U.S. patent application number 16/026901 was filed with the patent office on 2018-11-01 for nampt inhibitors for cancer therapy.
The applicant listed for this patent is Genzyme Corporation. Invention is credited to Kara Carter, Sanjay Danthi, Jill Gregory, Yiding Hu, Yinyin Huang, Alexandra Joseph, Paul Mason, Mohamud Mohamud, Andrew Papoulis, Yibin Xiang, Christopher Yee, Zhong Zhao.
Application Number | 20180312525 16/026901 |
Document ID | / |
Family ID | 53366297 |
Filed Date | 2018-11-01 |
United States Patent
Application |
20180312525 |
Kind Code |
A1 |
Mason; Paul ; et
al. |
November 1, 2018 |
NAMPT Inhibitors for Cancer Therapy
Abstract
The invention features compounds of Formula (I), Formula (I)-A
and Formula (I)-B as disclosed herein, as well as methods of
synthesis, therapy, diagnostics, and assays.
Inventors: |
Mason; Paul; (Natick,
MA) ; Carter; Kara; (Cambridge, MA) ; Joseph;
Alexandra; (Lexington, MA) ; Hu; Yiding;
(Lexington, MA) ; Gregory; Jill; (Acton, MA)
; Zhao; Zhong; (Wayland, MA) ; Yee;
Christopher; (Needham, MA) ; Mohamud; Mohamud;
(Columbia Heights, MN) ; Xiang; Yibin; (Dracut,
MA) ; Danthi; Sanjay; (Roxbury, MA) ; Huang;
Yinyin; (Chestnut Hill, MA) ; Papoulis; Andrew;
(Canton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Cambridge |
MA |
US |
|
|
Family ID: |
53366297 |
Appl. No.: |
16/026901 |
Filed: |
July 3, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15312482 |
Nov 18, 2016 |
10017523 |
|
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PCT/US2015/032170 |
May 22, 2015 |
|
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16026901 |
|
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62001905 |
May 22, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 35/02 20180101; A61P 31/16 20180101; C12N 5/0635 20130101;
A61P 3/10 20180101; A61P 31/20 20180101; C07D 491/107 20130101;
G01N 33/60 20130101; A61P 17/06 20180101; A61P 17/02 20180101; A61P
37/06 20180101; A61P 35/00 20180101; A61P 17/00 20180101; C07D
491/20 20130101; A61P 37/02 20180101; A61P 19/02 20180101; A61P
31/18 20180101; A61P 31/12 20180101; A61P 37/00 20180101; G01N
33/5008 20130101; A61P 43/00 20180101; C07D 498/10 20130101 |
International
Class: |
C07D 498/10 20060101
C07D498/10; G01N 33/60 20060101 G01N033/60; G01N 33/50 20060101
G01N033/50; C07D 491/107 20060101 C07D491/107; C07D 491/20 20060101
C07D491/20; C12N 5/0781 20060101 C12N005/0781 |
Claims
1. A compound of Formula (I): ##STR00035## or a pharmaceutically
acceptable salt thereof, wherein: Y.sub.1 is --CR.sub.14-- or N,
Y.sub.2 is --CR.sub.15-- or N, Y.sub.3 is --C(O)--, Y.sub.4 is
--CH.sub.2, or --N(R.sub.16)--, or Y.sub.3 and Y.sub.4 together are
--C(R.sub.17).dbd.C(R.sub.18)--, X is aryl, heteroaryl, arylalkyl,
heteroarylalkyl, or amide, Z is C.sub.2 or greater alkyl or
alkoxylalkyl, R is H or C.sub.1-C.sub.6 alkyl, R.sub.1-R.sub.18 are
independently H or C.sub.1-C.sub.6 alkyl, and Q is N; provided that
when R is H, X is not ##STR00036##
2. The compound of claim 1, wherein R is H.
3. The compound of claim 1, wherein X is heteroarylalkyl.
4. The compound of claim 3, wherein X is pyridinylmethyl,
substituted pyridinylmethyl, isoxazolylmethyl, substituted
isoxazolylmethyl, or thiazolylmethyl.
5. The compound of claim 4, wherein X is methylisoxazolylmethyl,
fluoropyridinylmethyl, or methylpyridinylmethyl.
6. The compound of claim 1, wherein Y.sub.1 is CH or N.
7. The compound of claim 1, wherein Y.sub.2 is CH or N.
8. The compound of claim 1, wherein Y.sub.4 is --CH.sub.2--.
9. The compound of claim 1, wherein Y.sub.3 and Y.sub.4 together
are --C(R.sub.17).dbd.C(R.sub.18)--.
10. The compound of claim 1, wherein Z is ethyl, propyl, isopropyl,
butyl, cyclobutylmethyl, methoxypropyl, or methoxybutyl.
11. The compound of claim 1, wherein R.sub.1-R.sub.18 are each
--H.
12. A pharmaceutical composition, comprising the compound of claim
1 and a pharmaceutically acceptable carrier.
13. A method of inhibition of activated and/or proliferating B
cells for therapeutic modulation of immunological disease,
comprising administering to a patient in need thereof a composition
comprising the compound of claim 1.
14. The method of claim 13, wherein the immunological disease is
lupus, rheumatoid arthritis, scleroderma, psoriasis, Sjogren's
syndrome, type I diabetes or multiple sclerosis.
15. A method of inducing, modulating or maintaining
immunosuppression for transplant, comprising administering to a
patient in need thereof a composition comprising the compound of
claim 1.
16. A method of suppressing or modulating an immune response to a
therapeutic biologic, comprising administering to a patient in need
thereof a composition comprising the compound of claim 1.
17. A method of inhibiting tumor cell growth wherein the tumor
cells are NAPRT deficient, comprising administering to a patient in
need thereof a composition comprising the compound of claim 1.
18. A method of treating leukemia and lymphoma, comprising
administering to a patient in need thereof a composition comprising
the compound of claim 1.
19. A method of treating a NAD-requiring viral infection,
comprising administering to a patient in need thereof a composition
comprising the compound of claim 1.
20. The method of claim 19, wherein the viral infection is selected
from influenza, RSV, HSV, HCV, HBV, HPV, HIV, CMV, EBOV, or EBV.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Utility
application Ser. No. 15/312,482, filed Nov. 18, 2016, which is a
U.S. National Phase application under 35 U.S.C. .sctn. 371 of
International Patent Application No. PCT/US2015/032170, filed May
22, 2015, which claims priority under 35 U.S.C. .sctn. 119(e) to
U.S. Provisional Application No. 62/001,905 filed May 22, 2014. The
contents of all parent applications are incorporated herein in
their entirety.
TECHNICAL FIELD
[0002] This invention relates generally to therapeutics and
treatments for various disorders, including, without limitation,
oncological disorders, immunological disorders, viral infections
and disorders related to nicotinamide phoshophoribosyltransferase
(NAMPT).
BACKGROUND
[0003] Two NAMPT inhibitors (FK866 and CHS828) have entered
clinical trials. See generally, Galli et al., "Medicinal Chemistry
of Nicotinamide Phosphoribosetransferase (NAMPT) Inhibitors", J.
Med. Chem. 2013 (56(16), pp 6279-6296.
SUMMARY
[0004] The present invention provides, inter alia, compounds of
Formula (I):
##STR00001##
or a pharmaceutically acceptable salt thereof, wherein: [0005]
Y.sub.1 is --CR.sub.14-- or N, [0006] Y.sub.2 is --CR.sub.15-- or
N, [0007] Y.sub.3 is --C(O)--, [0008] Y.sub.4 is --CH.sub.2, or
--N(R.sub.16)--, [0009] or Y.sub.3 and Y.sub.4 together are
--C(R.sub.17).dbd.C(R.sub.18)--, [0010] X is aryl, heteroaryl,
arylalkyl, heteroarylalkyl, or amide, [0011] Z is C.sub.2 or
greater alkyl or alkoxylalkyl, [0012] R is H or C.sub.1-C.sub.6
alkyl, [0013] R.sub.1-R.sub.18 are independently H or
C.sub.1-C.sub.6 alkyl, and [0014] Q is CH or N; provided that when
Q is N and R is H, X is not
##STR00002##
[0015] Preferably, X is phenyl or substituted phenyl, e.g.,
fluorophenyl; R is H; X is pyridinyl or substituted pyridinyl,
e.g., fluoropyridinyl or methylpyridinyl. Also preferably, Y.sub.1
is C; Y.sub.1 is N; Y.sub.2 is C Y.sub.2 is N; Y.sub.3 is --C(O)--;
Y.sub.4 is --C(H).dbd.. Also preferably Z is C.sub.2-C.sub.4 alkyl
or propyl, e.g., isopropyl. Also preferably, Z is alkoxyalkyl or
cyclobutylmethyl. Also preferably, R.sub.17 and R.sub.18, and most
preferably R.sub.1-R.sub.16 as well, are --H.
[0016] In other preferred embodiments, the compound has one of the
following Formulas (I)-A or (I)-B:
##STR00003##
[0017] The above compounds may be used to inhibit activated and/or
proliferating B cells for therapeutic modulation of immunological
disease by administering a composition comprising the compound to a
patient in need thereof. For example, the immunological disease may
be lupus, rheumatoid arthritis, scleroderma, psoriasis, Sjogren's
syndrome, type I diabetes or multiple sclerosis. The compounds may
be used for inducing, modulating or maintaining immunosuppression
for transplant by administering a composition comprising the
compound to a patient in need thereof. Other medical uses include:
suppressing or modulating an immune response to a therapeutic
biologic such as a recombinant protein, a nucleic acid, an antibody
or a peptide; inhibiting tumor cell growth, particularly for NAPRT
deficient tumor cells; treating leukemia (e.g., acute lymphocytic
leukemia) and lymphoma; treating a viral infection such as,
influenza, respiratory syncytial virus (RSV), herpes simplex virus
(HSV), hepatitis C virus (HCV), hepatitis B virus (HBV), human
papilloma virus (HPV), human immunodeficiency virus (HIV), human
cytomegalovirus (CMV), Ebolavirus (EBOV), and Epstein-Barr virus
(EBV). Other uses for the compounds include inhibiting
differentiation of B cells (e.g., in vivo or in vitro) into plasma
cells, by contacting the B cells with the compound. The presence of
a compound according to the invention in a sample (e.g., a sample
suspected of containing the compound) may be detected by contacting
the sample with a binding agent that binds to the compound and
detecting binding of the agent to the compound. The compound may
further comprise a detectable label, e.g., to facilitate such
detection.
[0018] In another aspect, the invention features synthesizing the
compound by reacting a benzoic acid derivative with carbonyl
diimidazole and a methanamine derivative.
[0019] These compounds have applications as therapeutics for
multiple immunological and oncological diseases including, but not
limited to, lupus, rheumatoid arthritis, Sjogren's syndrome, type I
diabetes, multiple sclerosis, transplant induction and maintenance,
scleroderma, acute lymphocytic leukemia, and cancer, particularly
NAPRT-deficient solid or liquid tumors. In addition, they are
therapeutics for NAD-requiring viral infections including, but not
limited to, influenza, RSV, HSV, HCV, HBV, HPV, HIV, CMV, EBOV, and
EBV.
[0020] The present invention also provides methods of treating the
above conditions by administering to a patient a therapeutically
effective amount of such a compound or a pharmaceutically
acceptable salt thereof. The present invention also provides use or
manufacture of the compound, or a pharmaceutically acceptable salt
thereof, for use in therapy. The present invention also provides
such a compound, or a pharmaceutically acceptable salt thereof, for
use in the treatment of a disease referenced herein.
[0021] While not wishing to bind ourselves to a specific mechanism
of action, the compounds selectively inhibit human primary B cells
while sparing T cells. In general, the compounds target NAMPT and
inhibit primary human B cell activation and proliferation while
selectively sparing primary human T cells, and they are useful for
treating B cell-mediated and proliferative disorders including
various immune-mediated diseases as well as cancer, in particular
B-cell malignancies, NAPRT-deficient solid or liquid tumors and
NAD-requiring viral infections including, but not limited to,
influenza, RSV, HSV, HCV, HBV, HPV, HIV, CMV, EBOV, and EBV.
[0022] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 are graphs demonstrating the mechanism of action of
the compounds described herein.
[0024] FIG. 2 is a graph showing the mean plasma concentration vs.
time for
1'-isopropyl-3-methyl-6-(4-(3-(thiazol-5-yl)propanoyl)phenyl)spiro-[b-
enzo[e][1,3]oxazine-2,4'-piperidin]-4(3H)-one following intravenous
or oral administration to rat.
[0025] FIG. 3 is a graph showing the mean plasma concentration vs.
time for
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methyl-
isoxazol-5-yl)methyl)benzamide following intravenous or oral
administration to rat.
[0026] FIG. 4 is a graph showing the mean plasma concentration vs.
time for
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methyl-
isoxazol-5-yl)methyl)benzamide following intravenous or oral
administration to mouse.
[0027] FIG. 5 is a graph showing in vivo assay results of
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide in NALM6 leukemia model.
[0028] FIG. 6 is a graph showing in vivo assay results of
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide in a HCT116 tumor model.
[0029] FIG. 7 is a graph showing in vivo assay results of
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide in another HCT116 tumor model.
DETAILED DESCRIPTION
I. Compounds
[0030] The present disclosure relates, inter alia, to a compound of
Formula (I), Formula (I)-A and Formula (I)-B, above.
General Definitions
[0031] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, can also be provided in combination in a single
embodiment. Conversely, various features of the invention which
are, for brevity, described in the context of a single embodiment,
can also be provided separately or in any suitable
subcombination.
[0032] For the terms "e.g." and "such as," and grammatical
equivalents thereof, the phrase "and without limitation" is
understood to follow unless explicitly stated otherwise. As used
herein, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. As used
herein, the term "about" means "approximately" (e.g., plus or minus
approximately 10% of the indicated value).
[0033] As used herein, the phrase "optionally substituted" means
unsubstituted or substituted. As used herein, the term
"substituted" means that a hydrogen atom is removed and replaced by
a substituent. In general all moieties may be substituted unless
otherwise indicated. It is to be understood that substitution at a
given atom is limited by valency. Throughout the definitions, the
term "C.sub.n-m" indicates a range which includes the endpoints,
wherein n and m are integers and indicate the number of carbons.
Examples include C.sub.1-4, C.sub.1-6, and the like.
[0034] The term "n-membered" where n is an integer typically
describes the number of ring-forming atoms in a moiety where the
number of ring-forming atoms is n. For example, piperidinyl is an
example of a 6-membered heterocycloalkyl ring, pyrazolyl is an
example of a 5-membered heteroaryl ring, pyridyl is an example of a
6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is
an example of a 10-membered cycloalkyl group.
[0035] As used herein, the term "C.sub.n-m alkyl", employed alone
or in combination with other terms, refers to a saturated
hydrocarbon group that may be straight-chain or branched, having n
to m carbons. In some embodiments, the alkyl group contains from 1
to 6 carbon atoms or from 1 to 4 carbon atoms, or from 1 to 3
carbon atoms. Examples of alkyl moieties include, but are not
limited to, chemical groups such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, s-butyl, and t-butyl.
[0036] As used herein, the term "C.sub.n-m alkoxy", employed alone
or in combination with other terms, refers to a group of formula
--O-alkyl, wherein the alkyl group has n to m carbons. Example
alkoxy groups include methoxy, ethoxy, and propoxy (e.g., n-propoxy
and isopropoxy). In some embodiments, the alkyl group has 1 to 3
carbon atoms.
[0037] As used herein, "C.sub.n-m alkenyl" refers to an alkyl group
having one or more double carbon-carbon bonds and having n to m
carbons. In some embodiments, the alkenyl moiety contains 2 to 6 or
2 to 4 carbon atoms. Example alkenyl groups include, but are not
limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl,
sec-butenyl, and the like.
[0038] As used herein, "C.sub.n-m alkynyl" refers to an alkyl group
having one or more triple carbon-carbon bonds and having n to m
carbons. Example alkynyl groups include, but are not limited to,
ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some
embodiments, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon
atoms.
[0039] As used herein, the term "C.sub.n-m alkylamino" refers to a
group of formula --NH(alkyl), wherein the alkyl group has n to m
carbon atoms. In some embodiments, the alkyl group has 1 to 6 or 1
to 4 carbon atoms.
[0040] As used herein, the term "di-C.sub.n-m-alkylamino" refers to
a group of formula --N(alkyl).sub.2, wherein the two alkyl groups
each has, independently, n to m carbon atoms. In some embodiments,
each alkyl group independently has 1 to 6 or 1 to 4 carbon
atoms.
[0041] As used herein, the term "C.sub.n-m alkylthio" refers to a
group of formula --S-alkyl, wherein the alkyl group has n to m
carbon atoms. In some embodiments, the alkyl group has 1 to 6 or 1
to 4 carbon atoms.
[0042] As used herein, the term "amino" refers to a group of
formula --NH.sub.2.
[0043] As used herein, the term "aryl", employed alone or in
combination with other terms, refers to a monocyclic or polycyclic
(e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbon, such as,
but not limited to, phenyl, 1-naphthyl, 2-naphthyl, anthracenyl,
phenanthrenyl, and the like. In some embodiments, aryl is
C.sub.6-10 aryl. In some embodiments, the aryl group is a
naphthalene ring or phenyl ring. In some embodiments, the aryl
group is phenyl.
[0044] As used herein, the term "carbonyl", employed alone or in
combination with other terms, refers to a --C(O)-- group.
[0045] As used herein, the term "cycloalkyl", employed alone or in
combination with other terms, refers to a non-aromatic cyclic
hydrocarbon moiety, which may optionally contain one or more
alkenylene groups as part of the ring structure. Cycloalkyl groups
can include mono- or polycyclic (e.g., having 2, 3 or 4 fused
rings) ring systems. Also included in the definition of cycloalkyl
are moieties that have one or more aromatic rings fused (i.e.,
having a bond in common with) to the cycloalkyl ring, for example,
benzo derivatives of cyclopentane, cyclopentene, cyclohexane, and
the like. One or more ring-forming carbon atoms of a cycloalkyl
group can be oxidized to form carbonyl linkages. In some
embodiments, cycloalkyl is C.sub.3-7 cycloalkyl, which is
monocyclic or bicyclic. Exemplary cycloalkyl groups include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl,
norbornyl, norpinyl, norcarnyl, and the like. In some embodiments,
the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or
cyclohexyl.
[0046] As used herein, "C.sub.n-m haloalkoxy" refers to a group of
formula --O-haloalkyl having n to m carbon atoms. An example
haloalkoxy group is OCF.sub.3. An additional example haloalkoxy
group is OCHF.sub.2. In some embodiments, the haloalkoxy group is
fluorinated only. In some embodiments, the alkyl group has 1 to 6
or 1 to 4 carbon atoms.
[0047] As used herein, the term "halo" refers to a halogen atom
selected from F, Cl, I or Br. In some embodiments, "halo" refers to
a halogen atom selected from F, Cl, or Br. In some embodiments,
exemplary halo groups are F.
[0048] As used herein, the term "C.sub.n-m haloalkyl", employed
alone or in combination with other terms, refers to an alkyl group
having from one halogen atom to 2s+1 halogen atoms which may be the
same or different, where "s" is the number of carbon atoms in the
alkyl group, wherein the alkyl group has n to m carbon atoms. In
some embodiments, the haloalkyl group is fluorinated only. In some
embodiments, the haloalkyl group is fluoromethyl, difluoromethyl,
or trifluoromethyl. In some embodiments, the haloalkyl group is
trifluoromethyl. In some embodiments, the alkyl group has 1 to 6 or
1 to 4 carbon atoms.
[0049] As used herein, the term "heteroaryl", employed alone or in
combination with other terms, refers to a monocyclic or polycyclic
(e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbon moiety,
having one or more heteroatom ring members selected from nitrogen,
sulfur and oxygen. In some embodiments, heteroaryl is 5- to
10-membered C.sub.1-9 heteroaryl, which is monocyclic or bicyclic
and which has 1, 2, 3, or 4 heteroatom ring members independently
selected from nitrogen, sulfur and oxygen. When the heteroaryl
group contains more than one heteroatom ring member, the
heteroatoms may be the same or different. The nitrogen atoms in the
ring(s) of the heteroaryl group can be oxidized to form N-oxides.
Example heteroaryl groups include, but are not limited to,
pyridine, pyrimidine, pyrazine, pyridazine, pyrrole, pyrazole,
azolyl, oxazole, isoxazole, thiazole, isothiazole, imidazole,
furan, thiophene, triazole, tetrazole, thiadiazole, quinoline,
isoquinoline, indole, benzothiophene, benzofuran, benzisoxazole,
imidazo[1,2-b]thiazole, purine, triazine or the like.
[0050] A 5-membered heteroaryl is a heteroaryl group having five
ring atoms comprising carbon and one or more (e.g., 1, 2, or 3)
ring atoms independently selected from N, O, and S. Exemplary
five-membered ring heteroaryls are thienyl, furyl, pyrrolyl,
imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl,
isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl,
1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl,
1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and
1,3,4-oxadiazolyl
[0051] A 6-membered heteroaryl is a heteroaryl group having six
ring atoms wherein one or more (e.g., 1, 2, or 3) ring atoms are
nitrogen. Exemplary six-membered ring heteroaryls are pyridyl,
pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.
[0052] As used herein, the term "heterocycloalkyl", employed alone
or in combination with other terms, refers to non-aromatic ring
system, which may optionally contain one or more alkenylene or
alkynylene groups as part of the ring structure, and which has at
least one heteroatom ring member independently selected from
nitrogen, sulfur and oxygen. When the heterocycloalkyl group
contains more than one heteroatom, the heteroatoms may be the same
or different. Heterocycloalkyl groups can include mono- or
polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems,
including spiro systems. Also included in the definition of
heterocycloalkyl are moieties that have one or more aromatic rings
fused (i.e., having a bond in common with) to the non-aromatic
ring, for example, 1,2,3,4-tetrahydro-quinoline, dihydrobenzofuran
and the like. The carbon atoms or heteroatoms in the ring(s) of the
heterocycloalkyl group can be oxidized to form a carbonyl, or
sulfonyl group (or other oxidized linkage) or a nitrogen atom can
be quaternized. In some embodiments, heterocycloalkyl is 5- to
10-membered C.sub.2-9 heterocycloalkyl, which is monocyclic or
bicyclic and which has 1, 2, 3, or 4 heteroatom ring members
independently selected from nitrogen, sulfur and oxygen. Examples
of heterocycloalkyl groups include 1,2,3,4-tetrahydro-quinoline,
dihydrobenzofuran, azetidine, azepane, pyrrolidine, piperidine,
piperazine, morpholine, thiomorpholine, and pyran.
[0053] The compounds described herein can be asymmetric (e.g.,
having one or more stereocenters). All stereoisomers, such as
enantiomers and diastereoisomers, are intended unless otherwise
indicated. Compounds of the present invention that contain
asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically inactive starting materials
are known in the art, such as by resolution of racemic mixtures or
by stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present invention. Cis and trans geometric
isomers of the compounds of the present invention are described and
may be isolated as a mixture of isomers or as separated isomeric
forms.
[0054] When the compounds of the invention contain a chiral center,
the compounds can be any of the possible stereoisomers. In
compounds with a single chiral center, the stereochemistry of the
chiral center can be (R) or (S). In compounds with two chiral
centers, the stereochemistry of the chiral centers can each be
independently (R) or (S) so the configuration of the chiral centers
can be (R) and (R), (R) and (S); (S) and (R), or (S) and (S). In
compounds with three chiral centers, the stereochemistry each of
the three chiral centers can each be independently (R) or (S) so
the configuration of the chiral centers can be (R), (R) and (R);
(R), (R) and (S); (R), (S) and (R); (R), (S) and (S); (S), (R) and
(R); (S), (R) and (S); (S), (S) and (R); or (S), (S) and (S).
[0055] Resolution of racemic mixtures of compounds can be carried
out by any of numerous methods known in the art. An example method
includes fractional recrystallization using a chiral resolving acid
which is an optically active, salt-forming organic acid. Suitable
resolving agents for fractional recrystallization methods are, for
example, optically active acids, such as the D and L forms of
tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid,
mandelic acid, malic acid, lactic acid or the various optically
active camphorsulfonic acids such as D-camphorsulfonic acid. Other
resolving agents suitable for fractional crystallization methods
include stereoisomerically pure forms of .alpha.-methylbenzylamine
(e.g., S and R forms, or diastereoisomerically pure forms),
2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,
cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.
[0056] Resolution of racemic mixtures can also be carried out by
elution on a column packed with an optically active resolving agent
(e.g., dinitrobenzoylphenylglycine). Suitable elution solvent
composition can be determined by one skilled in the art.
[0057] Compounds of the invention also include tautomeric forms.
Tautomeric forms result from the swapping of a single bond with an
adjacent double bond together with the concomitant migration of a
proton. Tautomeric forms include prototropic tautomers which are
isomeric protonation states having the same empirical formula and
total charge. Example prototropic tautomers include ketone-enol
pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic
acid pairs, enamine-imine pairs, and annular forms where a proton
can occupy two or more positions of a heterocyclic system, for
example, .sup.1H- and .sup.3H-imidazole, .sup.1H-, .sup.2H- and
.sup.4H-1,2,4-triazole, .sup.1H- and .sup.2H-isoindole, and
.sup.1H- and .sup.2H-pyrazole. Tautomeric forms can be in
equilibrium or sterically locked into one form by appropriate
substitution.
[0058] Compounds of the invention can also include all isotopes of
atoms occurring in the intermediates or final compounds. Isotopes
include those atoms having the same atomic number but different
mass numbers.
[0059] The term, "compound," as used herein is meant to include all
stereoisomers, geometric isomers, tautomers, and isotopes of the
structures depicted. Compounds herein identified by name or
structure as one particular tautomeric form are intended to include
other tautomeric forms unless otherwise specified (e.g., in the
case of purine rings, unless otherwise indicated, when the compound
name or structure has the 9H tautomer, it is understood that the 7H
tautomer is also encompassed).
[0060] All compounds, and pharmaceutically acceptable salts
thereof, can be found together with other substances such as water
and solvents (e.g., hydrates and solvates) or can be isolated.
[0061] In some embodiments, the compounds of the invention, or
salts thereof, are substantially isolated. By "substantially
isolated" is meant that the compound is at least partially or
substantially separated from the environment in which it was formed
or detected. Partial separation can include, for example, a
composition enriched in a compound of the invention. Substantial
separation can include compositions containing at least about 50%,
at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 97%, or at
least about 99% by weight of the compounds of the invention, or
salt thereof. Methods for isolating compounds and their salts are
routine in the art.
[0062] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0063] The expressions, "ambient temperature" and "room
temperature," as used herein, are understood in the art, and refer
generally to a temperature, e.g., a reaction temperature, that is
about the temperature of the room in which the reaction is carried
out, for example, a temperature from about 20.degree. C. to about
30.degree. C.
[0064] The present invention also includes pharmaceutically
acceptable salts of the compounds described herein. As used herein,
"pharmaceutically acceptable salts" refers to derivatives of the
disclosed compounds wherein the parent compound is modified by
converting an existing acid or base moiety to its salt form.
Examples of pharmaceutically acceptable salts include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines; alkali or organic salts of acidic residues such as
carboxylic acids; and the like. The pharmaceutically acceptable
salts of the present invention include the conventional non-toxic
salts of the parent compound formed, for example, from non-toxic
inorganic or organic acids. The pharmaceutically acceptable salts
of the present invention can be synthesized from the parent
compound which contains a basic or acidic moiety by conventional
chemical methods. Generally, such salts can be prepared by reacting
the free acid or base forms of these compounds with a
stoichiometric amount of the appropriate base or acid in water or
in an organic solvent, or in a mixture of the two; generally,
non-aqueous media like ether, ethyl acetate, alcohols (e.g.,
methanol, ethanol, iso-propanol, or butanol) or acetonitrile (MeCN)
are preferred. Lists of suitable salts are found in Remington's
Pharmaceutical Sciences, 17.sup.th Ed., (Mack Publishing Company,
Easton, 1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1),
1-19, and in Stahl et al., Handbook of Pharmaceutical Salts:
Properties, Selection, and Use, (Wiley, 2002). In some embodiments,
the compounds described herein include the N-oxide forms.
[0065] The following abbreviations may be used herein: AcOH (acetic
acid); Ac.sub.2O (acetic anhydride); aq. (aqueous); atm.
(atmosphere(s)); Boc (t-butoxycarbonyl); br (broad); Cbz
(carboxybenzyl); calc. (calculated); d (doublet); dd (doublet of
doublets); DCM (dichloromethane); DIAD (N,N'-diisopropyl
azidodicarboxylate); DIPEA (N,N-diisopropylethylamine); DMF
(N,N-dimethylformamide); Et (ethyl); EtOAc (ethyl acetate); g
(gram(s)); h (hour(s)); HATU
(N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium
hexafluorophosphate); HCl (hydrochloric acid); HPLC (high
performance liquid chromatography); Hz (hertz); J (coupling
constant); LCMS (liquid chromatography-mass spectrometry); m
(multiplet); M (molar); mCPBA (3-chloroperoxybenzoic acid);
MgSO.sub.4 (magnesium sulfate); MS (Mass spectrometry); Me
(methyl); MeCN (acetonitrile); MeOH (methanol); mg (milligram(s));
min. (minutes(s)); mL (milliliter(s)); mmol (millimole(s)); N
(normal); NaHCO.sub.3 (sodium bicarbonate); NaOH (sodium
hydroxide); Na.sub.2SO.sub.4 (sodium sulfate); NH.sub.4Cl (ammonium
chloride); NH.sub.4OH (ammonium hydroxide); nM (nanomolar); NMR
(nuclear magnetic resonance spectroscopy); OTf
(trifluoromethanesulfonate); Pd (palladium); Ph (phenyl); pM
(picomolar); POCl.sub.3 (phosphoryl chloride); RP-HPLC (reverse
phase high performance liquid chromatography); s (singlet); t
(triplet or tertiary); TBS (tert-butyldimethylsilyl); tert
(tertiary); tt (triplet of triplets); t-Bu (tert-butyl); TFA
(trifluoroacetic acid); THF (tetrahydrofuran); .mu.g
(microgram(s)); (microliter(s)); .mu.M (micromolar); wt % (weight
percent).
II. Synthesis
[0066] Compounds of the invention, including salts thereof, can be
prepared using known organic synthesis techniques and can be
synthesized according to any of numerous possible synthetic
routes.
[0067] The reactions for preparing compounds of the invention can
be carried out in suitable solvents which can be readily selected
by one of skill in the art of organic synthesis. Suitable solvents
can be substantially non-reactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, e.g., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected by the skilled artisan.
[0068] Preparation of compounds of the invention can involve the
protection and deprotection of various chemical groups. The need
for protection and deprotection, and the selection of appropriate
protecting groups, can be readily determined by one skilled in the
art. The chemistry of protecting groups can be found, for example,
in P. G. M. Wuts and T. W. Greene, Protective Groups in Organic
Synthesis, 4''.sup.h Ed., Wiley & Sons, Inc., New York (2006),
which is incorporated herein by reference in its entirety.
[0069] Reactions can be monitored according to any suitable method
known in the art. For example, product formation can be monitored
by spectroscopic means, such as nuclear magnetic resonance
spectroscopy (e.g., .sup.1H or .sup.13C), infrared spectroscopy,
spectrophotometry (e.g., UV-visible), mass spectrometry, or by
chromatographic methods such as high performance liquid
chromatography (HPLC), liquid chromatography-mass spectroscopy
(LCMS), or thin layer chromatography (TLC). Compounds can be
purified by those skilled in the art by a variety of methods,
including high performance liquid chromatography (HPLC)
("Preparative LC-MS Purification: Improved Compound Specific Method
Optimization" Karl F. Blom, Brian Glass, Richard Sparks, Andrew P.
Combs J. Combi. Chem. 2004, 6(6), 874-883, which is incorporated
herein by reference in its entirety) and normal phase silica
chromatography.
III. Specific Examples
[0070] The invention will be described in greater detail by way of
specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of noncritical parameters which can be changed or modified
to yield essentially the same results.
Example 1: Methods of Synthesis
Example 1-1:
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide
##STR00004##
[0072] To a solution of
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)benzoic
acid (100 mg) in DMF (2 mL) were added 1,1'-carbonyldiimidazole (64
mg, 1.5 eq) and (3-methylisoxazol-5-yl)methanamine (44 mg, 1.5 eq).
The mixture was stirred overnight at RT. The mixture was then
diluted with AcOEt and quenched with a saturated solution of
NaHCO.sub.3. The organic phase was washed with brine and dried over
MgSO.sub.4. After filtration, the residue was evaporated under
vacuum to give a crude yellow foam. This material was purified on
silicagel cartridge using a gradient of solvent 0-20% MeOH in DCM.
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide was obtained as a yellow foam (71.5 mg,
79%), LCMS RT=3.34 min.
Example 1-2:
N-((5-fluoropyridin-3-yl)methyl)-4-(1'-isopropyl-3-methyl-4-oxo-3,4-dihyd-
rospiro[benzo[e][1,3]oxazine-2,4'-piperidin]-6-yl)benzamide
##STR00005##
[0074]
4-(1'-isopropyl-2-methyl-1-oxo-2,4-dihydro-1H-spiro[isoquinoline-3,-
4'-piperidin]-7-yl)benzoic acid-lithium chloride salt (100 mg,
0.263 mmoles) and HATU (100 mg, 0.263 mmoles) were combined in 2 mL
DMF. DIEA (0.09 mL) was added to the solution. After the mixture
was stirred for 5 minutes at RT, (5-fluoropyridin-3-yl)methanamine
(0.289 mmoles) was added. The mixture was stirred at RT overnight
before it was quenched using a saturated solution of NaHCO.sub.3
and diluted with AcOEt. After the aqueous phase was extracted with
AcOEt, the combined organic phases were dried using
Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The
residue was purified on flash chromatography using a gradient 0-20%
MeOH in DCM. The reaction yielded 110 mg of
N-((5-fluoropyridin-3-yl)methyl)-4-(1'-isopropyl-3-methyl-4-oxo-3,4-dihyd-
rospiro[benzo[e][1,3]oxazine-2,4'-piperidin]-6-yl)benzamide.
Example 1-3:
3-fluoro-4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((6-m-
ethylpyridin-3-yl)methyl)benzamide
##STR00006##
[0076]
3-fluoro-4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-
-((6-methylpyridin-3-yl)methyl)benzamide was obtained using the
same method described for Example 1-1. (LCMS RT=2.65 min, MH+251.4,
m=30 mg, yield=21%).
Example 1-4:
6-(4-oxo-1'-propylspiro[chroman-2,4'-piperidin]-6-yl)-N-(thiazol-5-ylmeth-
yl)nicotinamide
##STR00007##
[0078]
6-(4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-(thiazol-5-ylmethyl)n-
icotinamide (150 mg, 0.345 mmoles) was combined with cesium
carbonate (135 mg, 0.41 mmoles) and 1-idopropane (58.6 mg, 0.345
mmoles) in 3 mL of DMF. The reaction mixture was stirred at RT for
72 hours. Then the mixture was quenched with water and diluted with
AcOEt. After extraction using AcOEt, the combined organic phases
were washed with brine and dried using MgSO.sub.4. After
filtration, the solvent was removed in vacuo to get a brown
semi-solid. The crude material was purified on silicagel cartridge
using a gradient 0-15% MeOH in DCM. The desired fractions were
combined and evaporated to get a tan solid (LCMS RT=3.07 min, 49
mg, 29%).
Example 1-5:
6-(1'-butyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-(thiazol-5-ylmethy-
l)nicotinamide
##STR00008##
[0080]
6-(1'-butyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-(thiazol-5-y-
lmethyl)nicotinamide was obtained using the same method described
for Example 1-4. (LCMS RT=3.25 min, MH+490.8, m=78 mg as a tan
solid, yield=48.4%).
Example 1-6:
N-((3-methylisoxazol-5-yl)methyl)-4-(4-oxo-1'-propylspiro[chroman-2,4'-pi-
peridin]-6-yl)benzamide
##STR00009##
[0082]
N-((3-methylisoxazol-5-yl)methyl)-4-(4-oxo-1'-propylspiro[chroman-2-
,4'-piperidin]-6-yl)benzamide was obtained using the same method
described for Example 1-1. (LCMS RT=3.27 min, m=11 mg,
yield=12%).
Example 1-7:
N-((3-methylisoxazol-5-yl)methyl)-6-(1'-propylspiro[chromene-2,4'-piperid-
in]-6-yl)nicotinamide
##STR00010##
[0084]
N-((3-methylisoxazol-5-yl)methyl)-6-(4-oxo-1'-propylspiro[chroman-2-
,4'-piperidin]-6-yl)nicotinamide (66 mg, 0.14 mmoles) was dissolved
in 1.5 mL of MeOH. Sodium borohydride (33 mg, 0.87 mmoles) was
added to the reaction mixture in one portion. Degassing happened
for several minutes then the yellowish mixture became colorless.
After 3 hours at RT, the reaction is completed. The mixture was
quenched with water and diluted with AcOEt. The aqueous phase was
extracted with AcOEt and the combined organic phases were washed
with brine twice. The organic phase was then dried using MgSO.sub.4
and concentrated under vacuum.
[0085] To the crude material was then added 8 mL of water and 2 mL
of concentrated H.sub.2SO.sub.4. The mixture was stirred vigorously
and heated at 80.degree. C. After 48 hours at this temperature, the
mixture was diluted with water, basified using solid
Na.sub.2CO.sub.3. The aqueous phase was extracted with AcOEt and
the combined organic phases were washed with brine twice. After
filtration and evaporation, the crude residue was purified on
silica gel cartridge using 20% MeOH in DCM. The desired fractions
were combined and concentrated under vacuum.
N-((3-methylisoxazol-5-yl)methyl)-6-(1'-propylspiro[chromene-2,4'-piperid-
in]-6-yl)nicotinamide was obtained as a white solid (21 mg, 33.3%,
LCMS RT=3.30 min, MH+ 459.3).
Example 1-8:
6-(1'-(3-methoxypropyl)spiro[chromene-2,4'-piperidin]-6-yl)-N-(thiazol-5--
ylmethyl)nicotinamide
##STR00011##
[0087]
6-(1'-(3-methoxypropyl)spiro[chromene-2,4'-piperidin]-6-yl)-N-(thia-
zol-5-ylmethyl)nicotinamide was obtained using the same method
described for Example 1-7.
Example 1-9:
6-(1'-isopropyl-3-methyl-4-oxo-3,4-dihydrospiro[benzo[e][1,3]oxazine-2,4'-
-piperidin]-6-yl)-N-((1-methyl-1H-pyrazol-4-yl)methyl)nicotinamide
##STR00012##
[0089]
6-(1'-isopropyl-3-methyl-4-oxo-3,4-dihydrospiro[benzo[e][1,3]oxazin-
e-2,4'-piperidin]-6-yl)-N-((1-methyl-1H-pyrazol-4-yl)methyl)nicotinamide
was obtained using the same method described for Example 1-2.
Example 1-10: 6-(1'-isopropyl-3-methyl-4-oxo-3,4
dihydrospiro[benzo[e][1,3]oxazine-2,4'-piperidin]-6-yl)-N-(thiazol-5-ylme-
thyl)nicotinamide
##STR00013##
[0091] 6-(1'-isopropyl-3-methyl-4-oxo-3,4
dihydrospiro[benzo[e][1,3]oxazine-2,4'-piperidin]-6-yl)-N-(thiazol-5-ylme-
thyl)nicotinamide was obtained using the same method described for
Example 1-2.
Example 1-11:
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((5-methylisox-
azol-3-yl)methyl)benzamide
##STR00014##
[0093]
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((5-meth-
ylisoxazol-3-yl)methyl)benzamide was obtained using the same method
described for Example 1-1. (LCMS RT=3.35 min, m=49.7 mg,
yield=39%).
Example 1-12:
6-(1'-isopropyl-3-methyl-4-oxo-3,4-dihydrospiro[benzo[e][1,3]oxazine-2,4'-
-piperidin]-6-yl)-N-((3-methylisoxazol-5-yl)methyl)nicotinamide
##STR00015##
[0095]
6-(1'-isopropyl-3-methyl-4-oxo-3,4-dihydrospiro[benzo[e][1,3]oxazin-
e-2,4'-piperidin]-6-yl)-N-((3-methylisoxazol-5-yl)methyl)nicotinamide
was obtained using the same method described for Example 1-2.
Example 1-13: Additional Compounds of Formula (I)
[0096] Additional compounds of Formula (I) were synthesized
according to the methods similar to those described above, which
are readily apparent to one of skill in the art. These compounds
are summarized in Table 1 below.
TABLE-US-00001 TABLE 1 Additional Compounds of Formula (I)
Structure Compound Name ##STR00016##
N-((5-fluoropyridin-3-yl)methyl)-4-(1'-isopropyl-4-
oxospiro[chroman-2,4'-piperidin]-6-yl)benzamide ##STR00017##
6-(1'-isopropyl-4-oxospiro[chroman-2,4'-
piperidin]-6-yl)-N-((1-methyl-1H-pyrazol-4- yl)methyl)nicotinamide
##STR00018## N-((5-fluoropyridin-3-yl)methyl)-4-(4-oxo-1'-
propylspiro[chroman-2,4'-piperidin]-6- yl)benzamide ##STR00019##
N-((3-methylisoxazol-5-yl)methyl)-6-(1'-((3-
methyloxetan-3-yl)methyl)-4-oxospiro[chroman-
2,4'-piperidin]-6-yl)nicotinamide ##STR00020##
6-(1'-ethyl-4-oxospiro[chroman-2,4'-piperidin]-6-
yl)-N-((3-methylisoxazol-5-yl)methyl)nicotinamide ##STR00021##
N-((2-fluoropyridin-3-yl)methyl)-4-(1'-isopropyl-4-
oxospiro[chroman-2,4'-piperidin]-6-yl)benzamide ##STR00022##
N-butoxy-6-(1'-isopropyl-4-oxospiro[chroman-
2,4'-piperidin]-6-yl)nicotinamide ##STR00023##
4-(1'-isopropyl-4-oxo-3,4-
dihydrospiro[benzo[e][1,3]oxazine-2,4'-piperidin]-
6-yl)-N-((3-methylisoxazol-5-yl)methyl)benzamide ##STR00024##
N-((3-ethylisoxazol-5-yl)methyl)-4-(1'-isopropyl-4-
oxospiro[chroman-2,4'-piperidin]-6-yl)benzamide ##STR00025##
6-(1'-isopropyl-4-oxo-3,4-
dihydrospiro[benzo[e][1,3]oxazine-2,4'-piperidin]-
6-yl)-N-((3-methylisoxazol-5- yl)methyl)nicotinamide ##STR00026##
6-(1'-(3-methoxypropyl)-4-oxospiro[chroman-
2,4'-piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
##STR00027## 4-(1'-isopropyl-3-methyl-4-oxo-3,4-
dihydrospiro[benzo[e][1,3]oxazine-2,4'-piperidin]-
6-yl)-N-((6-methylpyridin-3-yl)methyl)benzamide ##STR00028##
N-((3-methylisoxazol-5-yl)methyl)-6-(4-oxo-1'-
propylspiro[chroman-2,4'-piperidin]-6- yl)nicotinamide ##STR00029##
6-(1'-ethyl-4-oxospiro[chroman-2,4'-piperidin]-6-
yl)-N-(thiazol-5-ylmethyl)nicotinamide ##STR00030##
1'-isopropyl-3-methyl-6-(4-(3-(thiazol-5-
yl)propanoyl)phenyl)spiro[benzo[e][1,3]oxazine-
2,4'-piperidin]-4(3H)-one ##STR00031##
4-(1-(cyclobutylmethyl)-4'-oxo-3',4'-
dihydrospiro[piperidine-4,2'-pyrano[3,2-
b]pyridin]-6'-yl)-N-((5-fluoropyridin-3- yl)methyl)benzamide
##STR00032## N-((5-fluoropyridin-3-yl)methyl)-4-(1-isopentyl-4'-
oxo-3',4'-dihydrospiro[piperidine-4,2'-pyrano[3,2-
b]pyridin]-6'-yl)benzamide ##STR00033##
N-((5-fluoropyridin-3-yl)methyl)-6-(1'-(4-
methoxybutyl)-4-oxospiro[chroman-2,4'- piperidin]-6-yl)nicotinamide
##STR00034## N-((5-fluoropyridin-3-yl)methyl)-4-(1'-isopropyl-4-
oxospiro[chroman-2,4'-piperidin]-6-yl)benzamide
Example 2: In vitro Experiments
Example 2-1: Inhibition of NAMPT Enzymatic Activity
[0097] Recombinant NAMPT enzyme (MBL) was briefly pre-incubated in
the absence or presence of inhibitor followed by addition of
nicotinamide. Enzymatic reaction was allowed to proceed for 6 hours
at 37.degree. C. Reaction was terminated and NMN reaction product
detected using the method as described in: Zhang R Y, Qin Y, Lv X
Q, Wang P, Xu T Y, Zhang L, Miao C Y., Anal. Biochem. 2011 May 1;
412(1):18-25.
[0098] The 50% inhibitory concentration (IC.sub.50) is the
concentration at which 50% of the maximal activity was
inhibited.
[0099] The results from this assay for representative compounds are
presented in Table 2 below.
Example 2-2: Anti-Proliferative Activity on B Cells
[0100] Fresh primary human B cells obtained from Analytical
Biological Services (Wilmington, Del.) were diluted to
1.times.10.sup. 6 cells per ml in AIM-V medium (Invitrogen), and
stimulated with anti-IgM (10 ug/ml) (KPL) in the absence or
presence of inhibitor for 72 hours at 37.degree. C./5% CO.sub.2
prior to viability determination using CellTiterGlo (Promega) per
manufacturer's instruction.
[0101] The 50% inhibitory concentration (IC.sub.50) is the
concentration at which 50% of the maximal activity was
inhibited.
[0102] The results from this assay for representative compounds are
presented in Table 2 below.
Example 2-3: Anti-Proliferative Activity on T Cells
[0103] Fresh primary human T cells obtained from Analytical
Biological Services (Wilmington, Del.) were diluted to
1.times.10.sup. 6 cells per ml in AIM-V medium (Invitrogen), and
stimulated with CD3/CD28 beads (Invitrogen) in the absence or
presence of inhibitor for 72 hours at 37.degree. C./5% CO.sub.2
prior to viability determination using CellTiterGlo (Promega) per
manufacturer's instruction.
[0104] The 50% inhibitory concentration (IC.sub.50) is the
concentration at which 50% of the maximal activity was
inhibited.
[0105] The results from this assay for representative compounds are
presented in Table 2 below.
TABLE-US-00002 TABLE 2 NAMPT, B Cell and T Cell IC.sub.50 for
Selected Compounds Compound Name NAMPT IC.sub.50 (.mu.M) B CELL
IC.sub.50 (.mu.M) T CELL IC.sub.50 (.mu.M)
1'-isopropyl-3-methyl-6-(4-(3-(thiazol-5- 0.1661 0.0408 3.1938
yl)propanoyl)phenyl)spiro[benzo[e][1,3]oxazine-
2,4'-piperidin]-4(3H)-one 4-(1'-isopropyl-4-oxospiro[chroman-2,4'-
0.0009 0.0227 4.3750 piperidin]-6-yl)-N-((3-methylisoxazol-5-
yl)methyl)benzamide N-((5-fluoropyridin-3-yl)methyl)-4-(1'- 0.0055
0.0079 3.2994 isopropyl-4-oxospiro[chroman-2,4'-piperidin]-
6-yl)benzamide 6-(1'-isopropyl-4-oxospiro[chroman-2,4'- 0.0140
0.0598 5.5853 piperidin]-6-yl)-N-((1-methyl-1H-pyrazol-4-
yl)methyl)nicotinamide 6-(4-oxo-1'-propylspiro[chroman-2,4'- 0.0667
0.0152 1.0775 piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
(1'-butyl-4-oxospiro[chroman-2,4'-piperidin]- 0.0299 0.0248 1.8800
6-yl)-N-(thiazol-5-ylmethyl)nicotinamide
3-fluoro-4-(1'-isopropyl-4-oxospiro[chroman- 0.0020 0.0539 8.5138
2,4'-piperidin]-6-yl)-N-((6-methylpyridin-3- yl)methyl)benzamide
N-((3-methylisoxazol-5-yl)methyl)-4-(4-oxo- 0.0020 0.0129 0.6769
1'-propylspiro[chroman-2,4'-piperidin]-6- yl)benzamide
N-((5-fluoropyridin-3-yl)methyl)-4-(4-oxo-1'- 0.0030 0.0074 1.9653
propylspiro[chroman-2,4'-piperidin]-6- yl)benzamide
4-(1'-isopropyl-4-oxospiro[chroman-2,4'- 0.0010 0.0113 3.2428
piperidin]-6-yl)-N-((5-methylisoxazol-3- yl)methyl)benzamide
N-((3-methylisoxazol-5-yl)methyl)-6-(1'- 0.0010 0.0044 0.3417
propylspiro[chromene-2,4'-piperidin]-6- yl)nicotinamide
6-(1'-(3-methoxypropyl)spiro[chromene-2,4'- 0.0564 0.0455 5.7920
piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
N-((3-methylisoxazol-5-yl)methyl)-6-(1'-((3- 0.0030 0.0313 11.6673
methyloxetan-3-yl)methyl)-4- oxospiro[chroman-2,4'-piperidin]-6-
yl)nicotinamide N-((3-methylisoxazol-5-yl)methyl)-6-(1'-((3- 0.0014
0.0118 5.3998 methyloxetan-3-yl)methyl)-4-
oxospiro[chroman-2,4'-piperidin]-6- yl)nicotinamide
N-((2-fluoropyridin-3-yl)methyl)-4-(1'- 0.0010 0.0099 5.7883
isopropyl-4-oxospiro[chroman-2,4'-piperidin]- 6-yl)benzamide
N-butoxy-6-(1'-isopropyl-4- 0.0014 0.0450 7.3731
oxospiro[chroman-2,4'-piperidin]-6- yl)nicotinamide
N-((5-fluoropyridin-3-yl)methyl)-6-(1'-(4- 0.0042 0.0109 5.4532
methoxybutyl)-4-oxospiro[chroman-2,4'- piperidin]-6-yl)nicotinamide
4-(1'-isopropyl-4-oxo-3,4- 0.0040 0.0334 5.6776
dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((3-methylisoxazol-5- yl)methyl)benzamide
4-(1-(cyclobutylmethyl)-4'-oxo-3',4'- 0.0664 0.0830 3.9880
dihydrospiro[piperidine-4,2'-pyrano[3,2-
b]pyridin]-6'-yl)-N-((5-fluoropyridin-3- yl)methyl)benzamide
N-((3-ethylisoxazol-5-yl)methyl)-4-(1'- 0.0010 0.0213 1.7222
isopropyl-4-oxospiro[chroman-2,4'-piperidin]- 6-yl)benzamide
N-((5-fluoropyridin-3-yl)methyl)-4-(1- 0.1274 0.1068 9.6657
isopentyl-4'-oxo-3',4'-dihydrospiro[piperidine-
4,2'-pyrano[3,2-b]pyridin]-6'-yl)benzamide
N-((5-fluoropyridin-3-yl)methyl)-4-(1'- 0.0035 0.0060 0.9997
isopropyl-3-methyl-4-oxo-3,4-
dihydrospiro[benzo[e][1,3]oxazine-2,4'- piperidin]-6-yl)benzamide
6-(1'-isopropyl-4-oxo-3,4- 0.0030 0.0292 14.6143
dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((3-methylisoxazol-5- yl)methyl)nicotinamide
6-(1'-(3-methoxypropyl)-4-oxospiro[chroman- 0.1409 0.0438 3.2900
2,4'-piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
6-(1'-isopropyl-3-methyl-4-oxo-3,4 0.1071 0.0254 1.5503
dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
4-(1'-isopropyl-3-methyl-4-oxo-3,4- 0.0020 0.0153 4.6159
dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((6-methylpyridin-3- yl)methyl)benzamide
N-((3-methylisoxazol-5-yl)methyl)-6-(4-oxo- 0.0010 0.0059 0.7308
1'-propylspiro[chroman-2,4'-piperidin]-6- yl)nicotinamide
6-(1'-isopropyl-3-methyl-4-oxo-3,4- 0.0150 0.0977 6.1082
dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((1-methyl-1H-pyrazol-4- yl)methyl)nicotinamide
6-(1'-ethyl-4-oxospiro[chroman-2,4'- 0.2708 0.0509 3.3550
piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
6-(1'-isopropyl-3-methyl-4-oxo-3,4- 0.0020 0.0095 1.1806
dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((3-methylisoxazol-5- yl)methyl)nicotinamide
Example 2-4: Activity on Various Cell Lines
[0106] Leukemia/Lymphoma cell lines (ATCC) were diluted in
vendor-recommended media and incubated in the absence or presence
of inhibitor for 72 hours at 37.degree. C./5% CO.sub.2 prior to
viability determination using CellTiterGlo (Promega) per
manufacturer's instruction. The human cell lines studied include
acute T-cell leukemia (JURKAT), chronic myelogenous leukemia
(K562), and B-cell acute lymphocytic leukemia (NALM6 and
SUP-B15).
[0107] The anti-proliferative activity on these cell lines for
selected compounds is presented in Table 3 below.
TABLE-US-00003 TABLE 3 Anti-Proliferative Activity on Various Cell
Line Activity for Selected Compounds Compound Name JURKAT K562
NALM6 SUP-B15 1'-isopropyl-3-methyl-6-(4-(3-(thiazol-5- 0.034727
0.111998 0.018604 0.002847
yl)propanoyl)phenyl)spiro[benzo[e][1,3]oxazine-
2,4'-piperidin]-4(3H)-one 4-(1'-isopropyl-4-oxospiro[chroman-2,4'-
0.017937 0.068117 0.015026 0.002439
piperidin]-6-yl)-N-((3-methylisoxazol-5- yl)methyl)benzamide
N-((5-fluoropyridin-3-yl)methyl)-4-(1'- 0.020351 0.078792 0.008799
0.001097 isopropyl-4-oxospiro[chroman-2,4'-piperidin]-
6-yl)benzamide 6-(1'-isopropyl-4-oxospiro[chroman-2,4'- 0.209499
0.3045 0.102349 0.014141
piperidin]-6-yl)-N-((1-methyl-1H-pyrazol-4- yl)methyl)nicotinamide
6-(4-oxo-1'-propylspiro[chroman-2,4'- 0.016292 0.0357 0.01188
0.001894 piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
(1'-butyl-4-oxospiro[chroman-2,4'-piperidin]- 0.008845 0.01977
0.005734 0.00103 6-yl)-N-(thiazol-5-ylmethyl)nicotinamide
3-fluoro-4-(1'-isopropyl-4-oxospiro[chroman- 0.021286 1.734128
0.010666 0.002218 2,4'-piperidin]-6-yl)-N-((6-methylpyridin-3-
yl)methyl)benzamide N-((3-methylisoxazol-5-yl)methyl)-4-(4-oxo-
0.007779 0.024999 0.006739 0.002069
1'-propylspiro[chroman-2,4'-piperidin]-6- yl)benzamide
N-((5-fluoropyridin-3-yl)methyl)-4-(4-oxo-1'- 0.007074 0.018673
0.003155 0.000556 propylspiro[chroman-2,4'-piperidin]-6-
yl)benzamide 4-(1'-isopropyl-4-oxospiro[chroman-2,4'- 0.010663
0.063619 0.002695 0.001172 piperidin]-6-yl)-N-((5-methylisoxazol-3-
yl)methyl)benzamide N-((3-methylisoxazol-5-yl)methyl)-6-(1'-
0.00638 0.0258 0.00363 0.001036
propylspiro[chromene-2,4'-piperidin]-6- yl)nicotinamide
6-(1'-(3-methoxypropyl)spiro[chromene-2,4'- 0.0858 0.013857
0.001697 piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
N-((3-methylisoxazol-5-yl)methyl)-6-(1'-((3- 0.0393 0.829 0.024854
0.003771 methyloxetan-3-yl)methyl)-4-
oxospiro[chroman-2,4'-piperidin]-6- yl)nicotinamide
N-((3-methylisoxazol-5-yl)methyl)-6-(1'-((3- 0.013556 0.077811
0.008449 0.001764 methyloxetan-3-yl)methyl)-4-
oxospiro[chroman-2,4'-piperidin]-6- yl)nicotinamide
N-((2-fluoropyridin-3-yl)methyl)-4-(1'- 0.003659 0.00294 0.003481
0.00147 isopropyl-4-oxospiro[chroman-2,4'-piperidin]-
6-yl)benzamide N-butoxy-6-(1'-isopropyl-4- 0.0233 0.281968 0.0156
0.003019 oxospiro[chroman-2,4'-piperidin]-6- yl)nicotinamide
N-((5-fluoropyridin-3-yl)methyl)-6-(1'-(4- 0.012051 0.029438
0.00483 0.000847 methoxybutyl)-4-oxospiro[chroman-2,4'-
piperidin]-6-yl)nicotinamide 4-(1'-isopropyl-4-oxo-3,4- 0.042114
0.350422 0.023575 0.004112 dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((3-methylisoxazol-5- yl)methyl)benzamide
4-(1-(cyclobutylmethyl)-4'-oxo-3',4'- 0.154 0.332 0.0489 0.00729
dihydrospiro[piperidine-4,2'-pyrano[3,2-
b]pyridin]-6'-yl)-N-((5-fluoropyridin-3- yl)methyl)benzamide
N-((3-ethylisoxazol-5-yl)methyl)-4-(1'- 0.012171 0.044702 0.00742
0.002295 isopropyl-4-oxospiro[chroman-2,4'-piperidin]-
6-yl)benzamide N-((5-fluoropyridin-3-yl)methyl)-4-(1- 0.203 0.822
0.174 0.0129 isopentyl-4'-oxo-3',4'-dihydrospiro[piperidine-
4,2'-pyrano[3,2-b]pyridin]-6'-yl)benzamide
N-((5-fluoropyridin-3-yl)methyl)-4-(1'- 0.007037 0.028286 0.003564
0.000606 isopropyl-3-methyl-4-oxo-3,4-
dihydrospiro[benzo[e][1,3]oxazine-2,4'- piperidin]-6-yl)benzamide
6-(1'-isopropyl-4-oxo-3,4- 0.024793 0.875806 0.0171 0.003192
dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((3-methylisoxazol-5- yl)methyl)nicotinamide
6-(1'-(3-methoxypropyl)-4-oxospiro[chroman- 0.019627 0.054381
0.009382 0.001833 2,4'-piperidin]-6-yl)-N-(thiazol-5-
ylmethyl)nicotinamide 6-(1'-isopropyl-3-methyl-4-oxo-3,4 0.032986
0.075684 0.01763 0.002135 dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
4-(1'-isopropyl-3-methyl-4-oxo-3,4- 0.00767 0.028299 0.005259
0.000663 dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((6-methylpyridin-3- yl)methyl)benzamide
N-((3-methylisoxazol-5-yl)methyl)-6-(4-oxo- 0.003542 0.019439
0.002255 0.000706 1'-propylspiro[chroman-2,4'-piperidin]-6-
yl)nicotinamide 6-(1'-isopropyl-3-methyl-4-oxo-3,4- 0.18092
0.628968 0.121959 0.020856 dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((1-methyl-1H-pyrazol-4- yl)methyl)nicotinamide
6-(1'-ethyl-4-oxospiro[chroman-2,4'- 0.043571 0.180278 0.021589
0.00515 piperidin]-6-yl)-N-(thiazol-5- ylmethyl)nicotinamide
6-(1'-isopropyl-3-methyl-4-oxo-3,4- 0.016592 0.04761 0.006144
0.001508 dihydrospiro[benzo[e][1,3]oxazine-2,4'-
piperidin]-6-yl)-N-((3-methylisoxazol-5- yl)methy)nicotinamide
Example 2-5: Mechanism of Action
[0108] Sup-B15 cells (ATCC) were incubated in media lacking or
containing 200 .mu.M nicotinamide mononucleotide (NMN) (Sigma) in
the absence or presence of inhibitor for 72 hours at 37.degree.
C./5% CO.sub.2 prior to viability determination using CellTiterGlo
(Promega) per manufacturer's instruction.
[0109] Chemical proteomics. Sup-B15 cell extract was incubated with
a biotinylated derivative of
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide, followed by collection of
compound/protein complexes with streptavidin beads (Invitrogen).
Following extensive washing, bound proteins were eluted using free
(non-biotinylated)
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide or an inactive analog. Following
SDS-PAGE, one prominent band was excised and identified by LC/MS as
NAMPT.
[0110] The molecular target of
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide is nicotinamide phosphoribosyl
transferase (NAMPT), the product of which is nicotinamide
mononucleotide (NMN). The anti-proliferative activity of
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide is counteracted by NMN, and
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide potently inhibits NAMPT enzyme activity.
On-target, selective anti-proliferative activity was maintained
throughout the medicinal chemistry campaign, as all members of
Formula (I) 45 series are rescuable with NMN (FIG. 1).
Example 2-6: Activity on Influenza and RSV Infected Cells
[0111] To determine respiratory syncytial virus (RSV) inhibition,
Vero cells are plated in 96 well plates to a confluency of 80%.
Compounds are added at half log dilutions starting at 10 uM down to
3 nM in triplicate and allowed to incubate with cells for one hour.
Virus is added to the wells at an MOI of 10 and allowed to
replicate for 72 hours. Viable cells protected from virus induced
cytopathic effect (CPE) are detected using a fluorescent vital dye.
The EC.sub.50 of the inhibition of CPE is determined using a robust
fit methodology.
[0112] To determine influenza virus inhibition, MDCK cells are
plated in 96 well plates to a confluency of 80%. Compounds are
added at half log dilutions starting at 10 .mu.M down to 3 nM in
triplicate and allowed to incubate with cells for one hour. Virus
is added to the wells at an MOI of 10 and allowed to replicate for
72 hours. Viable cells protected from virus induced cytopathic
effect (CPE) are detected using a fluorescent vital dye. The
EC.sub.50 of the inhibition of CPE is determined using a robust fit
methodology.
Example 3: In vivo Experiments
Example 3-1: Pharmacokinetic Studies
Example 3-1-a: Oral and Intravenous Pharmacokinetics in
Sprague-Dawley Rats
[0113] The oral (PO) and intravenous (IV) pharmacokinetics of test
compounds,
1'-isopropyl-3-methyl-6-(4-(3-(thiazol-5-yl)propanoyl)phenyl)spiro[benzo[-
e][1,3]oxazine-2,4'-piperidin]-4(3H)-one and
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide, was evaluated in male Sprague-Dawley
Rats. An overview of the study design is presented in Table 4
below. Blood samples were collected prior to test compound dosing.
Animals were dosed at T.sub.0; subsequent blood samples were
collected 2, 5, 15, and 30 minutes and 1, 2, 4, 6, 8, and 24 hours
post-dosing. The blood samples were collected via jugular vein
cannula. The samples were centrifuged at 13,000 rpm at 4.degree. C.
for 5 minutes. Plasma samples were collected into a 96-well plate
after centrifugation of the blood samples. The plasma
concentrations of test compounds were analyzed by LC-MS/MS.
TABLE-US-00004 TABLE 4 Rat Pharmacokinetics Study Design Route of
Number Co- Adminis- of Dose Test Compound hort tration Animals
(mg/kg) 4-(1'-isopropyl-4- 1 IV 2 1
oxospiro[chroman-2,4'-piperidin]- 2 Oral 3 3
6-yl)-N-((3-methylisoxazol-5- yl)methyl)benzamide
1'-isopropyl-3-methyl-6-(4-(3- 3 IV 2 1 (thiazol-5- 4 Oral 3 3
yl)propanoyl)phenyl)spi- ro[benzo[e][1,3]oxazine-2,4'-
piperidin]-4(3H)-one
[0114] The test compounds, both intravenously and orally
administered, were well-tolerated. No adverse physical
abnormalities were observed. A graphical comparison of the oral
plasma concentrations compared to intravenous plasma
concentrations, as measured over time, for the test compounds are
presented in FIG. 2 and FIG. 3.
Example 3-1-b: Oral and Intravenous Pharmacokinetics in Mice
[0115] The oral and intravenous pharmacokinetics of test compound
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide was evaluated in female
NOD.Cg-Prkdc.sup.scidII2.sup.rgtm1MJ1/SzJ mice. An overview of the
study design is presented in Table 5 below. Blood samples were
collected prior to test compound dosing, at specified time points
after dosing, and after animal sacrifice. The in life blood samples
were collected by tail snip or facial vein; terminal blood samples
were collected via cardiac puncture following inhalation
anesthesia. The samples were collected into K2EDTA tubes and stored
on wet ice until proceeding to plasma by centrifugation at 3000 g
at 5.degree. C. within 1 hour of collection. Plasma samples were
collected into 96-well plates after centrifugation and stored at
-80.degree. C. The plasma concentrations of test compounds were
analyzed by LC-MS/MS.
TABLE-US-00005 TABLE 5 Mouse Pharmacokinetics Study Design Terminal
Route of Number In Life Sample Sample Co- Adminis- of Dose
Collection Time Collection hort tration Animals (mg/kg) Points Time
Point 1(a) IV 3 1 5 min, 30 min, 8 hours 2 hours 1(b) IV 3 1 10
min, 1 hour, 24 hours 4 hours 2(a) Oral 3 5 5 min, 30 min, 8 hours
2 hours 2(b) Oral 3 5 10 min, 1 hour, 24 hours 4 hours 3(a) Oral 3
10 5 min, 30 min, 8 hours 2 hours 3(b) Oral 3 10 10 min, 1 hour, 24
hours 4 hours 4(a) Oral 3 20 5 min, 30 min, 8 hours 2 hours 4(b)
Oral 3 20 10 min, 1 hour, 24 hours 4 hours
[0116] No significant clinical signs were observed during the
course of the study. A graphical comparison of the oral plasma
concentrations compared to intravenous plasma concentrations, as
measured over time, for the test compound is presented in FIG.
4.
Example 3-2: Efficacy Studies
Example 3-2-a: NALM6 Leukemia Study
[0117]
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-meth-
ylisoxazol-5-yl)methyl)benzamide was tested in the NALM6 leukemia
model.
[0118] As shown in FIG. 6, IP dosing of
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide (also known as GENZ-682945) produced a
significant survival benefit in this xenograft model, in excess of
the standard of care (clofarabine). 5.times.10.sup. 6NALM-6 cells
were injected via tail vein into female CB17SCID/Crl mice (Charles
River). One week later,
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide was administered by i.p. injection twice
daily for 35 days. Clofarabine was administered once daily by i.p.
injection once daily for 5 days for four weeks. Study endpoint was
survival time, and mice were sacrificed upon appearance of systemic
signs of severe disease such as paralysis or cachexia. The results
are shown in FIG. 5.
Example 3-2-b: HCT116 Tumor Growth Study
[0119] Tumor Growth Study: Female athymic nude mice
(Crl:NU(Ncr)-Foxn1.sub.nu, Charles River) (n=10/group) were
injected with 5.times.10.sup.6 HCT116 cells, and treatment was
initiated at average tumor size of 100 mm.sup.3.
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide and vehicle control were administered
orally twice daily for 8 days. Tumor size was measured twice weekly
with calipers.
[0120] Tumor NAD measurement: Female athymic nude mice
(Crl:NU(Ncr)-Foxn1.sub.nu, Charles River) (n=3/group) were injected
with 5.times.10.sup.6 HCT116 cells, and treatment was initiated at
tumor size of 250-400 mm.sup.3.
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide and vehicle control were administered
orally twice daily for 1.5 days (3 doses). Six hours post last
dose, tumors were excised, washed, sectioned, weighted, and snap
frozen. Tumor portions were extracted in ethanol/PBS (90:10) by
three cycles of rapid freeze/thaw/sonication, prior to analysis for
NAD content by LC/MS. The data are presented in FIG. 6.
[0121]
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-meth-
ylisoxazol-5-yl)methyl)benzamide was also tested in the HCT116
solid tumor model. Twice-daily oral dosing of
4-(1'-isopropyl-4-oxospiro[chroman-2,4'-piperidin]-6-yl)-N-((3-methylisox-
azol-5-yl)methyl)benzamide produced a significant tumor growth
delay (FIG. 7), which persisted for at least one week after dosing
was stopped due to 10% mortality in the treated group.
IV. Formulation, Dosage Forms and Administration
[0122] When employed as pharmaceuticals, the compounds of the
invention can be administered in the form of pharmaceutical
compositions. Thus the present disclosure provides a composition
comprising a compound of Formula (I), or a pharmaceutically
acceptable salt thereof, or any of the embodiments thereof, and at
least one pharmaceutically acceptable carrier. These compositions
can be prepared in a manner well known in the pharmaceutical art,
and can be administered by a variety of routes, depending upon
whether local or systemic treatment is indicated and upon the area
to be treated. Administration may be topical (including
transdermal, epidermal, ophthalmic and to mucous membranes
including intranasal, vaginal and rectal delivery), pulmonary
(e.g., by inhalation or insufflation of powders or aerosols,
including by nebulizer; intratracheal or intranasal), oral or
parenteral. Parenteral administration includes intravenous,
intraarterial, subcutaneous, intraperitoneal intramuscular or
injection or infusion; or intracranial, e.g., intrathecal or
intraventricular, administration. Parenteral administration can be
in the form of a single bolus dose, or may be, e.g., by a
continuous perfusion pump. Pharmaceutical compositions and
formulations for topical administration may include transdermal
patches, ointments, lotions, creams, gels, drops, suppositories,
sprays, liquids and powders. Conventional pharmaceutical carriers,
aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable.
[0123] This invention also includes pharmaceutical compositions
which contain, as the active ingredient, the compound of the
invention or a pharmaceutically acceptable salt thereof, in
combination with one or more pharmaceutically acceptable carriers
(excipients). In some embodiments, the composition is suitable for
topical administration. In making the compositions of the
invention, the active ingredient is typically mixed with an
excipient, diluted by an excipient or enclosed within such a
carrier in the form of, e.g., a capsule, sachet, paper, or other
container. When the excipient serves as a diluent, it can be a
solid, semi-solid, or liquid material, which acts as a vehicle,
carrier or medium for the active ingredient. Thus, the compositions
can be in the form of tablets, pills, powders, lozenges, sachets,
cachets, elixirs, suspensions, emulsions, solutions, syrups,
aerosols (as a solid or in a liquid medium), ointments containing,
e.g., up to 10% by weight of the active compound, soft and hard
gelatin capsules, suppositories, sterile injectable solutions and
sterile packaged powders.
[0124] In preparing a formulation, the active compound can be
milled to provide the appropriate particle size prior to combining
with the other ingredients. If the active compound is substantially
insoluble, it can be milled to a particle size of less than 200
mesh. If the active compound is substantially water soluble, the
particle size can be adjusted by milling to provide a substantially
uniform distribution in the formulation, e.g., about 40 mesh.
[0125] The compounds of the invention may be milled using known
milling procedures such as wet milling to obtain a particle size
appropriate for tablet formation and for other formulation types.
Finely divided (nanoparticulate) preparations of the compounds of
the invention can be prepared by processes known in the art see,
e.g., WO 2002/000196.
[0126] Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol; mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinyl pyrrolidone,
cellulose, water, syrup and methyl cellulose. The formulations can
additionally include: lubricating agents such as talc, magnesium
stearate and mineral oil; wetting agents; emulsifying and
suspending agents; preserving agents such as methyl- and
propylhydroxy-benzoates; and sweetening agents and flavoring
agents. The compositions of the invention can be formulated so as
to provide quick, sustained or delayed release of the active
ingredient after administration to the patient by employing
procedures known in the art.
[0127] In some embodiments, the pharmaceutical composition
comprises silicified microcrystalline cellulose (SMCC) and at least
one compound described herein, or a pharmaceutically acceptable
salt thereof. In some embodiments, the silicified microcrystalline
cellulose comprises about 98% microcrystalline cellulose and about
2% silicon dioxide w/w.
[0128] In some embodiments, the composition is a sustained release
composition comprising at least one compound described herein, or a
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable carrier. In some embodiments, the
composition comprises at least one compound described herein, or a
pharmaceutically acceptable salt thereof, and at least one
component selected from microcrystalline cellulose, lactose
monohydrate, hydroxypropyl methylcellulose and polyethylene oxide.
In some embodiments, the composition comprises at least one
compound described herein, or a pharmaceutically acceptable salt
thereof, and microcrystalline cellulose, lactose monohydrate and
hydroxypropyl methylcellulose. In some embodiments, the composition
comprises at least one compound described herein, or a
pharmaceutically acceptable salt thereof, and microcrystalline
cellulose, lactose monohydrate and polyethylene oxide. In some
embodiments, the composition further comprises magnesium stearate
or silicon dioxide. In some embodiments, the microcrystalline
cellulose is Avicel PH102.TM.. In some embodiments, the lactose
monohydrate is Fast-flo 316.TM.. In some embodiments, the
hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208
K4M (e.g., Methocel K4 M Premier.TM.) and/or hydroxypropyl
methylcellulose 2208 K100LV (e.g., Methocel KOOLV.TM.). In some
embodiments, the polyethylene oxide is polyethylene oxide WSR 1105
(e.g., Polyox WSR 1105.TM.).
[0129] In some embodiments, a wet granulation process is used to
produce the composition.
[0130] In some embodiments, a dry granulation process is used to
produce the composition. The compositions can be formulated in a
unit dosage form, each dosage containing from about 5 to about
1,000 mg (1 g), more usually about 100 mg to about 500 mg, of the
active ingredient. In some embodiments, each dosage contains about
10 mg of the active ingredient. In some embodiments, each dosage
contains about 50 mg of the active ingredient.
[0131] In some embodiments, each dosage contains about 25 mg of the
active ingredient. The term "unit dosage forms" refers to
physically discrete units suitable as unitary dosages for human
subjects and other mammals, each unit containing a predetermined
quantity of active material calculated to produce the desired
therapeutic effect, in association with a suitable pharmaceutical
excipient.
[0132] The components used to formulate the pharmaceutical
compositions are of high purity and are substantially free of
potentially harmful contaminants (e.g., at least National Food
grade, generally at least analytical grade, and more typically at
least pharmaceutical grade).
[0133] Particularly for human consumption, the composition is
preferably manufactured or formulated under Good Manufacturing
Practice standards as defined in the applicable regulations of the
U.S. Food and Drug Administration. For example, suitable
formulations may be sterile and/or substantially isotonic and/or in
full compliance with all Good Manufacturing Practice regulations of
the U.S. Food and Drug Administration.
[0134] The active compound may be effective over a wide dosage
range and is generally administered in a therapeutically effective
amount. It will be understood, however, that the amount of the
compound actually administered will usually be determined by a
physician, according to the relevant circumstances, including the
condition to be treated, the chosen route of administration, the
actual compound administered, the age, weight and response of the
individual patient, the severity of the patient's symptoms and the
like.
[0135] The therapeutic dosage of a compound of the present
invention can vary according to, e.g., the particular use for which
the treatment is made, the manner of administration of the
compound, the health and condition of the patient, and the judgment
of the prescribing physician. The proportion or concentration of a
compound of the invention in a pharmaceutical composition can vary
depending upon a number of factors including dosage, chemical
characteristics (e.g., hydrophobicity), and the route of
administration. For example, the compounds of the invention can be
provided in an aqueous physiological buffer solution containing
about 0.1 to about 10% w/v of the compound for parenteral
administration. Some typical dose ranges are from about 1 .mu.g/kg
to about 1 g/kg of body weight per day. In some embodiments, the
dose rang 5 e is from about 0.01 mg/kg to about 100 mg/kg of body
weight per day. The dosage is likely to depend on such variables as
the type and extent of progression of the disease or disorder, the
overall health status of the particular patient, the relative
biological efficacy of the compound selected, formulation of the
excipient, and its route of administration. Effective doses can be
extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0136] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical
excipient to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention. When
referring to these preformulation compositions as homogeneous, the
active ingredient is typically dispersed evenly throughout the
composition so that the composition can be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation is then subdivided into unit
dosage forms of the type described above containing from, e.g.,
about 0.1 to about 1000 mg of the active ingredient of the present
invention.
[0137] The tablets or pills of the present invention can be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol and cellulose acetate.
[0138] The liquid forms in which the compounds and compositions of
the present invention can be incorporated for administration orally
or by injection include aqueous solutions, suitably flavored
syrups, aqueous or oil suspensions, and flavored emulsions with
edible oils such as cottonseed oil, sesame oil, coconut oil, or
peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0139] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as described supra. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or sy 5 stemic effect. Compositions can be
nebulized by use of inert gases. Nebulized solutions may be
breathed directly from the nebulizing device or the nebulizing
device can be attached to a face mask, tent, or intermittent
positive pressure breathing machine. Solution, suspension, or
powder compositions can be administered orally or nasally from
devices which deliver the formulation in an appropriate manner.
Topical formulations can contain one or more conventional carriers.
In some embodiments, ointments can contain water and one or more
hydrophobic carriers selected from, e.g., liquid paraffin,
polyoxyethylene alkyl ether, propylene glycol, white Vaseline.RTM.
(petroleum jelly) and the like. Carrier compositions of creams can
be based on water in combination with glycerol and one or more
other components, e.g., glycerinemonostearate,
PEG-glycerinemonostearate and cetylstearyl alcohol. Gels can be
formulated using isopropyl alcohol and water, suitably in
combination with other components such as, e.g., glycerol,
hydroxyethyl cellulose and the like. In some embodiments, topical
formulations contain at least about 0.1, at least about 0.25, at
least about 0.5, at least about 1, at least about 2, or at least
about 5 wt % of the compound of the invention. The topical
formulations can be suitably packaged in tubes of, e.g., 100 g
which are optionally associated with instructions for the treatment
of the select indication, e.g., psoriasis or other skin
condition.
[0140] The amount of compound or composition administered to a
patient will vary depending upon what is being administered, the
purpose of the administration, such as prophylaxis or therapy, the
state of the patient, the manner of administration and the like. In
therapeutic applications, compositions can be administered to a
patient already suffering from a disease in an amount sufficient to
cure or at least partially arrest the symptoms of the disease and
its complications. Effective doses will depend on the disease
condition being treated as well as by the judgment of the attending
clinician depending upon factors such as the severity of the
disease, the age, weight and general condition of the patient and
the like.
[0141] The compositions administered to a patient can be in the
form of pharmaceutical compositions described above. These
compositions can be sterilized by conventional sterilization
techniques, or may be sterile filtered. Aqueous solutions can be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile aqueous carrier prior to
administration. The pH of the compound preparations typically will
be between 3 and 11, more preferably from 5 to 9 and most
preferably from 7 to 8. It will be understood that use of certain
of the foregoing excipients, carriers or stabilizers will result in
the formation of pharmaceutical salts.
[0142] The therapeutic dosage of a compound of the present
invention can vary according to, e.g., the particular use for which
the treatment is made, the manner of administration of the
compound, the health and condition of the patient, and the judgment
of the prescribing physician. The proportion or concentration of a
compound of the invention in a pharmaceutical composition can vary
depending upon a number of factors including dosage, chemical
characteristics (e.g., hydrophobicity), and the route of
administration. For example, the compounds of the invention can be
provided in an aqueous physiological buffer solution containing
about 0.1 to about 10% w/v of the compound for parenteral
administration. Some typical dose ranges are from about 1 .mu.g/kg
to about 1 g/kg of body weight per day. In some embodiments, the
dose range is from about 0.01 mg/kg to about 100 mg/kg of body
weight per day. The dosage is likely to depend on such variables as
the type and extent of progression of the disease or disorder, the
overall health status of the particular patient, the relative
biological efficacy of the compound selected, formulation of the
excipient, and its route of administration. Effective doses can be
extrapolated from dose-response curves derived from in vitro or
animal model test systems.
V. Labeled Compounds and Assay Methods
[0143] The compounds of the invention can further be useful in
investigations of biological processes as set forth in set forth in
Example 2: In vitro Experiments and Example 3: In vivo Experiments.
Thus, another aspect of the present invention relates to labeled
compounds of the invention (radio-labeled, fluorescent-labeled,
etc.) that would be useful not only in imaging techniques but also
in assays, both in vitro and in vivo, for localizing and
quantitating NAMPT in tissue samples, including human, and for
identifying NAMPT ligands by inhibition binding of a labeled
compound. Accordingly, the present invention includes NAMPT assays
that contain such labeled compounds.
[0144] The present invention further includes isotopically-labeled
compounds of the invention. An "isotopically" or "radio-labeled"
compound is a compound of the invention where one or more atoms are
replaced or substituted by an atom having an atomic mass or mass
number different from the atomic mass or mass number typically
found in nature (i.e., naturally occurring). Suitable radionuclides
(i.e., radioactive isotopes) that may be incorporated in compounds
of the present invention include but are not limited to .sup.3H
(also written as T for tritium), .sup.11C, .sup.13C, .sup.14C,
.sup.13N, .sup.15N, .sup.15O, .sup.17O, .sup.18O, .sup.18F,
.sup.35S, .sup.36Cl, .sup.82Br, .sup.75Br, .sup.76Br, .sup.77Br,
.sup.123I, .sup.124I, .sup.125I and .sup.131I. The radionuclide
that is incorporated in the instant radio-labeled compounds will
depend on the specific application of that radio-labeled compound.
For example, for in vitro Pim kinase labeling and competition
assays, compounds that incorporate .sup.3H, .sup.14C, .sup.82 Br,
.sup.125I, .sup.131I, .sup.35S or will generally be most useful.
For radio-imaging applications .sup.11C, .sup.18F, .sup.125I,
.sup.123I, .sup.124I, .sup.131I, .sup.75Br, .sup.76Br or .sup.77Br
will generally be most useful.
[0145] It is to be understood that a "radio-labeled" or "labeled
compound" is a compound that has incorporated at least one
radionuclide. In some embodiments the radionuclide is selected from
the group consisting of .sup.3H, .sup.14C, .sup.125I, .sup.35S and
.sup.82Br. In some embodiments, the compound incorporates 1, 2 or 3
deuterium atoms. Synthetic methods for incorporating radio-isotopes
into organic compounds are known in the art.
[0146] Specifically, a labeled compound of the invention can be
used in a screening assay to identify and/or evaluate compounds.
For example, a newly synthesized or identified compound (i.e., test
compound) which is labeled can be evaluated for its ability to bind
NAMPT by monitoring its concentration variation when contacting
with NAMPT, through tracking of the labeling. For example, a test
compound (labeled) can be evaluated for its ability to reduce
binding of another compound which is known to bind to NAMPT (i.e.,
standard compound). Accordingly, the ability of a test compound to
compete with the standard compound for binding to NAMPT directly
correlates to its binding affinity. Conversely, in some other
screening assays, the standard compound is labeled and test
compounds are unlabeled. Accordingly, the concentration of the
labeled standard compound is monitored in order to evaluate the
competition between the standard compound and the test compound,
and the relative binding affinity of the test compound is thus
ascertained.
VI. Kits
[0147] The present disclosure also includes pharmaceutical kits
useful, e.g., in the treatment or prevention NAMPT-associated
diseases or disorders, such as cancer, which include one or more
containers containing a pharmaceutical composition comprising a
therapeutically effective amount of a compound of formula (I), or
any of the embodiments thereof. Such kits can further include one
or more of various conventional pharmaceutical kit components,
e.g., containers with one or more pharmaceutically acceptable
carriers, additional containers, etc., as will be readily apparent
to those skilled in the art. Instructions, either as inserts or as
labels, indicating quantities of the components to be administered,
guidelines for administration, and/or guidelines for mixing the
components, can also be included in the kit.
[0148] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *