least one additional compound is selected from the group consisting of acetazolamide, almitrine, theophylline, caffeine, methyl progesterone, a serotinergic modulator, a cannabinoid and an ampakine. In yet another embodiment, the formulation is administered in conjunction with the use of a mechanical 20 ventilation device or positive airway pressure device on the subject. In yet another embodiment, the subject is a mammal. In yet another embodiment, the mammal is a human. In yet another embodiment, the formulation is administered to the subject by an inhalational, topical, oral, buccal, rectal, vaginal, intramuscular, subcutaneous, transdermal, intrathecal or intravenous route. 25 The invention also includes a method of reducing or minimizing the open channel fraction of the potassium maxi-K or BK channel in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound of formula (I). 30 The invention also includes a method of inhibiting the TASK-I (KCNK3) channel in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound of formula (I). - 44 - WO 2013/181217 PCT/US2013/043052 The invention also includes a method of increasing minute ventilation in a subject in need thereof. The method comprises administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound of formula (I). 5 In one embodiment, the compound of formula (I) is:
R
1
X-R
2 N b2 Z_ biY N R3 N R5 R4 H (I), wherein: R and R2 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, 10 heteroarylalkyl, substituted heteroarylalkyl, heteroaryl or substituted heteroaryl; or R and R2 combine as to form a biradical selected from the group consisting of 3 hydroxy-pentane-1,5-diyl, 6-hydroxy-cycloheptane-1,4-diyl, propane- 1,3-diyl, butane-1,4-diyl and pentane-1,5-diyl; R 3 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -NR 1
R
2 , -C(O)OR 1 , acyl, or aryl; 15 R 4 is H, alkyl, or substituted alkyl; R 5 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -OR', -NRR 2 , -C(O)OR 1 , acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic; or R3 and R combine as to form a biradical selected from the group consisting of 3,6,9-trioxa-undecane- 1,11 -diyl and 3,6-dioxa-octane- 1,8-diyl; R6 is H, 20 alkyl, substituted alkyl or alkenyl; X is a bond, 0 or NR 4 ; and, Y is N, CR 6 or C; wherein: if Y is N or CR 6 , then bond bl is nil and: (i) Z is H, bond b 2 is a single bond, and A is CH; or, (ii) Z is nil, bond b 2 is nil, and A is a single bond; and, if Y is C, then bond bl is a single bond, and: (i) Z is CH 2 , bond b2 is a single 25 bond, and A is CH; or, (ii) Z is CH, bond b2 is a double bond, and A is C; or a salt thereof. In another embodiment, the compound of formula (I) is selected from the group consisting of: N-(4,6-Bis-methylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, N-(4,6-Bis-ethylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl 30 hydroxylamine, N-(4-Cyclopropylmethyl)-N-(6-n-propylamino) [1,3,5]triazin-2-yl) - 45 - WO 2013/181217 PCT/US2013/043052 N,O-dimethyl-hydroxylamine, N-(4-Ethylamino)-N-(6-n-propylamino)-[1,3,5]triazin 2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2-methylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2,2 dimethylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6 5 (2,2-dimethylpropylamino))[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4 (Methoxy(methyl)amino)-6-(n-propylamino)-1,3,5-triazin-2-yl)propionamide, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-methyl-hydroxylamine, O-Allyl-N-(4,6-bis n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine, N-(4,6-Bis-n-propylamino 10 [1,3,5]triazin-2-yl)-hydroxylamine, 6-(Methoxy(methyl)amino)-N 2 -propyl-1,3,5 triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-isopropyl hydroxylamine, 6-[1,2]Oxazinan-2-yl-N,N'-dipropyl-[1,3,5]triazine-2,4-diamine, N 15 (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-N-methyl-hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-hydroxylamine, N (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-hydroxylamine, 6 ((Benzyloxy)(isopropyl) amino)-N 2
,N
4 -dipropyl-1,3,5-triazine-2,4-diamine, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-O-isopropyl-hydroxylamine, N-(4,6 20 Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl-N-methyl-hydroxylamine, 6 (Methyl(thiophen-2-ylmethoxy)amino)-N 2
,N
4 -dipropyl-1,3,5-triazine-2,4-diamine, N (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-cyclopropylmethyl-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-ethyl-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl) 25 hydroxylamine, 4-N-(2-Dimethylaminoethyl)amino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4-N-(3-(1-N-Methylimidazol-2-yl) propyl)-amino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, 4-N-(1-N-Methylimidazol-2-yl)-methylamino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-(N-(2 30 dimethylaminoethyl)amino)-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6 Bis-(N-(pyridin-4-yl-methyl)amino)-[1,3,5]triazin-2-yl)-N,0-dimethyl hydroxylamine, 4,6-Bis-[N-(3-methoxy-n-propyl)amino]-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine, 4,6-Bis-[N-(tetrahydropyran-4-yl-methyl)amino] [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(5,8,11 -Trioxa-2,14,16,18,19 - 46 - WO 2013/181217 PCT/US2013/043052 pentaazabicyclo[13.3. 1]nonadeca-1(18),15(19),16(17)-trien- 17-yl)-N,O dimethylhydroxylamine, 2,6-Bis-(N-n-propylamino)-[1,3]pyrimidin-4-yl)-N,O dimethyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N',N' dimethylhydrazine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl-N' 5 methylhydrazine, 2-(n-Propyl)amino-4-(i-propylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine, 2-(n-Propyl)amino-4-dimethylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine, 2-(n-Propyl)amino-4-methylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine, 2-(n-Propyl)amino-4-(i-propyl)amino-7-i-propyl-pyrrolidino[2,3 d]pyrimidine, 2,4-Bis-(n-propyl)amino-7H-pyrrolidino[2,3-d]pyrimidine, 2-(n 10 Propyl)amino-4-(4-hydroxypiperidin-1-yl)-7-methyl-pyrrolidino[2,3-d]pyrimidine, 8 (7-Methyl-2-(n-propylamino)-pyrrolidino[2,3-d]pyrimidin-4-yl)-8 azabicyclo[3.2.1 ]octan-3-ol, N-(2-Propylamino-7H-pyrrolo[2,3d]pyrimidin-4-yl) N,O-dimethyl-hydroxylamine, N-(2-(Propen-2-yl)amino-7-methyl pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-(Propen-2 15 yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine, N-(2-n Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) 20 hydrazine, N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4 yl)-hydrazine, N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine, N (2,4,6-Tris-n-propylamino)-[1,3,5]triazine, N-(2,4,6-Tris-n-propylamino)-1,3 pyrimidine, N-(2,4-Bis-n-propylamino)-N-(6-i-propylamino)-1,3-pyrimidine, a salt thereof and mixtures thereof. In another embodiment, the compound of formula (I) is 25 selected from the group consisting of: N-(4,6-Bis-n-propylamino-1,3,5]triazin-2-yl) O-methyl-hydroxylamine; N-(4,6-Bis-n-propylamino-1,3,5]triazin-2-yl)-N',N' dimethylhydrazine; N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine; N-[(2,6-Bis-n-propylamino)-pyrimidin-4-yl]-N,O-dimethyl-hydroxylamine; N,N Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine; a 30 salt thereof and mixtures thereof. In yet another embodiment, In one embodiment, the subject is a mammal. In another embodiment, the mammal is human. the compound of formula (I) is N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, or a salt thereof. - 47 - WO 2013/181217 PCT/US2013/043052 In one embodiment, the pharmaceutical formulation is administered via intravenous infusion. In another embodiment, the infusion dose of the pharmaceutical formulation is at least about 0.016 mg/kg/min. In yet another embodiment, the infusion dose of the pharmaceutical formulation is about 0.016 5 mg/kg/min. In yet another embodiment, the infusion dose of the pharmaceutical composition produces plasma concentrations of at least about 726 ng/mL in the subject. In yet another embodiment, the infusion dose of the pharmaceutical composition produces plasma concentrations of at least about 726 ng/mL in a mammal. 10 The present invention also includes a method of preventing or treating a breathing control disorder or disease in a subject in need thereof. The method includes administering to the subject an effective amount of a pharmaceutical formulation comprising at least a pharmaceutically acceptable carrier and at least one compound of formula (I):
R
1
X-R
2 N b 2 Z-bY N A 'N R5 1 1 15 R4 H (I), wherein
R
1 and R 2 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, heteroaryl or substituted heteroaryl; or R 20 and R2 combine as to form a biradical selected from the group consisting of 3 hydroxy-pentane-1,5-diyl, 6-hydroxy-cycloheptane-1,4-diyl, propane- 1,3-diyl, butane-1,4-diyl and pentane-1,5-diyl; R 3 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -NR 1
R
2 , -C(O)OR 1 , acyl, or aryl;
R
4 is H, alkyl, or substituted alkyl; R 5 is H, alkyl, substituted alkyl, cycloalkyl, 25 substituted cycloalkyl, alkenyl, substituted alkenyl, -OR', -NR 1
R
2 , -C(O)OR 1 , acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic; or R3 and R combine as to form a biradical selected from the group consisting of 3,6,9-trioxa-undecane- 1,11 -diyl and 3,6-dioxa-octane- 1,8-diyl; R6 is H, alkyl, substituted alkyl or alkenyl; X is a bond, 0 or NR 4 ; and, Y is N, CR 6 or C; 30 wherein: -48- WO 2013/181217 PCT/US2013/043052 if Y is N or CR 6 , then bond b' is nil and: (i) Z is H, bond b 2 is a single bond, and A is CH; or, (ii) Z is nil, bond b2 is nil, and A is a single bond; and, if Y is C, then bond bl is a single bond, and: (i) Z is CH 2 , bond b 2 is a single bond, and A is CH; or, (ii) Z is CH, bond b 2 is a double bond, and A is C; or a salt 5 thereof. The present invention also includes a method of preventing destabilization of or stabilizing breathing rhythm in a subject in need thereof. The method includes administering to the subject an effective amount of a pharmaceutical formulation comprising at least a pharmaceutically acceptable carrier and at least one 10 compound of formula (I) or a salt thereof. In one embodiment, administering the formulation of the invention stabilizes the breathing rhythm of the subject. In another embodiment, administering the formulation of the invention increases minute ventilation in the subject. In one embodiment, the destabilization is associated with a breathing 15 control disorder or disease. In one embodiment, the breathing disorder or disease is selected from the group consisting of narcotic-induced respiratory depression, anesthetic-induced respiratory depression, sedative-induced respiratory depression, anxiolytic-induced respiratory depression, hypnotic-induced respiratory depression, alcohol-induced 20 respiratory depression, analgesic-induced respiratory depression, sleep apnea, apnea of prematurity, obesity-hypoventilation syndrome, primary alveolar hypoventilation syndrome, dyspnea, altitude sickness, hypoxia, hypercapnia and chronic obstructive pulmonary disease (COPD). In another embodiment, the respiratory depression is caused by an anesthetic, a sedative, an anxiolytic agent, a hypnotic agent, alcohol or a 25 narcotic. In one embodiment, the subject is further administered at least one additional compound useful for treating the breathing disorder or disease. In another embodiment, the at least one additional compound is selected from the group consisting of acetazolamide, almitrine, theophylline, caffeine, methylprogesterone and 30 related compounds, a serotinergic modulator, a cannabinoid, and an ampakine. In yet another embodiment, the formulation is administered to the subject in conjunction with the use of a mechanical ventilation device or positive airway pressure device. In one embodiment, the formulation is administered to the subject by an inhalational, topical, oral, buccal, rectal, vaginal, intramuscular, subcutaneous, transdermal, - 49 - WO 2013/181217 PCT/US2013/043052 intrathecal or intravenous route. In another embodiment, the subject is a mammal including but not limited to mouse, rat, ferret, guinea pig, monkey, dog, cat, horse, cow, pig and other farm animals. In one embodiment, the subject is a human. In another embodiment, 5 the at least one compound of formula (I) is selected from the group consisting of: N (4,6-Bis-methylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4,6-Bis ethylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4 Cyclopropylmethyl)-N-(6-n-propylamino) [1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, N-(4-Ethylamino)-N-(6-n-propylamino)-[1,3,5]triazin-2-yl)-N,O 10 dimethyl-hydroxylamine, N-(Bis-4,6-(2-methylpropylamino)) [1,3,5]triazin-2-yl) N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2,2-dimethylpropylamino)) [1,3,5]triazin 2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2,2 dimethylpropylamino))[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4,6-Bis n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4 15 (Methoxy(methyl)amino)-6-(n-propylamino)-1,3,5-triazin-2-yl)propionamide, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-methyl-hydroxylamine, O-Allyl-N-(4,6-bis n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine, N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-hydroxylamine, 6-(Methoxy(methyl)amino)-N 2 -propyl-1,3,5 triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl 20 hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-isopropyl hydroxylamine, 6-[1,2]Oxazinan-2-yl-N,N'-dipropyl-[1,3,5]triazine-2,4-diamine, N (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-N-methyl-hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-hydroxylamine, N 25 (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-hydroxylamine, 6 ((Benzyloxy)(isopropyl) amino)-N 2
,N
4 -dipropyl-1,3,5-triazine-2,4-diamine, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-O-isopropyl-hydroxylamine, N-(4,6 Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl-N-methyl-hydroxylamine, 6 (Methyl(thiophen-2-ylmethoxy)amino)-N 2
,N
4 -dipropyl-1,3,5-triazine-2,4-diamine, N 30 (4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-cyclopropylmethyl-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-ethyl-N-methyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl) hydroxylamine, 4-N-(2-Dimethylaminoethyl)amino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4-N-(3-(1-N-Methylimidazol-2-yl) - 50 - WO 2013/181217 PCT/US2013/043052 propyl)-amino-6-N-(n-propyl)amino- [1,3,5 ]triazin-2-yl)-N,O-dimethyl hydroxylamine, 4-N-(1-N-Methylimidazol-2-yl)-methylamino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-(N-(2 dimethylaminoethyl)amino)-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6 5 Bis-(N-(pyridin-4-yl-methyl)amino)-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, 4,6-Bis-[N-(3-methoxy-n-propyl)amino]-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine, 4,6-Bis-[N-(tetrahydropyran-4-yl-methyl)amino] [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(5,8,11 -Trioxa-2,14,16,18,19 pentaazabicyclo[13.3.1 ]nonadeca-1(18),15(19),16(17)-trien-17-yl)-N,O 10 dimethylhydroxylamine, 2,6-Bis-(N-n-propylamino)-[1,3]pyrimidin-4-yl)-N,O dimethyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N',N' dimethylhydrazine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl-N' methylhydrazine, 2-(n-Propyl)amino-4-(i-propylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine, 2-(n-Propyl)amino-4-dimethylamino-7-methyl-pyrrolidino[2,3 15 d]pyrimidine, 2-(n-Propyl)amino-4-methylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine, 2-(n-Propyl)amino-4-(i-propyl)amino-7-i-propyl-pyrrolidino[2,3 d]pyrimidine, 2,4-Bis-(n-propyl)amino-7H-pyrrolidino[2,3-d]pyrimidine, 2-(n Propyl)amino-4-(4-hydroxypiperidin-1-yl)-7-methyl-pyrrolidino[2,3-d]pyrimidine, 8 (7-Methyl-2-(n-propylamino)-pyrrolidino[2,3-d]pyrimidin-4-yl)-8 20 azabicyclo[3.2.1 ]octan-3-ol, N-(2-Propylamino-7H-pyrrolo[2,3d]pyrimidin-4-yl) N,O-dimethyl-hydroxylamine, N-(2-(Propen-2-yl)amino-7-methyl pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-(Propen-2 yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine, N-(2-n Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, 25 N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4 yl)-hydrazine, N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine, N 30 (2,4,6-Tris-n-propylamino)-[1,3,5]triazine, N-(2,4,6-Tris-n-propylamino)-1,3 pyrimidine, N-(2,4-Bis-n-propylamino)-N-(6-i-propylamino)-1,3-pyrimidine, a salt thereof and mixtures thereof. -51 - WO 2013/181217 PCT/US2013/043052 Pharmaceutical Compositions and Formulations The invention also encompasses the use of pharmaceutical compositions of at least one compound of the invention or a salt thereof to practice the methods of the invention. 5 Such a pharmaceutical composition may consist of at least one compound of the invention or a salt thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one compound of the invention or a salt thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The at least one 10 compound of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art. In an embodiment, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of 15 between 1 ng/kg/day and 100 mg/kg/day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 500 mg/kg/day. The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of 20 the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient. Pharmaceutical compositions that are useful in the methods of the 25 invention may be suitably developed for inhalational, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, intrathecal, intravenous or another route of administration. A composition useful within the methods of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal. Other contemplated 30 formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of - 52 - WO 2013/181217 PCT/US2013/043052 the disease being treated, the type and age of the veterinary or human patient being treated, and the like. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of 5 pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit. As used herein, a "unit dose" is a discrete amount of the 10 pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 15 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such 20 compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to 25 which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. In one embodiment, the compositions of the invention are formulated 30 using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol and other - 53 - WO 2013/181217 PCT/US2013/043052 pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey). The carrier may be a solvent or dispersion medium containing, for 5 example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be 10 achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition 15 an agent that delays absorption, for example, aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not DMSO alone. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, 20 transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., 25 other analgesic agents. As used herein, "additional ingredients" include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier. The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. 30 Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid. - 54 - WO 2013/181217 PCT/US2013/043052 The composition preferably includes an antioxidant and a chelating agent which inhibit the degradation of the compound. Preferred antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.l1% by 5 weight by total weight of the composition. Preferably, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Particularly preferred chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0.l10% by weight by total weight of the composition. The 10 chelating agent is useful for chelating metal ions in the composition which may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art. 15 Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid 20 paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, 25 hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain 30 aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but - 55 - WO 2013/181217 PCT/US2013/043052 are not limited to, methyl, ethyl, or n-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol. 5 Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions 10 of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils 15 such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, 20 or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations. 25 A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as 30 gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. - 56 - WO 2013/181217 PCT/US2013/043052 These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents. Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of 5 depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. 10 Administration/Dosing The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a breathing disorder event. Further, several divided dosages, as 15 well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. Administration of the compositions of the present invention to a 20 patient, preferably a mammal or avian, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a breathing control disorder in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; 25 the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For 30 example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 0.01 mg/kg and 50 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the - 57 - WO 2013/181217 PCT/US2013/043052 determination regarding the effective amount of the therapeutic compound without undue experimentation. The compound can be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, 5 once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be 10 initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc. 15 Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. A medical doctor, e.g., physician or veterinarian, having ordinary skill 20 in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is 25 achieved. In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined 30 quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) - 58 - WO 2013/181217 PCT/US2013/043052 the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of breathing disorders in a patient. In one embodiment, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In another 5 embodiment, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from subject to subject 10 depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physical taking all other factors about the patient into account. 15 Compounds of the invention for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 pg to about 6,000 mg, about 100 pg to about 5,500 mg, about 200 pg to about 5,000 mg, about 400 pg to about 4,000 mg, about 800 pg to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 20 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments thereinbetween. In some embodiments, the dose of a compound of the invention is from 25 about 0.5 pg and about 5,000 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. 30 Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than - 59 - WO 2013/181217 PCT/US2013/043052 about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof. 5 In one embodiment, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of breathing disorder in a patient. 10 The term "container" includes any receptacle for holding the pharmaceutical composition. For example, in one embodiment, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged 15 pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased 20 functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating, preventing, or reducing a breathing disorder in a patient. 25 Routes of Administration Routes of administration of any of the compositions of the invention include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, 30 intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, intraperitoneal, intrathoracic, intrapleural and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, - 60 - WO 2013/181217 PCT/US2013/043052 granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood 5 that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein. Oral Administration For oral application, particularly suitable are tablets, dragees, liquids, 10 drops, suppositories, or capsules, caplets and gelcaps. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, a paste, a gel, toothpaste, a mouthwash, a coating, an oral rinse, or an emulsion. The compositions intended for oral use may be prepared according to any method known in the art and 15 such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients which are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. 20 Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods 25 described in U.S. Patents Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. Hard capsules comprising the active ingredient may be made using a 30 physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin. - 61 - WO 2013/181217 PCT/US2013/043052 Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil. 5 For oral administration, the compounds of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents; fillers; lubricants; disintegrates; or wetting agents. If desired, the tablets may be coated using suitable methods and coating materials such as OPADRYTM film coating systems available from Colorcon, 10 West Point, Pa. (e.g., OPADRY TM OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY TM White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by 15 conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl para-hydroxy benzoates or sorbic acid). Liquid formulations of a pharmaceutical composition of the invention 20 which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use. A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more 25 additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a 30 pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate. Known surface-active agents - 62 - WO 2013/181217 PCT/US2013/043052 include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn 5 starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc. Granulating techniques are well known in the pharmaceutical art for 10 modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation." For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent 15 under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated. Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the 20 absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be 25 provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution. U.S. Patent No. 5,169,645 discloses directly compressible wax containing granules having improved flow properties. The granules are obtained 30 when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) will melt. - 63 - WO 2013/181217 PCT/US2013/043052 The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds useful within the methods of the invention, and a further layer providing for the immediate release of one or more compounds useful within the methods of the invention. Using a wax/pH 5 sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release. Parenteral Administration As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching 10 of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In 15 particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques. Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically 20 acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited 25 to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or 30 granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may - 64 - WO 2013/181217 PCT/US2013/043052 comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable 5 diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained 10 release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Topical Administration An obstacle for topical administration of pharmaceuticals is the 15 stratum comeum layer of the epidermis. The stratum corneum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes cornified and living cells. One of the factors that limit the penetration rate (flux) of a compound through the stratum comeum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater 20 the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the 25 skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal. Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or 30 suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein. - 65 - WO 2013/181217 PCT/US2013/043052 Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, 5 ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone. One acceptable vehicle for topical delivery of some of the compositions of the invention may contain liposomes. The composition of the liposomes and their use are known in the art (for example, see Constanza, U.S. Patent 10 No. 6,323,219). In alternative embodiments, the topically active pharmaceutical composition may be optionally combined with other ingredients such as adjuvants, anti-oxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers, buffering agents, preservatives, and the like. In 15 another embodiment, a permeation or penetration enhancer is included in the composition and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum corneum with respect to a composition lacking the permeation enhancer. Various permeation enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, 20 polar lipids, or N-methyl-2-pyrrolidone, are known to those of skill in the art. In another aspect, the composition may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum corneum, and thus allows increased transport across the stratum corneum. Various hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those 25 of skill in the art. The topically active pharmaceutical composition should be applied in an amount effective to affect desired changes. As used herein "amount effective" shall mean an amount sufficient to cover the region of skin surface where a change is desired. An active compound should be present in the amount of from about 0.0001% 30 to about 15% by weight volume of the composition. More preferable, it should be present in an amount from about 0.0005% to about 5% of the composition; most preferably, it should be present in an amount of from about 0.001% to about 1% of the composition. Such compounds may be synthetically-or naturally derived. - 66 - WO 2013/181217 PCT/US2013/043052 Buccal Administration A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using 5 conventional methods, and may contain, for example, 0.1 to 20% (w/w) of the active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. 10 Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein. The examples of formulations described herein are not exhaustive and it is understood that the invention includes additional modifications of these and 15 other formulations not described herein, but which are known to those of skill in the art. Rectal Administration A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a 20 composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation. Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 20'C) and which is liquid at the rectal 25 temperature of the subject (i.e., about 37'C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives. 30 Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the - 67 - WO 2013/181217 PCT/US2013/043052 rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives. Additional Administration Forms Additional dosage forms of this invention include dosage forms as 5 described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this invention also include dosage forms as described in 10 PCT Applications Nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO 90/11757. 15 Controlled Release Formulations and Drug Delivery Systems Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology. In some cases, the dosage forms to be used can be provided as slow or controlled-release of one or more active ingredients therein using, for example, hydropropylmethyl 20 cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical 25 compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, that are adapted for controlled-release are encompassed by the present invention. Most controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. 30 Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be - 68 - WO 2013/181217 PCT/US2013/043052 used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects. Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and 5 gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various 10 inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "controlled-release component" in the context of the present invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the 15 active ingredient. In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations. The term sustained release is used in its conventional sense to refer to a 20 drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release that is longer that the same amount of agent administered in bolus form. 25 For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation. 30 In a preferred embodiment of the invention, the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation. The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some - 69 - WO 2013/181217 PCT/US2013/043052 delay following drug administration and that may, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours. The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to 5 produce pulsed plasma profiles of the drug after drug administration. The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration. As used herein, short-term refers to any period of time up to and 10 including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration. As used herein, rapid-offset refers to any period of time up to and 15 including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration. 20 Mechanical Devices In one aspect of the invention, a method of treating a patient lacking normal breathing and normal breathing control comprises administering the composition useful within the invention as described herein, and additionally treating the patient using a device for treatment of a lack of normal breathing. Such devices 25 include, but are not limited to, ventilation devices, CPAP and BiPAP devices. Mechanical ventilation is a method to mechanically assist or replace spontaneous breathing. Mechanical ventilation is typically used after an invasive intubation, a procedure wherein an endotracheal or tracheostomy tube is inserted into the airway. It is normally used in acute settings, such as in the operating room or 30 ICU, for a short period of time during surgery, medical procedure, or serious illness. It may also be used at home or in a nursing or rehabilitation institution, if patients have chronic illnesses that require long-term ventilation assistance. The main form of mechanical ventilation is positive pressure ventilation, which works by increasing the pressure in the patient's airway and thus forcing air into the lungs. Less common - 70 - WO 2013/181217 PCT/US2013/043052 today are negative pressure ventilators (for example, the "iron lung") that create a negative pressure environment around the patient's chest, thus sucking air into the lungs. Mechanical ventilation is often a life-saving intervention, but carries many potential complications including pneumothorax, airway injury, alveolar damage, and 5 ventilator-associated pneumonia. For this reason the pressure and volume of gas used is strictly controlled, and reduced as soon as possible. Types of mechanical ventilation are: conventional ventilation, high frequency ventilation, non-invasive ventilation (non-invasive positive pressure pentilation or NIPPV), proportional assist ventilation (PAV), adaptive support ventilation (ASV) and neurally adjusted 10 ventilatory assist (NAVA). Non-invasive ventilation refers to all modalities that assist ventilation without the use of an endotracheal tube. Non-invasive ventilation is primarily aimed at minimizing patient discomfort and the complications associated with invasive ventilation, and is often used in cardiac disease, exacerbations of chronic pulmonary 15 disease, sleep apnea, and neuromuscular diseases. Non-invasive ventilation refers only to the patient interface and not the mode of ventilation used; modes may include spontaneous or control modes and may be either pressure or volume modes. Some commonly used modes of NIPPV include: (a) Continuous positive airway pressure (CPAP): This kind of machine has 20 been used mainly by patients for the treatment of sleep apnea at home, but now is in widespread use across intensive care units as a form of ventilation. The CPAP machine stops upper airway obstruction by delivering a stream of compressed air via a hose to a nasal pillow, nose mask or full-face mask, splinting the airway (keeping it open under air pressure) so that unobstructed breathing becomes possible, reducing 25 and/or preventing apneas and hypopneas. When the machine is turned on, but prior to the mask being placed on the head, a flow of air comes through the mask. After the mask is placed on the head, it is sealed to the face and the air stops flowing. At this point, it is only the air pressure that accomplishes the desired result. This has the additional benefit of reducing or eliminating the extremely loud snoring that 30 sometimes accompanies sleep apnea. (b) Bi-level positive airway pressure (BIPAP): Pressures alternate between inspiratory positive airway pressure (IPAP) and a lower expiratory positive airway pressure (EPAP), triggered by patient effort. On many such devices, backup rates may be set, which deliver IPAP pressures even if patients fail to initiate a breath. -71 - WO 2013/181217 PCT/US2013/043052 (c) Intermittent positive pressure ventilation (IPPV), via mouthpiece or mask. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were 5 considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized 10 alternatives and using no more than routine experimentation, are within the scope of the present application. It is to be understood that, wherever values and ranges are provided herein, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. 15 Accordingly, all values and ranges encompassed by these values and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as 20 individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from I to 3, from I to 4, from I to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. 25 This applies regardless of the breadth of the range. The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein. 30 EXAMPLES The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein. - 72 - WO 2013/181217 PCT/US2013/043052 Materials: Unless otherwise noted, all remaining starting materials were obtained from commercial suppliers and used without purification. Final products are typically 5 isolated as hydrochloride acid addition salts unless noted otherwise. Example 1: N-(4,6-Bis-methylamino-[1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (XX)
H
3 C NO-CH 3 CI CIN N N CH 3
NH
2 N N CH 3
NH-OCH
3 'HCI N N HCI CI N CI H 3 C-N N N-CH 3 DIPEA, EtOH H 3 C-N N N-CH3 I I I I H H H H H XVIII XIX XX 10 Scheme 8 2-Chloro-N-(4,6-bis-methylamino)-[1,3,5]triazine (XIX) 2,4,6-Trichloro-1,3,5-triazine (XVIII) (5.0 g, 27 mmol) was dissolved 15 in acetone (35 mL) and poured into ice-water (50 mL) to form a very fine suspension. A solution of N-methylamine hydrochloride (3.66 g, 54 mmol) in water (20 mL) was added and the temperature maintained at approximately 0 'C. To this mixture, 2N NaOH (54 mL, 108 mmol) was added in a dropwise manner to keep the temperature between 0 'C and 5 'C. The mixture was stirred 30 min at ambient temperature for an 20 additional 60 min at 50 'C. The precipitate was filtered and washed with water (3 x 25 mL). After drying over anhydrous calcium chloride under high vacuum, 2-chloro N-(4,6-bis-methylamino)-[1,3,5]triazine (XIX) was isolated as a white powder (4.2 g, 89% yield). LCMS (ESI) m/z = 174 (M+H). 25 N-(4,6-Bis-methylamino-[1,3,5]triazin-2-yl)-N, 0-dimethyl-hydroxylamine hydrochloride (XX) A mixture of 2-chloro-N-(4, 6-bis-methylamino)-[1,3,5]triazine (XIX) (1.74 g, 10 mmol), N,O-dimethylhydroxylamine hydrochloride (3.88 g, 40 mmol) and DIPEA (7.74 g, 60 mmol) in EtOH (200 mL) was heated at 100 'C for 16 h, after - 73 - WO 2013/181217 PCT/US2013/043052 which the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (150 mL), washed with water (100 mL) and brine solution (100 mL), and then dried over Na 2
SO
4 . The solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (pet ether/ethyl acetate = 5/1 to 5 5/3) to yield 899 mg (23%) of the desired product. The isolated free amine (380 mg, 2 mmol) was placed into H 2 0 (10 mL) and 0.5 M aqueous HCl solution (6 mL) was added. The resultant solution was subjected to lyophilization to yield the desired product, N-(4,6-bis-methylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (XX), as a white solid (468 mg). LCMS (ESI) m/z = 199 (M+H). H 10 NMR (500 MHz, DMSO): 6 (ppm) 12.20-12.50 (br, 1H), 8.48-8.62 (m, 2H), 3.76 3.86 (m, 3H), 3.29-3.39 (m, 3H), 2.76-2.93 (m, 6H). Example 2: N-(4,6-Bis-ethylamino-[1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine 15 hydrochloride (XXII)
H
3 CI NO-CH 3 CI CI N N N CH 3
CH
2
NH
2 N ) N CH 3
NH-OCH
3 HCI N N .HCI CI N CI Et-N N N-Et DIPEA, EtOH Et-N N N-Et I I I I H H H H XVIII XXI XXII Scheme 9 2-Chloro-N-(4, 6-bis-ethylamino)-[1,3,5]triazine (XX7) 20 2,4,6-Trichloro-1,3,5-triazine (XVIII) (5.0 g, 27 mmol) was dissolved in acetone (35 mL) and poured into ice-water (50 mL) to form a very fine suspension. A solution of ethylamine (2.43 g, 54 mmol) in water (20 mL) was added and the temperature maintained at approximately 0 'C. To this mixture, 2N NaOH (27 mL, 54 mmol) was added in a dropwise manner to keep the temperature between 0 'C and 25 5 'C. The mixture was stirred for 30 min at ambient temperature, and for additional 60 min at 50 'C. The precipitate was filtered off, washed with water (3 x 25 mL). After drying over anhydrous calcium chloride under high vacuum, 2-chloro-N-(4, 6 bis-ethylamino)-[1,3,5]triazine (XXI) was isolated as a white powder (5.0 g, 92% yield). LCMS (ESI) m/z = 202 (M+H). 30 - 74 - WO 2013/181217 PCT/US2013/043052 N-(4,6-Bis-ethylamino-[1, 3,5]triazin-2-yl)-N, 0-dimethyl-hydroxylamine hydrochloride (XXII) A mixture of 2-chloro-N-(4, 6-bis-ethylamino)- [1,3,5]triazine (XXI) (4.03 g, 20 mmol), N,O-dimethylhydroxylamine hydrochloride (9.7 g, 100 mmol) and 5 DIPEA (1.806 g, 140 mmol) in EtOH (200 mL) was heated at 100 'C for 16 h. After this time, the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (400 mL), washed with water (100 mL) and a brine solution (100 mL), then dried over Na 2
SO
4 and concentrated. The crude product was purified by flash column chromatography (pet ether/ethyl acetate = 5/1 to 5/2) to yield 811 mg 10 (18%) of the desired product. The isolated free amine (811 mg, 3.58 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M HCl solution in H 2 0 (7.2 mL) was added. The resultant solution was lyophilized to yield the desired product, N-(4,6-bis-ethylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (XXII) as a white solid (938 mg). LCMS (ESI) m/z = 227 (M+H). H NMR (500 MHz, DMSO): 6 15 (ppm) 12.40-12.80 (br, 1H), 8.58-8.87 (m, 2H), 3.76-3.78 (m, 4H), 3.34-3.37 (m, 6H), 1.10-1.16 (m, 6H). Example 3: N-(4-Cyclopropylmethyl)-N-(6-n-propylamino) [1,3,51triazin-2-yl)-N,O-dimethyl 20 hydroxylamine (XXV) CI CI CI NN H2N N N H 2 N N N Cl ill N CI N N CI N N N H I I H H XVIII XXIII XXIV
H
3 C N O-CH 3
CH
3
NH-OCH
3 HCI N il N .HCI DIPEA, EtOH N N N I I H H XXV Scheme 10 2,4-Dichloro-N-(6-n-propylamino)-[1,3,5]triazine (XXIII) 25 2,4,6-Trichloro-1,3,5-triazine (XVIII) (20 g, 109 mmol) was dissolved in acetone (100 mL) and poured into ice-water (50 mL) to form a very fine - 75 - WO 2013/181217 PCT/US2013/043052 suspension. A solution of propan-1-amine (7.1 g, 120 mmol) in water (20 mL) was added and the temperature maintained at approximately 0 'C. To this mixture, 2N NaOH (60 mL, 120 mmol) was added in a dropwise manner to keep the temperature between -5 'C and 0 'C. The mixture was stirred at 0 'C for 60 min. The precipitate 5 was filtered off and washed with water (3 x 25 mL). After drying over calcium chloride under high vacuum, 2,4-dichloro-N-(6-n-propylamino)-[1,3,5]triazine (XXIII) was isolated as a white powder (18 g, 80% yield). LCMS (ESI) m/z = 208 (M+H)+. 10 2-Chloro-N-(4-cyclopropylmethyl)-N-(6-n-propylamino) [1,3,5]triazine (XXIV) 2,4-Dichloro-N-(6-n-propylamino)-[1,3,5]triazine (XXIII) (18 g, 87 mmol) was dissolved in acetone (100 mL) and poured into ice-water (50 mL) to form a very fine suspension. A solution of cyclopropylmethanamine (6.7 g, 95 mmol) in acetone (30 mL) was added and the temperature was maintained at approximately 0 15 'C. To this mixture, 2N NaOH (44 mL, 88 mmol) was added in a dropwise manner to keep the temperature between 0 'C and 5 'C. The mixture was stirred for 30 min at ambient temperature and for an additional 60 min at 50 'C. The precipitate was filtered off, washed with water (3 x 25 mL). After drying over anhydrous calcium chloride under high vacuum, 2-chloro-N-(4-cyclopropylmethyl)-N-(6-n-propylamino) 20 [1,3,5]triazine (XXIV) was isolated as a white powder (12 g, 57% yield). LCMS (ESI) m/z = 242 (M+H). N-(4-Cyclopropylmethyl)-N-(6-n-propylamino) [1,3,5]triazin-2-yl)-N,0-dimethyl hydroxylamine hydrochloride (XXV) 25 A mixture of 2-chloro-N-(4-cyclopropylmethyl)-N-(6-n-propylamino) [1,3,5]triazine (XXIV) (1.5 g, 6.2 mmol), N,O-dimethylhydroxylamine hydrochloride (3.0 g, 31.0 mmol) and DIPEA (6.5 g, 49.6 mmol) in EtOH (50 mL) was heated at 100 'C for 16 h, after the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (400 mL), washed with water (100 mL), then with a brine 30 solution (100 mL) and dried over Na 2
SO
4 . The crude was purified by flash column chromatography (pet ether/ethyl acetate = 5/1 to 5/2). The solvent was removed under reduced pressure to yield 500 mg (26%) of the desired product. The isolated free amine (500 mg, 1.88 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (4.0 mL) was added. The resultant solution was subjected was subjected - 76 - WO 2013/181217 PCT/US2013/043052 to lyophilization to yield the desired product, N-(4-cyclopropylmethyl)-N-(6-n propylamino) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (XXV, 520 mg) as a brown oil. LCMS (ESI) m/z = 267 (M+H)+. 1 H NMR (500 MHz, DMSO): 6 (ppm) 11.80-12.10 (br, 1H), 8.68-8.85 (m, 2H), 3.77 (s, 3H), 3.15-3.36 5 (m, 7H), 1.49-1.55 (m, 2H), 1.23 (s, 1H), 0.85-0.93 (m, 3H), 0.43-0.49 (m, 2H), 0.22 0.25 (m, 2H). Example 4: N-(4-Ethylamino)-N-(6-n-propylamino)-[1,3,51triazin-2-yl)-N,O-dimethyl 10 hydroxylamine (XXVII) CI CI CI N N H2N N N HCI N iN CIK'N CI N N CI N N N H I I H H XVIII XXIII XXVI
H
3 C N O-CH 3
CH
3
NH-OCH
3 HCI N N HCI DIPEA, EtOH N N N I I H H XXVII Scheme 11 2,4-Dichloro-N-(6-n-propylamino)-[1,3,5]triazine (XXIII) 15 2,4,6-Trichloro-1,3,5-triazine (XVIII) (20 g, 109 mmol) was dissolved in acetone (100 mL) and poured into ice-water (50 mL) to form a very fine suspension. A solution of propan-1-amine (7.1 g, 120 mmol) in water (20 mL) was added and the temperature maintained at approximately 0 'C. To this mixture was added 2 N NaOH (60 mL, 120 mmol) in a dropwise manner to keep the temperature 20 between -5 'C and 0 'C. The mixture was then stirred at 0 'C for 60 min. The precipitate was filtered off, washed with water (3 x 25 mL). After drying over anhydrous calcium chloride under high vacuum, 2,4-dichloro-N-(6-n-propylamino) [1,3,5]triazine (XXIII) was isolated as a white powder (18 g, 80% yield). LCMS (ESI) m/z = 208 (M+H). 25 - 77 - WO 2013/181217 PCT/US2013/043052 2-Chloro-N-(4-ethylamino)-N-(6-n-propylamino)-[1,3,5]triazine (XXVI) 2,4-Dichloro-N-(6-n-propylamino)-[1,3,5]triazine (XXIII) (4.0 g, 19.5 mmol) was dissolved in acetone (40 mL) and poured into ice-water (40 mL) to form a very fine suspension. A solution of ethanamine hydrochloride (1.91 g, 23.4 5 mmol) in water (10 mL) was added and the temperature was maintained at approximately 0 'C. A solution of NaOH (2.34 g, 58.5 mmol) in water (10 mL) was added in a dropwise manner to keep the temperature between 0 'C and 5 'C. The mixture was then stirred 40 min at room temperature and concentrated. The precipitate was filtered off, washed with water (3 x 25 mL). After drying over 10 calcium chloride under high vacuum, the desired product, 2-chloro-N-(4-ethylamino) N-(6-n-propylamino)-[1,3,5]triazine (XXVI) was isolated as a white powder (3.89 g, 92% yield). LCMS (ESI) m/z = 216 (M+H). N-(4-Ethylamino)-N-(6-n-propylamino) [1,3,5]triazin-2-yl)-N,0-dimethyl 15 hydroxylamine hydrochloride (XXVII) A mixture of 2-chloro-N-(4-ethylamino)-N-(6-n-propylamino) [1,3,5]triazine (XXVI) (2 g, 9.3 mmol), N,O-dimethylhydroxylamine hydrochloride (4.5 g, 46.5 mmol) and DIPEA (8.4 g, 65.1 mmol) in EtOH (20 mL) was heated at 100 'C for 16 h, after which time the solvent was removed under reduced pressure. 20 The residue was dissolved in EtOAc (150 mL), washed with water (100 mL), washed with a brine solution (100 mL) and then dried over Na 2
SO
4 . The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (pet ether/ethyl acetate = 10/1 to 2/1) to yield the desired product (820 mg, 37%). The isolated free amine (820 mg, 3.42 mmol) was dissolved in H 2 0 25 (10 mL), and 0.5 M aqueous HCl solution (11 mL) was added. The resultant solution was subjected to lyophilization to yield the desired product, N-(4-ethylamino)-N-(6-n propylamino) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (XXVII), as a colorless oil (944 mg). LCMS (ESI) m/z = 241 (M+H). 1 H NMR (500 MHz, DMSO): 6 (ppm) 12.25-12.75 (br, 1H), 8.71-8.75 (m, 2H), 3.75-3.92 (m, 30 6H), 3.25-3.37 (m, 4H), 1.50-1.55 (m, 2H), 1.09-1.16 (m, 3H), 0.87-0.94 (m, 3H). - 78 - WO 2013/181217 PCT/US2013/043052 Example 5: N-(Bis-4,6-(2-methylpropylamino)) [1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXIX) CI CI ,CI N O-H N N H 2 N Y N CH 3
NH-OCH
3 HCI N N .HCI C NN N DIPEA, EtOH N CI NCI NN N N N H H H H XVIII XXVIII XXIX 5 Scheme 12 2-Chloro-N-(4, 6-bis-(2-methylpropylamino)-[1,3,5]triazine (XXVIII) 2,4,6-Trichloro-1,3,5-triazine (XVIII) (5.0 g, 27 mmol) was dissolved in acetone (35 mL) and poured into ice-water (50 mL) to form a very fine suspension. 10 A solution of 2-methylpropan-1-amine (4.0 g, 54 mmol) in acetone (20 mL) was added and the temperature was maintained at approximately 0 0 C. To this mixture, 2 N NaOH (27 mL, 54 mmol) was added in a dropwise manner to keep the temperature between 0 0 C and 5'C. The mixture was stirred for 30 min at ambient temperature and for an additional 60 min at 50'C. The precipitate was filtered off, washed with water 15 (3 x 25 mL). After drying over calcium chloride under high vacuum, 2-chloro-N-(4, 6-bis-(2-methylpropylamino)-[1,3,5]triazine (XXVIII) was isolated as white powder (6.0 g, 87% yield). LCMS (ESI) m/z = 258 (M+H)+. N-(Bis-4,6-(2-methylpropylamino)) [1,3,5]triazin-2-yl)-N, 0-dimethyl-hydroxylamine 20 hydrochloride (XXIX) A mixture of 2-chloro-N-(4, 6-bis-(2-methylpropylamino) [1,3,5]triazine (XXVIII) (2.57 g, 10 mmol), N,O-dimethylhydroxylamine hydrochloride (1.94 g, 20 mmol) and DIPEA (5.16 g, 40 mmol) in EtOH (100 mL) was heated at 100 'C for 16 h, after which time the solvent was removed under 25 reduced pressure. The residue was dissolved in EtOAc (200 mL), washed with water (2 x 100 mL), washed with a brine solution (100 mL) and then dried over Na 2
SO
4 . The solvent was removed under reduced pressure. The residue was purified by flash column chromatography (pet ether/ethyl acetate = 5/1) to yield the desired product (920 mg, 33%). The isolated free amine (920 mg, 3.3 mmol) was dissolved in H 2 0 - 79 - WO 2013/181217 PCT/US2013/043052 (10 mL) and 0.5 M aqueous HCl solution (6.6 mL) was added. The resultant solution was subjected to lyophilization to yield the desired product, N-(bis-4,6-(2 methylpropylamino))[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (XXIX) as a white solid (1.0 g). LCMS (ESI) m/z = 283 (M+H). 1 H NMR (500 5 MHz, DMSO): 6 (ppm) 12.55-12.60 (br, 1H), 8.57-8.77 (br, 2H), 3.78 (s, 3H), 3.40 3.45 (m, 3H), 3.11-3.19 (m, 4H), 1.80-1.86 (m, 2H), 0.89-0.94 (m, 12H). Example 6: N-(Bis-4,6-(2,2-dimethylpropylamino)) [1,3,51triazin-2-yl)-N,O-dimethyl 10 hydroxylamine hydrochloride (XXXI)
H
3 C, NO-CH 3
H
3 C, NO-CH 3 N "N CH 3
NH-OCH
3 HCI NN H2N N N .HCI CIN <CI CI N CIN N N I I H H XVIII XXX XXXI Scheme 13 N-(4,6-Dichloro[1,3,5]triazin-2-yl)-N,0-dimethyl-hydroxylamine (XXX) 15 2,4,6-Trichloro-1,3,5-triazine (XVIII) (30 g, 163 mmol) was dissolved in acetone (300 mL), and N,O-dimethylhydroxylamine hydrochloride (15.8 g, 163 mmol) and DIPEA (42 g, 326 mmol) were added and the mixture then stirred at 0 0 C for 1 h. The solution was concentrated and the residue was treated with EtOAc (750 mL), washed with water (100 mL), and the organic layer was dried with Na 2 S04. The 20 volatiles were removed in vacuo and the residue was purified by flash column chromatography (pet ether/ethyl acetate = 50/1 to 10/1) to yield the desired product, N-(4,6-dichloro[1,3,5] triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXX), as a white solid (25 g, 73% yield). LCMS (ESI) m/z = 210 (M+H). 25 N-(Bis-4,6-(2,2-dimethylpropylamino)) [1,3,5]triazin-2-yl)-N,0-dimethyl hydroxylamine (XXI) A mixture of N-(4,6-dichloro[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine (XXX) (1 g, 4.78 mmol), 2,2-dimethylpropan-1-amine (832.5 mg, 9.57 mmol) and DIPEA (1.85 g, 14.34 mmol) in EtOH (20 mL) was heated at 100 C 30 for 16 h, after which time the solvent was removed under reduced pressure. The - 80 - WO 2013/181217 PCT/US2013/043052 residue was dissolved in EtOAc (40 mL), washed with water (20 mL) and with a brine solution (20 mL), dried over Na 2
SO
4 , and then concentrated. The crude product was purified by flash column chromatography (pet ether/ethyl acetate = 20/1 to 5/1) to yield the desired product (1.4 g, 95%). The isolated free amine (1.4 g, 4.52 mmol) 5 was dissolved in H 2 0 (10 mL) and 0.5 M HCl solution in H 2 0 (14.5 mL) was added, and the resultant solution was subjected to lyophilization to yield the desired product, N-(bis-4,6-(2,2-dimethylpropylamino))[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine hydrochloride, as a white solid (1.67 g). LCMS (ESI) m/z = 311 (M+H). 1 H NMR (500 MHz, DMSO): 6 (ppm) 12.40-12.70 (br, 1H), 8.52-8.81 (m, 10 2H), 3.75-3.79 (m, 3H), 3.33-3.36 (m, 3H), 3.14-3.21 (m, 4H), 0.89-0.96 (m, 18H). Example 7: 4,6-Bis-N-cyclopropylamino-[1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (XXXIII) CI HN C1 H 3 C NO-CH 3 N N H2iN N N CH 3
NH-OCH
3 HCI NNN.HCI CI N CI N N N DIPEA Z NA N NA I IN N N H H I I H H 15 XVIII XXXII XXXIII Scheme 14 2-Chloro-N-(4, 6-bis-(cyclopropylamino)-[1,3,5]triazine (XXII) 20 2,4,6-Trichloro-1,3,5-triazine (XVIII) (40 g, 217 mmol) was dissolved in 200 mL of acetone and poured into ice-water (250 mL) to form a very fine suspension. A solution of cyclopropanamine (24.8 g, 435 mmol) was added with stirring at 0 0 C. To this mixture, 2N NaOH (218 mL, 435 mmol) was added dropwise at a rate to keep the temperature between 0 0 C and 5'C. The resultant mixture was 25 stirred for 30 min at ambient temperature and then for an additional 60 min at 50'C. The precipitate was filtered off, washed with water (3 x 100 mL). After drying over calcium chloride under high vacuum, chloro-N-(4, 6-bis-(cyclopropylamino) [1,3,5]triazine (XXXII) was isolated as a white powder (46 g, 93% yield). LCMS (ESI) m/z = 226 (M+H). 30 -81 - WO 2013/181217 PCT/US2013/043052 4,6-Bis-N-cyclopropylamino-[1,3,5]triazin-2-yl)-N, 0-dimethyl-hydroxylamine (XXXIII) A mixture of chloro-N-(4, 6-bis-(cyclopropylamino)-[1,3,5]triazine (XXXII) (2.25 g, 10 mmol), N,O-dimethylhydroxylamine hydrochloride (1.94 g, 5 20 mmol) and DIPEA (5.16 g, 40 mmol) in EtOH (100 mL) was heated at 100 'C for 16 h, and the solvent was then removed under reduced pressure. The residue was dissolved in EtOAc (200 mL), washed with water (2 x 100 mL) and brine (100 mL) then dried over Na 2
SO
4 . The solvent was removed under reduced pressure. The residue was purified by flash column chromatography (pet ether/ethyl acetate = 3/1) 10 to yield 1.0 g (40%) of the desired product. The isolated free amine (1.0 g, 4.0 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (8 mL) and then the solution was lyophilized to yield N-(4,6-bis-cyclopropylamino-[ 1,3,5]triazin-2-yl) N,O-dimethyl-hydroxylamine hydrochloride (XXXIII) as a white solid (1.05 g). LCMS (ESI) m/z = 251 (M+H). 1 H NMR (500 MHz, DMSO): 6 (ppm) 12.00-12.80 15 (br, 1H), 8.70-9.50 (br, 2H), 3.76 (s, 3H), 3.28-3.38 (m, 3H), 2.69-2.89 (m, 2H), 0.59 0.81 (m, 8H). Example 8A: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) 20 Example 9A: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) CI ,--NH 2 C H 3
C-N-O-CH
3 N N NaOH/H 2 O N N NaOH N- acetone N N N- 1,4-dioxane CI N CI 50 0 C, 3h H H 60 0 C, 6 h XVIII XXXIV
H
3 CN 0-CH 3 H3C N o-CH3 NeO
H
2
SO
4 N 'N 0-S-O-H N ~N ~-"N SN N 1T4-dioxane ~-N N N~- RTO0.5hIII H H H H H XXXV XXXVI Scheme 15A 25 2-Chloro-N-(4,6-bis-(n-propylamino)-[1,3,5]triazine (XXXIV) - 82 - WO 2013/181217 PCT/US2013/043052 A 2 M NaOH solution (82 mL, 162.68 mmol) was added in a dropwise manner to a suspension of 2,4,6-trichloro-1,3,5-triazine (XVIII) (15.00 g, 81.34 mmol) and n-propylamine (13.4 mL, 162.68 mmol) in acetone (300 mL) and water (15 mL) at 0 0 C. The reaction mixture was heated at 50 0 C for 3 h and then cooled. 5 Water (100 mL) was added to the reaction mixture; the resultant precipitate was filtered, washed with water, ethyl ether and dried to yield 2-chloro-N-(4, 6-bis-(n propylamino)-[1,3,5]triazine (XXXIV) (15.88 g, 85% yield). N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-N, 0-dimethyl-hydroxylamine (XYXV) 10 A mixture of 2-chloro-N-(4, 6-bis-(n-propylamino)-[1,3,5]triazine (XXXIV) (10.00 g, 43.53 mmol), N,O-dimethylhydroxylamine hydrochloride (8.49 g, 87.06 mmol) and NaOH (3.13 g, 78.35 mmol) in 1,4-dioxane (120 mL) and water (30 mL) was heated at 60 0 C for 6 h, after which the volatiles were removed under reduced pressure. Saturated NaHCO 3 solution (500 mL) was added to the residue and 15 the mixture was extracted with EtOAc (3 x 200 mL). The combined organic extracts were washed with water (300 mL), then with a brine solution (300 mL) and dried over Na 2
SO
4 . The solvent was removed under reduced pressure and the resultant residue was filtered through silica gel using eluent CH 2 Cl 2 /EtOH (9/1 v/v) to yield N-(4,6-bis n-propylamino [1,3,5]triazine-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) (9.96 g, 20 90% yield). N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-N, 0-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) Concentrated (95%) H 2
SO
4 (0.72 mL, 12.74 mmol) was added in a 25 dropwise manner to a solution of N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine (XXXV, 3.24 g, 12.74 mmol) in 1,4-dioxane (100 mL) at 0 0 C. The mixture was stirred for 0.5 h at room temperature, volatiles were removed under reduced pressure. The residue was co-evaporated with dry toluene (3 x 25 mL). The resulting white residue was crystallized from ethanol/ethyl ether to yield N-(4,6 30 bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, hydrogen sulfate (XXXVI, 3.86g, 86% yield) as a white solid. 1 H NMR (400 MHz, DMSO): 6 (ppm) 12.0-11.2 (lH, br s), 8.7-8.3 (0.7 H, br s), 8.10 (0.3H, br s), 7.8-7.3 (lH, m), - 83 - WO 2013/181217 PCT/US2013/043052 3.78 (3H, s), 3.40-3.20 (7H, m), 1.61-1.45 (4H, m), 0.93-0.84 (6H, m). ESI-MS (m/z) 255 [M+H]+; melting point: 134-135 'C. Example 8B: 5 N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) Example 9B: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) H H 3 C, O.0, CI CI H N CH 3 N N NH2 N N H3C'N'O'CH 3 HCI N N CI N CI NaOH, H 2 0 , K2CO - N N N XVlll acetone H XXXV H N,N-dimethylacetamide XXXV 70-80 0 C
H
3 C, 0Os
H
2
SO
4 N CH 3 XXXV MEKN NN H 2 SO4 N- N N H H XXXVI 10 Scheme 15B Stage 1: 2-Chloro-N-(4,6-bis-(n-propvlamino)-f1,3,57triazine (XXXIV) Summary: Di-chloro displacement by 2 equivalents of n-propylamine. In one embodiment, two or more equivalence of n-propylamine produced only the 15 bis-propylamine derivative of the starting material. The reaction progress was monitored by HPLC and the desired intermediate (2-chloro-4,6-bis-n-propylamino-s triazine) was precipitated. In-process QC Tests were performed. A suitable glass reactor vessel equipped with a mechanical stirrer, thermocouple, condenser, addition funnel and a temperature control mantle was 20 charged with 8 L of acetone followed by 1 kg (5.42 moles) of cyanuric chloride. The stirring mixture was pre-cooled to 15 'C and n-propylamine (at ambient temperature) was added slowly via addition funnel, to maintain the temperature below 45'C. A 2M NaOH solution was prepared and was added to the mixture at a rate to maintain the temperature below 45'C. The pH of the mixture was acidic (approximately pH = 4) 25 and 6N NaOH was added to adjust the pH to 8-9. The mixture was stirred at 40-50'C for 0.5 h and the reaction was monitored for completion by IPC HPLC analysis. The - 84 - WO 2013/181217 PCT/US2013/043052 reaction was deemed complete when <2% of cyanuric chloride was detected. Analysis was repeated every hour until reaction was complete. After reaction was complete, WFI (sterile water) was added slowly to maintain temperature below 50'C. The resulting suspension was allowed to cool to 5 room temperature while stirring overnight. The solids were filtered through a polypropylene filter cloth and washed with acetone/water (1:2) followed by 1.5 L of MTBE. The solids were dried on the filter (assisted by vacuum) then placed in a vacuum oven (45±5 'C, > 29" Hg) for a minimum of 6 h to give <0.5% in change in weight loss. A sample was collected for analytical testing (QC HPLC analysis and 10 Karl Fisher analysis); 2 g was collected for QA retain. The product produced in Stage 1 was a white solid. This powder was transferred from drying pans to poly bag with nylon tie, double bagged and placed into fiber drum; label and submit to QA for quarantine storage. 15 Stage 2, Step 1: N-(4,6-Bis-n-propvlamino-f1, 3,5triazin-2-vl)-N, 0-dimethvl hydroxylamine (XYXV) Summary: Chloro displacement by 1 equivalent of N methoxymethylamine. The reaction progress was monitored by HPLC and precipitation of desired product free base was accomplished by adding water and 20 cooling . In-process QC Tests were pre-formed. The free base was converted to the corresponding sulfate salt with crystallization and the final product was subjected to vacuum oven drying. In-process QC Tests were performed as well as finished product QC testing. A suitable round-bottom flask equipped with a mechanical stirrer, 25 thermocouple, condenser and a heating mantle was charged with 6 L of N,N dimethylacetamide (DMA) followed by 1 kg (4.35 moles) of 6-chloro-N,N-dipropyl [1,3,5]-triazine-2,4-diamine (Stage 1 Product). Added to this stirring mixture at room temperature was K 2
CO
3 (1.2 kg, 6.53 moles, 2 eq.), with rinsing with a small quantity of additional DMA. To this, was added N,O-dimethylhydroxylamine hydrochloride 30 (0.637 kg, 8.71 moles, 1.5 eq.) in portions over ~5-10 minutes to reduce foaming (and while maintaining the temperature below 60'C) with rinsing using a small quantity of additional DMA. The mixture was heated to 75-80'C and stir for a minimum of 0.5 hours. Once at 75-80'C, the reaction was monitored for completion by HPLC. The mixture was cooled to below 65'C and water (12 L) was added. The resulting - 85 - WO 2013/181217 PCT/US2013/043052 suspension was allowed to cool to room temperature while stirring overnight (18 h). The resultant solids were filtered and washed with 1.2 L of water. The filter cake to air-dry one hour and a sample for OVI amine GC analysis was obtained. The remaining solids were vacuum oven dried (45'C, > 29" Hg) for a minimum of 6 hours 5 (NMT 1% weight change). The desired product (free base (XXXV)) was a dense white solid. An IPC sample for testing was obtained. Crystalline N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-N, 0 dimethyl-hydroxylaminefree base (XXXVj) A solution of (XXXV) free base in methylene chloride was rapidly 10 evaporated to dryness. The resulting solid was dried under vacuum at ca. 25C for ca. 45 minutes (XXXV freebase Form A). DSC: Sharp endotherm at ca. 95.2'C. XRPD: Figure 22. Stage 2, Step 2: Salt Formation and Crystallization of N-(4,6-Bis-n-propvlamino [1,3,5triazin-2-vl)-N, 0-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) 15 Methyl ethyl ketone (20L) was added to 2,800 g (12.2 moles) of substrate free base (XXXV). The mixture was stirred and heated to 40'C then polish filtered into a 72-L reactor through a medium porosity sintered glass funnel to yield a clear solution and a fluffy white residue (12 g, 0.43% of the free base weight). The mixture was warmed to 45'C and concentrated H 2
SO
4 (12.8 moles, 1.05 eq. based on 20 pre-filtered weight) was added slowly over lh via addition funnel. The mixture was allowed to stir overnight (18 h) while cooling (A solid precipitate was observed to begin forming at approximately 36'C; final temp = 27C). The mixture was further cooled to 15'C, stirred 0.5h and filtered. The solid product cake was washed with methyl ethyl ketone (6L), air dried on the 25 Blchner funnel (vacuum assisted) for 4 h, then placed in a vacuum oven (>29" Hg, 50-55'C) and dried for at least 6 h to give 3,252g (84%) of N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) as a white solid. H NMR (400 MHz, DMSO-d 6 ) 6 ppm 0.88 (quin., 6 H), 1.41-1.63 (m, 4 H), 3.16-3.40 (m, 7 H), 3.69-3.83 (m, 3 H), 7.50-8.56 (m, 2 H). In 30 Process Purity (HPLC (AUC @ 210nm) = 99.81%. Filter residue: 1 H NMR (400 MHz, methanol-d 4 ) 3.27-3.35 (m), 1.55-1.65 (q) 0.90-0.95 (t). MS m/z: 212 [M+H], consistent with data N-(4,6-bis(propylamino)-1,3,5-triazin-2-ol (CLXXVIII) (c.f., - 86 - WO 2013/181217 PCT/US2013/043052 Comparative Example 1). The isolated title compound contained no detectable (CLXXVIII). Stage 1: 5 IPC Test 1: Temperature Chart Recording during additions Maintain temperature control specified in process steps IPC Test 2: IPC HPLC analysis Reaction is complete when <2% of cyanuric chloride is detected. Repeat analysis every hour until reaction is complete. 10 Contact supervisor if reaction is not complete after third sample. Stage 1 Product QC testing: QC HPLC analysis Record results of assay of cyanuric chloride and 2-chloro-4,6-bis-n propylamino-s-triazine 15 Karl Fisher Record results Stage 2: Step 1: Reaction: 20 IPC Test 1: Temperature Chart Recording during additions Maintain temperature control specified in process steps IPC Test 2: IPC HPLC analysis Reaction is complete when <2% of 2-chloro-4,6-bis-n-propylamino-s-triazine is detected. 25 Repeat analysis every hour until reaction is complete. Contact supervisor if reaction is not complete after third sample. IPC Test 3: Residual amine analysis (by GC) n-Propylamine and N,O-dimethylhydoxylamine levels NMT 0.l1% IPC Test 4: Weight Change during drying 30 NMT 1% IPC Test 5: IPC HPLC analysis Purity by HPLC (AUC) record results Step 2: Salt Formation and Crystallization IPC Test 1: Temperature Chart Recording during concentrated sulfuric acid addition - 87 - WO 2013/181217 PCT/US2013/043052 Maintain temperature control specified in process step IPC Test 2: OVI of MEK and DMAc by GC NMT 800ppm each IPC Test 4: Weight Change during drying 5 NMT 1% Proton Nuclear Magnetic Resonance (NMR) Spectroscopv 1 H NMR data of N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine hydrogen sulfate (XXXVI) was obtained as a solution in 10 DMSO-d 6 at 400 MHz and is presented in Figure 10 and shift assignments are presented in Table 1. Table 1: 1 H Chemical shift assignments for (XXXVI) in DMSO-d 6 at 25 0 C Resonance Peak 6'H (ppm) Assignment 1 0.88 13, 17 2 1.53 12, 16 3 3.30 11,15 4 3.35 18 5 3.77 9 6 [7.42, 8.70] 10, 14 15 Carbon-1 3 NMR Spectroscopv "C NMR data of N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine hydrogen sulfate (XXXVI) was obtained as solutions in DMSO-d 6 at 100 MHz and is presented in Figure 11 and shift assignments are presented in Table 2. 20 Table 2: 13 C Chemical shift assignments for (XXXVI) in DMSO-d 6 at 25 0 C. Resonance peak 8 13 C (ppm) Assignment 1 11.25 13, 17 2 21.88 12, 16 3 34.08 18 4 42.24 11, 15 5 61.86 9 6 154.63 2 7 155.79 4,6 - 88 - WO 2013/181217 PCT/US2013/043052 Fourier Transform Infrared (FTIR) Spectroscopv The FTIR spectrum of N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl) N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) is presented in Table 3 and Figure 12. 5 Table 3: FTIR spectrum of (XXXVI) Wavenumber (cm 1 ) Assignment 3284 N-H stretch 2850-2960 C-H stretch 1615-1700 C=N bend 1536-1656 N-H bend 1020-1340 C-N stretch High and Low Resolution Mass Spectrometry The mass obtained from Liquid Chromatography-Mass Spectrometry 10 (LCMS) was 254 amu, which agrees with the theoretical mass of N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI). High-resolution results obtained through direct injection are presented in Table 4. Table 4: 15 Mass spec results for (XXXVI) Calculated Molecular Weight Theoretical Exact Mass Formula 254.1863 254.1855 CIIH 22
N
6 0 Chromatographic Puritv The HPLC chromatographic purity of the RS was determined to be 100.0% by area, with no related substances detected. 20 Water by Determination The water content was determined to be 0.04% by Karl Fischer titration. 25 Elemental Analvsis Elemental analysis of N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl) N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) was obtained and is presented in Table 5. - 89 - WO 2013/181217 PCT/US2013/043052 Table 5: Elemental analysis results for (XXXVI) Element Theoretical (%) Result (%) C 37.49 37.59 H 6.86 6.95 N 23.85 23.86 S 9.10 9.08 Thermal Analysis bv Differential Scanning Calorimetrv (DSC) 5 N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine hydrogen sulfate (XXXVI) was analyzed from 25 'C to 250 'C, at a rate of 10 'C per minute as per cUSP <891> / EP 2.2.34 and was found to have endotherms at 90.19 'C, 126.38 'C, and 138.30'C Figure 13. 10 X-rav Powder Diffraction XRPD diffraction pattern of N-(4,6-bis-n-propylamino-[1,3,5]triazin 2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) was obtained and is consistent with Form A; presented in Figure 14. 15 Counterion Content N-(4,6-bis-n-propylamino-[ 1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine hydrogen sulfate (XXXVI) was found to contain 27.13% sulfate content by titration. 20 pH ofAqueous Solution A 1% aqueous solution of N-(4,6-bis-n-propylamino-[1,3,5]triazin-2 yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) yielded a pH of 1.89. Physical Description 25 N-(4,6-bis-n-propylamino-[ 1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine hydrogen sulfate (XXXVI) was determined to be a white crystalline solid. Example 10: - 90 - WO 2013/181217 PCT/US2013/043052 N-(4-(Methoxy(methyl)amino)-6-(propylamino)- 1,3,5-triazin-2-yl)propionamide (XL) CI CI CI N N NH 3
/H
2 0 N N H 2 N N N CI'N CI H2N N CI H2N N N H XVIII XXXVII XXXVIII
H
3
C-N-O-CH
3
H
3 C N 0-CH3 O H3C N O-CH 3 H N NaOH "N N CI N N O DIPEA, EtOH - N < N NH 2 N 1 N N H I H H XXXIX XL Scheme 16 5 6-Amino-2,4-dichloro-[1,3,5]triazine (XXVII) 2,4,6-Trichloro-1,3,5-triazine (XVIII) (10.0 g, 55 mmol) was dissolved in acetone (80 mL) and poured into ice-water (80 mL) to form a very fine suspension. To this mixture, 1 N ammonium hydroxide solution (108 mL, 109.4 mmol) was added 10 at 0 0 C. The reaction was stirred for 30 min at ambient temperature and for additional 60 min at 25 'C. The precipitate was filtered off, washed with water (3 x 25 mL). After drying over calcium chloride under high vacuum, 6-amino-2,4-dichloro [1,3,5]triazine (XXXVII) was isolated as white powder (7.4 g, 82% yield). LCMS (ESI) m/z = 165 (M+H). 15 6-Amino-2-chloro-4-n-propylamino-[1,3,5]triazine (XXXVIII) 6-Amino-2,4-dichloro-[1,3,5]triazine (XXXVII) (30.0 g, 187 mmol) was dissolved in acetone (100 mL) and poured into ice-water (100 mL) to form a very fine suspension. To this mixture, a solution of propan-1-amine (11.0 g, 187 mmol) in 20 acetone (20 mL) was added at 0 0 C. To this reaction, 2 N NaOH (94 mL, 187 mmol) was added dropwise at a rate to keep the temperature between 0 0 C and 5'C. The mixture was stirred for 30 min at ambient temperature and for an additional 60 min at 50'C. The mixture was concentrated and then the precipitate was filtered off and washed with water (3 x 100 mL). After drying over calcium chloride under high 25 vacuum, 6-amino-2-chloro-4-n-propylamino-[1,3,5]triazine (XXXVIII) was isolated as a white powder (35 g, 100% yield). LCMS (ESI) m/z = 188 (M+H). -91 - WO 2013/181217 PCT/US2013/043052 N-(6-Amino-4-n-propylamino-[1, 3,5]triazin-2-yl)-N, 0-dimethyl-hydroxylamine (XX7X) A mixture of 6-amino-2-chloro-4-n-propylamino-[1,3,5]triazine 5 (XXXVIII) (5 g, 26.65 mmol), N,O-dimethylhydroxylamine hydrochloride (13 g, 133.24 mmol) and DIPEA (27.5 g, 213.2 mmol) in EtOH (100 mL) was heated at 100 'C for 16 h, after which time the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (400 mL), which was washed with water (200 mL) and then with a brine solution (200 mL) and finally dried over Na 2
SO
4 . The solvent 10 was removed under reduced pressure to yield N-(6-amino-4-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXIX) as a white solid (5 g, 89% yield). LCMS (ESI) m/z = 213 (M+H)+. N-(4-(Methoxy(methyl)amino)-6-(propylamino)-1, 3,5-triazin-2-yl)propionamide (XL) 15 N-(6-amino-4-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine (XXXIX) (5 g, 23.58 mmol) was dissolved in THF (50 mL). Propionyl chloride (3.25 g, 35.38 mmol) and DIPEA (5.47 g, 42.44 mmol) were added at 0 'C. The resultant mixture was stirred at ambient temperature for 10 min, then stirred at 70 'C for 16 h, after which time the solvent was removed under reduced 20 pressure. The residue was dissolved in EtOAc (250 mL), and this extract was washed with water (80 mL) and then with a brine solution (80 mL), and lastly dried over Na 2
SO
4 . The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography (pet ether/ethyl acetate = 5/1 to 2/1) to yield 930 mg (15%) of the desired product. The isolated free amine (930 mg, 3.47 mmol) 25 was dissolved in H 2 0 (10 mL) and 0.5 M HCl solution in H 2 0 (10.4 mL) and then the solution was lyophilized to yield N-(4-(methoxy(methyl)amino)-6-(n-propylamino) 1,3,5-triazin-2-yl)propionamide (XL) as a colorless oil (1.06 g). LCMS: (ESI) m/z = 269 (M+H). 1 H NMR (500 MHz, DMSO): 6 (ppm) 12.23 (s, 1H), 8.25-9.45 (m, 2H), 3.76-3.84 (m, 3H), 3.30-3.44 (m, 5H), 2.55-2.56 (m, 1H), 2.21-2.22 (m, 1H), 30 1.53-1.58 (m, 2H), 0.88-1.08 (m, 6H). Example 11: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-O-methyl-hydroxylamine (XLI) - 92 - WO 2013/181217 PCT/US2013/043052 Example 12: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-vl)-O-methyl-hydroxylamine hydrochloride (XLII) C1 ~NH 2 CI H-N-O-CH 3 N )N NaOH/H 2 0 N N NaOH 1) 1 -) '- I N)IIN N - ' 1,4-dioxane CI N C1 acetone H H XVIII XXXIV H, ,O-CH 3 H, N O-CH 3 N N N NH N N C N ), N ,, N Et 2 O / dioxane NN H H XLI XLII 5 Scheme 17 2-Chloro-N-(4, 6-bis-(n-propylamino)-[1,3,5]triazine (XXIV) A 2 M NaOH solution (163 mL, 325.36 mmol) was added in a dropwise manner to a suspension of 2,4,6-trichloro-1,3,5-triazine (XVIII) (30.0 g, 10 162.68 mmol) and n-propylamine (26.8 mL, 325.36 mmol) in acetone (600 mL) and water (30 mL) at 0 0 C (water - ice/NaCl bath). The ice bath was removed and the reaction mixture was heated at 50 'C for 3 h, then cooled. Water (200 mL) was added to the reaction mixture; the precipitate was filtered, washed with water (200 mL) and dried over P 2 0 5 at 40'C for 20 h to yield 2-chloro-N-(4, 6-bis-(n 15 propylamino)-[1,3,5]triazine (XXXIV, 33.6 g, 90% yield). 400 MHz H NMR (DMSO-d 6 , ppm) 7.80 (0.85H, t, J=5.5 Hz), 7.76-7.66 (lH, m), 7.49 (0.15H, t, J=5.5 Hz), 3.22-3.11 (4H, m), 1.55-1.42 (4H, m), 0.88-0.82 (6H, m). ESI-MS (m/z): 230, 232 [M+H]+. 20 N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-methyl-hydroxylamine (XLI) A mixture of 6-chloro-N,N'-dipropyl-[1,3,5]triazine-2,4-diamine (XXXIV) (2.30 g, 10.01 mmol), 0-methyl-hydroxylamine hydrochloride (1.67 g, 20.02 mmol) and NaOH (0.72 g, 18.00 mmol) in 1,4-dioxane (30 mL) and water (6 mL) was heated at 60 'C for 3 h. After this time, NaOH (0.72 g, 18.00 mmol) was 25 added and the reaction mixture was heated for another 3 h. The volatiles were removed under reduced pressure. Saturated NaHCO 3 solution (100 mL) was added to - 93 - WO 2013/181217 PCT/US2013/043052 the residue, the mixture was extracted with EtOAc (3 x 25 mL). The combined organic extracts were washed with water (50 mL), brine (50 mL) and dried over Na 2
SO
4 . The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography using gradient elution from CH 2 Cl 2 /EtOH 5 (99:1) to CH 2 Cl 2 /EtOH (95:5) to yield 2.17 g (90%) of N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-O-methyl-hydroxylamine (XLI). ESI-MS (m/z): 241 [M+H]+ N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-O-methyl-hydroxylamine hydrochloride (XLII) 10 A 2M HCl /ethyl ether (4.5 mL, 9.00 mmol) was added to the solution N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-O-methyl-hydroxylamine (XLI) (2.17 g, 9.03 mmol) in 1,4-dioxane (5 mL) at 0 0 C. The mixture was stirred for 0.5 h at 0 0 C, volatiles were removed under reduced pressure to yield N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-O-methyl-hydroxylamine hydrochloride (XLII) in quantitative 15 yield. 400 MHz 1 H NMR (DMSO-d 6 , ppm) 12.5-11.5 (2H, br s), 8.49 (lH, br s), 8.34 (lH, br s), 3.71 (3H, s), 3.34-3.16 (4H, m), 1.59-1.46 (4H, m), 0.94-0.83 (6H, m). ESI-MS (m/z) 241 [M+H]f. Example 13: 20 0-Allyl-N-(4,6-bis-n-propylamino-[1,3,51triazin-2-yl)-hydroxylamine (XLIII) Example 14: O-Allyl-N-(4,6-bis-n-propylamino-[1,3,51triazin-2-yl)-hydroxylamine hydrochloride salt (XLIV) CI H 2 N-O HN H N N N NaOH N )N HCI NAN C '-N N ''N 1,4-dioxane ,^ NNs Et 2 O / dioxane H H XXXIV XLIII XLIV 25 Scheme 18 O-Allyl-N-(4,6-bis-n-propylamino-[1, 3,5]triazin-2-yl)-hydroxylamine A mixture of 2-chloro-N-(4, 6-bis-(n-propylamino)-[1,3,5]triazine 30 (XXXIV) (2.00 g, 8.71 mmol), 0-allyl-hydroxylamine hydrochloride (1.91 g, 17.42 - 94 - WO 2013/181217 PCT/US2013/043052 mmol) and NaOH (0.70 g, 17.42 mmol) in 1,4-dioxane (25 mL) and water (5 mL) was heated at 60 'C for 4 h. The volatiles were removed under reduced pressure. Saturated NaHCO 3 solution (100 mL) was added to the residue and the mixture was extracted with EtOAc (3 x 25 mL). The combined organic extracts were washed with 5 water (50 mL), brine (50 mL) and dried over Na 2
SO
4 . The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography using gradient elution from CH 2 Cl 2 /EtOH (99:1) to CH 2 Cl 2 /EtOH (95:5) to yield O-allyl-N-(4,6-bis-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine (XLIII, 2.05 g, 88% yield). ESI-MS (m/z) 267 [M+H]f. 10 0-Allyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine hydrochloride O-Allyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine hydrochloride (XLIV) was prepared from O-allyl-N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-hydroxylamine (XLIII) and 2M HCl /ethyl ether as described in 15 Example 12. 400 MHz H NMR (DMSO-d 6 , ppm) 11.7-10.0 (lH, m), 7.9-7.1 (2H, m), 6.09-5.92 (1H, m), 5.39-5.18 (2H, m), 4.35 (2H, d, J=6.0 Hz), 3.28-3.11 (4H, m), 1.56-1.42 (4H, m), 0.91-0.81 (6H, m). ESI-MS (m/z): 267 [M+H]+. MP: 130 132 0 C. 20 Example 15: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-hydroxylamine (XLV) CI H 2 N-OH H N ,OH N )N NaOH N H 1,4-dioxane N N N H H XXXIV XLV Scheme 19 25 N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine (XLV) was prepared from 2-chloro-N-(4, 6-bis-(n-propylamino)-[1,3,5]triazine (XXXIV) and hydroxylamine hydrochloride as described in Example 13 (99% yield). 400 MHz H NMR (DMSO-d 6 , ppm) 9.0-8.6 (lH, br s), 8.39-8.14 (lH, s), 6.89-6.55 (2H, m), 3.23-3.06 (4H, m), 1.54-1.40 (4H, m), 0.84 (6H, t, J=7.4 Hz). ESI-MS (m/z): 227 30 [M+H]f. MP: 138-141 0 C. - 95 - WO 2013/181217 PCT/US2013/043052 Example 16 N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-vl)-N',N'-dimethylhydrazine (XLVI) N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N',N' 5 dimethylhydrazine (XLVI) may be prepared from 2-chloro-N-(4, 6-bis-(n propylamino)-[1,3,5]triazine (XXXIV) and N,N-dimethylhydrazine as described in Example 19. HN' N N ~N N N N H H XLVI Scheme 20 Example 17 10 6-(Methoxv(methyllamino)-N2-propyl-1,3,5-triazine-2,4-diamine (XLVII)
H
3 C N ,OCH 3 N N H2N N NW H Scheme 21 6-(Methoxy(methyl)amino)-N2-propyl-1,3,5-triazine-2,4-diamine (XLVII) may be prepared from 6-amino-2-chloro-4-n-propylamino-[1,3,5]triazine 15 (XXXVIII) and N,O-dimethylhydroxylamine as described in Example 10. Example 18: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N-methyl-hydroxylamine (XLVIII) C1 H 3 C-N-OH H 3 C, N ,OH N N H H NaOH N N N 1,4-dioxane H H XXXIV XLVIII 20 Scheme 22 N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine (XLVIII) was prepared from 2-chloro-N-(4, 6-bis-(n-propylamino) [1,3,5]triazine (XXXIV) and N-methyl-hydroxylamine hydrochloride as described in - 96 - WO 2013/181217 PCT/US2013/043052 Example 13 (90% yield). 400 MHz H NMR (DMSO-d 6 , ppm) 8.93 (1H, s), 6.92 6.43 (2H, m), 3.23-3.07 (7H, m), 1.55-1.38 (4H, m), 0.84 (6H, t, J=7.4 Hz). ESI-MS (m/z) 241 [M+H]+. 5 Example 19: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N,N'-dimethyl-hydrazine (XLIX) Example 20: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N,N'-dimethyl-hydrazine hydrogen sulfate (L) H H H C1 H 3
C-N-N-CH
3
H
3 C, N-CH 3
H
3 C, ,N-CH 3 AH N HN 8 N ), N NaOH )I H 2 S0 4 )I9 N N N N O-S-O-H N N N""'-- 1,4-dioxane N N N 1,4-dioxane -^ N H H 10 XXXIV XLIXL 10 Lxi u Scheme 23 N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,N'-dimethyl-hydrazine (XLIX) 15 A mixture of 2-chloro-N-(4, 6-bis-(n-propylamino)-[1,3,5]triazine (XXXIV) (2.50 g, 10.88 mmol), N,N'-dimethyl-hydrazine dihydrochloride (2.89 g, 21.76 mmol) and NaOH (2.18 g, 54.40 mmol) in 1,4-dioxane (40 mL) and water (20 mL) was heated at 60 0 C for 18 h. The volatiles were removed under reduced pressure. Saturated NaHCO 3 solution (100 mL) was added to the residue, the mixture 20 was extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with water (75 mL), brine (75 mL) and dried over Na 2 SO4. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography using gradient elution (CH 2 Cl 2 /EtOH (99:1) to CH 2 Cl 2 /EtOH (95:5)) to yield N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,N'-dimethyl-hydrazine (1.17 25 g, 42%). 200 MHz 1 H NMR (DMSO-d 6 , ppm): 6.81-6.44 (2H, m), 5.31 (1H, br s), 3.24-3.08 (4H, m), 3.05 (3H, s), 2.47-2.40 (3H, m), 1.57-1.37 (4H, m), 0.84 (6H, t, J=7.4 Hz). ESI-MS (m/z): 254 [M+H]+. N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,N'-dimethyl-hydrazine hydrogen 30 sulfate_(L) -97- WO 2013/181217 PCT/US2013/043052 95% H 2
SO
4 (0.26 mL, 4.62 mmol) was added dropwise to the solution of 6-(N,N'-dimethyl-hydrazino)-N,N'-dipropyl-[1,3,5]triazine-2,4-diamine (XLIX) (1.17 g, 4.62 mmol) in 1,4-dioxane (10 mL) at 0 0 C. The mixture was stirred for 0.5 h at room temperature; volatiles were removed under reduced pressure. The residue 5 was co-evaporated with dry toluene (3 x 25 mL) to yield N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-N,N'-dimethyl-hydrazine hydrogen sulfate (L) in quantitative yield. 400 MHz H NMR (DMSO-d 6 , ppm) 8.48-8.32 (lH, m), 7.9-7.7 (0.5H, br s), 7.70-7.61 (0.5H, m), 3.34-3.20 (4H, m), 3.21 (1.5H, s), 3.17 (1.5H, s), 2.52 (1.5H, s), 2.51 (1.5H, s, overlapped with DMSO), 1.59-1.46 (4H, m), 0.93-0.82 (6H, m). ESI 10 MS (m/z): 254 [M+H]+. Example 21: O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,51triazin-2-yl)-N-methyl-hydroxylamine (LIII) 15 Example 22: O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,51triazin-2-yll-N-methyl-hydroxylamine hydrogen sulfate salt (LIV) H2N'
H
2 C=O H2C N'O HCI HCI LI CI H3C N O
H
3 C + N N N N H3,N J -- NI 1NIl --- NaOHNN N NNN N H H 1,4-dioxane I L1l XXXIV LIII Scheme 24 20 Formaldehyde O-benzyl-oxime (LI) A NaOH (1.25 g, 31.32 mmol) solution in water (6 mL) was added to the mixture of O-benzyl-hydroxylamine hydrochloride (5.00 g, 31.32 mmol) and formaldehyde (~37 wt. % in H 2 0) (2.3 mL, 31.32 mmol) in toluene (40 mL). The 25 reaction mixture was stirred at room temperature for 1 h. After this time, the organic phase was separated and the water phase was extracted with dichloromethane (3 x 30 mL). The combined organic phases were dried over Na 2
SO
4 , and concentrated in vacuo to yield formaldehyde O-benzyl-oxime (LI, 4.15 g, 98%). 400 MHz H NMR - 98 - WO 2013/181217 PCT/US2013/043052 (CDCl 3 , ppm) 7.40-7.29 (5H, m), 7.09 (lH, d, J=8.2 Hz), 6.47 (lH, d, J=8.2 Hz), 5.14 (2H, s). O-Benzyl-N-methyl-hydroxylamine (LII) 5 A IM HCl/EtOH solution (50 mL) was added dropwise to the solution of formaldehyde O-benzyl-oxime (3.85 g, 28.48 mmol) in EtOH at 0 0 C. The mixture was stirred at room temperature for 1 hour, volatiles were removed in vacuo. The residue was dissolved in dichloromethane (100 mL), washed with saturated NaHCO 3 solution (75 mL), water (75 mL), and dried over Na 2
SO
4 . The product was purified 10 by flash column chromatography using gradient elution from petroleum ether/EtOAc (9:1) to petroleum ether/EtOAc (7:1) to yield O-benzyl-N-methyl-hydroxylamine (1.72 g, 44%). 200 MHz 1 H NMR (CDCl 3 , ppm) 7.40-7.27 (5H, m), 5.53 (1H, br s), 4.71 (2H, s), 2.73 (3H, s). 15 O-Benzyl-N-(4,6-bis-n-propylamino-[1, 3,5]triazin-2-yl)-N-methyl-hydroxylamine (LIII) 2-Chloro-N-(4, 6-bis-(n-propylamino)-[ 1,3,5]triazine (XXXIV) and 0 benzyl-N-methyl-hydroxylamine (LII) were reacted as described in Example 13 to yield O-benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl 20 hydroxylamine (LIII) (29% yield). 200 MHz 1 H NMR (DMSO-d 6 , ppm) 7.52-7.28 (5H, m), 7.07-6.67 (2H, m), 4.93 (2H, s), 3.26-3.03 (7H, m), 1.58-1.39 (4H, m), 0.85 (6H, t, J=7.2 Hz). ESI-MS (m/z): 331 [M+H]f. O-Benzyl-N-(4,6-bis-n-propylamino-[1, 3,5]triazin-2-yl)-N-methyl-hydroxylamine 25 hydrogen sulfate (LIV) O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine (LIII) was reacted with 95% H 2
SO
4 as described in Example 20 to yield O-benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine hydrogen sulfate (LIV) in quantitative yield. 400 MHz IH NMR 30 (DMSO-d 6 , ppm) 12.0-10.9 (lH, br s), 8.7-8.3 (lH, br s), 7.56-7.46 (2H, m), 7.46 7.37 (2.5H, m), 7.36-7.30 (0.5H, m), 5.07-4.95 (2H, m), 3.44-3.16 (7H, m), 1.61-1.45 (4H, m), 0.94-0.82 (6H, m). ESI-MS (m/z): 331 [M+H]+. - 99 - WO 2013/181217 PCT/US2013/043052 Example 23: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yll-N-isopropyl-hydroxylamine (LV) Example 24: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yll-N-isopropyl-hydroxylamine hydrogen 5 sulfate (LVI) ,N O N "N ",-N 'ilN 1,N- - LV Scheme 25 N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-isopropyl-hydroxylamine (LV) 2-Chloro-N-(4, 6-bis-(n-propylamino)-[ 1,3,5]triazine (XXXIV) and N 10 isopropyl-hydroxylamine hydrochloride were reacted as described in Example 13 to yield N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-isopropyl-hydroxylamine (LV) (61% yield). ESI-MS (m/z): 269 [M+H]. N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-isopropyl-hydroxylamine hydrogen sulfate (LVI) 15 N-(4,6-Bis-n-propylamino-[ 1,3,5]triazin-2-yl)-N-isopropyl hydroxylamine hydrogen sulfate (LVI) was prepared from N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N-isopropyl-hydroxylamine (LV) and 95% H 2
SO
4 as described in Example 20 (95% yield). 200 MHz H NMR (DMSO-d 6 , ppm) 11.5-11.1 (lH, br s), 10.66-10.40 (lH, m), 8.45 (lH, s), 7.75-7.36 (lH, m), 4.77-4.55 (lH, m), 3.30 -3.16 20 (4H, m), 1.61-1.44 (4H, m), 1.17 (6H, t, J=7.0 Hz), 0.89 (3H, t, J=7.3 Hz), 0.86 (3H, t, J=7.3 Hz). ESI-MS (m/z) 269 [M+H]f. M.P.: 154-156C. Example 25: 6-[1,21Oxazinan-2-yl-N,N'-dipropyl-[1,3,51triazine-2,4-diamine (LVII) 25 Example 26: 6-[1,21Oxazinan-2-yl-N,N'-di-n-propyl-[1,3,51triazine-2,4-diamine hydrogen sulfate (LVIII) - 100 - WO 2013/181217 PCT/US2013/043052 N Oe CI H N N )N DPEA N N H 2
SO
4 N N HSO 4 N N N- THF N N N dioxane N N N H H H H XXXIV LVll LVlll Scheme 26 6-[1,2]Oxazinan-2-yl-N,N'-di-n-propyl-[1,3,5]triazine-2,4-diamine (LVII) 5 An ACE* pressure tube was charged with 2-chloro-N-(4,6-bis-(n propylamino)-[1,3,5]triazine (XXXIV) (1.50 g, 6.53 mmol), N-ethyldiisopropylamine (19.59 mmol), 1,2-oxazinane hydrochloride (1.61 g, 13.06 mmol) and tetrahydrofuran. The reaction mixture was heated at 100 'C for 2 h, then cooled and poured into saturated NaHCO 3 solution (50 mL). The suspension was extracted with 10 EtOAc (3 x 25 mL). The combined organic extracts were washed with water (50 mL), brine (50 mL) and dried over Na 2 SO4. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography using gradient elution from CH 2 Cl 2 /EtOH (99:1) to CH 2 Cl 2 /EtOH (95:5) to yield 6 [1,2]oxazinan-2-yl-N,N'-di-n-propyl-[1,3,5]triazine-2,4-diamine (LVII) (1.63 g, 15 89%). ESI-MS (m/z): 281 [M+H]f. 6-[1,2]Oxazinan-2-yl-N,N'-di-n-propyl-[1,3,5]triazine-2,4-diamine hydrogen sulfate (LVIII) 6-[1,2]Oxazinan-2-yl-N,N'-di-n-propyl-[ 1,3,5]triazine-2,4-diamine 20 hydrogen sulfate (LVIII) was prepared from 6-[1,2]oxazinan-2-yl-N,N'-di-n-propyl [1,3,5]triazine-2,4-diamine (LVII) and 95% H 2
SO
4 as described in Example 20. Quantitative yield was isolated. 400 MHz 1 H-NMR (DMSO-d 6 , ppm) 11.6-11.3 (lH, br s), 8.61-8.41 (0.8H, m), 8.18-8.03 (0.2H, m), 7.63-7.28 (lH, m), 4.14-4.08 (2H, m), 3.92-3.81(2H, m, overlapped with water), 3.36-3.19 (4H,m), 1.86-1.78 (2H, m), 1.77 25 1.68 (2H, m), 1.6-1.45 (4H, m), 1.60-1.45 (6H, m). ESI-MS (m/z): 281 [M+H]f. M.P.: 134-137 0 C. - 101 - WO 2013/181217 PCT/US2013/043052 Example 27: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-O-isopropyl-N-methyl-hydroxylamine (LXIV) Example 28: 5 N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-O-isopropyl-N-methyl-hydroxylamine hydrogen sulfate (LXV) 2-Isopropoxy-isoindole-1,3-dione (LIX) Diethyl azodicarboxylate (14.5 mL, 73.56 mmol) was added dropwise 10 at 0 'C to a stirred suspension of propan-2-ol (4.7 mL, 61.30 mmol), triphenylphosphine (19.30 g, 73.56 mmol), and N-hydroxyphthalimide (10.00 g, 61.30 mmol) in THF (50 mL). The mixture was stirred at room temperature for 20 h and evaporated to dryness. The product was purified by flash column chromatography using gradient elution from petroleum ether/EtOAc (9:1) to petroleum ether/EtOAc 15 (5:1) to yield 2-isopropoxy-isoindole-1,3-dione (LIX, 10.92 g, 87%). 400 MHz H NMR (DMSO-d 6 , ppm): 7.86 (4H, s), 4.44 (lH, septet, J=6.2 Hz), 1.28 (6H, d, J=6.2 Hz). 0 DEAD, PPh 3 1) H 2
N-NH
2 CI O''Ph I N-OH - N-O H2N-O 2-PrOH / THF 2) HCI HCI LIX LX h0CH 3 -1, K 2 C0 3 HBr /AcOH Ph'O N-O Ph'0 N-O , HN-O HCI H acetone
CH
3 CH 3 LXI LXII LXIII CI LXIII H 3 C N 0 H 3 C N O N )N NaOH / H 2 0 N NN H 2
SO
4 N '1N N,--N N N--"-," 1,4-dioxane '^N N N'-- 1,4-dioxane N 'K N N H H H H XXXIV LXIV eO O-S-0-H LXV O 20 Scheme 27 0-Isopropyl-hydroxylamine hydrochloride (LX) - 102 - WO 2013/181217 PCT/US2013/043052 A mixture of 2-isopropoxy-isoindole-1,3-dione (LIX, 10.78 g, 52.50 mmol) and hydrazine monohydrate (5.1 mL, 105.00 mmol) in CH 2 Cl 2 (60 mL) was stirred at room temperature for 20 h. The reaction mixture was filtered. The filtrate was washed with water (70 mL), brine (70 mL) and dried over Na 2
SO
4 . After 5 removing the drying agent via filtration, 4M HCl/1,4-dioxane (13.8 mL, 55.00 mmol) was added and the volatiles was removed under reduced pressure to yield 0 isopropyl-hydroxylamine hydrochloride (LX, 3.91 g, 67% yield). 400 MHz IH NMR (DMSO-d 6 , ppm) 11.04 (3H, br s), 4.35 (lH, septet, J=6.2 Hz), 1.21 (6H, d, J=6.2 Hz). 10 O-Benzyl-N-isopropoxy carbamate (LXI) To a pre-cooled (0 'C) solution of O-isopropyl-hydroxylamine hydrochloride (3.89 g, 34.87 mmol) in CH 2 Cl 2 (150 mL) was added N,N-diisopropyl ethylamine (14.4 mL, 87.18 mmol) and benzyl chloroformate (5.0 mL, 34.87 mmol). 15 The resulting solution was stirred at room temperature for 5 h. At this time the solution was washed twice with saturated aqueous NaHCO 3 (30 mL) and dried over Na 2
SO
4 . The product was purified by flash column chromatography using gradient elution from petroleum ether/EtOAc (95:5) to petroleum ether/EtOAc (6:1) to yield O-benzyl-N-isopropoxycarbamate (LXI, 4.98 g, 68%). 400 MHz 1 H NMR (DMSO 20 d 6 , ppm) 10.22 (lH, s), 7.42-7.29 (5H, m), 5.07 (2H, s), 3.89 (lH, septet, J=6.2 Hz), 1.11 (6H, d, J=6.2 Hz). O-Benzyl-N-methyl-N-isopropoxy carbamate (LXII) An ACE* pressure tube was charged with benzyl isopropoxycarbamate 25 (4.98 g, 23.80 mmol), anhydrous K 2
CO
3 (4.94 g, 35.70 mmol), methyl iodide (6.7 mL, 107.10), and anhydrous acetone (30 mL). The reaction mixture was heated at 70 'C for 24 h. The reaction mixture was filtered, and the acetone was evaporated. The resulting slurry was dissolved in EtOAc, washed with water (3 x 50 mL), dried (Na 2 SO4), and filtered. The solvent was removed to yield benzyl 30 isopropoxy(methyl)carbamate (4.96 g, 93%). 400 MHz 1H-NMR (DMSO-d6, ppm) 7.41-7.30 (5H, m), 5.12 (2H, s), 4.08 (1H, septet, J=6.2 Hz), 3.08 (3H, s), 1.12 (6H, d, J=6.2 Hz). O-Isopropyl-N-methyl-hydroxylamine hydrochloride (LXIII) - 103 - WO 2013/181217 PCT/US2013/043052 O-Benzyl-N-methyl-Nisopropoxy carbamate (4.96 g, 22.22 mmol) and 33% HBr/AcOH (45 mL) were stirred at room temperature for 20 min. Saturated solution of NaHCO 3 (400 mL) was added, the suspension was extracted with CH 2 Cl 2 (3 x 150 mL). The combined organic extracts were dried over Na 2 S04. After 5 removal of the drying agent via filtration, 4M HCI/1,4-dioxane (6.7 mL, 26.65 mmol) was added, and the volatiles was removed under reduced pressure to yield 0 isopropyl-N-methyl-hydroxylamine hydrochloride (2.09 g, 75%). 400 MHz IH NMR (DMSO-d 6 , ppm) 12.3-11.7 (2H, br s), 4.49 (lH, septet, J=6.1 Hz), 2.77 (3H, s), 1.12 (6H, d, J=6.1 Hz). 10 N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-O-isopropyl-N-methyl-hydroxylamine (LXIV) A mixture of 2-chloro-N-(4,6-bis-(n-propylamino)-[1,3,5]triazine (XXXIV) (1.65 g, 16.61 mmol), 0-isopropyl-N-methyl-hydroxylamine hydrochloride 15 (LXIII, 2.09 g, 16.61 mmol) and NaOH (0.66 g, 16.61 mmol) in 1,4-dioxane (50 mL) and water (5 mL) was heated at 100 0 C for 16 h. The volatiles were then removed under reduced pressure. Saturated NaHCO 3 solution (50 mL) was added to the residue and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with water (50 mL), brine (50 mL) and dried over 20 Na 2
SO
4 . The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography using gradient elution from CH 2 Cl 2 /EtOH (99:1) to CH 2 Cl 2 /EtOH (95:5) to yield N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl) 0-isopropyl-N-methyl-hydroxylamine (LXIV, 1.93 g, 95%). ESI-MS (m/z): 283 [M+H]. 25 N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-O-isopropyl-N-methyl-hydroxylamine hydrogen sulfate (LXV) To a solution N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-O isopropyl-N-methyl-hydroxylamine (LXIV, 1.93 g, 6.83 mmol) in 1,4-dioxane (6 mL) 30 at 0 0 C was added 95% H 2
SO
4 (0.36 mL, 6.83 mmol) in a drop-wise manner. The mixture was stirred for 0.5 h at room temperature and then the volatiles were removed under reduced pressure. The residue was co-evaporated with dry toluene (3 x 25 mL) to yield N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-N-methyl hydroxylamine hydrogen sulfate (~quantitative yield). 400 MHz 1 H-NMR (DMSO - 104 - WO 2013/181217 PCT/US2013/043052 d 6 , ppm) 11.3-10.7 (lH, br s), 8.8-8.4 (1H, br s), 8.2-8.0 (0.3H, br s), 8.04-7.65 (0.7H, m), 4.43-4.28 (1H, m), 3.42-3.18 (7H, m), 1.64-1.44 (4H, m), 1.25 (6H, d, J=6.1 Hz), 0.94-0.82 (6H, m). ESI-MS (m/z): 283 [M+H]f. 5 Example 29: O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,51triazin-2-yl)-N-ethyl-hydroxylamine (LXVIII) Example 30: O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,51triazin-2-yll-N-ethyl-hydroxylamine 10 hydrogen sulfate (LXIX) 0 Ph H 3 C H Ph NaBH 3 CN Ph
H
2
N-O-
1 'd -- NO -HCI H 2 0 / MeOH H AcOH H LXVI LXVII Ph Ph CI 22 N OO N )N NaOH / H 2 0 A H 2
SO
4 N N N 1,4-dioxane , N Nl N 1,4-dioxane -^ N H H XXXXIV LXVIII eO O-S-0-H 6 LXIX Scheme 28 15 O-Benzyl-N-vinyl-hydroxylamine (LXVI) Acetaldehyde (24.7 mL, 44.2 mmol) was added dropwise to the cooled solution (0 0 C) of O-benzyl-hydroxylamine hydrochloride (7.00 g, 43.85 mmol) in water (100 mL) and MeOH (20 mL). The reaction mixture was stirred for 16 h. The volatiles were removed under reduced pressure, and the water suspension was 20 extracted with EtOAc (2 x 75 mL). The combined organic extracts were washed with brine, and then dried over Na 2
SO
4 and evaporated to yield O-benzyl-N-vinyl hydroxylamine (LXVI) in quantitative yield. 400 MHz 1 H NMR (DMSO-d 6 , ppm) 7.48 (0.5 H, q, J=5.8 Hz), 7.39-7.26 (5H, m), 6.86 (0.5 H, q, J=5.5 Hz), 5.06 (lH, s), 4.97 (lH, s), 1.78 (1.5H, d, J=5.5 Hz), 1.76 (1.5H, d, J=5.8 Hz). 25 - 105 - WO 2013/181217 PCT/US2013/043052 O-Benzyl-N-ethyl-hydroxylamine (LXVII) To a solution of O-benzyl-N-vinyl-hydroxylamine (LXVI, 6.52 g, 43.70 mmol) in AcOH (10 mL), NaCNBH 3 (11.00 g, 175.05 mmol) was added in portions. The reaction mixture was stirred at room temperature for 1 h. The mixture 5 was neutralized (pH 7) with IN NaOH and extracted with EtOAc (3 x 75 mL). The combined organic extracts were washed with saturated NaHCO 3 solution (2 x 100 mL), dried over Na 2
SO
4 , and purified by flash column chromatography (eluent: petroleum ether/EtOAc (9:1) to petroleum ether/EtOAc (1:4)) to yield O-benzyl-N ethyl-hydroxylamine (LXVII, 2.10 g, 32%). 400 MHz H NMR (DMSO-d 6 , ppm) 10 7.36-7.24 (5H, m), 6.50 (1H, t, J=6.4 Hz), 4.60 (2H, s), 2.81 (2H, qd, J=7.0, 6.4 Hz), 0.98 (3H, t, J=7.0 Hz). O-Benzyl-N-(4,6-bis-n-propylamino-[1, 3,5]triazin-2-yl)-N-ethyl-hydroxylamine (LXVIII) 15 2-Chloro-N-(4, 6-bis-(n-propylamino)-[ 1,3,5]triazine (XXXIV) and 0 benzyl-N-ethyl-hydroxylamine (LXVII) were reacted as described in Example 13 to afford O-benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl hydroxylamine (LXVIII, 38% yield). 400 MHz 1 H NMR (DMSO-d 6 , ppm) 7.51-7.45 (2H, m), 7.40-7.30 (3H, s), 6.97-6.85 (1H, m), 6.79-6.67 (1H, m), 4.97-4.87 (1H, m), 20 3.72-3.53 (2H, m), 3.23-3.11 (4H, m), 1.56-1.43 (4H, m), 1.13-1.00 (3H, m), 0.89 0.80 (6H, m). ESI-MS (m/z): 345 [M+H]+. O-Benzyl-N-(4,6-bis-n-propylamino-[1, 3,5]triazin-2-yl)-N-ethyl-hydroxylamine hydrogen sulfate (LXIX) 25 O-Benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl hydroxylamine (LXVIII) was reacted with 95% H 2
SO
4 as described in Example 9 to yield O-benzyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-methyl hydroxylamine hydrogen sulfate (LXIX) in quantitative yield. 400 MHz IH NMR (DMSO-d 6 , ppm) 12.0-11.0 (1H, br s), 8.7-8.0 (1H, m), 7.57-7.30 (5H, m), 5.07-4.95 30 (2H, m), 3.89-3.68 (2H, m), 3.39-3.14 (4H, m), 1.63-1.42 (4H, m), 1.23-1.07 (3H, m), 0.94-0.77 (6H, m). ESI-MS (m/z): 345 [M+H]+. Example 31: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-vl)-O-isopropyl-hydroxylamine (LXX) - 106 - WO 2013/181217 PCT/US2013/043052 Example 32: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yll-O-isopropyl-hydroxylamine hydrogen sulfate (LXXI) H N'0 N )IN N )IN N''' H H LXX Scheme 29 5 N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-hydroxylamine (LXX) 2-Chloro-N-(4,6-bis-(n-propylamino)-[1,3,5]triazine (XXXIV) and 0 isopropyl-hydroxylamine hydrochloride were reacted as described in Example 13 (80% yield). ESI-MS (m/z): 269 [M+H]f. 10 N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isopropyl-hydroxylamine hydrogen sulfate (LXXI) N-(4,6-Bis-n-propylamino-[ 1,3,5]triazin-2-yl)-O-isopropyl hydroxylamine (LXX) was reacted with 95% H 2
SO
4 as described in Example 20 15 (quantitative yield). 400 MHz H NMR (DMSO-d 6 , ppm) 11.5-10.7 (lH, m), 8.6-7.5 (3H, m), 4.08 (lH, septet, J=6.2 Hz), 3.38-3.13 (4H, m), 1.61-1.44 (4H, m), 1.21 (6H, d, J=6.2 Hz), 0.94-0.81 (6H, m). ESI-MS (m/z): 269 [M+H]+. Example 33: 2 4 20 6-((Benzyloxy)(isopropyl)amino)-N ,N 4 -dipropyl-1,3,5-triazine-2,4-diamine (LXXII) Example 34: 6-((Benzyloxy)(isopropyllamino)-N 2,N 4-dipropyl- 1,3,5-triazine-2,4-diamine hydrogen sulfate (LXXIII) - 107 - WO 2013/181217 PCT/US2013/043052 Ph N 1N ''~N' N N H H LXXII Scheme 30 6-((Benzyloxy)(isopropyl)amino)-N 2
,N
4 -dipropyl-1,3,5-triazine-2,4 diamine (LXXII) was prepared by reacting 2-chloro-N-(4,6-bis-(n-propylamino) [1,3,5]triazine (XXXIV) and O-benzyl-N-isopropyl-hydroxylamine as exemplified in 5 Example 13. The corresponding hydrogen sulfate (LXXIII) was prepared as described in Example 20. Example 35: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N-ethyl-O-isopropyl-hydroxylamine 10 (LXXVI) Example 36: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N-ethyl-O-isopropyl-hydroxylamine hydrogen sulfate (LXXVII) O Eti, K 2
CO
3 O O HBr/AcOH Ph'O N-O : Ph'O -- 1) -O , HN-O- -HCI H acetone Et Et LXI LXXIV LXXV CI LXXV EtN o EtN ,0 N )N NaOH / H 2 0 N ilN H 2
SO
4 N ilN N N N 1,4-dioxane N N N'-' 1,4-dioxane W-G'N N N "-, H H H H XXXIV LXXVI e O-S-0-H LXXVII 6 15 Scheme 31 O-Benzyl-N-ethyl-N-isopropoxy-carbamate (LXXIV) An ACE* pressure tube was charged with benzyl isopropoxycarbamate (4.08 g, 19.50 mmol), anhydrous K 2
CO
3 (4.04 g, 29.25 mmol), ethyl iodide (7.0 mL, 20 87.75 mmol), and anhydrous acetone (30 mL). The reaction mixture was heated at 70 'C for 24 h. Ethyl iodide (7.0 mL, 87.75 mmol) and K 2
CO
3 (4.04 g, 29.25 mmol) were added and the reaction mixture was heated for 24 h. The reaction mixture was - 108 - WO 2013/181217 PCT/US2013/043052 filtered, and the acetone was evaporated. The resulting slurry was dissolved in EtOAc (150 mL), washed with water (3 x 50 mL), dried (Na 2
SO
4 ), and filtered. The solvent was removed to yield O-benzyl-N-ethyl-N-isopropoxy-carbamate (3.86 g, 83%). 400 MHz 1 H NMR (DMSO-d 6 , ppm) 7.41-7.30 (5H, m), 5.12 (2H, s), 4.05 (lH, septet, 5 J=6.2 Hz), 3.46 (2H, q, J=7.0 Hz), 1.12 (6H, d, J=6.2 Hz), 1.06 (3H, t, J=7.0 Hz). N-Ethyl-O-isopropyl-hydroxylamine hydrochloride (LXYV) O-Benzyl-N-ethyl-N-isopropoxy-carbamate was reacted with HBr/AcOH as described for the preparation of compound LXIII in Example 27 (yield 10 71%). 400 MHz 1 H NMR (DMSO-d 6 , ppm) 11.7-11.2 (2H, br s), 4.41 (lH, septet, J=6.1 Hz), 3.16 (2H, q, J=7.2 Hz), 1.24 (6H, d, J=6.1 Hz), 1.19 (3H, t, J=7.2 Hz). N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-N-ethyl-O-isopropyl-hydroxylamine (LXXVI) 15 2-Chloro-N-(4, 6-bis-(n-propylamino)-[ 1,3,5]triazine (XXXIV) was reacted with N-ethyl-O-isopropyl-hydroxylamine hydrochloride (LXXV) as described in Example 13, yielding N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-O isopropyl-hydroxylamine (LXXVI) (88% yield). ESI-MS (m/z): 297 [M+H]f. 20 N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-N-ethyl-O-isopropyl-hydroxylamine hydrogen sulfate (LXXVII) N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-O-isopropyl hydroxylamine (LXXVI) was reacted with 95% H 2
SO
4 as described in Example 20 (quantitative yield). 400 MHz 1 H NMR (DMSO-d 6 , ppm) 12.0-10.8 (1H, m), 8.7-8.4 25 (lH, br s), 8.27-7.78 (lH, m), 4.39-4.25 (lH, m), 3.90-3.76 (2H, m), 3.39-3.15 (4H, m), 1.62-1.45 (4H, m), 1.25 (6H, d, J=6.1 Hz), 1.18-1.10 (3H, m), 0.95-0.82 (6H, m). ESI-MS (m/z): 297 [M+H]+. Example 37: 30 N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-vl)-O-isobutvl-N-methyl-hydroxylamine (LXXXII) Example 38: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-vl)-O-isobutvl-N-methyl-hydroxylamine hydrogen sulfate (LXXXIII) - 109 - WO 2013/181217 PCT/US2013/043052 benzyl chloroformate
H
2 N-O-iBu DIPEA LXXVIII 0 0 PhO N-'iBu CHA, K 2
CO
3 Ph O ,iBu HBr / AcOH iBu PhON-O Ph'0 N-O HN-O' HOI H acetone
CH
3 CH 3 LXXIX LXXX LXXXI CI LXXXI H 3 C N ,O-iBu H3C N ,O-iBu N iN NaOH / H 2 0 N )N H 2
SO
4 N )-N N 1,4-dioxane --- N N N ' 1,4-dioxane s/NNN H H H H XXXIV LXXXII eO O-S-O-H LXXXIII 6 Scheme 32 O-Benzyl-N-isobutoxy carbamate (LXXIX) 5 0-isobutyl-hydroxylamine hydrochloride (LXXVIII) was reacted with benzyl chloroformate as described for the preparation of compound LXI in Example 27, yielding O-benzyl-N-isobutoxy carbamate (LXXIX) (87% yield). 400 MHz H NMR (CDCl 3 , ppm) 7.36-7.23 (5H, m), 5.11 (2H, s), 3.58 (2H, d, J=6.6 Hz), 1.89 (lH, septet, J=6.7 Hz), 0.86 (6H, d, J=6.7 Hz). 10 O-Benzyl-N-methyl-N-isobutoxy carbamate (LXXX) O-Benzyl-N-isobutoxycarbamate was reacted with methyl iodide, as described for the preparation of compound LXII in Example 27, affording O-benzyl N-methyl-N-isobutoxycarbamate in 78% yield. 400 MHz IH-NMR (DMSO-d 6 , ppM) 15 7.41-7.30 (5H, m), 5.12 (2H, s), 3.59 (2H, d, J=6.6 Hz), 3.09 (3H, s), 1.80 (lH, septet, J=6.7 Hz), 0.87 (6H, d, J=6.7 Hz). O-Isobutyl-N-methyl-hydroxylamine hydrochloride (LXXYI) O-Benzyl-N-methyl-N-isobutoxycarbamate was reacted with 20 HBr/AcOH as described for the preparation of compound LXIII in Example 27 (38% yield). 200 MHz H NMR (DMSO-d 6 , ppm) 12.5-11.4 (2H, br s), 3.84 (2H, d, J=6.6 Hz), 2.81 (3H, s), 1.90 (lH, septet, J=6.7 Hz), 0.89 (6H, d, J=6.7 Hz). -110- WO 2013/181217 PCT/US2013/043052 N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl-N-methyl-hydroxylamine (LXXXII) 2-Chloro-N-(4, 6-bis-(n-propylamino)-[ 1,3,5]triazine (XXXIV) was reacted with 0-isobutyl-N-methyl-hydroxylamine hydrochloride as described in 5 Example 13, affording LXXXII in 82% yield. ESI-MS (m/z) 297 [M+H]f. N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl-N-methyl-hydroxylamine hydrogen sulfate (LXXYIII) N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl-N-methyl 10 hydroxylamine (LXXXII) was reacted with 95% H 2
SO
4 as described in Example 20 (quantitative yield). 400 MHz 1 H-NMR (DMSO-d 6 , ppm) 12.0-10.7 (1H, br s), 8.7 7.6 (2H, m), 3.82-3.72 (2H, m), 3.41-3.20 (7H, m), 2.11-1.82 (1H, m), 1.62-1.44 (4H, m), 1.00-0.82 (12H, m). ESI-MS (m/z): 297 [M+H]+. 15 Example 39: 2 4 6-(Methyl(thiophen-2-ylmethoxylamino)-N ,N -di-n-propyl-1,3,5-triazine-2,4 diamine (LXXXIV) Example 40: 6-(Methyl(thiophen-2-ylmethoxylamino)-N 2
,N
4 -di-n-propyl- 1,3,5-triazine-2,4 20 diamine hydrogen sulfate (LXXXV)
H
3 C, NO N N N N NN H H LXXXIV Scheme 33 6-(Methyl(thiophen-2-ylmethoxy)amino)-N 2
,N
4 -di-n-propyl-1,3,5 triazine-2,4-diamine (LXXXIV) may be prepared by reacting 2-chloro-N-(4,6-bis-(n propylamino)-[1,3,5]triazine (XXXIV) and O-(thiophen-2-yl-methyl)-N-methyl 25 hydroxylamine as exemplified in Example 13. Example 41: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-O-cyclopropylmethyl-N-methyl hydroxylamine (XCI) - 111 - WO 2013/181217 PCT/US2013/043052 Example 42: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-O-cyclopropylmethyl-N-methyl hydroxylamine hydrogen sulfate (XCII) 0 DEAD, PPh 3 1) H 2
N-NH
2 CI O'^ Ph N-OHN-O
-H
2 N-O DIPEA THF 0 2)HCI HCI cyclopropylmethanol LXXXVI LXXXVil P 0 CH 3 -1, K 2
CO
3 HBr AcOH Ph O N-0 Ph'^'0 N-0 HN-O -HCI H acetone
CH
3 CH 3 LXXXVIII LXXXIX XC CI XC H 3 C NO H 3 C N N N NaOH / H 2 0 N ilN H 2
SO
4 N )N N 1,4-dioxane N N-"'-- 1,4-dioxane / N N XXXIV XCI eO 0-S-0-H XCII 5 Scheme 34 2-Cyclopropylmethoxy-isoindole-1,3-dione (LXYXVI) N-Hydroxyphthalimide and cyclopropyl-methanol were reacted as 10 described for compound LIX in Example 27, to afford LXXXVI in 87% yield. 400 MHz H NMR (DMSO-d 6 , ppm) 7.86 (4H, s), 3.97 (2H, d, J=7.4 Hz), 1.22-1.11 (lH, m), 0.61-0.48 (2H, m), 0.34-0.22 (2H, m). O-Cyclopropylmethyl-hydroxylamine hydrochloride (LXXXVII) 15 2-Cyclopropylmethoxy-isoindole-1,3-dione was reacted with hydrazine as described for compound LX in Example 27 (LXXXVII, in 67% yield). 400 MHz H NMR (DMSO-d 6 , ppm) 10.95 (3H, br s), 3.83 (2H, d, J=7.4 Hz), 1.12 1.01 (lH, m), 0.62-0.50 (2H, m), 0.35-0.24 (2H, m). 20 O-Benzyl-N-cyclopropylmethoxy carbamate (LXXXVIII) 0-Cyclopropylmethyl-hydroxylamine hydrochloride was reacted with benzyl chloroformate as described for compound LXI in Example 27 (LXXXVIII, in -112- WO 2013/181217 PCT/US2013/043052 88% yield). 400 MHz H-NMR (DMSO-d 6 , ppm) 10.37 (lH, br s), 7.41-7.30 (5H, m), 5.08 (2H, s), 3.54 (2H, d, J=7.2 Hz), 1.06-0.92 (lH, m), 0.54-0.41 (2H, m), 0.25 0.13 (2H, m). 5 O-Benzyl-N-methyl-N-cyclopropylmethoxy-carbamate (LXXIX) O-Benzyl N-cyclopropylmethoxy carbamate and methyl iodide were reacted as described for compound LXII in Example 27 (LXXXIX, in 95% yield). 400 MHz H NMR (DMSO-d 6 , ppm) 7.42-7.30 (5H, m), 5.11 (2H, s), 3.62 (2H, d, J=7.2 Hz), 3.11 (3H, s), 1.06-0.93 (lH, m), 0.54-0.41 (2H, m), 0.26-0.13 (2H, m). 10 O-Cyclopropylmethyl-N-methyl-hydroxylamine hydrochloride (XC) O-Benzyl-N-methyl-N- cyclopropylmethoxy carbamate was reacted with HBr/AcOH as described for compound LXIII in Example 27, yielding XC in 77% yield. 400 MHz H NMR (DMSO-d 6 , ppm) 11.96 (2H, br s), 3.91 (2H, d, J=7.4 15 Hz), 2.80 (3H, s), 1.13-1.01 (lH, m), 0.63-0.50 (2H, m), 0.37-0.25 (2H, m). N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-O-cyclopropylmethyl-N-methyl hydroxylamine (XCI) 2-Chloro-N-(4, 6-bis-(n-propylamino)-[ 1,3,5]triazine (XXXIV) was 20 reacted with O-cyclopropylmethyl-N-methyl-hydroxylamine hydrochloride (XC) as described in Example 13, yielding (XCI) in 99% yield. 400 MHz IH NMR (DMSO d 6 , ppm) 6.91-6.77 (lH, m), 6.75-6.58 (1H, m), 3.77-3.64 (2H, m), 3.21-3.09 (7H, m), 1.54-1.41 (4H, m), 1.11-1.00 (lH, m), 0.88-0.80 (6H, m), 0.56-0.44 (2H, m), 0.32 0.20 (2H, m). ESI-MS (m/z) 295 [M+H]+. 25 N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-O-cyclopropylmethyl-N-methyl hydroxylamine hydrogen sulfate (XCII) N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-O-cyclopropylmethyl-N methyl-hydroxylamine (XCI) was reacted with 95% H 2
SO
4 as described in Example 30 20, yielding (XCII) in quantitative yield. 400 MHz IH-NMR (DMSO-d 6 , ppm) 11.6 11.0 (1H, br s), 8.7-8.4 (0.7H, br s), 8.2-8.0 (0.3H, br s), 7.89-7.42 (1H, m), 3.88-3.77 (2H, m), 3.42-3.18 (7H, m), 1.62-1.45 (4H, m), 1.24-1.13 (lH, m), 0.95-0.82 (6H, m), 0.61-0.52 (2H, m), 0.38-0.28 (2H, m). ESI-MS (m/z) 295 [M+H]+. - 113 - WO 2013/181217 PCT/US2013/043052 Example 43: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-vl)-O-ethyl-N-methyl-hydroxylamine (XCVI) Example 44: 5 N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-vl)-O-ethyl-N-methyl-hydroxylamine hydrogen sulfate (XCVII)
H
3 C N N N N ' N J N H H XCVI Scheme 35 tert-Butyl ethoxycarbamate (XCIII) 10 A solution of saturated Na 2
CO
3 solution (45.0 mL) was added dropwise at room temperature to a solution of O-ethyl-hydroxylamine (1.76 g, 18.0 mmol) and di-tert-butyl dicarbonate (5.13 g, 23.67 mmol) in dichloromethane (45.0 mL) and stirred for 24 h. Water was added, the mixture pH was adjusted to 2 by adding 6N HCl, and the resulting system was extracted with dichloromethane (3 x 50 15 mL). The combined organic extracts were dried over Na 2
SO
4 and evaporated. The crude product was purified by flash column chromatography using gradient elution from petroleum ether/EtOAc (98:2) to petroleum ether/EtOAc (95:5) to yield tert butyl ethoxycarbamate (XCIII) (2.62 g, 90%). 400 MHz 1 H NMR (DMSO-d 6 , ppm) 9.89 (lH, s) 3.72 (2H, q, J=7.0 Hz) 1.40 (9H, s) 1.10 (3H, t, J=7.0 Hz). 20 tert-Butyl ethoxy(methyl)carbamate (XCIV) A solution of tert-butyl ethoxycarbamate (XCIII, 2.48 g, 15.38 mmol) in DMF (10 mL) was added dropwise to the suspension of 60% sodium hydride (0.66 g, 16.92 mmol) in DMF (5 mL) at 0 C. After 30 min. methyl iodide (1.95 mL, 31.32 25 mmol) in DMF (10 mL) was added, and the reaction mixture was stirred at room temperature for 24 h. Water (100 mL) was added and the product was extracted with ethyl acetate (3 x 50 mL), dried over Na 2
SO
4 , and concentrated in vacuo to yield tert butyl ethoxy(methyl)carbamate (XCIV, 2.34 g, 87%). 400 MHz H NMR (DMSO-d 6 , ppm) 3.81 (2H, q, J=7.0 Hz), 3.00 (3H, s), 1.41 (9H, s), 1.12 (3H, t, J=7.0 Hz). 30 -114- WO 2013/181217 PCT/US2013/043052 O-Ethyl-N-methyl-hydroxylamine hydrochloride (XCV) A solution of 4M HCl/1,4-dioxane (25 mL) was added in portions to tert-butyl ethoxy(methyl)carbamate (XCIV, 2.34 g, 13.35 mmol) at 0 0 C. The mixture was stirred at room temperature for 4 h. The volatiles removed in vacuo and the 5 residue was triturated with ethyl ether and filtered to yield a solid (0-ethyl-N-methyl hydroxylamine hydrochloride, XCV, 1.35 g, 910%). N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-O-ethyl-N-methyl-hydroxylamine (XCVI) 10 2-Chloro-N-(4, 6-bis-(n-propylamino)-[ 1,3,5]triazine (XXXIV) was reacted with O-ethyl-N-methyl-hydroxylamine hydrochloride as described in Example 13, to yield XCVI in 93% yield. ESI-MS (m/z) 269 [M+H]f. N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-O-ethyl-N-methyl-hydroxylamine 15 hydrogen sulfate (XCVII) N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-O-ethyl-N-methyl hydroxylamine (XCVI) was reacted with 95% H 2
SO
4 as described in Example 20, to afford (XCVII) in 91% yield. 400 MHz 1H NMR (DMSO-d 6 , ppm) 11.7-10.8 (lH, br s), 8.79 -7.34 (2H, m), 4.09-3.98 (2H, m), 3.40-3.20 (7H, m), 1.61-1.46 9 (4H, m), 20 1.27 (3H, t, J=7.1 Hz), 0.94-0.84 (6H, m). 400 MHz 1 HNMR (D 2 0, ppm) 3.99-3.88 (2H, m), 3.34-3.13 (7H, m), 1.52-1.39 (4H, m), 1.14 (3H, t, J=7.1 Hz), 0.76 (6H, t, J=7.5 Hz). ESI-MS (m/z): 269 [M+H]f. M.P.: 84-86'C. Example 45: 25 N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-O-(2,2-difluoro-ethyl)-hydroxylamine Example 46: N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-O-(2,2-difluoro-ethyl)-hydroxylamine hydrogen sulfate (CI) -115- WO 2013/181217 PCT/US2013/043052 F HN' F N N '-'N N N '- H H C Scheme 36 2-(2,2-Difluoro-ethoxy)-isoindole-1,3-dione (XCVIII) N-Hydroxyphthalimide and 2,2-difluoro-ethanol were reacted as 5 described for compound LXI in Example 27, affording XCVIII in 52% yield. 400 MHz H NMR (DMSO-d 6 , ppm) 7.92-7.85 (4H, m), 6.34 (1H, tt, J=54.4, 3.9 Hz), 4.46 (2H, td, J=14.1, 3.9 Hz). O-(2,2-Difluoro-ethyl)-hydroxylamine hydrochloride (XCIX) 10 2-(2,2-Difluoro-ethoxy)-isoindole-1,3-dione was reacted with hydrazine as described for compound LX in Example 27, affording XCIX in 73% yield. 400 MHz H NMR (DMSO-d 6 , ppm) 12.3-10.3 (3H, br s), 6.38 (lH, tt, J=54.0, 3.3 Hz), 4.34 (2H, td, J=14.7, 3.3 Hz). 15 N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl)-hydroxylamine (C) 2-Chloro-N-(4, 6-bis-(n-propylamino)-[ 1,3,5]triazine (XXXIV) was reacted with O-(2,2-difluoro-ethyl)-hydroxylamine hydrochloride as described in Example 13, affording C in 59% yield. 400 MHz H NMR (DMSO-d 6 , ppm) 9.91 20 9.56 (lH, m), 7.00-6.90 (1H, m), 6.89-6.67 (1H, m), 6.48-6.13 (1H, m, 4.12-3.98 (2H, m), 3.20-3.09 (4H, m), 1.53-1.41 (4H, m), 0.88-0.80 (6H, m). ESI-MS (m/z) 291 [M+H]. N-(4,6-Bis-n-propylamino-[1, 3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl)-hydroxylamine 25 hydrogen sulfate (CI) N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl) hydroxylamine (C, 1.02 g, 3.51 mmol) was reacted with 95% H 2
SO
4 (0.19 mL, 3.51 mmol) in diethyl ether (3 mL) at 0 0 C. Two drops of EtOH were added, and the resultant crystals were filtered, washed with diethyl ether, and dried to yield N-(4,6 -116- WO 2013/181217 PCT/US2013/043052 bis-n-propylamino-[1,3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl)-hydroxylamine hydrogen sulfate (CI) (1.26 g,93%). 400 MHz H NMR (DMSO-d 6 , ppm) 11.8-10.5 (1H, m) 8.8-8.4 (0.3H, br s) 8.36-7.53 (1.7H, m) 6.50-6.08 (lH, m) 4.28-4.07 (2H, m) 3.39-3.13 (4H, m) 1.64-1.42 (4H, m) 0.97-0.78 (6H, m). ESI-MS (m/z) 291 [M+H]f. 5 M.P.: 91-93'C. Example 47: 4-N-(2-Dimethylaminoethyl)amino-6-N-(n-propyl)amino-[1,3,51triazin-2-yl)-N,O dimethyl-hydroxylamine (CIII) 10 Example 48: 4-N-(2-Dimethylaminoethyllamino-6-N-(n-propyllamino-[1,3,51triazin-2-yl)-N,O dimethyl-hydroxylamine hydrochloride (CIV) CI H3C N 10-CH 3 N N CH 3
NH-OCH
3 HCI N "N N N <CI DIPEA, EtOH N IN CI H H XXIII Cll
H
3 C, ,O-CH 3 H2NN N N N IiN N I I H H CIII Scheme 37 15 2-Chloro-6-N-(n-propyl) amino-[, 3,5]triazin-2-yl)-N, 0-dimethyl-hydroxylamine (CII) 2,4-Dichloro-N-(6-n-propylamino)-[1,3,5]triazine (XXIII) (18 g, 87 mmol) was dissolved in acetone (100 mL) and poured into ice-water (50 mL) to form a very fine suspension. A solution of N,O-dimethylhydroxylamine hydrochloride (9.3 20 g, 95 mmol) in water (30 mL) was added, while keeping the temperature at 0 'C (ice bath). To this mixture, 2N NaOH (44 mL, 88 mmol) was added dropwise at a rate adjusted to keep the temperature between 0 'C and 5 'C. The reaction was stirred for 30 min at ambient temperature and for additional 60 min at 50 'C. The resultant precipitate was filtered off, and washed with water (3 x 25 mL). After drying over 25 calcium chloride under high vacuum, 2-chloro-N-(6-n-propylamino)-[1,3,5]triazin-2 -117- WO 2013/181217 PCT/US2013/043052 yl)-N,O-dimethyl-hydroxylamine (CII, 12 g, 60%) was isolated as a white powder. LCMS (ESI) m/z = 232 (M+H). 4-N-(2-Dimethylaminoethyl)amino-6-N-(n-propyl)amino-[1, 3,5]triazin-2-yl)-N, 0 5 dimethyl-hydroxylamine (CIII) A mixture of 2-chloro-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine (CII, 1.5 g, 6.5 mmol), N,N-dimethylethane-1,2-diamine (3.5 g, 39 mmol) and DIPEA (2.5 g, 20 mmol) in EtOH (30 mL) was heated at 100 0 C for 16 h. The solvent was then removed under reduced pressure. The residue was 10 dissolved in EtOAc (80 mL), washed with water (2 x 50 mL) and then with a brine solution (50 mL) and lastly, dried over Na 2
SO
4 . The solvent was removed under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH=20/1 to 5/1) to yield 4-N-(2-dimethylaminoethyl)amino-6-N-(n propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (620 mg, 33%). 15 4-N-(2-Dimethylaminoethyl)amino-6-N-(n-propyl)amino-[1, 3,5]triazin-2-yl)-N, 0 dimethyl-hydroxylamine hydrochloride (CIV) 4-N-(2-dimethylaminoethyl)amino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (610 mg, 2.1 mmol) was dissolved 20 in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (6.6 mL) and the solution was lyophilized to yield 4-N-(2-dimethylaminoethyl)amino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (CIV, 630 mg) as a colorless oil. LCMS (ESI) m/z = 284 (M+H). 1 H NMR (500 MHz, DMSO) 6 (ppm) 10.55-10.88 (br, 1H), 8.70-9.10 (m, 2H), 4.37-4.43 (m, 5H), 3.76-3.87 (m, 7H), 2.84 25 2.88 (m, 6H), 1.60-1.64 (m, 2H), 0.94-1.02 (m, 3H). Example 49: 4-N-(3 -(1 -N-Methylimidazol-2-yl)-propyl)-amino-6-N-(n-propyllamino [1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine (CV) 30 Example 50: 4-N-(3 -(1 -N-Methylimidazol-2-yl)-propyl)-amino-6-N-(n-propyllamino [1,3,51triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (CVI) - 118- WO 2013/181217 PCT/US2013/043052
H
3 CN 0-CH 3 | H 3 CI O-CH3
H
2 N N N N N N N N CI EtOH -"-N N N III ) H H H N CII Cv Scheme 38 4-N-(3-(1-N-Methylimidazol-2-yl)-propyl)-amino-6-N-(n-propyl)amino-[1, 3,5]triazin 5 2-yl)-N,0-dimethyl-hydroxylamine (CV) 2-Chloro-6-N-(n-propylamino)-[ 1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine (CII) (400 mg, 2.9 mmol), DIPEA (5.16 g, 40 mmol) and 3-(1-methyl 1H-imidazol-2-yl)propan-1-amine (J. Heterocyclic Chem., 2005, 42:1011-15) (732 mg, 3.2 mmol) in EtOH (50 mL) were heated at 100 'C for 16 h. After this time, the 10 solvent was removed under reduced pressure. The residue was dissolved in DCM/MeOH (400 mL/200 mL), washed with water (50 mL) then dried over Na 2
SO
4 . The solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (DCM/MeOH=50/1 to 10/1) to yield 4-N-(3-(1-N methylimidazol-2-yl)-propyl)-amino-6-N-(n-propyl)amino-[ 1,3,5]triazin-2-yl)-N,0 15 dimethyl-hydroxylamine (CV, 210 mg, 22%). 4-N-(3-(1-N-Methylimidazol-2-yl)-propyl)-amino-6-N-(n-propyl)amino-[1, 3,5]triazin 2-yl)-N, 0-dimethyl-hydroxylamine hydrochloride (CVI) 4-N-(3-(1 -N-methylimidazol-2-yl)-propyl)-amino-6-N-(n 20 propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (CV) (210 mg, 0.63 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (1.3 mL), and the solution was lyophilized to yield 4-N-(3-(1-N-methylimidazol-2-yl)-propyl) amino-6-N-(n-propyl)amino-[ 1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrochloride (CVI, 210 mg) as a yellow oil. LCMS (ESI) m/z = 335 (M+H). H 25 NMR (500 MHz, DMSO) 6 (ppm) 14.40-14.55 (br, 1H), 8.60-8.80 (m, 2H), 7.57-7.62 (m, 2H), 3.77-3.83 (m, 6H), 3.28-3.42 (m, 7H), 2.80-2.84 (m, 2H), 1.95-2.10 (m, 2H), 1.53-1.56 (m, 2H), 0.89-0.93 (m, 3H). Example 51: - 119- WO 2013/181217 PCT/US2013/043052 4-N-(1 -N-Methylimidazol-2-yl)-methylamino-6-N-(n-propyllamino- [1,3,51triazin-2 yll-N,O-dimethyl-hydroxylamine (CVII) Example 52: 4-N-(1-N-Methylimidazol-2-yl)-methylamino-6-N-(n-propyllamino-[1,3,51triazin-2 5 yl)-N,0-dimethyl-hydroxylamine hydrochloride (CVIII) H3CN 0-CH 3 H 3 C,' N0-CH 3 H2N N 'N N N N N N I K 2
CO
3 / EtOH IJN N IN II I N H H H CII CVII Scheme 39 10 4-N-(1-N-Methylim idazol-2-yl)-methylamino-6-N-(n-propyl)amino-[1, 3,5]triazin-2 yl)-N,0-dimethyl-hydroxylamine (CVII) 2-Chloro-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine (CII) (1 g, 4.5 mmol), (1-methyl-1H-imidazol-2-yl)methanamine (600 mg, 5.4 mmol) and K 2
CO
3 (1.24 g, 9 mmol) in EtOH (50 mL) were heated at 100 C 15 for 16 h. The reaction mixture was filtered, and the volatiles were removed under reduced pressure. The residue was dissolved in EtOAc (100 mL), washed with water (30 mL) and then with a brine solution (30 mL), and lastly dried over Na 2
SO
4 . The solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (DCM/MeOH=20/1 to 8/1) to yield 4-N-(1-N 20 methylimidazol-2-yl)-methylamino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine (CVII, 650 mg, 47%). 4-N-(1-N-Methylim idazol-2-yl)-methylamino-6-N-(n-propyl)amino-[1, 3,5]triazin-2 yl)-N,0-dimethyl-hydroxylamine hydrochloride (CVIII) 25 4-N-(1 -N-methylimidazol-2-yl)-methylamino-6-N-(n-propyl)amino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (CVII, 650 mg, 2.1 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (6.3 mL). The resultant solution was subjected to lyophilization to yield 4-N-(1-N-methylimidazol-2-yl) methylamino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine 30 hydrochloride (CVIII, 389 mg) as a colorless oil. LCMS (ESI) m/z = 307 (M+H)+. - 120 - WO 2013/181217 PCT/US2013/043052 H NMR (500 MHz, DMSO) 6 (ppm) 14.65-14.85 (br, 1H), 8.70-9.20 (m, 2H), 7.62 7.70 (m, 2H), 4.87-4.91 (m, 2H), 3.75-3.89 (m, 9H), 3.31-3.40 (m, 3H), 1.55-1.56 (m, 2H), 0.85-0.96 (m, 3H). 5 Example 53: 4,6-Bis-(N-(2-dimethylaminoethyllamino)-[1,3,51triazin-2-yl)-N,O-dimethyl hydroxylamine (CIX) Example 54: 4,6-Bis-(N-(2-dimethylaminoethyllamino)-[1,3,51triazin-2-yl)-N,O-dimethyl 10 hydroxylamine hydrochloride (CX)
H
3 CII O-CH 3
H
3 C N,0-CH 3 ___L_ NN N N HN NN N CI N CI K 2 CO3 / EtOH N N N N N I I H H xxx CIX Scheme 40 4,6-Bis-(N-(2-dimethylaminoethyl)amino)-[1, 3,5]triazin-2-yl)-N, 0-dimethyl 15 hydroxylamine (CIX) N-(4,6-Dichloro[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXX) (7 g, 33.5 mmol), N,N-dimethyl-ethane-1,2-diamine (6.05 g, 68.7 mmol) and
K
2
CO
3 (10.2 g, 73.7 mmol) in THF (250 mL) were heated at 70 0 C for 5 h, after which time the solvent was removed under reduced pressure. The residue was 20 purified by reverse flash column chromatography to yield 4,6-bis-(N-(2 dimethylaminoethyl)amino)-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (270 mg, 3%). 4,6-Bis-(N-(2-dimethylaminoethyl)amino)-[1, 3,5]triazin-2-yl)-N, 0-dimethyl 25 hydroxylamine hydrochloride (CX) 4,6-Bis-(N-(2-dimethylaminoethyl)amino)-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine (270 mg, 0.86 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (2 mL), and the resultant solution was lyophilized to yield 4,6-bis-(N-(2-dimethylaminoethyl)amino)-[1,3,5]triazin-2-yl)-N,O-dimethyl 30 hydroxylamine hydrochloride (290 mg) as a colorless oil. LCMS (ESI) m/z = 313 - 121 - WO 2013/181217 PCT/US2013/043052 (M+H). 'H NMR (500 MHz, DMSO) 6 (ppm) 10.20-10.60 (br, 1H), 7.17-7.25 (m, 2H), 3.54-3.76 (m, 7H), 3.01-3.25 (m, 7H), 2.72 (s, 12H). Example 55: 5 4,6-Bis-(N-(pyridin-4-Vlmethyl)amino)-[ 1,3,51triazin-2-yl)-N,O-dimethyl hydroxylamine (CXI) Example 56: 4,6-Bis-(N-(pyridin-4-ylmethyllamino)-[ 1,3,51triazin-2-yl)-N,O-dimethyl hydroxylamine hydrochloride (CXII)
H
3 CN O-CH 3
H
2 N H3CN NO-CH3 N 1 1N _____N___ C) N. CI DIPEA / EtOH N N N N II I N- H H A ,N 10 XXX CXI Scheme 41 4,6-Bis-(N-(pyridin-4-ylmethyl)amino)-[1, 3,5]triazin-2-yl)-N, 0-dimethyl hydroxylamine (CXI) 15 N-(4,6-Dichloro[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXX) (1 g, 4.78 mmol), pyridin-4-ylmethanamine (1.14, 10.52 mmol) and DIPEA (1.85 g, 14.34 mmol) in EtOH (80 mL) were heated at 100 0 C for 16 h, after which the solvent was removed under reduced pressure. The crude product was purified by flash chromatography to yield 4,6-bis-(N-(pyridin-4-ylmethyl)amino)-[1,3,5]triazin-2 20 yl)-N,O-dimethyl-hydroxylamine (CXI) (450 mg, 27%). 4,6-Bis-(N-(pyridin-4-ylmethyl)amino)-[1, 3,5]triazin-2-yl)-N, 0-dimethyl hydroxylamine hydrochloride (CXII) 4,6-Bis-(N-(pyridin-4-ylmethyl)amino)-[1,3,5]triazin-2-yl)-N,O 25 dimethyl-hydroxylamine (CXI) (450 mg, 1.28 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (3.84 mL), and the resultant solution was lyophilized to yield 4,6-bis-(N-(pyridin-4-ylmethyl)amino)-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine-hydrochloride (497 mg) as a yellow solid. LCMS (ESI) m/z = 353 (M+H). 1 H NMR (500 MHz, MeOD) 6 (ppm) 8.79-8.89 (m, 4H), 7.98-8.20 (m, 4H), 30 5.04 (s, 2H), 4.85 (s, 2H), 3.90-3.95 (m, 3H), 3.50 (s, 1H), 3.33 (s, 2H). - 122 - WO 2013/181217 PCT/US2013/043052 Example 57: 4,6-Bis-[N-(3-methoxy-n-propyllaminol-[1,3,51triazin-2-yl)-N,O-dimethyl hydroxylamine (CXIII) 5 Example 58: 4,6-Bis-[N-(3-methoxy-n-propyl)aminol-[1,3,51triazin-2-yl)-N,O-dimethyl hydroxylamine hydrochloride salt (CXIV) H3C N ,0-CH 3 H3C N 0-CH3 N N H 2 N- N0-CH 3 N CI N CI DIPEA / EtOH CH 3 -0 -- N N N' '0-CH 3 I I H H xxx CXill Scheme 42 10 4,6-Bis-[N-(3-methoxy-n-propyl)amino]-[1, 3,5]triazin-2-yl)-N, 0-dimethyl hydroxylamine (CXIII) N-(4,6-Dichloro[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXX) (1 g, 4.78 mmol), 3-methoxypropan-1-amine (936 mg, 10.52 mmol) and 15 DIPEA (1.85 g, 14.34 mmol) in EtOH (50 mL) were heated at 100 'C for 16 h, after which time the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (200 mL), washed with water (50 mL) and then with a brine solution (50 mL) and lastly dried over Na 2 S04. The solvent was removed under reduced pressure. The crude product was purified by flash column chromatography 20 (pet ether/ethyl acetate = 5/1 to 1/2) to yield 4,6-bis-[N-(3-methoxy-n-propyl)amino] [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (CXIII) (1.4 g, 93%). 4,6-Bis-[N-(3-methoxy-n-propyl)amino]-[1, 3,5]triazin-2-yl)-N, 0-dimethyl hydroxylamine hydrochloride (CXIV) 25 4,6-Bis-[N-(3-methoxy-n-propyl)amino]-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine (CXIII, 1.4 g, 4.46 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (13.4 mL), and the resultant solution was lyophilized to yield 4,6-bis-[N-(3-methoxy-n-propyl)amino]-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine hydrochloride (1.56 g) as a colorless oil. LCMS (ESI) m/z = 315 - 123 - WO 2013/181217 PCT/US2013/043052 (M+H). 'H NMR (500 MHz, DMSO) 6 (ppm) 12.10-12.60 (br, 1H), 8.55-8.74 (m, 2H), 3.75-3.86 (m, 3H), 3.35-3.45 (m, 11H), 3.22-3.25 (m, 5H), 2.77 (s, 1H), 1.72 1.76 (m, 4H). 5 Example 59: 4,6-Bis-[N-(tetrahydropyran-4-ylmethyl)aminol-[1,3,51triazin-2-yl)-N,O-dimethyl hydroxylamine (CXV) Example 60: 4,6-Bis-[N-(tetrahydropyran-4-ylmethyllaminol-[1,3,51triazin-2-yl)-N,O-dimethyl 10 hydroxylamine hydrochloride (CXVI) H3CN O-CH 3
H
2 N H 3 C , N 3 IN N ___ININ IN DIPEA/ EtOH N N N CK~CI IN CI0 H xxx Cxv Scheme 43 4,6-Bis-[N-(tetrahydropyran-4-ylmethyl) amino]-[1,3,5]triazin-2-yl)-N, 0-dimethyl 15 hydroxylamine (CXV) N-(4,6-Dichloro[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXX) (1 g, 4.78 mmol), (tetrahydro-2H-pyran-4-yl)methanamine (1.21 g, 10.52 mmol) and DIPEA (1.85 g, 14.34 mmol) in EtOH (50 mL) were heated at 100 0 C for 16 h, after which time the solvent was removed under reduced pressure. The residue 20 was dissolved in EtOAc (200 mL), washed with water (50 mL) and then with a brine solution (50 mL) and lastly dried over Na 2 S04. The solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (PE/EA=5/1 to 1/1) to afford 4,6-bis-[N-(tetrahydropyran-4-ylmethyl)amino] [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (CXV) (1.5 g, 85%). 25 4,6-Bis-[N-(tetrahydropyran-4-ylmethyl)amino]-[1,3,5]triazin-2-yl)-N, 0-dimethyl hydroxylamine hydrochloride (CXVI) 4,6-Bis-[N-(tetrahydropyran-4-ylmethyl)amino]-[1,3,5]triazin-2-yl) N,O-dimethyl-hydroxylamine (CXV) (1.5 g, 4.1 mmol) was dissolved in H 2 0 (10 - 124 - WO 2013/181217 PCT/US2013/043052 mL) and 0.5 M aqueous HCl solution (12.3 mL), and the resultant solution was lyophilized to yield 4,6-bis-[N-(tetrahydropyran-4-ylmethyl)amino]-[1,3,5]triazin-2 yl)-N,O-dimethyl-hydroxylamine (CXVI) hydrochloride (1.65 g) as a white solid. LCM: (ESI) m/z = 367 (M+H). 1 H NMR (500 MHz, DMSO) 6 (ppm) 12.30-12.70 5 (br, 1H), 8.65-8.80 (m, 2H), 3.77-3.86 (m, 7H), 3.20-3.35 (m, 11H), 1.75-1.78 (m, 2H), 1.56-1.58 (m, 4H), 1.20-1.23 (m, 4H). Example 61: N-(5,8,11 -Trioxa-2,14,16,18,19-pentaazabicyclo[13.3. llnonadeca 10 1(18),15(19),16(17)-trien-17-yl)-N,O-dimethylhydroxylamine (CXVII) Example 62: N-(5,8,11 -Trioxa-2,14,16,18,19-pentaazabicyclo[13.3.llnonadeca 1(18),15(19),16(17)-trien-17-yl)-N,O-dimethylhydroxylamine hydrochloride (CXVIII)
NH
2
NH
2 H3C N'O-CH3
H
3 C,'N O-CH 3 N - N N N HN N NH CIA N CI 15 XXX CXVII Scheme 44 N-(5,8, 11-Trioxa-2,14,16,18,19-pentaazabicyclo[13.3. l]nonadeca 1(18),15(19),16(1 7)-trien-17-yl)-N,0-dimethylhydroxylamine (CXVII) 20 N-(4,6-Dichloro[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXX) (1.63 g, 7.8 mmol) in EtOH (100 mL) was added to 2,2'-(2,2'-oxybis(ethane 2,1-diyl)bis(oxy))diethanamine (Org. Biomol. Chem. 2005, 3:2255-61) (1.5 g, 7.8 mmol) and DIPEA (2.01 g, 15.6 mmol). The reaction was heated at 100 'C for 3 h, after which time the solvent was removed under reduced pressure. The residue was 25 dissolved in EtOAc (200 mL), washed with water (2 x 100 mL) and then with a brine solution (100 mL) and lastly dried over Na 2
SO
4 . The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (DCM/MeOH=50/1 to 20/1) to yield N-(5,8,1 1-trioxa-2,14,16,18,19 - 125 - WO 2013/181217 PCT/US2013/043052 pentaazabicyclo[13.3. 1]nonadeca-1(18),15(19),16(17)-trien- 17-yl)-N,O dimethylhydroxylamine (CXVII) (700 mg) as a colourless oil (yield 27%). N-(5,8,1 1-Trioxa-2,14,16,18,19-pentaazabicyclo[13.3. l]nonadeca 5 1(18),15(19),16(1 7)-trien-17-yl)-N,0-dimethylhydroxylamine hydrochloride (CXVIII) N-(5,8,11 -Trioxa-2,14,16,18,19-pentaazabicyclo[ 13.3.1 ]nonadeca 1(18),15(19),16(17)-trien-17-yl)-N,O-dimethylhydroxylamine (CXVII) (700 mg, 2.1 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (4.3 mL), and the resultant solution was lyophilized to yield N-(5,8,1 1-trioxa-2,14,16,18,19 10 pentaazabicyclo[13.3.1 ]nonadeca-1(18),15(19),16(17)-trien-17-yl)-N,O dimethylhydroxylamine hydrochloride (CXVIII) (750) mg as a colorless oil. LCMS: (ESI) m/z = 329 (M+H). 1 H NMR (500 MHz, DMSO) 6 (ppm) 11.50-12.60 (br, 1H), 8.61 (s, 2H), 3.77 (s, 3H), 3.30-3.62 (m, 16H), 3.30 (s, 3H). 15 Example 63: 2,6-Bis-(N-n-propylamino)-[1,31pyrimidin-4-yll-N,O-dimethyl-hydroxylamine (CXX) Example 64: 2,6-Bis-(N-n-propylamino)-[1,31pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine 20 hydrogen sulfate (CXXI) CI CI
H
3 C-N-0-CH 3
H
3 CN, 0--CH 3 NNH2N 1 NaOH CI N CI EtOH N N N N N N H H 1,4-dioxane H H CXIX CXX H2SO4 N3C' N0--3 N ~ O-S-O-H 1,4-dioxane N 0--O ",-NN N'vO CXXI Scheme 45 4-Chloro-2,6-bis-[N-n-propylamino]-1,3-pyrimidine (CXIX) 25 2,4,6-Trichloro-pyrimidine (5.00 g, 27.26 mmol) and n-propylamine (13.5 mL, 163.56 mmol) in EtOH were heated at 60 'C for 24 h and then cooled. The - 126 - WO 2013/181217 PCT/US2013/043052 volatiles were removed under reduced pressure. Water (100 mL) was added and the resulting suspension was extracted with CH 2 Cl 2 (3 x 75 mL). The combined organic extracts were washed with water (150 mL), then with a brine solution (100 mL) and dried over Na 2
SO
4 . The solvent was removed under reduced pressure to yield 4 5 chloro-2,6-bis-[N-n-propylamino]-1,3-pyrimidine (CXIX) (5.78 g, 93%). 200 MHz IH-NMR (DMSO-d 6 , ppm) 7.26-7.04 (lH, m) 7.04-6.81 (1H, m) 5.69 (1H, s) 3.26 3.01 (4H, m) 1.60-1.36 (4H, m) 0.87 (3H, t, J=7.4 Hz) 0.85 (3H, t, J=7.4 Hz); ESI-MS (m/z) 229, 231 [M+H]f. 10 (2,6-Bis-N-[n-propylamino]-pyrimidin-4-yl)-N, 0-dimethyl-hydroxylamine (CXX) 4-Chloro-2,6-bis-[N-n-propylamino]-1,3-pyrimidine (CXIX) (5.78 g, 25.27 mmol), N,O-dimethylhydroxylamine hydrochloride (4.93 g, 50.54 mmol) and NaOH (2.02 g, 50.54 mmol) in 1,4-dioxane (400 mL) and water (20 mL) were heated at 60 'C for 24 h. N,O-Dimethylhydroxylamine hydrochloride (4.93 g, 50.54 mmol) 15 and NaOH (3.03 g, 75.81 mmol) were added to the reaction mixture and heating was continued for 3 days at 1 10 0 C. The volatiles were removed under reduced pressure. Saturated NaHCO 3 solution (50 mL) was added to the residue and the mixture was extracted with CH 2 Cl 2 (3 x 75 mL). The combined organic extracts were washed with water (150 mL) and dried over Na 2
SO
4 . The solvent was removed under reduced 20 pressure, and the residue was purified by flash column chromatography
(CH
2 Cl 2 /EtOH (25/1)) to yield (2,6-bis-N-[n-propylamino]-pyrimidin-4-yl)-N,O dimethyl-hydroxylamine (CXX) (1.2 g, 19%). 200 MHz IH-NMR (DMSO-d 6 , ppm) 6.57-6.45 (lH, m) 6.13 (lH, t, J= 5.5 Hz) 5.34 (lH, s) 3.59 (3H, s) 3.20-3.04 (4H, m) 3.03 (3H, s) 1.58-1.37 (4H, m) 0.87 (3H, t, J=7.1 Hz) 0.84 (3H, t, J=7.1 Hz). 25 (2,6-Bis-N-[n-propylamino]-pyrimidin-4-yl)-N, 0-dimethyl-hydroxylamine hydrogen sulfate (CXX) (2,6-Bis-N-[n-propylamino]-pyrimidin-4-yl)-N,O-dimethyl hydroxylamine (1.20 g, 4.74 mmol) in 1,4-dioxane (15 mL) was treated with 95% 30 H 2
SO
4 (0.27 mL, 4.74 mmol) in a dropwise manner at 0 0 C. The mixture was stirred at room temperature for 0.5 h and then the volatiles were removed under reduced pressure. The resulting residue was co-evaporated with dry toluene (3 x 5 mL) to yield (2,6-bis-N-[n-propylamino]-pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (CXXI) in quantitative yield. 400 MHz H-NMR (DMSO-d 6 , ppm) - 127 - WO 2013/181217 PCT/US2013/043052 11.3-10.5 (1H, m), 8.29 (0.4H, br s), 7.38 (0.6H, br s), 5.48-5.20 (1H, m), 3.70 (3H, s), 3.36-3.21 (5H, m), 3.20-3.08 (2H, m), 1.61-1.48 (4H, m), 0.9 (6H, t, J=7.4 Hz). ESI-MS (m/z) 254 [M+H]f; melting point: 123-126 'C. 5 Example 65: 2-(n-Propyl)amino-4-(i-propylamino-7-methyl-pyrrolidino[2,3-dipyrimidine (CXXVI) Example 66: 2-(n-Propyllamino-4-(i-propylamino-7-methyl-pyrrolidino[2,3-dipyrimidine 10 hydrochloride (CXXVII) CI CI /1
NH
2 TBDMSOTf M NH 1. CN N DC-' N 'C N N 2. HCI H H CXXII CXXIII CI N HN N HN N NH H2N NH 10%Pd/C \ NH -N K 2
CO
3 , nBuOH N EtOAc AcOHN N N N CXXIV CXXV CXXVI Scheme 46 2-(t-Butyl-dimethylsilyl)amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (CXXII) 15 To a dichloromethane solution (100 mL) of 2-amino-4-chloro-7H pyrrolo[2,3-d]pyrimidine (8.7 g, 52 mmol) was added Et 3 N (26 g, 260 mmol) and the mixture was stirred at -30 C. After this time, TBDMSOTf (15.1 g, 57.2 mmol) was added in a slow dropwise manner and the resultant reaction was stirred at ambient temperature for 1.5 h. The observed solid material completely dissolved to form a 20 light brown solution. The mixture was then quenched with 1 N NaOH (100 mL) and extracted with DCM (250 mL). The organic layer was washed with H 2 0 (150 mL) and then with a brine solution (150 mL) and dried over Na 2
SO
4 . The solvents were removed in vacuo and the residue was purified by flash column chromatography (PE/EtOAc=10/1 to 5/1) to afford 2-(t-butyl-dimethylsilyl)amino-4-chloro-7H 25 pyrrolo[2,3-d]pyrimidine (CXXII, 11.4 g, 79%) as a light yellow solid. LCMS: (ESI) m/z = 283 (M+H). - 128 - WO 2013/181217 PCT/US2013/043052 2-(t-Butyl-dimethylsilyl)amino-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (CXXIII) 2-(t-Butyl-dimethylsilyl)amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (CXXII) (700 mg, 2.5 mmol), 0.18 mL (3.5 mmol) of methyl iodide in DMF (10 mL), 5 and K 2
CO
3 (552 mg, 4 mmol) were reacted at ambient temperature for 15 h. After addition of H 2 0 (10 mL), the reaction was extracted with EtOAc (100 mL), and the organic layer was washed with a brine solution (10 mL), and dried over Na 2
SO
4 . The solvents were removed in vacuo, and the residue was purified by flash column chromatography (PE/EtOAc=10/1 to 5/1) to afford 2-(t-butyl-dimethylsilyl)amino-4 10 chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (CXXIII) (750 mg, 100%) as a light yellow oil. LCMS (ESI) m/z = 297 (M+H). 2-(n-Propyl)amino-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (CXXIV) Under a nitrogen atmosphere 2-(t-butyl-dimethylsilyl)amino-4-chloro 15 7-methyl-pyrrolo[2,3-d]pyrimidine (CXXIII, 5.0 g, 17 mmol) and 1-iodopropane (4.3 g, 25 mmol) were dissolved in DMF (20 mL). The reaction mixture was cooled to 0 C with vigorous stirring, then NaH (1 g of a 60% dispersion in mineral oil, 21 mmol) was added. The mixture was stirred for 10 min. and water (50 mL) was slowly added to quench the reaction. The aqueous solution was extracted with EtOAc (200 mL), 20 and the organic layer was washed with water (50 mL) and with a brine solution (50 mL), and lastly, dried over anhydrous Na 2
SO
4 . After evaporation of volatiles, a yellow oily residue (5.7 g) was isolated. The residue was dissolved in Et 2 0 (50 mL) and concentrated hydrochloric acid (10 mL) was added at 0 C with stirring. The mixture was reacted for an additional 10 min. After this time, the solution was 25 extracted with EtOAc (200 mL) and IN NaOH (200 mL). The organic layer was washed with H 2 0 (150 mL) and then with a brine solution (150 mL) and lastly, dried over Na 2
SO
4 . The solvents were removed in vacuo and the resultant residue was purified by flash column chromatography (PE/EtOAc=10/1 to 5/1) to afford 2-(n propyl)amino-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (3.78 g, 100%) of the 30 desired product as a yellow solid. LCMS: (ESI) m/z = 225 (M+H). 2-(n-Propyl)amino-4-(i-propyl)amino-7-methyl-pyrrolo[2,3-d]pyrimidine (CXXV) To a solution of 2-(n-propyl)amino-4-chloro-7-methyl-pyrrolo[2,3 d]pyrimidine (1.6 g, 7.1 mmol) in n-butanol (10 mL) was added potassium carbonate - 129 - WO 2013/181217 PCT/US2013/043052 (4.9 g, 35.5 mmol), followed by propan-2-amine (632 mg, 10.7 mmol). The mixture was stirred in an autoclave equipped with a stirrer at 140 C for 16 h. After cooling to room temperature, water was added (20 mL), and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water and then 5 with a brine solution, and lastly dried over anhydrous Na 2
SO
4 . The volatiles were removed in vacuo and the residue was purified by flash column chromatography (PE/EtOAc=3/1) to yield 2-(n-propyl)amino-4-(i-propyl)amino-7-methyl-pyrrolo[2,3 d]pyrimidine (1.2 g, 75%) as a yellow solid. LCMS: (ESI) m/z = 248 (M+H). 10 2-(n-Propyl)amino-4-(i-propylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine (CXXVI) To the solution of 2-(n-propyl)amino-4-(i-propyl)amino-7-methyl pyrrolo[2,3-d]pyrimidine (CXXV, 1.0 g, 4 mmol) in EtOAc (50 mL) were added 10% Pd/C (1.0 g) and AcOH (2.43 g, 40 mmol). The mixture was attached to a hydrogenation apparatus and the system was evacuated and then refilled with 15 hydrogen. The reaction was stirred at ambient temperature for 48 h. After this time, the mixture was filtered over 10 g of silica-gel on a glass-filter. The filtrate was concentrated and the residue was purified by flash column chromatography (DCM/MeOH=50/1 to 10/1) to yield 2-(n-propyl)amino-4-(i-propylamino-7-methyl pyrrolidino[2,3-d]pyrimidine (CXXVI, 500 mg, 50%) as a yellow solid. 20 2-(n-Propyl)amino-4-(i-propylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine hydrochloride (CXXVHJ) The isolated free amine (CXXVI, 500 mg, 2 mmol) was dissolved in
H
2 0 (10 mL) and 0.5 M aqueous HCl solution (4 mL), and the solution was 25 lyophilized to yield 2-(n-propyl)amino-4-(i-propylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine hydrochloride (CXXVII, 525 mg) as a brown solid. LCMS (ESI) m/z = 250 (M+H). 1 H NMR (500 MHz, MeOD) 6 (ppm) 3.80 (s, 1H), 3.58 (t, J= 8.5 Hz, 2H), 3.23-3.29 (m, 2H), 2.88 (s, 3H), 2.77 (t, J= 8.5 Hz, 2H), 1.52-1.56 (m, 2H), 1.15 (d, J= 6.5 Hz, 6H), 0.89 (d, J= 7.5 Hz, 3H). 30 Example 67: 2-(n-Propyl)amino-4-dimethylamino-7-methyl-pyrrolidino [2,3-dipyrimidine (CXXVIII) - 130 - WO 2013/181217 PCT/US2013/043052 Example 68: 2-(n-Propyllamino-4-dimethylamino-7-methyl-pyrrolidino [2,3-dipyrimidine hydrochloride (CXXIX) CI H HCI -N N -N N S\-NH \NH 10%PdC \-NH -N K 2
CO
3 , nBuOH N EtOAc, AcOH N N N N CXXIV CXXVII CXXVIII 5 Scheme 47 2-(n-Propyl)amino-4-dimethylamino-7-methyl-pyrrolo[2,3-d]pyrimidine (CXXVII) To 2-(n-propyl)amino-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (1.0 g, 4.5 mmol) (CXXIV) in n-butanol (20 mL) was added potassium carbonate (3.7 10 g, 27 mmol) and dimethylamine hydrochloride (1.0 g (22 mmol). The mixture was stirred in an autoclave equipped with a stirrer at 120 C for 16 h. After cooling to room temperature, water was added (20 mL), and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water and then with a brine solution, and dried over anhydrous Na 2 SO4. The solvents were removed 15 in vacuo and the residue was purified by flash column chromatography (PE/EtOAc=6/1) to yield 2-(n-propyl)amino-4-dimethylamino-7-methyl-pyrrolo[2,3 d]pyrimidine (CXXVII, 800 mg, 76%) as a yellow solid. LCMS (ESI) m/z = 234 (M+H)+. 20 2-(n-Propyl)amino-4-dimethylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine (CXXVIII) To the solution of 2-(n-propyl)amino-4-dimethylamino-7-methyl pyrrolo[2,3-d]pyrimidine (CXXVII, 800 mg, 3.4 mmol) in EtOAc (30 mL) was added 10% Pd/C (1.0 g) and AcOH (3 mL), and the mixture was attached to a hydrogenation 25 apparatus. The system was evacuated and then refilled with hydrogen. The mixture was stirred at ambient temperature for 48 h. The mixture was filtered through 10 g of silica-gel on a glass-filter. The filtrate was concentrated and the residue was purified by flash column chromatography (EtOAc/MeOH=25/1) to yield 2-(n-propyl)amino-4 dimethylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine (CXXVIII, 600 mg, 75%). 30 - 131 - WO 2013/181217 PCT/US2013/043052 2-(n-Propyl)amino-4-dimethylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine hydrochloride (CXXIX) 2-(n-Propyl)amino-4-dimethylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine (CXXVIII, 600 mg, 2.6 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M 5 aqueous HCl solution (5.2 mL), and the solution was lyophilized to yield 2-(n propyl)amino-4-dimethylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine hydrochloride (CXXIX, 600 mg) as a yellow solid. LCMS (ESI) m/z = 236 (M+H). 1 H NMR (500 MHz, MeOD) 6 (ppm) 3.66 (t, J= 9.0 Hz, 2H), 3.40 (t, J= 7.0 Hz, 2H), 3.33 (s, 1H), 3.23 (t, J= 9.5 Hz, 2H), 3.17 (s, 5H), 3.00 (s, 3H), 1.63-1.67 (m, 2H), 1.00 (t, J= 7 10 Hz, 3H). Example 69: 2-(n-Propyllamino-4-methylamino-7-methyl-pyrrolidino[2,3-dipyrimidine (CXXXI) Example 70: 15 2-(n-Propyllamino-4-methylamino-7-methyl-pyrrolidino[2,3-dipyrimidine hydrochloride (CXXXII) HN HN \)-NH 1%Pd/C \ N N EtOAc, AcOH N CN CN CXXX CXXXI Scheme 48 2-(n-Propyl)amino-4-methylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine (CXXYI) 20 To a solution of 2-(n-propyl)amino-4-methylamino-7-methyl pyrrolo[2,3-d]pyrimidine (CXXX) (2.0 g, 9.13 mmol) in EtOAc (50 mL) was added 10% Pd/C (2.1 g) and AcOH (8 mL), and the mixture was attached to a hydrogenation apparatus. The system was evacuated and refilled with hydrogen. The mixture was stirred at ambient temperature for 48 h. The mixture was filtered through 10 g of 25 silica-gel on a glass-filter. The filtrate was concentrated and the residue was purified by flash column chromatography (DCM/MeOH=60/1 to 10/1) to yield 2-(n propyl)amino-4-methylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine (CXXXI, 700 mg, 43%) as a yellow solid. - 132 - WO 2013/181217 PCT/US2013/043052 2-(n-Propyl)amino-4-methylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine hydrochloride (CXXXII) 2-(n-Propyl)amino-4-methylamino-7-methyl-pyrrolidino[2,3 d]pyrimidine (CXXX, 700 mg, 3.2 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M 5 aqueous HCl solution (7 mL), and the solution was lyophilized to yield 2-(n propyl)amino-4-methylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine (717 mg) as a brown solid. LCMS: (ESI) m/z = 222 (M+H). H NMR (500 MHz, MeOD) 6 (ppm) 3.50 (t, J=8.5, 2H), 3.22 (t, J= 7.0 Hz, 2H), 2.79 (s, 6H), 2.68 (t, J= 9.0 Hz, 2H), 1.43-1.51 (m, 2H), 0.81 (t, J= 8.0 Hz, 3H). 10 Example 71: 2-(n-Propyl)amino-4-(i-propyl)amino-7- i-propyl-pyrrolidino[2,3-dipyrimidine (CXXXVI) Example 72: 15 2-(n-Propyllamino-4-(i-propyllamino-7- i-propyl-pyrrolidino[2,3-dipyrimidine hydrochloride salt (CXXXVII) CI N ( / NSi/ 1.1 )N? 1 I N -. C ~ I N S1 N 2. HCI H CXXII CXXXIII CI HN HN NH H 2 N ,)NH 10%Pd/C N N N K 2
CO
3 , nBuOH N N EtOAc, AcOH N N N CN CXXXIV CXXXV CXXXVI Scheme 49 20 2-(t-Butyl-dimethylsilyl)amino-4-chloro-7-i-propyl-pyrrolo[2,3-d]pyrimidine (CXXXIII) 2-(t-Butyl-dimethylsilanyl)amino-4-chloro-7H-pyrrolo[2,3 d]pyrimidine (CXXII) (5 g, 17.7 mmol), 2-iodopropane (4.5 g, 26.6 mmol) and
K
2
CO
3 (4.3 g, 26.6 mmol) in DMF (20 mL) were reacted at ambient temperature for 25 15 h. After addition of H 2 0 (50 mL) to the reaction mixture, the aqueous solution - 133 - WO 2013/181217 PCT/US2013/043052 was extracted with EtOAc (300 mL) and the organic layer was washed with a brine solution (50 mL) and dried with Na 2
SO
4 . The solvents were removed in vacuo and the residue was purified by flash column chromatography (pet ether/EtOAc=10/1 to 5/1) to afford 2-(t-butyl-dimethylsilyl)amino-4-chloro-7-i-propyl-pyrrolo[2,3 5 d]pyrimidine (CXXXIII, 5.7 g, 100%) as a light yellow oil. LCMS: (ESI) m/z = 325 (M+H). 2-(n-Propyl)amino-4-chloro-7-i-propyl-pyrrolo[2,3-d]pyrimidine (CXXXIV) Under nitrogen atmosphere, 2-(t-butyl-dimethylsilanyl)amino-4 10 chloro-7-i-propyl-pyrrolo[2,3-d]pyrimidine (5.7 g, 17.6 mmol) and 1-iodopropane (4.5 g (26.4 mmol) were dissolved in DMF (20 mL). The reaction mixture was cooled to 0 0 C with vigorous stirring, and NaH (1.06 g of a 60% dispersion in mineral oil, 26.4 mmol) of (60%) was added. The mixture was stirred for 10 min, and then water (50 mL) was slowly added to quench the reaction. The mixture was extracted 15 with EtOAc (300 mL) and the organic layer was washed with water (50 mL) and then with a brine solution (50 mL), and lastly, dried over anhydrous Na 2 SO4. After evaporation of the solvents, a yellow oily residue was isolated (6.5 g). The residue was dissolved in Et 2 0 (50 mL), concentrated hydrochloric acid (10 mL) was added at 0 0 C with stirring, and the mixture was stirred for an additional 10 min. After the 20 reaction was completed, the solution was extracted with EtOAc (200 mL) and IN NaOH (200 mL). The organic layer was washed with H 2 0 (150 mL) and then with a brine solution (150 mL) and dried over Na 2 SO4. The solvents in vacuo and the residue was purified by flash column chromatography (PE/EtOAc=10/1 to 5/1) to yield 2-(n-propyl)amino-4-chloro-7-i-propyl-pyrrolo[2,3-d]pyrimidine (CXXXIV, 4.5 25 g, !00%) as a yellow solid. LCMS (ESI) m/z = 253 (M+H). 2-(n-Propyl)amino-4-(i-propyl)amino-7-i-propyl-pyrrolo[2,3-d]pyrimidine (CXXV) To 2-(n-propyl)amino-4-chloro-7-i-propyl-pyrrolo[2,3-d]pyrimidine (CXXXIV, 3.0 g, 12 mmol) in n-butanol (10 mL) was added propan-2-amine (1.05 g, 30 18 mmol) and potassium carbonate (2.5 g, 18 mmol). The resulting mixture was stirred in an autoclave equipped with a stirrer at 140 0 C for 16 h. After cooling to room temperature, water was added (20 mL), and the mixture was extracted with EtOAc (3 x50 mL). The combined organic layers were washed with water and then with a brine solution and dried over anhydrous Na 2
SO
4 . After the solvents were - 134 - WO 2013/181217 PCT/US2013/043052 removed in vacuo, the residue was purified by flash column chromatography (PE/EtOAc=5/1 to 3/1) to yield 2-(n-propyl)amino-4-(i-propyl)amino-7-i-propyl pyrrolo[2,3-d]pyrimidine (CXXXV, 1.2 g, 36%) as a yellow solid. LCMS: (ESI) m/z = 276 (M+H)+. 5 2-(n-Propyl)amino-4-(i-propyl)amino-7-i-propyl-pyrrolidino[2,3-d]pyrimidine (CXXXVI) To a solution of 2-(n-propyl)amino-4-(i-propyl)amino-7-i-propyl pyrrolo[2,3-d]pyrimidine (1.0 g, 3.6 mmol) in EtOAc (10 mL) were added 10% Pd/C 10 (1.0 g) and AcOH (2.43 g, 40 mmol). The mixture was attached to a hydrogenation apparatus. The system was evacuated and refilled with hydrogen gas. The mixture was stirred at ambient temperature for 48 h, and filtered through 10 g of silica-gel on a glass-filter. The filtrate was concentrated and the residue was purified by flash column chromatography (DCM/MeOH=100/1 to 20/1) to yield 2-(n-propyl)amino-4 15 (i-propyl)amino-7-i-propyl-pyrrolidino[2,3-d]pyrimidine (CXXXVI) (800 mg, 79%) as a yellow solid. 2-(n-Propyl)amino-4-(i-propyl)amino-7-i-propyl-pyrrolidino[2,3-d]pyrimidine hydrochloride (CXYXVII) 20 2-(n-Propyl)amino-4-(i-propyl)amino-7-i-propyl-pyrrolo[2,3 d]pyrimidine (200 mg, 0.72 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (1.5 mL) and then the solution was lyophilized to yield 2-(n propyl)amino-4-(i-propyl)amino-7-i-propyl-pyrrolidino[2,3-d]pyrimidine hydrochloride (225 mg, 95%) as a yellow solid. LCMS (ESI) m/z = 278 (M+H). 1 H 25 NMR (500 MHz, MeOD) 6 (ppm) 4.36 (s, 1H), 3.90 (s, 1H), 3.67 (t, J= 8.5 Hz, 2H), 3.30 (t, J= 7.5 Hz, 2H), 2.84 (t, J= 8.5 Hz, 2H), 1.60-1.66 (m, 2H), 1.24 (d, J= 6.0 Hz, 6H), 1.21 (d, J= 6.0 Hz, 6H), 0.98 (t, J= 7.5 Hz, 3H). Example 73: N-(2-Propylamino-7H-pyrrolo[2,3dlpyrimidin-4-yll-N,O-dimethyl 30 hydroxylamine (CXLI) Example 74: N-(2-Propylamino-7H-pyrrolo[2,3dlpyrimidin-4-yll-N,O-dimethyl hydroxylamine hydrochloride (CXLII) - 135 - WO 2013/181217 PCT/US2013/043052 HO N H 2 O C I NH 2 POCI NH2 H < y
H
2 N H CXXXVIII CXXXIX 0
H
3 CI N 1O-CH 3 H H / N H 3 C-N-0-CH 3 N [RED] N N N NN N N H H H H CXL CXLI
H
3 C N O-CH 3 H-CI NG NN N I I I H H H CXLII Scheme 50 2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3d]pyrimidine (CXXXVIII) 5 To the solution of 2,4-diamino-6-hydroxypyrimidine (50 g, 397 mmol) in H 2 0 (750 mL) was added 2-chloroacetaldehyde (40% in H 2 0, 85 g, 437 mmol) in a dropwise manner at 0 C. The mixture was stirred at 65'C for 2 h and then heated at 100 0 C until the reaction was complete. The resultant solids were filtered and the remaining residue was heated at reflux in EtOH (750 mL). The additional 10 solids were filtered and the mother liquor was concentrated to afford 2-amino-4-oxo 4,7-dihydro-3H-pyrrolo[2,3d]pyrimidine(CXXXVIII) as a yellow solid 40 g (~67%, ~70% purity). LCMS (ESI) m/z = 151 (M+H)+. 2-Amino-4-chloro-7H-pyrrolo[2,3d]pyrimidine (CXXXIX) 15 2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3d]pyrimidine (CXXXVIII, 25 g, 167 mmol) was suspended in POCl 3 (200 mL) and cooled in an ice bath. The mixture was slowly warmed and heated up to 120 C for 3 h. After this time, the volatiles (excess POCl 3 ) were evaporated the under vacuum. To this residue was added ice water (200 mL) and the resultant solid was filtered to afford 2-amino-4 20 chloro-7H-pyrrolo[2,3d]pyrimidine (CXXXIX) as a yellow solid (20 g, ~71%, ~75% purity). LCMS (ESI) m/z = 169 (M+H)+. - 136 - WO 2013/181217 PCT/US2013/043052 4-Chloro-2-n-propylamino-7H-pyrrolo[2,3d]pyrimidine (CXL) To a solution of 2-amino-4-chloro-7H-pyrrolo[2,3d]pyrimidine (CXXXIX, 26 g, 155 mmol) and n-propionaldehyde (27 g, 464 mmol) in MeOH (600 5 mL) was added AcOH (50 mL). The reaction was stirred at ambient temperature for 30 min. After this time, NaBH 3 CN (49 g, 775 mmol) was added in portions at -20 C for 30 min. The resultant mixture was stirred at 80 C for 3 h and the volatiles were removed. The resultant residue was extracted with EtOAc (3 x 300 mL) and the combined organics were washed with a brine solution (2 x 100 mL). The organic 10 layer was dried over Na 2
SO
4 , concentrated and purified via silica-gel column chromatography (pet ether/EtOAc (5/1)) to afford 4-chloro-2-n-propylamino-7H pyrrolo[2,3d]pyrimidine (CXL) as a yellow solid (6 g, 18%). LCMS (ESI) m/z = 211 (M+H). 15 N-(2-Propylamino- 7H-pyrrolo[2,3d]pyrimidin-4-yl)-N, 0-dimethyl-hydroxylamine (CXLI) To a solution of 4-chloro-2-n-propylamino-7H pyrrolo[2,3d]pyrimidine (CXL, 1.0 g, 4.8 mmol) in n-BuOH (5 mL) was added potassium carbonate (3.3 g, 5.0 eq.) and N,O-dimethylhydroxylamine hydrochloride 20 (1.1 g, 4.0 eq.). The mixture was stirred in an autoclave equipped with a stirrer at 100 0 C for 8 h. After cooling to ambient temperature, water was added (20 mL) and the mixture was extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with water and then with a brine solution, and dried over anhydrous Na 2
SO
4 . The solvents were removed in vacuo and the resultant residue was purified 25 by flash column chromatography (pet ether/EtOAc=5/1) to yield N-(2-n-propylamino 7H-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine (CXLI, 500 mg, 45%). N-(2-Propylamino- 7H-pyrrolo[2,3d]pyrimidin-4-yl)-N, 0-dimethyl-hydroxylamine 30 hydrochloride (CXLII) N-(2-n-propylamino-7H-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl hydroxylamine (CXLI, 500 mg, 2.1 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (5 mL). The solution was then lyophilized to yield N-(2-n propylamino-7H-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine - 137 - WO 2013/181217 PCT/US2013/043052 hydrochloride as a white solid (CXLII, 550 mg). LCMS (ESI) m/z = 236 (M+H). H NMR (500 MHz, MeOD) 6 (ppm) 6.74 (d, J= 3.5 Hz, 1H), 6.41 (d, J= 4.0 Hz, 1H), 3.80 (s, 3H), 3.35 (s, 3H), 3.28-3.35 (m, 2H), 1.59-1.62 (m, 2H), 0.97 (t, J= 7.0 Hz, 3H). 5 Example 75: 2,4-Bis-(n-propyllamino-7H-pyrrolidino[2,3-dipyrimidine (CXLIX) Example 76: 2,4-Bis-(n-propyllamino-7H-pyrrolidino[2,3-dipyrimidine hydrochloride (CL) 10 5-Allyl-2-amino-4,6-dihydroxypyrimidine (CXLIII) Guanidine hydrochloride (27 g, 0.28 mol) was added to cold absolute EtOH (200 mL) and NaOEt (30% in ethanol) (200 mL), and the mixture was stirred at 0 C for 10 min. After this time, diethyl allylmalonate (55 g, 0.28 mol) was added. The reaction mixture was then stirred at room temperature for 18 h. Acidification 15 with 3N HCl precipitated the crude product (pH=6). The solid was collected by filtration and washed with ethanol. Recrystallization from water afforded 29 g (62 %) of pure 5-allyl-2-amino-4,6-dihydroxypyrimidine (CXLIII). LCMS (ESI) m/z = 168 (M+H). 00NH 0 CI C1 EtO OE H 2 N NH 2 HN POl 3 N 03 N tH 2 N IZN OH PCI 5
H
2 N JZ N CI H 2 N)N CI Dimethylaniline CXLIII CXLIV CXLV 0CIICI
PMBNH
2 I O N TFA N N N NH 2 N N N N N
CH
3 0 / \ PMB H H H CXLVI CXLVII CXLVIII
HN'
K2CO3 N N N N H H 20 CXLIX Scheme 51 5-Allyl-2-amino-4,6-chloropyrimidine (CXLIV) - 138 - WO 2013/181217 PCT/US2013/043052 5-Allyl-2-amino-4,6-dihydroxypyrimidine (4.9 g, 28.0 mmol) was added in small portions to a solution of PCl 5 (6.6 g, 29.5 mmol) in POCl 3 (180 mL) at 60 C, and diethylaniline (3 g) was added dropwise. The temperature was raised to 120 C. The reaction mixture was heated at reflux overnight before it was evaporated 5 to dryness. Hot water (100 'C) (100 mL) was added slowly to the residue, and the resulting suspension was cooled and extracted with CH 2 Cl 2 (2 x 100 mL). The combined organic layers were washed with cold water three times until the aqueous extract was above pH=5. The organic layer was dried (Na 2
SO
4 ) and evaporated to dryness in vacuo. The resultant residue was purified by column chromatography 10 (EtOAc/pet ether=l:10) to afford 5-allyl-2-amino-4,6-chloropyrimidine (CXLIV, 2.5 g, 42%). LCMS (ESI) m/z = 204 (M+H). 2-(2-Amino-4,6-chloropyrimidin-5-yl)ethanal (CXLV) 5-Allyl-2-amino-4,6-chloropyrimidine (1 g, 4.9 mmol) was dissolved 15 in ethyl acetate (40 mL) and reacted with ozone gas at -78 C for about 1 h (about 5% ozone at a rate of 1 L/min). The reaction was monitored by TLC (pet ether/AcOEt=3/l (v/v)), and once the starting material was consumed, the reaction mixture was flushed with oxygen for 10 min. At this time, Nal (3 g) and glacial acetic acid (3 mL) were added simultaneously to the cold reaction mixture, and the 20 temperature was allowed to warm up to 20 C with continuous stirring over a 60-min period. Sodium thiosulfate solution (67 g/100 mL of H 2 O) was added to the reaction mixture until it became colorless. The resulting mixture was diluted with water (30 mL) and extracted with CH 2 Cl 2 (4 x 70 mL). The combined organic extracts were washed successively with H 2 0 (4 x 30 mL), a saturated NaHCO 3 solution (30 mL) 25 and with a brine solution (30 mL) and lastly, dried over anhydrous Na 2
SO
4 ). After filtration and evaporation, 2-(2-amino-4,6-chloropyrimidin-5-yl)ethanal (CXLV, 1.1 g, 87%) was isolated as a white solid with ~80% purity. LCMS (ESI) m/z = 207 (M+H). 30 2-Amino-4-chloro-7-(4-methoxy)benzyl-pyrrolidino[2,3-d]pyrimidine (CXLVI) A solution of 2-(2-amino-4,6-chloropyrimidin-5-yl)ethanal (CXLV, 1.2 g, 5.8 mmol) and para-methoxybenzylamine (PMBNH 2 ) (1.6 g, 11.6 mmol) in THF (20 mL) and AcOH (2 mL) was stirred at ambient temperature for 30 min. To this mixture was added NaBH(OAc) 3 (6.2 g, 29 mmol) in portions and the reaction - 139 - WO 2013/181217 PCT/US2013/043052 was stirred overnight. The mixture was concentrated in vacuo, and extracted with EtOAc (3 x 80 mL). The combined organic extracts were then washed with a brine solution (2 x 50 mL), dried over anhydrous Na 2
SO
4 , filtered and concentrated in vacuum. The resultant residue was purified by flash column chromatography (pet 5 ether/EtOAc=10/1) to afford 2-amino-4-chloro-7-(4-methoxy)benzyl-pyrrolidino[2,3 d]pyrimidine (CXLVI, 950 mg, 52%) as a yellow solid (52%). LCMS (ESI) m/z = 291 (M+H)+. 2-n-Propylamino-4-chloro-7-(4-methoxy)benzyl-pyrrolidino[2,3-d]pyrimidine 10 (CXLVII) 2-Amino-4-chloro-7-(4-methoxy)benzyl-pyrrolidino[2,3-d]pyrimidine (CXLVI, 950 mg, 3.3 mmol) and propionaldehyde (575 mg, 16.5 mmol) in MeOH (30 mL) and AcOH (3 mL) were stirred at ambient temperature for 30 min. At this time, NaBH 3 CN (1.0 g, 16.5 mmol) was added in portions and the reaction was then 15 heated at 85 C for 16 h. After cooling, the mixture was evaporated, and extracted with EtOAc (2 x 50 mL). The combined organics were then washed with a brine solution (2 x50 mL). The organic layer was dried over anhydrous Na 2
SO
4 , filtered and concentrated in vacuum. The resultant residue was purified by flash column chromatography (PE/EtOAc=10/1) to afford 2-n-propylamino-4-chloro-7-(4 20 methoxy)benzyl-pyrrolidino[2,3-d]pyrimidine (CXLVII, 920 mg, 85%). LCMS (ESI) m/z = 333 (M+H). 2-n-Propylamino-4-chloro-7H-pyrrolidino[2,3-d]pyrimidine (CXLVIII) A solution of 2-n-propylamino-4-chloro-7-(4-methoxy)benzyl 25 pyrrolidino[2,3-d]pyrimidine (CXLVII, 920 mg, 3.2 mmol) in TFA (5 mL) was heated at 85 C for 3 h. After cooling the mixture was evaporated and extracted with EtOAc (2 x 50 mL). The combined organics were washed with a brine solution (2 x 50 mL), dried over Na 2
SO
4 , filtered and concentrated in vacuum. The resultant residue was purified by flash column chromatography (pet ether/EtOAc=5/1) to afford 30 2-n-propylamino-4-chloro-7H-pyrrolidino[2,3-d]pyrimidine (CXLVIII, 500 mg, 85%) as a colorless solid. LCMS (ESI) m/z = 213 (M+H). 2,4-Bis-(n-propyl)amino- 7H-pyrrolidino[2,3-d]pyrimidine (CXLIX) - 140 - WO 2013/181217 PCT/US2013/043052 2-n-Propylamino-4-chloro-7H-pyrrolidino[2,3-d]pyrimidine (CXLVIII, 500 mg, 2.38 mmol) was dissolved in n-butanol (5 mL), and potassium carbonate (1.64 g, 5.0 eq.) and propan-1-amine (923 mg, 4.0 eq.) were added. The mixture was stirred in an autoclave equipped with a stirrer at 100 0 C for 72 h. After 5 cooling to room temperature, 20 mL of water was added, and the mixture was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with water and with a brine solution, and dried over anhydrous Na 2
SO
4 . After removal of the solvents in vacuo, the resultant residue was purified by preparative HPLC to yield 2-n-propylamino-4-chloro-7H-pyrrolidino[2,3-d]pyrimidine (CXLIX, 210 mg, 37% 10 yield). 2,4-Bis-(n-propyl)amino-7H-pyrrolidino[2,3-d]pyrimidine hydrochloride (CL) 2-n-Propylamino-4-chloro-7H-pyrrolidino[2,3-d]pyrimidine (CXLIX, 210 mg, 0.89 mmol) was dissolved in H 2 0 (5 mL) and 0.5 M aqueous HCl solution 15 (2.0 mL), and the solution was lyophilized to yield 2,4-bis-(n-propyl)amino-7H pyrrolidino[2,3-d]pyrimidine hydrochloride (CL, 242 mg, 95% yield) as an orange solid. LCMS (ESI) m/z = 236 (M+H). 1 H NMR (500 MHz, MeOD) 6 (ppm) 3.72 (t, J= 9.0 Hz, 2H), 3.30-3.34 (m, 4H), 2.90 (t, J= 9.0 Hz, 2H), 1.61-1.65 (m, 4H), 0.96 0.99 (m, 6H). 20 Example 77: 2-(n-Propyl)amino-4-(4-hydroxypiperidin-1-vl)-7-methyl-pyrrolidino[2,3 dlpyrimidine (CLII) Example 78: 25 2-(n-Propyllamino-4-(4-hydroxypiperidin-1-vl)-7-methyl-pyrrolidino[2,3 dlpyrimidine hydrochloride (CLIII) OH HO HO CI N N HCI N N N \ -N H P./- r N N H \NH 10%Pd/C \ IN N K 2
CO
3 , nBuOH N N EtOAc, AcOH N'N CXXIV CLI CLII Scheme 52 - 141 - WO 2013/181217 PCT/US2013/043052 2-(n-Propyl)amino-4-(4-hydroxypiperidin-1-yl)- 7-methyl-pyrrolo[2,3-d]pyrimidine (CLI) 2-(n-Propyl)amino-4-chloro-7-methyl-pyrrolo[2,3 -d]pyrimidine (CXXIV) (1.6 g, 7.1 mmol) was added to n-butanol (10 mL) followed by potassium 5 carbonate (4.9 g, 35.5 mmol) and piperidin-4-ol hydrochloride (632 mg, 10.7 mmol). The mixture was stirred in an autoclave equipped with a stirrer at 130 C for 16 h. After cooling to room temperature, water was added (20 mL), and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water and then with a brine solution, and dried over anhydrous Na 2
SO
4 . The solvents 10 were removed in vacuo and the residue was purified by flash column chromatography (pet ether/EtOAc=3/1) to yield 2-(n-propyl)amino-4-(4-hydroxypiperidin-1-yl)-7 methyl-pyrrolo[2,3-d]pyrimidine (1.2 g, 75%) as a yellow solid. LCMS (ESI) m/z = 290 (M+H)+. 15 2-(n-Propyl)amino-4-(4-hydroxypiperidin-1-yl)- 7-methyl-pyrrolidino[2,3 dipyrimidine (CLII) To the solution of 2-(n-propyl)amino-4-(4-hydroxypiperidin- 1 -yl)-7 methyl-pyrrolo[2,3-d]pyrimidine (CLI, 1.0 g, 4 mmol) in EtOAc (50 mL) were added 10% Pd/C (1.0 g) and AcOH (2.43 g, 40 mmol). The mixture was attached to a 20 hydrogenation apparatus. The system was evacuated and refilled with hydrogen gas. The mixture was stirred at ambient temperature for 48 h. The mixture was filtered through 10 g of silica-gel on a glass-filter. The filtrate was concentrated and the residue was purified by flash column chromatography (DCM/MeOH=50/1 to 10/1) to yield 2-(n-propyl)amino-4-(4-hydroxypiperidin-1-yl)-7-methyl-pyrrolidino[2,3 25 d]pyrimidine (CLII, 500 mg, 50 % yield) as a yellow solid. 2-(n-Propyl)amino-4-(4-hydroxypiperidin-1-yl)- 7-methyl-pyrrolidino[2,3 dipyrimidine hydrochloride (CLIII) 2-(n-Propyl)amino-4-(4-hydroxypiperidin-1-yl)-7-methyl 30 pyrrolidino[2,3-d]pyrimidine (CLII, 500 mg, 2 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M HCl solution in H 2 0 (4 mL) and the solution was lyophilized to yield 2-(n propyl)amino-4-(4-hydroxypiperidin-1-yl)-7-methyl-pyrrolidino[2,3-d]pyrimidine hydrochloride (525 mg) as a yellow solid. LCMS (ESI) m/z = 292 (M+H). 1 H NMR (500 MHz, MeOD) 6 (ppm) 4.12-4.15 (m, 2H), 3.80-3.82 (m, 1H), 3.40 (t, J= 8.0 Hz, - 142 - WO 2013/181217 PCT/US2013/043052 2H), 3.29 (t, J= 7.0 Hz, 2H), 3.08-3.14 (m, 2H), 3.02 (t, J= 8.0 Hz, 2 H), 2.84 (s, 3H), 1.86-1.90 (m, 2H), 1.58-1.62 (m, 2H), 1.46-1.51 (m, 2H), 0.97 (t, J = 4.0 Hz, 3H). 5 Example 79: 8-(7-methyl-2-(n-propylamino)-pyrrolidino[2,3-dlpyrimidin-4-vl)-8 azabicyclo[3.2. lloctan-3-ol (CLV) Example 80: 8-(7-methyl-2-(n-propylamino)-pyrrolidino[2,3-dlpyrimidin-4-yl)-8 10 azabicyclo[3.2.lloctan-3-ol hydrochloride (CLVI) OH HO HO C N N HCI N -N 10%Pd/C N N
K
2 0NH 0,NH n HNNH N N K2CO3, nBuOH N EtOAc, AcOH NN CXXIV CLIV CLV Scheme 53 8-(7-Methyl-2-(n-propylamino)- 7H-pyrrolo[2,3-d]pyrimidin-4-yl)-8 15 azabicyclo[3.2. 1]octan-3-ol (CLIV) To a solution of 2-(n-propyl)amino-4-chloro-7-methyl-pyrrolo[2,3 d]pyrimidine (CXXIV) (1.0 g, 4.5 mmol) in n-butanol (10 mL) were added DIPEA (1.0 g, 7.8 mmol)and 8-aza-bicyclo[3.2.1]octan-3-ol hydrochloride (1.1 g, 6.7 mmol). The mixture was stirred at 125 C for 16 h. After cooling to room temperature, water 20 (20 mL) was added, and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water and then with a brine solution, and dried over anhydrous Na 2
SO
4 . The solvents were removed in vacuo and the resultant residue was purified by flash column chromatography (pet ether/EtOAc=6/1) to yield 8-(7-methyl-2-(n-propylamino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-8 25 azabicyclo[3.2.1]octan-3-ol (CLIV) (800 mg, 57% yield) as a yellow solid. LCMS (ESI) m/z = 316 (M+H). 8-(7-Methyl-2-(n-propylamino)-pyrrolidino[2,3-d]pyrimidin-4-yl)-8 azabicyclo[3.2. 1]octan-3-ol (CLV) - 143 - WO 2013/181217 PCT/US2013/043052 To a solution of 2-(n-propyl)amino-4-(4-hydroxy- 1 -aza bicyclo[3.2.1]octan-1-yl)-7-methyl-pyrrolo[2,3-d]pyrimidine (CLIV, 1.00 g, 3.2 mmol) in EtOAc (10 mL) were added 10% Pd/C (1.0 g) and AcOH (3 mL). The mixture was attached to a hydrogenation apparatus. The system was evacuated and 5 refilled with hydrogen. The mixture was stirred at ambient temperature for 48 h. The mixture was filtered through 10 g of silica-gel on a glass-filter. The filtrate was concentrated and the residue was purified by flash column chromatography (EtOAc/MeOH=50/1) to yield 8-(7-methyl-2-(n-propylamino)-pyrrolidino[2,3 d]pyrimidin-4-yl)-8-azabicyclo[3.2.1 ]octan-3-ol (CLV) (500 mg, 49% yield) as a 10 yellow solid. 8-(7-Methyl-2-(n-propylamino)-pyrrolidino[2,3-d]pyrimidin-4-yl)-8 azabicyclo[3.2. 1]octan-3-ol hydrochloride (CL VI) 8-(7-Methyl-2-(n-propylamino)-pyrrolidino[2,3-d]pyrimidin-4-yl)-8 15 azabicyclo[3.2.1]octan-3-ol (CLV, 200 mg, 0.63 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (1.3 mL), and the solution was lyophilized to yield 8-(7-methyl-2-(n-propylamino)-pyrrolidino[2,3-d]pyrimidin-4-yl)-8 azabicyclo[3.2.1]octan-3-ol hydrochloride (CLVI) (224 mg) as a yellow solid. LCMS (ESI) m/z = 318 (M+H). H NMR (500 MHz, DMSO) 6 (ppm) 4.55 (br, 2H), 3.90 20 (s, 1H), 3.60 (t, J= 8.0 Hz, 2H), 3.27 (t, J= 6.5 Hz, 2 H), 3.00 (t, J= 9.0 Hz, 2H), 2.92 (s, 3H), 2.27 (d, J= 7.0 Hz, 2H), 1.90-1.95 (m, 4H), 1.69 (s, 1H), 1.66 (s, 1H), 1.49-1.54 (m, 2H), 0.92 (t, J= 7.0 Hz, 3H). Example 81: 25 N-(2-(Propen-2-vl)amino-7-methyl-pyrrolo[2,3dlpyrimidin-4-vl)-N,O-dimethyl hydroxylamine (CLVIII) Example 82: N-(2-(Propen-2-yllamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl)-N,O-dimethyl hydroxylamine hydrochloride (CLIX) - 144 - WO 2013/181217 PCT/US2013/043052 0 CI CI -N' \ NH 1. I \-NH H HC \)-NH N2.HCI N K 2
CO
3 , nBuOH N N N N CXXIII CLVII CLVlI Scheme 54 2-(Propen-2-yl)amino-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (CLVII) 5 Under a nitrogen atmosphere, 2-(t-butyl-dimethylsilanyl)amino-4 chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (CXXIII) (7.5 g, 25 mmol) was dissolved in DMF (100 mL) and 3-iodoprop-1-ene (6.38 g, 38 mmol) was added. The mixture was cooled to 0 C with vigorous stirring, then NaH (1.5 g of a 60% dispersion in mineral oil, 38 mmol) was added. The mixture was stirred for 30 min, and water (50 10 mL) was slowly added to quench the reaction. The aqueous mixture was extracted with EtOAc (300 mL) and the organic layer was washed with water (100 mL) and then with a brine solution (100 mL), and dried over anhydrous Na 2
SO
4 . After evaporation, 8.6 g of a yellow oily residue was isolated. This material was dissolved in Et 2 0 (100 mL), and concentrated hydrochloric acid (20 mL) was added at 0 C with 15 stirring. The mixture was then stirred for an additional 10 min. The mixture was extracted with EtOAc (200 mL) and IN NaOH (200 mL). The organic layer was separated and washed with H 2 0 (150 mL) and with a brine solution (150 mL) and dried over anhydrous Na 2
SO
4 . The solvents were removed in vacuo and the resultant residue was purified by flash column chromatography (pet ether/EtOAc=10/1 to 5/1) 20 to yield 2-(propen-2-yl)amino-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (6 g, 100% yield) as a yellow solid. LCMS: (ESI) m/z = 223 (M+H). N-(2-(Propen-2-yl)amino- 7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N, 0-dimethyl hydroxylamine (CLVIII) 25 To 2-(propen-2-yl)amino-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (CLVII, 500 mg, 2.38 mmol) were added n-butanol (5 mL), potassium carbonate (1.64 g, 5.0 eq.) and N,O-dimethylhydroxylamine hydrochloride (923 mg, 4.0 eq.). The mixture was stirred in an autoclave equipped with a stirrer at 100 C for 8 h. After cooling to room temperature, water (20 mL) was added, and the mixture was 30 extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with - 145 - WO 2013/181217 PCT/US2013/043052 water and with a brine solution and dried over anhydrous Na 2
SO
4 . The solvents were then removed in vacuo and the resultant residue was purified by flash column chromatography (pet ether/EtOAc=3/1) to yield N-(2-(propen-2-yl)amino-7-methyl pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine (CLVIII, 310 mg, 55% 5 yield). N-(2-(Propen-2-yl)amino- 7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N, 0-dimethyl hydroxylamine hydrochloride (CLIX) N-(2-(Propen-2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O 10 dimethyl-hydroxylamine (310 mg, 1.32 mmol) was dissolved in H 2 0 (5 mL) and 0.5 M aqueous HCl solution (2.7 mL), and the solution was lyophilized to yield N-(2 (propen-2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl hydroxylamine hydrochloride (CLIX, 325 mg) as a white solid. LCMS (ESI) m/z = 248 (M+H). 1 H NMR (500 MHz, DMSO) 6 (ppm) 12.60- 14.80 (br, 1H), 7.70-8.70 15 (br, 1H), 7.06 (s, 1H), 6.55 (s, 1H), 5.90-5.94 (m, 1H), 5.31 (d, J= 17.0 Hz, 1H), 5.17 (d, J= 10.5 Hz, 1H), 4.06 (s, 2H), 3.84 (s, 3H), 3.70 (s, 3H), 3.53 (s, 3H). Example 83: N-(2-(Propen-2-vl)amino-7-methyl-pyrrolo[2,3dlpyrimidin-4-vl)-O-methyl 20 hydroxylamine (CLX) Example 84: N-(2-(Propen-2-yllamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl)-O-methyl hydroxylamine hydrochloride (CLXI) 0 HN N N N N CLX Scheme 55 25 N-(2-(Propen-2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl hydroxylamine (CLX) 2-(2-Propen-2-yl)amino-4-chloro-7-methyl-pyrrolo[2,3-d]pyrimidine (CLVII) (700 mg, 3.15 mmol) was dissolved in n-butanol, and potassium carbonate (2.2 g, 5.0 eq.) and O-methylhydroxylamine hydrochloride (768 mg, 4.0 eq.) were - 146 - WO 2013/181217 PCT/US2013/043052 added. The mixture was stirred in an autoclave equipped with a stirrer at 100 C for 12 h. After cooling to room temperature, water (20 mL) was added, and the mixture was extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with water and a brine solution, and dried over anhydrous Na 2
SO
4 . The solvents were 5 removed in vacuo and the resultant residue was purified by flash column chromatography (pet ether/EtOAc=5/1) to yield N-(2-(propen-2-yl)amino-7-methyl pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine (370 mg, 46% yield). N-(2-(Propen-2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl 10 hydroxylamine hydrochloride (CLXI) N-(2-(propen-2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O methyl-hydroxylamine (370 mg, 1.59mmol) was dissolved in H 2 0 (5 mL) and 0.5 M aqueous HCl solution (2 mL), and the solution was lyophilized to yield N-(2-(propen 2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine 15 hydrochloride (383 mg) as a white solid. LCMS (ESI) m/z = 234 (M+H). 1 H NMR (500 MHz, DMSO) 6 (ppm) 12.40-13.00 (br, 1H), 7.70-8.10 (br, 1H), 7.04 (d, J= 3.5 Hz, 1H), 6.49 (d, J= 3.0 Hz, 1H), 5.94-5.99 (m, 1H), 5.32 (d, J= 17.0 Hz, 1H), 5.17 (d, J= 10.5 Hz, 1H), 4.04 (s, 2H), 3.85 (s, 3H), 3.61 (s, 3H). 20 Example 85: N-(2-n-Propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl)-N,O-dimethyl hydroxylamine (CLXII) Example 86: N-(2-n-Propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl)-N,O-dimethyl 25 hydroxylamine hydrochloride (CLXIII) 0 -N N N N CN CLXII Scheme 56 N-(2-n-Propylamino- 7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N, 0-dimethyl hydroxylamine (CLXII) - 147 - WO 2013/181217 PCT/US2013/043052 The desired compound was prepared from 4-chloro-2-n-propylamino 7-methyl-pyrrolo[2,3d]pyrimidine and N,O-dimethyl-hydroxylamine as described in Example 73. LCMS: (ESI) m/z = 250 (M+H). 1 H NMR (500 MHz, CDCl 3 ) 6 (ppm) 7.70 (s, 1H), 7.02 (s, 1H), 6.52 (s, 1H), 3.82 (s, 3H), 3.67 (s, 3H), 3.53 (s, 3H), 3.34 (t, 5 J=8.5 Hz, 2H), 1.54-1.59 (m, 2H), 0.93 (t, J=8.5 Hz, 3H). Example 87: N-(2-n-Propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-vl)-O-methyl hydroxylamine (CLXIV) 10 Example 88: N-(2-n-Propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl)-O-methyl hydroxylamine hydrochloride (CLXV) 0 HN N /\)-NH N N CLXIV Scheme 57 15 N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine (CLXIV) The desired compound was prepared from 4-chloro-2-n-propylamino 7-methyl-pyrrolo[2,3d]pyrimidine and O-methyl-hydroxylamine as described in Example 73. LCMS (ESI) m/z = 236 (M+H). 1 H NMR (500 MHz, DMSO) 6 (ppm) 20 12.50 (s, 1H), 8.10 (s, 1H), 7.05 (s, 1H), 6.01 (s, 1H), 3.85 (s, 3H), 3.60 (s, 3H), 3.36 (s, 1H), 3.34 (t, J= 6.5 Hz, 2H), 1.58-1.62 (m, 2H), 0.96 (t, J= 7.5 Hz, 3H). Example 89: N-(2-n-Propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl)-hydrazine (CLXVI) 25 Example 90: N-(2-n-Propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl)-hydrazine hydrochloride (CLXVII) - 148 - WO 2013/181217 PCT/US2013/043052 C1
'NH
2 C N H 2
N-NH
2 HN - N ,/ -NH N I N \N CXXIV CLXVI Scheme 58 N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (CLXVI) To a solution of 4-chloro-2-n-propylamino-7-methyl 5 pyrrolo[2,3d]pyrimidine (CXXIV, 1.0 g, 4.5 mmol) in ethanol (70 mL) was added hydrazine (14 mL) and the mixture was heated at reflux for 3 h. After cooling to room temperature, the solvents were removed in vacuo. The residue was purified by flash column chromatography (CH 2 Cl 2 /MeOH=30/1) to yield N-(2-n-propylamino-7 methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (CLXVI, 1.0 g, 80% yield). 10 N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine hydrochloride (CLXVII) N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (460 mg, 2.0 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution 15 (4 mL), and the solution was lyophilized to yield N-(2-n-propylamino-7-methyl pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine hydrochloride (440 mg) as a brown solid. LCMS (ESI) m/z = 221 (M+H). 1 H NMR (500 MHz, DMSO) 6 (ppm) 10.63 (s, 1H), 7.56 (s, 2H), 6.97 (d, J= 3.0 Hz, 1H), 6.54 (d, J= 3.5 Hz, 1H), 3.58 (s, 3H), 3.33 (s, 2H), 1.56-1.61 (m, 2H), 0.95 (t, J= 8.0 Hz, 3H). 20 Example 91: N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl)-hydrazine (CLXVIII) Example 92: 25 N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl)-hydrazine hydrochloride (CLXIX) - 149 - WO 2013/181217 PCT/US2013/043052 Cl H 3 Cs /NH 2 / NH H 2
N-NH-CH
3 N >-NHN N >NH I N I N \NN CXXIV CLXVIII Scheme 59 N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (CLXVIII) 5 To a solution of 4-chloro-2-n-propylamino-7-methyl pyrrolo[2,3d]pyrimidine (CXXIV, 800 mg, 3.42 mmol) in n-butanol (5 mL) was added potassium carbonate (2.36 g, 5.0 eq.) and methylhydrazine (630 mg, 4.0 eq.). The mixture was stirred in an autoclave equipped with a stirrer at 100 C for 15 h. After cooling to room temperature, water was added (20 mL), and the mixture was 10 extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with water and with a brine solution, and dried over anhydrous Na 2
SO
4 . The solvents were removed in vacuo and the resultant residue was purified by flash column chromatography (pet ether/EtOAc=3/1 to DCM/MeOH=10/1) to yield N-methyl-N (2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (CLXVIII, 220 15 mg, 36% yield). N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine hydrochloride (CLXIX) N-methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) 20 hydrazine (220 mg, 0.94 mmol) was dissolved in H 2 0 (10 mL) and 0.5 M aqueous HCl solution (2 mL), and the solution was lyophilized to yield N-methyl-N-(2-n propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine hydrochloride (CLXIX, 238 mg) as a brown solid. LCMS (ESI) m/z = 235 (M+H). H NMR (500 MHz, DMSO) 6 (ppm) 7.50-8.10 (br, 4H), 7.05 (s, 1H), 6.67 (s, 1H), 3.60 (s, 3H), 25 3.58 (s, 3H), 3.33 (s, 2H), 1.55-1.62 (m, 2H), 0.95 (t, J= 7.5 Hz, 3H). Example 93: N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yl hydrazine (CLXX) - 150 - WO 2013/181217 PCT/US2013/043052 Example 94: N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3dlpyrimidin-4-yll hydrazine hydrochloride (CLXXI)
,N
C1 HN' C N H 2 N' H N \>NH \>-NH N N N N CXXIV CLXX Scheme 60 5 N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (CLXA) To a solution of 4-chloro-2-n-propylamino-7-methyl-pyrrolo[2,3d] pyrimidine (1.1 g, 5 mmol) and 1,1 -dimethylhydrazine (450 mg, 7.5 mmol) in 10 dioxane (30 mL) was added xphos (622 mg, 1 mmol), Pd 2 (dba) 3 (458 mg, 0.5 mmol) and Cs 2
CO
3 (2.45 g, 7.5 mmo). The solution was degassed by bubbling argon through it for 10 min using a syringe needle. The mixture was then stirred at 80 C for 2 h. After cooling to room temperature, water was added (20 mL) and the mixture was extracted with EtOAc (3 x 40 mL). The combined organic layers were washed 15 with water and with a brine solution, and dried over anhydrous Na 2
SO
4 . The solvents were removed in vacuo and the resultant residue was purified by flash column chromatography (DCM/MeOH=30/1 to 5/1) to yield N, N-dimethyl-N'-(2-n propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine (CLXX, 100 mg, 8% yield, ~85% purity). 20 N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine hydrochloride (CLXXI) N, N-dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin 4-yl)-hydrazine (100 mg, 0.4 mmol) was dissolved in H 2 0 (5 mL) and 0.5 M aqueous 25 HCl solution (1 mL) and the solution was lyophilized to yield N, N-dimethyl-N'-(2-n propylamino-7-methyl-pyrrolo[2,3-d]pyrimidin-4-yl)-hydrazine hydrochloride( 110 mg) as a yellow oil. LCMS (ESI) m/z = 249 (M+H)+. 1 H NMR (500 MHz, MeOD) 6 (ppm) 6.92 (d, J= 4.0 Hz, 1H), 6.50 (d, J= 3.0 Hz, 1H), 3.66 (s, 3H), 3.45 (t, J= 7.5 Hz, 2H), 2.79 (s, 6H), 1.67-1.72 (m, 2H), 1.01-1.04 (m, 3H). - 151 - WO 2013/181217 PCT/US2013/043052 Example 95: N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,51triazine (CLXXII)) Cl H 3
C-NH
2 .HCI H 3 C N H N N DIPEA N N N N THF 111 N ) H H TE-"N N N~ 70 0 C,48h H H XXX IV CLXXII 5 Scheme 61 A pressure tube was charged with 2-chloro-N-(4,6-bis-(n propylamino)-[1,3,5-triazine (XXXIV) (1.50 g, 6.53 mmol), N,N diisopropylethylamine (5.4 mL, 32.65 mmol), methylamine hydrochloride (2.2 g, 10 32.65 mmol) and tetrahydrofuran (20 mL). The reaction mixture was heated at 70 'C for 48 h. An additional amount of N,N-diisopropylethylamine (5.4 mL, 32.65 mmol) and methylamine hydrochloride (2.2 g, 32.65 mmol) were added, and the reaction mixture was heated at 70 'C for an additional 48 h. The mixture was poured into saturated NaHCO 3 solution (40 mL). The suspension was extracted with EtOAc (3 x 15 70 mL). The combined organic extracts were washed with water (70 mL), then with a brine solution (70 mL) and lastly dried over anhydrous Na 2
SO
4 . The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography using gradient elution from petroleum ether/EtOAc (9:1, (v/v)) to petroleum ether/EtOAc (1:4, (v/v)) to yield N-(2,4-bis-n-propylamino)-N-(6 20 methylamino)-[1,3,5]triazine_(0.35 g, 24%). 400 MHz 1 H NMR (DMSO-d 6 , ppm) 6.69-6.02 (3H, m), 3.22-3.02 (4H, m), 2.68 (3H, s), 1.55-1.38 (4H, m), 0.84 (6H, t, J=7.4 Hz). ESI-MS (m/z): 225 [M+H]f. Example 96: 25 N-(2,4,6-Tris-n-propylamino)-[1,3,51triazine (CLXXIII) Example 97: N-(2,4,6-Tris-n-propylamino)-[1,3,51triazine hydrogen sulfate (CLXXIV) - 152 - WO 2013/181217 PCT/US2013/043052 Cl n-Pr-NH 2 NH N )N NaOH N 'IN N N NTHE N l), 60 0 C,4h H H XXXIV CLXXIII Scheme 62 N-(2,4,6-Tris-n-propylamino)-[1,3,5]triazine (CLXXIII) 5 A mixture of 2-chloro-N-(4,6-bis-(n-propylamino)-[1,3,5-triazine (XXXIV) (1.00 g, 4.35 mmol), NaOH (348 mg, 8.70 mmol) and n-propylamine (1.4 mL, 17.40 mmol) in 1,4-dioxane (40 mL) was heated at 60 'C for 4 h. The volatiles were then removed under reduced pressure. Water (50 mL) was added to the residue and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic 10 extracts were washed with water (50 mL), then with a brine solution (50 mL) and lastly, dried over anhydrous Na 2
SO
4 . The solvent was removed under reduced pressure and the residue was purified by flash column chromatography using gradient elution from CH 2 Cl 2 /EtOH (99:1, (v/v)) to CH 2 Cl 2 /EtOH (97:3 (v/v)) to yield N (2,4,6-tris-n-propylamino)-[1,3,5]triazine (790 mg, 72%). 400 MHz IH-NMR 15 (CDCl 3 , ppm) 4.93-4.51 (3H, br s), 3.41-3.22 (6H, m), 1.62-1.49 (6H, m), 0.94 (9H, t, J=7.4 Hz). ESI-MS (m/z): 253 [M+H]+. N-(2,4,6-Tris-n-propylamino)-[1,3,5]triazine hydrogen sulfate (CLXXIV) The title compound was prepared from N-(2,4,6-tris-n-propylamino) 20 [1,3,5]triazine and 95% H 2
SO
4 in quantitative yield, as described in Example 8A. 400 MHz 1H-NMR (DMSO-d6, ppm) 8.26 (1H, br s) 8.10-7.99 (1H, m) 7.94-7.85 (1H, m) 3.33-3.16 (6H, m) 1.60-1.45 (6H, m) 0.88 (9H, t, J=7.5 Hz). ESI-MS (m/z): 253 [M+H]. 25 Example 98: N-(2,4,6-Tris-n-propylamino)-1,3-pyrimidine (CLXXV) Example 99: N-(2,4,6-Tris-n-propylamino)-1,3-pyrimidine hydrogen sulfate (CLXXVI) - 153 - WO 2013/181217 PCT/US2013/043052 CI n-Pr-NH 2 NH N DIPEA N N N pyr N N N H H CXIX 1300C, 48 h H H CLXXV Scheme 63 N-(2,4,6-Tris-n-propylamino)-1,3-pyrimidine (CLXXV) 5 A pressure tube was charged with 4-chloro-2,6-bis-(N-n-propylamino) 1,3-pyrimidine (CXIX) (1.53 g, 6.65 mmol), n-propylamine (2.7 mL, 33.25 mmol), N,N-diisopropylethylamine (2.3 mL, 13.30 mmol) and pyridine (15 mL). The reaction mixture was heated at 130 'C for 48 hours. The volatiles were removed under reduced pressure. Water (50 mL) was added to the residue and the mixture was 10 extracted with CH 2 Cl 2 (3 x 50 mL). The combined organic extracts were washed with water (100 mL) and dried over anhydrous Na 2
SO
4 . The solvent was removed under reduced pressure and the residue was purified by flash column chromatography using gradient elution from CH 2 Cl 2 /EtOH (99:1, (v/v)) to CH 2 Cl 2 /EtOH (95:5, (v/v)) to yield N-(2,4,6-tris-n-propylamino)-1,3-pyrimidine (0.90 g, 54%). 400 MHz H NMR 15 (DMSO-d 6 , ppm) 5.43 (1H, br s), 5.15 (1H, br s), 4.74 (1H, s), 3.27 (2H, td, J=6.9, 5.9 Hz), 3.16-3.09 (4H, m), 1.65-1.51 (6H, m), 0.96 (6H, t, J=7.4 Hz), 0.93 (3H, t, J=7.3 Hz). ESI-MS (m/z): 252 [M+H]+. N-(2,4,6-Tris-n-propylamino)-1,3-pyrimidine hydrogen sulfate (CLXXVI) 20 The title compound was prepared from N-(2,4,6-tris-n-propylamino)-1,3-pyrimidine and 95% H 2
SO
4 in quantitative yield, as described in Example 8A. 400 MHz 1H NMR (DMSO-d6, ppm) 10.81-10.58 (lH, br s) 7.9-7.7 (lH, m) 7.6-6.5 (3H, m) 5.01 (1H, s) 3.30-3.12 (4H, m) 3.09-2.94 (2H, m) 1.59-1.46 (6H, m) 0.94-0.84 (9H, m). ESI-MS (m/z): 252 [M+H]f. 25 Example 100: N-(4-i-Propylamino)-N-(2,6-bis-n-propylamino)-1,3-pyrimidine (CLXXVII) Example 101: - 154 - WO 2013/181217 PCT/US2013/043052 N-(4-i-Propylamino)-N-(2,6-bis-n-propylamino)-1,3-pyrimidine hydrogen sulfate (CLXXVIII) Cl i-Pr-NH 2 N' N DIPEA N NN N pyrI N ly CXX 1300C, 48 h H H CLXXVII Scheme 64 5 N-(4-i-Propylamino)-N-(2,6-bis-n-propylamino)-1,3-pyrimidine (CLXXVII) The title compound was prepared from 4-chloro-2,6-bis-(N-n propylamino)-1,3-pyrimidine (CXIX) and i-propylamine as described in Example 98 in 16% yield. 400 MHz 1 H NMR (DMSO-d6, ppm) 5.78 (1H, br s), 5.46 (1H, br s), 10 5.19 (lH, br s), 4.73 (lH, s), 3.83-3.71 (lH, m), 3.34-3.27 (2H, m), 3.19-3.10 (2H, m), 1.68-1.53 (4H, m), 1.23 (6H, d, J=6.4 Hz), 0.98 (3H, t, J=7.4 Hz), 0.94 (3H, t, J=7.4 Hz). ESI-MS (m/z): 252 [M+H]+. N-(4-i-Propylamino)-N-(2,6-bis-n-propylamino)-1,3-pyrimidine hydrogen sulfate 15 (CLXXVIII) The title compound was prepared from N-(4-i-propylamino)-N-(2,6 bis-n-propylamino)-1,3-pyrimidineand 95% H 2
SO
4 in quantitative yield, as described in Example 8A. 400 MHz 1 H-NMR (DMSO-d 6 , ppm) 0.76-10.40 (lH, m), 8.0 7.1(3H, m), 5.01 (lH, s), 4.18-3.93 (lH, m), 3.35-3.13 (3H, m), 3.12-2.94 (lH, m), 20 1.60-1.45 (4H, m), 1.19-1.11 (6H, m), 0.93-0.83 (6H, m). ESI-MS (m/z): 252 [M+H]. Comparative Example 1: 0 CI OH OH O N ~N N N N N O O N N N-"' -"--N N N N N H H H H H I H XXXIV CLXXVIII CLXXIX 25 Scheme 65 - 155 - WO 2013/181217 PCT/US2013/043052 N-(4,6-Bis(propvlamino)-1,3,5-triazin-2-ol (CLXYVIII) A vial was charged with 6-chloro-N 2
,N
4 -dipropyl- 1,3,5-triazine-2,4 diamine (XXXIV) (800 mg, 3.48 mmol), NaOH (696 mg, 17.40 mmol), water (3 mL) 5 and 1,4-dioxane (3 mL). The reaction mixture was heated at 140 'C for 30 min under microwave irradiation. The mixture was acidified with IN HCl to pH 5, precipitate were filtered, washed with water. The product was dissolved in 50% trifluoroacetic acid (30 mL), the indissoluble residue was filtered off. The filtrate was neutralized with saturated NaHCO 3 solution, the formed precipitate was filtered, washed with 10 water, acetone and dried to give 550 mg (75%) of 4,6-bis(propylamino)-1,3,5-triazin 2-ol (CLXXVIII). 400 MHz 1H NMR (DMSO-d 6 + CF 3 COOD, ppm) 3.18-3.34 (4H, m), 1.45-1.59 (4H, m), 0.86 (6H, t). ESI-MS (m/z): 212 [M+H]+. N-(4,6-Bis(propvlamino)-1,3,5-triazin-2-ol hydrogen sulfate (CLXXIX) 15 95% H 2
SO
4 (28pL, 0.52 mmol) was added to the suspension of 4,6 bis(propylamino)-1,3,5-triazin-2-ol (CLXXVIII) (110 mg, 0.52 mmol) in 1,4-dioxane (2 mL). The mixture was stirred for 30 min at room temperature. The resultant precipitate were filtered, washed with Et 2 0 and dried to afford 4,6-bis(propylamino) 1,3,5-triazin-2-ol hydrogen sulfate (CLXXIX) in quantitative yield. 400 MHz 1 H 20 NMR (CD 3 0D + CF 3 COOD, ppm) 3.35-3.45 (3.2H, m), 3.22-3.27 (0.8H, m), 1.58 1.70 (4H, m), 0.91-1.0 (6H, m). ESI-MS (m/z): 212 [M+H]+. Major Fragments (M+H) 170, 128, 86. Example 102: 25 Preparation of (XXXVI) Salt Form A (Figure 1) Method A: To (XXXV) in ethyl acetate at ambient temperature (ca. 151 mg/mL, clear solution) was added a solution of one molar equivalent of concentrated sulfuric acid in ethanol. The clear solution was stirred at ambient temperature for ca. 1 day 30 (clear), after which 6 volume equivalents of isopropyl ether (vs. ethyl acetate) were added, resulting in a cloudy mixture. The mixture was stirred for 3 days, then filtered to yield solid product (Hydrogen Sulfate Salt Form A). Method B: - 156 - WO 2013/181217 PCT/US2013/043052 To (XXXV) in methyl ethyl ketone at ca. 65 'C (ca. 104 mg/mL, clear solution) was added one molar equivalent concentrated sulfuric acid. A precipitate that immediately formed rapidly redissolved. The mixture was stirred at ca. 65 'C for ca.5 h and slowly cooled over ca. 1 h to ambient temperature. The mixture was 5 stirred for one day at ambient temperature, then filtered to yield sold product (61% yield) (Hydrogen Sulfate Salt Form A). Method C: To (XXXV) in methyl ethyl ketone at ca. 65 'C (ca. 100 mg/mL, clear solution) was added one molar equivalent of concentrated sulfuric acid. A precipitate 10 that formed immediately rapidly redissolved. The mixture was stirred at ca. 65 'C for ca. 1 h and slowly cooled over 1 h to ambient temperature, yielding solids in the form of rods. The mixture was stirred for ca. 4 h at ambient temperature. One volume equivalent of heptane (vs. methyl ethyl ketone) was added and the mixture was stirred for 1 day at ambient temperature, then was filtered to collect solid product (810% 15 yield) (Hydrogen Sulfate Salt Form A). Method D: To (XXXV) in methyl ethyl ketone at ca. 69 'C (ca. 200 mg/mL, clear solution) was added one molar equivalent of concentrated sulfuric acid. A precipitate that formed immediately rapidly redissolved. The mixture was stirred at ca. 69 'C for 20 ca. 1 h and quickly cooled to ambient temperature and stirred for ca. 0.5 h, yielding a thick paste. The mixture was stirred in an ice bath for ca. 45 minutes, then filtered to collect solid product (Hydrogen Sulfate Salt Form A). Method E: To (XXXV) in methyl ethyl ketone at ca. 67 'C (ca. 101 mg/mL, clear 25 solution) was added one molar equivalent of concentrated sulfuric acid. The mixture was stirred at ca. 67'C for ca. 1 h and slowly cooled to ambient temperature. The mixture was stirred for ca. 1.5 h at ambient temperature, then one volume equivalent (vs. methyl ethyl ketone) of methyl tert-butyl ether was added, the mixture was stirred for one day, then filtered to collect solid product (76% yield) (Hydrogen Sulfate Salt 30 Form A). Method F: To (XXXV) in acetone at ca. 50 'C (ca. 199 mg/mL, clear solution) was added one molar equivalent of concentrated sulfuric acid, forming a clear solution. The mixture was stirred at ca. 50 'C for ca. 1 h and slowly cooled to ambient - 157 - WO 2013/181217 PCT/US2013/043052 temperature, at which point solids had formed. To the mixture was added about two volume equivalents (vs. methyl ethyl ketone) of methyl tert-butyl ether. The mixture was stirred for one day at ambient temperature, then filtered to collect solid product (79% yield) (Hydrogen Sulfate Salt Form A). 5 Example 103: Preparation of a Mixture of (XXXVI) Salt Form A and (XXXVI) Salt Form B To (XXXV) in ethyl acetate at ca. 65'C (99 mg/mL, clear solution) was added one molar equivalent of concentrated sulfuric acid. A thick suspension 10 within 5 minutes along with a small amount of tacky material. The mixture was stirred at ca. 65 'C for about 5 h, slowly cooled to ambient temperature, stirred for 4 days then filtered to collect solid product (Hydrogen Sulfate Salt Form A as a Mixture with Form B), Figure 23 15 Example 104: Preparation of (XXXVI) Salt Form C A sample of (XXXVI) Form A was exposed to 97% relative humidity at ambient temperature for 4 days. The resulting solid form was designated Hydrogen Sulfate Form C. The resulting material under thermal gravimetric testing lost 4.6 20 wt% between about 30 and 50'C, consistent with approximately one molar equivalent of water (Figure 24). Example 105: Preparation of (XXXVI) Salt Form D 25 To (XXXV) in ethyl acetate at ca. 65 'C (ca.100 mg/mL) was added one molar equivalent of concentrated sulfuric acid. Tacky material immediately precipitated and more solids were observed after ca. 5 minutes. A subsample of these solids were removed with a pipette and filtered without cooling. These solid was designated Hydrogen Sulfate Salt Form D (Figure 25). 30 Example 106: Preparation of a Mixture of (XXXVI) Salt Form A and (XXXVI) Salt Form D To (XXXV) in isopropyl acetate at ca. 80 'C (203 mg / mL, clear solution) was added one molar equivalent of concentrated sulfuric acid as a solution - 158 - WO 2013/181217 PCT/US2013/043052 in isopropyl acetate. A precipitate formed. The mixture was stirred at ca. 80 'C for ca. 45 minutes, then was slowly cooled to ambient temperature, stirred for day , then filtered to yield the solid product (83% yield); Hydrogen Sulfate Form A in a Mixture with Hydrogen Sulfate Form D (Figure 26). 5 Example 107: Preparation of N-(4,6-Bis-n-propylamino-[1,3,51triazin-2-yl)-N,O-dimethyl hydroxylamine Phosphoric Acid Salt (CLXXX), Form A To (XXXV) in acetonitrile at ambient temperature were added two 10 molar equivalents of phosphoric acid. The mixture was stirred for 2 days, then the solid that was formed was collected by filtration and dried on the filter under a nitrogen stream (Phosphoric Acid Salt Form A; Figure 27). Example 108: 15 Preparation of (CLXXX) Form C To (XXXV) in methanol at ambient temperature (clear solution) was added one molar equivalent of phosphoric acid, resulting in a clear solution. The vial containing the solution was placed in an enclosed chamber with a vial of isopropyl ether and vapor diffused, forming a solid which was collected by filtration 20 (Phosphoric Acid Salt Form C, Figure 28). Example 109: Preparation of a Mixture of (CLXXX) Form A and (CLXXX) Form B To (XXXV) in acetonitrile (slurry) at ambient temperature was added 25 one molar equivalent of phosphoric acid. A thick slurry formed. The sturry was sonicated at ambient temperature for 3 days, then was filtered to collect the solid product (Phosphoric Acid Salt Form A in a Mixture with Form B, Figure 29). Example 110: 30 Preparation of a Mixture of (CLXXX) Form C and (CLXXX) Form D To (XXXV) in ethyl acetate (clear solution) were added two molar equivalents of phosphoric acid as a solution in ethanol. A precipitate formed. The mixture was further diluted with ethyl acetate, stirred for 3 days, then filtered to - 159 - WO 2013/181217 PCT/US2013/043052 collect solid product (a mixture of Phosphoric Acid Salt Form C and Phosphoric Acid Salt Form D, Figure 30). Example 111: 5 Preparation of a Mixture of (CLXXX) Form C and (CLXXX) Form E To (XXXV) in tetrahydrofuran (clear solution) at ambient temperature were added three molar equivalents of phosphoric acid. A precipitate formed. The mixture was stirred at ambient temperature for 3 days, then filtered to collect solid product (a mixture of Phosphoric Acid Salt Form C and Phosphoric Acid Salt Form E, 10 Figure 31). Example 112: Effect of Compound (XXXVI) on Opioid-Induced Respiratory Depression in the Rat Rats with pre-cannulated jugular vein (for administering drugs) were 15 acclimated to plethysmography chambers for a minimum of 60 minutes, or until animals were no longer restless. Each animal was dosed with morphine sulfate (10 mg/kg), dissolved in sterile water at a concentration of 10 mg/mL (supplied by Baxter Healthcare Corporation), via injection into the jugular vein catheter over a period of 5-10 seconds. 20 Compound (XXXVI) was dissolved in 20% hydroxypropyl cyclodextran (20%bcd on graph) at a concentration of 0.45 mg/mL at pH 5. After a period of 5 min, compound (XXXVI), labeled as cmpd (A), was administered via infusion into the jugular vein at a dose of 0.10 mg/kg/min for 20 min and then at a dose of 0.30 mg/kg/min for 20 min (e.g., 20 pL/min/0.3 kg rat to yield 0.03 25 mpk/min). After 20 minutes of infusion at this dose, the infusion pumps were turned off, and all animals were given a 20 minute recovery period, followed by a post-study analysis of rat health and behavior. The minute ventilation data indicate that compound (XXXVI) significantly reversed opioid-induced respiratory depression 30 in rat compared to vehicle. Results are illustrated in Figure 1. - 160 - WO 2013/181217 PCT/US2013/043052 Example 113: Effects of Morphine and Compound (XXXVI) on Blood Gases in the Rat Rats with pre-cannulated jugular vein and femoral arterial catheters (for administering drugs and obtaining blood samples respectively) were obtained 5 from Harlan laboratories and kept at the animal facility at Galleon Pharmaceuticals until the experimental procedures. Each animal was dosed with morphine sulfate (10 mg/kg), dissolved in saline at a concentration of 10 mg/mL, via injection into the jugular vein over a period of 20 seconds with a 20 second flush of 0.9% NaCl saline. Prior to morphine administration, two 250 pL samples of arterial blood were aspirated 10 from the femoral artery into a pre-heparinized syringe. The samples were analyzed on Radiometer's ABL Flex 800, where pO2, pCO2, pH, SaO2 and other parameters were recorded. Aspirated volumes of arterial blood were replaced by room temperature sterile saline (-300 pL) slowly flushed back into the femoral arterial catheter of the rodent to prevent hypovolemia. Morphine was then administered and 2 minutes later 15 another blood sample was taken. After a period of 5 min from the administration of morphine, compound (XXXVI), labeled as cmpd (A), was administered via infusion into the jugular vein at a dose of 0.1, 0.3 and 1.0 mg/kg/min (dissolved in PBS buffer). The infusion started at t = 15 minutes and ended at t =35 minutes. Arterial blood gas 20 analysis occurred at time points t = 12, 18, 25, 35, 40, 45, and 50 minutes. The data show that compound (XXXVI) significantly reverses opioid-induced respiratory depression in rat compared to vehicle. Results are illustrated in Figure 2a and Figure 2b and the accompanying Table 6. Table 6. Dose pH PaCO 2 PaO 2 SaO 2 Cmpd n (mg/kg/min) (% (% (% (% reversal) reversal) reversal) reversal) XXXVI 6 1 60 94 26 62 XXXVI 11 0.3 55 72 -16 43 XXXVI 10 0.1 37 47 16 51 XXXVI 6 0.03 20 18 -2.0 42 25 - 161 - WO 2013/181217 PCT/US2013/043052 Example 114: Hypoxic Ventilatory Response (HVR) and Effect of Compound (XXXVI) on HVR in the Rat Rats with a pre-cannulated jugular vein (for administering drugs) were 5 acclimated to plethysmography chambers for a minimum of 60 minutes, or until animals were no longer restless. Each animal was dosed with compound (XXXVI), labeled as cmpd (A), at 0.03 mg/kg/min via infusion into the jugular vein catheter for a period of 50 minutes. After a period of 20 minutes, an isocapnic hypoxic mixture (12% 02 balanced N 2 ) was administered into all chambers using a gas mixer (CWE 10 inc. GSM-3 gas mixer) for 20 minutes. After this time, the gas mixer was turned off, resulting in normal room air pumped into the chambers. Ten minutes later, the infusion pumps were turned off, and all animals were given a 20 minute recovery period, followed by a post-study analysis of rat health and behavior. The minute ventilation data show that compound (XXXVI) significantly potentiates the hypoxic 15 ventilatory response in the rat compared to vehicle. Results are illustrated in Figure 3. Example 115: Effect of Compound (XXXVI) on Opioid-Induced Respiratory Depression in the Monkey 20 Juvenile macaques (four-year-old Macacafascicularis, 2 to 5 kg, n=13) were used for the study. Animal husbandry was conducted under USDA guidelines and the protocols were approved by the Institutional Animal Care and Use Committee of East Carolina University. Anesthesia was induced with 5% isoflurane and then maintained with 25 1.5 to 2% isoflurane (100% 02, 2 L/min). Antebrachial veins were cannulated. Vivometrics Lifeshirts (Ventura, CA) were fitted to the animals for monitoring respiratory function by inductance plethysmography. Abdomen and rib cage deflections were calibrated using the Qualitative Diagnostic Calibration (QDC) procedure, and tidal volume was normalized to 15 mL, which represented a typical 30 tidal volume for these animals as previously measured using conventional techniques. Heart rate was monitored continuously by 3-lead ECG.
ETCO
2 was measured via a neonatal nasal cannula connected to a microstream CO 2 sensor (Cardell monitor, Model 9405). SpO2 and HR were monitored by pulse oximetry using a reflectance probe (Nelcor Max-Fast) positioned - 162 - WO 2013/181217 PCT/US2013/043052 on the inner aspect of the upper arm. HR was also determined from the ECG allowing measurement when animal activity compromised the integrity of pulse oximetry signals. BP was measured in anesthetized animals using a cuff positioned on the ankle. 5 Compound (XXXVI) was dissolved in 20% hydroxypropyl cyclodextran (HPBCD) and sterile filtered using a 2 p syringe filter. Compound (XXXVI), labeled as cmpd (A), was then delivered at a rate of 0.20 mg/kg/min for 5 minutes, followed by reduction of the infusion rate to 0.10 mg/kg/min for 10 minutes. Minute Ventilation and end-tidal CO 2 were monitored. Naloxone HCl (0.05 mg/kg 10 intravenous) was delivered to reverse morphine effects and conclude the experiment. The data showed that cmpd (A) produced a full reversal of end-tidal carbon dioxide increases caused by the opioid, and also increased minute ventilation (Figure 4). Example 116: 15 Dose-Dependent Effect of Compound (XXXVI) and (L) on Minute Ventilation (MV) in Naive Rats All surgical procedures were performed under anesthesia induced by 2% isoflurane in compressed medical grade air. With rats in supine position, the right femoral vein was catheterized using polyethylene tubing (PE-50). This catheter was 20 used for fluid and drug administration. Simultaneously, the right femoral artery was also catheterized for monitoring blood pressure. In order to measure the respiratory parameters in spontaneously breathing rats, trachea was intubated using 13 gauge tracheal tube (2.5mm ID, Instech Solomon, PA). After establishing a stable base-line at 1.5% isoflurane, cumulative 25 dose-dependent (0.01, 0.03, 0.1, 0.3, 1, 3, 10 mg/kg) ventilatory responses to compounds (XXXVI) and (L), labeled as cmpd (A) and cmpd (B) respectively, were generated from spontaneously breathing rats. Maximum peak minute ventilatory (MV) values at each dose of corresponding drug were calculated and used for generating ED 50 values. The results are shown in Figure 5. Both compounds 30 (XXXVI) and (L) increased minute ventilation in a dose-dependent manner with calculated ED 50 values of 0.14 and 0.13 mg/kg respectively. - 163 - WO 2013/181217 PCT/US2013/043052 Example 117: Effect of Compound (CXXI) on Dose-Dependent Minute Ventilation (MV) in Naive Rats Compound (CXXI), labeled as cmpd (C), was shown to increase 5 minute ventilation in a dose-dependent manner following the procedure above. Results are shown in Figure 6. Example 118: Effects of Compound (L) in the Opioid-Treated Rat 10 Following the procedure in Example 6, Compound (L), labeled as cmpd (B), was shown to reverse the effects of opioid on blood gases and pH in the rat by increasing pH, SaO 2 and PO2 and by decreasing pCO2 levels, as illustrated in Figures 7A-7D. 15 Example 119: Effects of Compound (CXLII) in the Opioid-Treated Rat Following the procedure in Example 6, Compound (CXLII), labeled as cmpd (D), was shown to reverse the effects of opioid on blood gases and pH in the rat by increasing PO2 levels, increasing (SaO 2 ) oxygen saturation, decreasing pCO 2 20 levels and raising pH, as illustrated in Figures 8A-8D. Example 120: Effects of Compound (CXLII) on Opioid-Induced Respiratory Depression in the Rat Following the procedure in Example 5, Compound (CXLII), labeled as 25 cmpd (D), was shown to reverse opioid-induced respiratory depression in the rat by increasing minute ventilation (MV) as determined by plethysmography. The results are illustrated in Figure 9. Example 121: 30 Effect of Compounds on Minute Ventilation (MV) and Cardiovascular Parameters in Naive Rats All surgical procedures were performed under anesthesia induced by 2% isoflurane in compressed medical grade air. With rats in supine position, the right femoral vein was catheterized using polyethylene tubing (PE-50). This catheter was - 164 - WO 2013/181217 PCT/US2013/043052 used for fluid and drug administration. Simultaneously, the right femoral artery was also catheterized for monitoring blood pressure and heart rate. In order to measure the respiratory parameters in spontaneously breathing rats, trachea was intubated using 13 gauge tracheal tube (2.5mm ID, Instech Solomon, PA). 5 After establishing a stable base-line at 1.5% isoflurane, compounds (typically at a dose of 1 mpk) were administered IV, and ventilatory parameters were generated from spontaneously breathing rats, along with cardiovascular output (mean arterial pressure (MAP) and heart rate). Maximum peak minute ventilatory (MV) responses (MPR), along with changes in minute ventilation versus baseline (AMV) 10 were obtained as shown in the tables below. Table 7. Cmpd Structure Formula Dose pH MV CV tion MPR AM MAP Heart V (mm Rate Hg) (B/min (1,5)-8-(4,6-bis(n- 0 20% lmpk 5 213 98 114 308 propylamino)- 1,3,5- HP$CD triazin-2-yl)-8 azabicyclo[3.2. 1]octa N n-3-one N 'N ""N 'ilN ),N-" H H (1,5)-8-(4,6- 0 20% lmpk 5 237 137 117 345 bis(isobutylamino)- HP$CD 1,3,5-triazin-2-yl)-8 azabicyclo[3.2. 1]octa N n-3-one N 'N N N N H H
N
4 -isopropyl-7- HN 20% lmpk 5 402 320 118 340 methyl-N -n-propy- HP$CD 7H-pyrroo[2,3-d] / N pyrimidine-2,4- N N N H diamine 6-(1-(2-(allylamino)- N N 1k 5 154 60 114 350 7-methyl-7H- N HP$CD pyrrolo[2,3-d] pyrimidin-4-yl) piperidin-4-yl)- N
N
2
,N
4 -di-n-propyl- / N N 1,3,5-triazine-2,4- / NH diamine - 165 - WO 2013/181217 PCT/US2013/043052 N 4-isopropyl-7- 20% lmpk 5 327 224 93 318 methyl-N 2 -n-propyl- HN HP$CD 6,7-dihydro-5H- N pyrrolo[2,3-d] N N N pyrimidine-2,4- H diamine CLX, 20% lmpk 5 182 103 96 336 CLXI NH / ' ITPfCD N N N H CXXXI, NH 20% lmpk 4 204 130 101 348 CXXXII NN HP$CD N N N H
N
4 ,7-diisopropyl-N 2 - 20% lmpk 6 250 155 103 356 n-propyl-7H- HN HP$CD pyrrolo[2,3-d] IN pyrimidine-2,4- N N N diamine H CXXXVI, NH 20% lmpk 4 238 143 98 389 CXXXVII HP$CD N N N H
N
4 -isopropyl-N 2 -n- 20% lmpk 4 259 172 96 387 propyl-7H- HN HP$CD pyrrolo[2,3-d] ' N pyrimidine-2,4- N N NN, diamine H XXIX N' 0 20% lmpk 4-6 253 161 92 371 NA N> HP$CD N N N H H CXLI, N'O 20% lmpk 4-6 264 163 93 336 CXLII N HP$CD N N N H H XX NON 20% lmpk 4-6 213 60 92 432 N 'N HP$CD ~'N N N H H XXII N' 0 , 20% lmpk 4-6 252 130 95 415 N N HP$CD N IIN ),N H H
N
2
,N
4 -di-I-propyl- 20% lmpk 4-6 336 164 94 427 7H-pyrrolo[2,3-d] HN HP$CD pyrimidine-2,4- N diamine Nl N NN H H XXV N' Os 20% lmpk 4-6 257 126 98 430 N N HP$CD '- N 'ilN -,N H HV -166 - WO 2013/181217 PCT/US2013/043052
N
2 -ethyl-6- H3 20% lmpk 4-6 208 117 95 445 (methoxyamino)-N4- HN.O HP$CD n-propyl-1,3,5- N N triazine-2,4-diamine /N iN N H H 7-methyl-N-n-propyl- 20% 1 4 175 83 93 372 4-(1,2,2-trimethyl- N HP$CD mpk hydrazinyl)-7H- / N pyrrolo[2,3-d] N N N 15/65/2 1 5 167 89 87 376 pyrimidin-2-amine / H 0 mpk
N
2
,N
4 -di-n-propyl- HN-' 20% 1 4 175 93 93 385 6,7-dihydro-5H- NHPCD mpk pyrrolo [2,3- N NHNf3 d]pyrimidine-2,4- H H 20% 1 6 263 167 99 381 diamine HP$CD mpk N 2-methyl-N4,N 6 -di- NH 15/65/2 lmpk 5 145 106 92 285 n-propyl-1,3,5- N N 0 triazine-2,4,6- N N N triamine H H 6-(1,2- N20% mpk 5 338 216 97 329 dimethylhydrazinyl)- N HP$CD 2 4 A HPf3CD N ,N4-di-n-propyl- N 'N 1,3,5-triazine-2,4- N N N diamine H H LIII, 20% lmpk 4-6 288 179 96 403 LIV
N
0 O HP$CD N A N N" N lN"" H H 6-(methoxy N O0, 20% lmpk 4-6 355 243 90 356 (methyl)amino) N 2,N4-di-n- N HPCD propylpyimidine- H H 2,4-diamine 6-(isopropoxy N 0 20% lmpk 271 209 98 378 (methyl)amino)- N HPCD N2 4 N HfC N2,N4-di-n-propyl- N N N 1,3,5-triazine-2,4- H H diamine 6-(ethyl(isopropoxy) K 20% 1 4 308 191 89 351 amino)-N 2 ,N4-di-n- N HP$CD mpk propyl-1,3,5-triazine- N N 2,4-diamine IN N N H H 6-(isobutoxy N 20% 1 4 248 133 87 351 (methyl)amino)- HPCD mpk N 2,N4-di-n-propyl- N N 1,3,5-triazine-2,4- -N N N diamine H H 6-(methyl(thiophen- N 20% 1 4 213 110 112 411 2-ylmethoxy)amino)- N HP$CD mpk N 2,N4-di-n-propyl- N )N 1,3,5-triazine-2,4- N IN )Ndiamine H H - 167 - WO 2013/181217 PCT/US2013/043052 6-((cyclopropyl N OA 20% 1 4 261 152 98 362 methoxy)(methyl) HP$CD mpk amino)-N 2
,N
4 -di-n- N "N propyl- 1,3,5-triazine- -N N )N*N -' 2,4-diamine H H CLII, OH 20% Impk 4 49 0.33 79 245 CLIII HPpCD N N N N- H XXXV, H 3 C NO-CH 3 15% 1 mpk 5-6 207 135 87 268 XXXVI N DMA N N N H H DSW XLVII N Os 20% lmpk 5 150. 61.4 109. 350.52 N )N HPpCD 73 8 6
H
2 N N HN H XLVIII N' OH 15/65/20 lmpk 5 114 30.1 98 300 N N N N N H H XXXV, H 3 C N 0-CH 3 20% lmpk 4-5 344 210 90 321 XXXVI NA HPpCD N N N H H CLV, OH 20% lmpk 4 170 57 111 349 CLVI HPpCD N N N N H XXXIIIN 20% lmpk 4-6 147 53 89 480 N HPpCD InN )IN -lN H H LIII, 20% lmpk 4-6 288 179 96 403 LIV H 3 C N"O HPPCD N -N -N 16N N -168 - WO 2013/181217 PCT/US2013/043052 Table 8. Compoun Formulat Dose pH MV CV d ion MPR DMV MAP HR (B/min) (mmHg) BL DE BL DE CXX, 20% 1 mpk 4.5 355 243 107 2.3 293 17 CXXI HPpCD CLXX, 20% 1 mpk 4 175 83 93 -1.0 372 -9.0 CLXXI HPpCD CXLIX, 20% 1 mpk 4 175 93 93 -1.7 385 -28 CL HPpCD CLXX, 15/65/20 1 mpk 5 167 89 87 1.0 376 -3.8 CLXXI XXXV, 20% 1 mpk 5 267 164 101 -1.7 335 43 XXXVI HPpCD CXLIX, 20% 1 mpk 6 263 167 99 -3 381 -18 CL HPpCD XXXI 20% 1 mpk 4 140 43 101 2.6 372 2.5 HPpCD CXIII, 20% 1 mpk 4 167.1 46 101 -4 368 23 CXIV HPpCD CXV, 15/65/20 1 mpk 4 155.7 33.8 97 -5 374 -5 CXVI CXVII, 20% 1 mpk 4.5 143.8 15 92 -8 365 -2 CXVIII HPpCD LXXII, 20% 1 mpk 4 176 82 358 8 108 -4 LXXIII HPpCD LXXVI, 20% 1 mpk 4 308 191 351 15 89 -2 LXXVII HPpCD LXXXII, 20% 1 mpk 4 248 133 351 19 87 10 LXXXIII HPpCD LXXXIV, 20% 1 mpk 4 213 110 411 15 112 -1 LXXXV HPjCD XCI, 20% 1 mpk 4 261 152 362 63 98 -2 XCII HPpCD Example 122: Effect of Compound (XXXVI) on Benzodiazepine-Induced Respiratory Depression 5 (BIRD) In one aspect, the objective of the current study was to evaluate the effects of an intravenous infusion of compound (XXXVI)_on respiratory depression, induced by midazolam in rats. - 169 - WO 2013/181217 PCT/US2013/043052 Procedures: Compound (XXXVI) was dissolved in 20% hydroxypropyl-beta cyclodextran in sterile water, and titrated to a pH of 4-8 using pH paper and NaOH or HCl, resulting in a clear, stable solution at a concentration of 1.5 mg/mL. Other 5 compounds: morphine sulfate, supplied as 10 mg/mL solution by (Baxter, Inc) and midazolam, supplied as 5 mg/mL solution (Hospira, Inc.) Materials: Male Sprague-Dawley rats (Harlan, Inc.), 250-350 g at time of dosing, surgically prepared by Harlan with jugular vein cannulas. 12 Chamber 10 Plethysmography System with temperature/humidity compensation (Epstein et al., 1980, J. Apply Physiol. 49:1107-1115); From Buxco, Inc. (PLY 3223; Buxco, Inc, Wilmington, NC, USA) Biosystem XA, software, v2.11.1. Customized 12 site automated infusion system (Harvard Apparatus; Instech, Inc) Methods: 15 Rat whole body plethysmography was used to evaluate and quantify minute ventilation and the pattern of breathing. A respiratory waveform was generated from the exchange of air between the animal and the chamber. This exchange induced changes in air volume that were measured with a pressure transducer, constituting the respiratory waveform. Atmospheric temperature and 20 humidity were also measured using temperature and humidity probes which sampled chamber conditions. A compensation factor was then determined and applied to the respiratory waveform using a standardized algorithm (Epstein et al., 1980, J. Apply Physiol. 49:1107-1115) to compensate for respiratory conditioning, which was reported as the parameter COMP (see appendix). 25 All animals were acclimated to plethysmography chambers for at least 1 hour, or until animals were no longer restless (up to 2 hours) prior to data collection. Bolus intravenous (IV) dosing was administered at a rate of 5-10 seconds per dose, and catheters were flushed with 350 pL of sterile saline to be sure of complete drug delivery. All 12 animals (6 vehicle; 6 drug treated) were dosed simultaneously within 30 one 60 second period. For intravenous infusions, vehicle or test compound was prepared at a 0.1 mg/mL stock as described, and administered with Harvard apparatus infusion pumps. Compound (XXXVI) was given via infusion over a 30 minute period at a rate of 20 pL/min/0.3 kg, to give a dose of 0.1 mg/kg/min, beginning 5 minutes after the bolus IV administration of midazolam. None of the included studies were - 170 - WO 2013/181217 PCT/US2013/043052 performed blinded, due to the logistics of simultaneously dosing multiple animals. All plethysmography data was recorded automatically by the Buxco equipment. Statistical Analysis: Respiratory data were collected on a breath-by-breath basis and 5 averaged into 1 min time bins for data analysis. For each designated acquisition phase, which is the time between doses, percent change from pre-treatment baseline values were calculated for each cohort on multiple ventilatory parameters including respiratory frequency (f), tidal volume (TV), accumulated volume (AV), minute ventilation (MV), inspiratory time (Ti), expiratory time (Te), peak inspiratory flow 10 (PIF), peak expiratory flow (PEF), relaxation time (RT), end inspiratory pause (EIP), end expiratory pause (EEP), delta volume (DV), expiratory flow at 50% TV (EF50), rejection index (Rinx), compensation (Comp), enhanced pause (Penh), pause (PAU), PEF rate (Rpef), relative humidity (RH), and atmospheric temperature (Temp). Each parameter was compared to vehicle in order to calculate percent 15 difference, using area under the curve (AUC) and peak response values for each defined acquisition period using a customized visual basic restructure analysis macro. Additionally, percent reversal of drug induced respiratory depression by compound was calculated using the mean respiratory depression derived from all vehicle (or untreated) animals, compared to the mean pre-treatment baselines of all animals in the 20 study. Results: Compound (XXXVI) (0.1 mg/kg/min) reversed the BIRD induced decrease in MV by 100 by the end of the infusion period (t = 38). Compound (XXXVI) had an effect to reverse the effects of midazolam on MV (Figure 15), TV 25 (Figure 16), AV, Te, PIF, PEF, and EEP, and had no obvious effect on f (Figure 17), Ti, RT, EIP, dv, EF50, Rinx, Comp, Penh, PAU, Rpef, RH, and Temp. Small increases in minute ventilation in all groups were often seen during IV bolus injections which were treated as injection artifacts due to animal arousal, and had no other apparent impact on ventilation. 30 Potential adverse events were monitored, and there were no adverse behavioral effects observed following administration of compound (XXXVI) at the doses tested. The results of these experiments demonstrated that compound (XXXVI) reverses midazolam induced-respiratory depression and appears to be well tolerated in rats at the doses tested. - 171 - WO 2013/181217 PCT/US2013/043052 Example 123: Effect of Compound (XXXVI) on Hypercapnia In one aspect, the objective of the study was to evaluate the effects of 5 Compound (XXXVI), administered by intravenous infusion, on the hypercapnic ventilatory response (HCVR) in rats. Vehicle, 20% hydroxypropyl-beta-cyclodextran in sterile water, was added to pre-weighed compound and mixed thoroughly, resulting in a clear solution. A 1.5 mg/mL stock was created to give an infusion dose of 0.10 mg/kg/min. A 10 separate 0.45 mg/mL stock was created to give an infusion dose of 0.03 mg/kg/min. All compound solutions and vehicles were titrated to have a pH between 4-8, using a pH paper and titrating with NaOH or HCl solution. Male Sprague-Dawley rats (Harlan, Inc.), 250-350 g at time of dosing, had been surgically prepared by Harlan to contain jugular vein cannulas. A 12 15 chamber plethysmography system with temperature/humidity compensation (Epstein et al., 1980, J. Apply Physiol. 49:1107-1115) from Buxco, Inc. (PLY 3223; Buxco, Inc, Wilmington, NC, USA) using Biosystem XA software, v2. 11.1 and a customized 12 site automated infusion system (Harvard Apparatus, Instech, Inc). Gas mixer (CWE Inc.) was used for this experiment to give concentrations of hypercapnia (3% 20 C0 2 , 21% 02, balance nitrogen). Three tanks containing 100% 02, C0 2 , and nitrogen were attached to the gas mixer, with the customized gas mixture fed to each plethysmograph at a rate of 2 L/min. Rat whole body plethysmography was used to evaluate and quantify minute ventilation and the pattern of breathing. A respiratory waveform was 25 generated from the exchange of air between the animal and the chamber. This exchange induced changes in air volume that were measured with a pressure transducer, constituting the respiratory waveform (Lomask M., 2005, "Respiration measurement in the whole body plethysmography," Buxco Inc., retrieved from http://www dot buxco dot com/ downloads/ LomaskWBP dot pdf). Atmospheric 30 temperature and humidity were also measured using temperature and humidity probes that sampled chamber conditions. A compensation factor was then determined and applied to the respiratory waveform using a standardized algorithm (Epstein et al., 1980, J. Apply Physiol. 49:1107-1115) to compensate for respiratory conditioning, which was reported as the parameter COMP. - 172 - WO 2013/181217 PCT/US2013/043052 All animals were acclimated to plethysmography chambers for at least 1 hour, or until animals were no longer restless (up to 2 hours prior to data collection). Minute ventilation (MV) was calculated by the Biosystem XA software from direct measurements of tidal volume (TV) and respiratory frequency (f) using the formula 5 MV = TV x f. Minute ventilation (mL/min) is a common endpoint for evaluating ventilatory performance. The protocol included compound (XXXVI) given as a 20 minute infusion, followed by a 20 minute exposure to 3% hypercapnia in addition to the compound (XXXVI) infusion. After 20 minutes of hypercapnia, the compound (XXXVI)_infusion continued for an additional 10 minutes, resulting in a total 10 compound (XXXVI) infusion time of 50 minutes. Each experiment included 6 animals receiving compound (XXXVI) (0.03 or 0.10 mg/kg/min) tested against 6 animals receiving vehicle, all of which were challenged with hypercapnia (3%). Animals were continuously monitored and observed for adverse behavioral effects. Such findings were recorded into laboratory notebooks for each individual animal 15 tested. Statistical Analysis Respiratory data was collected on a breath-by-breath basis and averaged into 1 min time bins for data analysis. For each designated acquisition phase, which is the time between doses, AUC percent change from pre-treatment 20 baseline values were calculated for each cohort on multiple ventilatory parameters including, respiratory frequency (f), tidal volume (TV), accumulated volume (AV), minute ventilation (MV), inspiratory time (Ti), expiratory time (Te), peak inspiratory flow (PIF), peak expiratory flow (PEF), relaxation time (RT), end inspiratory pause (EIP), end expiratory pause (EEP), delta volume (dV), expiratory flow at 50% TV 25 (EF50), rejection index (Rinx), compensation (Comp), enhanced pause (Penh), pause (PAU), PEF rate (Rpef), relative humidity (RH), and atmospheric temperature (Temp). Each parameter was compared to vehicle in order to calculate percent difference, using area under the curve (AUC) and peak response values for each defined acquisition period using a customized visual basic restructure analysis macro 30 (Lopotosky, S. Galleon Buxco Restructure tool, v5.2, 2008). All of the data analysis for the single, stand-alone studies was done using the restructure analysis macro (Lopotosky, S. Galleon Buxco Restructure tool, v5.2, 2008). All merged data analysis with both doses was performed in Graphpad Prism for MV, TV, and f only, for hypercapnia studies. Additionally, percent increase was calculated based on the - 173 - WO 2013/181217 PCT/US2013/043052 cohort average immediately prior to the hypercapnic challenge to the group's peak response. For merged hypercapnia studies, t=62 to t=67 was used. Results and Discussion: Administration of compound (XXXVI) stimulated respiration in naive 5 animals at 0.10 mg/kg/min, with little or no effect at 0.03 mg/kg/min. Furthermore, when animals received compound (XXXVI), exposure to hypercapnia (3% C0 2 ) resulted in an increased MV response in a dose dependent manner when compared to vehicle treatment alone. Compound (XXXVI), at 0.03 or 0.1 mg/kg/min IV, resulted in the augmentation of the hypercapnia ventilatory response (HCVR), at the higher 10 dose of 0.10 mg/kg/min, with no effect at 0.03 mg/kg/min. The 0.03 mg/kg/min and vehicle groups resulted in a 60% facilitation of the HCVR, from t=62 to t=67. The 0.10 mg/kg/min demonstrated an increase of 44% in MV during the HCVR challenge at the same time points, in addition to an elevated MV due to compound (XXXVI) alone. Compound (XXXVI) at 0.03 mg/kg/min against HCVR resulted in an effect 15 on PIF only, with no discernable change in f, TV, AV, MV, Ti, Te, PEF, RT, EIP, EEP, dv, EF50, Rinx, Comp, penh, PAU Rpef, RH, or Temp. The 0.10 mg/kg/min dose showed an effect on f, TV, AV, MV, Ti, Te, PIF, PEF, EIP, EF50, Comp, and RH, with no discernable change in EEP, dV, Rinx, Penh, PAU, Rpef, and Temp. These data suggest that compound (XXXVI), given as an infusion, may enhance the 20 ventilatory effects of hypercapnia, resulting in facilitation of ventilation during acute hypoxemia. In addition, there were no adverse clinical observations associated with compound (XXXVI) at the doses tested. Example 124: 25 Effect of Compound (XLII) on Respiratory Rate, Tidal Volume, Minute Volume and 5-min Average Change in Minute Volume in Rat General Methods: Adult male Sprague Dawley rats (250-350 g body weight; Harlan, Indianapolis, IN, USA) were anesthetized with isoflurane in oxygen. The femoral 30 vein was cannulated to administer compounds. Body temperature was maintained between 36.0 and 37.2 C throughout the experiment. The cervical portion of the trachea was cannulated and attached to a rat-sized pneumotachometer for measuring tracheal airflow. After surgical instrumentation was complete, isoflurane was slowly discontinued, while urethane (1.8 g/kg) was administered intravenously over 10-15 - 174 - WO 2013/181217 PCT/US2013/043052 minutes to maintain anesthesia. Rats were permitted to breathe spontaneously and inspire 100% oxygen. At least 30-45 minutes after discontinuing isoflurane, baseline tracheal airflow was recorded for 20-30 minutes prior to compound administration. Vehicle and test compound were administered intravenously over 15-25 seconds and 5 at a constant volume (1 mL/kg). At least 15-min elapsed between each dose of compound administered. Tracheal airflow was recorded continuously before, during and after compound administration. Tidal volume was derived from the integral of airflow, and respiratory rate was derived from the number of inspiratory cycles per minute. Minute volume was calculated as the product of tidal volume and respiratory 10 rate (Figure 32). Results: Compared to vehicle, Compound (XLII) increased both respiratory rate and tidal volume, and their product minute volume. 15 Example 125: Effect of Compound (CLXXII) on Respiratory Rate, Tidal Volume, Minute Volume and 5 min Average Change in Minute Volume in Rat General Methods: See Example 124 above Results 20 Compared to vehicle, Compound (CLXXII) increased both respiratory rate and tidal volume, and their product minute volume. Example 126: Effect of Compound (XXXV) on BK Potassium Channel Current in GH3 Cells 25 Experimental Procedures Stock solutions of the test article were prepared in dimethylsulfoxide (DMSO) and stored frozen. Test article concentrations were prepared fresh daily by diluting stock solutions into a physiological saline solution (composition in mM): KCl, 125; CaCl 2 , 8.5; HEPES, 10; EGTA, 11.25; pH adjusted to 7.2 with KOH 30 (prepared weekly and refrigerated until used). All test solutions contained 0.1% DMSO. GH3 cells were grown in culture using standard procedures. Ionic currents were measured from inside-out patches of GH3 cells. Channel activity was monitored in symmetrical K+ conditions with a holding potential of -60 mV. - 175 - WO 2013/181217 PCT/US2013/043052 Activities before and in the presence of each concentration of test articles were recorded for > 3 minutes, respectively. Data Analysis 5 Data acquisition and analyses were performed using the suite of pCLAMP (Ver. 8.2) programs (MDS-AT, Sunnyvale, CA. Percent inhibition by test article was calculated using the equation: 100*(NPocontroi- NPOtest)/NPOcontroi Results Compound (XXXV) was evaluated at 1 and 10 pM, and almitrine, a 10 positive control, was evaluated at 1 pM. Channel effects were estimated from the change in activities at steady-state after application of test articles. The effect of Compound (XXXV) is summarized in Table 9. A sample recording before and after 1 pM (XXXV) and all points histogram is showed in the Figure 21. 15 Table 9. Effect of Compound (XXXV) on the single BK potassium channel expressed in GH3 cells recorded in inside-out patches Channel NPo Inhibition (%) Cell ID Amplitude (XXXV) (mM) Almirite (XXXV) Almirite (pA) control (mM) (MI) (mM) 1 10 1 1 10 1 C 05 SP 101008 -14.3 0.013 0.0076 0.0057 0.0011 41.5 56.2 91.5 0000 E 05 SP 101008 -17.8 0.042 0.037 0.023 0.00090 11.9 45.2 97.9 0000 A 05 SP 101012 -15.5 0.037 0021. 0000 B 05 SP 101012 -16.6 0.066 0.05 22.7 0000 Average -16.0 0.040 0.014 0.020 0.0010 22.3 41.4 9. SD 1.4 0.022 0.016 0.027 0.0001 16.7 17.0 4.5 NPo: total channel open probability. 20 Inhibition (% ) = 100*(NPocontroi - NPotest)/NPocontroi Washout was performed to the patch C 05 SP 101007 0000 before and after application of 1 p.M almitrine, to the patch B 05 SP 101012 000 after application of 10 ptM (XXXV). - 176 - WO 2013/181217 PCT/US2013/043052 Summary and Conclusions As illustrated in Figure 34, Compound (XXXV) exerted concentration-dependent inhibition of the native BK channel expressed in GH3 cells. At the highest concentration tested (10 pM) Compound (XXXV) reduced total 5 channel open probability by 42%. Example 127: Effect of Compounds on Thallium Flux Through Rat TASK-I (KCNK1) and Human TASK-3 (KCNK9) Channels 10 Experimental procedure (a) Seed cell plates: CHO cells expressing recombinant tetracycline-inducible rat KCNK3/TASK-1 (KCNK3-CHO cells) were plated in poly-D-lysine coated 384-well plates at a density of 6,000 cells/well (50 pl/well of 120,000 cells/mL) and allowed to 15 incubate for 72 hrs in the presence of 5% CO 2 and 1 pg/mL tetracycline at 30'C. HEK cells expressing recombinant tetracycline-inducible human KCNK9/TASK-3 (KCNK9-HEK cells) were plated at a density of 15,000 cells per well (50 pl/well, 300,000 cells/mL) and incubated overnight at 37'C and 5% CO 2 in the presence of 1 pg/mL tetracycline. 20 (b) Preparation of compound containing plates: Drug plates were generated from 10 mM 100% DMSO mother plates. Compounds were transferred to 384-well drug plates and titrated with sequential 1:3 dilutions in 100% DMSO for a total of 9 dilutions. A dilution plate is made by taking 2.4 pl from the mother plate and adding to 17.6 pl of assay buffer for a 12% DMSO 25 dilution plate. From this dilution plate, a final daughter plate is generated by taking 1 pl of the dilution plate and adding to 40 pl of assay buffer for a final DMSO concentration of 0.3%. Each compound was evaluated in duplicate and the concentration range evaluated was 3 nM to 30 pM. Assay protocol: 30 When thallium was added to extracellular solution with a stimulus to open channels, TlV flowed down its concentration gradient into the cells and channel activity was detected with an indicator dye, FluxORTM , that increased in cytosolic fluorescence. The FluxORTM indicator dye was loaded into cells as a membrane - 177 - WO 2013/181217 PCT/US2013/043052 permeable acetoxymethyl ester. On the day of each assay, FluxORTM assay buffer was made fresh from Hank's Balanced Saline Solution (HBSS) buffered with 20 mM HEPES and pH adjusted to 7.4 with NaOH. Dye loading solution was prepared per manufacturer's instructions, and 25 pl of dye solution applied to each well with a 5 multidropper after plating medium was removed manually from cells. Dye was allowed to load for 90 minutes at room temperature in the dark. After the 90min dye loading incubation and subsequent manual dye removal, 24 pl of the drug daughter plate was transfer to cell plates by a cybi-well system and then incubated at room temperature for 20 minutes to allow for 10 equilibration within wells. Stimulation buffer was prepared from 5X chloride-free buffer, thallium, and potassium sulfate containing 5 times the desired final assay concentration of 10 mM K+ 2.8 mM Tl. Cell plates were then transferred to an FDSS 6000 kinetic imaging plate reader (Hamamatsu). Fluorescence at 525 nm (excitation 488 nm) was measured for 10 seconds at 1Hz to establish baseline for the entire plate. 15 After baseline was established, 6 pl/well of stimulation buffer was added and fluorescence signal recorded for another 140 seconds. Data analysis From the fluorescence signals in the absence and presence of Galleon compounds, Inhibitory concentration curves were plotted and IC 50 values were 20 estimated using a curve fitting algorithm. Results The results are summarized in Table 10. Using TlV flux as an indicator of rat TASK-I and human TASK-3 activity, some compounds displayed blockade of rTASK-1. 25 Table 10.
IC
50 values for inhibition of rat TASK-I and hTASK-3 Compound hTASK-3 rTASK-1
IC
50 (pLM) IC 50 (pLM) Cmpd (XXXVI) >30 25 Cmpd (XLII) >30 0.05 Cmpd (XLVI) >30 0.60 Cmpd (CLXXII) >30 0.01 Cmpd (CXXI) >30 0.22 Cmpd (CLXX) >30 0.16 - 178 - WO 2013/181217 PCT/US2013/043052 Example 128: Effect of Compound (XXXVI) on Ventilation in Human Volunteers N-(4,6-bis-n-propylamino-[ 1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine hydrogen sulfate (XXXVI) was administered to humans during two 5 single dose studies of similar design. The first study (GAL-021-101) was a single, ascending dose study in healthy subjects. Mild/moderate burning sensation at the infusion site was the only adverse event that differed from placebo from drug-treated subjects. At the highest dose (0.96 mg/kg/h) stimulation of minute ventilation and reduction of end tidal CO 2 10 (ETCO 2 ) were noted. The second study (GAL-021-102) extended the dose range explored during the initial study by a factor of 2 and established the maximum respiratory stimulatory dose in the healthy subjects without concomitant use of opioids or anesthetic agents. At the 0.96 mg/kg/h, similar changes in minute ventilation was 15 noted with greater effects at the highest dose (1.92 mg/kg/h). Phase I Data (a) Summary of Phase I Clinical Safety The first study (GAL-021-101) was a randomized, double-blind, placebo controlled, nine-period, ascending single dose study in three alternating 20 cohorts of healthy subjects. Three cohorts of 10 subjects each were randomized to treatment in a ratio of 4:1 (8 treated, 2 placebo) in a rotating panel design (Figure 35). Subjects in the first cohort participated in dosing periods 1, 4, and 7, with the second cohort participating in dosing periods 2, 5, and 8, and third cohort participating in dosing period 3, 6, and 9. The subjects who received placebo in the first dosing 25 period also received placebo in the second and third dosing periods. Similarly, drug treated subjects received drug in all three dosing periods. After an overnight fast, subjects received an intravenous infusion of N-(4,6-bis-n-propylamino-[1,3,5]triazin 2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) or placebo then PD, PK, and safety evaluations were conducted during the next 24 hours. 30 Eight intravenous dose levels were administered with the first seven doses being fixed rate infusions (0.1-0.96 mg/kg/h) for 1, 2, 3, or 4 hours and the last used a loading dose (0.72 mg/kg/h for 1 hour) followed by a maintenance dose (0.36 mg/kg/h for 3 hours). - 179 - WO 2013/181217 PCT/US2013/043052 The second study (GAL-021-102) was a randomized, double-blind, placebo-controlled, four-period, single-rising dose study in alternating cohorts of healthy subjects. Two cohorts of nine subjects each were randomized to treatment in a ratio of 2:1 (6 treated, 3 placebo) in a rotating panel design. Subjects in the first 5 cohort participated in dosing periods 1 and 3, with the second cohort participating in dosing periods 2 and 4. The subjects received placebo at most once during the study. After an overnight fast, subjects received an intravenous infusion of N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) or placebo then PD, PK, and safety evaluations were conducted during the 10 next 24 hours. Three intravenous dose levels were administered with fixed rate infusions (0.96-1.92 mg/kg/h) for 2 hours. During the 3 rd treatment period, two subjects had increasing ventilatory efforts and declining ETCO 2 (nadirs: 22 and 29 mmHg), and the infusions were stopped with rapid diminution of the respiratory 15 efforts. The stopping criteria for further dose escalation had been met. During the 4 th and final treatment period, subjects were reassigned to the two lower dose levels based on their previous treatment to increase the total number of subjects receiving these dose levels. A third study, a two-part, four-treatment period, double blinded, 20 placebo controlled, crossover, stepped infusion study, evaluated ventilatory PD effects of N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) on respiratory drive in a model of opioid induced respiratory depression. In Part 1, dynamic end-tidal CO 2 forcing technique was used. This is a progressive hypercapnia resulting in targeted minute ventilation to 20 L/min. 25 Once achieved maintain (clamp) the PCO 2 at the same level throughout the remainder of the treatment period to remove the confounding influence of CO 2 changes on minute ventilation. In each part, subjects were randomized to two treatments (placebo or N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen 30 sulfate (XXXVI)) with each subject receiving each treatment once. In Part 2, the treatment sequence and opioid doses from Part 1 were repeated under ambient air conditions and addition of pain assessments. All treatment periods were subdivided into 7 segments (treatment period details provided in Figure 36) with a sequential - 180 - WO 2013/181217 PCT/US2013/043052 design. Two dose levels of alfentanil and two dose levels of (XXXVI) were included in each treatment period to provide a preliminary assessment of the dose response. In Part 2, pain was assessed to determine if N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) 5 impaired the opioid pain reduction using transcutaneous electrical stimulation over the tibia. Twenty three subjects participated in the study. Twelve subjects completed both treatment periods of Part 1 and 8 subjects participated and completed both treatment periods of Part 2. Three subjects completed one treatment period of 10 Part 1. Of these subjects, 2 discontinued the study after completing the first treatment period and a 3 rd subject discontinued before completing segment 2 in the second treatment period. Of these 3 subjects, one successfully completed Part 2. Six subjects dropped out the study between just before starting the first segment to completion of segment 2. If segment 2 was completed subjects completed the remaining 5 segments 15 of the treatment period. The primary reason for drop outs by these subjects was procedure-related (e.g., arterial line placement, mask associated anxiety) or difficulty accommodating to the hyperventilation induced by the inspired supplemental
CO
2 induced hypercapnia. As illustrated in Figure 36, during each study part, subjects were 20 randomly assigned to either N-(4,6-bis-n-propylamino-[ 1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine hydrogen sulfate (XXXVI) or placebo during the first treatment period and received the other during the second treatment period. All subjects were pretreated with ondansetron and metoclopramide due severe nausea and vomiting in pilot study secondary to alfentanil administration. During Part 1 segment 25 1, baseline ventilatory parameter were assessed after freely breathing ambient air for ~20 minutes during a 10 minute assessment period at the end of each segment denoted by blue inverted triangles ( i). During segment 2, CO 2 concentrations were increased slowly using gas mixer until the subject achieved a minute ventilation of 20 L/min. Alfentanil was titrated in segment 3 to achieve 25-35% reduction in end tidal minute 30 ventilation and inhaled CO 2 adjusted to maintain the same end-tidal PCO 2 as at the end of segment 2. Similar CO 2 adjustments were made throughout the remaining segments. In segment 4, low dose N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine hydrogen sulfate (XXXVI) was administered initially by rapid infusion (2.0 mg/kg/min) and then maintenance infusion (0.4 mg/kg/h) to - 181 - WO 2013/181217 PCT/US2013/043052 approximately a low dose, steady state concentration. In segment 5, a second rapid infusion (2.0 mg/kg/h) and greater maintenance infusion (1.1 mg/kg/h) were used to assess the primary endpoint (minute ventilation) at high steady state concentration. Thereafter (segment 6), the individualized loading and maintenance dose of alfentanil 5 (from segment 3) were doubled and ventilatory parameters assessed. At the beginning of the final segment 7, the alfentanil and N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI)/placebo infusions were stopped and subjects observed. After establishing the individual alfentanil dose in Part 1, segment 1, the same infusion rates were used for all 10 remaining segment in Parts 1 and 2. For Part 2, the same segment progression was used while breathing ambient are without use of C02 clamping techniques. In Part 2, transcutaneous electrical stimulation (TENS) over the tibia was used to establish pain threshold for detection and maximum tolerability. During the pains testing, current progressive increase with subjects pushing a button to note onset of pain and 15 maximum tolerable pain, at point the current stopped. The pain assessments are performed during segments 1-6 before the ventilatory assessment interval. (b) Summary of Phase I Pharmacodynamics Pharmacodynamic (PD) evaluations included tidal volume, respiratory rate, minute ventilation, end-tidal C0 2 , and transcutaneous hemoglobin oxygen 20 saturation (SpO2). Baseline respiratory data was acquired for 1 hour before dosing and the parameters average over that period after removal of recorded regions contain artifacts by treatment-blinded polysomnographic experts before analysis. PD data was collected for ~1 hour after completing the infusion. The time weighted percent change from baseline was calculated for all respiratory parameters. The first 2 hours 25 of data from groups 6 and 7 (0.72 mg/kg/h for 2 hour or more) was pooled for PD analysis. At all doses, change in respiratory rate was small and usually < 10%. Tidal volume changes were also small with only the 0.96 mg/kg/h dose having a consistent increase < 10% (Figure 37). To further evaluate the respiratory changes, the responses were 30 integrated over the first 2 hours after the start of the infusion. Minute ventilation did not change significantly except at the two highest infusion rates with 7-16% increases noted (Figure 38A) using the Drager pneumotachometer-based system. End-tidal
CO
2 decreased in parallel to minute ventilation increases with an integrated reduction of -6% (Figure 38B). The onset of effects on minute ventilation and ETCO 2 were - 182 - WO 2013/181217 PCT/US2013/043052 rapid with > half maximal effect observed by the 7.5 minutes (first assessment point). A similar pattern of changes in minute ventilation, tidal volume and respiratory rate (Figures 38C-D) were observed with NOX-T3 inductance plethysmography-based system with ~24% increase in minute ventilation. Transcutaneous hemoglobin 02 5 tension was not significant different from placebo except at the 0.96 mg/kg/h dose with an average change of +0.85% vs. -0.05% (p<0.05). The second study was conducted at the same clinical site as the first study and similar PD assessments and equipment were utilized. The first dose had an identical infusion rate to the first study (0.96 mg/kg/h) with an extended duration of 2 10 hours. Minute ventilation increased rapidly with mean increase of 15.4% during the 2-hour infusion period, which yielded similar results to the first study (16.1%). The middle dose had a similar increase (13.6%), while the high dose had a greater increase (24.3%). The minute ventilation changes were statistically significant at all 3 dose levels (Figure 39A). The effects on tidal volume and respiratory rate underlying the 15 changes in minute ventilation were more variable with the low dose having a primary effect on tidal volume, the middle dose on respiratory rate, and the high dose on the combination of both. End tidal-CO 2 was also reduced at all dose levels with highest dose having mean reduction >8% over the infusion period (Figure 39B). Changes at the low and high dose were statistically different from placebo for ETCO 2 and tidal 20 volume. Half-maximal effects occurred before or just after the 5 minute assessment (first timepoint measured) in minute ventilation, tidal volume, and ETCO 2 (Figures 39B-D) A rapid initial response was previously observed at lower infusion rates in the first study, but tended to decline soon thereafter, while the PD responses were sustained at dose >0.72 mg/kg/h. 25 Similar to the highest infusion rate in the first study (0.96 mg/kg/h), hemoglobin 02 saturation (SpO2) increased rapidly after initiation of the infusion with statistically significant differences noted from 25 to 75 minutes for the low and high doses (Figure 40). The integrated effects over the two-hour infusion increased 0.60% and 0.76% for low and high doses, respectively, vs. placebo (0.13%, p < 0.05). For 30 the middle dose, the 1-2 hour period had a greater increase in minute ventilation (17.
2 %) than the first hour (10.0%) and also had a statistically significant increase in SPO2 vs. placebo (0.41% vs. -0.02%, p < 0.05). During the respiratory depression Proof-of-Concept study (GAL-021 104), ventilatory parameters were captured continuously, which were integrated over - 183 - WO 2013/181217 PCT/US2013/043052 the final ten-minute interval as the primary endpoints with segment 5 being the primary inferential statistical comparison. The initial CO 2 escalation (Segment 2) was performed slowly to increase minute ventilation to 20 liters/min (~2.5-fold baseline) (Figure 4 1A). Alfentanil was titrated thereafter with ~23 reduction in CO 2 stimulated 5 increment in minute ventilation (37% absolute reduction). With the additional of a minimally stimulatory (XXXVI) dose (0.4 mg/kg/h), there was trend in minute ventilation toward separation from placebo (p<0.07), which reached statistical significance at the higher infusion rate (1.1 mg/kg/h) (p<0.000 1). With the doubling of alfentanil loading dose and infusion rate in the sixth segment, minute ventilation 10 declined for both GAL-021 and placebo while statistically significant separations continued between the 2 treatments (p < 0.005). For tidal volume (Figure 41B) and respiratory rate (Figure 41C), similar patterns of the statistically significant difference were noted. During Part 2 of the study, subjects received the identical alfentanil 15 loading doses and infusion rates as in Part 1. Subjects were allowed to breathe ambient air and no attempt was made to regulate end-tidal CO 2 . or minute ventilation. Pain was also assessed using a transcutaneous electrical nerve stimulation device (TENS) placed over the anterior tibia area, so that minimal muscular effects were produced. During testing, current progressively increased producing a current 20 threshold associated with the onset of pain and maximum tolerable pain (Figure 36). With the administration of alfentanil, the pain onset threshold increased (Figure 42). N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) administration did not diminish the pain onset or maximum tolerable thresholds. 25 With the initiation of the alfentanil infusion, minute ventilation decreased (Figure 43A). Placebo treated subjects remained stable until the higher dose alfentanil in segment 6 when it further declined. Similar to the Part 1, minute ventilation separated from placebo treatment with low dose N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate 30 (XXXVI) treatment and reached statistical significance with high dose in segment 5. While the high dose of alfentanil diminished N-(4,6-bis-n-propylamino-[1,3,5]triazin 2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) stimulation in Part 1 (segment 6), there was no diminution in minute ventilation under room air conditions. Consistent with known opioid effects, end tidal CO 2 increase and further increased - 184 - WO 2013/181217 PCT/US2013/043052 with the high dose alfentanil dose (Figure 43B). N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) administration tended to reduce end tidal CO 2 concentrations with statistically significance noted with high dose alfentanil (segment 6). Respiratory rates were not 5 different (Figure 43C). Tidal volume increased with high dose N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) compared to placebo treatment with both the low and high dose alfentanil infusions (Figure 43D). During Part 1 and Part 2, N-(4,6-bis-n-propylamino-[1,3,5]triazin-2 10 yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) had consistent effects compared to placebo that were clinically and statistically significant at the higher infusion rate (1.1mg/kg/h). The effect was independent of the presence of exogenous
CO
2 or endogenous retention of CO 2 secondary to opioid administration. The low, minimally stimulatory dose of N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O 15 dimethyl-hydroxylamine hydrogen sulfate (XXXVI) separated from placebo treatment with minute ventilation changes approached statistical significant without achieving it. Similar low dose N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine hydrogen sulfate (XXXVI) trends were noted in both study parts. The increase in minute ventilation tended to be driven by increase in tidal 20 volume noted in both study parts. Changes in respiratory were noted only in Part 1. Overall, N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) improved ventilatory status after opioid administration consistent with previous observation in rats and non-human primates. The improved ventilatory status after N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl 25 hydroxylamine hydrogen sulfate (XXXVI) administration did not compromise the analgesic effects of alfentanil and suggesting that current difficulty balancing of opioid analgesia and respiratory depressive effects may be less nettlesome when the 2 pharmacologic agents are coadministered. (c) Summary of Phase I Pharmacokinetics 30 In general, the overall shape of the plasma concentration-time profile was similar in both studies. Plasma concentrations rose rapidly during the infusion and fell sharply initially and then gradually after the infusion. The mean maximum plasma concentrations (Cmax) were reached generally at the end of infusion or the end - 185 - WO 2013/181217 PCT/US2013/043052 of loading dose infusion in the case of the biphasic infusion, as illustrated in Figure 44. In the first study after the end of infusion, all plasma concentration time profiles displayed apparent bi-exponential decay with a steep distributional phase 5 and a gradual terminal phase with a mean terminal half-life (t ) ranging from 4.3 to 6.5 h (Figure 44A). The pharmacokinetics of N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) in healthy volunteers was best described by a two-compartmental model in general with a distributional half-life of less than 20 min. The mean systemic plasma clearance 10 values (CLp), were approximately half the hepatic blood flow rate in humans. The mean steady-state volume of distribution values (Vss), ranging from 1.7 to 2.5 L/kg, were roughly 2-3 times the total body water in humans, suggesting that N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) was highly distributed to tissues. The plasma concentration at one-hour 15 into infusion, Cih (range: 54.9-930 ng/mL), was dose-proportional at all tested infusion rates. Similarly, total systemic exposure (AUCo_) was also dose proportional. The Cih and AUC meet the statistical requirement for dose proportionality. The intra-subject variability is generally less than inter-subject variability for is, CL,, and Vs,. - 186 - WO 2013/181217 PCT/US2013/043052 7= I t CIA C A CIA'\O~ CI - ClA rl - \Cl Cl Cl C C It C ' fC Lf] \ 'C r l -j rCl r l - c -~t cl - "C: -4- r- 4 rcn, ~0 CIAl ~~Cl~Lf00~ 00 0 ~ ~ ~ I I l __ _ _ __ t '00 rq .q \00 !E -t =-0~ c- 0 c- 0 cn WO 2013/181217 PCT/US2013/043052 oD fn rl fn*C fn rql Cl . I-r CIACl0 I It 00 t WO 2013/181217 PCT/US2013/043052 During the second study, N-(4,6-bis-n-propylamino-[1,3,5]triazin-2 yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) was infused at 0.96, 1.44, and 1.92 mg/kg/h for 2-hours. In general, the plasma concentration-time profiles at higher doses were similar to those observed in the first-in-human study. 5 The mean maximum plasma concentrations (Cmax) were generally reached at the end of infusion as illustrated in Table 11 B and Figure 44B. Following the end of infusion, all plasma concentration-time profiles displayed apparent bi-exponential decay with a steep distributional phase and a gradual terminal phase with a mean terminal half-life (t ) ranging from 5.2 to 8.1 h. 10 Similar to those observed in the first-in-human study, the mean systemic plasma clearance values (CLp), ranging from 10.5 to 12.4 ml/min/kg, were approximately half the hepatic blood flow rate in humans. The mean steady-state volume of distribution values (Vss), ranging from 1.8 to 2.5 L/kg, were roughly 2-3 times the total body water in humans. 15 In general, N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine hydrogen sulfate (XXXVI) demonstrated well-behaved pharmacokinetics when administered intravenously to healthy volunteers in the dose range studied. The plasma concentration-time profiles fit well to a two compartmental model with a short distributional half-life of less than 20 min. With 20 Cmax and AUC 0 . being dose-proportional and t/, CLp, and Vss being dose independent in the first study (0.1 - 0.96 mg/kg/h), without obvious deviation from predicted linearity during the second study. Future dosage regimen adjustments could be simplified as a result of the linear pharmacokinetics. The terminal half-life (t ~6 h) suggest that little accumulation would occur after 24 hours, which is consistent 25 with low measurable plasma C24h. (d) Overall Conclusions from Phase I Clinical Trial(s) N-(4,6-bis-n-propylamino-[ 1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine hydrogen sulfate (XXXVI) was well tolerated during the initial clinical study with only the sensation of mild/moderate burning at the site of infusion 30 being different from placebo. Minute ventilation increased at the two highest infusion rates (0.72 and 0.96 mg/kg/h). During the second study, hyperventilation occurred at the high dose (1.92 mg/kg/h) to establish a PD maximum dose in healthy subject untreated with respiratory depressive agents. The stimulation of minute ventilation was similar in the two studies at the 0.96 mg/kg/h infusion rate (~16%) with greater - 189 - WO 2013/181217 PCT/US2013/043052 stimulation at the high dose (25%). The drug was well behaved pharmacokinetically with a rapid concentration rise during the initial infusion period and rapid decline with cessation of the infusion. The compound is dose proportional for both AUC and Clh with low inter- and intra-subject variability. 5 In the presence of opioid-induced respiratory depression, N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) administration was equally well tolerated compared to placebo with only mild burning sensation at infusion site in a few subjects. In the presence of clamped hypercapnia, minute ventilation, tidal volume and respiratory rate were increased to a 10 statistically and clinically significant extent with (XXXVI) dose of 1.1 mg/kg/h. When breathing room air, similar ventilatory improvements were noted for minute ventilation, end-tidal C0 2 , and tidal volume. Overall N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine hydrogen sulfate (XXXVI) has useful pharmacology in a setting simulation the postoperative setting and has potential 15 clinical utility in post-surgical respiratory depression. Example 129 Development of Liquid Formulation for (XXXVI) 20 A. Solubility of (XXXV) as a function of pH Methodology A solution of (XXXVI) of concentration ~107mg/mL in 100 mM citric acid at pH 2.5 was prepared. About 150pL aliquots of this solution were transferred into 6 separate glass HPLC vials. Each aliquot was titrated with 6N sodium 25 hydroxide to different pH values in the range 3-5.5 (Table 12A). All the samples, including the solution at pH 2.5, were shaken overnight at room temperature using a gyratory shaker. After shaking, the samples were evaluated visually for the presence of precipitate and were then spin filtered through 0.45pm syringe tip filters (4mm diameter) at 3,000 rpm for 20 min. The final pH values of the filtrates were 30 measured. The filtrates were then diluted appropriately and analyzed for (XXXV) concentration using the HPLC assay. Results The pH values of the samples prior to and after shaking overnight, appearance as well as (XXXV) concentrations of samples are illustrated in Table 12A. - 190 - WO 2013/181217 PCT/US2013/043052 Solutions prepared at target pH values of 2.5, 3.0 and 3.5 were clear (no excess solid present) and hence the (XXXV) concentration values for these samples do not represent saturation solubility values. The pH solubility profile is shown in Figure 19B. The closed diamonds are saturation solubility values and open diamonds are 5 measured concentrations in non-saturated samples (no excess solid present). The dotted line represents the best fit curve, which was plotted using Equation 1. This equation is a simplified representation of solubility equilibrium for weak bases that assumes that total solubility is much larger than the solubility of the free base in the pH range of interest: 10 log S = log So + log (1 + l 0 pKa-pH) Equation 1 where, 'S' is total solubility; 'So' is the solubility of the free base. Using the Microsoft Excel Solver function and a pKa value of 5.6 for (XXXVI), Equation 1 was fit to the experimentally determined pH-solubility profile. The solubility value of free base thus calculated was 0.18 mg/mL. Based on this 15 equation, solubility (expressed as the free base) is predicted to be 10 mg/mL at about pH 3.86, 25 mg/mL at about pH 3.46, and 50 mg/mL at about pH 3.16. Table 12A. pH Solubility Results target pH initial pH final pH after appearance (XXXVI) Shaking and afterwards concentration Filtration Shaking 2.5 2.51 2.42 Clear solution 104.1 3.0 3.05 3.00 Clear solution 99.8 3.5 3.51 3.51 Clear solution 101.6 4.0 4.00 3.79 Precipitate 20.8 4.5 4.55 4.37 Precipitate 3.46 5.0 4.93 4.52 Precipitate 2.73 5.5 5.63 5.02 Precipitate 0.99 20 B. Stability of (XXXV) as a Function of pH Methodology Buffer solutions containing ca. 10mM each of citric acid and glycine in the pH range 2-6 were prepared by titrating 250mL of a single stock containing both buffer agents with IN NaOH/IN HCl and removing 50mL aliquots at predetermined 25 pH values. XXXVI solutions at 1mg/mL were prepared in buffers of pH 2-5 by dissolving ~25mg of XXXVI in ~23mL of a given buffer, adjusting the pH to the target and making up the volume to 25.0 mL in a volumetric flask. (XXXVI) solution - 191 - WO 2013/181217 PCT/US2013/043052 at pH 6 was prepared similarly, but at a lower drug concentration of 0.25 mg/mL using 6.68 mg of (XXXVI). Each of the buffered XXXV solutions was filtered through a 0.2pm polyether sulfone syringe filter into a volumetric flask, which had been previously 5 rinsed with 70% ethanol and dried in a laminar flow hood. Following filtration, each buffered (XXXV) solution was split into 10 aliquots (9 aliquots of 2.5 mL each and 1 aliquot of 2 mL) in 5 mL serum vials previously rinsed with 70% ethanol and dried in a laminar flow hood. Filtration and preparation of aliquots were performed in a laminar flow hood. The 2 mL aliquot of each buffered (XXXV) solution was 10 submitted for TO analysis (HPLC assay and related substances). Of the nine 2.5mL aliquots, three aliquots were stored at each 40 0 C and 60 0 C, two aliquots were stored at 25 0 C and one aliquot wa s stored at -70 0 C. Samples stored at 40 0 C and at 60 0 C were evaluated for physical appearance, pH, assay and related substances after 1 week, 2 weeks and 4 weeks of storage. Samples 15 at 25 0 C and -70 0 C served as controls to be analyzed if necessary. Results The pH stability data are shown in Table 12B. All samples remained clear, colorless and particle free for up to 4 weeks of storage at both 40 0 C and at 60 0 C. The pH values for all the samples remained re latively constant for up to 4 weeks 20 at both storage temperatures. Both the assay and impurities data indicate that (XXXV) is significantly less stable at pH 2 as compared to the remaining pH values evaluated. No specific trends were observed in the pH range 3-6. The pH stability profiles at 60 0 C are shown in Figure 20A (% impurities). 25 Table 12B. pH Stability of (XXXV) (GAL 021) at pH 2-5 40 Storage 60 Storage Test Target initial 1 wk 2 wk 4 wk 1 wk 2 wk 4 wk pH pH 2 1.96 2.02 1.98 2.00 2.00 1.99 2.01 3 3.02 3.05 3.05 3.06 3.07 3.03 3.07 4 4.00 3.94 3.96 3.96 3.94 3.96 3.97 5 5.03 4.90 4.95 4.97 4.92 4.93 4.98 6 6.05 5.88 6.02 5.98 5.87 6.03 6.01 Assay 2 1.02 0.997 0.975 0.970 0.929 0.754 0.754 - 192 - WO 2013/181217 PCT/US2013/043052 3 0.984 0.984 0.987 0.991 0.971 0.952 0.952 4 1.01 0.999 1.006 1.012 0.992 0.979 0.979 5 1.01 0.989 0.992 0.977 0.977 0.947 0.947 6 0.260 0.257 0.253 0.251 0.247 0.234 0.234 Impurities 2 n.d. 0.58 1.48 2.49 3.83 9.89 14.34 3 n.d. n.d. 0.19 0.41 0.71 1.20 2.19 4 n.d. n.d. 0.17 0.22 n.d. 0.52 1.02 5 n.d. n.d. 0.26 n.d n.d. 0.62 1.10 6 n.d. n.d. 0.25 0.28 0.56 0.87 1.65 n.d. = not detected C. Stability of (XXXV) Across Narrow pH (pH 2-3.6) Methodology 5 Buffer solutions containing ca. 10mM each of citric acid and glycine in the pH range 2-3.6 were prepared by titrating 250mL of a single stock containing both buffer agents with IN NaOH/IN HCl and removing 50mL aliquots at predetermined pH values. (XXXVI) solutions at 1mg/mL were prepared in the buffers by dissolving ~25mg of (XXXVI) in ca. 23mL of a given buffer, adjusting the pH to the target and 10 making up the volume to 25.OmL in a volumetric flask. Each of the buffered (XXXV) solutions was filtered through a 0.2pm polyether sulfone syringe filter into a volumetric flask previously rinsed with 70% ethanol and dried in a laminar flow hood. Following filtration, each buffered (XXXV) solution was split into 9 aliquots in 5mL serum vials previously rinsed with 70% ethanol and dried in a laminar flow hood. 15 Filtration and preparation of aliquots were performed in a laminar flow hood. Of the nine aliquots, three aliquots were stored at each 40 'C and 60 'C, two aliquots wer e stored at 25 'C and one aliquot was submitted for TO testing. Samples were evaluated for pH, assay and related substances at TO and, following 1 week, 2 weeks and 4 weeks of storage at 40 'C and 60 'C. Samples stored at 25'C se rved as controls to be 20 analyzed if necessary Results Data are summarized in Table 12C and in Figure 20B. The most notable result from this study is that during both 40 'C and 60 'C storage, impurity levels decreased significantly y with increasing pH across the full pH range studied. - 193 - WO 2013/181217 PCT/US2013/043052 This means that any increase in the pH of the formulation led to an improvement in stability within the pH range in which an aqueous formulation is feasible. Table 12C. pH Stability of (XXXV) at pH 2-3.6 40CC Storage 60CC Storage Test Target initial 1 wk 2 wk 4 wk 1 wk 2 wk 4 wk pH 2.0 2.00 2.00 1.98 1.98 2.00 1.97 1.89 2.3 2.33 2.36 2.32 2.33 2.36 2.34 2.28 2.7 2.72 2.73 2.72 2.73 2.75 2.74 2.67 3.0 3.02 3.04 3.05 3.04 3.05 3.04 3.00 3.3 3.31 3.35 3.35 3.34 3.36 3.37 3.34 3.6 3.62 3.64 3.70 3.64 3.65 3.70 3.64 Assay 2.0 0.981 0.959 0.955 0.956 0.913 0.851 0.768 2.3 0.987 0.984 0.993 0.995 0.961 0.932 0.916 2.7 0.987 0.981 0.985 1.019 0.978 1.144 0.987 3.0 1.005 1.01 0.987 1.027 0.991 0.975 0.987 3.3 1.019 1.02 1.01 1.047 0.997 0.995 1.027 3.6 0.996 0.991 0.985 0.991 0.979 0.958 0.979 Impurities 2.0 0.14 0.99 1.55 2.84 3.87 7.56 13.99 2.3 0.09 0.47 0.63 1.70 1.81 3.53 6.30 2.7 n.d. 0.22 0.37 0.61 0.97 1.76 3.09 3.0 n.d. 0.21 0.34 0.58 0.61 1.27 1.82 3.3 n.d. n.d n.d 0.25 0.41 0.69 1.26 3.6 0.12 n.d n.d 0.17 0.36 0.62 0.99 5 n.d. = not detected D. Evaluation of the Solubility of (XXXV) in Solvents and IV Solutions Methodology The solubility of (XXXVI) and XXXV was evaluated in various 10 solvents and IV solutions. The solvents and target concentration for solubility evaluation are shown in Table 12D. Due to limits on the API supply at the time the study was conducted, most of the solvent systems were evaluated to a level of only 100 mg/mL even if the solution was not saturated at this level. Only in water was an attempt made to reach saturation solubility, regardless of how high that solubility 15 might be. Solubility of the salt/free base in each solvent was evaluated in duplicate. Evaluation of solubility of (XXXVI) in 0.9% NaCl, 5% dextrose, ethanol, 30% PEG 400, 70% PEG 400 and 100% PEG 400 was carried out at a target of 200mg/mL by adding ~100mg of drug to 500pL of solvent. The rest of the - 194 - WO 2013/181217 PCT/US2013/043052 solubility evaluations, except water, were carried out by adding ~50mg of drug (XXXVI or XXXV) to 500pL of solvent. For solubility in water, drug was added with intermittent shaking to ensure that excess undissolved drug is present indicating saturation 13 mm PTFE 0.2pm syringe filters. The filtrates were diluted appropriately 5 and analyzed for (XXXV) concentration by HPLC. Results The solubility results are shown in Table 12D. (XXXVI): The solubility of (XXXVI) was greater than 400 mg/mLa in water and at least 200 mg/mL in IV solutions (0.9% saline and 5% dextrose). During the 10 evaluation of solubility of the (XXXV) salt in water it was noted that -350-400 mg the API dissolved completely in 500pL of water; however, the sample formed a cloudy gel following ca. 2h of shaking at room temperature that dissolved upon addition of water (~80-250pL). Further shaking of this clear solution overnight at room temperature resulted in a cloudy gel formation. Addition of another small 15 aliquot of water (~60-220pL) resulted in a fluid suspension that could be filtered and analyzed for (XXXV) concentration. The exact reason for this behavior of the (XXXVI) in water was not clear but may suggest formation of a supersaturated solution. The salt was also freely soluble in ethanol and methanol and had a saturation solubility of about 68 mg/mL in acetonitrile. The solubility in 30%, 70% 20 and1 0 % propylene glycol was at least 90 mg/mL. The solubility in 100% PEG-400 reached saturation at ca. 36 mg/mL but (XXXVI) was soluble to at least 90 mg/mL in 70% PEG-400 and ~198 mg/mL in 30% PEG-400. (XXXV): The saturation solubility of (XXV) in water was ca. 0.2 1mg/mL, which is in agreement with the predicted value from the pH-solubility curve detailed 25 in Example 129A. The solubility in ethanol, methanol and acetonitrile were ~92mg/mL, ~102mg/mL and ~45mg/mL, respectively. The solubility of the free base in 5% Tween 80 dispersed system was ca. 2.7mg/mL. The solubility in PEG-400 and propylene glycol systems appears to increase with increasing levels of the cosolvent. The free base was more soluble in propylene glycol (~90mg/mL) than in PEG-400 30 (~73mg/mL). The data suggest that the solubility of XXXV free base in propylene glycol systems would be greater than in PEG-400 systems at similar pH values. - 195 - WO 2013/181217 PCT/US2013/043052 Table 12D. Solubility of (XXXV) in Solvents and IV Solutions (XXXV) hydrogen sulfate salt (XXXV) free base Solvent System Replicate Solubility Saturation Solubility Saturation pH (mg/mL) Solubility pH (mg/mL) Solubility Water 1 0.40 414.5 Y 8.80 0.206 Y 2 0.31 441.5 Y 8.67 0.206 Y 0.9% NaCl 1 0.61 >206.2 N 2 0.62 >201.4 N Not determined 5% Dextrose 1 0.61 >201.2 N 2 sample not recoverable Not determined Ethanol 1 NA >211.4 N NA 96.04 Y 2 NA >212.2 N NA 87.24 Y 30% PEG-400 1 0.84 >197.0 N 4.30 17.03 Y 2 0.82 >198.4 N 4.35 17.30 Y 70% PEG-400 1 1.68 >85.28 N 5.98 14.78 Y 2 1.65 >91.43 N 6.09 11.76 Y PEG-400 1 NA 35.74 Y NA 68.3 Y 2 sample not recoverable NA 77.8 Y 30% Propylene 1 1.06 >93.21 N 7.36 0.87 Y Glycol 2 1.07 >89.47 N 7.51 0.84 Y 70% Propylene 1 1.22 >87.80 N 7.93 12.4 Y Glycol 2 1.18 >92.43 N 8.25 12.1 Y Propylene 1 NA >92.09 N NA 87.0 Y Glycol 2 NA >94.65 N NA 93.2 Y Methanol 1 NA >108.9 N NA 94.82 Y 2 NA >98.52 N NA 108.38 Y Acetonitrile 1 NA 68.5 Y NA 49.4 Y 2 NA 67.1 Y NA 39.9 Y 5% Tween 80 1 7.54 2.69 Y 2 Not determined 7.61 2.68 Y * NA = not applicable. Samples listed as not recoverable were lost due to filtration failures. E. Stability of (XXXV) as a Function of Buffer Strength 5 Methodology A 10mg/mL (XXXVI) solution in 100 mM citrate buffer at pH 3.0 and a 500mM citrate buffer at pH 3.0 were prepared. Using these two stock solutions, (XXXVI) solutions at 1 mg/mL were prepared in citrate buffers of various concentrations in the range 10-250mM, as shown in Table 12E. - 196 - WO 2013/181217 PCT/US2013/043052 Table 12E. Solutions for Evaluation of Stability as a Function of Buffer Strength Solution Volume of Volume of Volume of 10 mg/mL (XXXVI) in 500 mM Citrate Water (mL) 100mM citrate pH 3.0 (mL) pH 3.0 (mL) lmg/mL (XXXV) 2 0 QS to 20.OmL in 10mM Citrate lmg/mL(XXXV) 2 0.4 QS to 20.OmL in 20mM Citrate lmg/mL (XXXV) 2 1.6 QS to 20.OmL in 50mM Citrate 1mg/mL (XXXV) 2 3.6 QS to 20.OmL in 100mM Citrate 1mg/mL (XXXV) 2 9.6 QS to 20.OmL in 250mM Citrate Each of the above solutions was stored in 5mL serum vials as ca. 2.5 5 3mL aliquots (total of 7 aliquots). Two aliquots were stored at each of 40 'C, 60 'C and 25 'C. Aliquots stored at 25 'C served as control s to be evaluated if necessary. At TO and after 2 weeks of storage at 40 'C and at 60 'C, samples were evaluated for pH, assay and related substances. Results 10 Data from the buffer effect study are summarized in Table 12F. Impurity levels increased noticeably at the highest buffer concentration, but there was no effect of buffer strength at 40 0 C within the range of buffer concentration relevant to the formulation. 15 Table 12F. Effect of Buffer Strength on (XXXV) Stability Buffer initial 40'C Storage 60'C Storage Test Strength 1 wk 2 wk 1 wk 2 wk 10 mM 3.16 3.26 3.21 3.3 3.2 pH 20 mM 3.27 3.33 3.30 3.3 3.3 50 mM 3.28 3.34 3.29 3.3 3.3 100 mM 3.30 3.32 3.29 3.3 3.2 250 mM 3.21 3.25 3.22 3.2 3.2 10 mM 0.971 0.953 0.947 0.953 0.956 Assay 20 mM 1.01 0.965 0.966 0.943 0.947 50 mM 0.976 0.959 0.949 0.936 0.943 100 mM 0.962 0.964 0.956 0.962 0.946 250 mM 0.947 0.972 0.945 0.955 0.936 10 mM n.d. n.d. n.d. 0.48 0.89 - 197 - WO 2013/181217 PCT/US2013/043052 Impurities 20 mM n.d. 0.13 n.d. 0.58 0.88 50 mM n.d. n.d. n.d. 0.59 0.87 100 mM n.d. 0.26 0.38 0.73 1.20 250 mM n.d. 0.21 0.56 1.03 1.80 n.d. = not detected F. Prototype Formulations Methodology 5 A total of nine (XXXV) prototypes were prepared that included a simple buffered solution, co-solvent systems as well as non-aqueous solutions. These are shown in Table 12G. Prototypes 6, 7 and 8 resulted in significant precipitation during preparation and hence were not evaluated further. The remaining prototypes were stored at 2-8 0 C, room temperature, 40 0 C and 60 0 C in serum vials. Samples 10 stored at 2-8 0 C and room temperature were analyzed at selected timepoints. Results Data gathered to date are provided in Table 12G.1-12G.6. The aqueous buffered formulation (Prototype #1) has been found to be stable at room temperature and under refrigeration, but some degradation has been observed under 15 accelerated conditions. Of special concern is that a very small amount of precipitate, described as one or two snowflake-like particles, has been observed after 2 weeks at 60 'C and after 8 weeks at 40 'C. Since the appearance of this precipitate during 60 'C storage coincided with an actual decrease in the primary impurity peak, the possibility of the degradant being insoluble is under investigation. 20 (XXXV) was unstable in Prototype #2, 50% PEG/50% water without pH adjustment. The instability was presumably due to the low pH of this formulation. The drug was found to be much more stable in Prototype #3, which has the same 50% PEG base but also contains sufficient sodium hydroxide to bring the apparent pH up to about 4.5. Similarly, good stability was seen in Prototype #9, 70% propylene 25 glycol/30% water with ammonium acetate buffer to bring the apparent pH to about 5.3. Good stability was also seen in simple solutions of the salt in PEG or propylene glycol, presumably due to the absence of water as a reactant. - 198 - WO 2013/181217 PCT/US2013/043052 Table 12G. (XXXVI) Prototypes for Stability Evaluation Prototype # Description 1 35 mg/mL (XXXV).H2SO4 in 50mM citrate, pH 3.0 2 35 mg/mL (XXXV).H2SO4 in 50% v/v PEG-400 in water 3 35 mg/mL (XXXV).H2S04 in 50% v/v PEG-400 in water adjusted to pH 4.4 with IN NaOH 4 30 mg/mL (XXXV).H2SO4 in 100% PEG-400 5 35 mg/mL (XXXV).H2SO4 in 100% propylene glycol 6 35 mg/mL (XXXV).H2SO4 in 50% v/v PEG-400 in acetate solution (~40mM sodium acetate and 1 00mM sodium hydroxide) 7 35 mg/mL (XXXV).H2SO4 in 50% v/v propylene glycol in acetate solution (~40mM sodium acetate and ~100mM sodium hydroxide) 8 35 mg/mL (XXXV).H2SO4 in 50% v/v PEG-400 in acetate solution (~40mM ammonium acetate and ~100mM ammonium hydroxide) 35 mg/mL (XXXV).H2SO4 in 70% v/v propylene glycol in acetate 9 solution (~40mM ammonium acetate and 1 00mM ammonium hydroxide) Table 12G.1. Stability of (XXXV) Formulation Prototype #1 Formulation #1: 35mg/mL (XXXV).H2SO4 in 50mM citrate, pH 3.0 Parameter Temperature Initial 1 week 2 weeks 4 weeks pH 2-8V 2.97 3.04 -- - RT 3.04 -- 3.07 40V 2.99 3.01 3.06 60V 3.01 3.02 2.97 Assay 2-8C 35.73 36.22 NA NA RT 35.93 -- 35.31 40V 34.83 35.40 33.96 60V 37.90 34.63 31.89 Impurities 2-8V n.d. n.d. -- - RT n.d. -- n.d. 40V n.d. 0.35 0.52 60V 0.73 0.49 0.51 5 Table 12G.2. Stability of (XXXV) Formulation Prototype #2 Formulation #2: 35mg/mL (XXXV).H2SO4 in 50% v/v PEG-400 in water Parameter Temperature Initial 1 week 2 weeks 4 weeks - 199 - WO 2013/181217 PCT/US2013/043052 pH RT 2.00 2.01 NA 2.07 40V 2.04 2.03 2.11 60V 2.04 2.14 2.36 Assay RT 33.93 35.26 NA 34.47 40V 35.30 34.81 33.92 60V 33.70 31.77 27.45 Impurities RT n.d. 0.39 0.39 1.10 40V 0.92 0.92 2.53 60V 2.11 2.11 11.99 Table 12G.3. Stability of (XXXV) Formulation Prototype #3 Formulation #3: 35mg/mL (XXXV).H2SO4 in 50% v/v PEG-400 in water adjusted to pH 4.4 with IN Parameter Temperature Initial 1 week 2 weeks 4 weeks pH 2-8C 4.38 4.51 NA NA R 4.40 NA 4.52 400 4.20 4.43 4.41 600 4.16-3.88 4.22 4.27 Assay 2-8C 22.17 37.31 NA NA R 36.23 NA 36.03 400 35.66 36.53 35.96 600 35.33 34.70 34.14 Impurities 2-8C n.d. n.d. NA NA R n.d. NA n.d. 400 n.d. n.d. 0.90 600 0.62 1.50 1.91 Table 12G.4. Stability of (XXXV) Formulation Prototype #4 Formulation #4: 30mg/mL (XXXV).H2SO4 in 100% PEG-400 Parameter Temperature Initial 1 week 2 weeks 4 weeks 2-8'C 27.94 30.18 NA NA Assay RT 30.20 NA 30.31 40V 29.91 29.89 30.19 60V 29.24 29.60 29.97 2-8'C n.d. n.d. NA NA Impurities RT n.d. NA n.d. 40V n.d. n.d. n.d. 60V 0.50 0.50 0.37 5 - 200 - WO 2013/181217 PCT/US2013/043052 Table 12G.5. Stability of (XXXV) Formulation Prototype #5 Formulation #5: 35mg/mL (XXXV).H2SO4 in 100% propylene glycol Parameter Temperature Initial 1 week 2 weeks 4 weeks Assay 2-8C 28.97 36.22 NA NA RT 36.23 NA 36.00 40 35.16 35.63 36.03 60V 35.56 35.33 35.56 Impurities 2-8C n.d. n.d. NA NA RT n.d. NA n.d. 40V n.d. 0.12 0.27 60V 0.23 0.74 0.99 Table 12G.6. Stability of (XXXV) Formulation Prototype #9 Formulation #9: 35mg/mL (XXXV).H2SO4 in 70% v/v propylene glycol in acetate solution (~40mM) Parameter Temperature Initial 1 week 2 weeks 4 weeks pH 2-8C 5.28 5.27 NA RT NA NA 40C 5.26 5.35 60C 5.25 5.34 Assay 2-8C 35.50 34.40 NA RT NA NA 40C 34.36 34.62 60C 34.69 34.55 Impurities 2-8C n.d. n.d. NA RT NA NA 40V n.d. n.d. 60C n.d. 0.39 5 G. Final Formulation and GMP Drug Product Stability Data The final formulation is shown in Table 12H. Stability data for drug product formulation in accord with Table 12H are shown in Table 121.1 and 121.2 at 25 'C/60 %RH and 2-8'C, respectively. The levels of N-(4,6-Bis(propylamino) 1,3,5-triazin-2-ol (CLXXVIII) measured during the stability study are shown in Table 10 12J. -201- WO 2013/181217 PCT/US2013/043052 Table 12H. Final Formulation for (XXXV) Component Function Concentration % w/w (XXXV) Active 10 mg/mL 1.386 (as free base) (as salt) Citric acid, Anhydrous, USP Buffer 20 mM 0.384 1 N Sodium Hyrdoxide adjust pH to 3.2 q.s. q.s. Sterile Water for Injection, solvent q.s. q.s. USP Table 121.1. Long-term stability results (25 C/60%RH) for Formulated (XXXVI), GMP lot# 0411051 la Test Specification Months 0 1 (I)* 3 (I)* 6 (I)* 6 (U)** 9 (I)* Appearance b. ( 3 )b ( 2 )b ( 2 )b ( 2 )b ( 2 )b ( 2 )b of Solution pH 2.7-3.6 (3)3.1, (2)3.1, (2)3.1, (2)3.1, (2)3.1, 3.1, 3.1 3.1, 3.1 3.1 3.1 3.2 3.2 Osmolality Report (3) 126, (2) 125, 2) 128, 2)127, 2) 127, 125,125 Results, 126, 125 128 127 128 mOsm/kg 127 Vial d. (2)e (2)e (2)e (2)e (2)e (2)e Appearance Identification Retention (3) (2) 1.00 (2) 1.00, (2) 1.00, (2) 1.00, 1.00 by Retention time between 0.99. 1.00 1.00 1.00 1.00 1.00 Time 0.98 and 1.02 0.99, relative to 0.99 standard Assay 90 - 110% of (3) (2) (2) (2) (2) (2) label claim 99.1, 97.9%, 97.5%, 99.3% 98.2% 96.2%, 99.1, 97.9% 98.1% 97.4% 98.3 96.2% 99.3 Related NMT 1.00% (3) (2) RRT 2) RRT 2) RRT 2) RRT RRT Substances for any single <0.10% 0.672: 0.672: 0.672: 0.672: 0.672: related <010% 0.15% 0.44% 0.82% 0.81% 1.16 substance <0.10% RRT RRT RRT RRT RRT Totals 0.672: 0.672: 0.672: 0.672: 0.672: NMT 3.00% <0.10 0.15% 0.44% 0.82% 0.81% 1.16 total related all Total: Total: Total: Total: Total: substances 0.15 0.44 0.82 0.81% 1.16 Total: Total: Total: Total: Total: 0.15 0.44 0.82 0.81% 1.16 Subvisible f. (3) (3) (3) Particulates 207, 37 418, 0 78, 0 122, 7 306, 0 99,0 177,0 282,0 54,0 Weight Loss NMT 2.5 g (5) 0.0 all (5) 0.0 all (5) 0.0 all (5 vials) loss in weight - 202 - WO 2013/181217 PCT/US2013/043052 a. Package components for GMP batch: 50 mL clear, 20 mm opening, USP Type 1 glass (Wheaton 223745); Gray butyl teflon 2 coated stopped. 4416/50 (West 1014 4937); flip off seal, 20 mm, 3766 white, 8-bridge (West 5420 3028). b. Colorless to pale yellow liquid. Clear and particle free 5 c. Colorless liquid. Clear and particle free d. Vial free of cracks, stopper securely in place and no sign of leakage e. Both vials free from crack stoppers securely in place, no sign of leakage f. For each container: NMT 6000 > 10 pm particles; NMT 600 > 25 pm *(I) = inverted; ** (U) = upright 10 Table 121.2. Long-term stability results (5 0 C/Ambient) for Formulated (XXXVI), GMP lot# 0411051 la Test Specification Months 0 1(I)* 3 (I)* 6 (I)* 6 ( U ) * * 9 (I)* 10 Appearance b. (2)" (2)" (2)" (2))b (2)b (2)b of Solution pH 2.7-3.6 3.1,3.1 (2)3.1, (2)3.1, (2)3.1, (2)3.1, (2) 3.1 3.1 3.1 3.1 3.1, 3.1 Osmolality Report (2) 124 (2) 124, (2) 127, (2) 128, (2) 128, (2) Results 124 124 127 128 128 kg 124, mOsm/kg 124 Vial d. (2)e (2)e (2)e (2)e (2)e (2)e Appearance Identification Retention 1.00 (2) 1.00 (2) 1.00 (2) 1.00 (2) 1.00 (2) by Retention time between 1.00 1.00 1.00 1.00 1.00 1.00 Time 0.98 and 1.02 1.00 relative to standard Assay 90 - 110% of 97.8, (2) (2) (2) (2) (2) label claim 97.8 98.3, 99.0, 100.1, 99.2, 97.8, 98.3 99.0 100.2 99.2 97.8 Related NMT 1.00% RRT (2) (2) (2) (2) (2) Substances for any single 0.672: <0.10%, <0.10%, <0.10%, <0.10%, RRT related 0.11 <0.10% <0.10% <0.10% <0.10% 0.672: substance RRT Total: Total: Total: Total: 0.11 0.672: <0.10% <0.10% <0.10% <0.10% RRT NMT 3.00% 0.11 Total: Total: Total: Total: 0.672: total related Total: <0.10% <0.10 <0.10 <0.10 0.11 substances 0.11 Total: Total: 0.11 0.11 Total: 0.11 Subvisible f. (3) (3) (3) (3) (3) (I)* Particulates 224, 0 224, 0 27, 0 156, 0 133, 0 150,0 150,0 75,0 150,0 160,0 71,0 71,0 139,0 146,0 68,3 Weight Loss NMT 2.5 g (5) (5) (5) (5) (5) (5) (5 vials) loss in 0.0 all 0.0 all 0.0 all 0.0 all 0.0 all 0.0 all weight LAL* NMT (I) 4EU/mL <0.5 EU/mL Sterility* - 203 - WO 2013/181217 PCT/US2013/043052 a. Package components for GMP batch: 50 mL clear, 20 mm opening, USP Type 1 glass (Wheaton 223745); Gray butyl teflon 2 coated stopped. 4416/50 (West 1014 4937); flip off seal, 20 mm, 3766 white, 8-bridge (West 5420 3028). b. Colorless to pale yellow liquid. Clear and particle free 5 c. Colorless liquid. Clear and particle free d. Vial free of cracks, stopper securely in place and no sign of leakage e. Both Vials free from crack stoppers securely in place, no sign of leakage f. For each container: NMT 6000 > 10 pm particles; NMT 600 > 25 pm *(I) = inverted; ** (U) = upright 10 Table 12J. Level of Degradant 4,6-Bis(propylamino)-1,3,5-triazin-2-ol (CLXXVIII) GMP StabilityStudy, Lot # GMP lot# 04110511 Release Testing T = T = T = T= (Drug Product 1 month 3 month 6 month 9 month Production lot Sample Pull) Beg. Mid. End Vial Vial Vial Vial Vial Vial Vial Vial 1 2 1 2 1 2 1 2 5-C, amb. 0.03 0.03 0.06 0.06 0.08 0.08 0.11 0.11 RH Ivre 0.00 0.00 0.00 amb. NA NA NA NA 0.08 0.08 NA NA RH Upright 15 The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the 20 invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations. - 204 -

Claims (52)

  1. 2. The composition of claim 1, wherein the salt is hydrogen sulfate, hydrochloride, phosphate, hydrogen phosphate or dihydrogen phosphate.
  2. 3. The composition of claim 1, further comprising at least one pharmaceutically acceptable carrier. 15 4. A method of preventing or treating a breathing control disorder or disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound selected from the group consisting of: N-(2,4-bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine 20 (CLXXII), N-(2,4,6-tris-n-propylamino)-[1,3,5]triazine (CLXXIII), N-(2,4,6-tris-n propylamino)-1,3-pyrimidine (CLXXIV), N-(2,4-bis-n-propylamino)-N-(6-i propylamino)-1,3-pyrimidine (CLXXV), a salt thereof, and any combinations thereof.
  3. 5. The method of claim 4, wherein the breathing control disorder or disease is selected from the group consisting of respiratory depression, sleep apnea, 25 apnea of prematurity, obesity-hypoventilation syndrome, primary alveolar hypoventilation syndrome, dyspnea, hypoxia, and hypercapnia.
  4. 6. The method of claim 5, wherein the respiratory depression is caused by an anesthetic, a sedative, an anxiolytic agent, a hypnotic agent, alcohol or a narcotic. - 205 - WO 2013/181217 PCT/US2013/043052
  5. 7. The method of claim 4, wherein the subject is further administered a composition comprising at least one additional compound useful for treating the breathing control disorder or disease.
  6. 8. The method of claim 7, wherein the at least one additional 5 compound is selected from the group consisting of acetazolamide, almitrine, theophylline, caffeine, methyl progesterone, a serotinergic modulator, a cannabinoid and an ampakine.
  7. 9. The method of claim 4, wherein the formulation is administered in conjunction with the use of a mechanical ventilation device or positive airway 10 pressure device on the subject.
  8. 10. The method of claim 4, wherein the subject is a mammal.
  9. 11. The method of claim 10, wherein the mammal is a human.
  10. 12. The method of claim 4, wherein the formulation is administered to the subject by an inhalational, topical, oral, buccal, rectal, vaginal, intramuscular, 15 subcutaneous, transdermal, intrathecal, intraperitoneal, intrathoracic, intrapleural or intravenous route.
  11. 13. A method of preventing destabilization or stabilizing breathing rhythm in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical formulation comprising at least one 20 pharmaceutically acceptable carrier and at least one compound selected from the group consisting of: N-(2,4-bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine (CLXXII), N-(2,4,6-tris-n-propylamino)-[1,3,5]triazine (CLXXIII), N-(2,4,6-tris-n propylamino)-1,3-pyrimidine (CLXXIV), N-(2,4-bis-n-propylamino)-N-(6-i propylamino)-1,3-pyrimidine (CLXXV), a salt thereof, and any combinations thereof. 25 14. The method of claim 13, wherein the destabilization is associated with a breathing control disorder or disease selected from the group consisting of respiratory depression, sleep apnea, apnea of prematurity, obesity hypoventilation syndrome, primary alveolar hypoventilation syndrome, dyspnea, hypoxia, and hypercapnia. - 206 - WO 2013/181217 PCT/US2013/043052
  12. 15. The method of claim 14, wherein the respiratory depression is caused by an anesthetic, a sedative, an anxiolytic agent, a hypnotic agent, alcohol or a narcotic.
  13. 16. The method of claim 15, wherein the subject is further 5 administered a composition comprising at least one additional compound useful for treating the breathing control disorder or disease.
  14. 17. The method of claim 16, wherein the at least one additional compound is selected from the group consisting of acetazolamide, almitrine, theophylline, caffeine, methyl progesterone, a serotinergic modulator, a cannabinoid 10 and an ampakine.
  15. 18. The method of claim 13, wherein the formulation is administered in conjunction with the use of a mechanical ventilation device or positive airway pressure device on the subject.
  16. 19. The method of claim 13, wherein the subject is a mammal. 15 20. The method of claim 19, wherein the mammal is a human.
  17. 21. The method of claim 13, wherein the formulation is administered to the subject by an inhalational, topical, oral, buccal, rectal, vaginal, intramuscular, subcutaneous, transdermal, intrathecal, intraperitoneal, intrathoracic, intrapleural or intravenous route. 20 22. A composition comprising N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) or a salt thereof, wherein 4,6-bis-(n-propylamino)-1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 1% (w/w) with respect to (XXXV) or the salt thereof.
  18. 23. The composition of claim 22, wherein 4,6-bis-(n-propylamino) 25 1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 0.5% (w/w) with respect to (XXXV) or the salt thereof. - 207 - WO 2013/181217 PCT/US2013/043052
  19. 24. The composition of claim 23, wherein 4,6-bis-(n-propylamino) 1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 0.3% (w/w) with respect to (XXXV) or the salt thereof.
  20. 25. The composition of claim 24, wherein 4,6-bis-(n-propylamino) 5 1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 0.2% (w/w) with respect to (XXXV) or the salt thereof.
  21. 26. The composition of claim 25, wherein 4,6-bis-(n-propylamino) 1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 0.15% (w/w) with respect to (XXXV) or the salt thereof. 10 27. The composition of claim 26, wherein 4,6-bis-(n-propylamino) 1,3,5-triazin-2-ol (CLXXVIII) is present at a level equal to or lower than about 0.1% (w/w) with respect to (XXXV) or the salt thereof.
  22. 28. The composition of claim 27, wherein 4,6-bis-(n-propylamino) 1,3,5-triazin-2-ol (CLXXVIII) is essentially absent from the composition. 15 29. A composition comprising N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV) or a salt thereof, further comprising a buffer and a liquid carrier.
  23. 30. The composition of claim 29, wherein the salt of (XXXV) is the hydrogen sulfate salt (XXXVI). 20 31. The composition of claim 29, further comprising citric acid.
  24. 32. The composition of claim 29, wherein the pH of the composition is adjusted with a base.
  25. 33. The composition of claim 32, wherein the base comprises sodium hydroxide. 25 34. The composition of claim 29, wherein the liquid carrier comprises water. - 208 - WO 2013/181217 PCT/US2013/043052
  26. 35. The composition of claim 29, wherein the pH of the composition ranges from about 2.5 to about 6.
  27. 36. The composition of claim 35, wherein the pH of the composition ranges from about 2.5 to about 5. 5 37. The composition of claim 36, wherein the pH of the composition ranges from about 3 to about 4.
  28. 38. The composition of claim 29, wherein the concentration of (XXXV) or the salt thereof in the composition is about 1-10 mg/mL.
  29. 39. The composition of claim 38, wherein the concentration of 10 (XXXV) or the salt thereof in the composition is about 5-10 mg/mL.
  30. 40. The composition of claim 39, wherein the concentration of (XXXV) or the salt thereof in the composition is about 10 mg/mL.
  31. 41. The composition of claim 29, comprising less than 0.5% (w/w) (CLXXVIII) with respect to (XXXV). 15 42. The composition of claim 41, wherein less than 0.5% (w/w) of N-(4,6-Bis-n-propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) forms over a six-month period of storage of the composition at 2-8'C.
  32. 43. The composition of claim 42, wherein less than 0.5% (w/w) of N-(4,6-Bis-n-propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) 20 forms over a twelve-month period of storage of the composition at 2-8'C.
  33. 44. The composition of claim 43, wherein less than 0.5% (w/w) of N-(4,6-Bis-n-propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) forms over an eighteen-month period of storage of the composition at 2-8'C.
  34. 45. The composition of claim 44, wherein less than 0.5% (w/w) of 25 N-(4,6-Bis-n-propylamino)-[1,3,5]-triazin-2-ol (CLXXVIII) with respect to (XXXV) forms over a twenty-four-month period of storage of the composition at 2-8'C. - 209 - WO 2013/181217 PCT/US2013/043052
  35. 46. A crystalline free base of N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine (XXXV), Form A, with a XRPD spectrum as per Figure 22.
  36. 47. At least one crystalline salt of N-(4,6-Bis-n-propylamino 5 [1,3,5]triazin-2-yl)-N.O-dimethyl-hydroxylamine (XXXV) selected from the group consisting of: (i) crystalline hydrogen sulfate salt (XXXVI) Form A, with a XRPD spectrum as per Figure 14; (ii) crystalline hydrogen sulfate salt (XXXVI) comprising a mixture of Form A and 10 Form B, with a XRPD spectrum as per Figure 23; (iii) crystalline hydrogen sulfate salt (XXXVI) Form C, with a XRPD spectrum as per Figure 24; (iv) crystalline hydrogen sulfate salt (XXXVI) Form D, with a XRPD spectrum as per Figure 25; 15 (v) crystalline hydrogen sulfate salt (XXXVI) comprising a mixture of Form A and Form D, with a XRPD spectrum as per Figure 26; (vi) crystalline phosphoric acid salt (CLXXX) Form A, with a XRPD spectrum as per Figure 27; (vii) crystalline phosphoric acid salt (CLXXX) Form C, with a XRPD spectrum as 20 per Figure 28; (viii) crystalline phosphoric acid salt (CLXXX) comprising a mixture of Form A and Form B, with a XRPD spectrum as per Figure 29; (ix) crystalline phosphoric acid salt (CLXXX) comprising a mixture of Form C and Form D, with a XRPD spectrum as per Figure 30; 25 (x) crystalline phosphoric acid salt (CLXXX) comprising a mixture of Form C and Form E, with a XRPD spectrum as per Figure 31; and any mixtures thereof.
  37. 48. A salt of N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl) hydroxylamine (XXXV), comprising approximately one mole equivalent of sulfuric 30 acid.
  38. 49. The salt of claim 48, further comprising one mole equivalent of water. - 210 - WO 2013/181217 PCT/US2013/043052
  39. 50. A salt of N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N.O dimethyl-hydroxylamine (XXXV), comprising at least one mole equivalent of phosphoric acid.
  40. 51. The salt of claim 50, comprising about one mole equivalent of 5 phosphoric acid.
  41. 52. A method of reducing or minimizing the open channel fraction of the potassium maxi-K or BK channel in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least 10 one compound of formula (I): R 1 X-R 2 N b 2 b R3 N R5 R4 H (I), wherein R and R2 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, 15 heteroarylalkyl, substituted heteroarylalkyl, heteroaryl or substituted heteroaryl; or R1 and R 2 combine as to form a biradical selected from the group consisting of 3 hydroxy-pentane-1,5-diyl, 6-hydroxy-cycloheptane-1,4-diyl, propane- 1,3-diyl, butane-1,4-diyl and pentane- 1,5-diyl; R3 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, 20 substituted alkenyl, -NR 1 R 2 , -C(O)OR 1 , acyl, or aryl; R is H, alkyl, or substituted alkyl; R 5 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -OR', -NR 1 R 2 , -C(O)OR 1 , acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic; or R3 and Ri combine 25 as to form a biradical selected from the group consisting of 3,6,9-trioxa-undecane 1,1 1-diyl and 3,6-dioxa-octane-1,8-diyl; R6 is H, alkyl, substituted alkyl or alkenyl; X is a bond, 0 or NR 4 ; and, -211- WO 2013/181217 PCT/US2013/043052 Y is N, CR or C; wherein: if Y is N or CR 6 , then bond b 1 is nil and: (i) Z is H, bond b 2 is a single bond, and A is CH; or, (ii) Z is nil, bond b 2 is nil, and A is a single bond; 5 and, if Y is C, then bond bl is a single bond, and: (i) Z is CH2, bond b2 is a single bond, and A is CH; or, (ii) Z is CH, bond b2 is a double bond, and A is C; or a salt thereof. 10 53. The method of claim 52, wherein the compound of formula (I) is selected from the group consisting of: N-(4,6-Bis-methylamino-[1,3,5]triazin-2 yl)-N,O-dimethyl-hydroxylamine, N-(4,6-Bis-ethylamino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine, N-(4-Cyclopropylmethyl)-N-(6-n-propylamino) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4-Ethylamino)-N-(6-n 15 propylamino)-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2 methylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6 (2,2-dimethylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N (Bis-4,6-(2,2-dimethylpropylamino))[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl 20 hydroxylamine, N-(4-(Methoxy(methyl)amino)-6-(n-propylamino)-1,3,5-triazin-2 yl)propionamide, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-methyl hydroxylamine, O-Allyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl) hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine, 6 (Methoxy(methyl)amino)-N 2 -propyl-1,3,5-triazine-2,4-diamine, N-(4,6-Bis-n 25 propylamino-[1,3,5]triazin-2-yl)-N-methyl-hydroxylamine, O-Benzyl-N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N-methyl-hydroxylamine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N-isopropyl-hydroxylamine, 6-[1,2]Oxazinan-2-yl N,N'-dipropyl-[1,3,5]triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2 yl)-O-isopropyl-N-methyl-hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino 30 [1,3,5]triazin-2-yl)-N-ethyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin 2-yl)-O-isopropyl-hydroxylamine, 6-((Benzyloxy)(isopropyl) amino)-N 2 ,N 4 -dipropyl 1,3,5-triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-O isopropyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl - 212 - WO 2013/181217 PCT/US2013/043052 N-methyl-hydroxylamine, 6-(Methyl(thiophen-2-ylmethoxy)amino)-N 2 ,N 4 -dipropyl 1,3,5-triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O cyclopropylmethyl-N-methyl-hydroxylamine, N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-O-ethyl-N-methyl-hydroxylamine, N-(4,6-Bis-n-propylamino 5 [1,3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl)-hydroxylamine, 4-N-(2 Dimethylaminoethyl)amino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, 4-N-(3-(1-N-Methylimidazol-2-yl)-propyl)-amino-6-N-(n propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4-N-(1-N Methylimidazol-2-yl)-methylamino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O 10 dimethyl-hydroxylamine, 4,6-Bis-(N-(2-dimethylaminoethyl)amino)-[1,3,5]triazin-2 yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-(N-(pyridin-4-yl-methyl)amino) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-[N-(3-methoxy-n propyl)amino]-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-[N (tetrahydropyran-4-yl-methyl)amino]-[1,3,5]triazin-2-yl)-N,O-dimethyl 15 hydroxylamine, N-(5,8,11 -Trioxa-2,14,16,18,19-pentaazabicyclo[13.3.1]nonadeca 1(18),15(19),16(17)-trien-17-yl)-N,O-dimethylhydroxylamine, 2,6-Bis-(N-n propylamino)-[ 1,3 ]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N',N'-dimethylhydrazine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N-methyl-N'-methylhydrazine, 2-(n-Propyl)amino 20 4-(i-propylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4 dimethylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4 methylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4-(i propyl)amino-7-i-propyl-pyrrolidino[2,3-d]pyrimidine, 2,4-Bis-(n-propyl)amino-7H pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4-(4-hydroxypiperidin-1-yl)-7 25 methyl-pyrrolidino[2,3-d]pyrimidine, 8-(7-Methyl-2-(n-propylamino) pyrrolidino[2,3 -d]pyrimidin-4-yl)-8-azabicyclo[3.2.1 ]octan-3 -ol, N-(2-Propylamino 7H-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-(Propen-2 yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2 (Propen-2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine, 30 N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O methyl-hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4 - 213 - WO 2013/181217 PCT/US2013/043052 yl)-hydrazine, N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine, N (2,4,6-Tris-n-propylamino)-[1,3,5]triazine, N-(2,4,6-Tris-n-propylamino)-1,3 pyrimidine, N-(2,4-Bis-n-propylamino)-N-(6-i-propylamino)-1,3-pyrimidine, a salt thereof and mixtures thereof. 5 54. The method of claim 52, wherein the subject is a mammal.
  42. 55. The method of claim 54, wherein the mammal is human.
  43. 56. A method of inhibiting the TASK-I (KCNK3) channel in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical formulation comprising at least one 10 pharmaceutically acceptable carrier and at least one compound of formula (I): R 1 X-R 2 N b 2 Z-bY N R ' N R5 R4 H (I), wherein R and R2 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, 15 heteroarylalkyl, substituted heteroarylalkyl, heteroaryl or substituted heteroaryl; or R1 and R2 combine as to form a biradical selected from the group consisting of 3 hydroxy-pentane-1,5-diyl, 6-hydroxy-cycloheptane-1,4-diyl, propane- 1,3-diyl, butane-1,4-diyl and pentane- 1,5-diyl; R 3 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, 20 substituted alkenyl, -NR 1 R 2 , -C(O)OR 1 , acyl, or aryl; R 4 is H, alkyl, or substituted alkyl; R 5 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -OR', -NR 1 R 2 , -C(O)OR 1 , acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic; or R 3 and R combine 25 as to form a biradical selected from the group consisting of 3,6,9-trioxa-undecane 1,1 1-diyl and 3,6-dioxa-octane-1,8-diyl; R 6 is H, alkyl, substituted alkyl or alkenyl; - 214 - WO 2013/181217 PCT/US2013/043052 X is a bond, 0 or NR 4 ; and, 66 Y isN, CRo or C; wherein: if Y is N or CR, then bond b' is nil and: (i) Z is H, bond b 2 is a single bond, and A is CH; or, 5 (ii) Z is nil, bond b 2 is nil, and A is a single bond; and, if Y is C, then bond bl is a single bond, and: (i) Z is CH2, bond b2 is a single bond, and A is CH; or, (ii) Z is CH, bond b2 is a double bond, and A is C; 10 or a salt thereof.
  44. 57. The method of claim 56, wherein the compound of formula (I) is selected from the group consisting of: N-(4,6-Bis-methylamino-[1,3,5]triazin-2 yl)-N,O-dimethyl-hydroxylamine, N-(4,6-Bis-ethylamino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine, N-(4-Cyclopropylmethyl)-N-(6-n-propylamino) 15 [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4-Ethylamino)-N-(6-n propylamino)-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2 methylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6 (2,2-dimethylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N (Bis-4,6-(2,2-dimethylpropylamino))[1,3,5]triazin-2-yl)-N,O-dimethyl 20 hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, N-(4-(Methoxy(methyl)amino)-6-(n-propylamino)-1,3,5-triazin-2 yl)propionamide, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-methyl hydroxylamine, O-Allyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl) hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine, 6 25 (Methoxy(methyl)amino)-N 2 -propyl-1,3,5-triazine-2,4-diamine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N-methyl-hydroxylamine, O-Benzyl-N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N-methyl-hydroxylamine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N-isopropyl-hydroxylamine, 6-[1,2]Oxazinan-2-yl N,N'-dipropyl-[1,3,5]triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2 30 yl)-O-isopropyl-N-methyl-hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N-ethyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin 2-yl)-O-isopropyl-hydroxylamine, 6-((Benzyloxy)(isopropyl) amino)-N 2 ,N 4 -dipropyl 1,3,5-triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-O - 215 - WO 2013/181217 PCT/US2013/043052 isopropyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl N-methyl-hydroxylamine, 6-(Methyl(thiophen-2-ylmethoxy)amino)-N 2 ,N 4 -dipropyl 1,3,5-triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O cyclopropylmethyl-N-methyl-hydroxylamine, N-(4,6-Bis-n-propylamino 5 [1,3,5]triazin-2-yl)-O-ethyl-N-methyl-hydroxylamine, N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl)-hydroxylamine, 4-N-(2 Dimethylaminoethyl)amino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, 4-N-(3-(1-N-Methylimidazol-2-yl)-propyl)-amino-6-N-(n propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4-N-(1-N 10 Methylimidazol-2-yl)-methylamino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine, 4,6-Bis-(N-(2-dimethylaminoethyl)amino)-[1,3,5]triazin-2 yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-(N-(pyridin-4-yl-methyl)amino) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-[N-(3-methoxy-n propyl)amino]-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-[N 15 (tetrahydropyran-4-yl-methyl)amino]-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, N-(5,8,11 -Trioxa-2,14,16,18,19-pentaazabicyclo[13.3.1]nonadeca 1(18),15(19),16(17)-trien-17-yl)-N,O-dimethylhydroxylamine, 2,6-Bis-(N-n propylamino)-[ 1,3 ]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N',N'-dimethylhydrazine, N-(4,6-Bis-n 20 propylamino-[1,3,5]triazin-2-yl)-N-methyl-N'-methylhydrazine, 2-(n-Propyl)amino 4-(i-propylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4 dimethylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4 methylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4-(i propyl)amino-7-i-propyl-pyrrolidino[2,3-d]pyrimidine, 2,4-Bis-(n-propyl)amino-7H 25 pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4-(4-hydroxypiperidin-1-yl)-7 methyl-pyrrolidino[2,3-d]pyrimidine, 8-(7-Methyl-2-(n-propylamino) pyrrolidino[2,3 -d]pyrimidin-4-yl)-8-azabicyclo[3.2.1 ]octan-3 -ol, N-(2-Propylamino 7H-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-(Propen-2 yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2 30 (Propen-2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O methyl-hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) - 216 - WO 2013/181217 PCT/US2013/043052 hydrazine, N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4 yl)-hydrazine, N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine, N (2,4,6-Tris-n-propylamino)-[1,3,5]triazine, N-(2,4,6-Tris-n-propylamino)-1,3 pyrimidine, N-(2,4-Bis-n-propylamino)-N-(6-i-propylamino)-1,3-pyrimidine, a salt 5 thereof and mixtures thereof.
  45. 58. The method of claim 57, wherein the compound of formula (I) is selected from the group consisting of: N-(4,6-Bis-n-propylamino-1,3,5]triazin-2 yl)-O-methyl-hydroxylamine; N-(4,6-Bis-n-propylamino-1,3,5]triazin-2-yl)-N',N' dimethylhydrazine; N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine; 10 N-[(2,6-Bis-n-propylamino)-pyrimidin-4-yl]-N,O-dimethyl-hydroxylamine; N,N Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-hydrazine; a salt thereof and mixtures thereof.
  46. 59. The method of claim 56, wherein the subject is a mammal.
  47. 60. The method of claim 59, wherein the mammal is human. 15 61. A method of increasing minute ventilation in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical formulation comprising at least one pharmaceutically acceptable carrier and at least one compound of formula (I): R 1 X-R 2 N A N R5 R4 H (I), wherein 20 R and R2 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroarylalkyl, substituted heteroarylalkyl, heteroaryl or substituted heteroaryl; or R1 and R2 combine as to form a biradical selected from the group consisting of 3 25 hydroxy-pentane-1,5-diyl, 6-hydroxy-cycloheptane-1,4-diyl, propane- 1,3-diyl, butane-1,4-diyl and pentane- 1,5-diyl; - 217 - WO 2013/181217 PCT/US2013/043052 R 3 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, -NRR 2 , -C(O)OR', acyl, or aryl; R 4 is H, alkyl, or substituted alkyl; R 5 is H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, 5 substituted alkenyl, -OR', -NRR 2 , -C(O)OR 1 , acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, or substituted heterocyclic; or R 3 and R combine as to form a biradical selected from the group consisting of 3,6,9-trioxa-undecane 1,1 1-diyl and 3,6-dioxa-octane-1,8-diyl; R 6 is H, alkyl, substituted alkyl or alkenyl; 10 X is a bond, 0 or NR 4 ; and, Y is N, CR 6 or C; wherein: if Y is N or CR, then bond bl is nil and: (i) Z is H, bond b2 is a single bond, and A is CH; or, (ii) Z is nil, bond b2 is nil, and A is a single bond; 15 and, if Y is C, then bond bl is a single bond, and: (i) Z is CH 2 , bond b 2 is a single bond, and A is CH; or, (ii) Z is CH, bond b 2 is a double bond, and A is C; or a salt thereof. 20 62. The method of claim 61, wherein the compound of formula (I) is selected from the group consisting of: N-(4,6-Bis-methylamino-[1,3,5]triazin-2 yl)-N,O-dimethyl-hydroxylamine, N-(4,6-Bis-ethylamino-[1,3,5]triazin-2-yl)-N,O dimethyl-hydroxylamine, N-(4-Cyclopropylmethyl)-N-(6-n-propylamino) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(4-Ethylamino)-N-(6-n 25 propylamino)-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6-(2 methylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N-(Bis-4,6 (2,2-dimethylpropylamino)) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, N (Bis-4,6-(2,2-dimethylpropylamino))[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl 30 hydroxylamine, N-(4-(Methoxy(methyl)amino)-6-(n-propylamino)-1,3,5-triazin-2 yl)propionamide, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-methyl hydroxylamine, O-Allyl-N-(4,6-bis-n-propylamino-[1,3,5]triazin-2-yl) hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-hydroxylamine, 6 - 218 - WO 2013/181217 PCT/US2013/043052 (Methoxy(methyl)amino)-N 2 -propyl-1,3,5-triazine-2,4-diamine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N-methyl-hydroxylamine, O-Benzyl-N-(4,6-bis-n propylamino-[1,3,5]triazin-2-yl)-N-methyl-hydroxylamine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N-isopropyl-hydroxylamine, 6-[1,2]Oxazinan-2-yl 5 N,N'-dipropyl-[1,3,5]triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2 yl)-O-isopropyl-N-methyl-hydroxylamine, O-Benzyl-N-(4,6-bis-n-propylamino [1,3,5]triazin-2-yl)-N-ethyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin 2-yl)-O-isopropyl-hydroxylamine, 6-((Benzyloxy)(isopropyl) amino)-N 2 ,N 4 -dipropyl 1,3,5-triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N-ethyl-O 10 isopropyl-hydroxylamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O-isobutyl N-methyl-hydroxylamine, 6-(Methyl(thiophen-2-ylmethoxy)amino)-N 2 ,N 4 -dipropyl 1,3,5-triazine-2,4-diamine, N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-O cyclopropylmethyl-N-methyl-hydroxylamine, N-(4,6-Bis-n-propylamino [1,3,5]triazin-2-yl)-O-ethyl-N-methyl-hydroxylamine, N-(4,6-Bis-n-propylamino 15 [1,3,5]triazin-2-yl)-O-(2,2-difluoro-ethyl)-hydroxylamine, 4-N-(2 Dimethylaminoethyl)amino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl hydroxylamine, 4-N-(3-(1-N-Methylimidazol-2-yl)-propyl)-amino-6-N-(n propyl)amino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4-N-(1-N Methylimidazol-2-yl)-methylamino-6-N-(n-propyl)amino-[1,3,5]triazin-2-yl)-N,O 20 dimethyl-hydroxylamine, 4,6-Bis-(N-(2-dimethylaminoethyl)amino)-[1,3,5]triazin-2 yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-(N-(pyridin-4-yl-methyl)amino) [1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-[N-(3-methoxy-n propyl)amino]-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, 4,6-Bis-[N (tetrahydropyran-4-yl-methyl)amino]-[1,3,5]triazin-2-yl)-N,O-dimethyl 25 hydroxylamine, N-(5,8,11 -Trioxa-2,14,16,18,19-pentaazabicyclo[13.3.1]nonadeca 1(18),15(19),16(17)-trien-17-yl)-N,O-dimethylhydroxylamine, 2,6-Bis-(N-n propylamino)-[ 1,3 ]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N',N'-dimethylhydrazine, N-(4,6-Bis-n propylamino-[1,3,5]triazin-2-yl)-N-methyl-N'-methylhydrazine, 2-(n-Propyl)amino 30 4-(i-propylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4 dimethylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4 methylamino-7-methyl-pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4-(i propyl)amino-7-i-propyl-pyrrolidino[2,3-d]pyrimidine, 2,4-Bis-(n-propyl)amino-7H pyrrolidino[2,3-d]pyrimidine, 2-(n-Propyl)amino-4-(4-hydroxypiperidin-1-yl)-7 - 219 - WO 2013/181217 PCT/US2013/043052 methyl-pyrrolidino[2,3-d]pyrimidine, 8-(7-Methyl-2-(n-propylamino) pyrrolidino[2,3 -d]pyrimidin-4-yl)-8-azabicyclo[3.2.1 ]octan-3 -ol, N-(2-Propylamino 7H-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2-(Propen-2 yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl-hydroxylamine, N-(2 5 (Propen-2-yl)amino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O-methyl-hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-N,O-dimethyl hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl)-O methyl-hydroxylamine, N-(2-n-Propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) hydrazine, N-Methyl-N-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4-yl) 10 hydrazine, N,N-Dimethyl-N'-(2-n-propylamino-7-methyl-pyrrolo[2,3d]pyrimidin-4 yl)-hydrazine, N-(2,4-Bis-n-propylamino)-N-(6-methylamino)-[1,3,5]triazine, N (2,4,6-Tris-n-propylamino)-[1,3,5]triazine, N-(2,4,6-Tris-n-propylamino)-1,3 pyrimidine, N-(2,4-Bis-n-propylamino)-N-(6-i-propylamino)-1,3-pyrimidine, a salt thereof and mixtures thereof. 15 63. The method of claim 62, wherein the compound of formula (I) is N-(4,6-Bis-n-propylamino-[1,3,5]triazin-2-yl)-N,O-dimethyl-hydroxylamine, or a salt thereof.
  48. 64. The method of claim 61, wherein the pharmaceutical formulation is administered via intravenous infusion. 20 65. The method of claim 64, wherein the infusion dose of the pharmaceutical formulation is at least about 0.016 mg/kg/min.
  49. 66. The method of claim 65, wherein the infusion dose of the pharmaceutical formulation is about 0.0 16 mg/kg/min.
  50. 67. The method of claim 61, wherein the infusion dose of the 25 pharmaceutical composition produce plasma concentrations of at least about 726 ng/mL in the subject.
  51. 68. The method of claim 61, wherein the subject is a mammal.
  52. 69. The method of claim 68, wherein the mammal is human. - 220 -
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