Sommaire du brevet 3209557 - Base de données sur les brevets canadiens (2024)

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CARDIAC SARCOMERE INHIBITORS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Patent Application
No. 63/156,853, filed March 4, 2021, the disclosure of which is hereby
incorporated herein
by reference in its entirety.
FIELD
[0002] Provided herein are heterocyclic compounds, pharmaceutical
compositions
comprising such compounds, and methods of treating various cardiac diseases
and conditions
with such compounds.
BACKGROUND
[0003] The disclosure relates to certain chemical entities that
selectively modulate
the cardiac sarcomere, and specifically to certain chemical entities,
pharmaceutical
compositions and methods for treating various cardiac diseases and conditions.
[0004] The cardiac sarcomere is composed of a network of
contractile and structural
proteins that regulate cardiac muscle function. The components of the cardiac
sarcomere present targets for the treatment of various cardiac diseases and
conditions, for
example by increasing contractility or facilitating complete relaxation to
modulate systolic
and diastolic function, respectively. The force and speed of cardiac muscle
contraction is a
major determinant of organ function and is modulated by the cyclical
interactions of actin and
myosin. Regulation of actin and myosin binding is determined by a network of
myofilament
regulatory proteins and the level of intracellular Ca2+. The troponin complex
and tropomyosin
are thin filament proteins which govern the availability of actin binding
sites, and the
essential and regulatory light chains, and myosin binding protein C modulate
the position and
mechanical properties of myosin.
[0005] Abnormalities in the cardiac sarcomere have been identified
as the driving cause
for a variety of cardiac diseases and conditions, such as hypertrophic
cardiomyopathy (HCM)
and heart failure with preserved ejection fraction (HFpEF). Mutations in the
proteins of the
sarcomere, cause disease by rendering the cardiac muscle either 'hyper' or
'hypo' contractile.
Modulators of the cardiac sarcomere can be used to rebalance contractility and
stop or reverse
the course of disease.
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[0006] Current agents that target the cardiac sarcomere, such as
inotropes (drugs that
increase the contractile ability of the heart) are poorly selective for
cardiac tissue, which leads
to recognized adverse effects that limit their use. These adverse effects
include cell damage
caused by an increased rate of energy expenditure, exacerbation of relaxation
abnormalities,
and potential arrhythmogenic side effects that may result from increased
cytosolic Ca2+ and
cyclic AMP concentrations in the inotropically stimulated myocardium. Given
the
limitations of current agents, new approaches are needed to improve cardiac
function in HCM
and HFpEF.
[0007] There remains a great need for agents that exploit new
mechanisms of action and
which may have better outcomes in terms of relief of symptoms, safety, and
patient mortality,
both short-term and long-term. New agents with an improved therapeutic index
over current
agents will provide a means to achieve these clinical outcomes. The
selectivity of agents
directed at the cardiac sarcomere (for example, by targeting cardiac myosin)
has been
identified as an important means to achieve this improved therapeutic index.
The present
disclosure provides such agents (particularly cardiac sarcomere inhibitors)
and methods for
their use. These agents are allosteric inhibitors of cardiac myosin. Benefits
of these
compounds include a wider therapeutic index, less impact on cardiac
relaxation, better
pharmaco*kinetics, and better safety. Agents of the present disclosure are also
useful in that
they may allow for once-daily dosing for the methods of use described herein.
[0008] The present disclosure provides chemical entities,
pharmaceutical compositions
and methods for the treatment of heart failure including HCM and HFpEF. The
compositions
are inhibitors of the cardiac sarcomere, for example, inhibitors of cardiac
myosin.
BRIEF SUMMARY
[0009] In one aspect, provided is a compound of Formula (I):
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R1
R2
0
,
R'
R4
0 (I),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing, wherein:
R1 is halo or Ci_6haloalkyl;
R2 is H, halo, or Ci 6alkyl;
R3 is:
(i) cyclohexyl, wherein the cyclohexyl is optionally substituted with one
or more
independently selected Ci_6alky1 or C1_6haloalkyl substituents, or
(ii) Ci 6 alkyl; and
R4 is:
(i) -C(0)H,
(ii) -C(0)NH2,
õA-
(iii) , or
N
(iv)
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provided that:
N
(I) when R3 is Ct_6alkyl, then R4 is
(2) when R3 is iso-propyl, then the total number of halo atoms in R1 and R2 is
at least
two, and
(3) when R3 is cyclohexyl substituted with one or more independently selected
C1_
6haloalkyl substituents, then R4 is other than -C(0)H.
[0010] In one aspect, provided herein is a compound of Formula (I-
A):
R1
R2
0
,
R'
0
(I-A),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing, wherein R1. R2. and R3 are as described herein for a compound of
Formula (I).
[0011] In one aspect, provided herein is a compound of Formula (I-
B):
R1
R2, 0
3
0
NH2 (I-B),
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or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing, wherein RI, R2, and R.3 are as described herein for a compound of
Formula (I).
[0012] In one aspect, provided herein is a compound of Formula (1-
C):
R1
R2, 0
,
0
(LC),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing, wherein RI, R2. and R3 are as described herein for a compound of
Formula (I).
[0013] In one aspect, provided herein is a compound of Formula (I-
D):
R1
0
3
0
N=.N
(I-D),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing, wherein R1, R2, and R3 are as described herein for a compound of
Formula (I).
[0014] Provided in some aspects are compounds selected from the
group consisting of
compounds of Table 1, or stereoisomer or tautomer thereof, or a
pharmaceutically acceptable
salt of any of the foregoing.
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[0015] Provided in some aspects is a pharmaceutical composition,
comprising (i) a
compound of Formula (I), or any variation or embodiment thereof, or a
stereoisomer or
tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing, and (ii) one or
more pharmaceutically acceptable excipients.
[0016] Provided in some aspects are methods of treating heart
disease in a subject in need
thereof, the method comprising administering to the subject a compound of
Formula (I), or
any variation thereof, or stereoisomer or tautomer thereof, or a
pharmaceutically acceptable
salt of any of the foregoing, or a pharmaceutical composition containing a
compound of
Formula (I), or any variation thereof or stereoisomer or tautomer thereof, or
a
pharmaceutically acceptable salt of any of the foregoing. In some embodiments,
the heart
disease is hypertrophic cardiomyopathy (HCM). In some embodiments, the HCM is
obstructive or nonobstructive or is caused by sarcomeric and/or non-sarcomeric
mutations.
In some embodiments, the heart disease is heart failure with preserved
ejection fraction
(HFpEF). In some embodiments, the heart disease is selected from the group
consisting of
diastolic dysfunction, primary or secondary restrictive cardiomyopathy,
myocardial
infarction, angina pectoris, and left ventricular outflow tract obstruction.
In some
embodiments, the heart disease is hypertensive heart disease, congenital heart
disease, cardiac
ischcmia, coronary heart disease, diabetic heart disease, congestive heart
failure, right heart
failure, cardiorenal syndrome, or infiltrative cardiomyopathy. In some
embodiments, the
heart disease is a condition that is or is related to cardiac senescence
and/or diastolic
dysfunction due to aging. In some embodiments, the heart disease is a
condition that is or is
related to left ventricular hypertrophy and/or concentric left ventricular
remodeling.
[0017] Provided in other aspects are methods of treating a disease
or condition associated
with HCM in a subject in need thereof, wherein the method comprises
administering to the
subject a compound of Formula (I), or any variation thereof, or stercoisomer
or tautomer
thereof, or a pharmaceutically acceptable salt of any of the foregoing, or a
pharmaceutical
composition containing a compound of Formula (I), or any variation thereof or
stereoisomer
or tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing. In some
embodiments, the disease or condition is selected from the group consisting of
Fabry's
Disease, Danon Disease, mitochondrial cardiomyopathies, and Noonan Syndrome.
[0018] Provided in some aspects are methods of treating a disease
or condition that is
associated with secondary left ventricular wall thickening in a subject in
need thereof,
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wherein the method comprises administering to the subject a compound of
Formula (I), or
any variation thereof, or stereoisomer or tautomer thereof, or a
pharmaceutically acceptable
salt of any of the foregoing, or a pharmaceutical composition containing a
compound of
Formula (I), or any variation thereof or stereoisomer or tautomer thereof, or
a
pharmaceutically acceptable salt of any of the foregoing. In some embodiments,
the disease
or condition is selected from the group consisting of hypertension, valvular
heart diseases
(such as aortic stenosis and Mitral valve regurgitation), metabolic syndromes
(such as
diabetes and obesity), end stage renal disease, scleroderma. sleep apnea,
amyloidosis, Fabry's
disease, Friedreich Ataxia, Danon disease, Noonan syndrome, and Pompe disease.
[0019] Provided in other aspects are methods of treating a disease
or condition that is
associated with small left ventricular cavity and cavity obliteration,
hyperdynamic left
ventricular contraction, myocardial ischemia, or cardiac fibrosis. Also
provided are methods
of treating muscular dystrophies (e.g., duch*enne muscular dystrophy) or
glycogen storage
diseases.
[0020] Also provided are methods of inhibiting the cardiac
sarcomere, wherein the
method involves contacting the cardiac sarcomere with a compound of Formula
(I), or any
variation thereof, or stereoisomer or tautomer thereof, or a pharmaceutically
acceptable salt
of any of the foregoing, or a pharmaceutical composition containing a compound
of Formula
(I), or any variation thereof or stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing.
DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows an experimental X-ray powder diffraction (XRPD)
pattern of an
amorphous form of 5-(3,4-difluorobenzy1)-8-((1r,4r)-4-methylcyclohexyl)-6,9-
dioxo-2,5,8-
triazaspiro[3.5]nonane-2-carbaldehyde.
DETAILED DESCRIPTION
Definitions
[0022] As used in the present specification, the following words
and phrases are
generally intended to have the meanings as set forth below, except to the
extent that the
context in which they are used indicates otherwise.
[0023] Throughout this application, unless the context indicates
otherwise, references to a
compound of Formula (I) includes all subgroups of Formula (I) defined herein,
such as
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Formula (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1),
including all
substructures, subgenera, preferences, embodiments, examples and particular
compounds
defined and/or described herein. References to a compound of Formula (I) and
subgroups
thereof, such as Formula (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1). (I-C2),
(I-D), or (I-D1),
include ionic forms, polymorphs, pseudopolymorphs, amorphous forms, solvates,
co-crystals,
chelates, isomers, tautomers, oxides (e.g., N-oxides, S-oxides), esters,
prodrugs, isotopes
and/or protected forms thereof. In some embodiments, references to a compound
of Formula
(I) and subgroups thereof, such as Formula (I-A), (I-A1), (I-B), (I-B1), (I-
C), (I-C1), (I-C2),
(I-D), or (I-D1), include polymorphs, solvates, co-crystals, isomers,
tautomers and/or oxides
thereof. In some embodiments, references to a compound of Formula (I) and
subgroups
thereof, such as Formula (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1). (I-C2),
(I-D), or (I-D1),
include polymorphs, solvates, and/or co-crystals thereof. In some embodiments,
references
to a compound of Formula (I) and subgroups thereof, such as Formula (I-A), (I-
A1), (I-B), (I-
B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), include isomers, tautomers
and/or oxides thereof.
In some embodiments, references to a compound of Formula (I) and subgroups
thereof, such
as Formula (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-
D1), include solvates
thereof.
[0024] -Alkyl" encompasses straight and branched carbon chains
having the indicated
number of carbon atoms, for example, from 1 to 20 carbon atoms, or 1 to 8
carbon atoms, or
1 to 6 carbon atoms. For example, C1_6 alkyl encompasses both straight and
branched chain
alkyl of from 1 to 6 carbon atoms. When an alkyl residue having a specific
number of
carbons is named, all branched and straight chain versions having that number
of carbons are
intended to be encompassed; thus, for example, "propyl" includes n-propyl and
isopropyl;
and "butyl" includes n-butyl, sec-butyl, isobutyl and t-butyl. Examples of
alkyl groups
include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl,
sec-butyl, tert-butyl,
pentyl, 2-pentyl, 3-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and
3-methylpentyl.
[0025] When a range of values is given (e.g., C1_6 alkyl), each
value within the range as
well as all intervening ranges are included. For example, "Ci_6 alkyl"
includes C1, C2, C3,
-37 C4,-
C5, C6, C1-6, C2-6, C3-6, C4-6, C5-6, C1-5, C2-5, C3-5, C4-5, C1-4, C24, C3-4,
C1-3, C2-3, and C1_2 alkyl.
[0026] "Halogen" or "halo" refers to fluoro, chloro, bromo, or
iodo.
[0027] "Haloalkyl" refers to an alkyl moiety, as defined herein,
wherein one or more of
the hydrogen atoms in the alkyl moiety are replaced by one or more
independently selected
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halo moieties. Examples of haloalkyl moieties include, but are not limited to,
-CH2F, -CHF2,
-CF3, -CH2-CH2C1, -CH2-CHC12, -CH/-CC13, and -CHF-CH2C1.
[0028] -Cyclohexyl" refers to a moiety.
[0029] Unless otherwise indicated, compounds disclosed and/or
described herein include
all possible enantiomers, diastereomers, meso isomers and other stereoisomeric
forms,
including racemic mixtures, optically pure forms and intermediate mixtures
thereof.
Enantiomers, diastereomers, meso isomers, and other stereoisomeric forms can
be prepared
using chiral synthons or chiral reagents, or resolved using conventional
techniques. Unless
specified otherwise, when the compounds disclosed and/or described herein
contain olefinic
double bonds or other centers of geometric asymmetry, it is intended that the
compounds
include both E and Z isomers. When the compounds described herein contain
moieties
capable of tautomerization, and unless specified otherwise, it is intended
that the compounds
include all possible tautomers.
[0030] "Protecting group" has the meaning conventionally associated
with it in organic
synthesis, i.e., a group that selectively blocks one or more reactive sites in
a multifunctional
compound such that a chemical reaction can be carried out selectively on
another unprotected
reactive site, and such that the group can readily be removed after the
selective reaction is
complete. A variety of protecting groups are disclosed, for example, in T.H.
Greene and P.
G. M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley
& Sons,
New York (1999). For example, a -hydroxy protected form" contains at least one
hydroxyl
group protected with a hydroxyl protecting group. Likewise, amines and other
reactive
groups may similarly be protected.
[0031] The term "pharmaceutically acceptable salt" refers to a salt
of any of the
compounds herein which are known to be non-toxic and are commonly used in the
pharmaceutical literature. In some embodiments, the pharmaceutically
acceptable salt of a
compound retains the biological effectiveness of the compounds described
herein and are not
biologically or otherwise undesirable. Examples of pharmaceutically acceptable
salts can be
found in Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences,
January 1977, 66(1),
1-19. Pharmaceutically acceptable acid addition salts can be formed with
inorganic acids and
organic acids. Inorganic acids from which salts can be derived include, for
example,
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and
phosphoric acid. Organic
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acids from which salts can be derived include, for example, acetic acid,
propionic acid,
glycolic acid, pyruvic acid, lactic acid, oxalic acid, malic acid, maleic
acid, malonic acid,
succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic
acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethylsulfonic acid,
p-
toluenesulfonic acid, stearic acid and salicylic acid. Pharmaceutically
acceptable base
addition salts can be formed with inorganic and organic bases. Inorganic bases
from which
salts can be derived include, for example, sodium, potassium, lithium,
ammonium, calcium,
magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from
which salts
can be derived include, for example, primary, secondary, and tertiary amines;
substituted
amines including naturally occurring substituted amines; cyclic amines; and
basic ion
exchange resins. Examples of organic bases include isopropylamine,
trimethylamine,
diethylamine, triethylamine, tripropylamine, and ethanolamine. In some
embodiments, the
pharmaceutically acceptable base addition salt is selected from ammonium,
potassium,
sodium, calcium, and magnesium salts.
[0032] If the compound described herein is obtained as an acid
addition salt, the free base
can be obtained by basifying a solution of the acid salt. Conversely, if the
compound is a free
base, an addition salt, particularly a pharmaceutically acceptable addition
salt, may be
produced by dissolving the free base in a suitable organic solvent and
treating the solution
with an acid, in accordance with conventional procedures for preparing acid
addition salts
from base compounds (see, e.g., Berge et al., Pharmaceutical Salts, J.
Pharmaceutical
Sciences, January 1977, 66(1), 1-19). Those skilled in the art will recognize
various synthetic
methodologies that may be used to prepare pharmaceutically acceptable addition
salts.
[0033] A "solvate" is formed by the interaction of a solvent and a
compound. Suitable
solvents include, for example, water and alcohols (e.g., ethanol). Solvates
include hydrates
having any ratio of compound to water, such as monohydrates, dihydrates and
hemi-hydrates.
[0034] The term "substituted" means that the specified group or
moiety bears one or
more substituents, e.g., alkyl substituents or haloalkyl substituents. The
term "unsubstituted"
means that the specified group bears no substituents. Where the term
"substituted" is used to
describe a structural system, the substitution is meant to occur at any
valency-allowed
position on the system. When a group or moiety bears more than one
substituent, it is
understood that the substituents may be the same or different from one
another. In some
embodiments, a substituted group or moiety bears from one to five
substituents. In some
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embodiments, a substituted group or moiety bears one substituent. In some
embodiments, a
substituted group or moiety bears two substituents. In some embodiments, a
substituted
group or moiety bears three substituents. In some embodiments, a substituted
group or
moiety bears four substituents. In some embodiments, a substituted group or
moiety bears
five substituents.
[0035] By "optional" or "optionally", it is meant that the
subsequently described event or
circ*mstance may or may not occur, and that the description includes instances
where the
event or circ*mstance occurs and instances in which it does not. For example,
"optionally
substituted cyclohexyl" encompasses both unsubstitued cyclohexyl and
substituted
cyclohexyl, as defined herein. It will be understood by those skilled in the
art, with respect to
any group containing one or more substituents, that such groups are not
intended to introduce
any substitution or substitution patterns that are sterically impractical,
synthetically non-
feasible, and/or inherently unstable. It will also be understood that where a
group or moiety
is optionally substituted, the disclosure includes both embodiments in which
the group or
moiety is substituted and embodiments in which the group or moiety is
unsubstituted.
[0036] The compounds disclosed and/or described herein can be
enriched isotopic forms,
e.g., enriched in the content of 2H, 3H, 11C, 13C and/or "C. In one
embodiment, the
compound contains at least one deuterium atom. Such deuterated forms can be
made, for
example, by the procedure described in U.S. Patent Nos. 5,846,514 and
6,334.997. Such
deuterated compounds may improve the efficacy and increase the duration of
action of
compounds disclosed and/or described herein. Deuterium substituted compounds
can be
synthesized using various methods, such as those described in: Dean, D.,
Recent Advances in
the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery
and
Development, Curr. Pharm. Des., 2000; 6(10); Kabalka, G. et al., The Synthesis
of
Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989,
45(21),
6601-21; and Evans, E., Synthesis of radiolabeled compounds, J. Radioanal.
Chem., 1981,
64(1-2), 9-32.
[0037] The term "pharmaceutically acceptable carrier" or
"pharmaceutically acceptable
excipient" includes any and all solvents, dispersion media, coatings,
antibacterial and
antifungal agents, isotonic and absorption delaying agents and the like. The
use of such media
and agents for pharmaceutically active substances is well known in the art.
Except insofar as
any conventional media or agent is incompatible with the active ingredient,
its use in
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pharmaceutical compositions is contemplated. Supplementary active ingredients
can also be
incorporated into the pharmaceutical compositions.
[0038] The terms "patient," "individual," and "subject" refer to an
animal, such as a
mammal, bird, or fish. in some embodiments, the patient or subject is a
mammal. Mammals
include, for example, mice, rats, dogs, cats, pigs, sheep, horses, cows and
humans. In some
embodiments, the patient or subject is a human, for example a human that has
been or will be
the object of treatment, observation or experiment. The compounds,
compositions and
methods described herein can be useful in both human therapy and veterinary
applications.
[0039] As used herein, the term "therapeutic" refers to the ability
to modulate the cardiac
sarcomere. As used herein, "modulation" refers to a change in activity as a
direct or indirect
response to the presence of a chemical entity as described herein, relative to
the activity of in
the absence of the chemical entity. The change may be an increase in activity
or a decrease in
activity, and may be due to the direct interaction of the chemical entity with
the a target or
due to the interaction of the chemical entity with one or more other factors
that in turn affect
the target's activity. For example, the presence of the chemical entity may,
for example,
increase or decrease the target activity by directly binding to the target, by
causing (directly
or indirectly) another factor to increase or decrease the target activity, or
by (directly or
indirectly) increasing or decreasing the amount of target present in the cell
or organism.
[0040] The term "therapeutically effective amount" or "effective
amount" refers to that
amount of a compound disclosed and/or described herein that is sufficient to
affect treatment,
as defined herein, when administered to a patient in need of such treatment. A

therapeutically effective amount of a compound may be an amount sufficient to
treat a
disease responsive to modulation of the cardiac sarcomere. The therapeutically
effective
amount will vary depending upon, for example, the subject and disease
condition being
treated, the weight and age of the subject, the severity of the disease
condition, the particular
compound, the dosing regimen to be followed, timing of administration, the
manner of
administration, all of which can readily be determined by one of ordinary
skill in the art. The
therapeutically effective amount may be ascertained experimentally, for
example by assaying
blood concentration of the chemical entity, or theoretically, by calculating
bioavailability.
[0041] "Treatment" (and related terms, such as "treat", "treated",
"treating") includes one
or more of: inhibiting a disease or disorder; slowing or arresting the
development of clinical
symptoms of a disease or disorder; and/or relieving a disease or disorder
(i.e., causing relief
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from or regression of clinical symptoms). The term covers both complete and
partial
reduction of the condition or disorder, and complete or partial reduction of
clinical symptoms
of a disease or disorder. Thus, compounds described and/or disclosed herein
may prevent an
existing disease or disorder from worsening, assist in the management of the
disease or
disorder, or reduce or eliminate the disease or disorder.
[0042] "ATPase" refers to an enzyme that hydrolyzes ATP. ATPases
include proteins
comprising molecular motors such as the myosins.
[0043] As used herein, "selective binding" or "selectively binding"
refers to preferential
binding to a target protein in one type of muscle or muscle fiber as opposed
to other types.
For example, a compound selectively binds to fast skeletal troponin C if the
compound
preferentially binds troponin C in the troponin complex of a fast skeletal
muscle fiber or
sarcomere in comparison with troponin C in the troponin complex of a slow
muscle fiber or
sarcomere or with troponin C in the troponin complex of a cardiac sarcomere.
[0044] It is understood that embodiments described herein as
"comprising" include
"consisting of' and "consisting essentially of' embodiments.
Compounds
[0045] Compounds and salts thereof (such as pharmaceutically
acceptable salts) are
detailed herein, including in the Brief Summary and in the appended claims.
Also provided
are the use of all of the compounds described herein, including any and all
stereoisomers,
including geometric isomers (cis/trans), E/Z isomers, enantiomers,
diastereomers, and
mixtures thereof in any ratio including racemic mixtures, salts and solvates
of the compounds
described herein, as well as methods of making such compounds. Any compound
described
herein may also be referred to as a drug.
[0046] In one aspect, provided are compounds of Formula (I):
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R1
R2
0
,
R'
R4
0
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing, wherein:
R1 is halo or Ci_6haloalkyl;
R2 is H, halo, or Ci 6alkyl;
R3 is:
(i) cyclohexyl, wherein the cyclohexyl is optionally substituted with one
or more
independently selected Ci_6alky1 or C1_6haloalkyl substituents, or
(ii) Ci 6 alkyl; and
R4 is:
(i) -C(0)H,
(ii) -C(0)NH2,
õA-
(iii) , or
N
(iv)
14
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provided that:
N
(I) when R3 is Ct_6alkyl, then R4 is
(2) when R3 is iso-propyl, then the total number of halo atoms in R1 and R2 is
at least
two, and
(3) when R3 is cyclohexyl substituted with one or more independently selected
C1_
6haloalkyl substituents, then R4 is other than -C(0)H.
[0047] In some embodiments, provided herein is a compound of
Formula (I), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R4 is -C(0)H. In some embodiments, provided herein is a
compound of
Formula (I), or a stereoisomer or tautomer thereof, or a pharmaceutically
acceptable salt of
any of the foregoing, wherein the compound is of Fatinula (I-A):
R1
R2
0
3
0
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing.
[0048] In some embodiments, provided herein is a compound of
Formula (I), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R4 is -C(0)N1-1/. In some embodiments, provided herein is a
compound
of Formula (I), or a stereoisomer or tautomer thereof, or a pharmaceutically
acceptable salt of
any of the foregoing, wherein the compound is of Fatinula (I-B):
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R1
R2, 0
R3
0
NH2 (I-B),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing.
[0049] In some embodiments, provided herein is a compound of
Formula (I), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R4 is N . In some embodiments, provided herein
is a
compound of Formula (I), or a stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing, wherein the compound is of Formula (I-
C):
R1
R2
0
R3
NJ
N
(I-C),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing.
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[0050] In some embodiments, provided herein is a compound of
Formula (I), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R4 is N . In some embodiments, provided
herein is a
compound of Formula (I), or a stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing, wherein the compound is of Formula (I-
D):
R1
R2, 0
3
NJ
(I-D).
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing.
[0051] In some embodiments, provided herein is a compound of
Formula (I), such as a
compound of Formula (I-A), (I-B), (I-C), or (I-D), or a stereoisomer or
tautomer thereof, or a
pharmaceutically acceptable salt of any of the foregoing, wherein R3 is
cyclohexyl, wherein
the cyclohexyl is optionally substituted with one or more independently
selected Ci_6alkyl or
CI_6haloalkyl substituents. In some embodiments, R3 is unsubstituted
cyclohexyl. In some
embodiments, R3 is cyclohexyl, wherein the cyclohexyl is substituted with one
or more
independently selected C 1_6a1ky1 or Ci_6haloalkyl substituents.
[0052] In some embodiments, R3 is cyclohexyl, wherein the
cyclohexyl is optionally
substituted with one or more independently selected C1_6alkyl substituents. In
some
embodiments, R3 is cyclohexyl, wherein the cyclohexyl is optionally
substituted with one or
more independently selected Ci_3alkyl substituents. In some embodiments, R3 is
cyclohexyl,
wherein the cyclohexyl is optionally substituted with one or more
independently selected
methyl or ethyl substituents. In some embodiments, R3 is cyclohexyl, wherein
the cyclohexyl
17
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is optionally substituted with one or more methyl susbtituents. In some
embodiments, R3 is
cyclohexyl, wherein the cyclohexyl is optionally substituted with one or more
ethyl
susbtituents. In some embodiments, R3 is cyclohexyl, wherein the cyclohexyl is
optionally
substituted with one methyl susbtituent. In some embodiments, R3 is
cyclohexyl, wherein the
cyclohexyl is optionally substituted with one ethyl susbtituent. In some
embodiments, R3 is
5411a or . In some embodiments, R3 is
.
[0053] In some embodiments, R3 is cyclohexyl, wherein the
cyclohexyl is substituted
with one or more independently selected C1_6haloa1kyl substituents. In some
embodiments,
R3 is cyclohexyl, wherein the cyclohexyl is substituted with one or more
independently
s&ar.õF
selected Ci_3haloalkyl substituents. In some embodiments, R3 is F
.
[0054] In some embodiments, R3 is - or
54-10,T,,F
F
'
[0055] In some embodiments, provided herein is a compound of
Formula (I) or Formula
(I-A), or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable
salt of any of
the foregoing, wherein R3 is ICTCL'Rx , wherein Rx is C1_6alkyl, wherein the
compound is of Fat _____ iaula (I-A1):
18
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R1
R2
0
0 (IRx
(I-A1),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing. In some embodiments, Rx is methyl.
[0056] In some embodiments, provided herein is a compound of
Formula (I) or Formula
(1-B), or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable
salt of any of
the foregoing, wherein R3 is ICIC:LRx , wherein Rx is Ci_6alky1 or
Ci_ohaloalkyl,
wherein the compound is of Formula (I-B1):
W
R2
011 0
N
0
NH2 (I-B1),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing. In some embodiments, provided herein is a compound of Formula (I-
B1). or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein 12' is Ci_oarkyl. In some embodiments, IV is methyl.
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[0057] In some embodiments, provided herein is a compound of
Formula (I) or Formula
(I-C), or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable
salt of any of
the foregoing, wherein R3 is ICCL-Rx , wherein Rx is C1_6alky1 or
CI_6haloalkyl,
wherein the compound is of Formula (I-C1):
R1
R2
0
N
N
0 ORx
N
(I-C1),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing. In some embodiments, provided herein is a compound of Formula (I-
C1), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein Rx is C1_6alkyl. In some embodiments, Rx is methyl.
[0058] In some embodiments, provided herein is a compound of
Formula (I) or Formula
a-D), or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable
salt of any of
the foregoing, wherein R3 is Rx , wherein Rx is C1_6alky1 or
Ci_6ha1oalkyl,
wherein the compound is of Formula (I-D1):
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R1
R2
4101 0
0
Rx
(I-D1),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing. In some embodiments, provided herein is a compound of Formula (I-
D1), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein IV is Ci 6alkyl. In some embodiments, Rx is methyl.
[0059] In some embodiments of the foregoing. R3 is
Rx , wherein the
A'aRx moiety is attached to the remainder of the molecule in the (1r,4r)
stereochemical configuration. In some embodiments, R3 is
/Rx . In some
cziNw
embodiments, R3 is Rx
[0060]
In some embodiments, provided herein is a compound of Formula (I), such as
a
compound of Formula (I-C), or a stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing, wherein R3 is Ci_6alkyl. In some
embodiments, R3 is
Ci_5alkyl. In some embodiments. R3 is Ci_4alkyl. In some embodiments, R3 is
C1_3alkyl. In
some embodiments, R3 is C3_6alkyl. In some embodiments, R3 is C3_5alkyl. In
some
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embodiments, R3 is , or . In some embodiments,
R3 is
=
[0061] In some embodiments of the foregoing, R3 is Ci_6alkyl,
wherein the CI,6a11y1
moiety of R3 is attached to the remainder of the molecule in the S
stereochemical
configuration. In some embodiments, R3 is
[0062] In some embodiments, provided herein is a compound of
Formula (I) or Formula
(I-C), or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable
salt of any of
the foregoing, wherein R3 is iso-propyl, wherein the compound is of Formula (I-
C2):
R1
R2
0
0
(I-C2),
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing. In some embodiments, provided herein is a compound of Formula (I-
C2), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein then the total number of halo atoms in RI and R2 is at
least two. In some
embodiments, the total number of halo atoms in RI and R2 is two. In some
embodiments, the
total number of halo atoms in fe and R2 is at least three. In some
embodiments, the total
number of halo atoms in R1 and R2 is at three. In some embodiments, the total
number of
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halo atoms in RI and R2 is at least four. In some embodiments, the total
number of halo
atoms in RI and R2 is four.
[0063]
In some embodiments, provided herein is a compound of Formula (1), such as
a
compound of Formula (1-A), (1-A1), (1-B), (1-B1), (1-C), (1-C1), (1-C2). (1-
D), or (1-D1), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R1 is halo. In some embodiments, R1 is fluoro or chloro. In
some
embodiments, R1 is fluoro. In other embodiments. R1 is chloro.
[0064]
In some embodiments, provided herein is a compound of Formula (I), such as
a
compound of Formula (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2). (I-
D), or (I-D1), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R1 is Ci_6haloalkyl. In some embodiments, R1 is
C1_6ha10a1ky1. In some
embodiments, RI is C1_9haloalkyl. In some embodiments, RI is -CF3.
[0065]
In some embodiments, provided herein is a compound of Formula (I), such as
a
compound of Formula (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2). (I-
D), or (I-D1), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R2 is H.
[0066]
In some embodiments, provided herein is a compound of Formula (I), such as
a
compound of Formula (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2). (I-
D), or (I-D1), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R2 is halo. In some embodiments, R2 is fluoro or chloro. In
some
embodiments, R2 is fluoro. In other embodiments. R2 is chloro.
[0067]
In some embodiments, provided herein is a compound of Formula (I), such as
a
compound of Formula (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2). (I-
D), or (I-D1), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R2 is Ci 6a1ky1. In some embodiments, R2 is Ci 3alkyl. In
some
embodiments, R2 is C1_3alkyl. In some embodiments, R2 is Ci_2alkyl. In some
embodiments,
R2 is methyl.
[0068]
In some embodiments, provided herein is a compound of Formula (1), such as
a
compound of Formula (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2). (I-
D), or (I-D1), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R1 is halo or C1_6haloalky1 and R2 is H. In some
embodiments, R1 is halo
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or Ci_6haloalkyl and R2 is halo. In some embodiments, RI is halo or
Ci_6haloalkyl and R2 is
chloro or fluoro. In some embodiments, RI is halo or C1_6ha1oalkyl and R2 is
C1_6a11y1. In
some embodiments, Rl is halo or Ci_6haloalkyl and R2 is methyl.
[0069] In some embodiments, provided herein is a compound of
Formula (1), such as a
compound of Formula (I-A), (I-A1), (I-13), (I-B1), (I-C), (I-C1), (I-C2), (I-
D), or (I-D1), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein R1 is halo and R2 is H. In some embodiments, R1 is fluoro
or chloro and
R2 is H. In some embodiments, le is fluoro and R2 is H. In some embodiments,
RI is chloro
and R2 is H. In some embodiments, RI is C1_6haloalkyl and R2 is H. In some
embodiments,
R1 is -CF3 and R2 is H.
[0070] In some embodiments, RI is halo and R2 is halo. In some
embodiments. R1 is
fluoro or chloro and R2 is halo. In some embodiments, RI is fluoro or chloro
and R2 is fluoro
or chloro. In some embodiments, R1 is fluoro and R2 is fluoro. In some
embodiments, R1 is
chloro and R2 is chloro. In some embodiments, RI is fluoro and R2 is chloro.
In some
embodiments, R1 is chloro and R2 is fluoro. In some embodiments, R1 is
Ci_6haloa1ky1 and
R2 is halo. In some embodiments, RI is -CF3 and R2 is halo.
[0071] In some embodiments, Rl is halo and R2 is Ci_olkyl. In some
embodiments, R1 is
fluoro or chloro and R2 is C1_6a1ky1. In some embodiments, RI is fluoro or
chloro and R2 is
methyl. In some embodiments. RI- is fluoro and R2 is methyl. In some
embodiments, RI is
chloro and R2 is methyl. In some embodiments, R1 is Ci_6haloalkyl and R2 is
Ci_6alkyl. In
some embodiments, RI is -CF3 and R2 is Ci_6alky1.
[0072] In some embodiments, provided herein are compounds and salts
thereof described
in Table 1.
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Table 1
Compound
Structure Name
No.
CI
5-(4-chlorobenzy1)-8-
5 0 ((lr,40-4-ethylcyclohexyl)-
1 NA1 6,9-dioxo-2,5,8-
triazaspiro[3.51nonane-2-
IO carboxamide
O,NIN
NH2
I
F
8-41r,40-4-
(1111 0 ethylcyclohexyl)-5-(4-
2 N)1 fluorobenzy1)-6,9-
dioxo-
N 1\1d"r 2,5,8-
triazaspiro[3.5]nonane-
,.. N
2-carboxamide
I 0
NH2
I
CI
F 0
0
5-(4-chloro-3-fluorobenzy1)-
8-((1r,4r)-4-
methylcyclohexyl)-6,9-
N dioxo-2,5,8-
0... NI rN
triazaspiro[3.51nonane-2-
./J
I 0 carboxamide
NH2
F
F 0
0
5-(3,4-difluorobenzy1)-8-
((lr,40-4-
4
N -IL) methylcyclohexyl)-6,9-
dioxo-2,5,8-
triazaspiro[3.5[nonane-2-
ON/JYNI'.a. carbaldehyde
1 0
H
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F
0
5-(3,4-difluorobenzy1)-8-
(01%40-4-
methylc yclohexyl)-6,9-
dioxo-2,5,8-
triazaspiro[3.51nonane-2-
0NN
0 carboxamide
NH2
IP 05-(4-fluoro-3-methylbenzy1)-
8-((lr,4r)-4-
methylcyclohexyl)-6,9-
6
N dioxo-2,5,8-
,NriY N triazaspiro13.51nonane-2-
04.10 1 0 carboxamide
NH2
CI
o5-(3-chloro-4-fluorobenzy1)-
8-((1r,40-4-
methylcyclohexyl)-6,9-
7
N dioxo-2,5,8-
O. N N
triazaspiro[3.51nonane-2-
0 carboxamide
NH2
CI
F
5-(4-chloro-3-fluorobenzy1)-
o 8-isopropy1-2-
(pyridazin-3-
8 y1)-2,5,8-
triazaspiro13.5]nonane-6,9-
, N N dione
N 0
CI
5-(3-chloro-4-fluorobenzy1)-
0 8-isopropy1-2-
(pyridazin-3-
9

NH y1)-2,5,8-
)
triazaspiro13.51nonane-6,9-
N,-
dionc
N N 0
Nfj.Y
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CI
S 05-(4-chlorobenzy1)-8-
(01..40-4-
N methylcyclohexyl)-
2-
(pyridazin-3- y1)-2,5,8-
tri azaspiro13.51nonane-6,9-
dione
0
F
5-(3,4-difluorobenzy1)- 8-
0 ((lr,40-4-
ethylcyclohexyl)-
11
N)-H 6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-2-
0..N IN carboxamide
0
NH2
F =
5-(3,4-difluorobenzy1)- 8-
(01%40-4-
0
N methylcyclohexyl)-
2-
12
(pyridazin-3- y1)-2,5,8-
triazaspiro [3.51nonane-6,9-
NI/j)1
N 4I3 dione
0
F F
I. 08-isopropy1-2-(pyridazin-3-
y1)-5-(4-
13
(trifluoromethyl)benzy1)-
NH2,5,8-triazaspiro [3 .51nonane-
6,9-dione
N 0
F
(S)-8-(sec-butyl)-5-(3,4-
o difluorobenzy1)-2-
14
Nit.) (pyri dazi n-3-
y1)-2,5, g-
triazaspiro [3.51nonane-6,9-
,1\1 1\11(
N,
dione
N 0
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F
5-(3,4-difluorobenzy1)-8-
0 (pentan-3-y1)-2-(pyridazin-3-
NA1 y1)-2,5,8-
triazaspiro[3.51nonane-6,9-
dione
N 0
(S)-8-(sec-butyl)-5-(4-
1:1*1 0 fluoro-3-methylbenzy1)-2-
16
NA1 (pyridazin-3-y1)-
2,5,8-
triazaspiro[3.51nonane-6,9-
,N
N, dione
N 0
1161 05-(4-fluorobenzy1)-8-
((1r,40-4-
N )L1 methylcyclohexy1)-2-
17
(pyridazin-3-y1)-2,5,8-
triazaspiro13.5]n0nane-6,9-
, N Nrj-11 N dione
0
CI
0
5-(4-chlorobenzy1)-8-
((lr,4r)-4-
N )1.) methylcyclohexyl)-6,9-
18
dioxo-2,5,8-
triazaspiro13.5]nonane-2-
0,, N 1'. a carboxamide
1 0
NH2
11.1 0 5-(4-fluorobenzy1)-8-
((lr,40-4-
methylcyclohexyl)-2-
19
N (pyridazin-4-y1)-
2,5,8-
N4.0
triazaspiro13.51nonane-6,9-
l dione
Nfjir
0
N
28
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11 1 8-41r,4r)-4-
0
(difluoromethyl)cyclohexyl)-
5-(4-fluorobenzy1)-2-
N -)L-) (pyridazin-4-y1)-
2,5,8-
N
triazaspiro[3.5]nonane-6,9-
N
- NI
0 F
dione
***0
N
[0073] In some embodiments, provided herein is a compound of
Formula (I), or a
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein the compound is selected from the group consisting of
5-(4-chlorobenzy1)-8-(4-ethylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-2-
carboxamide;
8-(4-ethylcyclohexyl)-5-(4-fluorobenzy1)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonanc-2-
carboxamide;
5-(4-chloro-3-fluorobenzy1)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-
2-carboxamide;
5-(3,4-difluorobenzy1)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-2-
carbaldehyde;
5-(3,4-difluorobenzy1)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-2-
carboxamide;
5-(4-fluoro-3-methylbenzy1)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-
2-carboxamide;
5-(3-chloro-4-fluorobenzy1)-8-(4-nacthylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-
2-carboxamide;
5-(4-chloro-3-fluorobenzy1)-8-isopropy1-2-(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-
dione;
5-(3-chloro-4-fluorobenzy1)-8-isopropy1-2-(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-
dione;
5-(4-chlorobenzy1)-8-(4-methylcyclohexyl)-2-(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-
6,9-dione;
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5-(3,4-difluorobenzy1)-8-(4-ethylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-2-
carboxamide;
5-(3,4-difluorobenzy1)-8-(4-methylcyclohexyl)-2-(pyriciazin-3-y1)-2,5,8-
triazaspiro13.5Jnonane-6,9-dione;
8-isopropy1-2-(pyridazin-3-y1)-5-(4-(trifluoromethypbenzy1)-2,5,8-
triazaspiro[3.5]nonane-
6,9-dione;
8-(sec-buty1)-5-(3,4-difluorobenzy1)-2-(pyridazin-3-y1)-2,5,8-
triazaspiro[3.51nonane-6,9-
dione;
5-(3,4-difluorobenzy1)-8-(pentan-3-y1)-2-(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-
dione;
8-(sec-buty1)-5-(4-fluoro-3-methylbenzy1)-2-(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-
6,9-dione;
5-(4-fluorobenzy1)-8-(4-methylcyclohexyl)-2-(pyridazin-3-y1)-2,5,8-
triazaspiro13.5Jnonane-
6,9-dione;
5-(4-chlorobenzy1)-8-(4-methylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro13.51nonane-2-
carboxamide;
5-(4-fluorobenzy1)-8-(4-methylcyclohexyl)-2-(pyridazin-4-y1)-2,5,8-
triazaspiro[3.5]nonane-
6,9-dione; and
8-(4-(difluoromethyl)cyclohexyl)-5-(4-fluorobenzy1)-2-(pyridazin-4-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione,
or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing.
[0074]
In some variations, any of the compounds described herein, such as a
compound
of Formula (I), (I-A), (I-Al),(T-B), (I-B1), (I-C). (I-C1), (I-C2), (I-D), or
(I-Dl), or any
variation thereof, or a compound of Table 1 may be deuterated (i.e., one or
more hydrogen
atoms are replaced by one or more deuterium atoms). In some of these
variations, the
compound is deuterated at a single site. In other variations, the compound is
deuterated at
multiple sites. Deuterated compounds can be prepared from deuterated starting
materials in a
manner similar to the preparation of the corresponding non-deuterated
compounds.
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Hydrogen atoms may also be replaced with deuterium atoms using other method
known in
the art.
100751 Any formula given herein, such as Formula (1), (1-A), (1-
A1), (1-B), (1-B1), (1-C),
(1-C1), (1-C2), (1-D), or (1-D1), is intended to represent compounds having
structures depicted
by the structural formula as well as certain variations or forms. In
particular, compounds of
any formula given herein may have asymmetric centers and therefore exist in
different
enantiomeric or diastereomeric forms. All optical isomers and stereoisomers of
the
compounds of the general formula, and mixtures thereof in any ratio, are
considered within
the scope of the foimula. Thus, any formula given herein is intended to
represent a racemate,
one or more enantiomeric forms, one or more diastereomeric forms, one or more
atropisomeric forms, and mixtures thereof in any ratio. Where a compound of
Table 1 is
depicted with a particular stereochemical configuration, also provided herein
is any
alternative stereochemical configuration of the compound, as well as a mixture
of
stereoisomers of the compound in any ratio. For example, where a compound of
Table 1 has a
stereocenter that is in an "S" stereochemical configuration, also provided
herein is
enantiomer of the compound wherein that stereocenter is in an "R"
stereochemical
configuration. Likewise, when a compound of Table 1 has a stereocenter that is
in an
configuration, also provided herein is enantiomer of the compound in an -S"
stereochemical
configuration. Also provided are mixtures of the compound with both the "S"
and the "R"
stereochemical configuration. Additionally, if a compound of Table 1 has two
or more
stereocenters, also provided are any enantiomer or diastereomer of the
compound. For
example, if a compound of Table 1 contains a first stereocenter and a second
stereocenter
with "R" and "R" stereochemical configurations, respectively, also provided
are
stereoisomers of the compound having first and second stereocenters with "S"
and "S"
stereochemical configurations, respectively, "S and "ir stereochemical
configurations,
respectively, and -R" and "S" stereochemical configurations, respectively. If
a compound of
Table 1 contains a first stereocenter and a second stereocenter with -S" and
"S"
stereochemical configurations, respectively, also provided are stereoisomers
of the compound
having first and second stereocenters with "R" and -R" stereochemical
configurations,
respectively, "S" and "R" stereochemical configurations, respectively, and "R"
and "S"
stereochemical configurations, respectively. If a compound of Table 1 contains
a first
stereocenter and a second stereocenter with -S" and -R" stereochemical
configurations,
respectively, also provided are stereoisomers of the compound having first and
second
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stereocenters with "R" and "S" stereochemical configurations, respectively,
"R" and "R"
stereochemical configurations, respectively, and "S" and "S" stereochemical
configurations,
respectively. Similarly, if a compound of Table 1 contains a first
stereocenter and a second
stereocenter with "R" and "S" stereochemical configurations, respectively,
also provided are
stereoisomers of the compound having first and second stereocenters with "S"
and -R"
stereochemical configurations, respectively, "R" and "R" stereochemical
configurations,
respectively, and -S" and "S" stereochemical configurations, respectively.
Furthermore,
certain structures may exist as geometric isomers (i.e., cis and trans
isomers), as tautomers,
or as atropisomers. Additionally, any formula given herein is intended to
refer also to any
one of hydrates, solvates, and amorphous and polymorphic forms of such
compounds, and
mixtures thereof, even if such forms arc not listed explicitly. In some
embodiments, the
solvent is water and the solvates are hydrates.
[0076] Representative examples of compounds detailed herein,
including intermediates
and final compounds, are depicted in the tables and elsewhere herein. It is
understood that in
one aspect, any of the compounds may be used in the methods detailed herein,
including,
where applicable, intermediate compounds that may be isolated and administered
to an
individual or subject.
[0077] The compounds depicted herein may be present as salts even
if salts are not
depicted, and it is understood that the compositions and methods provided
herein embrace all
salts and solvates of the compounds depicted here, as well as the non-salt and
non-solvate
form of the compound, as is well understood by the skilled artisan. In some
embodiments,
the salts of the compounds provided herein are pharmaceutically acceptable
salts.
[0078] In one variation, the compounds herein are synthetic
compounds prepared for
administration to an individual or subject. In another variation, compositions
are provided
containing a compound in substantially pure form. In another variation,
provided are
pharmaceutical compositions comprising a compound detailed herein and a
pharmaceutically
acceptable carrier. In another variation, methods of administering a compound
arc provided.
The purified forms, pharmaceutical compositions and methods of administering
the
compounds are suitable for any compound or form thereof detailed herein.
[0079] Any variation or embodiment of R1, R2, R3, and R4 provided
herein can be
combined with every other variation or embodiment of R1, R2, R3, and R4, the
same as if each
combination had been individually and specifically described.
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[0080] As used herein, when any variable occurs more than one time
in a chemical
formula, its definition on each occurrence is independent of its definition at
every other
occurrence.
[00811 Formula (1) includes all subformulae thereof. For example,
Formula (1) includes
compounds of Formula (1-A), (T-Al),(1-B), (I-B1), (1-C), (1-C1), (1-C2), (I-
D), or (1-D1).
[0082] Certain compound names provided herein, including in Table
1, are provided by
ChemBioDraw Professional 15Ø0.106. One of skilled in the art would
understand that the
compounds may be named or identified using various commonly recognized
nomenclature
systems and symbols. By way of example, the compounds may be named or
identified with
common names, systematic or non-systematic names. The nomenclature systems and

symbols that are commonly recognized in the art of chemistry include, for
example,
Chemical Abstract Service (CAS), ChemBioDraw Ultra, and International Union of
Pure and
Applied Chemistry (IUPAC).
[0083] In some embodiments, the compounds of the disclosure, or a
pharmaceutically
acceptable salt thereof, may have advantages related to one or more of the
following: hERG
profile, toxicity profile, safety window, selectivity, off-target profile,
favorable drug/drug
interaction profile, PK parameters including bioavailability, clearance and
half life,
mechanism of action, CYP inhibition and time dependent inhibition profile,
permeability
and/or efflux, solubility, metabolism, unbound fraction, adequate human dose,
and ease of
synthesis on a large scale.
Compositions
[0084] Also provided are compositions, such as pharmaceutical
compositions, that
include a compound disclosed and/or described herein and one or more
additional medicinal
agents, pharmaceutical agents, adjuvants, carriers, excipients, and the like.
Suitable
medicinal and pharmaceutical agents include those described herein. In some
embodiments,
the pharmaceutical composition includes a pharmaceutically acceptable
excipient or adjuvant
and at least one chemical entity as described herein. Examples of
pharmaceutically
acceptable excipients include, but are not limited to, mannitol, lactose,
starch, magnesium
stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose,
glucose, gelatin,
sucrose, and magnesium carbonate. In some embodiments, provided are
compositions, such
as pharmaceutical compositions that contain one or more compounds described
herein. or
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stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing.
[0085] In some embodiments, provided is a pharmaceutically
acceptable composition
comprising a compound of Formula (1), (1-A), (1-A1), (1-B), (1-B1), (1-C), (1-
C1), (1-C2), (I-
D), or (T-D1), or a compound of Table 1, or stereoisomer or tautomer thereof,
or a
pharmaceutically acceptable salt of any of the foregoing. In some aspects, a
composition
may contain a synthetic intermediate that may be used in the preparation of a
compound
described herein. The compositions described herein may contain any other
suitable active or
inactive agents.
[0086] Any of the compositions described herein may be sterile or
contain components
that are sterile. Sterilization can be achieved by methods known in the art.
Any of the
compositions described herein may contain one or more compounds or conjugates
that are
substantially pure.
[0087] Also provided are packaged pharmaceutical compositions,
comprising a
pharmaceutical composition as described herein and instructions for using the
composition to
treat a patient suffering from a disease or condition described herein.
Methods of Use
[0088] The compounds and pharmaceutical compositions herein may be
used to treat or
prevent a disease or condition in an individual or subject.
[0089] When used in a prophylactic manner, the compounds disclosed
and/or described
herein may prevent a disease or disorder from developing or lessen the extent
of a disease or
disorder that may develop in an individual or subject at risk of developing
the disease or
disorder.
[0090] Without being bound by theory, the compounds and
pharmaceutical compositions
disclosed herein are believed to act by inhibiting myosin. This inhibition
potentially
decreases the number of independent myosin heads interacting with actin
filaments reducing
the amount of contraction. Reducing contraction of cardiac muscle can be
important for the
treatment of heart diseases in which over-contraction is an issue. In some
embodiments,
provided are methods of treating or preventing heart disease in an individual
or subject,
comprising administering to the individual or subject in need thereof a
compound of Formula
(I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or
a compound of Table
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1, or stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing. In some embodiments, provided are methods of treating or preventing
heart
disease in a subject in need thereof comprising administering to the subject a
therapeutically
effective amount of at least one chemical entity as described herein. In some
embodiments,
provided are methods of treating heart disease in a subject in need thereof
comprising
administering to the subject a therapeutically effective amount of at least
one chemical entity
as described herein. In some embodiments, provided are methods of treating an
established
or diagnosed heart disease in a subject in need thereof comprising
administering to the
subject a therapeutically effective amount of at least one chemical entity as
described herein.
In some embodiments, provided are methods of preventing heart disease in a
subject in need
thereof comprising administering to the subject a therapeutically effective
amount of at least
one chemical entity as described herein.
[0091] Also provided herein is the use of a compound of Formula
(I), (I-A), (I-A1), (I-B),
(I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or
tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing, in the
manufacture of a medicament for treatment of a heart disease in a subject. In
some aspects,
provided is a compound or composition as described herein for use in a method
of treatment
of the human or animal body by therapy. In some embodiments, provided herein
are
compounds of Formula (I), (I-A). (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2),
(I-D), or (I-D1),
or a compound of Table 1, or stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing, for use in a method of treatment of
the human or
animal body by therapy. In some embodiments, provided herein are compounds of
Formula
(I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or
a compound of Table
1, or stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing, for use in treating or preventing heart disease. In some
embodiments, provided
herein are compounds of Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-
C1), (I-C2), (I-D),
or (I-D1), or a compound of Table 1, or stereoisomer or tautomer thereof, or a

pharmaceutically acceptable salt of any of the foregoing, for use in treating
heart disease. In
some embodiments, provided herein are compounds of Formula (I), (I-A), (I-A1),
(I-B), (I-
B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or
tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing, for use in
treating an established or diagnosed heart disease. In other embodiments,
provided herein are
compounds of Formula (1), (1-A), (1-A1), (1-B), (1-B1), (1-C), (1-C1), (1-C2),
(1-D), or (1-D1),
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or a compound of Table 1, or stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing, for use in preventing heart disease.
In some
embodiments, provided herein are compounds of Formula (I), (I-A), (I-A1), (I-
B), (I-B1), (I-
C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or tautomer
thereof, or a pharmaceutically acceptable salt of any of the foregoing, for
use in treating a
disease or condition associated with HCM. In some embodiments, provided herein
are
compounds of Formula (I), (I-A). (I-A1), (I-B), (I-B1), (I-C), (I-CI), (I-C2),
(I-D), or (I-D1),
or a compound of Table 1. or stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing, for use in treating a disease or
condition associated
with secondary left ventricular wall thickening. In some embodiments, provided
herein arc
compounds of Formula (I), (I-A). (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2),
(I-D), or (I-D1),
or a compound of Table 1, or stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing, for use in ameliorating a symptom
associated with
heart disease. In other embodiments, provided herein are compounds of Formula
(I), (I-A), (I-
A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of
Table 1, or
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, for use in reducing the risk of a symptom associated with heart
disease. In other
embodiments, provided herein are compounds of Formula (I), (I-A), (I-A1), (I-
B), (I-B1), (I-
C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or tautomer
thereof, or a pharmaceutically acceptable salt of any of the foregoing, for
use in treating a
disease or condition associated with small left ventricular cavity, cavity
obliteration,
hyperdynamic left ventricular contraction, obstruction of blood flow out of
the left ventricle,
cardiac hypertrophy, small cardiac stroke volume, impaired relaxation of the
left ventricle,
high left ventricle filling pressure, myocardial ischemia, or cardiac
fibrosis. In certain
embodiments, provided herein are compounds of Formula (1), (1-A), (1-A1), (1-
B), (1-B1), (I-
C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or tautomer
thereof, or a pharmaceutically acceptable salt of any of the foregoing, for
use in treating a
disease or condition associated with small left ventricular cavity and cavity
obliteration,
hyperdynamic left ventricular contraction, myocardial ischemia, or cardiac
fibrosis. In some
embodiments, provided herein are compounds of Formula (I), (I-A), (I-A1), (I-
B), (I-B1), (I-
C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or tautomer
thereof, or a pharmaceutically acceptable salt of any of the foregoing, for
use in treating
muscular dystrophies. In some embodiments, provided herein are compounds of
Formula (I),
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(I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a
compound of Table 1,
or stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of
any of the
foregoing, for use in treating a glycogen storage disease. In other
embodiments, provided
herein are compounds of Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-
C1), (I-C2), (I-D),
or (I-D1), or a compound of Table 1, or stereoisomer or tautomer thereof, or a

pharmaceutically acceptable salt of any of the foregoing, for use in
modulating the cardiac
sarcomere, such as inhibiting the cardiac sarcomere. In yet other embodiments,
provided
herein are compounds of Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-
C1), (I-C2), (I-D),
or (I-D1), or a compound of Table 1, or stereoisomer or tautomer thereof, or a

pharmaceutically acceptable salt of any of the foregoing, for use in
potentiating cardiac
myosin.
[0092] In some embodiments, the subject is a mammal. In some
embodiments, the
subject is a mouse, rat, dog, cat, pig, sheep, horse, cow, or human. In some
embodiments, the
subject is a human. In some embodiments, the subject has an established or
diagnosed heart
disease. In some embodiments, the subject has established or diagnosed
hypertrophic
cardionayopathy (HCM). In some embodiments, the subject is at risk for
developing heart
disease. In some embodiments, the subject has a mutation that increases risk
for heart
disease. In some embodiments, the subject has a mutation that increases risk
for hypertrophic
cardiomyopathy (HCM). In some embodiments, the mutation is a sarcomeric
mutation. In
some embodiments, the mutation is a mutation in myosin heavy chain 13 (m-Hc-
I3), cardiac
muscle troponin T (cTnT), tropomyosin alpha-1 chain (TPM1), myosin-binding
protein C
cardiac-type (MYBPC3), cardiac troponin I (cTnI), myosin essential light chain
(ELC), titin
(TTN), myosin regulatory light chain 2 ventricular/cardiac muscle isoform (MLC-
2), cardiac
muscle alpha actin, muscle LEVI protein (MLP), or protein kinase AMP-activated
non-
catalytic subunit gamma 2 (PRKAG2). In some embodiments, the mutation is a
mutation in
MHC-P. In some embodiments, the subject has established or diagnosed
hypertrophic
cardionayopathy without a confirmed genetic etiology.
[0093] ln some embodiments, the subject has a high risk of
progressive symptoms. In
some embodiments, the subject has a high risk of atrial fibrillation,
ventricular
tachyarrhythmias, stroke, and/or sudden death. In some embodiments, the
subject has a
reduced exercise capacity. In some embodiments, the reduced exercise capacity
is as
compared to an age-matched control population. In some embodiments, the
subject is
eligible for surgical intervention or percutaneous ablation to treat the heart
disease.
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[0094] In some embodiments, the heart disease is hypertrophic
cardiomyopathy (HCM).
In some embodiments, the heart disease is obstructive HCM. In some
embodiments, the
heart disease is nonobstructive HCM. In some embodiments, the HCM is
associated with a
sarcomeric mutation. In some embodiments, the HCM is associated with a non-
sarcomeric
mutation. In some embodiments, the heart disease is obstructive or
nonobstructive HCM
caused by sarcomeric and/or non-sarcomeric mutations. In some embodiments, the

sarcomeric mutation is a mutation in a myosin heavy chain f3 (MHC-I3), cardiac
muscle
troponin T (cTnT), troponayosin alpha-1 chain (TPM1), myosin-binding protein C
cardiac-
type (MYBPC3), cardiac troponin I (cTnI), myosin essential light chain (ELC),
titin (TTN),
myosin regulatory light chain 2 ventricular/cardiac muscle isoform (MLC-2),
cardiac muscle
alpha actin, or muscle LIM protein (MLP). In some embodiments, the sarcomeric
mutation is
a mutation in MHC-13. In some embodiments, the non-sarcomeric mutation is a
mutation in
protein kinase AMP-activated non-catalytic subunit gamma 2 (PRKAG2).
[0095] In some embodiments, provided herein are methods of treating
a disease or
condition associated with HCM, comprising administering to the individual or
subject in need
thereof a compound of Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-
C1), (I-C2), (I-D), or
(I-D1), or a compound of Table 1, or stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing. In some embodiments, the disease or
condition is
Fabry's Disease, Danon Disease, mitochondrial cardiomyopathies, or Noonan
Syndrome.
[0096] Also provided herein is the use of a compound of Formula
(I), (I-A), (I-A1), (I-B),
(I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or
tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing, in the
manufacture of a medicament for treatment of a disease or condition associated
with HCM.
[0097] In some embodiments, the heart disease is heart failure with
preserved ejection
fraction (HFpEF). In some embodiments, the heart disease is diastolic
dysfunction. In some
embodiments, the heart disease is cardionayopathy. In some embodiments, the
heart disease is
primary or secondary restrictive cardionayopathy. In some embodiments, the
heart disease is
condition or symptoms caused by coronary artery disease. In some embodiments,
the heart
disease is myocardial infarction or angina pectoris. In some embodiments, the
heart disease is
left ventricular outflow tract obstruction. In some embodiments, the heart
disease is
hypertensive heart disease. In some embodiments, the heart disease is
congenital heart
disease. In some embodiments, the heart disease is cardiac ischemia and/or
coronary heart
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disease. In some embodiments, the heart disease is diabetic heart disease. In
other
embodiments, the heart disease is congestive heart failure. In some
embodiments, the heart
disease is right heart failure. In other embodiments, the heart disease is
cardiorenal syndrome.
In some embodiments, the heart disease is infiltrative cardiomyopathy. In some

embodiments, the heart disease is a condition that is or is related to cardiac
senescence or
diastolic dysfunction due to aging. In some embodiments, the heart disease is
a condition that
is or is related to left ventricular hypertrophy and/or concentric left
ventricular remodeling.
[0098] In some embodiments, the provided are methods of treating a
disease or condition
associated with secondary left ventricular wall thickening in an individual or
subject,
comprising administering to the individual or subject in need thereof a
compound of Formula
(I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or
a compound of Table
1, or stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing. In some embodiments, the disease is hypertension, valvular heart
diseases (aortic
stenosis, Mitral valve regurgitation), metabolic syndromes (diabetes,
obesity), end stage renal
disease, scleroderma, sleep apnea, amyloidosis, Fabry's disease, Friedreich
Ataxia, Danon
disease, Noonan syndrome, or Pompe disease.
[0099] Also provided herein is the use of a compound of Formula
(I), (I-A), (I-A1), (I-B),
(I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or
tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing, in the
manufacture of a medicament for treatment of a disease or condition associated
with
secondary left ventricular wall thickening.
[0100] In some embodiments, provided are methods of ameliorating a
symptom
associated with heart disease in a subject, comprising administering to the
individual or
subject in need thereof a compound of Formula (I), (I-A), (I-A1), (I-B), (I-
B1), (I-C). (I-C1),
(I-C2), (I-D), or (I-D1), or a compound of Table 1, or stereoisomer or
tautomer thereof, or a
pharmaceutically acceptable salt of any of the foregoing, wherein the symptom
is one or
more selected from poor or reduced cardiac elasticity, poor or reduced
diastolic left
ventricular relaxation, abnormal left atrial pressure (e.g., abnomally high
left atrial pressure),
paroxysmal or permanent atrial fibrillation, increased left atrial and
pulmonary capillary
wedge pressures, increased left ventricular diastolic pressures, syncope,
ventricular relaxation
during diastole, ventricular fibrosis, left ventricular hypertrophy, left
ventricular mass,
increased left ventricular wall thickness, left ventricular mid-cavity
obstruction, increased
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systolic anterior motion of mitral valve, left ventricular outflow tract
obstruction, chest pain,
exertional dyspnea, pre-syncope, abnormal exercise capacity, and fatigue.
[0101] In some embodiments, the provided are methods of reducing
the risk of a
symptom associated with heart disease in a subject, comprising administering
to the
individual or subject in need thereof a compound of Formula (I), (I-A), (I-
A1), (LB), (I-B 1),
(I-C), (I-Cl), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or tautomer
thereof, or a pharmaceutically acceptable salt of any of the foregoing,
wherein the symptom
is one or more selected from sudden cardiac death, poor or reduced cardiac
elasticity, poor or
reduced diastolic left ventricular relaxation, abnormal left atrial pressure
(e.g., abnomally
high left atrial pressure), paroxysmal or permanent atrial fibrillation,
increased left atrial and
pulmonary capillary wedge pressures, increased left ventricular diastolic
pressures, syncope,
ventricular relaxation during diastole, ventricular fibrosis, left ventricular
hypertrophy, left
ventricular mass, increased left ventricular wall thickness, left ventricular
mid-cavity
obstruction, increased systolic anterior motion of mitral valve, left
ventricular outflow tract
obstruction, chest pain, exertional dyspnea, pre-syncope, abnormal exercise
capacity. and
fatigue.
[0102] In some embodiments, the provided are methods of treating a
disease or condition
associated with small left ventricular cavity, cavity obliteration,
hyperdynamic left ventricular
contraction, obstruction of blood flow out of the left ventricle, cardiac
hypertrophy, small
cardiac stroke volume, impaired relaxation of the left ventricle, high left
ventricle filling
pressure, myocardial ischemia, or cardiac fibrosis in an individual or
subject, comprising
administering to the individual or subject in need thereof a compound of
Formula (I), (I-A),
(I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound
of Table 1, or
stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing.
[0103] In some embodiments, provided are methods of treating a
disease or condition
associated with small left ventricular cavity and cavity obliteration,
hyperdynamic left
ventricular contraction, myocardial ischemia, or cardiac fibrosis in an
individual or subject,
comprising administering to the individual or subject in need thereof a
compound of Formula
(I), (I-A), (I-A1), (I-B), (I-B 1), (I-C), (I-C1), (I-C2), (1-D), or (I-D1),
or a compound of Table
1, or stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt
of any of the
foregoing.
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[0104] Also provided herein is the use of a compound of Formula
(I), (I-A), (I-A1), (I-B),
(I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or
tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing, in the
manufacture of a medicament for treatment of a disease or condition associated
with small
left ventricular cavity and cavity obliteration, hyperdynamic left ventricular
contraction,
myocardial ischemia, or cardiac fibrosis.
[0105] In some embodiments, the provided are methods of treating
muscular dystrophies
in an individual or subject (e.g., duch*enne muscular dystrophy), comprising
administering to
the individual or subject in need thereof a compound of Formula (I), (I-A), (I-
A1), (I-B). (I-
B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or
tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing. Also provided
herein is the use of a compound of Formula (I), (I-A), (I-A1), (I-B), (I-B1),
(I-C), (I-C1), (I-
C2), (I-D), or (I-D1), or a compound of Table 1, or stereoisomer or tautomer
thereof, or a
pharmaceutically acceptable salt of any of the foregoing, in the manufacture
of a medicament
for treatment of muscular dystrophies (e.g., duch*enne muscular dystrophy).
[0106] In some embodiments, the provided are methods of treating a
glycogen storage
disease in an individual or subject, comprising administering to the
individual or subject in
need thereof a compound of Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C),
(I-C1), (I-C2), (I-
D), or (I-D1), or a compound of Table 1, or stereoisomer or tautomer thereof,
or a
pharmaceutically acceptable salt of any of the foregoing. Also provided herein
is the use of a
compound of Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2),
(I-D), or (I-D1),
or a compound of Table 1, or stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing, in the manufacture of a medicament
for treatment of a
glycogen storage disease.
[0107] Also provided are methods for modulating the cardiac
sarcomere in an individual
or subject which method comprises administering to an individual or subject in
need thereof a
therapeutically effective amount of at least one chemical entity as described
herein. In some
embodiments, provided are methods of inhibiting the cardiac sarcomere,
comprising
contacting the cardiac sarcomere with at least one chemical entity as
described herein, such
as a compound of Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-
C2), (I-D), or (I-
DI), or a compound of Table 1, or stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing. Additionally provided herein is the
use of at least one
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chemical entity as described herein, such as a compound of Formula (I), (I-A),
(I-A1), (I-B),
(I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or
tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing, in the
manufacture of a medicament for inhibiting the cardiac sarcomere of an
individual or subject.
[0108] Also provided are methods for potentiating cardiac myosin in
an individual or
subject which method comprises administering to an individual or subject in
need thereof a
therapeutically effective amount of at least one chemical entity as described
herein such as a
compound of Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2),
(I-D), or (I-D1),
or a compound of Table 1, or stereoisomer or tautomer thereof, or a
pharmaceutically
acceptable salt of any of the foregoing. Additionally provided herein is the
use of at least one
chemical entity as described herein, such as a compound of Formula (I), (I-A),
(I-A1), (I-B),
(I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of Table 1, or
stereoisomer or
tautomer thereof, or a pharmaceutically acceptable salt of any of the
foregoing, in the
manufacture of a medicament for potentiating cardiac myosin in an individual
or subject.
[0109] In some embodiments, the methods provided herein further
comprise monitoring
the effectiveness of the treatment. Examples of indicators include, but are
not limited to
improvement in one or more of the following: New York Heart Association (NYHA)

Functional Classification, exercise capacity, cardiac elasticity, diastolic
left ventricular
relaxation, left atrial pressure, paroxysmal or permanent atrial fibrillation,
left atrial and
pulmonary capillary wedge pressures, left ventricular diastolic pressures,
syncope, ventricular
relaxation during diastole, ventricular fibrosis, left ventricular
hypertrophy, left ventricular
mass, left ventricular wall thickness, left ventricular mid-cavity obstruction
systolic anterior
motion of mitral valve, left ventricular outflow tract obstruction. chest
pain, exertional
dyspnea, pre-syncope, abnormal exercise capacity, and fatigue. These
indicators can be
monitored by techniques known in the art including self-reporting; ECG,
including
ambulatory ECG; echocardiography; cardiac MR1; CT; biopsy; cardiopulmonary
exercise
testing (CPET); and actigraphy.
[0110] In some embodiments, the compound reduces the contractility
of a cardiomyocyte.
In some embodiments, the compound reduces the contractility of a cardiomyocyte
by greater
than 40%, such as greater than 45%, 50%, 60%, 70%, 80%, or 90%. In some
embodiments,
the compound reduced the contractility of a cardiomyocyte 40%-90%, such as 40%-
80%, 40-
70%, 50%-90%, 50%-80% or 50%-70%. In some embodiments, the compound does not
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significantly alter calcium transients in the cardiomyocyte. In some
embodiments, the
compound decreases the ATPase activity in a cardiamyocyte. Methods of
measuring
contractility, ATPase activity, and calcium transients are known in the art,
for example, by
calcium labeling, electrophysiological recordings, and microscopic imaging. In
some
embodiments, the compound does not significantly inhibit or induce a
cytochrome P450
(CYP) protein.
[0111] In some embodiments, provided herein are compounds of
Formula (I). (I-A), (I-
A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-D1), or a compound of
Table 1, or a
stereosiomer or tautomer thereof, or a pharmaceutically acceptable salt of any
of the
foregoing, wherein the elimination half-life (t1/2; calculated as ln(2)/k,
wherein the elimination
rate constant, k, is calculated as the absolute value of the slope of the
linear regression of
logarithm of the concentration versus time for the last three data points of a
concentration-
time profile) is < 30 hours in a human. In some embodiments, 10 hours < t1/2 <
30 hours in a
human. In some embodiments, t1/2 is between about 10 hours and about 30 hours,
between
about 10 hours and about 25 hours. between about 15 hours and about 30 hours.
or between
about 15 hours and about 25 hours. In some embodiments, t1/2 is about 12, 15,
18, 21, 24, 27,
or 30 hours. In some embodiments, the elimination half-life of a compound
provided herein
is such that the compound is suitable for once-daily dosing.
[0112] In some embodiments, the subject has a left ventricular wall
that is thicker than
normal prior to treatment. In some embodiments, the subject has a left
ventricular wall
thickness that is greater than 15 mm, such as greater than 18 mm, 20 mm, 22
mm, 25 mm, or
30 mm prior to treatment. In some embodiments, the left ventricular wall
thickness is
reduced by greater than 5%, such as greater than 8%, 10%, 12%, 15%, 20%, or
30%
following treatment. Left ventricular wall thickness can be measured by
methods known in
the art, such as by echocardiography, CT scan, or a cardiac MRI.
[0113] In some embodiments, the subject has abnormal cardiac
fibrosis prior to
treatment. In some embodiments, the abnormal cardiac fibrosis is reduced by
greater than
5%, such as greater than 8%, 10%, 12%, 15%, 20%, or 30% following treatment.
Cardiac
fibrosis can be measured by methods known in the art, such as by biopsy or a
cardiac MRT.
[0114] In some embodiments, the subject has reduced exercise
capacity prior to
treatment. In some embodiments, the exercise capacity of the subject is
increased by greater
than 5%, such as greater than 8%, 10%, 12%, 15%, 20% or 30% following
treatment. In
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some embodiments, the exercise capacity is measured by cardiopulmonary
exercise testing
(CPET). CPET measures changes in oxygen consumption (V02 max). Methods of
measuring CPET and V02 max are well known in the art (Malhotra et al., JACC:
Heart
Failure, 2016, 4(8): 607-616; Guazzi et al., J Amer College Cardiol, 2017, 70
(13): 1618-
1636; Rowin et al., JACC: Cariovasc Imaging, 2017, 10(11):1374-1386). In some
embodiments, V02 max is improved by more than 1 mL/kg/m2, such as more than
1.2
mL/kg/m2. 1.4 mL/kg/m2, 1.5 mL/kg/m2, 1.7 mL/kg/m2, 2 mL/kg/m2, 2.2 mL/kg/m2,
2.5
mL/kg/m2. 3 mL/kg/m2, 3.2 mL/kg/m2, or 3.5 mL/kg/m2 following treatment.
[0115] In some embodiments, the subject has a New York Heart
Association (NYHA)
Functional Classification of II, III, or IV prior to treatment. In some
embodiments, the
subject has a New York Heart Association (NYHA) Functional Classification of
III or IV
prior to treatment. In some embodiments, the subject has a New York Heart
Association
(NYHA) Functional Classification of IV prior to treatment. In some
embodiments, the
subject remains in the same NYHA functional class or has a reduced NYHA
functional class
following treatment.
[0116] In some embodiments, V09 max is improved by more than 1
mL/kg/m2, such as
more than 1.2 mL/kg/m2, 1.4 mL/kg/m2, 1.5 mL/kg/m2, 1.7 mL/kg/m2, or 2
mL/kg/m2 and the
subject has a reduced NYHA functional class following treatment. In some
embodiments.
V02 max is improved by more than 2.5 mL/kg/m2, 3 mL/kg/m2, 3.2 mL/kg/m2, or
3.5
mL/kg/m2 and the subject remains in the same NYHA functional class or has a
reduced
NYHA functional class following treatment.
[0117] In some embodiments, daily function and/or activity level of
the subject is
improved following treatment. Improved daily function and/or activity level
may be
measured, for example, by journaling or actigraphy, such as a FitBit or FitBit-
like monitors.
[0118] In some embodiments, the subject has one or more of
decreased shortness of
breath, decreased chest pain, decreased arrhythmia burden, such as atrial
fibrillation and
ventricular arrhythmias, decreased incidence of heart failure, and decreased
ventricular
outflow obstruction following treatment.
Dosages
[0119] The compounds and compositions disclosed and/or described
herein are
administered at a therapeutically effective dosage, e.g., a dosage sufficient
to provide
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treatment for the disease state. While human dosage levels have yet to be
optimized for the
chemical entities described herein, generally, a daily dose ranges from about
0.01 to 100
mg/kg of body weight; in some embodiments, from about 0.05 to 10.0 mg/kg of
body weight,
and in some embodiments, from about 0.10 to 1.4 mg/kg of body weight. Thus,
for
administration to a 70 kg person, in some embodiments, the dosage range would
be about
from 0.7 to 7000 mg per day; in some embodiments, about from 3.5 to 700.0 mg
per day, and
in some embodiments, about from 7 to 100.0 mg per day. The amount of the
chemical entity
administered will be dependent, for example, on the subject and disease state
being treated,
the severity of the affliction, the manner and schedule of administration and
the judgment of
the prescribing physician. For example, an exemplary dosage range for oral
administration is
from about 5 mg to about 500 mg per day, and an exemplary intravenous
administration
dosage is from about 5 mg to about 500 mg per day, each depending upon the
compound
pharmaco*kinetics.
[0120] A daily dose is the total amount administered in a day. A
daily dose may be, but
is not limited to be, administered each day, every other day, each week, every
2 weeks, every
month, or at a varied interval. In some embodiments, the daily dose is
administered for a
period ranging from a single day to the life of the subject. In some
embodiments, the daily
dose is administered once a day. In some embodiments, the daily dose is
administered in
multiple divided doses, such as in 2, 3, or 4 divided doses. In some
embodiments, the daily
dose is administered in 2 divided doses.
[0121] Administration of the compounds and compositions disclosed
and/or described
herein can be via any accepted mode of administration for therapeutic agents
including, but
not limited to, oral, sublingual, subcutaneous, parenteral, intravenous,
intranasal, topical,
transderrnal, intraperitoneal, intramuscular, intrapulmonary, vagin*l, rectal,
or intraocular
administration. In some embodiments, the compound or composition is
administered orally or
intravenously. In some embodiments, the compound or composition disclosed
and/or
described herein is administered orally.
[0122] Pharmaceutically acceptable compositions include solid, semi-
solid, liquid and
aerosol dosage forms, such as tablet, capsule, powder, liquid, suspension,
suppository, and
aerosol forms. The compounds disclosed and/or described herein can also be
administered in
sustained or controlled release dosage forms (e.g., controlled/sustained
release pill, depot
injection, osmotic pump, or transdermal (including electrotransport) patch
forms) for
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prolonged timed, and/or pulsed administration at a predetermined rate. In some
embodiments,
the compositions are provided in unit dosage forms suitable for single
administration of a
precise dose.
[0123] The compounds disclosed and/or described herein can be
administered either
alone or in combination with one or more conventional pharmaceutical carriers
or excipients
(e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine,
talcum, cellulose,
sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate). If
desired, the
pharmaceutical composition can also contain minor amounts of nontoxic
auxiliary substances
such as wetting agents, emulsifying agents, solubilizing agents, pH buffering
agents and the
like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives,
sorbitan monolaurate,
triethanolamine acetate, triethanolamine oleate). Generally, depending on the
intended mode
of administration, the pharmaceutical composition will contain about 0.005% to
95%, or
about 0.5% to 50%, by weight of a compound disclosed and/or described herein.
Actual
methods of preparing such dosage forms are known, or will be apparent, to
those skilled in
this art; for example, see Remington 's Pharmaceutical Sciences, Mack
Publishing Company,
Easton, Pennsylvania.
[0124] In some embodiments, the compositions will take the form of
a pill or tablet and
thus the composition may contain, along with a compounds disclosed and/or
described
herein, one or more of a diluent (e.g., lactose, sucrose, dicalcium
phosphate), a lubricant (e.g.,
magnesium stearate), and/or a binder (e.g., starch, gum acacia,
polyvinylpyrrolidine, gelatin,
cellulose, cellulose derivatives). Other solid dosage forms include a powder,
marume,
solution or suspension (e.g., in propylene carbonate, vegetable oils or
triglycerides)
encapsulated in a gelatin capsule.
[0125] Liquid pharmaceutically administrable compositions can, for
example, be
prepared by dissolving, dispersing or suspending etc. a compound disclosed
and/or described
herein and optional pharmaceutical additives in a carrier (e.g., water,
saline, aqueous
dextrose, glycerol, glycols, ethanol or the like) to form a solution or
suspension. Injectablcs
can be prepared in conventional forms, either as liquid solutions or
suspensions, as
emulsions, or in solid forms suitable for dissolution or suspension in liquid
prior to injection.
The percentage of the compound contained in such parenteral compositions
depends, for
example, on the physical nature of the compound, the activity of the compound
and the needs
of the subject. However, percentages of active ingredient of 0.01% to 10% in
solution are
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employable, and may be higher if the composition is a solid which will be
subsequently
diluted to another concentration. In some embodiments, the composition will
comprise from
about 0.2 to 2% of a compound disclosed and/or described herein in solution.
[0126] Pharmaceutical compositions of the compounds disclosed
and/or described herein
may also be administered to the respiratory tract as an aerosol or solution
for a nebulizer, or
as a microfine powder for insufflation, alone or in combination with an inert
carrier such as
lactose. In such a case, the particles of the pharmaceutical composition may
have diameters
of less than 50 microns, or in some embodiments, less than 10 microns.
[0127] In addition, pharmaceutical compositions can include a
compound disclosed
and/or described herein and one or more additional medicinal agents,
pharmaceutical agents,
adjuvants, and the like. Suitable medicinal and pharmaceutical agents include
those
described herein.
Kits
[0128] Also provided are articles of manufacture and kits
containing any of the
compounds or pharmaceutical compositions provided herein. The article of
manufacture may
comprise a container with a label. Suitable containers include, for example,
bottles, vials,
and test tubes. The containers may be formed from a variety of materials such
as glass or
plastic. The container may hold a pharmaceutical composition provided herein.
The label on
the container may indicate that the pharmaceutical composition is used for
preventing,
treating or suppressing a condition described herein, and may also indicate
directions for
either in vivo or in vitro use.
In one aspect, provided herein are kits containing a compound or composition
described
herein and instructions for use. The kits may contain instructions for use in
the treatment of a
heart disease in an individual or subject in need thereof. A kit may
additionally contain any
materials or equipment that may be used in the administration of the compound
or
composition, such as vials, syringes, or IV bags. A kit may also contain
sterile packaging.
Combinations
[0129] The compounds and compositions described and/or disclosed
herein may be
administered alone or in combination with other therapies and/or therapeutic
agents useful in
the treatment of the aforementioned disorders, diseases, or conditions.
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[0130] The compounds and compositions described and/or disclosed
herein may be
combined with one or more other therapies to treat a heart disease, such as
HCM or HFpEF.
In some embodiments, the one or more therapies include therapies that retard
the progression
of heart failure by down-regulating neurohormonal stimulation of the heart and
attempt to
prevent cardiac remodeling (e.g., ACE inhibitors, angiotensin receptor
blockers (ARB s). 13-
blockers, aldosterone receptor antagonists, or neural endopeptidase
inhibitors). In some
embodiments, the one or more therapies include therapies that improve cardiac
function by
stimulating cardiac contractility (e.g., positive inotropic agents, such as
the I3-adrenergic
agonist dobutamine or the phosphodiesterase inhibitor milrinone). In other
embodiments, the
one or more therapies include therapies that reduce cardiac preload (e.g.,
diuretics, such as
furosemide) or afterload (vasodilators of any class, including but not limited
to calcium
channel blockers, phosphodiesterase inhibitors, endothelin receptor
antagonists, renin
inhibitors, or smooth muscle myosin modulators).
[0131] The compounds and compositions described and/or disclosed
herein may be
combined with one or more other therapies to treat HCM or HFpEF. In some
embodiments,
the compounds and/compositions may be combined with a P-blocker, verapamil,
and/or
disopyramide.
General Synthetic Methods
[0132] Compounds of Formula (1), (1-A), (1-A1), (1-B), (1-B1), (1-
C), (I-C1), (1-C2), (1-
D), or (I-D1) will now be described by reference to illustrative synthetic
schemes for their
general preparation below and the specific examples that follow. Artisans will
recognize
that, to obtain the various compounds herein, starting materials may be
suitably selected so
that the ultimately desired substituents will be carried through the reaction
scheme with or
without protection as appropriate to yield the desired product. Alternatively,
it may be
necessary or desirable to employ, in the place of the ultimately desired
substituent, a suitable
group that may be carried through the reaction scheme and replaced as
appropriate with the
desired substituent. In addition, one of skill in the art will recognize that
protecting groups
may be used to protect certain functional groups (amino, carboxy, or side
chain groups) from
reaction conditions, and that such groups are removed under standard
conditions when
appropriate. Unless otherwise specified, the variables are as defined above in
reference to
Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D), or (I-
D1).
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[0133] Where it is desired to obtain a particular enantiomer of a
compound, this may be
accomplished from a corresponding mixture of enantiomers using any suitable
conventional
procedure for separating or resolving enantiomers. Thus, for example,
diastereomeric
derivatives may be produced by reaction of a mixture of enantiomers, e.g. a
racemate, and an
appropriate chiral compound. The diastereomers may then be separated by any
convenient
means, for example by crystallization and the desired enantiomer recovered. In
another
resolution process, a racemate may be separated using chiral High Performance
Liquid
Chromatography. Alternatively, if desired a particular enantiomer may be
obtained by using
an appropriate chiral intermediate in one of the processes described.
[0134] Chromatography, recrystallization and other conventional
separation procedures
may also be used with intermediates or final products where it is desired to
obtain a particular
isomer of a compound or to otherwise purify a product of a reaction.
[0135] General methods of preparing compounds described herein are
depicted in
exemplified methods below. Variable groups in the schemes provided herein are
defined as
for Formula (I), (I-A), (I-A1), (I-B), (I-B1), (I-C), (I-C1), (I-C2), (I-D),
or (I-D1), or any
variation thereof. Other compounds described herein may be prepared by similar
methods.
[0136] In some embodiments, compounds provided herein may be
synthesized according
to Scheme 1.
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Scheme 1
R1
R2 R1 R1
0 R2, R2
0
1110
NH2 C1CI 0
AcOH, NaCN ___________________________ .
NH
N.J.L.C1
0 THF-H20, 60 C >ro...õ,N TEA,
CH2Cl2CN 0 C to rt >r0,1rNICN
II
0 0
H2N10 Rx R1 R1
R2 R2
l
110 0
(HCI salt) AcOH, Et0H, 80 C
Njii\j'0 N'il
DIEA, MeCN, 65 C No,
0N ,,ICN Rx
0
'Rx
Oil 0
R1 0 R1
R2 HO)1-1<F R2
F 0
0 F (.1 0
TEA, CH2Cl2 H)L0----'t F3
NJH N jj'l
DIEA, CH2Cl2
HNij'IrN40 OyNdYNO
or 0
0
'Rx ONa RY
N!'
AcOH, MeCN, 55 C
[0137] In Scheme 1, R1 and R2 are as defined elsewhere herein for a
compound of
Formula (I), or any variation or embodiment thereof, or a stereoisomer or
tautomer thereof, or
a pharmaceutically acceptable salt of any of the foregoing; Rx is C1_6alkyl or
C1_6haloalkyl;
and R3' is H or -NH).
[0138] In some embodiments, compounds provided herein may be
synthesized according
to Scheme 2.
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Scheme 2
R1
R1
R1
R2,
R2
0
410 NH2 j R2 ,--0
Br)-1..õ,,,,.Br 140 0
0 ' NH
Boc--Nijir STAB, AcOH, 0 DOE, , o/n (0----\ K2003,
DCM/H20
rt
Boo 'N
0,, _
0 0 C¨rt '

Boo"- 0
R1 R1
R2 R2
H2N4.0
trans
1111 0 0
TFA
___________________________ ' ____ N)L-1 _________________________ N)L1
trans
TEA, ACN, rt-80 C trans
N
13oC"Nrjy IN ARO DCM
HNri'lr 4.0
0 0
R1
R2 0
0 0 0
_________________________________________________ õ.
N,11.1trans
reflux
0,..,NY N40
or
1 0
NaCN, AcOH/THF RY
or TMSCN, TEA, THF
[0139] In Scheme 2, RI and R2 are as defined elsewhere herein for a
compound of
Formula (I), or any variation or embodiment thereof, or a stereoisomer or
tautomer thereof, or
a pharmaceutically acceptable salt of any of the foregoing; and RY is H or -
NH2.
[0140] In some embodiments, compounds provided herein may be
synthesized according
to Scheme 3.
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Scheme 3
R1
R1
R2 0
R2, 0
NH2 j Br'.&-Br
0
0 ,.. ___________________________
,..
Boc,Nd'Y STAB, AcOH, DCE, it, o/n NH
0
0----..,, K2CO3, DCIVI/H20, 0 C--rt
Boc.õNrir
0
R1 R1
R2 R1
R2
0 0 H2N,r-
el R2
)1,,,,õõBr ________________________________ .- 0 TFA 110 0
N _________________________________________________________________ >
TEA, ACN, rt-80 C Nj(.1 Boc DCM N)(-1
Boc,NdY '"---
,Nii N'T-'-
0 1 HNI-j-
IiN-T-
0 0
R1
R2 =NUN
Br 0
N7

0
N)1
TFA, CH2Cl2 -) _____________________________________________________________
.-
Pd-PEPPSI-IPentC1 2-methylpyridine(o-plcoline),
Cs2CO3, dioxane, reflux
N
,N Nd---ir N.N.r-
,
,N CI
R1 0 1. NI `- R1
0 .--
R2 R2,
011 HO)Li(F
F
0

N Al 0
K2003, IPA, 120 C
2. Li0H, THF N.TD
H NI N 3. PhOPh, 180 C
0
(TFA salt)
[0141] In Scheme 3, RI and R2 are as defined elsewhere herein for a
compound of
Formula (I), or any variation or embodiment thereof, or a stereoisomer or
tautomer thereof, or
a pharmaceutically acceptable salt of any of the foregoing.
[0142] Particular non-limiting examples are provided in the Example section
below.
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EXAMPLES
[0143] The following examples are offered to illustrate but not to
limit the compositions,
uses, and methods provided herein. The compounds are prepared using the
general methods
described above.
[0144] The following abbreviations may be used throughout the
Examples: TEA
(trimethylamine), DCM (dichloromethane), (Boc)20 (di-tert-butyl decarbonate),
EA (Ethyl
acetate), PE (Petroleum ether, DMF (N,N-dimethylformamide), DIEA (N-ethyl-N-
isopropylpropan-2-amine), HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-
b]pyridinium 3-oxid hexafluorophosphate), HOAt (1-Hydroxy-7-azabenzotriazole),
HOBt
(Hydroxybenzotriazole), EDCI (1-Ethyl-3-(3-dimethylanainopropyl)carbodiimide),
Me0H
(methanol), Et0H (ethanol). WA (iPrOH; propan-2-ol), NMP (1-methylpyrrolidin-2-
one),
STAB (sodium triacetoxyhydroborate), ACN (acetonitrile), TFA (trifluoroacetic
acid), DPPA
(Diphenylphosphoryl azide), DBU (1,8-Diazabicyclo(5.4.0)undec-7-ene), TI-IF
(tetrahydrofuran), PPh3 (triphenylphosphane), SM (starting material), Hex
(hexane), NCS (N-
chlorosuccinimide), r.t. (room temperature), DCE (dichloroethane), FA (formic
acid), CHC13
(Chloroform), BnBr (benzyl bromide). HC1 (hydrogen chloride), equiv
(equivalent), RT
(retention time). SEC (supercritical fluid chromatography), and DSC (bis(2,5-
dioxopyrrolidin-l-y1) carbonate).
[0145] XRPD diffractograms were collected using the following
parameters:
[0146] XRPD Machine: Rigaku MiniFlex600 6G Benchtop X-ray
Diffraction System;
X-ray Generator: Delivering 600W of Power (40 kV / 15 mA); Sealed-off X-ray
Tube:
Toshiba A-21-Cu Tube w/ Normal Focus; X-ray radiation: CuKa; Software:
Smartlab Studio
II x64 ver. 4.5.352.0 (data collection & analysis); Incident Soller Slit:
2.5'; Divergence
Slit:1.25'; Length Limiting Slit: 10; Sample Stage: ASC-8 PM_MF; Filter: Cu
beta X1.5;
Receiving Soller Slit: 2.5'; Scattering Slit: 8.0 mm; Receiving Slit: 0.3 mm;
Detector: High
Speed D/tex Utra2 ME RAC; Power: 40 kV / 15 mA; Scanning Rate: 2 0 / min;
Step: 0.01'0;
Scanning Range: 3' ¨ 30' 0 or 3' ¨ 40' 0; Sample Holder: Zero Background Si
Sample
Holder w/ 0.2 mm Indent.
Example 1
Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-
2,5,8-
triazaspiro[3.5]nonane-2-carbaldehyde
(Compound 4)
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Step 1: Synthesis of 1-(tert-butyl) 3-ethyl 34(3,4-
difluorobenzypamino)azetidine-1,3-
dicarboxylate:
F
F
0
NH2 j
NH
Boc,Nrilro STAB, AcOH, DCE, rt, o/n
0
4-a
[0147] To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1,3-
dicarboxylate (4.0 g,
16.4 mmol, 1.0 equiv) and 3,4-difluorobenzaldehyde (2.4 g, 19.6 mmol, 1.2
equiv) in DCE
(40.0 mL) at 0 C were added STAB (7.0 g, 32.8 mmol, 2.0 equiv) and AcOH (2.0
g, 32.8
mmol, 2.0 equiv). The resulting mixture was stiffed at r.t. overnight,
adjusted the pH to 8
with ammonium hydroxide, added water (50.0 mL) and extracted with DCM (50.0
mL)
twice. The combined organic layers were washed with brine (50 mL) twice, dried
over
anhydrous Na2SO4, and concentrated under reduced pressure to afford 6.0 g of 1-
tert-butyl 3-
ethyl 3-(3,4-difluorobenzyl)amino)azetidine-1,3-dicarboxylate as a yellow oil.
LRMS (ES)
m/z 315 (M+H-56).
Step 2: Synthesis of 1-(tert-butyl) 3-ethyl 3-(2-bromo-N-(3,4-
difluorobenzyl)acetamido)azetidine-1,3-dicarboxylate:
F
0
Br le) 0
NHBr
K2CO3, DCM/H20, 0 C-rt
Boc 0 Boc-
"Nrj'ir
0
4-a 4-b
[0148] To a solution of 1-tert-butyl 3-ethyl
3-[[(3,4-
difluorophenyl)methyl]amino]azetidine-1,3-dicarboxylate (6.0 g, 16.2 nunol,
1.0 equiv) in
DCM (60.0 mL) at 0 C were added a solution of K2CO3 (3.4 g, 24.3 mmol, 1.50
equiv) in
water (30 mL), and then bromoacetyl bromide (3.9 g, 19.4 mmol, 1.2 equiv)
dropwise over a
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period of 10 min. The resulting mixture was stirred at r.t. overnight and
extracted with DCM
(50.0 mL) twice. The combined organic layers were washed with brine (100 mL)
twice, dried
over anhydrous Na2SO4, and concentrated under reduced pressure to afford 8.0 g
of 1-(tert-
butyl) 3-ethyl 3- (2-bromo-N-(3 ,4-difluorob enzyl)acetamido) azetidine- 1,3 -
dic arboxylate as a
yellow oil. LRMS (ES) m/z 435 (M+H-56).
Step 3: Synthesis of tert-butyl 5 -(3 ,4-difluorobenzy1)- 8-((1r,4 r)-4-
methylc yclohexyl)-6,9-
dioxo-2,5 ,8-triazaspiro[3 .5] nonane-2-carboxylate :
Si 0 H2N.3/40
trans F
..,õ
N)*Li trans
TEA, ACN, rt-80 C
Boc Boc'NdiN ))
0 0
4-b
4-c
[0149] To a solution of 1-(tert-butyl) 3-ethyl
3 -(2-bro mo-N-(3 ,4-
difluorobenzyl)acetamido)azetidine-1,3-dicarboxylate (8.0 g, 16.3 mmol, 1.0
equiv) in ACN
(80 mL) were added TEA (4.9 g, 48.4 mmol, 3.0 cquiv) and trans-(1r,40-4-
methylcyclohexan-
1-amine (2.8 g, 24.7 mmol, 1.5 equiv). The resulting mixture was stirred at
r.t. for 1 h,
gradually warmed to 80 C, and stirred at 80 C overnight. The mixture was
cooled to r.t.,
concentrated under reduced pressure, and triturated with a mixture of PE and
EA (7/1; 80 mL)
to afford 7 g (-80% purity) of tert-butyl 5-(3,4-difluorobenzy1)-8-((lr,40-4-
methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate as an
off-white
solid. LRMS (ES) m/z 422 (M+H-56).
Step 4: Synthesis of 5 -(3,4-difluorobenzy1)- 8-((lr,40-4-meth ylc yclohexyl)-
2,5,8-
triaz aspiro [3 .5 ] nonane-6,9-dione:
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F
161 0 0
TFA
N j.L) trans
trans
Boc
Nri-T 1`14.0 DCM
HNri'lr N41/40
0 0
4-c 4-d
[0150] To a stirred solution of tert-butyl 5-(3,4-difluorobenzy1)-8-((1r,4r)-4-

methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (7.0 g,
14.7 mmol,
1.0 equiv) in DCM (70.0 mL) was added TFA (18.0 mL). The resulting mixture was
stirred at
r.t. for 3h, diluted with water (100.0 mL), adjusted the pH to 13-14 with
aqueous NaOH
solution (2 N), and extracted with DCM (100 mL) twice. The combined organic
layers were
washed with brine (100.0 mL) twice, dried over anhydrous Na2SO4, and
concentrated under
reduced pressure to afford 4.5 g (-80% purity) of 5-(3,4-difluorobenzy1)-8-
((1r,4r)-4-
methylcyclohexyl)-2,5,8-triazaspiro113.51nonane-6.9-dione as a yellow semi-
solid. LRMS (ES)
m/z 378 (M+H).
Step 5: Synthesis of 5-(3,4-difluorobenzy1)-8-((1r,40-4-methylcyclohexyl)-6,9-
dioxo-2,5,8-
triazaspiro[3.5]nonane-2-carbaldehyde (Compound 4):
F
0
111111 0
0 0
N ).L1 trans N-it)trans
reflux
0 440
0
4-d
Compound 4
[0151] A solution of tert-butyl 5 -(3 ,4-difluorobenzy1)- 8-((1r,4
r)-4-methylc yclohexyl)-6,9-
di oxo-2,5,8-tri azaspiroP .5Thonane-2-carboxy I ate (1.5 g, 4.0 mmol, 1 .0
equiv) in ethyl formate
(15.0 mL) was stirred at 80 C overnight. The mixture was cooled to r.t.,
concentrated under
reduced pressure, and purified by C18 column chromatography, eluted with a
mixture of water
(0.05% NH4HCO3)/CH3CN (3:2) to afford 1.3 g (81%) of 5-(3,4-difluorobenzy1)-8-
((lr,40-
4-methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde as
an amorphous
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white solid. An experimental X-ray powder diffraction (XRPD) pattern of this
amorphous
white solid is shown in FIG. 1 LRMS (ES) na/z 406 (M+H); IFI NMR (300 MHz,
DMSO-d6)
6 7.96 (s. 1H). 7.47 - 7.29 (m, 2H), 7.10 (ddd, J = 9.4, 4.4, 2.0 Hz, 1H),
4.82 (s, 2H), 4.50 (d,
J = 9.6 Hz, 1H), 4.15-4.28 (m, J = 3H), 4.01 (s, 2H), 3.96 (d, J = 10.8 Hz,
1H). 1.80- 1.69 (m,
2H), 1.65 - 1.48 (m, 4H), 1.35 (d, J = 10.9 Hz, 1H), 1.13 - 0.93 (m, 2H), 0.88
(d, J = 6.5 Hz,
3H).
Example 2
Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-
2,5,8-
triazaspiro[3.5]nonane-2-carboxamide
(Compound 5)
Step 1: Synthesis of 5-(3,4-difluorobenzy1)-8-((lr,4r)-4-methylcyclohexyl)-6,9-
dioxo-2,5,8-
triazaspiro[3.5]nonane-2-carboxamide (Compound 5):
F
I -.0 F
0 0
NA1 trans N).1.) trans
Hiv TEA, THF, rt, o/n
rirN40
0 I 0
NH2
4-d Compound 5
[0152] To a stirred solution of tert-butyl 5-(3,4-difluorobenzy1)-8-((lr,4r)-4-

methylcyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (1.5 g,
4.0 mmol, 1.0
equiv) in THF (15.0 mL) at 0 C were added TEA (1.2 g, 11.9 mmol, 3.0 equiv)
and
isocyanatotrimethylsilane (685 mg. 6.0 mmol, 1.5 equiv) dropwise over a period
of 5 min. The
resulting mixture was stirred at r.t. overnight, concentrated under reduced
pressure, and
triturated first with a mixture of PE and EA (5/1; 20 mL) and then with hexane
(20 mL) to
afford 1.4 g (84%) of 5-(3,4-difluorobenzy1)-8-((1r,40-4-methylcyclohexyl)-6,9-
dioxo-2,5,8-
triazaspiro[3.5]nonanc-2-carboxamide as an off-white solid. LRMS (ES) m/z 421
(M+H); 114
NMR (400 MHz, DMSO-d6) 6 7.55 - 7.30 (m, 2H), 7.09 (dd, J = 8.0, 4.3 Hz, 1H),
6.00 (s,
2H), 4.82 (s, 2H), 4.20 (t, J = 9.1 Hz, 3H), 3.99 (s, 2H), 3.84 (d, J = 9.3
Hz, 2H), 1.74 (d. J =
12.9 Hz, 2H), 1.58 (dtt, J = 20.6, 12.1, 6.1 Hz, 4H), 1.42 - 1.29 (m, 11-1),
1.04 (qd, J = 12.2, 4.5
Hz, 2H), 0.88 (d, J = 6.5 Hz, 3H).
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Compounds in the following table were prepared in a similar manner as Compound
5.
No. Structure Name and Data
5-(4-chlorobenzy1)-8-((1r,40-4-
ethylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-2-carboxamide.
CI
LRMS (ES) m/z 433.1 (M+H); 11-1 NMR (400
MHz, Methanol-d4) 6 7.35 (d, J = 8.2 Hz, 2H),
0
1
N ).H 7.28 (d, J = 8.2 Hz, 2H), 4.95
(s, 2H), 4.40 (d, J
= 9.4 Hz. 2H), 4.30 (tt, J = 12.3, 3.9 Hz, 1H),
ON o 4.10 - 4.06 (m, 4H), 1.91 (d, J = 12.0 Hz, 2H),
NH2 I 1.78 - 1.70 (m, 2H), 1.58 (qd, J = 12.0, 2.6 Hz,
2H), 1.27 (p, J = 7.1 Hz, 2H), 1.20 - 1.02 (m,
3H), 0.92 (t, J = 7.4 Hz, 3H).
8-((lr,40-4-ethylcyclohexyl)-5-(4-
F fluorobenzy1)-6,9-dioxo-2,5,8-
S triazaspiro[3.5]nonane-2-
carboxamide.
LRMS (ES) m/z 417.2 (M+H). 1H NMR (400
0 MHz, Methanol-d4) 6 7.31 (dd, J =
8.4, 5.5 Hz,
2
N ).L1 2H), 7.07 (t, J = 8.5 Hz, 2H),
4.96 (s, 2H), 4.39
(d, J = 9.4 Hz, 2H), 4.31 (td, J = 12.1, 6.0 Hz,
1H), 4.09 (d, J = 10.1 Hz, 4H), 1.91 (d, J = 12.5
Coy Ndli
Hz, 2H), 1.74 (d, J = 10.3 Hz, 2H), 1.59 (qd, J =
NH2
12.0, 2.8 Hz, 2H), 1.27 (p, J = 7.1 Hz, 2H), 1.21
- 1.02 (m, 3H), 0.92 (t, J = 7.4 Hz, 3H).
Example 3
Synthesis of 5-(4-chloro-3-fluorobenzy1)-8-isopropyl-2-(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione
(Compound 8)
Step 1: Synthesis of 1-(tert-butyl) 3-ethyl 3-(4-chloro-3-
fluorobenzyl)amino)azetidine-1,3-
dicarboxylate:
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CI
CI
F
F
NH2 j
H
NH
Boc,Nrir STAB, AcOH, DCE, rt,
o/n
0
Boc,NrjT
0
8-a
[0153] To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1.3-
dicarboxylate (2.0 g.
8.2 mmol, 1.0 equiv) and 4-chloro-3-fluorobenzaldchyde (2.0 g, 12.3 mmol, 1.5
cquiv) in
DCE (20.0 mL) at 0 C were added ACOH (984 mg, 16.4 mmol, 2.0 equiv.) and STAB
(3.5
g, 16.5 mmol, 2.0 equiv) in portions. The resulting mixture was stirred at
r.t. overnight,
adjusted the pH to 8 with ammonium hydroxide, added water (30.0 mL) and
extracted twice
with DCM (30.0 mL). The combined organic layers were washed with brine (30.0
mL) twice,
dried over anhydrous Na2SO4, and concentrated under reduced pressure to afford
3.0 g of 1-
tert-butyl 3-ethyl 3-(4-chloro-3-fluorobenzyl)amino)azetidine-1,3-
dicarboxylate as a yellow
oil. LRMS (ES) miz 331 (M+H-56).
Step 2: Synthesis of 1-(tert-butyl) 3-ethyl 3-(2-bromo-N-(4-chloro-3-
fluorobenzyl)acetamido)azetidine-1,3-dicarboxylate:
CI CI
F
0
Br)1..õ,..,Br 0 lel
NH
Boc If K2CO3, DCM/H20, 0 C-
rt
Boc,NdY
0 0
8-a 8-b
[0154] To a stirred solution of 1-tert-butyl 3-ethyl 3-(4-chloro-3-
fluorobenzyl)amino)azetidine-1,3-dicarboxylate (3.0 g, 7.8 mmol, 1.0 equiv) in
DCM (30.0
mL) at 0 C were added a solution of K2CO3 (1.7 g, 12.3 mmol, 1.50 equiv) in
water (15 mL),
and then bromoacetyl bromide (1.9 g, 9.4 mmol, 1.2 equiv) dropwise over a
period of 5 mm.
The resulting mixture was stirred at r.t. overnight and extracted with DCM
(40.0 mL) twice.
The combined organic layers were washed with brine (40 mL) twice, dried over
anhydrous
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Na2SO4, concentrated under reduced pressure, and purified by C18 column
chromatography,
eluted with water (0.5% ammonium carbonate) and ACN (1/4) to afford 3.9 g
(89%) of 1-(tert-
butyl) 3-ethyl 3-(2-bromo-N-(4- chloro -3 -fluorobenzyl)ac etamido)azetidine-
1,3 -dic arbox ylate
as a yellow oil. LRMS (ES) miz 451 (M+H-56).
Step 3: Synthesis of tert-butyl 5-(4-chloro-3-fluorobenzy1)-8-isopropy1-6,9-
dioxo-2,5,8-
triazaspiro [3 .5] nonane-2-c arboxylate:
F Br CI CI
41111 0 4110 0
-J.L1
TEA, ACN, rt-80 C N
Boc,NOY
Boc,NrjYNT--
0 0
8-b 8-c
[0155] To a solution of 1-(tert-butyl) 3-ethyl 3-(2-bromo-N-(4-chloro-3-
fluorobenzyl)acetamido)azetidine-1,3-dicarboxylate (1.5 g, 3.0 mmol, 1.0
equiv) in ACN (20
mL) were added TEA (898 mg, 8.9 mmol, 3.0 equiv) and isopropylamine (262 mg,
4.4 mmol,
1.5 equiv). The resulting mixture was stirred at r.t. for 1 h, gradually
warmed to 80 C, and
stirred at 80 C overnight. The mixture was cooled to r.t. and added water
while stirring. The
precipitated solids were collected by filtration and washed with water (50.0
mL) to afford 1.1
g (85%) of tert-butyl 5-(4-chloro-3 -fluorobenzy1)-
84 soprop y1-6,9- dioxo-2,5, 8-
triazaspiro[3.5]nonanc-2-carboxylate as an off-white solid. LRMS (ES) m/z 384
(M+H-56).
Step 4: Synthesis of 5 -(4-chloro-3-flu orobenzy1)-8-isoprop y1-2,5 ,8 -
triazaspiro [3 .5] nonane-
6,9-di one:
CI
CI
0 TFA
N
DCM Wit)
NrjYN-r-
DOG'. HNIJYN-r-
0 0
8-c 8-d
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[0156] To a stirred solution of tert-b utyl 5-(4-chloro-3-
fluorobenzy1)-8-isopropy1-6,9-
dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (1.1 g, 2.4 mmol, 1.0 equiv)
in DCM (12.0
mL) was added TFA (3.0 nth). The resulting mixture was stirred at r.t. for 3
h, diluted with
water (20.0 mL), adjusted the pH to 13-14 with aqueous NaOH solution (2N), and
extracted
with DCM (20 mL) twice. The combined organic layers were washed with brine
(20.0 mL)
twice, dried over anhydrous Na2SO4, and concentrated under reduced pressure to
afford 950
mg of 5-(4-chloro-3 -fluorobenzy1)- 8-isopropyl-2,5, 8-triazaspiro [3.5]
nonane- 6,9-dione as a
yellow solid. LRMS (ES) m/z 340 (M-FH).
Step 5: Synthesis of 5 -(4-chloro-3 -flu orobenzy1)-8-isoprop y1-2-(p yrid
azin- 3 -y1)-2,5,8-
triazaspiro [3.5]nonane-6,9-dione (Compound 8):
CI
CI
N rB
141111
0
0 ____________________________________________________________
N-A-1
Pd-PEPPS1-1PentC1 2-methylpyridine(o-plcoline),
Cs2CO3, dioxane, reflux
HdY N 0
0
8-d Compound
8
[0157] To a stirred solution of 5-(4-chloro-3 - fluorobenzyl) - 8-
is oprop y1-2,5 ,8-
triazaspiro [3.5]nonane-6,9-dione (950 mg, 2.8 mmol, 1.0 equiv) and 3-
bromopyridazine (662.2
mg, 4.2 mmol, 1.5 equiv) in dioxane (10 mL) were added Cs2CO3 (1.8 g, 5.5
mmol, 2.0 equiv)
and Pd-PEPPSI-IPentC12-methylpyridine (o-picoline) (117.6 mg, 0.14 mmol, 0.05
equiv). The
resulting mixture was stirred at 90 "C overnight under nitrogen atmosphere.
The mixture was
allowed to cool down to r.t., filtered to remove solids, and purified by C18
column
chromatography, eluted with water (0.05% ammonium carbonate)/ACN (2:1) to
afford 695 mg
(59%) of 5-(4-chloro-3-fluorobenzy1)-8-isopropy1-2,5,8-triazaspiro[3.5]nonane-
6,9-dione as a
yellow solid. LRMS (ES) m/z 418 (M+H); 1H NMR (400 MHz, DMSO-d6) 6 8.59 (dd, J
= 4.6,
1.3 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.43 - 7.34 (m, 2H), 7.14 (dd, J = 8.3,
2.0 Hz, 1H), 6.84
(dd, J = 9.0, 1.4 Hz, 1H), 4.95 (s, 2H), 4.60 (h, J = 6.8 Hz, 1H), 4.47 (d, J
= 9.5 Hz, 2H), 4.18
(d, J = 9.5 Hz, 2H), 4.03 (s, 2H), 1.15 (d, J = 6.8 Hz, 6H).
[0158] Compounds in the following table were prepared in a similar
manner as Compound
8.
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No. Structure Name and Data
5-(3-chloro-4-fluorobenzy1)-8-isopropy1-2-
(pyridazin-3-y1)-2,5,8-
CI triazaspiro[3.5]nonane-6,9-dione.
LRMS (ES)
m/z 418.1 (M+H). 1H NMR (400 MHz, DMS0-
d6) 6 8.59 (dd, J = 4.5, 1.3 Hz, 1H). 7.51 (dd, J
9 = 7.0, 2.2 Hz, 1H), 7.38 (dd, J =
9.0, 4.5 Hz,
N
1H), 7.35 (t, J = 9.1 Hz, 1H), 7.29 - 7.24 (m,
N N/
1H), 6.84 (dd, J = 8.9, 1.3 Hz, 11-1), 4.93 (s, 2H),
,J-r)
N 4.65 - 4.54 (m, 1H), 4.46 (d, J =
9.5 Hz, 2H),
4.18 (d, J = 9.5 Hz, 2H), 4.03 (s, 2H), 1.14 (d, J
= 6.8 Hz, 6H).
F F
8-isopropyl-2-(pyridazin-3-y1)-5-(4-
(trifluoromethyl)benzy1)-2,5,8-
1010 triazaspiro[3.5]nonane-6,9-dione.
LRMS (ES)
m/z 434.1 (M+H). 111 NMR (400 MHz,
Methanol-d4) 6 8.58 (s, 1H), 7.64 (d, J = 8.1
13
Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.46 (s, 1H),
N 6.89 (d, J = 9.0 Hz, 1H), 5.17
(s, 2H), 4.75 (h,
N N/
= 6.9 Hz, 1H), 4.63 (d, J = 9.5 Hz, 2H), 4.33 (d,
,J-()
N J = 9.5 Hz, 2H), 4.15 (s, 2H), 1.26 (d, J = 6.8
0
Hz, 6H).
(S)-8-(sec-buty1)-5-(3,4-difluorobenzy1)-2-
(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione. LRMS (ES)
m/z 416.1 (M+H). 1H NMR (400 MHz,
11111 0 Methanol-d4) 6 8.58 (s, 1H), 7.47 (dd, J = 9.5,
14 4.2 Hz, 1H), 7.29 -7.17 (m, 2H), 7.14 -7.07
(m, 1H), 6.92 (d, J = 9.0 Hz, 1H), 5.06 (s, 2H),
N,k,õõNrisY.--- 4.63 (dd, J = 9.5, 5.8 Hz, 2H),
4.59 - 4.49 (m,
0 1H), 4.37 (dd, J = 11.3,9.5 Hz, 2H), 4.09 (q, J
= 15.8 Hz, 2H), 1.72 - 1.52 (m, 2H), 1.23 (d, J
= 6.9 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H).
5-(3,4-difluorobenzy1)-8-(pentan-3-y1)-2-
(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione. LRMS (ES)
m/z 430.1 (M+H). 1H NMR (400 MHz,
o Methanol-d4) 6 8.57 (d, J = 4.6 Hz, 1H), 7.46
NA1 (dd, J = 9.1, 4.5 Hz, 1H), 7.25 -
7.16 (in, 2H),
7.08 (ddd, J = 9.0, 4.2, 1.9 Hz, 1H), 6.92 (dd, J
= 9.1, 1.3 Hz, 1H), 5.07 (s, 2H), 4.65 (d, J = 9.5
N

0 Hz, 2H), 4.45 - 4.35 (m, 1H), 4.40 (d, J = 9.5
Hz, 2H), 4.04 (s, 2H), 1.70- 1.52 (m, 4H), 0.91
(t, J = 7.4 Hz, 6H).
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(S)-8-(sec-buty1)-5-(4-fluoro-3-
methylbenzy1)-2-(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione. LRMS (ES)
0 m/z 412.1 (M+H). 1H NMR (400 MHz,

Methanol-d4) 6 8.57 (s, 1H), 7.53 (dd, J = 9.1,
WI) 4.4 Hz, 1H), 7.15 (dd, = 7.1, 2.2
Hz, 1H), 7.14
16
- 7.08 (m, 1H), 7.01 - 6.93 (in, 2H), 5.06 (d, J
1\11"( =
= 16.2 Hz, 1H), 5.00 (d, J = 16.2 Hz, 1H), 4.63
(d, J = 9.7 Hz, 2H), 4.60 -4.50 (m, 1H), 4.46 -
N 0
4.38 (m, 2H), 4.16 - 4.01 (m, 2H), 2.20 (d, J =
1.9 Hz, 3H), 1.73 - 1.54 (m, 2H), 1.23 (d, J =
6.9 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H).
5-(4-fluorobenzy1)-8-((1r,4r)-4-
methylcyclohexyl)-2-(pyridazin-3-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione. LRMS (ES)
0 m/z 438.2 (M+H). 1H NMR (400 MHz,

Methanol-d4) 6 8.62 - 8.50 (m, 1H), 7.45 (dd,
= 9.1, 4.5 Hz, 1H), 7.36 -7.29 (m, 2H), 7.08 -
17
7.00 (m, 2H), 6.89 (dd, J = 9.1, 1.2 Hz, 1H),
-N N -r Nd
N, 5.06 (s, 2H), 4.60 (d, J = 9.5
Hz, 2H), 4.35 (d, J
= 9.5 Hz, 3H), 4.14 (s, 2H), 1.88- 1.81 (m,
0
2H), 1.78 - 1.71 (m, 2H), 1.64 (qd, J = 12.3, 3.6
Hz, 2H), 1.49 - 1.34 (m, 1H), 1.12 (qd, J =
12.7, 3.6 Hz, 2H), 0.94 (d, J = 6.6 Hz, 3H).
5-(4-fluorobenzy1)-8-((1r,40-4-
methylcyclohexyl)-2-(pyridazin-4-y1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione. LRMS (ES)
0 m/z 438.2 (M+H). 1H NMR (400 MHz,
DMSO-
d6) 6 8.61 (d, J = 6.0 Hz, 1H), 8.47 (d, J = 2.9
Hz, 1H), 7.27 (dd, J = 8.5, 5.6 Hz, 2H), 7.17 -
19
7.10 (m, 2H), 6.53 (dd, J = 6.1, 3.0 Hz, 1H),
4.89 (s, 2H), 4.38 (d, J = 9.5 Hz, 2H), 4.22 -
N Nr N
4.11 (m, 1H), 4.20 (d, J = 9.4 Hz, 2H), 4.03 (s,
II
0 , 2H), 1.77 - 1.68 (m, 2H), 1.64 -
1.51 (m, 4H),
N
1.39- 1.27 (m, 1H), 1.01 (qd, J= 12.4, 11.8,
5.3 Hz, 2H), 0.87 (d, J = 6.5 Hz, 3H).
8-41r,4r)-4-(difluoromethyl)cyclohexyl)-5-(4-
F fluorobenzy1)-2-(pyridazin-4-y1)-
2,5,8-
triazaspiro[3.5]nonane-6,9-dione. LRMS (ES)
m/z 474.2 (M+H). 1H NMR (400 MHz, DMSO-
o d6) 6 8.61 (dd, J = 6.1, 0.9 Hz,
1H), 8.47 (dd, J
20 = 3.1, 1.0 Hz, 1H), 7.30 - 7.24
(m, 2H), 7.17 -
7.10 (m, 2H), 6.53 (dd, J = 6.1, 3.1 Hz, 1H),
5.87 (td, J = 57.2, 4.3 Hz, 1H), 4.89 (s, 2H),
N 4.38 (d, J = 9.5 Hz, 2H), 4.22 -
4.12 (m, 1H),
0 F
r,
4.20 (d, J = 9.4 Hz, 2H), 4.05 (s, 2H), 1.87 -
F 1.56 (m, 7H), 1.30- 1.18 (qd, J =
12.5, 3.2 Hz,
2H).
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Example 4
Synthesis of 5-(3,4-difluorobenzyl)-8-((1r,4r)-4-methylcyclohexyl)-2-
(pyridazin-3-y1)-
2,5,8-triazaspiro[3.51nonane-6,9-dione
(Compound 12)
Step 1: Synthesis of tert-butyl 3 -c yano-3 -((3 ,4-difluorobenzyl)amino)
azetidine-1-
carboxylate:
F
NH
NH2
õOyN'---/
AcOH, NaCN
0 THF-H20, 65 C
>- yN ON
0
[0159] To a solution of tert-butyl 3-oxoazetidine-1-carboxylate
(3.1 g, 18.2 mmol, 1.3
equiv) in THF (12.0 mL) were added acetic acid (1.0 g. 16.8 mmol, 1.2 equiv)
and (3,4-
difluorophenyl)methanamine (2.0 g, 14.0 mmol, 1.0 equiv) in water (6.0 mL).
After stirring at
r.t. for 5 minutes, to the mixture was added a solution of sodium cyanide (5.7
g, 116.9 mmol,
1.0 equiv) in water (2.8 mL). The mixture was heated at 60 C for 15 h in an
oil bath, cooled
to rt, neutralized by addition of a saturated aqueous solution of sodium
bicarbonate, and
extracted with ethyl acetate (30.0 mL X 2). The combined organic layers were
washed with
brine, dried over magnesium sulfate, and concentrated under reduced pressure.
To the resulting
yellow solid was added diethyl ether (30.0 mL), sonicated for 1 minute, cooled
to 0 C, and
filtered. The resulting white precipitate was washed with ice cold diethyl
ether (15.0 mL) and
dried overnight to provide 3.4 g (76%) of tert-butyl 3-cyano-3-((3,4-
difluorobenzyl)amino)azetidine-1-carboxylate. LRMS (ES) rn/z 297.1 (M+H-27).
Step 2: Synthesis of tert-butyl 3-(2-chloro-N-(3,4-difluorobenzyl)acetamido)-3-

cyanoazetidine-1-carboxylate:
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F
0
116 0
NH
N)-Ls,C1
DI PEA, CH2Cl2
0 C to rt >ray
Nrj'CN
0 0
[0160] To a solution of tert-butyl 3-cyano-3-((3,4-
difluorobenzyl)amino)azetidine-1-
carboxylate (3.1 g, 9.7 mmol, 1.0 equiv) and N,N-diisopropylethylamine (5.0
mL, 29.0 mmol,
3.0 equiv) in DCM (125.0 mL) cooled to 0 C was added chloroacetyl chloride
(1.9 mL, 24.2
mmol, 2.5 equiv) dropwise over 20 minutes. The mixture was stirred at 0 C for
15 min,
warmed to rt, and stirred for 2 hours, diluted with saturated sodium
bicarbonate, and
extracted with DCM. The combined organic layers were dried over sodium
sulfate,
concentrated, and purified by silica gel column chromatography (0%-40%
Et0Ac/hexanes
gradient) to provide 3.3g (86%) of tert-butyl 3-(2-chloro-N-(3,4-
difluorobenzyl)acetamido)-
3-cyanoazetidine-1-carboxylate. LRMS (ES) na/z 400.1 (M+H).
Step 3: Synthesis of tert-butyl 3-cyano-3-(N-(3,4-difluorobenzy1)-2-(((1r,46-4-

methylcyclohexyflanaino)acetamido)azetidine-1-carboxylate:
HAL.
461 0 111 I 0 H
DIEA, MeCN, reflux
>01(NCN >raliNrj-CN
0 0
[0161] To a solution of tert-butyl 3-(2-chloro-N-(3,4-di
fluorobenzyl)acetamido)-3-
cyanoazetidine-l-carboxylate (1.5 g, 3.8 mmol, 1.0 equiv) in acetonitrile
(15.0 mL) were added
(1r,4r)-4-methylcyclohexan-l-amine (641.6 mg, 5.7 mmol, 1.5 equiv) and D1PEA
(2.0 nth,
11.3 mmol, 3.0 equiv). The solution was heated at 75 C for 2 hours,
concentrated and purified
by silica gel chromatography using a gradient of 30 to 100% Et0Ac in hexanes
as eluent to
provide 1.3 g (70%) of tert-butyl 3-cyano-3-(N-(3,4-difluorobenzy1)-2-
(((lr,4r)-4-
methylcyclohexyl)anaino)acetamido)azetidine-1 -carboxylate. LRMS (ES) m/z
477.3 (M+H).
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Step 4: Synthesis of tert-butyl 5-(3,4-difluorobenzy1)-8-(( 1r,40-4-
methylcyclohexyl)-6,9-
dioxo-2,5 ,8-triazaspiro[3 .5] nonane-2-carboxylate :
F
0
0
AcOH, Et01-1, reflux
a3/40
N N
rj-CN >,OT 4I0
0
0
[0162]
To a solution of tert-butyl 3-cyano-3-(N-(3,4-difluorobenzy1)-2-4(1r,40-
4-
methylcyclohexyl)amino)acetamido)azetidine-1-carboxylate (1.3 g, 2.6 mmol, 1.0
equiv) in
ethanol (15.0 mL) was added acetic acid (2.3 mL, 40.0 mmol, 15.0 equiv). The
reaction was
heated at 75 C for 15 h, then 90 C for 3 h, cooled to rt, and sonicated for
10 minutes. The
precipitates were collected by filtration, washed with ice cold ethanol, and
dried to afford 953.9
mg
(76%) of tert-butyl 543 ,4 -difluorobenz y1)-84(1 r,4r)-4-methylc
yclohex y1)-6,9-diox o-
2,5,8-triazaspirol 3.5]nonane-2-carboxylate. LRMS (ES) m/z 422.2 (M+H-56).
Step 5: Synthesis of 5-(3,4-difluorobenzy1)-8-((lr,4r)-4-methylcyclohexyl)-
2,5,8-
triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate:
0
HOAl<F 0 TFA, CH2Cl2 0 F
NA1
Nr
N4.10 isY
0
0 (TFA salt)
[0163]
To a solution of tert-butyl 5-(3,4-dilluorobenzy1)-8-((1r,4r)-4-
methylcyclohexyl)-
6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (953.9 mg, 2.0 mmol, 1.0
equiv) in
DCM (2.0 mL) was added TFA (2.0 mL) at r.t. The mixture was stirred for 30
minutes,
concentrated, and dried under high vacuum to afford 981.0 mg (99%) of 543,4-
difluorobenzyl) - 8-((1r,4 r)-4-methylc yclohexyl)-2,5 ,8-triaz aspiro [3.5]
nonane-6,9-dione 2,2,2-
trifluoroacctatc. LRMS (ES) m/z 378.20 (M+H).
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Step 6: Synthesis of 5 -(3,4-difluorobenzy1)- 8-((lr,4 r)-4-meth ylc
yclohexyl)-2-(p yridazin-3 -
y1)-2,5,8-triazaspiro [3 .5[nonane-6,9-dione (Compound 12):
1. N,NCI
0
F =
HOJYF
F
0
0 K2003, IPA, 120 C =
N j1-) 2. LOH, THE NA1
H /j..1rN,..0 3. PhOPh, 180 C
,N NIJYN441C1
NO"
0 0
(TFA salt) Compound 12
[0164] To a solution of 5-(3,4-difluorobenzy1)-8-((lr,40-4-
methylcyclohexyl)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate (302.8 mg, 0.62 mmol,
1.0 equiv) in
1PA (4.0 mL) were added potassium carbonate (171.5 mg, 1.2 mmol, 2.0 equiv)
and methyl 6-
chloropyridazine-3-carboxylate (159.5 mg, 0.92 mmol, 1.5 equiv). The reaction
vial was
capped, heated at 120 C for 30 mm, and cooled to r.t. To the mixture was
added LiOH solution
(1 M, 1.8 mL, 1.8 mmol, 3.0 equiv) and the mixture was stirred for 10 minutes,
diluted with
water, acidified with HC1 (1.0 M) to pH 3, and extracted with DCM. The
combined organic
layers were dried over sodium sulfate and concentrated. To the resulting solid
was added
diphenyl ether (4.0 mL) and the mixture was heated at 180 C for 5 min, cooled
to rt, and
purified by silica gel column chromatography using a gradient of 0 to 10% Me0H
in DCM to
provide 75.1 mg (27%) of 5-(3 ,4-difluorobenz y1)- 8-((1 r,4r)-4-methylc
yclohexyl)-2-
(pyrid azin-3-y1)-2,5,8-triazaspiro[3.5]nonane-6,9-dione. LRMS (ES) miz 456.2
(M-FH). I H
NMR (400 MHz, DMSO-d6) 6 8.58 (dd, J= 4.5, 1.3 Hz, 1H), 7.41 -7.32 (m, 3H),
7.12 - 7.06
(m, 1H), 6.83 (dd, J = 9.0, 1.4 Hz, 1H), 4.92 (s, 2H), 4.45 (d, J = 9.5 Hz,
2H), 4.20- 7.11 (m,
1H), 4.18 (d, J= 9.5 Hz, 2H), 4.04 (s, 2H), 1.73 (d, J= 13.1 Hz, 2H), 1.64-
1.49 (m, 4H), 1.40
- 1.28 (m, 1H), 1.01 (qd, J= 12.1, 4.6 Hz, 2H), 0.87 (d, J = 6.5 Hz, 3H).
[0165] Compounds in the following table were prepared in a similar
manner as Compound
12.
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No. Structure Name and Data
5-(4-chlorobenzy1)-8-((lr,40-4-
C I methylcyclohexyl)-2- (p yridazin-
3-y1)- 2,5,8-
triazaspiro[3.5]nonane- 6,9-dione. LRMS
0 (ES) m/z 454.20 (M+H). 1H NMR
(400
MHz, DMSO-d6) 6 8.59 (dd, J = 4.6, 1.3 Hz,
Nj-Li 1H), 7.41 - 7.33 (m, 3H), 7.26
(d, J = 8.4 Hz,

2H), 6.82 (dd, J = 9.0, 1.3 Hz, 1H), 4.93 (s,
2H), 4.45 (d, J = 9.5 Hz, 2H), 4.22 - 4.11 (m,
1H), 4.19 (d, J = 9.7 Hz, 2H), 4.04 (s. 2H),
1.73 (d, J = 12.2 Hz, 2H), 1.63- 1.51 (m,
4H), 1.40- 1.28 (m, 1H), 1.01 (ddd, J = 23.9,
12.3, 4.9 Hz, 2H), 0.87 (d, J = 6.5 Hz, 3H).
Example 5
Synthesis of 5-(4-chlorobenzy1)-8-((1r,4r)-4-methylcyclohexyl)-6,9-dioxo-2,5,8-

triazaspiro[3.5]nonane-2-carboxamide
(Compound 18)
Step 1: Synthesis of tert-butyl 3-((4-chlorobenzyl)amino)-3-cyanoazetidine-1-
carboxylate:
CI
0111 CI
NH2
NH
AcOH, NaCN
0 THF-H20, 65 C NfiC N
0
[0166] To a solution of tert-butyl 3-oxoazetidine-1-carboxylate
(20.0g. 116.9 mmol, 1.0
equiv) in TI-IF (60.0 mL) were added acetic acid (8.0 mL, 140.2 mmol, 1.2
equiv) and (4-
chlorophenyl)methanamine (17.1 mL, 140.2 mmol, 1.2 equiv) in water (45.0 mL).
After
stirring at r.t. for 5 minutes, to the mixture was added a solution of sodium
cyanide (5.7 g,
116.9 mmol, 1.0 equiv) in water (10.0 mL). The mixture was heated at 65 C for
15 h in an oil
bath, cooled to rt, neutralized by addition of a saturated aqueous solution of
sodium
bicarbonate, and extracted with ethyl acetate (150.0 mL x 3). The combined
organic layers
were washed with brine, dried over sodium sulfate, filtered, and concentrated
under reduced
pressure. To the resulting yellow solid was added diethyl ether/hexanes (140.0
mL, 2:1) and
the solution was sonicated for 5 minutes, cooled to 0 "C, and filtered. The
resulting white
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precipitate was washed with ice cold diethyl ether (50.0 mL) and dried
overnight to provide
29.7 g (92%) of tert-butyl 3-((4-chlorobenzyl)amino)-3-cyanoazetidine-1-
carboxylate. LRMS
(ES) ink 295.2 (M+H-27).
Step 2: Synthesis of tert-butyl 3-(2-chloro-N-(4-chlorobenzyl)acetamido)-3-
cyanoazetidine-
1-carboxylate:
CI CI
so
0
CI
NH N-11-C1
DIPEA, CH2Cl2
0 CN 0 C to it 0 Nrj'-CN
y y
0 0
[0167] To a solution of tert-butyl 34(4-chlorobenzyl)amino)-3-
cyanoazetidine-1-
carboxylate (14.0 g, 43.5 mmol, 1.0 equiv) and N,N-diisopropylethylamine (22.8
mL, 130.1
mmol, 3.0 equiv) in DCM (150.0 mL) cooled to 0 C was added 2-chloroacetyl
chloride (8.7
mL, 108.8 mmol, 2.5 equiv) in DCM (50.0 mL) dropwisc over 25 minutes. The
mixture was
stirred at 0 C for 15 min, warmed to rt, and stirred for 20 h. The reaction
mixture was diluted
with saturated sodium bicarbonate and extracted with DCM. The combined organic
layers were
dried over sodium sulfate, concentrated and purified by silica gel column
chromatography (0%-
40% Et0Ac/hexanes gradient) to provide 14.1 g (81%) of tert-butyl 3-(2-ehloro-
N-(4-
chlorobenzyl)acetamido)-3-cyanoazetidine-1-carboxylate. LRMS (ES) na/z 398.1
(M+H).
Step 3: Synthesis of tert-butyl 3-(N-(4-chlorobenzy1)-2-(((lr,40-4-
methylcyclohexyl)amino)acetamido)-3-cyanoazetidine-l-carboxyl ate:
CI CI
H2N46.0
110
lel 0 0
N--11C1 _________________________________________
N4,0
DIEA, MeCN, reflux
Nij'C N 0 NT-CN
Y
0 0
[0168] To a solution of ler-I-butyl
3-(2-chloro-N-(4-chlorobenzyl)acetanaido)-3-
cyanoazetidine-1-carboxylate (11.0 g, 27.6 mmol, 1 equiv) in acetonitrile
(90.0 mL) were
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added (1r,40-4-methylcyclohexan-1-amine (3.6 g, 30.4 mmol, 1.0 equiv) and
DIPEA (9.6 mL,
69.1 mmol, 2.5 equiv). The solution was heated at reflux temperature for 3.5 h
and more
(1r,40-4-methylcyclohexan-1-amine (362.1 mg, 3.0 mmol, 0.1 equiv) was added.
The mixture
was heated with stirring for 1 hour, cooled, diluted with water, and extracted
with ethyl acetate.
The combined organic layers were washed with brine, dried over sodium sulfate,
and
concentrated to provide 12.6 g (96%) of tert-butyl 3-(N-(4-chlorobenzy1)-2-(((
1r,40-4-
methylcyclohexyl)amino)acetamido)-3-cyanoazetidine-1-carboxylate. LRMS (ES)
na/z 475.2
(M+H).
Step 4: Synthesis of tert-butyl 5-(4-chlorobenzy1)-8-((1r,40-4-
methylcyclohexyl)-6,9-dioxo-
2,5,8-triazaspiro [3. 5]n0nane-2-carboxylate:
a
a
N AcOH, Et0H, reflux
N
4.0
0
I 01
[0169] To a solution of tert-butyl
3 -(N-(4-chlorobenzy1)-2-(((lr,40-4-
methylcyclohexyl)amino)acetamido)-3-c yanoazetidine-l-carboxylate (12.6 g,
26.4 mmol, 1.0
equiv) in ethanol (88.0 mL) was added acetic acid at 0 h (15.1 mL, 264.7 mmol,
10.0 equiv),
14.5 h (7.6 mL. 132.4 mmol, 5.0 equiv), and 20.5 h (7.6 mL, 132.4 mmol, 5.0
equiv). The
reaction was stirred at reflux temperature for a total of 21 h, cooled to rt,
and sonicated for 10
minutes. The solid was filtered, washed with ice cold ethanol, and dried. The
filtrate was
concentrated, dissolved in ethanol, and sonication/filtration was repeated 3
more times (4 in
total) to obtain 9.8 g (78%) of tert-butyl 5 -(4-chlorobenzy1)-8-((lr,40-4-
methylcyclohexyl)-
6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate. LRMS (ES) m/z 420.1 (MA-
H-56).
Step 5: Synthesis of 5-(4-chlorobenzy1)-8-((1r,40-4-methylcyclohexyl)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate:
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CI CI
(11101 0
0 )
0 TFA, CH2Cl2 nelHOY
HNIJY
8 0 0 ..,õ
(TFA salt)
[0170] To a solution of tert-butyl 5-(4-chlorobenzy1)-8-((1r,40-4-
methylcyclohexyl)-6,9-
dioxo-2,5,8-triazaspiro[3.5]nonanc-2-carboxylatc (15.1 g, 31.8 mmol, 1.0
cquiv) in DCM (55.0
mL) was added TFA (55.0 mL) at r.t. The mixture was stirred for 30 minutes,
concentrated,
and dried under high vacuum to afford 15.6 g (99%) of 5-(4-chlorobenzy1)-8-
((lr,40-4-
methylcyclohexyl)-2,5,8-triazaspiro[3.5]nonane-6.9-dione 2.2,2-
trifluoroacetate. LRMS (ES)
m/z 376.2 (M+H).
Step 6: Synthesis of 5-(4-chlorobenzy1)-8-((lr,40-4-methylcyclohexyl)-6,9-
dioxo-2,5,8-
triazaspiro[3.51nonane-2-carboxamide (Compound 18):
C
CI I
0
IONION HOA1<F ONa
0
0
N)Li
NA1 AcOH, MeCN,
HNTJTN110
NH2
(TFA salt) Compound 18
[0171] To a solution of 5 -(4-chlorob enzy1)-8-((lr,4 r)-4-methylc
yclohexyl)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate (15.6 g, 31.8 mmol,
1.0 equiv) in
acetonitrile (102.0 mL) were added acetic acid (175.0 _tL, 3.1 mmol, 0.1
equiv) and sodium
cyanate (4.0 g, 61.2 mmol, 1.9 equiv). The mixture was heated at 55 'C for 80
min, cooled to
rt, diluted with water and saturated sodium bicarbonate, and extracted with
ethyl acetate. The
combined organic layers were washed with brine, dried over sodium sulfate, and
concentrated
to afford precipiates which was sonicated in ether (100.0 mL). The resulting
precipitaes were
collected by filtration, washed with ice cold ether, and dried to give a white
solid, which was
sonicated again in ethanol (100.0 mL). The precipitates were collected again
by filtration,
washed with ice cold ether, and dried to provide 8.7 g (65%) of 5-(4-
chlorobenzy1)-8-41r ,4r)-
7 1
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4-methyleyclohexyl)-6,9-dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxamide as a
white solid.
LRMS (ES) tu/z 419.2 (M+H). 1H NMR (400 MHz, DMSO-d6) 6 7.39 (d, J= 8.4 Hz,
2H), 7.24
(d, J = 8.4 Hz, 2H), 6.01 (s, 2H), 4.82 (s, 2H), 4.22 - 4.12 (m, 1H), 4.19 (d,
J = 9.3 Hz, 2H),
3.99 (s, 2H), 3.83 (d, J = 9.3 Hz, 2H), 1.77 - 1.69 (m, 2H), 1.63 - 1.48 (m,
4H), 1.41 - 1.27
(m, 1H), 1.03 (qd, J= 12.3, 4.1 Hz, 2H), 0.87 (d, J= 6.4 Hz, 3H).
[0172] Compounds in the following table were prepared in a similar
manner as Compound
18.
No. Structure Name and Data
5-(4-chloro-3-fluorobenzy1)-8-((lr,4r)-4-
methylcyclohexyl)-6,9-dioxo-2,5,8-
CI
triazaspiro[3.51nonane-2-carboxamide.
1101 LRMS (ES) m/z 437.15 (M+H). 1H
NMR (400
MHz, DMSO-d6) 6 7.55 (t, J = 8.0 Hz, 1H),
0
3
N -"1.1\ 7.35 (dd, J = 10.6, 2.0 Hz, 1H),
7.11 (dd, J =
8.3, 1.5 Hz, 1H), 5.99 (s, 2H), 4.83 (s. 2H),
4.20 (d, J = 9.2 Hz, 2H), 4.17 (tt, J = 11.4, 4.7
13 1\ 1Jr Ntl Hz. 1H), 3.98 (s, 2H),
3.82 (d, J = 9.3 Hz, 2H),
0 1.73 (d, J = 12.8 Hz, 2H), 1.66-
1.52 (m, 4H),
N H2
1.39 - 1.27 (m, 1H), 1.04 (Id, J = 12.3, 4.2 Hz,
2H), 0.87 (d, J = 6.5 Hz, 3H).
5-(4-fluoro-3-methylbenzy1)-841r,40-4-
methylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.51nonane-2-carboxamide.
LRMS (ES) m/z 417.20 (M+H). 1H NMR (400
MHz, DMSO-d6) 6 7.16 -7.10 (m, 1H), 7.08
0
6
!Tit) (d, J = 9.3 Hz, 1H), 7.05 - 6.99 (m, 1H), 6.00
(s, 2H), 4.78 (s, 2H), 4.20 - 4.12 (m, 1H), 4.18
N
(d, J = 9.3 Hz, 2H), 3.98 (s, 2H), 3.86 (d, J =
rds.)-r 440 9.2 Hz, 2H), 2.20 (d, J = 1.9 Hz, 3H), 1.73 (d,
1 0 = 13.0 Hz, 2H), 1.66 - 1.47 (m,
4E1), 1.39 -
N H2
1.27 (m, 1H), 1.10 - 0.96 (m, 2H). 0.87 (d, J =
6.5 Hz, 3H).
5-(3-chloro-4-fluorobenzyl)-8-((lr,4r)-4-
methylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-2-carboxamide.
CI
LRMS (ES) m/z 437.20 (M+H). 1H NMR (400
MHz, DMSO-d6) 6 7.50 (dd, J = 7.1, 2.2 Hz,
0
7
111), 7.38 (t, J = 8.9 Hz, 1H), 7.23 (ddd, J =
8.5, 4.6, 2.0 Hz, 1H), 6.00 (s, 2H), 4.81 (s,
N
2H), 4.20 (d, J = 9.3 Hz, 2H), 4.22 - 4.12 (m,
41/41:73 1H), 3.98 (s, 2H), 3.83 (d, J = 9.3 Hz, 2H),
NH
0 1.73 (d, J = 12.8 Hz, 2H), 1.63 -
1.49 (m, 4H),
2
1.39 - 1.28 (m, 1H), 1.04 (ddd, J = 12.8, 8.5,
3.9 Hz, 2H), 0.87 (d, J = 6.5 Hz, 3H).
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5-(3,4-difluorobenzy1)-8-((lr,4r)-4-
F
ethylcyclohexyl)-6,9-dioxo-2,5,8-
triazaspiro[3.51nonane-2-carboxamide.
F
LRMS (ES) m/z 435.20 (M+H). 1H NMR (400
MHz, DMSO-d6) 6 7.48 -7.31 (m, 2H), 7.10
0
Nrit'l -7.04 (m, 1H), 6.00 (s, 2H), 4.81 (s, 2H), 4.20
11
(d, J = 9.3 Hz, 2H), 4.18 (tt, J = 12.2, 3.7 Hz,
1H), 3.98 (s, 2H), 3.82 (d, J = 9.3 Hz, 2H),
0.,.. NdY N 1.80 (d, J = 12.5 Hz, 2H), 1.67 - 1.58 (m, 2H),
1.52 (qd, J = 12.3, 2.7 Hz, 2H), 1.21 (p, J = 7.0
NH2
Hz, 2H), 1.15 - 1.05 (in, 1H), 0.99 (qd, J =
11.0,2.8 Hz, 2H), 0.86 (t, J = 7.3 Hz, 3H).
Example 6
Synthesis of 5-(4-chlorobenzy1)-8-(2,4-difluoropheny1)-6,9-dioxo-2,5,8-
triazaspiro[3.5]nonane-2-carbaldehyde
(Comparator A)
CI
0 CI F CI
lell H 0
H2 N
\./ IP SI
0-....--' STAB F
0 NJYNH2 ..- _________________ ,
NH F
Y 0 AcOH, DCE -...,..- NH
0 LiHMDS -.- /....4.111
THF, 0 C
86% 0 N- i n
0 Ni-JY
II
y 0 84% y - 8
0
F
0
CI
CI
0
00 K2CO3
NC 010
F
CI I)
__________________________ --....-- f... _____________ .. F
TEA, THF 0 y N4_' ---ni F1 DMF, rt 'N'=-='-'
0 C tort
73% 0 0 Nilf-N
41110
0y 0
87 %
0
F
CI 0 CI
0 HO)IF 0
0
0 F F
AO...-CF.-... 0
TEA, DCM
V F II.) H 3

__________________________ . N F
quant. A1 DIPEA, MeCN
HNIJ-IN
Oil 66% 0 Nir N
0 Y 0 0
F
F
H
Comparator A
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Step 1: Synthesis of 1-(tert-butyl) 3-ethyl 34(4-chlorobenzypamino)azetidine-
1,3-
dicarboxylate
CI
CI
H 0
NH
2
STAB
AcOH, DCE NH
0
0 86%
0
0
[0173]
To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1,3-dicarboxylate
(10.6 g,
43.3 mmol, 1.0 equiv) and 4-chlorobenzaldehyde (6.1 g, 43.3 mmol, 1.0 equiv)
in DCE
(120.0 mL) at 0 C were added AcOH (5.0 mL). Upon warming to r.t. and stirred
at rt, to the
mixture was added additional AcOH (5.0 mL) twice at 4 h, 8.5 h, and 9 h (total
of 20.0 mL).
To this mixture was added STAB (11.0g. 52.0 mmol, 1.2 equiv.). The mixture was
stirred at
r.t. overnight, diluted with aqueous sodium bicarbonate, and extracted with
DCM three times.
The combined organic layers were dried over sodium sulfate and concentrated.
The obtained
oil was dissolved in warmed hexanes and EA (dropwise until the mixture went
into
hom*ogeneous solution) at 60 C with stirring. The mixture was then cooled to 0
C and
precipitation was collected by filtration and washed with cold hexanes. The
filtrate was
concentrated and repeated the process again. The combined solid was dried
under vacuum to
afford 13.8 g (86%) of 1-(tert-butyl) 3-ethyl 34(4-
chlorobenzyl)amino)azetidine-1,3-
dicarboxylate as a white solid. LRMS (ES) m/z 313.1 (M+H-56).
Step 2: Synthesis of tert-butyl 34(4-chlorobenzyl)amino)-34(2,4-
di fluorophenyl )carbamoyl)azetidine-l-carboxyl ate
CI
cl
401 H2N
NH
NH LiHMDS
THF, 0 C
84% 0
I I 0
0
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[0174] To a stirred solution of 1-(tert-butyl) 3-ethyl 3-((4-
chlorobenzyl)amino)azetidine-
1,3-dicarboxylate (10.0 g, 27.1 mmol, 1.0 equiv) and 2,4-difluoroaniline (3.0
mL. 29.8 mmol,
1.1 equiv.) in THF (200.0 mL) at 0 C under N2 were added a solution of LHMDS
(54.2 mL,
1 M in THF, 54.2 mmol, 2.0 equiv) dropwise over a period of 20 min. The
mixture was
stirred at 0 C for 20 mm, quenched with water (50 mL, acidified to pH 5 using
aqueous HC1
(0.5 N), and extracted with EA (100 mL) twice. The combined organic layer were
washed
with brine, dried over sodium sulfate, concentrated to afford 11.3 g of tert-
butyl 3-((4-
chlorobenzyl)amino)-3-42,4-difluorophenyl)carbamoyDazetidine-1-carboxylate,
which was
used for next step without purification. LRMS (ES) m/z 396.1 (M+H-56).
Step 3: Synthesis of tert-butyl 3-(2-chloro-N-(4-chlorobenzyl)acetamido)-3-
((2,4-
difluorophenyl)carbamoyl)azetidine-1-carboxylate
CI ci
0
CI,Jce,-C1 F
NH
/j
0,,,,Nfj-Y TEA, THF ,Ir
0 0 C to rt I I 0 101
0
[0175] To a solution of tert-butyl 3-((4-chlorobenzyl)amino)-34(2,4-

difluorophenyl)carbamoyl)azetidine-1-carboxylate (11.3 g, 23.2 mmol, 1.0
equiv) in THF (20
mL) cooled to 0 C were added TEA (4.8 mL, 34.7 mmol, 1.5 equiv) and 2-
chloroacetyl
chloride (1.5 mL, 27.8 mmol, 1.2 cquiv). Upon stirring at 0 C. for 30 mm,
additional TEA
(4.8 mL, 34.7 mmol, 1.5 equiv) and 2-chloroacetyl chloride (1.8 mL, 23.2 mmol,
1.0 equiv)
were added into the mixture and the mixture was stirred at 0 "C. for 30 min.
To this mixture
was added additional 2-chloroacetyl chloride (0.6 mL, 6.9 mmol, 0.3 equiv).
The mixture was
gradually warmed to rt, stirred for 45min, cooled to 0 C, and quenched with
aqueous sodium
bicarbonate. The mixture was extracted with EA three times. The combined
organic layers
were washed with bring, dried over sodium sulfate, and concentrated to afford
13.2 g of tert-
butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-((2,4-
difluorophenyl)carbamoyl)azetidine-1-carboxylate, which was used for next step
without
further purification. LRMS (ES) m/z 528.1 (M+H).
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Step 4: Synthesis of tert-butyl 5-(4-chlorobenzy1)-8-(2,4-difluoropheny1)-6,9-
dioxo-2,5,8-
triazaspiro[3.5]nonane-2-carboxylate
a
101
0 0
NCI F
K2CO3 NI)L1
0-õNOFY DMF, rt 0Nd-r"
0 11011 73% 0
0
0
[0176] To a stirred solution of tert-butyl 3-(2-chloro-N-(4-
(trifluoromethyl)benzyl)acetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-
1-
carboxylate (13.2 g, 20.1 mmol, 1.0 equiv) in DMF (40.0 mL) under N2 was added
K2CO3
(4.2 g, 30.1 mmol, 1.5 equiv.). The resulting mixture was stirred at r.t. for
3.5 h, diluted with
water, and extracted with EA twice. The combined organic layers were washed
with water
twice and brine once, dried over sodium sulfate, and concentrated to afford
11.7 g of tert-
butyl 5-(4-chlorobenzy1)-8-(2,4-difluoropheny1)-6,9-dioxo-2,5,8-
triazaspiro113.5]nonane-2-
carboxylate. LRMS (ES) na/z 436.1 (M+H-56).
Step 5: Synthesis of 5-(4-chlorobenzy1)-8-(2,4-difluoropheny1)-2,5,8-
triazaspiro[3.5]nonane-
6,9-dione 2,2,2-trifluoroacetate
01 ci 0
lb 0 H0)-H<FF
0
N1)1."1 TFA, DCM
quant N )1'1 F
HNIJYN 40/
0 0
0
[0177] To a stirred solution of tert-butyl 5-(4-chlorobenzy1)-8-
(2,4-difluoropheny1)-6,9-
dioxo-2,5,8-triazaspiro[3.5]nonane-2-carboxylate (14.7 g, 29.8 mmol, 1.0
equiv) in DCM
(50.0 mL) was added TEA (25.0 mL). The resulting mixture was stirred at r.t.
for 3 h and
concentrated to dryness to afford 15.1 g of 5-(4-chlorobenzy1)-8-(2,4-
difluoropheny1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate, which was used for
next step without
further purification. LRMS (ES) na/z 392.1 (M-FH).
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Step 6: Synthesis of 5-(4-chlorobenzy1)-8-(2,4-difluoropheny1)-6,9-dioxo-2,5,8-

triazaspiro[3.5]nonane-2-carbaldehyde
ci 0
F
Hoiy
0
101
0 0
NA1
DIPEA, MeCN F
HNIdiN 66%
0 OyNd-Tor
Comparator A
[0178] To a solution of 5-(4-chlorobenzy1)-8-(2,4-difluoropheny1)-
2,5,8-
triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacctatc (15.1 g, 29.9 mmol, 1
equiv) and N,N-
diisopropylethylamine (15.7 mL, 89.7 mmol, 3 equiv) in ACN (50 mL) was added
2,2.2-
trifluoroethyl formate (5.8 mL, 59.8 mmol, 2 equiv). The reaction was stirred
at r.t. for 1
hour, diluted with water, and extracted with DCM. The combined organic layers
were dried
over sodium sulfate, concentrated, and purified by silica gel chromatography
using 0 to 100%
Et0Ac gradient, then 0-10% Me0H, DCM gradient. The solid was suspended in
Et0H/MTBE 1:1, heated to 80 'C, cooled on ice, filtered, washed with MTBE, and
dried to
afford 8.3 g (66%) of 5-(4-chlorobenzy1)-8-(2,4-difluoropheny1)-6,9-dioxo-
2,5,8-
triazaspiro[3.5]nonane-2-carbaldehyde LRMS (ES) m/z 420.1 (M+H). 1-E1 NMR (400
MHz,
DMSO-d6) 6 7.98 (s, 1H), 7.65 (td, J = 8.8, 6.0 Hz, 1H), 7.48 - 7.41 (m, 1H),
7.43 (d, J = 8.5
Hz, 2H), 7.34 (d, J= 8.4 Hz, 2H), 7.22 (td, J= 8.4, 2.4 Hz, 1H), 4.91 (s, 2H),
4.55 (d, J= 9.7
Hz, 1H), 4.43 (d, J= 1.6 Hz, 2H), 4.37 (d. J= 9.8 Hz, 1H), 4.28 (d, J= 10.8
Hz, 1H), 4.08 (d,
J= 10.8 Hz, 1H).
Example 7
Synthesis of 8-(2,4-difluoropheny1)-6,9-dioxo-5-(4-(trifluoromethyl)benzy1)-
2,5,8-
triazaspiro[3.5]nonane-2-carbaldehyde
(Comparator B)
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CF3
CF3
0
IP OF
NH2 H2N
H 0 F
,1\111CL NH ..
Boc 0 STAB, AcOH, DCE, rt, 0/fl 0,õ LHMDS, THF
Boc--N 0
CF3
CF3
0 0
=0CI,
IL ,..,C1 K2CO3
NH F
H N H F Boc _____________ .-
,NrjyN F 0101 TEA, THF, 0 C
Boc,Nd-r" 0
0
0
F DMF
CF3 CF3
CF3
I.1 0 Si 0y0H
0 0
W CF3 NaOCN
TFA
NA1 F
DCM N-Th F AcOH, THF
Boc,Nij)f N 0
HNTJYN so
0 1 0
F 0
F NH2
F
Comparator B
Synthesis of 1-(Ie/1-butyl) 3-ethyl 34(4-
(trifluoromethyl)henzyl)amino)azetidine-1,3-
dicarboxylate
CF3
11101 CF3
(110
NH2
H 0
NI-Jr() NH
Boc" 0 STAB, AcOH, DCE, rt, o/n
/j..,irØõ.
Boc'N 0
[0179]
To a solution of 1-tert-butyl 3-ethyl 3-aminoazetidine-1,3-dicarboxylate
(15.0 g,
61.4 mmol, 1.0 equiv) and 4-(trifluoromethyl)benzaldehyde (11.8 g, 67.5 mmol,
1.1 equiv) in
DCE (60.0 mL) at 0 C were added AcOH (7.4 g, 122.8 mmol, 2.0 equiv.) and STAB
(19.5 g,
92.1 mmol, 1.5 equiv) in portions. The resulting mixture was stirred at r.t.
overnight, adjusted
the pH to 8 with ammonium hydroxide, added water (100.0 mL) and extracted
twice with
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DCM (300.0 mL). The combined organic layers were washed with brine twice,
dried over
anhydrous Na/SO4, and concentrated under reduced pressure to afford 29.6 g of
1-(iert-butyl)
3-ethyl 3-((4-(trifluoromethyl)benzyl)amino)azetidine-1,3-dicarboxylate. which
was sued for
next step without purification. LRMS (ES) m/z 347.1 (M+H-56)
Synthesis of tert-butyl 34(2,4-difluorophenyl)carbamoy1)-3-((4-
(trifluoromethyl)benzyl)amino)azetidine-1-carboxylate
C F3 C F3
H 2 N (10
11101
N H N H
Boc r
LHMDS, THF
BocIV
,IN
0 0
[0180] To a stirred solution of 1-(tert-butyl) 3-ethyl 3-((4-
(trifluoromethyl)benzypamino)azetidine-1,3-dicarboxylatc (29.6 g, 44.1 mmol,
1.0 cquiv)
and 2,4-difluoroanilinc (4.9 mL, 48.5 mmol, 1.1 equiv.) in THF (200.0 mL) at 0
C under N2
were added a solution of LHMDS (88.3 mL, 1 M in THE, 88.3 mmol, 2.0 equiv)
dropwise
over a period of 20 min. The mixture was stirred at 0 C for 20 min, quenched
with water (50
mL, acidified to pH 5 using aqueous HC1 (3N), and extracted with EA (100 mL)
twice. The
combined organic layer were washed with brine, dried over sodium sulfate,
concentrated to
afford 36.2 g of teri-butyl 34(2,4-difluorophenyl)carbamoy1)-3-((4-
(trifluoromethyl)benzyl)amino)azetidine-l-carboxylate, which was used for next
step without
purification. LRMS (ES) m/z 430.1 (M+H-56).
Synthesis of tert-butyl 3-(2-chloro-N-(4-(trifluoromethyl)benzyflacetamido)-3-
((2,4-
difluorophenyl)carbamoyl)azetidine-1-carboxylate
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CF3
CF3
110 0
CI 0
N H F
NH F TEA, THF, 0 C
Boc,NirN
Boc,Nd'Y 0
o
[0181] To a solution of tert-butyl 34(2,4-difluorophenyl)carbamoy1)-
34(4-
(trifluoromethyl)benzypamino)azetidine-l-carboxylate (36.2 g, 40.6 mmol, 1.0
equiv) in
THF (100 mL) cooled to 0 C were added TEA (8.5 mL, 60.9 mmol, 1.5 equiv) and
2-
chloroacetyl chloride (3.9 mL, 48.7 mmol, 1.2 equiv). Upon stirring at 0 'C.
for 30 min,
additional TEA (8.5 mL, 60.9 mmol. 1.5 equiv) and 2-chloroacetyl chloride (3.3
mL, 40.6
mmol, 1.0 equiv) were added into the mixture and the mixture was stirred at 0
C. for 30 min.
To this mixture was added additional 2-chloroacetyl chloride (1.0 mL, 12.2
mmol, 0.3 equiv).
The mixture was gradually warmed to rt, stirred for 45min, cooled to 0 C, and
quenched
with aqueous sodium bicarbonate. The mixture was extracted with EA three
times. The
combined organic layers were washed with bring, dried over sodium sulfate, and

concentrated to afford 42.0 g of tert-butyl 3-(2-chloro-N-(4-
(trifluoromethyl)benzypacetamido)-34(2,4-difluorophenyl)carbamoyl)azetidine-1-
carboxylate, which was used for next step without further purification. LRMS
(ES) m/z 562.1
(M+H).
Synthesis of tert-butyl 8-(2,4-difluoropheny1)-6,9-dioxo-5-(4-
(trifluoromethypbenzy1)-2,5,8-
triazaspiro[3.5]nonane-2-carboxylate
CF3 CF3
so
0
11õ..C1 K2CO3
N H F F
DMF
Boc,Nrj-YN
Boc,Nrf
0 0
[0182] To a stirred solution of ter/-butyl 3-(2-chloro-N-(4-
(trifluoromethyl)benzypacetamido)-3-((2,4-difluorophenyl)carbamoyl)azetidine-1-

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carboxylate (42.0 g, 33.4 mmol, 1.0 equiv) in DMF (80.0 mL) under N2 was added
K2CO3
(7.0 g, 50.1 mmol, 1.5 equiv.). The resulting mixture was stirred at r.t. for
3.5 h, diluted with
water, and extracted with EA twice. The combined organic layers were washed
with water
twice and brine once, dried over sodium sulfate, concentrated, and purified by
silica gel using
a gradient of 0-60% EA in hexanes as eluent to afford 12.0 g (37% over 4
steps) of tert-butyl
8-(2,4-difluoropheny1)-6,9-dioxo-5-(4-(trifluoromethyl)benzy1)-2,5.8-
triazaspiro[3.5]nonane-
2-carboxylate. LRMS (ES) m/z 469.7 (M+H-56).
Synthesis of 8-(2,4-difluoropheny1)-5-(4-(trifluoromethyl)benzy1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate
CF3 CF3
110 =0y0 H
0 C F3
T FA
N F N F
DC M N
Bac, H NlisT
0 0
[0183] To a stirred solution of tert-butyl 8-(2,4-difluoropheny1)-
6,9-dioxo-5-(4-
(trifluoromethy1)ben7y1)-2,5,8-triazaspiro[3.5]nonane-2-carboxy1ate (12.0 g.
22.8 mmol, 1.0
equiv) in DCM (12.0 mL) was added TFA (40.0 mL). The resulting mixture was
stirred at r.t.
for 3 h and concentrated to dryness to afford 12.3 g of 8-(2,4-difluoropheny1)-
5-(4-
(trifluoromethyl)benzy1)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2,2-
trifluoroacetate, which
was used for next step without further purification. LRMS (ES) m/z 426.1 (M-
FH).
Synthesis of 8-(2,4-difluoropheny1)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-
2,5,8-
triazaspiro[3.5]nonane-2-carbaldehyde
CF3
CF3
O
0y0H
0
CF3 NaOCN
N F
H AcOH, THF
1\111-N 1 0 Nd-IN
0 101 NH2
Comparator B
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[0184] To a stirred solution of 8-(2,4-difluoropheny1)-5-(4-
(trifluoromethyl)benzy1)-
2,5,8-triazaspiro[3.51n0nane-6,9-dione 2,2,2-trifluoroacete (12.3 g, 22.8
nunol, 1.0 equiv) in
THF (50.0 mL) were added sodium cyanate (4.4 g, 68.4 mmol, 3.0 equiv.) and a
few drops of
acetic acid. The mixture was stirred at r.t. for 30 min, concentrated, and
purified by silica
column chromatography using a gradient of 0-10% Me0H in DCM as eluent to
afford 8.8 g
(73% over two steps) of 8-(2,4-difluoropheny1)-6,9-dioxo-5-(4-
(trifluoromethyl)benzy1)-
2,5,8-triazaspiro[3.5]n0nane-2-carbaldehyde. LRMS (ES) m/z 469.1 (M+H); 1H NMR
(400
MHz, Methanol-d4) 7.70 (d, J = 8.1 Hz, 2H), 7.61 - 7.53 (m, 3H), 7.20 (ddd, J
= 10.4, 8.8,
2.8 Hz, 1H), 7.12 (dddd, J = 9.1, 8.0, 2.8, 1.4 Hz, 1H), 5.15 (s, 2H), 4.53 -
4.49 (m, 4H), 4.20
(d, J = 9.5 Hz, 2H).
Example 8
Synthesis of 8-((lr,4r)-4-(difluoromethypeyelohexyl)-6,9-dioxo-5-(4-
(trifluoromethyl)benzyl)-2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde
(Comparator E)
Step 1: Synthesis of tert-butyl 3-cyano-3-((4-
(trifluoromethyl)benzyl)amino)azetidine-1-
carboxylate:
FEE
F F
14111
N Niit H2
NH
>,0y
AcOH, NaCN
0 THF-H20, 60 00 fjCN
"Y N
0
[0185] To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (25
g, 146.0 mmol, 1.0
equiv) in THF (90 mL) were added acetic acid (10.5 g, 175.2 mmol, 1.2 equiv)
and (4-
(trifluoromethyl)phenyemethanamine (31.7 g, 181.1 mmol, 1.2 equiv) in water
(40.0 mL).
After stirring at r.t. for 5 minutes, to the mixture was added a solution of
sodium cyanide (7.2
g, 146.0 mmol, 1.0 equiv) in water (10 mL) was added. The mixture was heated
at 60 C for
18 h in an oil bath, cooled to rt, neutralized by addition of a saturated
aqueous solution of
sodium bicarbonate, and extracted with ethyl acetate (150 mL x 2). The
combined organic
layers were washed with brine, dried over magnesium sulfate, and concentrated
under
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reduced pressure. To the resulting yellow solid was added diethyl
ether/hexanes (200 mL,
1:2) and the solution was sonicated for a minute, cooled to 0 C, and
filtered. The resulting
white precipitate was washed with ice cold diethyl ether (50 mL) and dried
overnight to
provide tert-butyl 3-cyano-34(4-(trifluoromethypbenzyl)amino)azetidine-1-
carboxylate (43.9
g, 85% yield). LRMS (ES) m/z 329.2 (M+H-27). 1H NMR (400 MHz, DMSO-do) 6 7.71
(d,
J= 8.0 Hz, 2H), 7.60 (d, J= 8.0 Hz, 2H), 4.16 (d, J= 8.8 Hz, 2H), 3.92 (t, J=
7.2 Hz, 1H),
3.84 (d, J= 9.2 Hz, 2H), 3.81 (dd, J=7.3 Hz, 2H), 1.39 (s, 9H).
Step 2: Synthesis of tert-butyl 3-(2-chloro-N-(4-
(trifluoromethypbenzyl)acetamido)-3-
cyanoazetidine-l-carboxylate:
F F F F
0
s
NH
TEA, CH2Cl2
>,.Ø1r,Nrj'CN 0 C to rt 0 NI-CN
Y
0 0
[0186] To a solution of tert-butyl 3-cyano-3-((4-
(trifluoromethyl)benzyl)amino)azetidine-
1-carboxylate (3.0 g, 8.4 mmol, 1.0 equiv) and triethylamine (1.3 g, 12.7
mmol, 1.5 equiv) in
DCM (0.2 M) cooled to 0 'V was added chloroacetyl chloride (0.95 g, 8.4 mmol,
1.0
equiv). The mixture was stirred at 0 C for 15 min, warmed to rt, and stirred
for 2 h. To the
mixture were added additional 2-chloroacetyl chloride (0.95 g, 8.4 mmol, 1.0
equiv) and
triethylamine (1.3 g, 12.7 mmol, 1.5 equiv). The reaction was stirred for 2 h,
quenched with a
saturated aqueous solution of ammonium chloride, and separated the layers. The
aqueous
layer was extracted with DCM once. The combined organic layers were dried over

magnesium sulfate, concentrated, and purified by silica gel column
chromatography (5%-
70% Et0Ac/hexanes, Rf =0.24 (20% Et0Ac/hexanes) to afford 3.4 g (92%) of tert-
butyl 3-
(2-chloro-N-(4-(trifluoromethyl)benzyl)acetamido)-3-cyanoazetidine-1-
carboxylate. LRMS
(ES) m/z 432.1 (M+H). 1H NMR (400 MHz, DMSO-d6) 6 7.77 (d, J = 8.1 Hz, 2H),
7.60 (d, J
= 8.0 Hz, 2H), 4.95 (s, 2H), 4.52 (s, 2H), 4.15 (s, 4H), 1.35 (s, 9H).
Step 3: Synthesis of tert-butyl 3-cyano-3-(2-(((1r,4r)-4-
(difluoromethyl)cyclohexyl)amino)-N-
(4-(trifluoromethyl)benzyl)acetamido)azetidine-1-carboxylate:
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F F F F
1110
I. 0 0
N)-C1 (HCI salt)
DIEA, MeCN, 65 C
F
0 NriCN ty--
> yN CN
0 0
101871
To a solution of tert-butyl 3-(2-chloro-N-(4-
(trifluoromethyl)benzyl)acetamido)-3-
cyanoazetidine-1 -carboxylate (0.8 g, 1.9 mmol, 1 equiv) in acetonitrile (15
mL) were added
(1r,4r)-4-(difluoromethyl)cyclohexan-1-amine hydrochloride (0.51 g, 2.8 rnmol,
1.5 equiv)
and DIPEA (1.2 g, 9.3 mmol, 5 equiv). The solution was heated at 65 C for 4 h
at which point
LCMS indicated completion of reaction. The reaction was diluted with ethyl
acetate and water
(1:1, 80 mL) and the aqueous layer was extracted with ethyl acetate. The
combined organic
layers were washed with brine, dried over magnesium sulfate, concentrated, and
purified with
silica gel chromatography using a gradient of 25% to 100% ethyl acetate in
hexanes as eluent
to afford 0.65 g (64%) of
tert-butyl 3-cy ano-3-(2 -(41r,4r)-4-
(difluoro meth yl)cyclohex yl)amino)-N-(4-(trifluoromethyl)benz
yl)acetamido)azetidine- 1-
carboxylate as a pale yellow oil. Rf =0.55 (100% ethyl acetate, silica). LRMS
(ES) na/z 545.0
(M+H).
Step 4: Synthesis of iert-butyl 8-((1r,4r)-4-(difluoromethypcyclohexyl)-6,9-
dioxo-5-(4-
(trifluoromethyl)benzy1)-2,5 ,8-triaz aspiro [3.5] nonane-2-c arboxylate :
F F
F F
11 1 0
[Si 0
AcOH Et0H, 80 C
= F
>.(DyNiCN
0 F
0
0
[0188] To a solution of tert-butyl
3-cyano-3-(2-(((lr,4r)-4-
(difluoro meth yl)cyclohex yl)amino)-N-(4-(trifluoromethyl)benz
yl)acetamido)azetidine- 1-
carboxylate (0.27 g, 0.50 mmol, 1.0 equiv) in ethanol (2 mL) was added acetic
acid (0.18 g,
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3.0 mmol, 6.0 equiv). The reaction was heated at 70 C for 15 hours, cooled to
rt, and diluted
with hexanes (1.0 naL). The precipitation was collected by filtration, washed
with ethanol-
hexanes (1:2, 2 mL), and dried to afford 186 mg (69%) of tert-butyl 8-41r,4r)-
4-
(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-(trifluoromethyl)benzyl)-2,5,8-
triazaspiro[3.5]nonane-2-carboxylate as a pale yellow solid. LRMS (ES) m/z
490.2 (M+H-56).
1H NMR (400 MHz, DMSO-d6) 6 7.71 (d, J = 8.1 Hz, 2H), 7.48 (d, J = 8.0 Hz,
2H), 5.89 (td,
J = 56.7, 3.8 Hz, 1H), 4.93 (s, 2H), 4.29 ¨ 4.15 (m. 3H). 4.02 (s, 2H), 3.93
(d, J = 9.5 Hz, 2H),
1.91 ¨ 1.55 (m, 7H), 1.35 (s, 9H), 1.38 ¨ 1.20 (m, 2H).
Step 5: Synthesis of 8-((1r,4r)-4-(difluoromethypcyclohexyl)-5-(4-
(trifluoromethyl)benzy1)-
2,5,8-triazaspiro[3.5]nonanc-6,9-dione 2,2,2-triflu oro acetate:
F F
F F
11101 0
0
TFA, CH2Cl2 0N)1 HO)-
H
I\12 F F
oYNNF
HI\lNakT3
0 0
(TFA salt)
101891 To a solution of tert-butyl 8-((1r,4r)-4-
(dif1uoromethyl)cyclohexyl)-6,9-dioxo-5-
(4-(trifluoromethyl)benzy1)-2,5,8-triazaspiro[3.5]nonane-2-carboxyl ate (200
mg, 0.37 mmol,
1.0 equiv) in DCM (1.5mL) was added TFA (1.5mL) at r.t. The mixture was
stirred at r.t. for
1 h, concentrated under reduced pressure, and dried under high vacuum to
afford 190 mg
(94%) of 8-((1r,4r)-4-(difluoromethyl)cyclohexyl)-5-(4-
(trifluoromethyl)benzy1)-2,5,8-
triazaspiro[3.5]nonane-6,9-dione 2,2,2-trifluoroacetate, which was used
without further
purification. LRMS (ES) m/z 446.2 (M+H).
Step 6: Synthesis of 8-((lr,4r)-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-
(trifluoromethyl)benzy1)-2,5,8-triazaspiro[3.51nonane-2-carbaldehyde
(Comparator E):
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F F
F F
0
0
HO)YF
0 0
HACY--NCF3
N DIEA, CH2Cl2 N
HNIJNY m40 OyNDLir 41/40
0
(TFA salt) F Comparator E F
[0190] To a solution of
8-((lr,4r)-4- (difluoromethyl)c yclohexyl)-5 -(4-
(trifluoromethyl)benzy1)-2,5,8-triazaspiro[3.5]nonane-6,9-dione 2,2.2-
trifluoroacetate (54.0
mg, 0.10 mmol) in acetonitrile (0.6 mL) were added D1PEA (37.0 mg, 0.29 mmol,
3.0 equiv)
and 2,2,2-trifluoroethyl formate (124.0 mg. 0.97 mmol, 10.0 equiv). The
mixture was heated
in a microwave reactor at 110 C for 20 min, concentrated, and purified by
HPLC using a
gradient of 10% to 100% ACN in water (both with 0.1% HCOOH) as eluent to
afford 21.0 mg
(46%) of 84(1 r,40-4-(difluoromethyl)cyclohexyl)-6,9-dioxo-5-(4-
(trifluoromethyl)benzyl)-
2,5,8-triazaspiro[3.5]nonane-2-carbaldehyde as a foam. LRMS (ES) m/z 473.9
(M+H). 1H
NMR (400 MHz, DMSO-d6) 6 7.96 (s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.47 (d, J =
8.0 Hz, 2H),
5.89 (td, J= 56.4, 4.5 Hz, 1H), 4.94 (s, 2H), 4.51 (d, J= 9.6 Hz, 1H), 4.30 -
4.15 (m, 3H), 4.04
(s. 2H), 3.96 (d, J= 10.7 Hz, 1H), 1.95 - 1.49 (m, 7H), 1.41 - 1.06 (m, 2H).
Biological Example B-1: Myofibril Assay
[0191] To evaluate the effect of compounds on the ATPase activity
of full-length cardiac
myosin in the context of the native sarcomere, skinned myofibril assays were
performed.
Bovine cardiac myofibrils were obtained by hom*ogenizing bovine cardiac left
ventricular
tissue in the presence of a detergent such as triton X-100. Such treatment
removes
membranes and a majority of the soluble cytoplasmic proteins but leaves intact
the cardiac
sarcomeric acto-myosin apparatus. Myofibril preparations retain the ability to
hydrolyze
ATP in a Ca2 regulated manner. ATPase activities of such myofibril
preparations in the
presence and absence of compounds were assayed at Ca2+ concentrations
activating to a
defined fraction of the maximal rate (i.e., 25%, 75%). Small molecule agents
were assessed
for their ability to inhibit the steady-state ATPase activity of bovine
cardiac myofibrils using
pyruvate kinase and lactate dehydrogenase (PK/LDH)-coupled enzyme system. This
assay
regenerates myosin-produced ADP into ATP by oxidizing NADH, producing an
absorbance
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change at 340 nm. Prior to testing small molecule agents, the bovine cardiac
myofibrils were
assessed for their calcium responsiveness and the calcium concentration that
achieves either a
50% (pCaso) or 75% (pCa75) activation of the myofibril system was chosen as
the final
condition for assessing the inhibitory activity of the small molecule agents.
All enzymatic
activity was measured in a buffered solution containing 12 mM PIPES
(piperazine-N,Ni-
bis(2-ethanesulfonic acid), 2 mM magnesium chloride at pH 6.8 (PM 12 buffer).
Final assay
conditions were 1 mg/mL of bovine cardiac myofibrils, 4 U/mL pyruvate kinase,
6 U/mL
lactate dehydrogenase, 50 laM ATP, 0.1 nag/mL BSA (bovine serum albumin), 10
ppm
antifoam, 1 mM DTT, 0.5 mM NADH, 1.5 mM PEP, 0.6 mM EGTA, and an amount of
CaCl2sufficient to achieve either 50% or 75% activation of the myofibril
ATPase activity.
Results for compounds tested are provided in Table A. Compounds tested were
prepared in
accordance with the synthetic procedures described herein.
Table A
Compound No. CDMF IC15 ( M)
1 0.80
2 2.1
3 0.7
4 1.5
1.3
6 1.4
7 0.8
8 1.3
9 1.6
0.3
11 0.7
12 0.4
13 2.0
14 1.3
1.4
16 0.9
17 0.9
18 1.2
Comparator A 1.8
Comparator B 1.1
Comparator C 1.1
Comparator D 1.1
Comparator E E3
[0192] Comparator C and Comparator D have the following structures:
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Compound Structure Name
F F
(S )- 1-(5-chloro-3-
fluoropyridin-2-y1)-3-1161
Comparator C (oxetan-3-y1)-4-(4-
N )1) F (trifluoromethyl)benzyl)pip
erazine-2,5-dione
N
01-1 0 N ci
F F

4-(2-acetyl-6,9-dioxo-5-(4-
(trifluoromethyl)benzy1)-
Comparator D o 2,5,8-
F triazaspiro[3.5]nonan-8-
N
õTN/J-1s- so y1)-3-fluorobenzonitrile
N
10193] The preparation of Comparator C and Comparator D is
described in
W02020/047447A1.
Biological Example B-2: Pharmaco*kinetics Single Dose Studies
Mouse Single Dose Studies
[0194] Male C57BL/6 mice (18-25g, 6-8 weeks old) were obtained from
Zhejiang Vital
River Laboratory Animal Technology Co., Ltd. All animals for IV administration
had free
access to food and water. The IV dosing was performed through tail vein. The
IV dose
solution for the Test Articles was prepared in 10% DMA/20% PG/70% HPf3CD
solution
(40% w/v aqueous HPf3CD) at a concentration of 0.1 mg/mL. The oral dosing
suspension was
prepared by suspending Test Article in 0.5% HPMC/0.1% Tween 80 in water at a
concentration 0.2 mg/mL. Concentrations of IV and PO doses were measured at
the end of
the study. Pharmaco*kinetic (PK) parameters were calculated using the nominal
dose values if
the measured values were within 20% of the nominal values. A group of 15 mice
received the
IV dose where the volume was 5 mL/kg. Another group of 15 mice received by
oral gavage
of Test Article at 1 mg/kg. The oral dose volume was 5 mL/kg. Sparse blood
samples were
collected from groups of three mice via retro-orbital bleeding, placed into a
K,EDTA
microtainer tube and maintained on ice until centrifugation to obtain plasma.
Each designated
group of mice were bled at two-time points. The time points were predose (PO
only), 5 (IV
only). 15, 30 minutes, 1, 2, 4, 6, 8 and 24 hours postdose. Blood samples were
centrifuged
and the collected plasma was stored at -80 C until analysis. Plasma samples
were analyzed
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for Test Article concentrations using an LC/MS/MS method. Briefly, a 50 pL
aliquot of each
plasma sample was mixed with 100 1.11_, of acetonitrile that contained an
internal standard (IS).
The mixture was vortexed and centrifuged. Ten (10) fiL of the resulting
solution was injected
onto a reverse-phase C18 column and the resultant peaks detected on a LC/MS/MS
equipped
with a turbo ionspray ionization source. Sample concentrations below the limit
of
quantification (BLQ) were treated as zero for PK calculations. Composite PK
parameters
were estimated from a maximum of two sampling points per mouse and three mice
per
sampling point and the sparse data option of WinNonlin was used for
noncompartmental
analysis of the concentration-time data (Phoenix WinNonLin software, version
64; Pharsight,
Mountain View, CA). The elimination rate constant (k) was calculated as the
absolute value
of the slope of the linear regression of logarithm of the concentration versus
time for the last
three data points of the concentration-time profiles. Apparent elimination
half-life (t1/2) values
were calculated as ln(2)/k. Area under the concentration-time curve (AUC)
values were
estimated using linear trapezoidal method. AUC t values were calculated from
the dosing time
to the last measurable concentration. AUC., values were calculated as the sum
of the
corresponding AUC t and the ratio of the last detectable concentration divided
by k. Plasma
clearance (CL) was calculated from Dose/AUC,. Mean resident time (MRT) was
estimated
by moment analysis. Volume of distribution at steady state (Vss) was
calculated from
MRTx CL. Maximum concentration (C,,,ax) and time to reach C max (tmax) were
recorded as
observed. Bioavailability was calculated dAUC.,po/dAUC.,tv x100% where dAUC
was the
dose normalized AUC value. Data for compounds tested are provided in Table B.
Compounds tested were prepared in accordance with the synthetic procedures
described
herein.
Table B
CL
Compound No. -612 (h) Vss (L/kg)
(mL/min/kg)
4 28.4 1.77 4.2
18.9 3.08 4.08
6 28.2 1.27 2.52
7 15 2.5 2.95
8 132 0.66 5.14
13 101 0.46 2.25
18 21 1.18 2.95
Comparator B 21.45 2.96 4.51
Comparator C 4.67 2.6 1.39
Comparator E 11.6 2.8 3.28
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Rat Single Dose Studies
[0195]
Male Sprague Dawley rats were obtained from Zhejiang Vital River
Laboratory
Animal Technology Co.. Ltd. The animals in IV group had free access to water
and food. The
animals in PO group were fasted overnight before dosing and provided with food
2 hours
post dosing. The IV dose solution was prepared in 10% DMA/50% PG/40% HPI3CD
solution
(40% w/v aqueous HPI3CD) at a concentration of 1 mg/mL. . The oral dosing
suspension was
prepared by suspending Test Article in 0.5% HPMC/0.1% Tween 80 in water at a
concentration 0.2 mg/mL. Concentrations of IV and PO doses were measured at
the end of
the study. Pharmaco*kinetic parameters were calculated using the nominal dose
values if the
measured values were within 20% of the nominal values. Three rats were dosed
IV via a
bolus injection via caudal vein. Three rats per dose group were dosed by oral
gavage. Blood
samples were collected from the jugular vein cannula at predose, 5 (IV
only),15, 30 minutes,
and 1, 2, 4, 6, and 24 hours post-dose. Blood volumes were replaced with an
equal amount of
sterile 0.9% saline. Blood samples were centrifuged and the collected plasma
was stored at -
80 C for subsequent analysis. Plasma samples were analyzed for Test Article
concentrations
using a LC/MS/MS method. Briefly, a 50 pL aliquot of each plasma sample was
mixed with
100 L of acetonitrile that contained an internal standard. The mixture was
vortexed and
centrifuged. Ten (10)1..LL of the resulting solution was injected onto a
reverse-phase C18
column and the resultant peaks detected on a LC/MS/MS equipped with a turbo
ionspray
ionization source. Sample concentrations below the limit of quantification
(BLQ) were
treated as zero for phai __ liaco*kinetic calculations. Pharmaco*kinetic
parameters were estimated
from individual animals using noncompartmental analysis of the concentration-
time data
(Phoenix WinNonLin software, version 64; Pharsight, Mountain View, CA). The
elimination
rate constant (k) was calculated as the absolute value of the slope of the
linear regression of
logarithm (log) of the concentration versus time for the last three data
points of the
concentration-time profiles. Apparent elimination half-life (t1/2) values were
calculated as
ln(2)/k. Area under the concentration-time curve (AI V) values were estimated
using linear
trapezoidal method. AUCt values were calculated from the dosing time to the
last measurable
concentration. AUC. values were calculated as the sum of the corresponding
AUCt and the
ratio of the last detectable concentration divided by k (AUCt_.). Plasma
clearance (CL) was
calculated from Dose/AUC. Mean resident time (MRT) was estimated by moment
analysis.
Volume of distribution at steady state (Vss) was calculated from MRT. x CL.
Maximum
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concentration (Cmax) and time to reach Cmax (tmax) were recorded as observed.
Bioavailability
was calculated from the ratio of dose normalized AUC,0 of individual rats/mean
dAUC, x
100% where dAUC was the dose normalized AUC value. Data for compounds tested
are
provided in Table C. Compounds tested were prepared in accordance with the
synthetic
procedures described herein.
Table C
Compound No. (mL/ (h) Vss (L/kg)
miCLn/kg) t112
3 78.49 2.6 14.7
4 52.57 3.34 12.98
71.07 3.04 13.7
6 76.6 2 10.1
7 61.93 2.1 12.6
8 79.91 1.2 5.22
9 27.4 0.925 1.64
13 38.9 0.95 2.17
18 63.6 2.15 9.48
Comparator A 29.59 6.48 15.05
Comparator B 41.86 5.87 16.58
Comparator C 20.23 3.3 5.2
Comparator D 8.23 4.84 2.98
Comparator E 18.75 5.4 8.23
Dog Single Dose Studies
[0196] Non-naïve male beagle dogs (8 months -3 years of age, body
weight 8-13 kg)
were used in this study. All animals for IV administration had free access to
food and water;
all animals for PO were fasted overnight prior to dosing and were fed
approximately 6 hours
after dosing. For the animals in PO group, pentagastrin (6.0 ng/kg, i.m.) was
administrated 20
minutes before dosing PO formulation and 1.5 hours after the first
pentagastrin dosing. The
dosing volume was 0.024 mL/kg, the concentration was 250 ug/mL in DMSO/1 N
NaOH/PBS. 10 mL of 0.001 N HC1 was used to wash the gavage catheter for each
animal.
The IV dose solution was prepared in 10% DMA/50% PG/40% HP13CD solution (40%
w/v
aqueous HPI3CD) at a concentration of 1.0 mg/mL. The oral dose suspension was
prepared by
suspending the compound in 0.5% HPMC/0.1% Tween 80 in distilled water at a
concentration of 0.2 mg/mL. Concentrations of IV and PO doses were measured at
the end of
the study. PK parameters were calculated using the nominal dose values if the
measured
values were within 20% of the nominal values. Blood samples were collected by
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venipuncture of peripheral veins except the dosing vein at pre-dose, 5 15, 30
min. 1, 2, 4, 6,
8, 24 and 48 h post-dose. Blood samples were centrifuged and resultant plasma
was frozen
for bioanalysis. Plasma samples were stored at -80 C before analysis. Plasma
samples were
analyzed for compound concentrations using the LC/MS/MS method. Briefly, a 50
IaL
aliquot of each plasma sample was mixed with 100 IaL of acetonitrile that
contained an
internal standard. The mixture was vortexed and centrifuged. Ten (10) uL of
the resulting
solution was injected onto a reverse-phase C18 column and the resultant peaks
detected on a
LC/MS/MS equipped with a turbo ionspray ionization source. Sample
concentrations below
the limit of quantification (BLQ) were treated as zero for PK calculations. PK
parameters
were estimated from individual animals using noncompartmental analysis of the
concentration-time data (Phoenix WinNonLin software. version 64; Pharsight,
Mountain
View, CA). The elimination rate constant (k) was calculated as the absolute
value of the slope
of the linear regression of logarithm (log) of the concentration versus time
for the last three
data points of the concentration-time profiles. Apparent elimination half-life
(t1/2) values were
calculated as ln(2)/k. Area under the concentration-time curve (AUC) values
were estimated
using linear trapezoidal method. AUC t values were calculated from the dosing
time to the last
measurable concentration. AUC. values were calculated as the sum of the
corresponding
AUC t and the ratio of the last detectable concentration divided by k. Plasma
clearance (CL)
was calculated from Dose/AUC.. Mean resident time extrapolated to infinity
(MRT.) was
estimated by moment analysis. Võ was calculated from MRT. x CL. Maximum
concentration (Cm) and time to reach Cma, (ti-flax) were recorded as observed.
As this was a
cross-over study, bioavailability was calculated dAUC,,po/dAUC,,iv X 100%
where dAUC
was the dose normalized AUC value from the same animal given IV and PO dose.
Data for
compounds tested are provided in Table D. Compounds tested were prepared in
accordance
with the synthetic procedures described herein.
Table D
CL
Compound No. fin (h) Vss (L/kg)
(mL/min/kg)
4 7.57 9.54 4.23
13 7.45 6.44 2.85
18 18.8 5.8 5.73
Comparator A 1.59 39.17 5.2
Comparator B 2.39 45.88 6.51
Comparator C 4.92 14.66 5.91
Comparator D 3.55 23.69 6.7
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Comparator E 1.49 33.1 4.1
Monkey Single Dose Studies
[0197] Non-naive Male Cynomolgus Monkey (2-5 years of age, body
weight 2-5 kg)
used in this study were obtained from Topgene Biotechnology. All animals for
IV
administration had free access to food and water; all animals for PO were
fasted overnight
prior to dosing and were fed approximately 6 hours after dosing. The IV dose
solution was
prepared in 10% DMA/50% PG/40% HPI3CD solution (40% w/v aqueous HP13CD) at a
concentration of 1.0 mg/mL. The oral dose suspension was prepared by
suspending the
compound in 0.5% HPMC/0.1% Tween 80 in distilled water at a concentration of
0.2 mg/mL.
Concentrations of IV and PO doses were measured at the end of the study. PK
parameters
were calculated using the nominal dose values if the measured values were
within 20% of the
nominal values. Blood samples were collected by venipuncture of peripheral
veins except the
dosing vein at pre-dose, 5 15, 30 min, 1, 2, 4, 6, 8, 24 and 48 h post-dose.
Blood samples
were centrifuged and resultant plasma was frozen for bioanalysis. Plasma
samples were
stored at -80 C before analysis. Plasma samples were analyzed for compound
concentrations
using the LC/MS/MS method. Briefly, a 50 pL aliquot of each plasma sample was
mixed
with 100 pL of acetonitrile that contained an internal standard. The mixture
was vortexed and
centrifuged. Ten (10) pL of the resulting solution was injected onto a reverse-
phase C18
column and the resultant peaks detected on a LC/MS/MS equipped with a turbo
ionspray
ionization source. Sample concentrations below the limit of quantification
(BLQ) were
treated as zero for PK calculations. PK parameters were estimated from
individual animals
using noncompartmental analysis of the concentration-time data (Phoenix
WinNonLin
software, version 64; Pharsight, Mountain View, CA). The elimination rate
constant (k) was
calculated as the absolute value of the slope of the linear regression of
logarithm (log) of the
concentration versus time for the last three data points of the concentration-
time profiles.
Apparent elimination half-life (t1/2) values were calculated as ln(2)/k. Area
under the
concentration-time curve (AUC) values were estimated using linear trapezoidal
method.
AUCt values were calculated from the dosing time to the last measurable
concentration.
AUC., values were calculated as the sum of the corresponding AUCt and the
ratio of the last
detectable concentration divided by k. Plasma clearance (CL) was calculated
from
Dose/AUC.,. Mean resident time extrapolated to infinity (MRT) was estimated by
moment
analysis. V,s was calculated from MRT,, x CL. Maximum concentration (C.) and
time to
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reach Cmax ((max) were recorded as observed. As this was a cross-over study,
bioavailability
was calculated dAUC.,po/dAUC.,iv x 100% where dAUC was the dose normalized AUC

value from the same animal given IV and PO dose. Data for compounds tested are
provided
in Table E. Compounds tested were prepared in accordance with the synthetic
procedures
described herein.
Table E
Compound No. (mL/ tin (h) Vss (L/kg)
miCLn/kg)
4 11.6 9.94 8.89
13 25.8 2.05 3.98
18 7.91 10.9 6.08
Comparator B 8.72 16.22 9.96
Comparator C 13.78 11.56 12.03
Comparator D 6.92 18.12 12.43
Comparator E 3.4 16 4.35
Projected Single-Dose CL and Vss values in Humans
[0198] Allometric scaling for the prediction of human Clearance and
Volume of
Distribution was based on interspecies simple allometric scaling of mouse,
rat, dog, and
cynomolgus monkey intravenous pharmaco*kinetic parameters (Boxenbaum, J
Pharmaco*kinet
Biopharna 10:201-27, 1982). Prediction of human CL was performed by
extrapolating pre-
clinical species' plasma intravenous clearance. The 'rule of exponents'
(Mahmood & Balian,
Life Sci 59:579-85, 1996) was examined in this prediction, where it has been
proposed that,
when the exponent of simple allometry lies between 0.71 and 0.99, a correction
factor based
on Maximum Life Span (MLP) of this species can be applied and, when the
exponent of
simple allometry is greater than 1.0, a correction factor based on Brain
Weight (BrW) can be
applied, or a protein binding correction can be applied when available. In a
similar manner,
simple allometric scaling was employed to predict human Volume of
Distribution. This
method has been used successfully for varied drugs (Ward & Smith, Drug Metab
Dispos
32:612-19, 2004; McGinnity et al., Curr Drug Metab 8:463-79, 2007). Projected
data for
selected compounds are provided in Table F.
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Table F
Projected CL Projected tin. Projected tin
Projected Vss
Compound No.
(mL/min/kg) (h) Method
(L/kg)
SA, ROE, MLP
4 1.84 28.3 correction, fu
4.51
correction
fu ROE,
13 4.66 8.51 SA,
3.43
correction
SA, ROE, BrW
18 3.3 25
7.1
correction
Comparator A 1.44 38.7 SA.fu 41.2
correction
SA, ROE, fu
Comparator B 1.96 54.5 9.25
correction
SA, ROE, MLP
Comparator C 3.9 41 correction, fu 13.9
correction
SA, ROE, MLP
Comparator D 0.96 62 correction. fu 5.16
correction
SA, ROE, fu
Comparator E 1.2 51.7 5.5
correction
I-01991 Key for Table F: SA = Simple Allometry; ROE = Rule of
Exponents; fu
correction = function unbound in plasma correction; MLP correction = Maximum
Life Span
Correction; BrW correction = Brain Weight correction
Dog Cassette Dosing
[0200] Non-naïve male beagle dogs (8 months to 3 years of age, body
weight 8-14 kg)
sourced from Jiangsu Johnsen Bioresource CO. and/or Beijing Rixinkeji CO., LTD
and/or
Beijing Marshall Biotechnology CO., LTD were used in this study. All animals
for IV
administration had free access to food and water. The IV dose solution was
prepared in
10% DMA/50% PG/40% HPI3CD solution (40% w/v aqueous FIII3CD) at a
concentration of
0.2 mg/mL. Concentrations of IV dose was measured at the end of the study. PK
parameters
were calculated using the nominal dose values if the measured values were
within 20% of the
nominal values. Blood samples were collected by venipuncture of peripheral
veins except the
dosing vein at pre-dose, 5 15, 30 min, 1, 2, 4, 6, 8 and 24 h post-dose. Blood
samples were
centrifuged and resultant plasma was frozen for bioanalysis. Plasma samples
were stored
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at -80 C before analysis. Plasma samples were analyzed for compound
concentrations using
the LC/MS/MS method. Briefly, a 50111_, aliquot of each plasma sample was
mixed with
100 iaL of acetonitrile that contained an internal standard. The mixture was
vortexed and
centrifuged. Ten (10) L of the resulting solution was injected onto a reverse-
phase C18
column and the resultant peaks detected on a LC/MS/MS equipped with a turbo
ionspray
ionization source. Sample concentrations below the limit of quantification
(BLQ) were
treated as zero for PK calculations. PK parameters were estimated from
individual animals
using noncompartmental analysis of the concentration-time data (Phoenix
WinNonLin
software, version 64; Pharsight, Mountain View, CA). The elimination rate
constant (k) was
calculated as the absolute value of the slope of the linear regression of
logarithm (log) of the
concentration versus time for the last three data points of the concentration-
time profiles.
Apparent elimination half-life (t1/2) values were calculated as ln(2)/k. Area
under the
concentration-time curve (AUC) values were estimated using linear trapezoidal
method.
AUCt values were calculated from the dosing time to the last measurable
concentration.
AUCc, values were calculated as the sum of the corresponding AUC t and the
ratio of the last
detectable concentration divided by k. Plasma clearance (CL) was calculated
from
Dose/AUC,. Mean resident time extrapolated to infinity (MRT) was estimated by
moment
analysis. Vss was calculated from MRT,, x CL. Maximum concentration (Cm) and
time to
reach Ciõaõ (tmax) were recorded as observed. As this was a cross-over study,
bioavailability
was calculated dAUC,,p,/dAUC,,i, x 100% where dAUC was the dose normalized AUC

value from the same animal given IV and PO dose. Data for compounds tested are
provided
in Table G. Compounds tested were prepared in accordance with the synthetic
procedures
described herein.
Table G
CL
Compound No. (mL/min/kg) t112 (h) Vss (L/kg)
1 3.65 12.5 3.44
2 4.06 7.34 1.99
3 7.16 13.1 6.9
4 5.12 11.8 4.48
9.26 9.86 6.17
6 9.08 7.11 3.98
7 9.22 10.9 6.33
8 15.6 5.66 6.26
9 13.9 3.65 3.08
36.3 9.38 18.6
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11 6.3 9.66 4.42
12 34.4 7.39 17.8
13 11.5 4.16 3.72
14 36.5 2.43 6.44
15 24.2 1.49 2.84
16 33 3.07 7.08
17 41.9 3.02 8.25
18 7.07 9.08 4.29
Monkey Cassette Dosing
[0201] Non-naive male beagle dogs (2-5 years of age, body weight 2-
5 kg) sourced from
Topgene Biotechnology were used in this study. All animals for IV
administration had free
access to food and water. The IV dose solution was prepared in 10% DMA/50%
PG/40%
HP13CD solution (40% w/v aqueous HP(3CD) at a concentration of 0.2 mg/mL.
Concentrations of IV dose was measured at the end of the study. PK parameters
were
calculated using the nominal dose values if the measured values were within
20% of the
nominal values. Blood samples were collected by venipuncture of peripheral
veins except the
dosing vein at pre-dose, 5 15, 30 min, 1, 2, 4, 6, 8 and 24 h post-dose. Blood
samples were
centrifuged and resultant plasma was frozen for bioanalysis. Plasma samples
were stored
at -80 C before analysis. Plasma samples were analyzed for compound
concentrations using
the LC/MS/MS method. Briefly, a 50 ittL aliquot of each plasma sample was
mixed with
100 it L of acetonitrile that contained an internal standard. The mixture was
vortexed and
centrifuged. Ten (10) 1., of the resulting solution was injected onto a
reverse-phase C18
column and the resultant peaks detected on a LC/MS/MS equipped with a turbo
ionspray
ionization source. Sample concentrations below the limit of quantification
(BLQ) were
treated as zero for PK calculations. PK parameters were estimated from
individual animals
using noncompartmental analysis of the concentration-time data (Phoenix
WinNonLin
software, version 64; Pharsight, Mountain View, CA). The elimination rate
constant (k) was
calculated as the absolute value of the slope of the linear regression of
logarithm (log) of the
concentration versus time for the last three data points of the concentration-
time profiles.
Apparent elimination half-life (t1/2) values were calculated as ln(2)/k. Area
under the
concentration-time curve (AUC) values were estimated using linear trapezoidal
method.
AUCt values were calculated from the dosing time to the last measurable
concentration.
AUCc, values were calculated as the sum of the corresponding AUCt and the
ratio of the last
detectable concentration divided by k. Plasma clearance (CL) was calculated
from
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Dose/AUC.,. Mean resident time extrapolated to infinity (MRT) was estimated by
moment
analysis. Vss was calculated from MRT,,, x CL. Maximum concentration (C.) and
time to
reach Cmax max, (t 1 were recorded as observed. As this was a cross-over
study, bioavailability
"
was calculated dAUC,po/dAUC.,i, X 100% where dAUC was the dose normalized AUC
value from the same animal given IV and PO dose. Data for compounds tested are
provided
in Table H. Compounds tested were prepared in accordance with the synthetic
procedures
described herein.
Table H
CL
Compound No. (mL/minikg) t112 (h) Vss (L/kg)
4 13.1 8.69 8.8
19 8.2 10.2
6 12.3 8.07 6.29
7 10.2 12.5 9.12
8 20.2 3.72 4.76
9 15.2 1.59 1.88
11 33.1 3.03 7.26
13 21.4 1.83 3.15
14 17.1 1.1 1.48
18 12.5 8.04 10.2
Projected Cassette-Dosing CL and Vss Values in Humans
[0202]
For PK data derived from Cassette IV dosing, predictions of Human Clearance
and Volume of Distribution were performed using Single Species allometry. In
this case
values were predicted from plasma intravenous Clearance of Dog and Monkey PK
by
applying protein binding correction (Tang, Drug Metab Dispos 33:1294-96, 2005;
Patel,
Journal of Pharmaceutical Research International, 22(3): 1-7, 2018). Projected
data for
selected compounds are provided in Table J and Table K.
Table J
Projected Human Projected Human
Projected Human
Single Species Single Species Single Species
Compound
No. Allometry: Dog Allometry: Dog Allometry:
Dog
CL (mL/min/kg) tin (h) Vss
(L/kg)
1 1.5 11.2
24.1
2 1.3 5.8
10.8
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3 3.2 28.8
52.5
4 1.5 25.8 22
4.6 19.8 52
6 1.5 13.2
11.1
7 3.6 20.4 42
8 5.98 12.00 41
9 5 2.6
18.8
14.9 6.1 130.2
11 2.6 8.3
30.9
12 14.1 6.1
124.6
13 4.8 9.6
26.8
14 15 2.1
45.1
9.92 1.4 19.9
16 13.51 2.5
49.6
17 17.17 2.3
57.8
18 3.6 18.00
36.9
Table K
Projected Human Projected Human Projected Human
Single Species Single Species Single
Species
Compound
No Allometry: Monkey Allometry: Monkey Allometry: Monkey
.
CL (mL/min/kg) tin (h) Vss (L/kg)
4 3.1 15.84 71
5 6.4 12.72
118
6 1.9 12.24
32.6
7 3.5 21.36
108
8 6.8 5.52 55
9 4.8 2.88
20.3
11 13.5 5.2
101.6
13 6.7 3.6
33.6
14 7 2.1
20.7
18 4.3 19.44
121.4
Biological Example B-3: Echocardiography assessment of acute pharmacodynarnic
effect in
rat cardiac contractility
[0203]
Assessment of in vivo cardiac function by echocardiography was performed in
male Sprague Dawlcy rats under isoflurane (1-3%) anesthesia. 2-D M-mode images
of the
left ventricle were acquired in the parastemal long-axis view before, during,
and after
administration of compounds. In vivo fractional shortening was determined by M-
mode
image analysis with the following calculation: ((End diastolic diameter - end
systolic
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diameter)/ end diastolic diameter x 100). Three pre-dose baseline M-Mode
images were taken
at 1-minute intervals prior to compound administration. Compounds were
formulated in a
0.5% hydroxypropyl methylcellulose 2910 (HPMC 2910): 0.1% Tween 80 suspension
and
delivered as a single dose (5 mL/kg) by oral gavage. At one and four hours
post-dose, rats
were lightly anesthetized for M-mode echocardiography measurement. Concurrent
with
echocardiography measurements, blood samples were taken to determine the
corresponding
compound plasma concentrations. The resulting plasma concentrations were used
to estimate
the IC50 and ICio values, which is the concentration at which fractional
shortening is 50% and
10% of the pre-dose baseline contractility, respectively.
Table L: IC50, IC10 values and IC50/IC10 ratio
Compound No. ICio ( M) ICso ( M) ICsoaCio
4 0.284 2.763 9.73
1.17 4.439 3.79
8 0.508 2.253 4.44
13 0.317 1.172 3.7
18 0.892 2.835 3.18
Biological Example B-4: In Vitro Determination of Time-Dependent inhibition of
CYP450
enzymes
[0204]
An assessment of the time-dependent Inhibitory Potential of the test
compounds
against Principal Human Cytochrome P450 Isozymes using Human Liver Microsomes
was
also carried out using standard methods (Grimm et al, Drug Metab. Divas., Jul;
37(7):1355-
70. doi: 10.1124/dmc1.109.026716, 2009). Pooled human microsomes and selective
CYP
probe substrates were used for in vitro assessment of test compounds at 25 and
50 p.M as
time-dependent inhibitors of seven human hepatic cytochrome P450 isozymes (CYP
IA2,
2B6, 2C9, 2C19, 2D6, and 3A4). LC-MS/MS was used to quantify metabolite
formation.
The inhibition of each P450 enzyme in human liver microsomes was measured as
the
percentage decrease in the activity of marker metabolite formation as measured
by LC-
MS/MS compared to non-inhibited controls (= 100% activity) at time zero and
after 30
minutes incubation. The occurrence of any time dependent inhibition was then
expressed as
the fold change in enzyme activity at time zero relative to the activity after
30 minutes
incubation.
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Table M: Time Dependent Inhibition for tested compounds against CYP1A2,
CYP2B6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4-M and CYP3A4-T
CYP1A2 CYP2B6
(%
( %
Activity
Activity
NADPH
NADPH
% % TO) /(% % % TO)
/(%
Activity Activity Activity Activity Activity Activity
Compound Conc NADPH NADPH NADPH NADPH NADPH NADPH
No. ( M) TO T30 T30) TO T30
T30)
Comparator 25 NA NA NA NA NA NA
C 50 NA NA NA NA NA NA
18 25 85 99 0.9 80 103
0.8
50 92 90 1.0 76 83
0.9
25 89 87 1.0 79 78 1.0
50 99 88 1.1 83 76
1.1
4 25 95 90 1.1 95 84
1.1
50 86 85 1.0 76 76
1.0
6 25 97 85 1.1 95 83
1.1
50 95 99 1.0 87 77
1.1
7 25 81 95 0.9 70 74
0.9
50 79 100 0.8 78 70
1.1
8 25 82 96 0.9 83 97
0.9
50 86 101 0.9 88 100
0.9
13 25 88 99 0.9 102 116
0.9
50 92 110 0.8 90 81
1.1
CYP2C9 CYP2C19
( %
( %
Activity
Activity
NADPH
NADPH
% % TO) /(% % % TO)
/(%
Activity Activity Activity Activity Activity Activity
Compound Conc NADPH NADPH NADPH NADPH NADPH NADPH
No. ( M) TO T30 T30) TO T30
T30)
Comparator 25 NA NA NA NA NA
NA
C 50 NA NA NA NA NA NA
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18 25 44 47 0.9 103 99
1.0
50 32 27 1.2 91 84
1.1
25 62 79 0.8 101 83 1.2
50 62 64 1.0 102 77
1.3
4 25 83 84 1.0 117 86
1.4
50 63 71 0.9 84 75
1.1
6 25 48 41 1.2 105 89
1.2
50 34 33 1.0 100 92
1.1
7 25 94 75 1.3 87 87
1.0
50 87 61 1.4 82 83
1.0
8 25 104 107 1.0 94 99
0.9
50 112 109 1.0 93 92
1.0
13 25 107 114 0.9 108 108
1.0
50 111 109 1.0 113 119
0.9
CYP2D6 CYP2C8
(%
(%
Activity
Activity
NADPH
NADPH
% % TO) /(% % % TO) /(%
Activity Activity Activity Activity Activity Activity
Conc NADPH NADPH NADPH NADPH NADPH NADPH
Compound ( M) TO T30 T30) TO T30 T30)
Comparator 25 NA NA NA NA NA
NA
C 50 NA NA NA NA NA NA
18 25 103 105 1.0 65 100
0.7
50 104 91 1.1 100 76
1.3
5 25 98 89 1.1 87 88
1.0
50 94 84 1.1 104 53
2.0
4 25 118 90 1.3 79 80
1.0
50 93 88 1.1 97 79
1.2
6 25 112 87 1.3 79 88
0.9
50 107 92 1.2 86 80
1.1
7 25 80 88 0.9 72 64
1.1
50 87 95 0.9 109 103
1.1
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8 25 92 98 0.9 117 117
1.0
50 87 91 1.0 110 112
1.0
13 25 96 100 1.0 116 98
1.2
50 98 72 1.4 106 106
1.0
CYP3A4-T CYP3A4-M
(%
(%
Activity
Activity
NADPH
NADPH
% % TO) /( % % % TO) /( %
Activity Activity Activity Activity Activity Activity
Conc NADPH NADPH NADPH NADPH NADPH NADPH
Compound ( M) TO T30 T30) TO T30 T30)
Comparator 25 41 23 1.8 75 48 1.6
C 50 35 19 1.8 57 33
1.7
18 25 100 85 1.2 125 71
1.8
50 85 75 1.1 126 102
1.2
25 100 87 1.1 75 69 1.1
50 98 68 1.4 67 35
1.9
4 25 80 90 0.9 70 88
0.8
50 83 98 0.8 57 78
0.7
6 25 95 76 1.3 89 89
1.0
50 89 63 1.4 72 86
0.8
7 25 100 100 1.0 100 100
1.0
50 100 100 1.0 100 100
1.0
8 25 100 100 1.0 100 100
1.0
50 100 100 1.0 100 100
1.0
13 25 74 77 1.0 87 88
1.0
50 56 65 0.9 72 97
0.7
[0205] NA: not available; CYP3A4-T : CYP3A4 activity as measured by
the testosterone
probe substrate; CYP3A4-M : CYP3A4 activity as measured by the midazolam probe

substrate
[0206]
For 3A4, measuring the % activity using both midalzolam and testosterone as
probes, compound 13, compound 8, compound 7 and compound 4 do not show any
indication
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WO 2022/187501
PCT/US2022/018725
of time dependent inhibition since the activity of the enzyme did not change
by greater than
1.2 fold. Comparator C however did show a change in fold activity of > 1.5
fold for both
probes at the 25 and 501.11\4 concentrations. Compound 18, compound 5, and
compound 6
also showed some change in the fold activity > 1.2 fold either at one of the
concentrations
tested or with one of the probe substrates studies suggesting that there may
be some change in
3A4 activity when tested in time dependent format for these compounds. All the
compounds
tested showed no change in activity for 1A2 and 2B6 when tested in this
format. For 2C9,
compound 7 only showed a 1.3 fold change in activity at the 25 i_tM
concentration and a 1.3
fold change in activity at the 50 pM concentration. For 2C19, compound 5
showed a 1.3 fold
change in activity at the 50 uM concentration and compound 4 showed a 1.4 fold
change in
activity at the 25 uM concentration, although no change in activity was
observed for
compound 4 at the 50 [I M concentration. For 2D6, compound 13 showed a 1.4
fold change in
activity at the 50 1i1\4 concentration and compound 6 and compound 4 showed a
1.3 fold
change in activity at the 25 uM concentration, although no change in activity
was observed
for these compounds at the 50 IVI concentration. For 2C8, compound 18 showed
a 1.3 fold
change in activity at the 50 uM concentration and compound 5 showed a 2.0 fold
change in
activity at the 50 uM concentration.
[0207] While the foregoing written description of the compounds,
uses, and methods
described herein enables one of ordinary skill to make and use the compounds,
uses, and
methods described herein, those of ordinary skill will understand and
appreciate the existence
of variations, combinations, and equivalents of the specific embodiment,
method, and
examples herein. The compounds, uses, and methods provided herein should
therefore not be
limited by the above-described embodiments, methods, or examples, but rather
encompasses
all embodiments and methods within the scope and spirit of the compounds,
uses, and
methods provided herein.
[0208] All references disclosed herein are incorporated by
reference in their entirety.
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