U.S. patent application number 13/886351 was filed with the patent office on 2014-02-20 for mst1 kinase inhibitors and methods of their use.
The applicant listed for this patent is David John AUGERI, Jeffrey Thomas BAGDANOFF, Simon David Peter BAUGH, Marianne CARLSEN, Kenneth Gordon CARSON, John Anthony GILLERAN, Wei HE, Tamas ORAVECZ, Konstantin SALOJIN, Leonard SUNG. Invention is credited to David John AUGERI, Jeffrey Thomas BAGDANOFF, Simon David Peter BAUGH, Marianne CARLSEN, Kenneth Gordon CARSON, John Anthony GILLERAN, Wei HE, Tamas ORAVECZ, Konstantin SALOJIN, Leonard SUNG.
Application Number | 20140051681 13/886351 |
Document ID | / |
Family ID | 45852728 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140051681 |
Kind Code |
A1 |
AUGERI; David John ; et
al. |
February 20, 2014 |
MST1 KINASE INHIBITORS AND METHODS OF THEIR USE
Abstract
Compounds for the inhibition of mammalian Ste20-like kinase 1
(MST1) are disclosed, along with compositions comprising them and
methods of their use in the treatment, management or prevention of
an inflammatory or autoimmune diseases or disorders.
Inventors: |
AUGERI; David John;
(Princeton, NJ) ; BAGDANOFF; Jeffrey Thomas;
(Martinez, CA) ; BAUGH; Simon David Peter;
(Ringoes, NJ) ; CARLSEN; Marianne; (Yardley,
PA) ; CARSON; Kenneth Gordon; (Princeton, NJ)
; GILLERAN; John Anthony; (Bridgewater, NJ) ; HE;
Wei; (Beijing, CN) ; ORAVECZ; Tamas; (The
Woodlands, TX) ; SALOJIN; Konstantin; (Conroe,
TX) ; SUNG; Leonard; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AUGERI; David John
BAGDANOFF; Jeffrey Thomas
BAUGH; Simon David Peter
CARLSEN; Marianne
CARSON; Kenneth Gordon
GILLERAN; John Anthony
HE; Wei
ORAVECZ; Tamas
SALOJIN; Konstantin
SUNG; Leonard |
Princeton
Martinez
Ringoes
Yardley
Princeton
Bridgewater
Beijing
The Woodlands
Conroe
Cambridge |
NJ
CA
NJ
PA
NJ
NJ
TX
TX
MA |
US
US
US
US
US
US
CN
US
US
US |
|
|
Family ID: |
45852728 |
Appl. No.: |
13/886351 |
Filed: |
May 3, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13410407 |
Mar 2, 2012 |
8440652 |
|
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13886351 |
|
|
|
|
61449171 |
Mar 4, 2011 |
|
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Current U.S.
Class: |
514/211.05 ;
514/213.01; 514/221; 514/255.05; 514/301; 514/309; 514/333;
514/335 |
Current CPC
Class: |
C07D 417/14 20130101;
A61P 17/04 20180101; A61P 19/02 20180101; C07D 409/14 20130101;
A61K 31/553 20130101; C07D 401/14 20130101; C07D 405/14 20130101;
A61K 31/497 20130101; C07D 413/04 20130101; A61P 17/06 20180101;
A61K 31/5513 20130101; A61P 21/04 20180101; C07D 403/04 20130101;
A61P 1/00 20180101; A61P 29/00 20180101; A61P 37/02 20180101; A61K
31/444 20130101; C07D 401/04 20130101; A61K 31/4725 20130101; A61P
1/16 20180101; A61P 11/06 20180101; A61K 31/4418 20130101; C07D
495/04 20130101; A61P 25/00 20180101; A61P 37/06 20180101; A61P
9/00 20180101; A61P 1/04 20180101; A61P 37/00 20180101; A61P 43/00
20180101; C07D 471/04 20130101; A61K 31/55 20130101; A61P 7/04
20180101; A61K 31/4365 20130101; A61P 3/10 20180101; A61P 17/00
20180101 |
Class at
Publication: |
514/211.05 ;
514/309; 514/255.05; 514/333; 514/221; 514/301; 514/213.01;
514/335 |
International
Class: |
C07D 401/04 20060101
C07D401/04; C07D 417/14 20060101 C07D417/14; C07D 401/14 20060101
C07D401/14; C07D 471/04 20060101 C07D471/04; C07D 405/14 20060101
C07D405/14; C07D 403/04 20060101 C07D403/04; C07D 413/04 20060101
C07D413/04; C07D 409/14 20060101 C07D409/14; C07D 495/04 20060101
C07D495/04 |
Claims
1. A method of treating an inflammatory or autoimmune disease,
comprising administering to a patient in need thereof a
therapeutically effective amount of a compound of the formula:
##STR00051## or a pharmaceutically acceptable salt thereof,
wherein: A is aryl or 4-7-membered heterocycle; D is a 4-7-membered
heterocycle; X is N or CH; Y.sub.1 and Y.sub.2 are each
independently S, N or CH, provided that at least one of Y.sub.1 and
Y.sub.2 is N or CH; each R.sub.1 is independently R.sub.1A,
--(R.sub.1B).sub.nSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nSO.sub.pN(R.sub.1C).sub.2,
--(R.sub.1B).sub.nNR.sub.1CSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nC(O)N(R.sub.1C).sub.2, or
--(R.sub.1B).sub.nNR.sub.1CC(O)R.sub.1C, or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more of R.sub.1A; each
R.sub.1A is independently amino, alkoxyl, carboxyl, cyano, halo, or
hydroxyl; each R.sub.1B is independently C.sub.1-12 hydrocarbyl
optionally substituted with one or more of amino, alkoxyl,
carboxyl, cyano, halo, or hydroxyl; each R.sub.1C is independently
hydrogen or optionally substituted C.sub.1-12 hydrocarbyl or
2-12-membered heterocarbyl, which optional substitution is with one
or more of amino, alkoxyl, carboxyl, cyano, halo, or hydroxyl; each
R.sub.3A is independently amino, alkoxyl, carboxyl, cyano, halo, or
hydroxyl; m is 0-3; n is 0 or 1; p is 0-2; and Q is 0-2.
2. (canceled)
3. (canceled)
4. (canceled)
5. The method of claim 1, wherein the compound is of the formula:
##STR00052## wherein Z is N or CR.sub.1.
6. The method of claim 5, wherein the compound is of the formula:
##STR00053## wherein Y.sub.1 and Y.sub.2 are each independently N
or CH.
7. The method of claim 5, wherein the compound is of the formula:
##STR00054##
8. The method of claim 5, wherein the compound is of the formula:
##STR00055##
9. The method of claim 5, wherein the compound is of the formula:
##STR00056## wherein: R.sub.4 is hydrogen or alkyl; and r is 1 or
2.
10. The method of claim 5, wherein the compound is of the formula:
##STR00057##
11. (canceled)
12. (canceled)
13. The method of claim 1, wherein X is N.
14. The method of claim 1, wherein Y.sub.1 is CH.
15. The method of claim 1, wherein Y.sub.2 is CH.
16. The method of claim 5, wherein Z is N.
17. The method of claim 5, wherein Z is CR.sub.1.
18. The method of claim 1, wherein R.sub.1 is
--(R.sub.1B).sub.nSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nSO.sub.pN(R.sub.1C).sub.2,
--(R.sub.1B).sub.nNR.sub.1CSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nC(O)N(R.sub.1C).sub.2, or
--(R.sub.1B).sub.nNR.sub.1CC(O)R.sub.1C.
19. The method of claim 18, wherein n=0.
20. The method of claim 18, wherein p=2.
21. The method of claim 20, which is of the formula:
##STR00058##
22-24. (canceled)
25. The use of claim 1, wherein the autoimmune disease or disorder
is achlorhydra autoimmune, Addison's Disease, ankylosing
spondylitis, anti-phospholipid syndrome, asthma (e.g., bronchial
asthma), atopic dermatitis, autoimmune atrophic gastritis, Behest's
disease, Celiac Disease, Crohn's Disease, Cushing's Syndrome,
dermatomyositis, Goodpasture's Syndrome, graft-vs-host disease,
Grave's Disease, Hashimoto's thyroiditis, hepatitis (e.g.,
inflammatory and alcohol-induced), idiopathic adrenal atrophy,
idiopathic thrombocytopenia, Kawasaki syndrome, Lambert-Eaton
Syndrome, lupus erythematosus, multiple sclerosis, myasthenia
gravis, pemphigoid, pemphigus vulgaris, pernicious anemia,
pollinosis, polyarteritis nodosa, primary biliary cirrhosis,
primary sclerosing cholangitis, psoriasis, psoriatic arthritis,
Raynauds, Reiter's Syndrome, relapsing polychondritis, rheumatoid
arthritis, Schmidt's Syndrome, scleroderma, Sjogren's Syndrome
sympathetic ophthalmia, Takayasu's Arteritis, temporal arteritis,
thyrotoxicosis, transplant rejection (e.g., of organ, cell or bone
marrow), type 1 diabetes, ulcerative colitis, uveitis, and
Wegener's granulomatosis.
26. The use of claim 25, wherein the autoimmune disease or disorder
is Crohn's disease, graft-versus-host disease, psoriasis,
rheumatoid arthritis, or ulcerative colitis.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/410,407 filed Mar. 2, 2012, which claims
priority to U.S. provisional patent application No. 61/449,171,
filed Mar. 4, 2011, the entireties of which are incorporated herein
by reference.
1. FIELD OF THE INVENTION
[0002] This invention is directed to compounds useful as inhibitors
of mammalian Step 20-like kinase 1 (MST1), compositions comprising
them, and methods of their use.
2. BACKGROUND OF THE INVENTION
[0003] Mammalian Step 20-like kinase 1 (MST1) is a component of the
"Hippo" signaling pathway, and "has been implicated in regulating
the cell cycle, apoptosis and cellular responses to oxidative
stress." Choi, J., et al., Plos One 4(11):e8011, 1 (2009).
MST1-deficient mice reportedly display an accumulation of mature
lymphocytes in the thymus and a decrease of lymphocytes in the
blood and peripheral lymphoid tissues. Dong, Y., et al., J.
Immunology 183(6):3865-3872, 3865 (2009). See also, Katagiri, K.,
et al., Nat Immunol. (9):919-28 (2006); Ling, P., et al., Cell
Signal. 20(7):1237-47 (2008). MST1 is also known as
serine/threonine kinase 4 (STK4) and kinase responsive to stress 2
(KRS2).
3. SUMMARY OF THE INVENTION
[0004] This invention is directed to compounds useful for the
inhibition of MST1. One embodiment of the invention encompasses
compounds of the formula:
##STR00001##
and pharmaceutically acceptable salts thereof, wherein: A is aryl
or 4-7-membered heterocycle; B is aryl or 4-7-membered heterocycle;
X is N or CH; Y.sub.1 and Y.sub.2 are each independently S, N or
CH, provided that at least one of Y.sub.1 and Y.sub.2 is N or CH;
each R.sub.1 is independently R.sub.1A,
--(R.sub.1B).sub.nSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nSO.sub.pN(R.sub.1C).sub.2,
--(R.sub.1B).sub.nNR.sub.1CSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nC(O)N(R.sub.1C).sub.2, or
--(R.sub.1B).sub.nNR.sub.1CC(O)R.sub.1C, or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more of R.sub.1A; each
R.sub.1A is independently amino, alkoxyl, carboxyl, cyano, halo, or
hydroxyl; each R.sub.1B is independently C.sub.1-12 hydrocarbyl
optionally substituted with one or more of amino, alkoxyl,
carboxyl, cyano, halo, or hydroxyl; each R.sub.1C is independently
hydrogen or optionally substituted C.sub.1-12 hydrocarbyl or
2-12-membered heterocarbyl, which optional substitution is with one
or more of amino, alkoxyl, carboxyl, cyano, halo, or hydroxyl;
R.sub.2 and R.sub.3 are taken together to form a 5-7-membered
heterocycle optionally substituted with one or more of R.sub.3A,
or: R.sub.2 is hydrogen or C.sub.1-4 alkyl; and R.sub.3 is hydrogen
or optionally optionally substituted C.sub.1-12 hydrocarbyl or
2-12-membered heterocarbyl, which optional substitution is with one
or more of R.sub.3A; each R.sub.3A is independently amino, alkoxyl,
carboxyl, cyano, halo, or hydroxyl; k is 0 or 1; m is 0-3; n is 0
or 1; and p is 0, 1, or 2;
[0005] Another embodiment encompasses formulation comprising a
compound of the invention and a pharmaceutically acceptable
excipient. Another encompasses a method of using a compound of the
invention for the inhibition of MST1.
[0006] Another embodiment encompasses a method of using a compound
of the invention for the treatment, management or prevention of an
inflammatory or autoimmune disease or disorder.
4. BRIEF DESCRIPTION OF THE FIGURES
[0007] Certain aspects of the invention may be understood from the
attached figures, described below:
[0008] FIGS. 1A and 1B show the difference between MST1-/- mice
(n=13) and their wild-type littermates (n-11) in a collagen-induced
arthritis disease model (CIA).
[0009] FIGS. 2A and 2B show the effect of different doses of a
compound of the invention when administered prophylactically (FIG.
2A) and therapeutically (FIG. 2B) to mice in an experimental
autoimmune encephalomyelitis (EAE) disease model.
[0010] FIG. 3 shows the effect of different doses of a compound of
the invention when administered therapeutically to rats in an EAE
disease model.
[0011] FIGS. 4A and 4B show the effect of different doses of a
compound of the invention when administered to mice in a CIA
disease model. FIG. 4A shows the cumulative arthritis score over
the course of the experiment; FIG. 4B shows the change in ankle
thickness over the course of the experiment.
[0012] FIGS. 5A and 5B show the effect of different doses of a
compound of the invention when administered to rats in a CIA
disease model. FIG. 5A shows the cumulative arthritis score over
the course of the experiment; FIG. 5B shows the change in ankle
thickness over the course of the experiment.
[0013] FIG. 6 shows the effect of short-term treatment with a
compound of the invention on liver enzymes and cytokine response in
mice subjected to a concanavalin A (ConA)-induced hepatitis disease
model.
[0014] FIGS. 7A and 7B show the effect of different doses of a
compound of the invention when administered therapeutically to rats
in an EAE disease model. FIG. 7A shows the clinical score as a
function of time; FIG. 7B shows the disease development as a
function of compound dose (or vehicle).
[0015] FIG. 8 shows the effect of short-term treatment with a
compound of the invention on liver enzymes and cytokine response in
mice subjected to a ConA-induced hepatitis disease model.
[0016] FIGS. 9A and 9B show the effect of different doses of a
compound of the invention when administered therapeutically to mice
in an EAE disease model. FIG. 9A shows the clinical score as a
function of time for mice to whom the compound (and vehicle) were
administered subcutaneously; FIG. 9B shows the clinical score as a
function of time for mice to whom the compound (and vehicle) were
administered orally.
[0017] FIGS. 10A and 10B show the effect of a compound of the
invention in a mouse CIA model as a function of dose and method of
delivery. FIGS. 10A and 10B show the effect of the compound on
cumulative scores and change in ankle thickness when compound (and
vehicle control) were administered subcutaneously. FIGS. 10C and
10D show the results obtained when the compound (and vehicle
control) was administered orally.
5. DETAILED DESCRIPTION OF THE INVENTION
[0018] This invention is based, in part, on the discovery that MST1
knockout mice are significantly more resistant to animal autoimmune
and inflammatory disease models than their wild-type littermates.
This finding prompted the discovery of novel compounds that inhibit
MST1, the most preferred of which are effective in animal disease
models and exhibit desirable toxicological and pharmacokinetic
properties.
5.1. Definitions
[0019] Unless otherwise indicated, the phrases "compounds of the
invention," "compounds of the present disclosure," and the like
refer to the compounds disclosed herein, particularly compounds of
Formula I and salts thereof.
[0020] Unless otherwise indicated, the term "hydrocarbyl" means an
aliphatic or alicyclic moiety having an all-carbon backbone and
consisting of carbon and hydrogen atoms. Examples of hydrocarbyl
groups include those having 1-20, 1-12, 1-6, and 1-4 carbon atoms
(referred to as C.sub.1-20 hydrocarbyl, C.sub.1-12 hydrocarbyl,
C.sub.1-6 hydrocarbyl, and C.sub.1-4 hydrocarbyl, respectively).
Particular examples include alkyl, alkenyl, alkynyl, aryl, benzyl,
cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkenylalkyl,
napthyl, phenyl, and phenylethyl.
[0021] Examples of alkyl moieties include straight-chain and
branched moieties having 1-20, 1-12, 1-6, 1-4 and 1-3 carbon atoms
(referred to as C.sub.1-20 alkyl, C.sub.1-12 alkyl, C.sub.1-6
alkyl, C.sub.1-4 alkyl and C.sub.1-3 alkyl, respectively).
Particular examples include methyl, ethyl, propyl, isopropyl,
n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,
4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl,
undecyl and dodecyl.
[0022] Examples of alkenyl moieties include straight-chain and
branched C.sub.2-20, C.sub.2-12 and C.sub.2-6 alkenyl. Particular
examples include vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl,
1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,
2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,
1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl,
3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl
and 3-decenyl.
[0023] Examples of alkynyl moieties include straight-chain and
branched C.sub.2-20, C.sub.2-12 and C.sub.2-6 alkynyl. Particular
examples include ethynyl and 2-propynyl (propargyl).
[0024] Examples of aryl moieties include anthracenyl, azulenyl,
fluorenyl, indan, indenyl, naphthyl, phenyl and phenanthrenyl.
[0025] Examples of cycloalkyl moieties include C.sub.3-12,
C.sub.3-7, C.sub.4-6 and C.sub.6 cycloalkyl. Particular examples
include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
adamantyl.
[0026] Unless otherwise indicated, the term "halo" encompass
fluoro, chloro, bromo, and iodo.
[0027] Unless otherwise indicated, the term "heterocarbyl" refers
to a moiety having a backbone made up of one or more carbon atoms
and one or more heteroatoms. Particular heteroatoms are nitrogen,
oxygen and sulfur. A heterocarbyl moieties can be thought of as a
hydrocarbyl moiety wherein at least one carbon atom, CH, CH.sub.2,
or CH.sub.3 group is replaced with one or more heteroatoms and the
requisite number of hydrogen atoms to satisfy valencies. Examples
of heterocarbyl include 2-20, 2-12, 2-8, 2-6 and 2-4 membered
heterocarbyl moieties, wherein the number range refers to the sum
total of carbon, nitrogen, oxygen, and/or sulfur atoms in the
moiety. The term "2-12 membered heterocarbyl" thus refers to a
heterocarbyl moiety having a total of 2-12 carbon, nitrogen,
oxygen, and/or sulfur atoms. Particular heterocarbyl moieties
include straight chain and branched heteroalkyl, heteroalkenyl, and
heteroalkynyl, as well as heterocycle and heteroaryl.
[0028] Examples of heteroalkyl moieties include 2-8-membered,
2-6-membered and 2-4-membered heteroalkyl moieties. Particular
examples include alkoxyl, acyl (e.g., formyl, acetyl, benzoyl),
alkylamino (e.g., di-(C.sub.1-3-alkyl)amino), arylamino, aryloxime,
carbamates, carbamides, alkylcarbonyl, arylcarbonyl, aminocarbonyl,
alkylaminocarbonyl, alkylsulfanyl, arylsulfanyl, alkylsulfinyl,
arylsulfinyl, alkylsulfonyl, arylsulfonyl, alkylsulfonylamino, and
arylsulfonylamino.
[0029] Unless otherwise indicated, the term "heterocycle" refers to
a cyclic (monocyclic or polycyclic) heterocarbyl moiety which may
be aromatic, partially aromatic or non-aromatic. Heterocycles
include heteroaryls. Examples include 4-10-membered, 4-7-membered,
6-membered, and 5-membered heterocycles. Particular examples
include benzo[1,3]dioxolyl, 2,3-dihydro-benzo[1,4]dioxinyl,
cinnolinyl, furanyl, hydantoinyl, morpholinyl, oxetanyl, oxiranyl,
piperazinyl, piperidinyl, pyrrolidinonyl, pyrrolidinyl,
tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,
tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl
and valerolactamyl. Because the term "heterocycle" refers to a
ring, standing alone it does not encompass moieties such as
oxazolidinone and imidazolidinone: such moieties are considered
substituted heterocycles, viz. heterocycles substituted with
oxo.
[0030] Examples of heteroaryl moieties include acridinyl,
benzimidazolyl, benzofuranyl, benzoisothiazolyl, benzoisoxazolyl,
benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl, imidazolyl,
indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl,
phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl,
pyrimidinyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl,
tetrazolyl, thiazolyl, and triazinyl.
[0031] Unless otherwise indicated, the term "include" has the same
meaning as "include, but are not limited to," and the term
"includes" has the same meaning as "includes, but is not limited
to." Similarly, the term "such as" has the same meaning as the term
"such as, but not limited to."
[0032] Unless otherwise indicated, the terms "manage," "managing"
and "management" encompass preventing the recurrence of the
specified disease or disorder in a patient who has already suffered
from the disease or disorder, and/or lengthening the time that a
patient who has suffered from the disease or disorder remains in
remission. The terms encompass modulating the threshold,
development and/or duration of the disease or disorder, or changing
the way that a patient responds to the disease or disorder.
[0033] Unless otherwise indicated, the term "MST1 inhibitor" means
a compound that inhibits MST1 in vitro with an IC.sub.50 of less
than 1 .mu.m, 0.5 .mu.m or 0.25 .mu.m as determined by the assay
described herein.
[0034] Unless otherwise indicated, the term "pharmaceutically
acceptable salts" refers to salts prepared from pharmaceutically
acceptable non-toxic acids or bases including inorganic acids and
bases and organic acids and bases. Suitable pharmaceutically
acceptable base addition salts include metallic salts made from
aluminum, calcium, lithium, magnesium, potassium, sodium and zinc
or organic salts made from lysine, N,N'-dibenzylethylenediamine,
chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine
(N-methylglucamine) and procaine. Suitable non-toxic acids include
inorganic and organic acids such as acetic, alginic, anthranilic,
benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic,
formic, fumaric, furoic, galacturonic, gluconic, glucuronic,
glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic,
maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,
pantothenic, phenylacetic, phosphoric, propionic, salicylic,
stearic, succinic, sulfanilic, sulfuric, tartaric acid, and
p-toluenesulfonic acid. Specific non-toxic acids include
hydrochloric, hydrobromic, phosphoric, sulfuric, and
methanesulfonic acids. Examples of specific salts thus include
hydrochloride and mesylate salts. Others are well-known in the art.
See, e.g., Remington's Pharmaceutical Sciences, 18.sup.th ed. (Mack
Publishing, Easton Pa.: 1990) and Remington: The Science and
Practice of Pharmacy, 19.sup.th ed. (Mack Publishing, Easton Pa.:
1995).
[0035] Unless otherwise indicated, the term "substituted," when
used to describe a chemical structure or moiety, refers to a
derivative of that structure or moiety wherein one or more of its
hydrogen atoms is substituted with an atom, chemical moiety or
functional group such as, but not limited to, alcohol, aldehyde,
alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl,
ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (--OC(O)alkyl),
amide (--C(O)NH-alkyl- or -alkylNHC(O)alkyl), amidinyl
(--C(NH)NH-alkyl or --C(NR)NH.sub.2), amine (primary, secondary and
tertiary such as alkylamino, arylamino, arylalkylamino), aroyl,
aryl, aryloxy, azo, carbamoyl (--NHC(O)O-alkyl- or
--OC(O)NH-alkyl), carbamyl (e.g., CONH.sub.2, CONH-alkyl,
CONH-aryl), carbonyl, carboxyl, carboxylic acid, carboxylic acid
anhydride, carboxylic acid chloride, cyano, ester, epoxide, ether
(e.g., methoxy, ethoxy), guanidino, halo, haloalkyl (e.g.,
--CCl.sub.3, --CF.sub.3, --C(CF.sub.3).sub.3), heteroalkyl,
hemiacetal, imine (primary and secondary), isocyanate,
isothiocyanate, ketone, nitrile, nitro, oxygen (i.e., to provide an
oxo group), phosphodiester, sulfide, sulfonamido (e.g.,
SO.sub.2NH.sub.2), sulfone, sulfonyl (including alkylsulfonyl,
arylsulfonyl and arylalkylsulfonyl), sulfoxide, thiol (e.g.,
sulfhydryl, thioether) and urea (--NHCONH-alkyl-). In a particular
embodiment, the term substituted refers to a derivative of that
structure or moiety wherein one or more of its hydrogen atoms is
substituted with alcohol, alkoxy, alkyl (e.g., methyl, ethyl,
propyl, t-butyl), amide (--C(O)NH-alkyl- or -alkylNHC(O)alkyl),
amidinyl (--C(NH)NH-alkyl or --C(NR)NH.sub.2), amine (primary,
secondary and tertiary such as alkylamino, arylamino,
arylalkylamino), aryl, carbamoyl (--NHC(O)O-alkyl- or
--OC(O)NH-alkyl), carbamyl (e.g., CONH.sub.2, as well as
CONH-alkyl, CONH-aryl), halo, haloalkyl (e.g., --CCl.sub.3,
--CF.sub.3, --C(CF.sub.3).sub.3), heteroalkyl, imine (primary and
secondary), isocyanate, isothiocyanate, thiol (e.g., sulfhydryl,
thioether) or urea (--NHCONH-alkyl-).
[0036] Unless otherwise indicated, a "therapeutically effective
amount" of a compound is an amount sufficient to provide a
therapeutic benefit in the treatment or management of a disease or
condition, or to delay or minimize one or more symptoms associated
with the disease or condition. A "therapeutically effective amount"
of a compound means an amount of therapeutic agent, alone or in
combination with other therapies, that provides a therapeutic
benefit in the treatment or management of the disease or condition.
The term "therapeutically effective amount" can encompass an amount
that improves overall therapy, reduces or avoids symptoms or causes
of a disease or condition, or enhances the therapeutic efficacy of
another therapeutic agent.
[0037] Unless otherwise indicated, the terms "treat," "treating"
and "treatment" contemplate an action that occurs while a patient
is suffering from the specified disease or disorder, which reduces
the severity of the disease or disorder, or retards or slows the
progression of the disease or disorder.
[0038] Unless otherwise indicated, one or more adjectives
immediately preceding a series of nouns is to be construed as
applying to each of the nouns. For example, the phrase "optionally
substituted alky, aryl, or heteroaryl" has the same meaning as
"optionally substituted alky, optionally substituted aryl, or
optionally substituted heteroaryl."
[0039] It should be noted that a chemical moiety that forms part of
a larger compound may be described herein using a name commonly
accorded it when it exists as a single molecule or a name commonly
accorded its radical. For example, the terms "pyridine" and
"pyridyl" are accorded the same meaning when used to describe a
moiety attached to other chemical moieties. Thus, the two phrases
"XOH, wherein X is pyridyl" and "XOH, wherein X is pyridine" are
accorded the same meaning, and encompass the compounds
pyridin-2-ol, pyridin-3-ol and pyridin-4-ol.
[0040] It should also be noted that if the stereochemistry of a
structure or a portion of a structure is not indicated with, for
example, bold or dashed lines, the structure or the portion of the
structure is to be interpreted as encompassing all stereoisomers of
it. Similarly, names of compounds having one or more chiral centers
that do not specify the stereochemistry of those centers encompass
pure stereoisomers and mixtures thereof. Moreover, any atom shown
in a drawing with unsatisfied valences is assumed to be attached to
enough hydrogen atoms to satisfy the valences. In addition,
chemical bonds depicted with one solid line parallel to one dashed
line encompass both single and double (e.g., aromatic) bonds, if
valences permit. This invention encompasses tautomers and solvates
(e.g., hydrates) of the compounds disclosed herein.
5.2. Compounds of the Invention
[0041] This invention encompasses compounds of the formula:
##STR00002##
and pharmaceutically acceptable salts thereof, wherein: A is aryl
or 4-7-membered heterocycle; B is aryl or 4-7-membered heterocycle;
X is N or CH; Y.sub.1 and Y.sub.2 are each independently S, N or
CH, provided that at least one of Y.sub.1 and Y.sub.2 is N or CH;
each R.sub.1 is independently R.sub.1A,
--(R.sub.1B).sub.nSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nSO.sub.pN(R.sub.1C).sub.2,
--(R.sub.1B).sub.nNR.sub.1CSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nC(O)N(R.sub.1C).sub.2, or
--(R.sub.1B).sub.nNR.sub.1CC(O)R.sub.1C, or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more of R.sub.1A; each
R.sub.1A is independently amino, alkoxyl, carboxyl, cyano, halo, or
hydroxyl; each R.sub.1B is independently C.sub.1-12 hydrocarbyl
optionally substituted with one or more of amino, alkoxyl,
carboxyl, cyano, halo, or hydroxyl; each R.sub.1C is independently
hydrogen or optionally substituted C.sub.1-12 hydrocarbyl or
2-12-membered heterocarbyl, which optional substitution is with one
or more of amino, alkoxyl, carboxyl, cyano, halo, or hydroxyl;
R.sub.2 and R.sub.3 are taken together to form a 5-7-membered
heterocycle optionally substituted with one or more of R.sub.3A,
or: R.sub.2 is hydrogen or C.sub.1-4 alkyl; and R.sub.3 is hydrogen
or optionally optionally substituted C.sub.1-12 hydrocarbyl or
2-12-membered heterocarbyl, which optional substitution is with one
or more of R.sub.3A; each R.sub.3A is independently amino, alkoxyl,
carboxyl, cyano, halo, or hydroxyl; k is 0 or 1; m is 0-3; n is 0
or 1; and p is 0, 1, or 2. Preferred compounds are MST1
inhibitors.
[0042] Particular compounds of the invention are of the
formula:
##STR00003##
wherein D is a 4-7-membered heterocycle; and q is 0-2.
[0043] Particular compounds are of the formula:
##STR00004##
wherein Y.sub.1 and Y.sub.2 are each independently S, N, NH, CH or
CH.sub.2.
[0044] Particular compounds are of the formula:
##STR00005##
wherein Y.sub.1 and Y.sub.2 are each independently S, N or CH,
provided that at least one of Y.sub.1 and Y.sub.2 is N or CH; and k
is 0 or 1. Particular compounds are such that R.sub.3 is not
hydrogen when X is CH. More particular compounds are such that
R.sub.3 is not hydrogen when X is CH, Y.sub.1 is CH and Y.sub.2 is
CH. More particular compounds are such that R.sub.3 is not hydrogen
when X is CH, Y.sub.1 is CH, Y.sub.2 is CH, and R.sub.2 is
hydrogen.
[0045] Particular compounds are of the formula:
##STR00006##
wherein Y.sub.1 and Y.sub.2 are each independently N or CH.
[0046] Other compounds of the invention of the formula:
##STR00007##
wherein Y.sub.1 and Y.sub.2 are each independently N or CH. Others
are of the formula:
##STR00008##
wherein Y.sub.1 and Y.sub.2 are each independently N or CH; and Z
is N or CR.sub.1.
[0047] Particular compounds of the invention are of the
formulae:
##STR00009##
wherein Y.sub.1 and Y.sub.2 are each independently N or CH; r is 1
or 2, and R.sub.4 is hydrogen or alkyl.
[0048] Other compounds of the invention are of the formula:
##STR00010##
wherein Y.sub.2 is N or CH. Others are of the formula:
##STR00011##
wherein Y.sub.1 is N or CH.
[0049] Where applicable to the formulae disclosed herein,
particular compounds of the invention are such that X is N.
[0050] Where applicable to the formulae disclosed herein,
particular compounds of the invention are such that Y.sub.1 is
CH.
[0051] Where applicable to the formulae disclosed herein,
particular compounds of the invention are such that Y.sub.2 is
CH.
[0052] Where applicable to the formulae disclosed herein,
particular compounds of the invention are such that Z is N.
[0053] Where applicable to the formulae disclosed herein,
particular compounds of the invention are such that Z is
CR.sub.1.
[0054] Where applicable to the formulae disclosed herein,
particular compounds of the invention are such that R.sub.1 is
--(R.sub.1B).sub.nSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nSO.sub.pN(R.sub.1C).sub.2,
--(R.sub.1B).sub.nNR.sub.1CSO.sub.pR.sub.1C,
--(R.sub.1B).sub.nC(O)N(R.sub.1C).sub.2, or
--(R.sub.1B).sub.nNR.sub.1CC(O)R.sub.1C.
[0055] Particular compounds of the invention are of the
formula:
##STR00012##
[0056] Other compounds are of the formula:
##STR00013##
[0057] Compounds of the invention can be prepared by methods known
in the art and by methods described herein. In general, compounds
of the invention can be prepared as shown below:
##STR00014##
wherein the starting 2-amino heteroaryl bromide 1 is coupled with
an appropriate aromatic boronic ester under standard Suzuki
coupling conditions to provide, after treatment with an
electrophilic brominating reagent, 2-amino hetero-biaryl bromide 2.
Subsequent Suzuki coupling of 2 with another aromatic boronic ester
displaying an amide functionality provides final product 3.
5.3. Methods of Use
[0058] This invention encompasses a method of inhibiting MST1,
which comprises contacting MST1 (in vitro or in vivo) with an
effective amount of a compound of the invention.
[0059] Another embodiment encompasses a method of suppressing
immune response in a patient (e.g., a human), which comprises
administering to the patient an effective amount of a compound of
the invention.
[0060] Another embodiment encompasses a method of treating,
managing or preventing an autoimmune or inflammatory disease or
disorder, which comprises administering to a patient in need
thereof a therapeutically or prophylactically effective amount of a
compound of the invention. Examples of diseases and disorders
include achlorhydra autoimmune, Addison's Disease, ankylosing
spondylitis, anti-phospholipid syndrome, asthma (e.g., bronchial
asthma), atopic dermatitis, autoimmune atrophic gastritis, Behcet's
disease, Celiac Disease, Crohn's Disease, Cushing's Syndrome,
dermatomyositis, Goodpasture's Syndrome, graft-vs-host disease,
Grave's Disease, Hashimoto's thyroiditis, hepatitis (e.g.,
inflammatory and alcohol-induced), idiopathic adrenal atrophy,
idiopathic thrombocytopenia, Kawasaki syndrome, Lambert-Eaton
Syndrome, lupus erythematosus, multiple sclerosis, myasthenia
gravis, pemphigoid, pemphigus vulgaris, pernicious anemia,
pollinosis, polyarteritis nodosa, primary biliary cirrhosis,
primary sclerosing cholangitis, psoriasis, psoriatic arthritis,
Raynauds, Reiter's Syndrome, relapsing polychondritis, rheumatoid
arthritis, Schmidt's Syndrome, scleroderma, Sjogren's Syndrome
sympathetic ophthalmia, Takayasu's Arteritis, temporal arteritis,
thyrotoxicosis, transplant rejection (e.g., of organ, cell or bone
marrow), type 1 diabetes, ulcerative colitis, uveitis, and
Wegener's granulomatosis.
[0061] The amount, route of administration and dosing schedule of a
compound will depend upon factors such as the specific indication
to be treated, prevented, or managed, and the age, sex and
condition of the patient. The roles played by such factors are well
known in the art, and may be accommodated by routine
experimentation. In a particular embodiment, a compound of the
invention is administered to a human patient in an amount of about
1-50, 1-25, or 2.5-15, or 5-10 mpk.
[0062] Compounds of the invention can be administered in
combination with other immunosuppressant or anti-inflammatory
drugs. The drugs can be administered at the same or at different
times.
[0063] Examples of immunosuppressants include aminopterin,
azathioprine, cyclosporin A, D-penicillamine, gold salts,
hydroxychloroquine, leflunomide, methotrexate, minocycline,
rapamycin, sulfasalazine, tacrolimus (FK506), and pharmaceutically
acceptable salts thereof. A particular immunosuppressant is
methotrexate.
[0064] Additional examples include anti-TNF antibodies, such as
adalimumab, certolizumab pegol, etanercept, and infliximab. Others
include interleukin-1 blockers, such as anakinra. Others include
anti-B cell (CD20) antibodies, such as rituximab. Others include T
cell activation blockers, such as abatacept.
[0065] Additional examples include inosine monophosphate
dehydrogenase inhibitors, such as mycophenolate mofetil (CellCeptS)
and mycophenolic acid (Myfortic.RTM.).
[0066] Examples of anti-inflammatory drugs include glucocorticoids
and NSAIDs.
[0067] Examples of glucocorticoids include aldosterone,
beclometasone, betamethasone, cortisone, deoxycorticosterone,
dexamethasone, fludrocortisones, hydrocortisone,
methylprednisolone, prednisolone, prednisone, triamcinolone, and
pharmaceutically acceptable salts thereof.
[0068] Examples of NSAID include salicylates (e.g., aspirin,
amoxiprin, benorilate, choline magnesium salicylate, diflunisal,
faislamine, methyl salicylate, magnesium salicylate, salicyl
salicylate, and pharmaceutically acceptable salts thereof),
arylalkanoic acids (e.g., diclofenac, aceclofenac, acemetacin,
bromfenac, etodolac, indometacin, nabumetone, sulindac, tolmetin,
and pharmaceutically acceptable salts thereof), arylpropionic acids
(e.g., ibuprofen, carprofen, fenbufen, fenoprofen, flurbiprofen,
ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, tiaprofenic
acid, suprofen, and pharmaceutically acceptable salts thereof),
arylanthranilic acids (e.g., meclofenamic acid, mefenamic acid, and
pharmaceutically acceptable salts thereof), pyrazolidine
derivatives (e.g., azapropazone, metamizole, oxyphenbutazone,
phenylbutazone, sulfinprazone, and pharmaceutically acceptable
salts thereof), oxicams (e.g., lornoxicam, meloxicam, piroxicam,
tenoxicam, and pharmaceutically acceptable salts thereof), COX-2
inhibitors (e.g., celecoxib, etoricoxib, lumiracoxib, parecoxib,
rofecoxib, valdecoxib, and pharmaceutically acceptable salts
thereof), and sulphonanilides (e.g., nimesulide and
pharmaceutically acceptable salts thereof).
5.4. Pharmaceutical Compositions
[0069] This invention encompasses pharmaceutical compositions
comprising one or more compounds of the invention. Certain
pharmaceutical compositions are single unit dosage forms suitable
for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or
rectal), parenteral (e.g., subcutaneous, intravenous, bolus
injection, intramuscular, or intraarterial), or transdermal
administration to a patient. Examples of dosage forms include, but
are not limited to: tablets; caplets; capsules, such as soft
elastic gelatin capsules; cachets; troches; lozenges; dispersions;
suppositories; ointments; cataplasms (poultices); pastes; powders;
dressings; creams; plasters; solutions; patches; aerosols (e.g.,
nasal sprays or inhalers); gels; liquid dosage forms suitable for
oral or mucosal administration to a patient, including suspensions
(e.g., aqueous or non-aqueous liquid suspensions, oil-in-water
emulsions, or a water-in-oil liquid emulsions), solutions, and
elixirs; liquid dosage forms suitable for parenteral administration
to a patient; and sterile solids (e.g., crystalline or amorphous
solids) that can be reconstituted to provide liquid dosage forms
suitable for parenteral administration to a patient.
[0070] The formulation should suit the mode of administration. For
example, the oral administration of a compound susceptible to
degradation in the stomach may be achieved using an enteric
coating. Similarly, a formulation may contain ingredients that
facilitate delivery of the active ingredient(s) to the site of
action. For example, compounds may be administered in liposomal
formulations in order to protect them from degradative enzymes,
facilitate transport in circulatory system, and effect their
delivery across cell membranes.
[0071] Similarly, poorly soluble compounds may be incorporated into
liquid dosage forms (and dosage forms suitable for reconstitution)
with the aid of solubilizing agents, emulsifiers and surfactants
such as, but not limited to, cyclodextrins (e.g.,
.alpha.-cyclodextrin, .beta.-cyclodextrin, Captisol.RTM., and
Encapsin.TM. (see, e.g., Davis and Brewster, Nat. Rev. Drug Disc.
3:1023-1034 (2004)), Labrasol.RTM., Labrafil.RTM., Labrafac.RTM.,
cremafor, and non-aqueous solvents, such as, but not limited to,
ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate,
benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene
glycol, dimethyl formamide, dimethyl sulfoxide (DMSO),
biocompatible oils (e.g., cottonseed, groundnut, corn, germ, olive,
castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol,
polyethylene glycols, fatty acid esters of sorbitan, and mixtures
thereof (e.g., DMSO:cornoil).
[0072] The composition, shape, and type of a dosage form will
typically vary depending with use. For example, a dosage form used
in the acute treatment of a disease may contain larger amounts of
one or more of the active ingredients it comprises than a dosage
form used in the chronic treatment of the same disease. Similarly,
a parenteral dosage form may contain smaller amounts of one or more
of the active ingredients it comprises than an oral dosage form
used to treat the same disease. How to account for such differences
will be apparent to those skilled in the art. See, e.g.,
Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing,
Easton Pa. (1990).
[0073] 5.4.1. Oral Dosage Forms
[0074] Pharmaceutical compositions of the invention suitable for
oral administration can be presented as discrete dosage forms, such
as, but are not limited to, tablets (e.g., chewable tablets),
caplets, capsules, and liquids (e.g., flavored syrups). Such dosage
forms contain predetermined amounts of active ingredients, and may
be prepared by methods of pharmacy well known to those skilled in
the art. See generally, Remington's Pharmaceutical Sciences, 18th
ed., Mack Publishing, Easton Pa. (1990).
[0075] Typical oral dosage forms are prepared by combining the
active ingredient(s) in an intimate admixture with at least one
excipient according to conventional pharmaceutical compounding
techniques. Excipients can take a wide variety of forms depending
on the form of preparation desired for administration.
[0076] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit forms. If
desired, tablets can be coated by standard aqueous or non-aqueous
techniques. Such dosage forms can be prepared by conventional
methods of pharmacy. In general, pharmaceutical compositions and
dosage forms are prepared by uniformly and intimately admixing the
active ingredients with liquid carriers, finely divided solid
carriers, or both, and then shaping the product into the desired
presentation if necessary. Disintegrants may be incorporated in
solid dosage forms to facility rapid dissolution. Lubricants may
also be incorporated to facilitate the manufacture of dosage forms
(e.g., tablets).
[0077] 5.4.2. Parenteral Dosage Forms
[0078] Parenteral dosage forms can be administered to patients by
various routes including subcutaneous, intravenous (including bolus
injection), intramuscular, and intraarterial. Because their
administration typically bypasses patients' natural defenses
against contaminants, parenteral dosage forms are specifically
sterile or capable of being sterilized prior to administration to a
patient. Examples of parenteral dosage forms include solutions
ready for injection, dry products ready to be dissolved or
suspended in a pharmaceutically acceptable vehicle for injection,
suspensions ready for injection, and emulsions.
[0079] Suitable vehicles that can be used to provide parenteral
dosage forms of the invention are well known to those skilled in
the art. Examples include: Water for Injection USP; aqueous
vehicles such as Sodium Chloride Injection, Ringer's Injection,
Dextrose Injection, Dextrose and Sodium Chloride Injection, and
Lactated Ringer's Injection; water-miscible vehicles such as ethyl
alcohol, polyethylene glycol, and polypropylene glycol; and
non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil,
sesame oil, ethyl oleate, isopropyl myristate, and benzyl
benzoate.
6. EXAMPLES
6.1. Experimental Autoimmune Encephalomyelitis (EAE) Disease
Model
[0080] Knockout mice and compounds were tested in an EAE disease
model, which was generally carried out as described below.
[0081] Mouse Model. Here, an adaptation of the method of Bettelli,
et al., J. Immunol. 161:3299 (1998) was implemented. Eight to 12
weeks old C57Bl/6-Albino/129SvEv littermates were immunized
subcutaneously with a total of 300 .mu.g MOGp35-55 peptide
(MEVGWYRSPFSRVVHLYRNGK) emulsified in complete Freund's adjuvant
(CFA) containing 250 .mu.g heat-inactivated Mycobacterium
tuberculosis H37 Ra (Difco Laboratories). The antigen was divided
equally across two injection sites in the abdominal flanks.
Immediately following the injection of the emulsified antigen (day
0), each mouse received one intravenous injection of 500 ng of
Pertussis toxin (List Biological Laboratories). Animal weights were
recorded prior to the start of the experiment and monitored
throughout the experiment, 2-3 times per week. Disease severity was
scored on a scale as follows: 0=asymptomatic, no detectable sign of
disease; 1=slight weakness of the tail/floppy tail and/or slumped
hindquarters; 2=definite total paralysis of the tail; 3=mild
waddle/distorted gait and total paralysis of the tail and/or mild
impaired righting reflex; 4=heavy waddle with impaired
control-weakness of hind limbs and/or impaired righting reflex;
5=paralysis of one of the hind limbs possibly with mild forelimb
weakness; 6=paralysis of both hind limbs and/or moderate to severe
forelimb weakness; 7=quadriplegia/paralysis of all limbs--hind and
fore; 8=moribund/dead. When the severity of EAE was scored a "2" or
greater, an additional water source consisting of Napa Nectar.TM.,
and pre-moistened food were placed on the cage floor. Animals with
severe onset of the disease were given subcutaneous fluid therapy
consisting of 1 cc of normal saline a minimum of once a day.
Animals displaying scores of 7-8 without signs of recovery for more
than 2 days were sacrificed.
[0082] Rat Model. EAE was elicited in rats according to Mannie, M.
D., et al., Proc Natl. Acad. Sci. U.S.A. 82:5515-5519 (1985). A
synthetic peptide consisting of a sequence analogous to the
reported minimal length encephalitogenic determinant of the bovine
myelin basic protein (MBP) molecule (MBP68-82: YGSLPQKAQRPQDEN).
Lewis rats (150-200 g, female; Charles River Laboratories,
Wilmington, Mass.) were injected subcutaneously in both sides of
the dorsal tail root with 0.1 ml of an emulsion consisting of 100
.mu.g of encephalitogenic MBP peptide in complete Freund's adjuvant
containing 200 .mu.g of Mycobacterium tuberculosis H37Rv, Jamaican
strain. Immunized animals were monitored daily for disease onset
and progression, starting one week after immunization. Disease
severity was scored as described above.
[0083] Animals displaying scores of 7-8 without signs of recovery
for more than 2 days were sacrificed.
6.2. Collagen-Induced Arthritis (CIA) Disease Model
[0084] Knockout mice and compounds were tested in a CIA disease
model, which was generally carried out as described below.
[0085] Mouse Model. Eight to 16 weeks old DBA/1 mice were immunized
intradermally (i.d.) at several sites into the base of the tail
with 100 .mu.g of chicken type II collagen (CII; Sigma Chemical
Co.) in Freund's complete adjuvant (CFA; Difco, Detroit, Mich.)
containing 2.5 mg/ml M. tuberculosis, followed by a repeat booster
i.d. injection of CII (100 .mu.g emulsified in CFA) given 3 weeks
after the primary immunization. Mice were monitored daily for signs
of arthritis, and disease severity scores were assessed by a visual
scoring of 0 to 4 according to the following scale: 0=no erythema
and swelling; 1=erythema and mild swelling of the mid-foot or ankle
joint; 2=erythema and mild swelling extending from the ankle to the
mid-foot; 3=erythema and moderate swelling extending from the ankle
to the metatarsal joints; 4=erythema and severe swelling of the
ankle, foot, and digits. Total disease severity scores were
recorded as a sum of visual scores for four limbs. In addition to
visual scoring, paw thickness was measured with a micrometer
caliper.
[0086] Rat Model. CIA was elicited in rats according to Rosloniec,
E. F., et al., Curr Protoc Immunol Chapter 15:Unit 15, pp. 11-25.
Here, Lewis rats (150-200 g, female; Charles River Laboratories,
Wilmington, Mass.) were injected intradermally at the base of the
tail with a total of 300 .mu.l of a 1:1 emulsion of bovine type II
collagen (CII; 150 .mu.g total; Sigma-Aldrich) and incomplete
Freund's adjuvant (IFA; 150 .mu.l total; Sigma-Aldrich), followed
by a repeat booster injection of the same emulsion 7 days after the
primary immunization. Rats were monitored for signs of arthritis by
clinical scoring (visual observation) of each rat paw, using the
rating scale described above. The extent of swelling was calculated
by subtracting the baseline values of the first measurement from
the values of subsequent measurements.
6.3. Concanavalin A (ConA)-Induced Hepatitis Disease Model
[0087] This model was generally conducted as follows.
C57Bl/6-Albino/129SvEv mice were injected intravenously (i/v) via
the lateral tail vein with a single sublethal dose of Concanavalin
A from Canavalia ensiformis (Jack bean, Type IV-S, lyophilized
powder, aseptically processed; Sigma) administered at 10-16 mg/kg
mouse body weight in a total volume of 0.1-0.3 ml of pyrogen-free
PBS. The tail vein injections of mice restrained in a Plexiglas
mouse restrainer were performed without anesthesia, using a 1 ml
syringe with a 27 gauge needle. At six and 24 hr post-injection,
blood samples were collected by retro-orbital bleeding. The animals
were sacrificed and sera from the blood samples were analyzed for
the presence of IL-12, TNF-.alpha., MCP-1, IFN-.gamma., IL-10, and
IL-6 using a mouse inflammation cytometric bead array (CBA) kit (BD
Biosciences, Mountain View, Calif.), according to the
manufacturer's instructions. Data were acquired with a FACSCalibur
flow cytometer and analyzed with BD CBA Software (BD Biosciences).
Biochemical markers of liver failure were assessed by measuring
serum liver damage enzymes, aspartate aminotransferase (AST) and
alanine aminotransferase (ALT), using a standard clinical
biochemical analyzer. Livers of ConA-treated mice were sectioned
and stained with H&E to evaluate the degree of T cell-mediated
immune inflammation.
6.4. MST1 Knockout Mice
[0088] MST1 knockout mice (-/-) and their wild-type
(C57Bl/6-Albino/129SvEv) littermates were bred and evaluated in
EAE, CIA, and ConA-induced hepatitis disease models. In the EAE
model, eight to 12 week-old MST1-/- and wild-type (+/+)
(C57Bl/6-Albino/129SvEv) littermates were tested. In the wild-type
mice, EAE began as early as day 8 post-immunization, with a mean
onset at day 11.4.+-.0.78 and 12.3.+-.0.9 (experiment #1 and
experiment #2, respectively). The onset of disease was
significantly delayed in the MST1-deficient animals (day
16.6.+-.1.41 in experiment #1 and day 15.1.+-.1.1 in experiment #2;
p=0.005 and 0.06, respectively). In the acute post-immunization
phase of EAE (days 7-22), there was a 4- to 6-fold decrease in the
mean accumulative disease score in MST1-/- mice compared with their
wild-type littermates. During this phase of the disease, the mean
clinical score was significantly lower in the MST1-/- mice than in
+/+ animals. In addition, the mean peak EAE score observed in the
MST1+/+ animals on day 17 post-immunization was significantly
higher than the mean peak EAE score in the MST1-/- group. Thus,
homozygous deficiency of the MST1 gene significantly delayed the
onset of EAE in mice and alleviated the severity of EAE in the
acute post-immunization phase. Together, these results demonstrate
that MST1-/- mice develop less severe EAE with significantly lower
disease scores, and they are less susceptible to disease when
compared with their wild-type littermates.
[0089] In the CIA model, eight to 16 week old MST1-deficient and
wild-type (C57Bl/6-Albino/129SvEv) control mice were tested. As
shown in FIGS. 1A and 1B, the MST1-deficient mice displayed a
markedly decreased incidence of arthritis when compared with their
wild-type littermates (* indicates p<0.03). Joint swelling and
clinical signs of inflammation in the ankle and wrist joints were
evident in wild-type controls from day 21 after the initial
immunization, whereas the first signs of arthritis as measured by
arthritis severity scores of 2 in the MST1-deficient mice developed
only on day 42. The mean cumulative arthritis scores in the
wild-type control group remained higher than 3.5 throughout the
experiment. The severity of arthritis was significantly decreased
in the MST1-deficient mice with a maximum mean cumulative arthritis
score of 1.8 and a maximum mean .DELTA. of ankle thickness of 70
.mu.m reached by day 49, whereas arthritis in the control group
began as earlier as at day 25 post-immunization, with a mean
cumulative arthritis score of 3.5 at day 28, a maximum mean
cumulative arthritis score of 6.2 at day 46 and a maximum mean
.DELTA. of ankle thickness of 485 .mu.m reached by day 32. This
significant decrease in the severity of arthritis in the MST1-/-
mice persisted until day 53 after the first immunization.
[0090] In the Con-A hepatitis model, -/- and control wild-type
(C57Bl/6-Albino/129SvEv) mice were tested. Analysis of Con
A-induced cytokine production revealed that the serum levels of
IL-12, TNF-.alpha., and MCP-1 were significantly decreased in
MST-1-/- mice compared to wild-type littermates. Similarly, release
of liver damage enzymes (ALT and AST) in MST-1-/- mice was lower in
MST-1-deficient than wild-type mice in two independent
experiments.
6.5. General Synthetic Method A and Synthesis of
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-N-cy-
clopropylbenzenesulfonamide
[0091] The captioned compound was prepared by general method A,
represented below:
##STR00015##
[0092] In particular, a room temperature slurry of
3-bromo-2-nitropyridine (6.4 g, 31.57 mmol),
1-oxo-1,2,3,4-tetrahydroisoquinolin-6-ylboronic acid (6.63 g, 34.73
mmol), PdCl.sub.2dppf-DCM (1.031 g, 1.26 mmol) and Cs.sub.2CO.sub.3
(15.43 g, 47.35 mmol) in DMF was sonicated for 20 min while
iteratively evacuating the reaction mixture and back-filling with
dry N.sub.2 to effect degassing. The degassed slurry was then
heated to 100.degree. C. and the reaction was monitored by LC/MS.
At completion, the reaction was filtered then concentrated under
vacuum. The resulting crude material was redissolved in MeOH,
treated with silica gel (15 g) then concentrated under vacuum to
dryness, then azeotroped with toluene to remove trace methanol. The
powder was slurried in DCM, then flashed over a tall plug of
silica, eluting with 4-10% MeOH:DCM. Pure fractions were combined
and concentrated. The resulting brown solid was slurried in DCM (15
mL), then filtered to remove colored impurities to provide
6-(2-nitropyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one as a tan
solid (7.4 g, 87% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 8.63 (d, J=4.5 Hz, 1H), 8.23 (d, J=7.8 Hz, 1H), 8.04
(br. s., 1H), 7.86-7.98 (m, 2H), 7.38 (s, 1H), 7.35 (d, J=9.0 Hz,
1H), 3.37-3.44 (m, 2H), 2.95 (t, J=6.3 Hz, 2H); .sup.13C NMR (101
MHz, DMSO-d.sub.6) .delta. ppm 163.78, 156.77, 148.11, 142.04,
139.99, 137.26, 129.76, 128.46, 128.33, 127.63, 127.02, 126.19,
27.52; MS (EI) m/z: 270 [M+H].sup.+; HRMS calcd for
C.sub.14H.sub.12N.sub.3O.sub.3 [M+H].sup.+ 270.0879, found
270.0870.
[0093] A solution of
6-(2-nitropyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one (7.4 g,
27.50 mmol) in methanol (100 mL) and DMF (200 mL) was degassed by
iteratively evacuating the reaction flask and backfilling with dry
nitrogen (5.times.). To the degassed solution was added 10% Pd on
carbon (Degussa type, 50 wt % water, 1.5 g). The reaction was
charged with hydrogen (.about.1 atm) by iteratively evacuating the
reaction vessel and back-filling with hydrogen (3.times.). The
reaction was maintained at room temperature with vigorous stirring
and monitored by LC/MS. At completion, the reaction was degassed
and purged with nitrogen as before, then heated to 70.degree. C.
and filtered while hot over a pad of celite. The pad was rinsed
with DMF (100 mL) that was heated to 120.degree. C. The combined
organics were concentrated under vacuum, and azeotroped with
toluene (2.times.150 mL) to remove residual DMF.
[0094] The obtained solid was treated with methanol (200 mL) then
heated and sonicated to provide milky slurry. The slurry was cooled
to 0.degree. C. and treated with bromine (1.48 mL, 28.88 mmol) by
careful addition. The reaction was monitored by LC/MS, and, after
completion (15 min) was concentrated under vacuum. The resulting
material was stored under high vacuum overnight to remove trace
bromine to provide
6-(2-amino-5-bromopyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one
hydrobromide as a brown solid (11.16 g, 100% yield), which was used
without further purification. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 8.31 (d, J=2.3 Hz, 1H), 8.03 (br. s., 1H), 8.00 (d,
J=2.0 Hz, 1H), 7.95 (d, J=8.3 Hz, 1H), 7.45 (s, 1H), 7.44 (s, 2H),
3.38-3.45 (m, 2H), 2.96 (t, J=6.6 Hz, 2H); .sup.13C NMR (101 MHz,
DMSO-d.sub.6) .delta. ppm 163.89, 151.88, 144.15, 140.08, 137.31,
136.47, 130.22, 128.00, 127.79, 127.10, 126.28, 104.76, 48.52,
27.70; MS (EI) m/z: 318, 320 [M+H].sup.+; HRMS calcd for
C.sub.14H.sub.12BrN.sub.3O [M+H].sup.+ 318.0242, found
318.0234.
[0095] A slurry of the aryl bromide
6-(2-amino-5-bromopyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one
hydrobromide (6.50 g, 16.25 mmol),
4-(N-cyclopropylsulfamoyl)phenylboronic acid (6.57 g, 17.88 mmol),
Pd(PPh.sub.3).sub.4 (565 mg, 0.49 mmol) and 2.0 M aq
Na.sub.2CO.sub.3 (4.3 g, 48.75 mmol) in n-BuOH (180 mL) was
degassed by bubbling a stream of anhydrous nitrogen for 10 min. The
reaction mixture was then heated to 100.degree. C. for 3 h. At
completion, the hot reaction was diluted with DMF (300 mL) and
heated to 125.degree. C. before filtering the hot slurry over a pad
of celite dampened with hot DMF. The organics were cooled to room
temperature, then treated with silica gel (40 g) and the solvent
was removed under vacuum. The dry powder was slurried in DCM and
loaded on top of a tall plug of silica gel slurried in DCM and the
system was eluted with 10-20% MeOH. The product containing
fractions were concentrated, and the resulting solid was slurried
in hot DMF, then filtered. The cake was rinsed with MeOH, then
dried under vacuum to provide
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3--
yl)-N-cyclopropylbenzenesulfonamide as a white powder (5.3 g, 75%
yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.62 (d,
J=2.0 Hz, 1H), 8.34 (d, J=2.0 Hz, 1H), 8.13 (br. s., 2H), 8.07 (br.
s., 1H), 8.04 (d, J=8.6 Hz, 2H), 7.99 (d, J=8.3 Hz, 1H), 7.88 (d,
J=8.3 Hz, 2H), 7.57 (s, 1H), 7.56 (s, 2H), 3.43 (t, J=5.7 Hz, 2H),
2.99 (t, J=6.3 Hz, 2H), 2.12 (tt, J=6.8, 3.5 Hz, 1H), 0.44-0.53 (m,
2H), 0.36-0.44 (m, 2H); MS (EI) m/z: 435 [M+H].sup.+.
6.6. General Synthetic Method B and Synthesis of
5-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-N-et-
hylthiophene-2-sulfonamide
[0096] The captioned compound was prepared by general method B,
represented below:
##STR00016##
[0097] A solution of
6-(2-amino-5-bromopyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one
hydrobromide (2.00 g, 5.0 mmol), obtained as described in Example
5.2, was cooled to 0.degree. C. in DCM (50 mL) and was slowly
treated with a 1 N aq. NaHCO.sub.3 solution (25 mL). The cooled
solution was stirred vigorously for 5 min, and then the layers were
separated. The organic layer was dried over MgSO.sub.4, filtered
and concentrated then dried thoroughly under vacuum. The resulting
tan solid (1.54 g, 5.0 mmol) was dissolved in THF (500 mL), cooled
to -78.degree. C. and treated with a 1.6M MeLi solution in
Et.sub.2O (10.3 mL, 16.5 mL). The reaction was maintained for 15
min, before the dropwise addition of 1.7M t-BuLi (8.8 mL, 14.96
mmol) over 10 min. The cooled solution was stirred vigorously for
an additional 45 min before the addition of triisopropylborate (5.2
mL, 22.5 mmol). The reaction was allowed to warm to room
temperature for 30 min before quenching with sat. aq. NH.sub.4Cl (1
mL) followed by water (5 mL). The resulting precipitate was
filtered off and washed with cold water, then dried overnight under
vacuum to provide
(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)boronic
acid (1.10 g, 78% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 8.35 (d, J=2.3 Hz, 1H), 7.97 (br. s., 1H), 7.92 (d,
J=7.8 Hz, 1H), 7.76 (t, J=5.6 Hz, 1H), 7.67 (d, J=2.5 Hz, 1H), 7.50
(d, J=3.8 Hz, 1H), 7.43-7.47 (m, 2H), 6.21 (s, 2H), 2.96 (t, J=6.4
Hz, 2H), 2.85-2.92 (m, 2H); MS (EI) m/z: 284 [M+H].sup.+.
[0098] A mixture of
(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)boronic
acid (291 mg, 1.02 mmol), 5-bromo-N-ethylthiophene-2-sulfonamide
(278 mg, 1.02 mmol), and PdCl.sub.2(PPh.sub.3).sub.2 (35 mg, 0.05
mmol) in n-BuOH (3 mL) was treated with 2.0 M aq. Na.sub.2CO.sub.3
solution (1 mL) then microwave heated at 140.degree. C. for 6 min.
The reaction was then filtered, and purified by HPLC to provide
5-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-N-et-
hylthiophene-2-sulfonamide as a white solid after preparatory HPLC
purification. .sup.1H NMR (300 MHz, MeOH) .delta. ppm 8.31 (d,
J=2.3 Hz, 1H), 8.07 (d, J=8.0 Hz, 1H), 7.72 (d, J=2.3 Hz, 1H), 7.54
(dd, J=3.8, 2.1 Hz, 1H), 7.52 (d, J=1.7 Hz, 1H), 7.48 (s, 1H), 7.34
(d, J=3.8 Hz, 1H), 3.57 (dd, J=13.4, 6.5 Hz, 1H), 2.93-3.15 (m,
2H), 1.85-2.03 (m, 3H), 1.13 (dd, J=14.5, 7.2 Hz, 3H); MS (EI) m/z:
429 [M+H].sup.+.
6.7. General Synthetic Method C and Synthesis of
6-(2-amino-5-(4-(ethylsulfonyl)phenyl)pyridin-3-yl)-3,4-dihydroisoquinoli-
n-1(2H)-one
[0099] The captioned compound was prepared by General Method C,
represented below:
##STR00017##
[0100] In particular, a slurry of 2-amino-5-bromopridine (1.24 g,
4.67 mmol), 4-(ethylsulfonyl)phenylboronic acid (1.10 g, 5.14
mmol), and Pd(PPh.sub.3).sub.4 (0.16 g, 0.13 mmol) in n-BuOH (20
mL) was treated with 2.0 M aq. Na.sub.2CO.sub.3 (4.5 mL). The
reaction was sparged with dry N.sub.2 under sonication for 10 min
to degas, and then refluxed for 16 h at 100.degree. C. At
completion, the reaction was cooled to room temperature and
concentrated to dryness. The residue was partitioned between EtOAc
(50 mL) and water (30 mL). The layers were separated and the
aqueous layer was extracted with EtOAc (2.times.50 mL). The
combined organics were washed with brine (20 mL), dried over
Na.sub.2SO.sub.4 and concentrated. The crude material was purified
by flash chromatography over silica gel (30-100% EtOAc:hexane
eluent) to provide 5-(4-(ethylsulfonyl)phenyl)pyridin-2-amine as a
white solid (0.57 g, 47% yield). .sup.1H NMR (300 MHz, MeOH)
.delta. ppm 8.27-8.31 (m, 1H), 7.91-7.98 (m, 2H), 7.79-7.89 (m,
3H), 6.71 (dd, J=8.7, 0.7 Hz, 1H), 3.30-3.35 (m, 2H), 3.24 (q,
J=7.4 Hz, 2H), 1.25 (t, J=7.4 Hz, 3H); MS (EI) m/z: 263
[M+H].sup.+.
[0101] A room temperature solution of
5-(4-(ethylsulfonyl)phenyl)pyridin-2-amine (0.56 g, 2.14 mmol) in
DCM (15 mL) was treated with NBS (0.42 g, 2.35 mmol) and the
reaction was maintained for 1.5 h. At completion, mixture was
washed with sat. aq. Na.sub.2S.sub.2O.sub.4 solution (10 mL),
followed by sat. aq. NaHCO.sub.3 (10 mL) and brine (5 mL). The
organic layer was dried over Na.sub.2SO.sub.4, then filtered and
concentrated to 3-bromo-5-(4-(ethylsulfonyl)phenyl)pyridin-2-amine
(560 mg, 77% yield) as an orange solid. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. ppm 8.37-8.49 (m, 1H), 8.15-8.25 (m, 1H),
7.90-7.96 (m, 2H), 7.85-7.90 (m, 2H), 6.60 (br. s, 2H), 3.34 (q,
J=7.4 Hz, 2H), 1.11 (t, J=7.4 Hz, 3H); MS (EI) m/z: 343, 341
[M+H].sup.+.
[0102] A mixture of the
3-bromo-5-(4-(ethylsulfonyl)phenyl)pyridin-2-amine (100 mg, 0.29
mmol), 1-oxo-1,2,3,4-tetrahydroisoquinolin-6-ylboronic acid (75 mg,
0.29 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (7.0 mg, 0.01 mmol) and
Na.sub.2CO.sub.3 in 3:1 MeCN:H.sub.2O was microwave heated at
150.degree. C. for 3 min. At completion, the reaction mixture was
filtered, then purified by preparatory HPLC to provide
6-(2-amino-5-(4-(ethylsulfonyl)phenyl)pyridin-3-yl)-3,4-dihydroisoquinoli-
n-1(2H)-one as a pale yellow solid (48 mg, 41% yield) after reverse
phase preparatory HPLC purification. .sup.1H NMR (400 MHz, MeOD)
.delta. ppm 8.37 (d, J=2.5 Hz, 1H), 8.06 (d, J=8.0 Hz, 1H), 7.94
(d, J=8.5 Hz, 2H), 7.89 (d, J=8.5 Hz, 2H), 7.81 (d, J=2.5 Hz, 1H),
7.53 (d, J=7.8 Hz, 1H), 7.49 (s, 1H), 3.56 (t, J=6.7 Hz, 2H),
3.19-3.27 (m, 2H), 3.07 (q, J=6.5 Hz, 2H), 1.25 (t, J=7.3 Hz, 3H);
MS (EI) m/z: 408 [M+H].sup.+.
6.8. Synthesis of
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-N,N--
diethylbenzenesulfonamide
##STR00018##
[0104] General method A was applied to
4-(N,N-diethylsulfamoyl)phenylboronic acid, providing
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-N,N--
diethylbenzenesulfonamide after preparatory HPLC purification.
.sup.1H NMR (400 MHz, MeOD) .delta. ppm 8.33 (d, J=2.3 Hz, 1H),
8.06 (d, J=8.0 Hz, 1H), 7.85 (m, J=8.5 Hz, 2H), 7.76-7.82 (m, 3H),
7.53 (dd, J=8.0, 1.5 Hz, 1H), 7.48 (s, 1H), 4.10 (q, J=7.1 Hz, 4H),
3.56 (t, J=6.7 Hz, 2H), 3.26 (q, J=7.0 Hz, 6H), 3.07 (t, J=6.7 Hz,
2H); MS (EI) m/z: 451 [M+H].sup.+.
6.9. Synthesis of
4-(6-amino-5-(1-hydroxyisoquinolin-6-yl)pyridin-3-yl)-N-ethylbenzenesulfo-
namide
##STR00019##
[0106] General method C was applied to
4-(N-ethylsulfamoyl)phenylboronic acid and
6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoquinolin-1-ol to
provide
4-(6-amino-5-(1-hydroxyisoquinolin-6-yl)pyridin-3-yl)-N-ethylbenzenesulfo-
namide. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 11.31 (d,
J=5.3 Hz, 1H), 8.47 (d, J=2.3 Hz, 1H), 8.30 (d, J=8.3 Hz, 1H), 8.12
(s, 1H), 7.96 (d, J=8.3 Hz, 2H), 7.80-7.86 (m, 3H), 7.64 (dd,
J=8.3, 1.5 Hz, 1H), 7.58 (dd, J=11.3, 5.5 Hz, 1H), 7.23 (dd,
J=12.8, 6.8 Hz, 1H), 6.60 (d, J=7.0 Hz, 1H), 2.75-2.84 (m, 2H),
0.98 (t, J=7.3 Hz, 3H); MS (EI) m/z: 421 [M+H].sup.+.
6.10. Synthesis of
4-(2-amino-5-(4-(N-cyclopropylsulfamoyl)phenyl)pyridin-3-yl)benzamide
##STR00020##
[0108] General method B was applied to
4-bromo-N-cyclopropylbenzenesulfonamide (41 mg, 0.15 mmol),
6-amino-5-(4-carbamoylphenyl)pyridin-3-ylboronic acid (53 mg, 0.15
mmol), to provide give
4-(2-amino-5-(4-(N-cyclopropylsulfamoyl)phenyl)pyridin-3-yl)benzamide
as a white solid (29 mg, 41% yield). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.42 (d, J=2.3 Hz, 1H), 8.03 (br. s.,
1H), 7.98 (d, J=8.3 Hz, 2H), 7.90 (d, J=8.5 Hz, 2H), 7.87 (d, J=2.5
Hz, 1H), 7.80 (d, J=8.5 Hz, 2H), 7.76 (d, J=1.5 Hz, 1H), 7.61 (d,
J=8.3 Hz, 2H), 7.37 (br. s., 1H), 6.06 (br. s., 2H), 2.06-2.13 (m,
1H), 0.42-0.50 (m, 2H), 0.35-0.42 (m, 2H); MS (EI) m/z: 409
[M+H].sup.+.
6.11. Synthesis of
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-N-et-
hylbenzenesulfonamide
##STR00021##
[0110] General method B was applied to
4-(6-amino-5-bromopyridin-3-yl)-N-ethylbenzenesulfonamide (50 mg,
0.141 mmol) and 4-(N-ethylsulfamoyl)phenylboronic acid (64 mg,
0.336 mmol) to provide
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3--
yl)-N-ethylbenzenesulfonamide as a white solid (28 mg, 47% yield)
after purification by reverse phase preparatory HPLC. .sup.1H NMR
(400 MHz, METHANOL-d.sub.4) .delta. ppm 8.35 (d, J=2.3 Hz, 1H),
8.08 (d, J=7.8 Hz, 1H), 7.90 (d, J=8.5 Hz, 2H), 7.79-7.84 (m, 3H),
7.48-7.58 (m, 2H), 3.57 (t, J=6.8 Hz, 2H), 3.08 (t, J=6.8 Hz, 2H),
2.93 (q, J=7.8 Hz, 2H), 1.08 (t, J=7.3 Hz, 3H); MS (EI) m/z: 423
[M+H].sup.+.
6.12. Synthesis of
2'-amino-5'-(4-(N-cyclopropylsulfamoyl)phenyl)-2,3'-bipyridine-5-carboxam-
ide
##STR00022##
[0112] General method B was applied to
4-(6-amino-5-bromopyridin-3-yl)-N-cyclopropylbenzenesulfonamide
(112 mg, 0.304 mmol) and 5-carbamoylpyridin-2-ylboronic acid (75
mg, 0.453 mmol) to provide
2'-amino-5'-(4-(N-cyclopropylsulfamoyl)phenyl)-[2,3'-bipyridine]-5-carbox-
amide as a white solid (35 mg, 28% yield) after purification by
reverse phase preparatory HPLC. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 9.07-9.16 (m, 1H), 8.53 (d, J=2.3 Hz, 1H), 8.47 (d,
J=2.3 Hz, 1H), 8.32 (dd, J=14.1, 10.8 Hz, 2H), 8.21 (br. s., 1H),
8.01 (d, J=8.5 Hz, 2H), 7.91 (d, J=2.5 Hz, 1H), 7.85 (d, J=8.3 Hz,
2H), 7.79 (br. s., 2H), 7.61 (br. s., 1H), 2.06-2.18 (m, 1H),
0.34-0.55 (m, 4H); MS (EI) m/z: 410 [M+H].sup.+.
6.13. Synthesis of
6-(2-amino-5-(1,1-dioxido-2,3-dihydrobenzo[d]isothiazol-5-yl)pyridin-3-yl-
)-3,4-dihydroisoquinolin-1(2H)-one
[0113] The captioned compound was prepared by the method shown
below.
##STR00023##
[0114] To a solution of 4-bromo-2-methylbenzenesulfonamide (496 mg,
2.0 mmol) in toluene (10 mL) at room temperature was added AIBN
(383 mg, 2.20 mmol) and N-bromosuccinimide (389 mg, 2.20 mmol). The
mixture was heated to 90.degree. C. for 2 h. At completion, the
reaction was cooled to room temperature. Water (20 mL) was added
and the layers were separated. The aqueous layer was extracted with
EtOAc (3.times.20 mL). The combined organics were washed with brine
(20 mL), dried over Na.sub.2SO.sub.4, and concentrated under
reduced pressure to provide a pale yellow solid (170 mg, 26% yield)
which was taken up in DMF (5 mL) and treated with K.sub.2CO.sub.3
(196 mg, 2.0 mmol). The reaction was stirred at room temperature
overnight. After filtration and concentration, the residue was
purified by flash chromatography over silica gel (4-10% MeOH/DCM
eluent) to afford 5-bromo-2,3-dihydrobenzo[d]isothiazole
1,1-dioxide as a white solid (108 mg, 85% yield).
[0115] General method B was applied to
5-bromo-2,3-dihydrobenzo[d]isothiazole 1,1-dioxide (108 mg, 0.44
mmol) and (140 mg, 0.48 mmol) to provide
6-(2-amino-5-(1,1-dioxido-2,3-dihydrobenzo[d]isothiazol-5-yl)pyridin-3-yl-
)-3,4-dihydroisoquinolin-1(2H)-one after purification by reverse
phase preparatory HPLC. .sup.1H NMR (400 MHz, METHANOL-d.sub.4)
.delta. ppm 8.35 (br. s, 1H), 8.05-8.14 (m, 2H), 7.86 (s, 2H), 7.82
(s, 1H), 7.57 (s, 1H), 7.53 (s, 1H), 4.52 (s, 2H), 3.57 (t, J=6.8
Hz, 2H), 3.09 (t, J=6.5 Hz, 2H); MS (EI) m/z=407.5 [M+H].sup.+
6.14. General Method D and Synthesis of
6'-amino-N-cyclopropyl-N-methyl-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
-yl)-[2,3-bipyridine]-5-sulfonamide
[0116] The captioned compound was prepared by general method D,
represented below:
##STR00024##
[0117] 6-chloro-N-ethyl-N-methylpyridine-3-sulfonamide (2.34 g,
10.0 mmol), tetramethyl tin (1.08 g, 10.00 mmol) and
Pd(PPh.sub.3).sub.4 (572 mg, 0.50 mmol) were mixed in dioxane (5
mL). After degassing, the mixture was heated to 90.degree. C. At
completion, the reaction was cooled to room temperature and
concentrated. The residue was then flashed over alumina (20-100%
chloroform/hexane eluent) to afford
N-ethyl-N-methyl-6-(trimethylstannyl)pyridine-3-sulfonamide (1.46
g, 40% yield).
[0118] A mixture of
N-ethyl-N-methyl-6-(trimethylstannyl)pyridine-3-sulfonamide (225
mg, 0.6 mmol) and
6-(2-amino-5-bromopyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-on- e
(190 mg, 0.6 mmol) and Pd(PPh.sub.3).sub.4 (34 mg, 0.03 mmol) in
dioxane (3 mL) was degassed and heated to 90.degree. C. At
completion, the reaction was cooled to room temperature and
concentrated. The residue was purified by flash chromatography over
silica gel to provide
6'-amino-N-ethyl-N-methyl-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-[-
2,3'-bipyridine]-5-sulfonamide as a yellow solid (134 mg, 50%
yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.88 (dd,
J=17.9, 2.1 Hz, 2H), 8.17-8.22 (m, 1H), 8.10-8.16 (m, 2H), 7.94 (d,
J=7.8 Hz, 2H), 7.43-7.51 (m, 2H), 3.41 (s, 2H), 3.30 (s, 3H), 2.97
(t, J=6.4 Hz, 2H), 0.86 (t, J=7.3 Hz, 3H); MS (EI) m/z=438.5
[M+1].sup.+.
6.15. Synthesis of
6-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-et-
hyl-N,4-dimethylpyridine-3-sulfonamide
##STR00025##
[0120] General method D was applied to
N-ethyl-N,4-dimethyl-6-(trimethylstannyl)pyridine-3-sulfonamide
(233 mg, 0.6 mmol) and
6-(2-amino-5-bromopyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one
(190 mg, 0.6 mmol) to give
6-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-et-
hyl-N,4-dimethylpyridine-3-sulfonamide as a yellow solid (41 mg,
33% yield) after purification by preparatory reverse phase HPLC.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.96 (s, 1H), 8.84
(s, 1H), 8.14 (s, 1H), 7.93-8.04 (m, 2H), 7.73 (d, J=8.0 Hz, 1H),
7.70 (s, 1H), 3.39-3.47 (m, 2H), 3.23 (q, J=7.3 Hz, 2H), 3.01 (t,
J=6.5 Hz, 2H), 2.81 (s, 3H), 2.61 (s, 3H), 1.09 (t, J=7.2 Hz, 3H);
MS (EI) m/z=453 [M+1].sup.+.
6.16. Synthesis of
6'-amino-N-ethyl-N-methyl-5'-(6-oxo-5,6-dihydro-4H-thieno[2,3-c]pyrrol-2--
yl)[2,3'-bipyridine]-5-sulfonamide
[0121] The captioned compound was prepared by the method shown
below:
##STR00026##
[0122] To a solution of methyl 3-methylthiophene-2-carboxylate
(10.0 g, 64.0 mmol) in CCl.sub.4 (100 mL) was added AIBN (180 mg,
1.1 mmol) and N-bromosuccinimide (11.4 g, 64.0 mmol). The reaction
mixture was refluxed for 10 min, and then an additional portion of
AIBN (460 mg, 2.8 mmol) was added. This reaction was heated
overnight at 90.degree. C. under a reflux condenser. At completion,
the reaction was filtered over a short plug of silica gel (100% DCM
eluent). The filtrate was concentrated and purified by flash
chromatography over silica gel (2% EtOAc/hexane eluent) to afford
methyl 3-(bromomethyl)thiophene-2-carboxylate (9.5 g, 63% yield) as
a white solid. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) .delta. ppm
7.66 (d, J=5.0 Hz, 1H), 7.23 (d, J=5.0 Hz, 1H), 4.95 (s, 2H), 3.89
(s, 3H); MS (EI) m/z=236.1 [M+1].sup.+.
[0123] To a solution of 3-(bromomethyl)thiophene-2-carboxylate (9.3
g, 39.6 mmol) in DMF (150 mL) was added 7.0 N NH.sub.3 in MeOH (150
mL). After stirring at room temperature for 1 h, the reaction was
concentrated and purified by flash chromatography over silica gel
(5%-10% MeOH/DCM) to provide methyl
3-(aminomethyl)thiophene-2-carboxylate (4.86 g, 72% yield) as a
white solid. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) .delta. ppm
7.83 (d, J=5.0 Hz, 1H), 7.28 (d, J=5.0 Hz, 1H), 4.45 (s, 2H), 3.94
(s, 3H); MS (EI) m/z=172.2 [M+1].sup.+.
[0124] A mixture of methyl 3-(aminomethyl)thiophene-2-carboxylate
(4.5 g, 26.3 mmol) and K.sub.2CO.sub.3 (3.64 g, 26.3 mmol) in 1:1
MeOH:EtOH (600 mL) was heated overnight under reflux. The reaction
was concentrated and purified by flash chromatography over silica
gel (50-100% EtOAc/hexane) to yield
4H-thieno[2,3-c]pyrrol-6(5H)-one (2.2 g, 60% yield) as a white
solid. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) .delta. ppm 7.87 (d,
J=4.8 Hz, 1H), 7.17 (d, J=4.8 Hz, 1H), 4.39 (s, 2H); MS (EI)
m/z=140.2 [M+1].sup.+.
[0125] A solution of 4H-thieno[2,3-c]pyrrol-6(5H)-one (1.0 g, 7.2
mmol) in acetic acid (9 mL) and water (7 mL) was cooled to
0.degree. C. Bromine (407 .mu.L, 7.92 mmol) was added and the
reaction was maintained at 0.degree. C. for 1.5 h. At completion,
water (30 mL) was added and the mixture was and extracted with
EtOAc (2.times.50 mL). The combined organics were washed with 5%
aq. Na.sub.2SO.sub.3 (30 mL), sat. aq. NaHCO.sub.3 (30 mL) and
brine (30 mL), then dried over Na.sub.2SO.sub.4, filtered,
concentrated. The crude material was purified by flash
chromatography over silica gel (5% MeOH/DCM) to yield
2-bromo-4H-thieno[2,3-c]pyrrol-6(5H)-one (1.3 g, 83% yield) as a
white solid. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) .delta. ppm
7.28 (s, 1H), 4.39 (s, 3H); MS (EI) m/z=140.2 [M+1].sup.+.
[0126] A solution of tert-butyl
(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)carba
mate (736 mg, 2.3 mmol), 2-bromo-4H-thieno[2,3-c]pyrrol-6(5H)-one
(500 mg, 2.3 mmol) and Pd(PPh.sub.3).sub.4 (40 mg, 0.035 mmol) in
n-BuOH (5 mL) and 2.0 M Na.sub.2CO.sub.3 (4.6 mL) was degassed and
the resulting reaction mixture was refluxed at 110.degree. C. for 2
h. Upon completion, the reaction was cooled to room temperature,
filtered, and washed with MeOH (10 mL) and water (2.times.10 mL).
The solid was dried under high vacuum yielding
2-(2-aminopyridin-3-yl)-4H-thieno[2,3-c]pyrrol-6(5H)-one (383 mg,
72% yield) as a light brown solid. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.40 (br. s., 1H), 8.00 (dd, J=5.0, 1.8
Hz, 1H), 7.52 (dd, J=7.3, 1.8 Hz, 1H), 7.36 (s, 1H), 6.66 (dd,
J=7.4, 4.9 Hz, 1H), 5.99 (s, 2H), 4.32 (s, 2H); MS (EI) m/z=232.3
[M+1].sup.+.
[0127] To a solution of
2-(2-aminopyridin-3-yl)-4H-thieno[2,3-c]pyrrol-6(5H)-one (350 mg,
1.52 mmol) in DMF (5 mL) was added NBS (297 mg, 1.67 mmol). At
completion, the reaction was concentrated and the residue was taken
up in 20% MeOH/DCM (10 mL) then filtered and concentrated. After
drying under high vacuum,
2-(2-aminopyridin-3-yl)-4H-thieno[2,3-c]pyrrol-6(5H)-one (313 mg,
66% yield) was isolated as a brown solid. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.44 (br. s., 1H), 8.08 (d, J=2.5 Hz,
1H), 7.67 (d, J=2.3 Hz, 1H), 7.39 (s, 1H), 6.27 (br. s., 2H), 4.32
(s, 2H); MS (EI) m/z=311.2 [M+1].sup.+.
[0128] General method D was applied to
N-ethyl-N-methyl-6-(trimethylstannyl)pyridine-3-sulfonamide (218
mg, 0.6 mmol) and
2-(2-amino-5-bromopyridin-3-yl)-4H-thieno[2,3-c]pyrrol-6(5H)-on- e
(186 mg, 0.6 mmol) to give
6'-amino-N-ethyl-N-methyl-5'-(6-oxo-5,6-dihydro-4H-thieno[2,3-c]pyrrol-2--
yl)-[2,3'-bipyridine]-5-sulfonamide as a yellow solid (94 mg, 38%
yield) after purification by preparatory reverse phase HPLC.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.91 (d, J=1.5 Hz,
1H), 8.87 (d, J=2.3 Hz, 1H), 8.44 (s, 1H), 8.26 (d, J=2.3 Hz, 1H),
8.10-8.20 (m, 2H), 7.43 (s, 1H), 6.65 (s, 2H), 4.35 (s, 2H), 3.30
(s, 4H), 3.09 (dd, J=14.3, 7.0 Hz, 2H), 2.72 (s, 3H), 1.06 (t,
J=7.0 Hz, 3H); MS (EI) m/z=430 [M+1].sup.+.
6.17. Synthesis of
1-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)ph-
enyl)cyclopentanecarbonitrile
##STR00027##
[0130] General method A was applied to
1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)cyclopentanecarb-
onitrile (149 mg, 0.5 mmol) and
6-(3-amino-6-bromopyrazin-2-yl)-3,4-dihydroisoquinolin-1(2H)-one
(180 mg, 0.5 mmol) to give
1-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)ph-
enyl)cyclopentanecarbonitrile as a yellow solid (89 mg, 0.22 mmol,
44% yield) after purification by preparatory reverse phase HPLC.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.60 (s, 1H), 8.02
(d, J=8.3 Hz, 3H), 7.96 (d, J=8.0 Hz, 1H), 7.72-7.78 (m, 1H), 7.70
(s, 1H), 7.57 (m, J=8.5 Hz, 3H), 3.38-3.45 (m, 2H), 2.99 (t, J=6.4
Hz, 2H), 2.37-2.46 (m, 2H), 2.03-2.16 (m, 2H), 1.84-1.94 (m, 4H);
MS (EI) m/z=410.5 [M+1].sup.+.
6.18. Synthesis of
6'-amino-N-(cyclopropylmethyl)-N-methyl-5'-(1-oxo-1,2,3,4-tetrahydroisoqu-
inolin-6-yl)-[2,3'-bipyridine]-5-sulfonamide
##STR00028##
[0132] General method D was applied to
N-(cyclopropylmethyl)-N-methyl-6-(trimethylstannyl)pyridine-3-sulfonamide
(233 mg, 0.6 mmol) and
6-(2-amino-5-bromopyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one
(190 mg, 0.6 mmol) to give
6'-amino-N-(cyclopropylmethyl)-N-methyl-5'-(1-oxo-1,2,3,4-tetrahydroisoqu-
inolin-6-yl)-[2,3'-bipyridine]-5-sulfonamide as a yellow solid (127
mg, 0.27 mmol, 46% yield) after purification by reverse phase
preparatory HPLC. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
8.87 (d, J=1.5 Hz, 1H), 8.84 (d, J=2.3 Hz, 1H), 8.05-8.19 (m, 3H),
7.90-7.98 (m, 2H), 7.43-7.50 (m, 2H), 6.34 (s, 2H), 3.38-3.45 (m,
2H), 2.97 (t, J=1.0 Hz, 2H), 2.91 (d, J=6.8 Hz, 2H), 2.80 (s, 3H),
0.85-0.96 (m, 1H), 0.43-0.51 (m, 2H), 0.16-0.21 (m, 2H); MS (EI)
m/z=464.5 [M+1].sup.+.
6.19. Synthesis of
6'-amino-N-ethyl-N,4-dimethyl-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-y-
l)-[2,3'-bipyridine]-5-sulfonamide
##STR00029##
[0134] General method D was applied to
N-ethyl-N,4-dimethyl-6-(trimethylstannyl)pyridine-3-sulfonamide
(226 mg, 0.6 mmol) and
6-(2-amino-5-bromopyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one
(190 mg, 0.6 mmol) to give
6'-amino-N-ethyl-N,4-dimethyl-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-y-
l)-[2,3'-bipyridine]-5-sulfonamide as a yellow solid (41 mg, 33%
yield) after purification by reverse phase preparatory HPLC.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.84-8.88 (m, 2H),
8.56 (s, 1H), 8.23 (s, 1H), 8.06 (br. s., 1H), 8.00 (d, J=7.8 Hz,
1H), 7.49-7.56 (m, 2H), 3.38-3.48 (m, 2H), 3.23 (q, J=7.0 Hz, 2H),
3.00 (t, J=6.3 Hz, 2H), 2.81 (s, 3H), 2.61 (s, 3H), 1.09 (t, J=7.0
Hz, 3H); MS (EI) m/z=452.5[M+1].sup.+.
6.20. General Method E and Synthesis of
(S)-4-(6-amino-5-(3-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e]-[1,4]diaz-
epin-8-yl)pyridin-3-yl)-N-cyclopropylbenzenesulfonamide
[0135] The captioned compound was prepared by general method E,
represented below:
##STR00030##
[0136] A slurry of methyl 4-bromo-2-fluorobenzoate (4.66 mg, 20.0
mmol) (S)-propane-1,2-diamine (2.22 g, 30 mmol) and K.sub.2CO.sub.3
(2.94 g, 3.0 mmol) was microwave heated at 140.degree. C. for 15
min. After filtration and concentration, the residue was purified
by flash chromatography on silica (2-10% MeOH/DCM eluent) to
provide (S)-methyl 2-((2-aminopropyl)amino)-4-bromobenzoate (2.66
g, 9.20 mmol) as a white oily solid. This material was dissolved in
methanol (160 mL) and treated with NaOH (13.5 g, 200.0 mmol), then
heated to 70.degree. C. overnight. At completion, the reaction was
neutralized to pH .about.7 with conc. aq. HCl and the solid was
filtered and washed with MeOH. The combined filtrate was
concentrated to provide (S)-2-((2-aminopropyl)amino)-4-bromobenzoic
acid as a white solid. This solid was used without further
purification (2.5 g, 46% yield, 2 steps).
[0137] To a solution of the crude
(S)-2-((2-aminopropyl)amino)-4-bromobenzoic acid (544 mg, 2.0 mmol)
and Et.sub.3N (731 mg, 7.24 mmol) in DMF was added HATU (829 mg,
2.18 mmol). After 30 min, the reaction mixture was concentrated and
purified by flash chromatography on silica gel (2-10% MeOH/DCM
eluent) to provide
(S)-8-bromo-3-methyl-3,4-dihydro-1H-benzo[e][1,4]diazepin-5(2H)-one
as a white oily solid (464 mg, 91% yield). This material was
dissolved in dioxane (20 mL), then treated with Pd.sub.2(dba).sub.3
(83 mg, 0.09 mmol), PCy.sub.3 (121 mg, 0.43 mmol), diboron pinacol
ester (595 mg, 2.35 mmol) and KOAc (532 mg, 5.43 mmol), then heated
at 90.degree. C. for 2 h. At completion, the reaction was cooled to
room temperature, then filtered and concentrated. The crude
material was flashed over silica gel (2-10% MeOH/DCM eluent) to
provide
(S)-3-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro--
1H-benzo[e][1,4]diazepin-5(2H)-one (527 mg, 96% yield) as a yellow
solid.
[0138] General method C was applied to
(S)-3-methyl-8-(4,4,5,5-tetra
methyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[e][1,4]diazepin-5(2H-
)-one (303 mg, 0.7 mmol) and
4-(6-amino-5-bromopyridin-3-yl)-N-cyclopropylbenzenesulfonamide to
give
(S)-4-(6-amino-5-(3-methyl-5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diaze-
pin-8-yl)pyridin-3-yl)-N-cyclopropylbenzenesulfonamide as a yellow
solid (180 mg, 33% yield) after purification by reverse phase
preparatory HPLC. MS (EI) m/z=464.6 [M+1].sup.+.
6.21. Synthesis of
6'-amino-N-ethyl-N-methyl-5'-(7-oxo-4,5,6,7-tetrahydrothieno[2,3-c]pyridi-
n-2-yl)-[2,3'-bipyridine]-5-sulfonamide
[0139] The captioned compound was prepared the method shown
below:
##STR00031##
[0140] To a suspension of P.sub.2O.sub.5 (25.4 g, 179.0 mmol) in
methanesulfonic acid (100 mL) was added 3-(thiophen-3-yl)propanoic
acid (5.0 g, 32.0 mmol) and the reaction was stirred at room
temperature for 1 h. At completion, the reaction was concentrated
and the residue was purified by flash chromatography (20-30%
EtOAc/hexane eluent) to give 4H-cyclopenta[b]thiophen-6(5H)-one
(1.34 g, 30% yield) as a brown solid. .sup.1H NMR (300 MHz, MeOH)
.delta. ppm 8.14 (d, J=4.8 Hz, 1H), 7.17 (d, J=4.8 Hz, 1H),
2.94-3.15 (m, 4H); MS (EI) m/z=139.2 [M+1].sup.+.
[0141] A solution of 4H-cyclopenta[b]thiophen-6(5H)-one (1.34 g,
9.7 mmol), hydroxylamine HCl (1.45 g, 20.8 mmol) and NaOAc (7.3 g,
89.0 mmol) in MeOH (150 mL) was stirred overnight at room
temperature. The reaction was then concentrated under reduced
pressure, taken up in EtOAc (100 mL) and filtered over a plug of
silica gel, eluting with EtOAc. The filtrate was concentrated,
taken up in polyphosphoric acid (100 g) and heated for 2 h at
130.degree. C. Upon completion, the reaction was quenched by
pouring over ice water (100 mL). The aqueous mixture was extracted
with DCM (2.times.100 mL) and the combined extracts were washed
with 0.1 M NaOH (100 mL), dried over Na.sub.2SO.sub.4, then
filtered and concentrated.
5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one (837 mg, 55% yield) was
isolated as a white solid after purification by flash
chromatography over silica gel (20-30% EtOAc/hexane eluent).
.sup.1H NMR (300 MHz, MeOH) .delta. ppm 7.69 (d, J=5.0 Hz, 1H),
7.06 (d, J=5.0 Hz, 1H), 3.57 (t, J=7.1 Hz, 2H), 2.94 (t, J=7.1 Hz,
2H); MS (EI) m/z=154.2 [M+1].sup.+.
[0142] To a 0.degree. C. solution of
5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one (820 mg, 5.36 mmol) in
HOAc (7 mL) and (5 mL) water was added bromine (303 .mu.L, 5.9
mmol) and the reaction was stirred 1.5 h. The reaction was diluted
in water (30 mL) and the aqueous mixture was extracted with EtOAc
(2.times.50 mL). The combined organics were washed with 5% aq.
Na.sub.2SO.sub.3 (40 mL), sat. aq. NaHCO.sub.3 (40 mL), and brine
(40 mL). The organic was dried over Na.sub.2SO.sub.4, then filtered
and concentrated. After purification by flash chromatography over
silica gel (50-100% EtOAc/hexane eluent),
2-bromo-5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one (906 mg, 73%
yield) was isolated as a white solid. .sup.1H NMR (400 MHz,
METHANOL-d.sub.4) .delta. ppm 7.10 (s, 1H), 3.55 (t, J=7.0 Hz, 2H),
2.88 (t, J=7.0 Hz, 2H); MS (EI) m/z=233.1 [M+1].sup.+.
[0143] A solution of tert-butyl (3-(4,4,5,5-tetra
methyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)carbamate (416 mg, 1.3
mmol), 2-bromo-5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one (300 mg,
1.3 mmol) and Pd(PPh.sub.3).sub.4 (23 mg, 0.02 mmol) in n-BuOH (3
mL) and 2M Na.sub.2CO.sub.3 (3 mL) was degassed under bubbling
nitrogen and the reaction was heated 2 h at 100.degree. C. Upon
completion, the reaction was filtered and concentrated under
reduced pressure, then partitioned between DCM (10 mL) water (10
mL). The layers were separated and the aqueous layer was extracted
with DCM (3.times.10 mL). The combined organics were dried over
Na.sub.2SO.sub.4 and concentrated. The concentrate was slurried in
EtOAc, sonicated and filtered to afford
2-(2-aminopyridin-3-yl)-5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one
(281 mg, 88% yield) as a white solid. .sup.1H NMR (300 MHz, MeOH)
.delta. ppm 7.99 (dd, J=5.0, 1.3 Hz, 1H), 7.62 (dd, J=7.5, 1.4 Hz,
1H), 7.25 (s, 1H), 6.76 (dd, J=7.5, 5.1 Hz, 1H), 3.61 (t, J=7.0 Hz,
2H), 2.97 (t, J=7.1 Hz, 2H); MS (EI) m/z=246.3 [M+1].sup.+.
[0144] To a solution of
2-(2-aminopyridin-3-yl)-5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one
(250 mg, 1.02 mmol) in DMF (3 ml) was added NBS (199 mg, 1.12
mmol). The reaction was stirred at room temperature for 1 h, then
concentrated and taken up in DCM (10 mL). The organic solution was
washed with a sat. aq. Na.sub.2S.sub.2O.sub.3, then dried over
Na.sub.2SO.sub.4, and concentrated to give
2-(2-amino-5-bromopyridin-3-yl)-5,6-dihydrothieno[2,3-c]pyridin-7(4H)-one
(320 mg, 97% yield) as an orange solid. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.06 (d, J=2.0 Hz, 1H), 7.73 (br. s.,
1H), 7.66 (d, J=1.8 Hz, 1H), 7.29 (s, 1H), 6.23 (br. s., 2H),
3.40-3.50 (m, 2H), 2.83 (t, J=6.9 Hz, 2H); MS (EI) m/z=325.2
[M+1].sup.+.
[0145] The final step of general method D was applied to
N-ethyl-N-methyl-6-(trimethylstannyl)pyridine-3-sulfonamide (233
mg, 0.6 mmol) and
2-(2-amino-5-bromopyridin-3-yl)-5,6-dihydrothieno[2,3-c]pyridin-
-7(4H)-one (194 mg, 0.6 mmol) to give
6'-amino-N-ethyl-N-methyl-5'-(7-oxo-4,5,6,7-tetrahydrothieno[2,3-c]pyridi-
n-2-yl)-[2,3'-bipyridine]-5-sulfonamide as a yellow solid (140 mg,
0.31 mmol, 53% yield) after purification by reverse phase
preparatory HPLC. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
8.91 (d, J=2.0 Hz, 1H), 8.85 (d, J=2.3 Hz, 1H), 8.26 (d, J=2.3 Hz,
1H), 8.10-8.19 (m, 2H), 7.73 (br. s, 1H), 7.33 (s, 1H), 6.62 (s,
2H), 3.44-3.50 (m, 2H), 3.09 (s, 2H), 2.86 (t, J=1.0 Hz, 1H), 2.72
(s, 3H), 1.01-1.09 (m, 3H); MS (EI) m/z=444.5 [M+1].sup.+.
6.22. General Method F and Synthesis of
4-(4-(5-amino-6-(5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)p-
yrazin-2-yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile
[0146] The captioned compound was prepared by general method F,
represented below:
##STR00032##
[0147] To a stirred suspension of 2-(4-chlorophenyl)acetonitrile
(3.06 g, 20.3 mmol) at 0.degree. C. in DMSO (200 mL) was added NaH
(1.79 g, 60% in mineral oil, 44.6 mmol). The reaction was stirred
at 0.degree. C. for 15 min then at room temperature for 30 min,
resulting in a dark purple solution. To this solution was added
1-chloro-2-(2-chloroethoxy)ethane (3.18 g, 22.33 mmol) dropwise.
The resulting mixture was stirred at room temperature overnight
before diluting with 50 mL water and neutralizing to pH.about.7.0
with 1.0 M aq. HCl. The reaction was extracted with Et.sub.2O
(3.times.200 mL) and the combined organics were washed with brine
(200 mL), dried over Na.sub.2SO.sub.4, and concentrated. The
residue was purified by flash chromatography on silica gel (10-30%
EtOAc/hexane eluent) to provide
4-(4-chlorophenyl)tetrahydro-2H-pyran-4-carbonitrile as a yellow
crystalline solid (4.01 g, 89% yield) (Note: This compound does not
ionize well on LC/MS. It does not have intense UV absorption at 220
nM. The TLC R.sub.f is 0.6 in 30% EA/Hexane).
[0148] A solution of
4-(4-chlorophenyl)tetrahydro-2H-pyran-4-carbonitrile (889 mg, 4.0
mmol), Pd(dba).sub.2 (138 mg, 0.24 mmol), PCy.sub.3 (268 mg, 0.96
mmol), diboron pinacol ester (1.21 g, 4.8 mmol) and KOAc (1.18 g,
12.0 mmol) in 16 mL dioxane was microwave heated to 150.degree. C.
for 15 min. The resulting mixture was filtered, then concentrated
and purified by flash chromatography on silica (2-5% MeOH/DCM
eluent) to provide
4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)tetrahydro-2H-py-
ran-4-carbonitrile as a yellow crystalline solid (1.01 g, 81%
yield).
[0149] A solution of
4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)tetrahydro-2H-py-
ran-4-carbonitrile (1.53 g, 4.86 mmol), Pd(PPh.sub.3).sub.4 (271
mg, 0.24 mmol), 2.0 M aq. Na.sub.2CO.sub.3 (4.9 mL) and the
5-bromopyrazin-2-amine (846 mg, 4.86 mmol) were combined in n-BuOH
(20 mL). The slurry was degassed for 10 min under a bubbling stream
of anhydrous nitrogen, then heated at 90.degree. C. for 2 h. The
resulting mixture was filtered, concentrated and purified by flash
chromatography on silica (1-5% MeOH/DCM eluent) to provide
4-(4-(5-aminopyrazin-2-yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile
(1.08 g, 79% yield).
[0150] To a solution of
4-(4-(5-aminopyrazin-2-yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile
(1.08 m, 3.84 mmol) in DMF (30 mL) was added N-bromosuccinimide
(747 mg, 4.22 mmol) in one portion. The reaction was then stirred
at room temperature for 1 h. At completion, the mixture was poured
over ice (50 mL) and stirred for 20 min. After filtration and
washing with water and drying under vacuum,
4-(4-(5-amino-6-bromopyrazin-2-yl)phenyl)tetrahydro-2H-pyran-4-carbonitri-
le was obtained as obtained as a dark brown solid (1.37 g, 100%
yield).
[0151] General method C was applied to
4-(4-(5-amino-6-bromopyrazin-2-yl)phenyl)tetrahydro-2H-pyran-4-carbonitri-
le (215 mg, 0.6 mmol) and 8-(4,4,5,5-tetra
methyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[e][1,4]diazepin-5(2H-
)-one (173 mg, 0.6 mmol) to provide
4-(4-(5-amino-6-(5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-8-yl)p-
yrazin-2-yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile as a yellow
solid (87 mg, 33% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta. ppm 8.47 (s, 1H), 8.14 (d, J=8.3 Hz, 1H), 8.01 (d, J=8.3
Hz, 2H), 7.58 (d, J=8.3 Hz, 2H), 7.21 (d, J=8.3 Hz, 1H), 7.04 (s,
1H), 6.40 (br. s., 1H), 4.93 (br. s., 2H), 4.57 (br. s., 1H), 4.12
(d, J=8.8 Hz, 2H), 3.94 (t, J=11.7 Hz, 2H), 3.50-3.73 (m, 4H),
2.02-2.26 (m, 4H); MS (EI) m/z=441.5 [M+1].sup.+.
6.23. Synthesis of
2'-amino-5'-(4-(N-cyclopropylsulfamoyl)phenyl)-[2,3'-bipyridine]-5-carbox-
amide
##STR00033##
[0153] A mixture of 2-amino-5-bromopyridine (3.58 g, 20.7 mmol),
N-cyclopropyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzenesul-
fonamide (5.00 g, 20.7 mmol), sodium carbonate (3.29 g, 31.05
mmol), and PdCl.sub.2(PPh.sub.3).sub.2 (0.436 g, 0.62 mmol) in 3:1
acetonitrile:water was microwave heated at 80.degree. C. for 20
min, and then at 100.degree. C. for 15 min. The reaction was then
heated in an oil bath at 80.degree. C. for 16 h. The reaction was
cooled to room temperature and filtered. The solid was washed with
methanol (10 mL), and the combined filtrates were concentrated. The
crude material was purified by flash chromatography over silica gel
(0-5% MeOH/DCM) to give
4-(6-aminopyridin-3-yl)-N-cyclopropylbenzenesulfonamide (2.08 g,
35% yield). .sup.1H NMR (400 MHz, METHANOL-d.sub.4) .delta. ppm
8.27 (d, J=2.01 Hz, 1H), 7.88-7.95 (m, 2H), 7.85 (d, J=2.51 Hz,
1H), 7.81-7.84 (m, 1H), 7.71-7.79 (m, 2H), 6.45-6.88 (m, 1H),
1.89-2.41 (m, 1H), 0.13-0.82 (m, 4H); MS (EI) m/z: 290 [M+H].sup.+.
HPLC (Sunfire C18 4.6 mm.times.50 mm, 10-90% MeCN:10 mM aq.
NH.sub.4OAc, 2 min gradient) t.sub.R=1.34 min, 88% integrated
area.
[0154] To a solution of
4-(6-amino-pyridin-3-yl)-N-cyclopropylbenzenesulfonamide (1.04 g,
3.59 mmol) in DCM (12 mL) was added portionwise N-bromosuccinimide
(0.64 g, 3.59 mmol). The reaction was stirred for 1 h, then was
filtered. The filtrate was washed with sat. aq.
Na.sub.2S.sub.2O.sub.4 (20 mL), sat. aq. NaHCO.sub.3 (10 mL), then
dried over Na.sub.2SO.sub.4 and concentrated to give
4-(6-amino-5-bromo-pyridin-3-yl)-N-cyclopropyl-benzenesulfonamide
(1.07 g, 81% yield). .sup.1H NMR (400 MHz, METHANOL-d.sub.4)
.delta. ppm 8.30 (d, J=2.01 Hz, 1H), 8.12 (d, J=2.01 Hz, 1H), 7.92
(d, J=8.53 Hz, 2H), 7.76 (d, J=8.53 Hz, 2H), 2.19 (tt, J=6.71, 3.58
Hz, 1H), 0.17-0.83 (m, 4H). MS (EI) m/z: 368, 370 [M+H].sup.+. HPLC
(Sunfire C18 4.6 mm.times.50 mm, 10-90% MeCN:10 mM aq. NH.sub.4OAc,
2 min gradient) t.sub.R=1.68 min, 90% integrated area.
[0155] A mixture of
4-(6-amino-5-bromo-pyridin-3-yl)-N-cyclopropyl-benzenesulfonamide
(100 mg, 0.27 mmol), diboron pinacol ester (103 mg, 0.41 mmol),
potassium acetate (40 mg, 0.41 mmol), Pd.sub.2(dba).sub.3 (8 mg,
0.008 mmol), and PCy.sub.3 (5 mg, 0.016 mmol) in 1,4-dioxane (2 mL)
was heated at 80.degree. C. to provide
4-[6-amino-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin-3-yl]-
-N-chclopropyl-benzenesulfonamide (112 mg, 100% yield) after
purification by preparatory HPLC. This material was combined with
6-chloro nicotinamide (42 mg, 0.27 mmol), Na.sub.2CO.sub.3 (43 mg,
0.41 mmol), and PdCl.sub.2(PPh.sub.3).sub.4 (6 mg, 0.008 mmol) in
3:1 acetonitrile:water (2 mL). The reaction mixture was microwave
heated at 150.degree. C. for 6 min. At completion, the reaction
mixture was concentrated and purified by reverse phase preparatory
HPLC to give
2'-amino-5'-(4-(N-cyclopropylsulfamoyl)phenyl)-[2,3'-bipyridine]-5-carbox-
amide (7 mg, 6% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
ppm 9.11 (d, J=1.25 Hz, 1H), 8.53 (d, J=2.26 Hz, 1H), 8.47 (d,
J=2.26 Hz, 1H), 8.26-8.39 (m, 2H), 8.21 (br. s., 1H), 8.00 (d,
J=8.53 Hz, 3H), 7.91 (d, J=2.51 Hz, 1H), 7.76-7.87 (m, 4H), 7.62
(br. s., 1H), 2.12 (dd, J=6.53, 3.51 Hz, 1H), 0.46-0.54 (m, 2H),
0.37-0.45 (m, 2H). MS (EI) m/z: 410 [M+H].sup.+. HPLC (Sunfire C18
4.6 mm.times.50 mm, 10-90% MeCN:10 mM aq. NH.sub.4OAc, 2 min
gradient) t.sub.R=1.39 min, 96% integrated area.
6.24. Synthesis of
6'-amino-N-cyclopropyl-N-methyl-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
-yl)-[3,3'-bipyridine]-6-sulfonamide
##STR00034##
[0157] General Method C was applied to
6'-amino-5'-bromo-N-cyclopropyl-[3,3'-bipyridine]-6-sulfonamide
(650 mg, 1.76 mmol) and
6'-amino-5'-bromo-N-cyclopropyl-[3,3'-bipyridine]-6-sulfonamide
(410 mg, 2.11 mmol) to provide
6'-amino-N-cyclopropyl-N-methyl-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
-yl)-[3,3'-bipyridine]-6-sulfonamide (130 mg, 16% yield) after
purification by preparative HPLC. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 9.11 (d, J=1.8 Hz, 1H), 8.51 (d, J=2.3
Hz, 1H), 8.39 (dd, J=8.2, 2.1 Hz, 1H), 7.93 (s, 3H), 7.86 (d, J=2.3
Hz, 1H), 7.46-7.56 (m, 2H), 6.19 (s, 2H), 3.36-3.46 (m, 2H), 2.96
(t, J=6.4 Hz, 2H), 2.88 (s, 3H), 2.23-2.31 (m, 1H), 0.72-0.79 (m,
2H), 0.62-0.69 (m, 2H); MS (EI) m/z=450.5 [M+1].sup.+.
6.25. Synthesis of
N-(4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)ph-
enyl)ethanesulfonamide
##STR00035##
[0159] General method B was applied to
6-(2-amino-5-bromopyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one
hydrobromide (150 mg, 0.38 mmol) and
4-(ethylsulfonamide)phenylboronic acid (96 mg, 0.42 mmol) to give
N-(4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)ph-
enyl)ethanesulfonamide (58 mg, 35% yield) after purification by
reverse phase preparatory HPLC. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 9.79 (s, 1H), 8.28 (d, J=2.51 Hz, 1H), 7.92 (d, J=8.03
Hz, 2H), 7.58-7.69 (m, 3H), 7.44-7.53 (m, 2H), 7.26 (d, J=8.78 Hz,
2H), 5.96 (br. s., 2H), 3.41 (td, J=6.53, 2.51 Hz, 2H), 3.16 (s,
2H), 3.09 (q, J=7.28 Hz, 2H), 2.96 (t, J=6.40 Hz, 2H), 1.20 (t,
J=7.28 Hz, 3H); MS (EI) m/z: 422 [M+H].sup.+. HPLC (Sunfire C18 4.6
mm.times.50 mm, 10-90% MeOH:10 mM aq. Ammonium formate, 2 min
gradient) t.sub.R=1.94 min, 97% integrated area.
6.26. Synthesis of
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-me-
thyl-N-((1R,2R)-2-methylcyclopropyl)benzenesulfonamide
[0160] The captioned compound was prepared by the method shown
below:
##STR00036##
[0161] To a 0.degree. C. solution of ethyl
2-(diethoxyphosphoryl)acetate (16.90 mL, 84.37 mmol) in
methyltetrahydrofuran (150 mL) was added n-BuLi (33.0 mL, 82.5
mmol, 2.5 M in hexane). The reaction was maintained at 0.degree. C.
for 30 minutes before the addition of (S)-2-methyloxirane. The
reaction mixture was then transferred to a teflon lined steel
reactor, sealed and heated to 150.degree. C. for 18 h. The reactor
was cooled to room temperature, then treated with water:30% aq.
NaOH (2:1) and refluxed at 100.degree. C. for 5 h. The biphasic
mixture was again cooled to room temperature and transferred to a
separatory funnel. The layers were separated and the aqueous phase
was acidified with conc. aq. HCl (100 mL) and extracted with
isopropyl acetate (3.times.200 mL) while maintaining the aqueous
phase at pH .about.3. The combined organics were washed with 10%
NaCl (2.times.100 mL), filtered over celite and concentrated to
provide (1R,2R)-2-methylcyclopropanecarboxylic acid (7.9 g, 92%
yield), which was used directly in the next reaction. .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta. ppm 1.40-1.52 (m, 1H), 1.30-1.39
(m, 1H), 1.20-1.28 (m, 1H), 1.13 (d, J=6.0 Hz, 3H), 0.76 (ddd,
J=7.8, 6.5, 4.0 Hz, 1H).
[0162] To a 0.degree. C. solution of
(1R,2R)-2-methylcyclopropanecarboxylic acid (2.0 g, 20.0 mmol) in
anhydrous acetone (100 mL) was added triethylamine (2.87 mL, 20.6
mmol) and ethyl chloroformate (1.96 mL, 20.6 mL). The cold bath was
removed and the reaction was allowed to warm to room temperature
for 30 min before the addition of sodium azide (1.36 g, 21.00 mmol)
in water (20 mL). The reaction was maintained at room temperature
for 1 h, then diluted with DCM (300 mL) and water (200 mL). The
layers were separated and the organic was washed with water
(2.times.100 mL) and brine (100 mL) then dried over MgSO.sub.4 and
concentrated to provide (1R,2R)-2-methylcyclopropanecarbonyl azide
(1.8 g, 72% yield) which was used directly in the next reaction.
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.47-1.58 (m, 1H),
1.35-1.42 (m, 1H), 1.28-1.34 (m, 1H), 1.14 (d, J=6.0 Hz, 3H),
0.79-0.87 (m, 1H).
[0163] A solution of (1R,2R)-2-methylcyclopropanecarbonyl azide
(1.80 g, 14.4 mmol) in toluene (100 mL) was heated to 90.degree. C.
for 1 h. After gas evolution had subsided, the solution was
cannulated into a flask containing a slurry of benzyl alcohol (4.5
mL, 43.2 mmol) and copper(I) chloride (750 mg, 7.5 mmol) in DMF
(100 mL). The reaction was maintained at room temperature for 1 h,
then diluted with Et.sub.2O (200 mL) then washed with brine, dried
over MgSO.sub.4 and concentrated. The crude product was purified by
flash chromatography over silica gel (20-30% EtOAc/hexane eluent)
to provide benzyl ((1R,2R)-2-methylcyclopropyl)-carbamate (1.68 g,
57% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm
7.27-7.46 (m, 5H), 5.11 (br. s., 2H), 4.80-4.99 (m, 1H), 2.28 (d,
J=3.0 Hz, 1H), 1.07 (d, J=5.5 Hz, 3H), 0.82-0.97 (m, 1H), 0.67
(ddd, J=9.0, 5.3, 3.8 Hz, 1H), 0.51 (q, J=5.9 Hz, 1H).
[0164] A solution of benzyl ((1R,2R)-2-methylcyclopropyl)carbamate
(1.68 g, 8.2 mmol) in DCM (55 mL) was charged with 10 wt % Pd/C,
then degassed and charged with hydrogen (50 PSI). The reaction was
complete after 30 min and so was filtered over celite, rinsing with
DCM (15 mL). The liquor was cooled to 0.degree. C., and treated
with triethylamine (3.43 mL, 24.6 mmol) and
4-bromobenzene-1-sulfonyl chloride (2.20 g, 8.61 mmol). The cold
bath was removed and the reaction was stirred for 1 h. At
completion, the reaction was washed with 0.5 M aq. HCl (2.times.20
mL) and brine (20 mL), dried over MgSO.sub.4 and concentrated to
provide 4-bromo-N-((1R,2R)-2-methylcyclopropyl)benzenesulfonamide
(2.06 g, 87% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.
ppm 7.74-7.80 (m, 1H), 7.66-7.72 (m, 1H), 4.90 (br. s., 1H), 1.94
(dt, J=3.5, 1.8 Hz, 1H), 1.41 (t, J=7.3 Hz, 1H), 0.89-1.01 (m, 3H),
0.70-0.81 (m, 1H), 0.35-0.45 (m, 1H).
[0165] The appropriate procedure specified in general method F was
applied to the conversion of
4-bromo-N-((1R,2R)-2-methylcyclopropyl)benzenesulfonamide (2.06 g,
7.1 mmol) to N-((1R,2R)-2-methylcyclopropyl)-4-(4,4,5,5-tetra
methyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamide (1.38 g, 58%
yield) which was obtained after flash chromatography over silica
gel (20-40% EtOAc/hexane eluent). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 7.96 (s, 2H), 7.85-7.92 (m, 2H),
4.75-4.80 (m, 1H), 1.88-1.93 (m, 1H), 1.38 (s, 12H), 1.27 (s, 1H),
0.95 (d, J=1.3 Hz, 3H), 0.70-0.79 (m, 1H), 0.33-0.40 (m, 1H).
[0166] The appropriate procedure specified in general method F was
applied to the conversion of
N-((1R,2R)-2-methylcyclopropyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
-2-yl)benzenesulfonamide (676 mg, 1.69 mmol) to
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-((-
1R,2R)-2-methylcyclopropyl)benzenesulfonamide hydrochloride after
treatment with aq. HCl (350 mg, 45% yield). .sup.1H NMR (400 MHz,
METHANOL-d.sub.4) .delta. ppm 8.34 (d, J=6.5 Hz, 2H), 8.11-8.16 (m,
1H), 7.97-8.03 (m, 2H), 7.89-7.96 (m, 2H), 7.57-7.61 (m, 1H),
7.54-7.56 (m, 1H), 3.55-3.61 (m, 2H), 3.07-3.14 (m, 2H), 1.84-1.89
(m, 1H), 0.92-0.97 (m, 3H), 0.80-0.89 (m, 1H), 0.64-0.70 (m, 1H),
0.31-0.38 (m, 1H); MS (EI) m/z=450.5 [M+1].sup.+.
[0167] To a room temperature slurry of
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-((-
1R,2R)-2-methylcyclopropyl)benzenesulfonamide hydrochloride (110
mg, 0.227 mmol) and cesium carbonate (222 mg, 0.681 mmol) was added
methyl iodide (16.0 .mu.L, 0.251 mmol). At completion, the reaction
was filtered and concentrated, then flashed over silica gel (5-10%
MeOH/DCM eluent) to provide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2--
yl)-N-methyl-N-((1R,2R)-2-methylcyclopropyl)-benzenesulfonamide (90
mg, 86% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm
8.55 (s, 1H), 8.25 (d, J=7.8 Hz, 1H), 8.16 (d, J=8.5 Hz, 2H), 7.92
(d, J=8.5 Hz, 2H), 7.83 (dd, J=8.0, 1.5 Hz, 1H), 7.70 (s, 1H), 5.95
(br. s, 1H), 5.14 (br. s, 2H), 3.66 (dt, J=7.0, 2.3 Hz, 2H), 3.14
(t, J=7.0 Hz, 2H), 2.76 (s, 3H), 1.48-1.53 (m, 1H), 1.27-1.35 (m,
1H), 1.06 (d, J=6.3 Hz, 3H), 0.96-1.02 (m, 1H), 0.47 (dd, J=12.3,
6.0 Hz, 1H); MS (EI) m/z=464.5 [M+1].sup.+.
6.27. Synthesis of
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-et-
hyl-N,2-dimethylbenzenesulfonamide
[0168] The captioned compound was prepared by the method shown
below:
##STR00037##
[0169] To a 0.degree. C. solution of
4-bromo-2-methylbenzenesulfonyl chloride (1.00 g, 3.71 mmol) in
CH.sub.2Cl.sub.2 (10 mL) was added N-ethyldiisopropylamine (0.71
mL, 4.31 mmol) and N-methylethylamine (0.96 mL, 11.13 mmol). The
cold bath was removed and the reaction was stirred for 2 h. At
completion, the reaction was concentrated, and the crude material
was dissolved in CH.sub.2Cl.sub.2 (20 mL). The mixture was washed
with 0.5 N aqueous HCl (3.times.10 mL), sat. aq. NaHCO.sub.3
(2.times.10 mL), water (10 mL), and saturated aqueous NaCl (5 mL),
dried (Na.sub.2SO.sub.4), and concentrated to give
4-bromo-N-ethyl-2,N-dimethyl-benzenesulfonamide (0.91 g, 3.11 mmol,
85% yield). This material was directly dissolved in 1,4-dioxane (10
mL) and treated with bisboron pinacol ester (0.87 g, 3.43 mmol),
PdCl.sub.2(dppf)-DCM (69 mg, 0.093 mmol), and KOAc (0.92 g, 9.34
mmol). The reaction was heated at 85.degree. C. for 2 h. At
completion, the reaction was cooled to room temperature and
concentrated. The crude material was treated with CH.sub.2Cl.sub.2
(30 mL) and water (10 mL). The organic layer was washed with water
(2.times.10 mL) and brine (5 mL), dried over Na.sub.2SO.sub.4 and
concentrated. The crude material was purified by flash
chromatography over silica gel (5-20% EtOAc:hexane eluent) to give
N-ethyl-2,N-dimethyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-ben-
zenesulfonamide (0.64 g, 60% yield). .sup.1H NMR (300 MHz, MeOH)
.delta. ppm 7.80-7.88 (m, 1H), 7.68-7.77 (m, 2H), 3.26 (q, J=7.12
Hz, 2H), 2.83 (s, 3H), 2.61 (s, 3H), 1.37 (s, 12H), 1.17 (t, J=7.15
Hz, 3H). MS (EI) m/z: 340 [M+H].sup.+. HPLC (Sunfire C18 4.6
mm.times.50 mm, 10-90% MeCN:10 mM aq NH.sub.4OAc, 2 min gradient)
t.sub.R=2.48 min, 91% integrated area.
[0170] A solution of
N-ethyl-2,N-dimethyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-ben-
zenesulfonamide (140 mg, 0.42 mmol),
6-(3-amino-6-bromopyrazin-2-yl)-3,4-dihydroisoquinolin-1(2H)-one
(150 mg, 0.38 mmol) Pd(PPh.sub.3).sub.4 (13.2 mg, 0.011 mmol) and
2.0 M aq. Na.sub.2CO.sub.3 (0.38 mL) in n-butanol (2 mL) was
microwave heated at 150.degree. C. for 10 min to provide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-et-
hyl-N,2-dimethylbenzenesulfonamide (49 mg, 29% yield) after
purification by reverse phase preparatory HPLC. .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 8.69 (s, 1H), 8.04 (s, 1H),
7.93-8.02 (m, 3H), 7.82 (d, J=8.28 Hz, 1H), 7.67-7.77 (m, 2H), 6.62
(s, 2H), 3.43 (td, J=6.40, 2.51 Hz, 2H), 3.18 (q, J=6.94 Hz, 3H),
3.00 (t, J=6.53 Hz, 2H), 2.76 (s, 3H), 2.59 (s, 3H), 1.08 (t,
J=7.15 Hz, 3H). MS (EI) m/z: 452 [M+H].sup.+. HPLC (Sunfire C18 4.6
mm.times.50 mm, 10-90% MeCN:10 mM aq NH.sub.4OAc, 4 min gradient)
t.sub.R=2.14 min, 100% integrated area.
6.28. Synthesis of
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-cy-
clopropyl-N-methylbenzenesulfonamide
##STR00038##
[0172] General method C was applied to
4-(5-amino-6-bromopyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
(383 mg, 1.00 mmol) and
6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydroisoquinolin-1(-
2H)-one (300 mg, 1.1 mmol) to provide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-cy-
clopropyl-N-methylbenzenesulfonamide (382 mg, 85% yield) after
purification by preparatory HPLC. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.73 (s, 1H), 8.26 (d, J=8.5 Hz, 2H),
8.03 (br. s., 1H), 7.97 (d, J=8.0 Hz, 1H), 7.84 (d, J=8.5 Hz, 2H),
7.74 (d, J=8.0 Hz, 1H), 7.70 (s, 1H), 3.42 (t, J=5.3 Hz, 2H), 2.99
(t, J=6.4 Hz, 2H), 2.67 (s, 3H), 1.77-1.86 (m, 1H), 0.61-0.81 (m,
4H); MS (EI) m/z=450.5 [M+1].sup.+.
6.29. Synthesis of
2-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)ph-
enyl)-2-methylpropanenitrile
##STR00039##
[0174] General method B was applied to
4-(2-cyanopropan-2-yl)phenylboronic acid (79 mg, 0.42 mmol) and
6-(3-amino-6-bromopyrazin-2-yl)-3,4-dihydroisoquinolin-1(2H)-one
(150 mg, 0.38 mmol) in n-butanol (2 mL) to give
2-{4-[5-amino-6-(1-oxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-pyrazin-2-yl]-
-phenyl}-2-methyl-propionitrile (119 mg, 82% yield) after
purification by column chromatography on silica (2-5% MeOH/DCM
eluent).
[0175] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.59 (s,
1H), 8.02 (d, J=8.53 Hz, 2H), 7.96 (d, J=8.03 Hz, 2H), 7.75 (dd,
J=8.03, 1.25 Hz, 1H), 7.70 (s, 1H), 7.58 (d, J=8.53 Hz, 2H), 6.45
(s, 2H), 3.43 (td, J=6.40, 2.51 Hz, 2H), 2.99 (t, J=6.40 Hz, 2H),
1.71 (s, 6H). MS (EI) m/z: 384 [M+H].sup.+. HPLC (Shim-Pack VP ODS
4.6 mm.times.50 mm, 10-90% 95% MeOH, 5% water with 0.1% TFA, 4 min
gradient) t.sub.R=3.22 min, 99% integrated area.
6.30. General Method G and Synthesis of
4-(5-amino-6-(1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-7-yl)pyrazin-2-y-
l)-N-ethyl-N-methylbenzenesulfonamide
[0176] The captioned compound was prepared by general method G,
represented below:
##STR00040##
[0177] To a room temperature solution of
7-hydroxy-2,3,4,5-tetrahydro-1H-benzo[c]azepin-1-one (2.3 g, 12.9
mmol) in THF (100 mL) was added NaH (671 mg, 16.7 mmol), followed
by
1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide
(5.98 g, 16.77 mmol). The reaction was stirred at room temperature
for 2 h, then quenched with sat. aq. NH.sub.4Cl and extracted with
EtOAc (3.times.50 mL). The combined organics were washed with
brine, dried over Na.sub.2SO.sub.4 and concentrated. The crude
material was purified by flash chromatography over silica gel (20%
EtOAc/hexane eluent) to provide
1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-7-yl
trifluoromethanesulfonate (12.9 mmol, 77% yield) MS (EI) m/z=310
[M+1].sup.+.
[0178] A degassed slurry of
1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-7-yl
trifluoromethanesulfonate (4.0 g, 12.9 mmol) diboron pinacol ester
(9.9 g, 39.0 mmol), KOAc (1.9 g, 19.5 mmol), and Pd(dppf)Cl.sub.2
(963 mg, 1.3 mmol) in dioxane (8 mL) was heated to 90.degree. C.
for 1.5 h. At completion, the reaction was filtered and
concentrated, then purified by flash chromatography over silica gel
(30% EtOAc/hexanes eluent) to provide.
7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3,4,5-tetrahydro-1H-ben-
zo[c]azepin-1-one (1.5 g, 41% yield). MS (EI) m/z=288
[M+1].sup.+.
[0179] General method C was applied to
7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3,4,5-tetrahydro-1H-ben-
zo[c]azepin-1-one (95 mg, 0.33 mmol) and
4-(5-amino-6-bromopyrazin-2-yl)-N-ethyl-N-methylbenzenesulfonamide
(108 mg, 0.29 mmol) to provide
4-(5-amino-6-(1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-7-yl)pyrazin-2-y-
l)-N-ethyl-N-methylbenzenesulfonamide (33 mg, 22% yield) after
purification by preparatory HPLC. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 8.70 (s, 1H), 8.22 (d, J=8.5 Hz, 2H),
8.12 (t, J=5.8 Hz, 1H), 7.80 (d, J=8.5 Hz, 2H), 7.71 (dd, J=8.0,
1.5 Hz, 1H), 7.62-7.66 (m, 2H), 6.66 (br. s, 2H), 2.93-3.09 (m,
4H), 2.83 (t, J=1.0 Hz, 2H), 2.67 (s, 3H), 1.94 (dd, J=12.3, 6.0
Hz, 2H), 1.03 (t, J=7.2 Hz, 3H); MS (EI) m/z=452.5 [M+1].sup.+.
6.31. Synthesis of
6-(3-amino-6-(4-(1-cyclopropylethoxy)phenyl)pyrazin-2-yl)-3,4-dihydroisoq-
uinolin-1(2H)-one
[0180] The captioned compound was prepared by the method shown
below:
##STR00041##
[0181] To a 0.degree. C. slurry of NaH (460 mg, 11.4 mmol, 60 wt %
dispersion in mineral oil) in DMF (14 mL) was added
1-cyclopropylethanol (0.79 mL, 7.41 mmol) dropwise. The cold bath
was removed for 30 min before the addition of
1-bromo-4-fluorobenzene (1.00 g, 5.7 mmol). The resulting reaction
mixture was heated to 100.degree. C. for 3 h. At completion, the
reaction was cooled to room temperature and concentrated. The
resulting oil was diluted with water (30 mL) and extracted into
EtOAc (3.times.30 mL). The combined organics were washed with brine
(20 mL), then dried over MgSO.sub.4 and concentrated to provide
1-bromo-4-(1-cyclopropylethoxy)benzene (1.30 g, 95% yield). The
resulting oil was used directly without further purification.
[0182] A slurry of 1-bromo-4-(1-cyclopropylethoxy)benzene (1.30 g,
5.39 mmol),
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (1.77
g, 7.01 mmol), PdCl.sub.2dppf-DCM (197 mg, 0.27 mmol), KOAc (1.06
g, 10.78 mmol) in DMF (12 mL) was heated to 100.degree. C. for 2 h.
At completion, the reaction was concentrated, then purified by
flash chromatography over silica gel (20-40% EtOAc/hexane eluent)
to provide
2-(4-(1-cyclopropylethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(1.38 g, 88% yield).
[0183] General method A was applied to
2-(4-(1-cyclopropylethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
(639 mg, 2.00 mmol), providing
6-(3-amino-6-(4-(1-cyclopropylethoxy)phenyl)pyrazin-2-yl)-3,4-dihydroisoq-
uinolin-1(2H)-one (321 mg, 40% yield) after purification by reverse
phase preparatory HPLC. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
ppm 8.50 (s, 1H), 8.00 (br. s., 1H), 7.95 (d, J=8.0 Hz, 1H), 7.88
(d, J=8.8 Hz, 2H), 7.72-7.77 (m, 1H), 7.70 (s, 1H), 6.97 (d, J=8.8
Hz, 2H), 6.30 (s, 2H), 3.94-4.04 (m, 1H), 3.42 (td, J=6.4, 2.5 Hz,
2H), 2.99 (t, J=6.5 Hz, 2H), 1.29 (d, J=6.0 Hz, 3H), 1.09 (dt,
J=8.0, 4.9 Hz, 1H), 0.43-0.53 (m, 2H), 0.25-0.39 (m, 2H); MS (EI)
m/z=401.5 [M+1].sup.+.
6.32. Synthesis of
6-(2-amino-5-(4-(2-(dimethylamino)propan-2-yl)phenyl)pyridin-3-yl)-3,4-di-
hydroisoquinolin-1(2H)-one
[0184] The captioned compound was prepared by the method shown
below:
##STR00042##
[0185] To a solution of 2-(4-bromophenyl)propan-2-amine (500 mg,
2.34 mmol) in DCM (30 mL) was added 37% aqueous formaldehyde (525
.mu.L, 7.0 mmol), Na.sub.2SO.sub.4 (100 mg) and STAB (2.98 g, 14.0
mmol). After stirring 2 h at room temperature, the reaction was
partitioned between sat. aq. NaHCO.sub.3 (20 mL) and DCM (20 mL).
The layers were separated and the aqueous layer was extracted with
DCM (2.times.30 mL). The combined organics were dried over
Na.sub.2SO.sub.4, filtered and concentrated to provide
2-(4-bromophenyl)-N,N-dimethylpropan-2-amine (464 mg, 82% yield) as
a yellow oil. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) .delta. ppm
7.45 (d, J=6.0 Hz, 4H), 2.16 (s, 6H), 1.40 (s, 6H); MS (EI)
m/z=243.2 [M+1].sup.+.
[0186] General method G was applied to
2-(4-bromophenyl)-N,N-dimethylpropan-2-amine (450 mg, 1.86 mmol),
to provide
N,N-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ph-
enyl)propan-2-amine (300 mg, 56% yield) after purification by flash
chromatography over silica gel (50-100% EtOAc/hexanes). MS (EI)
m/z=290.2 [M+1].sup.+.
[0187] General Method C was applied to
N,N-dimethyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pro-
pan-2-amine (109 mg, 0.38 mmol) and
6-(2-amino-5-bromopyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one
hydrobromide (125 mg, 0.31 mmol) to afford
6-(2-amino-5-(4-(2-(dimethylamino)propan-2-yl)phenyl)pyridin-3-yl)-3,4-di-
hydroisoquinolin-1(2H)-one (14 mg, 11% yield) as a white solid
after purification by flash chromatography. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. ppm 8.32 (s, 1H), 7.81-8.11 (m, 2H),
7.37-7.79 (m, 7H), 5.87 (s, 2H), 3.41 (m, 2H), 2.85-3.10 (m, 2H),
2.11 (s, 6H), 1.30 (s, 6H); MS (EI) m/z=401.5 [M+1].sup.+.
6.33. Synthesis of
4-(5-amino-6-(5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-8-yl)pyrazin--
2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
[0188] The captioned compound was prepared by the method shown
below:
##STR00043##
[0189] A solution of 7-bromochroman-4-one (340 mg, 1.5 mmol) and
methanesulfonic acid (3 mL, 23.0 mmol) in DCM was cooled to
0.degree. C. and treated with NaN.sub.3 (146 mg, 2.25 mmol)
portionwise, while maintaining the internal temperature below
5.degree. C. The reaction was then stirred at 0.degree. C. for 4 h.
At completion, the reaction was slowly neutralized with 8 N aq.
NaOH, then diluted with DCM (20 mL) and water (20 mL). The layers
were separated and the organics were dried over MgSO.sub.4,
filtered and evaporated to afford
8-bromo-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (370 mg, 100%
yield). MS (EI) m/z=242.1 [M+1].sup.+.
[0190] General methods G and C were successively applied to
8-bromo-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one (370 mg, 1.5
mmol) to provide
4-(5-amino-6-bromopyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulf-
onamide (241 mg, 41% yield, 2 steps) after purification of the
crude reaction mixture over silica gel (2-10% MeOH/DCM eluent.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.72 (s, 1H), 8.43
(dd, J=10.3, 4.8 Hz, 1H), 8.26 (d, J=8.8 Hz, 2H), 7.90 (d, J=8.0
Hz, 1H), 7.85 (d, J=8.3 Hz, 2H), 7.54 (dd, J=8.0, 1.8 Hz, 1H), 7.41
(d, J=1.5 Hz, 1H), 4.31-4.40 (m, 2H), 3.56 (s, 3H), 3.33-3.43 (m,
1H), 2.47-2.52 (m, 2H), 0.65-0.79 (m, 4H); MS (EI) m/z=466.5
[M+1].sup.+.
6.34. Synthesis of
4-(5-amino-6-(1-oxo-3-hydro-4,4-dideutero-isoquinolin-6-yl)pyrazin-2-yl)--
N-cyclopropyl-N-Trideuteromethylbenzenesulfonamide
[0191] The captioned compound was prepared by the method shown
below:
##STR00044##
[0192] AlCl.sub.3 (4.9 g, 36.7 mmol) and LiAlD.sub.4 (770 mg, 18.4
mmol) were suspended in anhydrous Et.sub.2O (50 mL) and cooled to
0.degree. C. 6-methoxy-2,3-dihydro-1H-inden-1-one (744 mg, 4.59
mmol) was added slowly to the stirred slurry. The reaction was
removed from the cold bath and stirred overnight while gradually
warming to room temperature. At completion, the reaction was
quenched sequentially by addition of water (5.6 mL), 15% aq. KOH
(5.6 mL), and water (16.8 mL). Na.sub.2SO.sub.4 was added and the
mixture was filtered. The organics were concentrated then purified
by flash chromatography over silica gel (30% EtOAc/hexanes eluent)
to provide 5-methoxy-3,3-dideutero-2-hydro-1H-indene as a clear oil
(500 mg, 73% yield) .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm
7.38 (dd, J=9.00 Hz, 1H), 7.18-7.22 (m, 2H), 3.85 (s, 3H),
3.06-3.11 (m, 2H), 2.71-2.75 (m, 2H).
[0193] 5-methoxy-3,3-dideutero-2-hydro-1H-indene (500 mg, 3.33
mmol) was dissolved in acetic acid (10 mL) and a solution of
CrO.sub.3 (1.33 g, 13.32 mmol) in 50% aq. acetic acid (10 mL) was
slowly added. The reaction was stirred at 50.degree. C. for 0.5 h,
then cooled to 0.degree. C. and quenched with isopropanol (3 mL).
The reaction mixture was partitioned between 0.25 N aq. NaOH (50
mL) and EtOAc (50 mL). The layers were separated and the organics
were washed with brine, dried over MgSO.sub.4 and concentrated to
afford 5-methoxy-3,3-dideutero-2-dihydro-inden-1-one (545 mg, 100%
yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 7.70 (br.
s., 1H), 6.91 (br. s., 2H), 3.89 (br. s., 3H), 2.67 (br. s., 2H);
MS (EI) m/z=165.4 [M+1].sup.+.
[0194] Methanesulfonic acid (1.0 mL, 15.4 mmol) was added to a
solution of 5-methoxy-3,3-dideutero-2-dihydro-inden-1-one (600 mg,
3.65 mmol) in DCM (40 mL), and the solution cooled to 0.degree. C.
Sodium azide (356 mg, 5.48 mmol) was slowly added portion-wise to
the stirred solution. After 6 h at 0.degree. C. the reaction was
quenched with 8N aq. NaOH (20 mL). The layers were separated, and
the aqueous layer was extracted with DCM (3.times.20 mL). The
combined organics were washed with brine (30 mL), dried over
Na.sub.2SO.sub.4 and concentrated to afford
6-methoxy-4,4-dideutero-3-hydroisoquinolin-1(2H)-one (500 mg, 76%
yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 7.99 (d,
J=8.78 Hz, 1H), 6.88 (dd, J=8.78, 2.51 Hz, 1H), 6.74 (d, J=2.51 Hz,
1H), 3.88 (s, 3H), 3.60 (s, 2H); MS (EI) m/z=180.4 [M+1].sup.+.
[0195] To a -78.degree. C. solution of
6-methoxy-4,4-dideutero-3-hydroisoquinolin-1(2H)-one (2.9 g, 16.2
mmol) in DCM (150 mL) was added a freshly prepared 1.0 M solution
of BBr.sub.3 in DCM (32.4 mL, 32.4 mmol). The reaction was allowed
to gradually warm to room temperature overnight. At completion, the
reaction was cooled to 0.degree. C. and carefully quenched with
MeOH (10 mL) then concentrated and purified by flash chromatography
over silica gel (10% MeOH/DCM eluent) to provide
6-hydroxy-4,4-dideutero-3-hydroisoquinolin-1(2H)-one as a bright
orange solid (2.33 g, 86% yield). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 7.66 (d, J=8.53 Hz, 1H), 7.60 (br. s.,
1H), 6.68 (dd, J=8.53, 2.51 Hz, 1H), 6.61 (d, J=2.26 Hz, 1H); MS
(EI) m/z=166.4 [M+1].sup.+.
[0196] A solution room temperature of
6-hydroxy-4,4-dideutero-3-hydroisoquinolin-1(2H)-one (2.75 g, 16.2
mmol) in THF (150 mL) was treated with NaH (1.62 g of 60 wt. %
dispersion in mineral oil, 40.5 mmol). The reaction was stirred for
0.5 h, then treated with PhNTf.sub.2 (17.36 g, 48.6 mmol) and the
reaction was maintained at room temperature for 2 h. At completion,
the reaction was quenched with sat. aq. NH.sub.4Cl (50 mL), then
diluted with water (50 mL) and extracted with EtOAc (3.times.100
mL). The combined organics were washed with brine (100 mL), then
dried over Na.sub.2SO.sub.4 and concentrated. The crude material
was purified by flash chromatography over silica gel (2-10%
MeOH/DCM eluent to provide
1-oxo-3-hydro-4,4-dideutero-isoquinolin-6-yl
trifluoromethanesulfonate (3.31 g, 69% yield) as a slightly red
solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.14 (br.
s., 1H), 7.99 (d, J=8.53 Hz, 1H), 7.52 (d, J=2.51 Hz, 1H), 7.45
(dd, J=8.66, 2.64 Hz, 1H), 3.38 (d, J=2.76 Hz, 2H); MS (EI)
m/z=298.2[M+1].sup.+.
[0197] A slurry of 1-oxo-3-hydro-4,4-dideutero-isoquinolin-6-yl
trifluoromethanesulfonate (3.30 g, 11.1 mmol) diboron pinacol ester
(8.45 g, 33.3 mmol), KOAc (1.63 g, 16.7 mmol) and Pd(dppf)Cl.sub.2
(814 mg, 1.1 mmol) in dioxane (50 mL) was degassed then heated to
90.degree. C. for 1.5 h. At completion, the reaction was filtered
and concentrated, then purified by flash chromatography over silica
gel (30% EtOAc/hexanes eluent) to provide
6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-hydro-4,4-dideutero-iso-
quinolin-1(2H)-one (2.2 g, 72% yield). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 7.99 (br. s, 1H), 7.80 (dd, J=1.00 Hz,
1H), 7.61 (dd, J=7.78, 5.77 Hz, 2H), 7.59 (dd, J=14.00 Hz, 2H),
3.34 (d, J=2.26 Hz, 2H), 1.29 (s, 12H); MS (EI) m/z=276.4
[M+1].sup.+. Method C was applied to
6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-hydro-4,4-dideutero-iso-
quinolin-1(2H)-one (1.14 g, 4.15 mmol) and
3,5-dibromopyrazin-2-amine (1.0 g, 3.95 mmol). At completion, the
crude reaction mixture was filtered then purified by flash
chromatography over silica gel (5-10% MeOH/DCM eluent) to provide
4-(5-amino-6-(1-oxo-3-hydro-4,4-dideutero-isoquinolin-6-yl)pyrazin-2-yl)--
N-cyclopropyl-N-Trideuteromethylbenzenesulfonamide (435 mg, 23%
yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.72 (s,
1H), 8.26 (d, J=8.53 Hz, 2H), 8.02 (br. s., 1H), 7.97 (d, J=8.03
Hz, 1H), 7.84 (d, J=8.53 Hz, 2H), 7.74 (dd, J=8.03, 1.76 Hz, 1H),
7.70 (d, J=1.51 Hz, 1H), 3.41 (d, J=2.51 Hz, 2H), 1.78-1.86 (m,
1H), 0.72-0.79 (m, 2H), 0.64-0.72 (m, 2H); MS (EI)
m/z=455.1[M+1].sup.+.
6.35. Synthesis of
4-(6-amino-5-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrid-
in-3-yl)-N-cyclopropylbenzenesulfonamide
[0198] The captioned compound was prepared by the method shown
below:
##STR00045##
[0199] A 1.0 M solution of TiCl.sub.4 in DCM (50 mL, 49.8 mmol) was
added to a reaction vessel containing DCM (40 mL) cooled to
-40.degree. C. Then, a 2.0 M solution of dimethyl zinc in toluene
(35.5 ml, 71.1 mmol) was added slowly and the solution was stirred
for 20 minutes at -40.degree. C. To this mixture was added a
solution of 6-bromo-2,3-dihydro-1H-inden-1-one (5.0 g, 23.7 mmol)
in DCM (40 ml) and the reaction was allowed to warm gradually to
room temperature overnight. At completion, the reaction was cooled
to 0.degree. C. and quenched with MeOH (10 mL), then diluted with
water (50 mL) and DCM (50 mL). The layers were separated and the
organic layer was washed with brine (50 mL), dried over
Na.sub.2SO.sub.4, filtered and concentrated.
6-bromo-1,1-dimethyl-2,3-dihydro-1H-indene (4.14 g, 78% yield) was
isolated as a clear oil after purification by flash chromatography
over silica gel (hexane eluent). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 7.22-7.33 (m, 2H), 7.07 (d, J=8.5 Hz,
1H), 2.86 (t, J=7.3 Hz, 2H), 1.95 (t, J=7.3 Hz, 2H), 1.28 (s, 6H);
MS (EI) m/z=226.2 [M+1].sup.+.
[0200] To a solution of 6-bromo-1,1-dimethyl-2,3-dihydro-1H-indene
(3.6 g, 16 mmol) in acetic acid (50 mL) was added to a solution of
CrO.sub.3 (9.6 g, 96.0 mmol) in 50% aqueous acetic acid (50 mL).
This solution was heated for 3 h at 60.degree. C. At completion,
the reaction mixture was quenched by the addition of isopropanol
(20 mL). The quenched reaction was partitioned between EtOAc (200
mL) and a 0.25 M aqueous NaOH (100 mL). The layers were separated
and the organics were extracted with EtOAc (2.times.100 mL). The
combined organics were washed with brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated. After purification by
flash chromatography over silica gel (50% EtOAc/hexanes eluent),
5-bromo-3,3-dimethyl-2,3-dihydro-1H-inden-1-one (2.91 g, 76% yield)
was isolated as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta. ppm 7.65 (s, 1H), 7.47-7.58 (m, 2H), 2.58 (s, 2H), 1.42 (s,
6H); MS (EI) m/z=240.1 [M+1].sup.+.
[0201] 5-bromo-3,3-dimethyl-2,3-dihydro-1H-inden-1-one (1.6 g, 6.7
mmol) was dissolved in DCM (12 mL) and cooled to 0.degree. C. Then
MeSO.sub.3H (1.83 ml, 28.1 mmol) was added, followed by the
portion-wise addition of NaN.sub.3 (653 mg, 10.1 mmol) over
.about.10 min, while maintaining the internal reaction temperature
below 5.degree. C. The reaction was allowed to warm to room
temperature and was stirred 3 h under nitrogen. At completion, the
reaction was cooled to 0.degree. C. and quenched with 8 M aq. NaOH.
After stirring 30 minutes at room temperature, the quenched
reaction was partitioned between DCM (50 mL) and water (50 mL). The
layers were separated and the aqueous layer was extracted with DCM
(2.times.30 mL). The combined organics were washed with brine (50
mL), dried over Na.sub.2SO.sub.4, filtered and concentrated.
6-bromo-4,4-dimethyl-3,4-dihydroisoquinolin-1(2H)-one (711 mg, 40%
yield) was isolated as a white solid after purification by flash
chromatography over silica gel (eluting in 50-100% EtOAc/hexane).
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.02 (br. s., 1H),
7.77 (d, J=8.0 Hz, 1H), 7.46-7.64 (m, 2H), 3.16 (d, J=3.0 Hz, 2H),
1.27 (s, 6H); MS (EI) m/z=255.1 [M+1].sup.+.
[0202] A solution of
6-bromo-4,4-dimethyl-3,4-dihydroisoquinolin-1(2H)-one (500 mg, 1.97
mmol), diboron pinacol ester (752 mg, 2.96 mmol), KOAc (773 mg,
7.88 mmol) and Pd(dppf)Cl.sub.2 (43 mg, 0.06 mmol) in dioxane (7
mL) was heated for 2 hours at 100.degree. C. under reflux. At
completion the reaction was cooled to room temperature, filtered
and concentrated the purified by flash chromatography over silica
gel, (2-5% DCM/MeOH eluent). The product containing fractions were
concentrated, taken up in hexanes, sonicated and filtered to afford
4,4-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydroi-
soquinolin-1(2H)-one (511 mg, 86% yield) as a white solid. .sup.1H
NMR (300 MHz, CHLOROFORM-d) .delta. ppm 8.09 (d, J=7.6 Hz, 1H),
7.71-7.87 (m, 2H), 3.33 (d, J=3.1 Hz, 2H), 1.39 (m, J=8.0 Hz, 18H);
MS (EI) m/z=302.2 [M+1].sup.+.
[0203] General method C was applied to
4-(6-amino-5-bromopyridin-3-yl)-N-cyclopropylbenzenesulfonamide
hydrobromide (92 mg, 0.25 mmol) and
4,4-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydroi-
soquinolin-1(2H)-one (75 mg, 0.25 mmol) to give
4-(6-amino-5-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrid-
in-3-yl)-N-cyclopropylbenzenesulfonamide (43 mg, 37% yield) as a
white solid after purification by flash chromatography over silica
gel (2-5% MeOH/DCM eluent). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 8.61 (s, 1H), 8.06 (d, J=8.5 Hz, 2H), 7.96-8.02 (m,
2H), 7.80 (d, J=1.5 Hz, 1H), 7.75 (dd, J=7.5, 1.5 Hz, 1H), 7.62 (d,
J=8.5 Hz, 2H), 6.00 (s, 2H), 3.20 (d, J=2.0 Hz, 2H), 2.06-2.18 (m,
1H), 1.33 (s, 6H), 0.33-0.55 (m, 4H); MS (EI) m/z=463.6
[M+1].sup.+.
6.36. Synthesis of
1-(4-(5-amino-6-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquin-
olin]-6'-yl)pyrazin-2-yl)phenyl)cyclopropanecarbonitrile
[0204] The captioned compound was prepared by the method shown
below:
##STR00046##
[0205] A 2.5 M solution of n-BuLi in hexanes (23.6 mL, 59 mmol) was
added dropwise to a 0.degree. C. slurry of
methyltriphenylphosphonium bromide (16.8 g, 47.0 mmol) in THF (200
mL). After 1 h, a solution of 6-bromo-2,3-dihydro-1H-inden-1-one
(10.0 g, 47.0 mmol) in THF (50 mL) was added to the resulting
solution and the reaction cold bath was removed. Upon completion,
the reaction was quenched with water (40 mL). The layers were
separated and the aqueous layer was extracted with EtOAc
(2.times.40 mL). The combined organics were washed with brine (50
mL), dried over Na.sub.2SO.sub.4, filtered and concentrated to
afford 6-bromo-1-methylene-2,3-dihydro-1H-indene (6.06 g, 62%
yield) as a clear oil after purification by flash chromatography
over silica gel (hexane eluent). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 7.61 (d, J=1.5 Hz, 1H), 7.32 (dd, J=8.0,
1.8 Hz, 1H), 7.13 (d, J=8.0 Hz, 1H), 5.45 (t, J=2.5 Hz, 1H), 5.08
(t, J=2.1 Hz, 1H), 2.88-2.98 (m, 2H), 2.78-2.86 (m, 2H); MS (EI)
m/z=210.1 [M+1].sup.+.
[0206] A 1.1 M toluene solution of diethyl zinc (104.0 mL, 114.8
mmol) was added to a reaction vessel containing DCM (100 mL) and
cooled to 0.degree. C. TFA (8.9 ml, 114.8 mmol) was added to the
resulting solution and the reaction was stirred at 0.degree. C. for
15 min. To the cooled solution was added CH.sub.2I.sub.2 (9.26 mL,
114.8 mmol), and the reaction was stirred for an additional 15
minutes at 0.degree. C. Then, a solution of
6-bromo-1-methylene-2,3-dihydro-1H-indene (6.0 g, 28.7 mmol) in DCM
(90 mL) was added and the reaction was maintained 0.degree. C. for
another 15 min, then allowed to gradually warm to room temperature.
Upon completion, the reaction was quenched with a sat. aq.
NH.sub.4Cl (100 mL) and diluted with DCM (200 mL). The layers were
separated and the aqueous layer was washed with DCM (2.times.100
mL). The combined organics were washed with brine (100 mL), dried
over Na.sub.2SO.sub.4, then filtered and concentrated.
6'-bromo-2',3'-dihydrospiro[cyclopropane-1,1'-indene (5.9 g, 93%
yield) was obtained after purification by flash chromatography
(hexanes eluent) as a clear oil. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 7.22 (dd, J=7.9, 1.9 Hz, 1H), 7.05 (d,
J=7.8 Hz, 1H), 6.78 (d, J=1.8 Hz, 1H), 2.99 (t, J=7.5 Hz, 2H),
2.06-2.20 (t, 2H), 0.83-1.03 (m, 4H); MS (EI) m/z=224.1
[M+1].sup.+.
[0207] To a solution of
6'-bromo-2',3'-dihydrospiro[cyclopropane-1,1'-indene] (5.7 g, 25.6
mmol) in acetone (78 mL) and 1.5 M aq. MgSO.sub.4 (29 mL) was added
KMnO.sub.4 (4.46 g, 28.2 mmol). The reaction was stirred overnight
at room temperature. Upon completion, the reaction was filtered
over celite and concentrated. The residue was partitioned between
water (100 mL) and EtOAc (200 mL), and the layers were separated.
The organic was washed with brine, dried over Na.sub.2SO.sub.4,
then filtered and concentrated.
6'-bromospiro[cyclopropane-1,1'-inden]-3'(2'H)-one (2.11 g, 39%
yield) was isolated as a white solid after flash chromatography
over silica gel (5% EtOAc/hexane eluent). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 7.59 (d, J=8.3 Hz, 1H), 7.45 (dd, J=8.0,
1.5 Hz, 1H), 7.14 (d, J=1.3 Hz, 1H), 2.76 (s, 2H), 1.15-1.34 (m,
4H); MS (EI) m/z=238.1 [M+1].sup.+.
[0208] A solution of
6'-bromospiro[cyclopropane-1,1'-inden]-3'(2'H)-one (2.1 g, 8.86
mmol), hydroxylamine HCl (1.23 g, 17.7 mmol) and NaOAc (4.32 g,
53.1 mmol) in MeOH (160 mL) was stirred overnight at room
temperature. The reaction was concentrated, slurried water (40 mL),
then sonicated and filtered. The precipitate was dried under high
vacuum to give
(E)-6'-bromospiro[cyclopropane-1,1'-inden]-3'(2'H)-one oxime (2.23
g, 100% yield) as a white solid. MS (EI) m/z=253.1[M+1].sup.+.
[0209] To a 0.degree. C. solution of
(E)-6'-bromospiro[cyclopropane-1,1'-inden]-3'(2'H)-one oxime (500
mg, 1.98 mmol) in THF (5 mL) was added triethylamine (332 .mu.L,
2.38 mmol) followed by slow addition of MsCl (170 .mu.L, 2.18
mmol). The reaction was stirred 10 minutes at 0.degree. C. then
concentrated. The residue was triturated from methanol (5 mL) to
afford (E)-6'-bromospiro[cyclopropane-1,1'-inden]-3'(2'H)-one
O-methylsulfonyl oxime (653 mg, 100% yield) as a white solid after
drying under high vacuum. .sup.1H NMR (300 MHz, CHLOROFORM-d)
.delta. ppm 7.64 (d, J=8.4 Hz, 1H), 7.42 (dd, J=8.3, 1.6 Hz, 1H),
6.98 (d, J=1.5 Hz, 1H), 3.26 (s, 3H), 3.15 (s, 2H), 1.06-1.42 (m,
4H); MS (EI) m/z=331.2 [M+1].sup.+.
[0210] A solution of
(E)-6'-bromospiro[cyclopropane-1,1'-inden]-3'(2'H)-one-O-methylsulfonyl
oxime (653 mg, 1.98 mmol) in DCM (10 mL) was cooled to 0.degree. C.
BF.sub.3.MeOH (362 .mu.L, 4.36 mmol) was added to the cooled
solution, followed by slow addition of TiCl.sub.4 (304 .mu.L, 2.77
mmol) and the reaction was maintained at 0.degree. C. for 4 h. At
completion, the reaction was quenched with water (10 mL). The
layers were separated and aqueous layer was washed with DCM
(2.times.10 mL). The combined organics were dried over
Na.sub.2SO.sub.4, filtered and concentrated.
6'-bromo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinolin]-1'-one
(228 mg, 0.90 mmol, 46% yield) was isolated as a white solid after
purification by flash chromatography (50-100% EtOAc/hexane eluent).
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 7.98 (d, J=8.3 Hz,
1H), 7.46 (dd, J=8.3, 1.8 Hz, 1H), 7.01 (d, J=1.8 Hz, 1H), 5.94
(br. s., 1H), 3.37 (d, J=2.8 Hz, 3H), 0.94-1.20 (m, 4H); MS (EI)
m/z=253.1 [M+1].sup.+.
[0211] General Method G was applied to
6'-bromo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinolin]-1'-one
(228 mg, 0.90 mmol) to provide after purification by flash
chromatography over silica gel (50-100% EtOAc/hexanes),
6'-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2',3'-dihydro-1'H-spiro[-
cyclopropane-1,4'-isoquinolin]-1'-one (199 mg, 74% yield) as a
white solid. .sup.1H NMR (300 MHz, CHLOROFORM-d) .delta. ppm 8.12
(d, J=7.6 Hz, 1H), 7.77 (dd, J=7.6, 1.0 Hz, 1H), 7.30 (s, 1H), 3.37
(d, J=2.3 Hz, 2H), 1.36 (s, 12H), 0.94-1.22 (m, 4H); MS (EI)
m/z=300.2[M+1].sup.+.
[0212] General method C was applied to
1-(4-(5-amino-6-bromopyrazin-2-yl)phenyl)cyclopropanecarbonitrile
(95 mg, 0.30 mmol) and
6'-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2',3'-dihydro-1'H-spiro]-
[cyclopropane-1,4'-isoquinolin]1'-one (100 mg, 0.33 mmol) to
provide
1-(4-(5-amino-6-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquin-
olin]-6'-yl)pyrazin-2-yl)phenyl)cyclopropanecarbonitrile (44 mg,
36% yield) after flash chromatography over silica gel (2-5%
MeOH/DCM eluent). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
0.76-1.07 (m, 4H) 1.08-1.35 (m, 4H) 1.44-1.63 (m, 2H) 1.65-1.86 (m,
2H) 6.37 (s, 1H) 7.10-7.51 (m, 3H) 7.68 (d, J=7.78 Hz, 1H)
7.83-8.28 (m, 4H) 8.57 (s, 1H). MS (EI) m/z: 408 [M+H].sup.+. HPLC
(Sunfire C18 4.6 mm.times.50 mm, 10-90% MeCN:10 mM aq NH.sub.4OAc,
2 min gradient) t.sub.R=1.69 min, 100% integrated area.
6.37. Synthesis of
4-(5-amino-6-(3-oxospiro[cyclopropane-1,1'-isoindolin]-6-Y.sub.1)pyrazin--
2-yl)-N-cyclopropylbenzenesulfonamide
[0213] The captioned compound was prepared by the method shown
below:
##STR00047##
[0214] To a solution of methyl 4-chloro-2-cyanobenzoate (2.0 g,
10.2 mmol) in Et.sub.2O (40 mL) was added Ti(OiPr).sub.4 (3.43 ml,
11.7 mmol). After cooling to 0.degree. C., a 3.0 M solution of
EtMgBr in Et.sub.2O (6.8 ml, 20.4 mmol) was added slowly. The
reaction was warmed to room temperature and stirred for 3 h. Upon
completion, the reaction was quenched with 1.0 M HCl (20 mL) then
filtered over celite. The layers were separated and the aqueous
layer was washed with EtOAc (2.times.20 mL). The combined organics
were dried over Na.sub.2SO.sub.4, then filtered and concentrated.
The crude product was purified by flash chromatography over silica
gel (2-5% MeOH/DCM eluent) to provide
6'-chlorospiro[cyclopropane-1,1'-isoindolin]-3'-one (400 mg, 21%
yield) as a yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 8.75 (br. s., 1H), 7.66 (d, J=8.5 Hz, 1H), 7.40-7.54
(m, 2H), 1.47 (dt, J=11.2, 2.8 Hz, 4H); MS m/z C.sub.10H.sub.8ClNO
[M+1].sup.+=194.6.
[0215] A solution of
6'-chlorospiro[cyclopropane-1,1'-isoindolin]-3'-one (380 mg, 1.96
mmol), bis(pinacolato)diboron (808 mg, 3.18 mmol), KOAc (677 mg,
6.9 mmol), P(Cy).sub.3 (137 mg, 0.49 mmol) and Pd(dba).sub.3 (180
mg, 0.20 mmol) in dioxane (8 ml) was heated overnight at
100.degree. C. under a reflux condenser. At completion, the
reaction was allowed to cool to room temperature, diluted in DCM
(40 mL), sonicated and filtered. The filtrate was purified by flash
chromatography over silica gel (50/100% EtOAc/hexane eluent). The
product containing fractions were taken up in hexanes, then
sonicated and filtered to afford
6'-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)spiro[cyclopropane-1,1'-i-
soindolin]-3'-one (520 mg, 93% yield) as a yellow solid. .sup.1H
NMR (400 MHz, METHANOL-d.sub.4) .delta. ppm 7.70-7.91 (m, 2H), 7.56
(s, 1H), 1.46-1.68 (m, 4H), 1.15-1.43 (m, 12H); MS (EI) m/z=286.1
[M+1].sup.+.
[0216] General method B was applied to
4-(5-amino-6-bromo-pyrazin-2-yl)-N-cyclopropyl-benzenesulfonamide
(140 mg, 0.38 mmol) and
6'-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)spiro[cyclopropane-1-1'-i-
soindolin]3'-one (120 mg, 0.42 mmol) to provide
4-(5-amino-6-(3'-oxospiro[cyclopropane-1,1'-isoindolin]-6'-yl)pyrazin-2-y-
l)-N-cyclopropylbenzenesulfonamide (58 mg, 34% yield) after
purification by flash chromatographyl over silica gel (2-5%
MeOH/DCM eluent). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
0.29-0.58 (m, 4H) 1.36-1.68 (m, 4H) 2.13 (d, J=2.51 Hz, 1H) 6.62
(s, 1H) 7.62 (s, 1H) 7.74-7.96 (m, 5H) 8.21 (d, J=8.53 Hz, 2H) 8.70
(s, 1H) 8.75 (s, 1H) 8.96-9.32 (m, 1H). MS (EI) m/z: 448
[M+H].sup.+. HPLC (Sunfire C18 4.6 mm.times.50 mm, 10-90% MeCN:10
mM aq NH.sub.4OAc, 2 min gradient) t.sub.R=1.47 min, 97% integrated
area.
6.38. Synthesis of
5-(3-amino-6-(4-(N-cyclopropyl-N-methylsulfamoyl)phenyl)pyrazin-2-yl)-N-m-
ethylindoline-1-carboxamide
[0217] The captioned compound was prepared by the method shown
below:
##STR00048##
[0218] To a solution of 5-bromoindoline (1.0 g, 5.05 mmol) and DIEA
(1.9 mL, 11.11 mmol) in THF (40 mL) was added methylisocyanate (346
mg, 6.06 mmol). The reaction was maintained at room temperature for
4 h. The resulting precipitate was filtered and dried under vacuum
to afford 5-bromo-N-methylindoline-1-carboxamide (935 mg, 72%
yield) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 7.75 (d, J=8.78 Hz, 1H), 7.17-7.31 (m, 2H), 6.60 (d,
J=4.27 Hz, 1H), 3.85 (t, J=8.78 Hz, 2H), 3.10 (t, J=8.66 Hz, 2H),
2.65 (d, J=4.27 Hz, 3H); MS (EI) m/z=255.1[M+1].sup.+.
[0219] General method G was applied to
5-bromo-N-methylindoline-1-carboxamide (935 mg, 3.67 mmol) to
provide
N-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline-1-carbox-
amide as a white foam (1.10 g, 100% yield) after purification by
flash chromatography (5% MeOH/DCM). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 1.34 (s, 12H) 3.10-3.22 (m, 2H) 3.90 (s,
2H) 4.54-4.66 (m, 1H) 7.60 (br. s, 1H) 7.66 (br. d, J=1.00 Hz, 1H)
7.85 (br. d, J=1.00 Hz, 1H) 7.84-7.91 (m, 1H); MS (EI) m/z=301.0
[M-1].sup.-.
[0220] Method C was applied to
N-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline-1-carbox-
amide (85 mg, 0.28 mmol) and
4-(5-amino-6-bromopyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
(100 mg, 0.26 mmol). The organic layer was decanted then triturated
with Et.sub.2O to provide
5-(3-amino-6-(4-(N-cyclopropyl-N-methylsulfamoyl)phenyl)pyrazin-2-yl)-N-m-
ethylindoline-1-carboxamide as a yellow solid (35 mg, 28% yield).
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.58-8.66 (m, 1H),
8.20-8.31 (m, 1H), 7.92-7.99 (m, 1H), 7.79-7.88 (m, 2H), 7.47-7.64
(m, 2H), 6.61-6.71 (m, 1H), 6.60-6.65 (m, 1H), 6.58-6.71 (m, 2H),
6.42-6.49 (m, 2H), 3.88-3.98 (m, 2H), 3.13-3.25 (m, 2H), 2.68 (s,
3H), 2.08 (s, 3H), 1.79-1.88 (m, 1H), 0.62-0.81 (m, 4H); MS (EI)
m/z=479.1[M+1].sup.+.
6.39. Synthesis of
4-(5-amino-6-(8-oxo-5,6,7,8-tetrahydro-2,7-naphthyridin-3-yl)pyrazin-2-yl-
)-N-cyclopropylbenzenesulfonamide
[0221] The captioned compound was prepared by the method shown
below:
##STR00049##
[0222] To a solution of
6-chloro-1,2,3,4-tetrahydro-2,7-naphthyridine hydrochloride (5.0 g,
24.4 mmol) and ditertbutyl dicarbonate (8.0 g, 36.6 mmol) in DCM
(100 mL) at room temperature was added triethylamine (10.2 mL, 73.2
mmol). Upon completion, the reaction was diluted in DCM (100 mL),
washed with a sat. aq. NaHCO.sub.3, dried over Na.sub.2SO.sub.4,
filtered and concentrated. This residue was purified by flash
chromatography over silica gel (20% EtOAc/hexane eluent) to afford
tert-butyl 6-chloro-3,4-dihydro-2,7-naphthyridine-2(1H)-carboxylate
(4.0 g, 61% yield) as a clear oil. .sup.1H NMR (400 MHz,
METHANOL-d.sub.4) .delta. ppm 8.17 (s, 1H), 7.30 (s, 1H), 4.60 (s,
2H), 3.66 (t, J=5.9 Hz, 2H), 2.88 (t, J=5.8 Hz, 2H), 1.43-1.61 (m,
9H); MS (EI) m/z=269.7 [M+1].sup.+.
[0223] To a solution of NaIO.sub.4 (9.56 g, 44.7 mmol) and
RuCl.sub.3 (927 mg, 4.47 mmol) in water (50 mL), DCM (50 mL) and
MeCN (2 mL) was added a solution of tert-butyl
6-chloro-3,4-dihydro-2,7-naphthyridine-2(1H)-carboxylate (4.0 g,
14.9 mmol) in DCM (25 mL). This reaction was stirred at room
temperature for 2 h. At completion, the reaction was and was
quenched with isopropanol (10 mL), filtered over celite and the
filter cake was washed with DCM (20 mL). The filtrate was
partitioned in a separatory funnel and the layers were separated.
The organic layer was extracted with DCM (2.times.20 mL) and the
combined organics were dried over Na.sub.2SO.sub.4, then filtered
and concentrated. The residue was purified by flash chromatography
over silica gel (20-50% EtOAc/hexane eluent) to give tert-butyl
6-chloro-1-oxo-3,4-dihydro-2,7-naphthyridine-2(1H)-carboxylate (3.0
g, 71% yield) as a yellow crystalline solid. .sup.1H NMR (400 MHz,
METHANOL-d.sub.4) .delta. ppm 8.92 (s, 1H), 7.47 (s, 1H), 4.03 (t,
J=6.3 Hz, 2H), 3.09 (t, J=6.1 Hz, 2H), 1.54-1.62 (m, 9H); MS (EI)
m/z=283.7 [M+1].sup.+.
[0224] To a solution of tert-butyl
6-chloro-1-oxo-3,4-dihydro-2,7-naphthyridine-2(1H)-carboxylate (3.0
g, 10.6 mmol) in DCM (20 mL) was added 4N HCl solution in dioxane
(10 mL). After stirring 20 minutes at room temperature, the
reaction was diluted with DCM (50 mL, then sonicated and filtered
to give 6-chloro-3,4-dihydro-2,7-naphthyridin-1(2H)-one
hydrochloride (2.22 g, 93% yield) as a white solid. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. ppm 8.70 (s, 1H), 8.23 (br. s.,
1H), 7.57 (s, J=0.6 Hz, 1H), 3.40 (t, J=6.6, 2.8 Hz, 2H), 2.96 (t,
J=6.6 Hz, 2H); MS (EI) m/z=183.6 [M+1].sup.+.
[0225] A solution of
6-chloro-3,4-dihydro-2,7-naphthyridin-1(2H)-one hydrochloride (800
mg, 3.7 mmol), hexamethylditin (1.83 g, 5.6 mmol) and
Pd(PPh.sub.3).sub.4 (214 mg, 0.19 mmol) in dioxane (40 mL) was
degassed under bubbling nitrogen then heated at 110.degree. C. for
4 h. At completion, the reaction was concentrated and purified by
flash chromatography over neutral alumina (100% EtOAc eluent, then
5% MeOH/DCM eluent). This yielded
6-(trimethylstannyl)-3,4-dihydro-2,7-naphthyridin-1(2H)-one (1.22
g, 100% yield) as a brown oil that was used without further
purification. MS (EI) m/z [M+1].sup.+=312.
[0226] A solution of
4-(5-amino-6-bromopyrazin-2-yl)-N-cyclopropylbenzenesulfonamide
(150 mg, 0.41 mmol),
6-(trimethylstannyl)-3,4-dihydro-2,7-naphthyridin-1(2H)-one (150
mg, 0.48 mmol) and Pd(PPh.sub.3).sub.4 (23 mg, 0.02 mmol) in
dioxane (2 mL) was heated in the microwave 20 min at 150.degree. C.
At completion, the reaction mixture was purified by reverse phase
preparatory HPLC to provide
4-(5-amino-6-(8-oxo-5,6,7,8-tetrahydro-2,7-naphthyridin-3-yl)pyrazin-2-yl-
)-N-cyclopropylbenzenesulfonamide (8 mg, 5% yield) as an orange
solid after lyophilization. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 9.02 (s, 1H), 8.87 (s, 1H), 8.59 (s, 1H), 8.35 (d,
J=8.5 Hz, 2H), 8.17 (br. s., 1H), 7.84-7.99 (m, 3H), 3.51 (t, 2H),
(t, J=6.3 Hz, 2H), 0.29-0.57 (m, 4H); MS (EI) m/z=437.5
[M+1].sup.+.
6.40. Synthesis of
4-(5-amino-6-(4-bromo-1-hydroxyisoquinolin-6-yl)pyrazin-2-yl)-N-cycloprop-
yl-N-methylbenzenesulfonamide
[0227] The captioned compound was prepared by the method shown
below:
##STR00050##
[0228] To a room temperature slurry of
4-(5-amino-6-(1-hydroxyisoquinolin-6-yl)pyrazin-2-yl)-N-cyclopropyl-N-met-
hylbenzenesulfonamide hydrochloride (1.00 g, 2.07 mmoL, prepared by
general method C) in DMF (10 mL) was added
1,1-dimethoxy-N,N-dimethylmethanamine (1 mL). Within minutes, the
slurry discharged to a solution. Then, N-bromosuccinimide (386 mg,
2.17 mmol) was added in one portion. After 1 h the reaction was
concentrated and flashed over silica gel (4-8% MeOH/DCM eluent) to
provide
4-(5-amino-6-(4-bromo-1-hydroxyisoquinolin-6-yl)pyrazin-2-yl)-N-cycloprop-
yl-N-methylbenzenesulfonamide hydrochloride (480 mg, 43% yield).
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 11.59-11.67 (br. s,
1H), 8.77 (s, 1H), 8.36 (d, J=8.3 Hz, 1H), 8.24-8.30 (m, 2H), 8.17
(d, J=1.5 Hz, 1H), 7.99 (dd, J=8.3, 1.5 Hz, 1H), 7.86 (d, J=8.5 Hz,
2H), 7.62 (d, J=6.0 Hz, 1H), 6.76 (br. s, 2H), 2.69 (s, 3H),
1.80-1.90 (m, 1H), 0.76 (s, 2H), 0.65-0.73 (m, 2H); MS (EI)
m/z=527.4 [M+1].sup.+.
6.41. Additional Compounds
[0229] Numerous additional compounds were prepared, some of which
are listed below in Table 1.
TABLE-US-00001 TABLE 1 IC.sub.50 (.mu.M) Compound 1 .mu.M ATP 50
.mu.M ATP 4-(6-amino-6'-fluoro-[3,3'-bipyridin]-5-yl)benzamide --
-- 4-(2-amino-5-(1H-pyrazol-4-yl)pyridin-3yl)benzamide -- *
4-(2-amino-5-(4-sulfamoylphenyl)pyridin-3-yl)benzamide -- *
4-(2-amino-5-(1-(N,N-dimethylsulfamoyl)-1H-imidazol-4-yl)pyridin-3-
-- -- yl)benzamide
4-(5-(4-(1H-imidazol-1-yl)phenyl)-2-aminopyridin-3-yl)benzamide --
* 4-(2-amino-5-(4-benzoylphenyl)pyridin-3-yl)benzamide -- *
4-(2-amino-5-(4-fluorophenyl)pyridin-3-yl)-2-ethoxybenzamide -- *
3-amino-N-(pentan-2-yl)-6-(3,4,5-trimethoxyphenyl)pyrazine-2- -- *
carboxamide 3-amino-N-cyclohexyl-6-(3,4-dimethoxyphenyl)pyrazine-2-
-- * carboxamide
4-(2-amino-5-(4-(2-oxo-2-(pyrrolidin-1-yl)ethyl)phenyl)pyridin-3-
-- * yl)benzamide
4-(2-amino-5-(4-(N-methylsulfamoyl)phenyl)pyridin-3-yl)benzamide --
* 3-amino-N-cyclohexyl-6-(4-(methoxymethyl)phenyl)pyrazine-2- -- *
carboxamide
4-(2-amino-5-(4-(benzylcarbamoyl)phenyl)pyridin-3-yl)-N- -- *
hydroxybenzamide
6-(2-amino-5-(4-fluorophenyl)pyridin-3-yl)-3,4-dihydroisoquinolin-
* ** 1(2H)-one
4-(6-amino-5-(4-hydroxyquinazolin-7-yl)pyridin-3-yl)-N,N- ** ***
diethylbenzenesulfonamide
4-(2-amino-5-(4-(N,N-diethylsulfamoyl)phenyl)pyridin-3-yl)-2- -- **
fluorobenzamide
4-(2-amino-5-(4-(N-cyclopropylsulfamoyl)phenyl)pyridin-3- ** **
yl)benzamide
4-(6-amino-5-(3-hydroxy-1H-indazol-6-yl)pyridin-3-yl)-N,N- -- *
diethylbenzenesulfonamide
3-amino-N-cyclohexyl-6-(4-(N-ethylsulfamoyl)phenyl)pyrazine-2- * **
carboxamide
4-(6-amino-5-(2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-7- -- *
yl)pyridin-3-yl)-N,N-diethylbenzenesulfonamide
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
*** *** N-cyclopropylbenzenesulfonamide
2'-amino-5'-(4-(N,N-diethylsulfamoyl)phenyl)-[2,3'-bipyridine]-5-
** *** carboxamide
4-(6-amino-5-(3-oxo-2,3-dihydrobenzo[d]isoxazol-6-yl)pyridin-3-yl)-
-- * N,N-diethylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N,N-di(trideuteromethyl)benzenesulfonamide
4-(5-(4-(N-acetylsulfamoyl)phenyl)-2-aminopyridin-3-yl)benzamide --
* 4-(6-amino-5-(4-hydroxyquinazolin-7-yl)pyridin-3-yl)-N- ** ***
ethylbenzenesulfonamide
2'-amino-5'-(4-(N-ethylsulfamoyl)phenyl)-[2,3'-bipyridine]-5- **
*** carboxamide
4-(2-amino-5-(2-chloro-4-(N-cyclopropylsulfamoyl)phenyl)pyridin-3-
-- * yl)benzamide
4-(2-amino-5-(4-(2-(dimethylamino)-2-oxoethoxy)phenyl)pyridin-3- --
* yl)benzamide 3-amino-N-(4-(aminomethyl)cyclohexyl)-6-(4-(N- **
*** cyclopropylsulfamoyl)phenyl)pyrazine-2-carboxamide
4-(2-amino-5-(4-propoxyphenyl)pyridin-3-yl)benzamide -- *
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-cyclopropyl-N-trideuteromethylbenzenesulfonamide
6-(2-amino-5-(4-(ethylsulfonyl)phenyl)pyridin-3-yl)-3,4- ** **
dihydroisoquinolin-1(2H)-one
4-(5-amino-6-(5-phenyl-1H-imidazol-2-yl)pyrazin-2-yl)-N- -- **
cyclopropylbenzenesulfonamide
5-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
* ** N-(2,2,2-trifluoroethyl)thiophene-2-carboxamide
6-(2-amino-5-(5-methylthiophen-2-yl)pyridin-3-yl)-3,4- ** ***
dihydroisoquinolin-1(2H)-one
6-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-cyclopropyl-N-trideuteromethylpyridine-3-sulfonamide
6-(2-amino-5-(2-(dimethylamino)thiazol-4-yl)pyridin-3-yl)-3,4- --
** dihydroisoquinolin-1(2H)-one
(E)-4-(6-amino-5-(5-(hydroxyimino)-5,6,7,8-tetrahydronaphthalen-2-
** *** yl)pyridin-3-yl)-N-ethylbenzenesulfonamide
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
*** *** N-cyclopropyl-N-methylbenzenesulfonamide
N-cyclopropyl-4-(3-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-1H-
** *** pyrrolo[2,3-b]pyridin-5-yl)benzenesulfonamide methyl
2-(4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6- * **
yl)pyridin-3-yl)-N-methylphenylsulfonamido)acetate
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
*** *** N-ethyl-N-methylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-cyclopropyl-N-methylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-ethyl-N-methylbenzenesulfonamide
6'-amino-N-ethyl-N-methyl-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-
*** *** 6-yl)-[2,3'-bipyridine]-5-sulfonamide
6-(3-amino-6-(4-((cyclopropylmethyl)sulfonyl)phenyl)pyrazin-2-yl)-
** *** 3,4-dihydroisoquinolin-1(2H)-one
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
** *** N-cyclopropyl-N-ethylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-(cyclopropylmethyl)-N-ethylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
** *** N-(2,2-difluoroethyl)-N-methylbenzenesulfonamide
6-(5-(4-(4-azaspiro[2.4]heptan-4-ylsulfonyl)phenyl)-2-aminopyridin-
** *** 3-yl)-3,4-dihydroisoquinolin-1(2H)-one
5-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
** *** N-ethyl-N-methylthiophene-2-sulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-methyl-N-propylbenzenesulfonamide
6-(2-amino-5-(1,1-dioxido-2,3-dihydrobenzo[d]isothiazol-5- ** ***
yl)pyridin-3-yl)-3,4-dihydroisoquinolin-1(2H)-one
4-(5-amino-6-(1-oxo-1,2-dihydroisoquinolin-6-yl)pyrazin-2-yl)-N-
*** *** cyclopropyl-N-methylbenzenesulfonamide
4-(5-amino-6-(1-hydroxyisoquinolin-6-yl)pyrazin-2-yl)-N-ethyl-N-
*** *** methylbenzenesulfonamide
6'-amino-N-cyclopropyl-N-methyl-5'-(1-oxo-1,2,3,4- *** ***
tetrahydroisoquinolin-6-yl)-[2,3'-bipyridine]-5-sulfonamide
4-(6-amino-5-(7-oxo-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2- **
*** yl)pyridin-3-yl)-N-ethyl-N-methylbenzenesulfonamide
6-(3-amino-6-(4-(piperidin-1-ylsulfonyl)phenyl)pyrazin-2- *** ***
yl)isoquinolin-1(2H)-one
4-(6-amino-5-(1-oxo-1,2-dihydroisoquinolin-6-yl)pyridin-3-yl)-N-
*** *** cyclopropyl-N-methylbenzenesulfonamide
6'-amino-N-cyclopropyl-N-methyl-5'-(1-oxo-1,2,3,4- *** ***
tetrahydroisoquinolin-6-yl)-[3,3'-bipyridine]-6-sulfonamide
4-(3-amino-6-(4-(N-cyclopropyl-N-methylsulfamoyl)phenyl)pyrazin- **
*** 2-yl)-2-isopropoxybenzamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-trideuteromethyl-N-(2-dideutero-propyl)benzenesulfonamide
N-(4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-
** *** yl)phenyl)ethanesulfonamide
6'-amino-N-cyclopropyl-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-
*** *** [2,3'-bipyridine]-5-sulfonamide tert-butyl
6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-5',6'- * **
dihydro-[3,4'-bipyridine]-1'(2'H)-carboxylate
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-methyl-N-((1R,2R)-2-methylcyclopropyl)benzenesulfonamide
6-(2-amino-5-(2-oxoindolin-5-yl)pyridin-3-yl)-3,4-dihydroisoquinolin-
** ** 1(2H)-one
6-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-ethyl-N-methylpyridine-3-sulfonamide
6-amino-N,N-dimethyl-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-
* ** 5',6'-dihydro-[3,4'-bipyridine]-1'(2'H)-sulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-cyclopropyl-2-fluoro-N-methylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-cyclobutyl-N-methylbenzenesulfonamide
6-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-cyclopropyl-N-methylpyridine-3-sulfonamide
4-(2-amino-1'-(2-cyanoacetyl)-1',2',3',6'-tetrahydro-[3,4'-bipyridin]-
* * 5-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
5-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
** *** N-cyclopropyl-N-methylpyridine-2-sulfonamide
6'-amino-N-methyl-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-N-
*** *** propyl-[2,3'-bipyridine]-5-sulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-cyclopropylbenzenesulfonamide
6-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
* ** N-((1S,2S)-2-(methoxymethyl)cyclopropyl)-N-methylpyridine-3-
sulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** 2-chloro-N-ethyl-N-methylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-ethyl-N,2-dimethylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-ethylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
** *** yl)benzonitrile
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
** *** N-cyclobutyl-N-(2,2,2-trifluoroethyl)benzenesulfonamide
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
*** *** N-cyclobutylbenzenesulfonamide
2-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
** *** yl)phenyl)-2-methylpropanenitrile
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
*** *** 2-chloro-N-cyclopropylbenzenesulfonamide
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
*** *** N-cyclopropyl-2-fluorobenzenesulfonamide
6-(3-amino-6-(1-methyl-1H-pyrazol-4-yl)pyrazin-2-yl)-3,4- * **
dihydroisoquinolin-1(2H)-one
N-allyl-4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6- ***
*** yl)pyridin-3-yl)-N-methylbenzenesulfonamide
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
*** *** N-cyclopropyl-N,2-dimethylbenzenesulfonamide
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** 2-chloro-N-methyl-N-propylbenzenesulfonamide
6-(3-amino-6-(4-(2,2,2-trifluoroacetyl)phenyl)pyrazin-2-yl)-3,4- *
** dihydroisoquinolin-1(2H)-one
4-(5-amino-6-(4,4-dideutero-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
*** *** yl)pyrazin-2-yl)-N-cyclopropyl-N-trideuteromethyl-
benzenesulfonamide
4-(5-amino-6-((2-hydroxypyridin-4-yl)ethynyl)pyrazin-2-yl)-N- * **
cyclopropyl-N-methylbenzenesulfonamide
6-(2-amino-5-(4-((cyclopropyl(methyl)amino)methyl)phenyl)pyridin-
** *** 3-yl)-3,4-dihydroisoquinolin-1(2H)-one
6'-amino-N-cyclopropyl-4-methyl-5'-(1-oxo-1,2,3,4- *** ***
tetrahydroisoquinolin-6-yl)-[2,3'-bipyridine]-5-sulfonamide
6-(3-amino-6-(4-(tert-butyl)phenyl)pyrazin-2-yl)-3,4- ** ***
dihydroisoquinolin-1(2H)-one
6-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
*** *** N-ethyl-N,4-dimethylpyridine-3-sulfonamide
1-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
*** *** yl)phenyl)cyclopentanecarbonitrile
4-(6-amino-5-(1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-7- *** ***
yl)pyridin-3-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
1-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
** *** yl)phenyl)cyclopropanecarbonitrile
4-(5-amino-6-(1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-7- ** ***
yl)pyrazin-2-yl)-N-ethyl-N-methylbenzenesulfonamide
1-(4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-
** ** yl)phenyl)cyclopropanecarbonitrile
2-(4-(5-amino-6-(1-oxo-1,2-dihydroisoquinolin-6-yl)pyrazin-2- **
*** yl)phenyl)-2-methylpropanenitrile
6-(3-amino-6-(4-(1-cyclopropylethoxy)phenyl)pyrazin-2-yl)-3,4- **
*** dihydroisoquinolin-1(2H)-one
6-(2-amino-5-(4-(2-(dimethylamino)propan-2-yl)phenyl)pyridin-3-yl)-
** *** 3,4-dihydroisoquinolin-1(2H)-one
2-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
** *** yl)phenoxy)-2-methylpropanamide ethyl
2-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6- * **
yl)pyrazin-2-yl)phenyl)acetate
2-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
* ** yl)-1H-pyrazol-1-yl)acetonitrile
4-(5-amino-6-(1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-7- *** ***
yl)pyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
2-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
** *** yl)-2-methylphenyl)-2-methylpropanenitrile
2-(6'-amino-5'-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)-[3,3'- **
** bipyridin]-6-yl)-2-methylpropanenitrile
2-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
* ** yl)phenoxy)acetonitrile
4-(5-amino-6-(5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-8- ***
*** yl)pyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
4-(6-amino-5-(2-oxo-1,2,3,4,5,6-hexahydrobenzo[b]azocin-8- * **
yl)pyridin-3-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
4-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
** *** yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile
4-(5-amino-6-(3-methyl-1-oxo-1,2,3,4-tetrahydrophthalazin-6- ** ***
yl)pyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
6-(3-amino-6-(4-(2-hydroxypropan-2-yl)phenyl)pyrazin-2-yl)-3,4- **
*** dihydroisoquinolin-1(2H)-one
4-(5-amino-6-(2-methyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6- * **
yl)pyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
4-(5-amino-6-(5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-
*** *** 8-yl)pyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
4-(6-amino-5-(5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-
*** *** 8-yl)pyridin-3-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
1-(4-(5-amino-6-(1-oxo-1,2-dihydroisoquinolin-6-yl)pyrazin-2- **
*** yl)phenyl)cyclopropanecarbonitrile
2-(5-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
** *** yl)thiophen-2-yl)acetonitrile
5-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-
** *** 1-methyl-2,3-dihydro-1H-indene-1-carbonitrile
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridazin-3-
** *** yl)-N-cyclopropyl-N-methylbenzenesulfonamide
1-(4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridazin-
* * 3-yl)phenyl)cyclopropanecarbonitrile
4-(5-amino-6-(4,4-dideutero-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
*** *** yl)pyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
2-(5-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
** *** yl)thiophen-2-yl)-2-methylpropanenitrile
(1R,5S,6s)-6-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
** ***
yl)pyrazin-2-yl)phenyl)-3-oxabicyclo[3.1.0]hexane-6-carbonitrile
N-(4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-
** *** yl)benzyl)acetamide
6-(3-amino-6-(4-(4-hydroxytetrahydro-2H-pyran-4-yl)phenyl)pyrazin-
** *** 2-yl)-3,4-dihydroisoquinolin-1(2H)-one
4-(6-amino-5-(2-(2-cyanoethyl)-1-oxo-1,2,3,4-tetrahydroisoquinolin-
* ** 6-yl)pyridin-3-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
(R)-4-(6-amino-5-(3-methyl-5-oxo-2,3,4,5- *** ***
tetrahydrobenzo[f][1,4]oxazepin-8-yl)pyridin-3-yl)-N-cyclopropyl-N-
methylbenzenesulfonamide
6-(2-amino-5-(4-(2-morpholinopropan-2-yl)phenyl)pyridin-3-yl)-3,4-
** *** dihydroisoquinolin-1(2H)-one
4-(6-amino-5-(5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-8- ***
*** yl)pyridin-3-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
5-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-
* *** 1-methyl-2,3-dihydro-1H-indene-1-carbonitrile
4-(4-(5-amino-6-(5-oxo-2,3,4,5-tetrahydro-1H- ** ***
benzo[e][1,4]diazepin-8-yl)pyrazin-2-yl)phenyl)tetrahydro-2H-pyran-
4-carbonitrile 4-(5-amino-6-(3-methyl-5-oxo-2,3,4,5-tetrahydro-1H-
*** *** benzo[e][1,4]diazepin-8-yl)pyrazin-2-yl)-N-cyclopropyl-N-
methylbenzenesulfonamide
2-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-
** *** yl)phenyl)cyclopropanecarbonitrile
6-(3-amino-6-(4-propionylphenyl)pyrazin-2-yl)-3,4- ** ***
dihydroisoquinolin-1(2H)-one
4-(5-amino-6-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
*** *** yl)pyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
4-(5-amino-6-(5-oxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-
*** *** 8-yl)pyrazin-2-yl)-N-cyclopropylbenzenesulfonamide
4-(6-amino-5-(5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-8- ***
*** yl)pyridin-3-yl)-N-cyclopropylbenzenesulfonamide
4-(5-amino-6-(5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-8- ***
*** yl)pyrazin-2-yl)-N-cyclopropylbenzenesulfonamide
4-(6-amino-5-(1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-7- *** ***
yl)pyridin-3-yl)-N-cyclopropylbenzenesulfonamide
6-(3-amino-6-(4-(1-(methylamino)cyclopropyl)phenyl)pyrazin-2-yl)-
*** *** 3,4-dihydroisoquinolin-1(2H)-one
4-(6-amino-5-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
*** *** yl)pyridin-3-yl)-N-cyclopropylbenzenesulfonamide
1-(4-(5-amino-6-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-
*** *** 6-yl)pyrazin-2-yl)phenyl)cyclopropanecarbonitrile
4-(5-amino-6-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
*** *** yl)pyrazin-2-yl)-N-cyclopropylbenzenesulfonamide
6-(3-amino-6-(4-(pentafluorosulfanyl)phenyl)pyrazin-2-yl)-3,4- * **
dihydroisoquinolin-1(2H)-one
(S)-4-(6-amino-5-(3-methyl-5-oxo-2,3,4,5- ** ***
tetrahydrobenzo[f][1,4]oxazepin-8-yl)pyridin-3-yl)-N-cyclopropyl-N-
methylbenzenesulfonamide
4-(4-(5-amino-6-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-
*** *** 6-yl)pyrazin-2-yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile
4-(4-(5-amino-6-(1-oxo-2,3,4,5-tetrahydro-1H-benzo[c]azepin-7- **
*** yl)pyrazin-2-yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile
4-(4-(6-amino-5-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-
*** *** 6-yl)pyridin-3-yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile
(R)-4-(6-amino-5-(3-((benzyloxy)methyl)-5-oxo-2,3,4,5- * ***
tetrahydrobenzo[f][1,4]oxazepin-8-yl)pyridin-3-yl)-N-cyclopropyl-N-
methylbenzenesulfonamide
(S)-4-(6-amino-5-(3-(hydroxymethyl)-5-oxo-2,3,4,5- *** ***
tetrahydrobenzo[f][1,4]oxazepin-8-yl)pyridin-3-yl)-N-cyclopropyl-N-
methylbenzenesulfonamide
4-(5-amino-6-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'- ***
*** isoquinolin]-6'-yl)pyrazin-2-yl)-N-cyclopropyl-N-
methylbenzenesulfonamide
1-(4-(5-amino-6-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-
*** ***
isoquinolin]-6'-yl)pyrazin-2-yl)phenyl)cyclopropanecarbonitrile
4-(5-amino-6-(5-oxo-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepin-8- **
*** yl)pyrazin-2-yl)-N-cyclopropyl-2-methylbenzenesulfonamide
4-(5-amino-6-(4,4-dimethyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
*** *** yl)pyrazin-2-yl)-N-cyclopropyl-2-methylbenzenesulfonamide
4-(5-amino-6-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'- ***
*** isoquinolin]-6'-yl)pyrazin-2-yl)-N-cyclopropyl-2-
methylbenzenesulfonamide
4-(5-amino-6-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'- ***
***
isoquinolin]-6'-yl)pyrazin-2-yl)-N-cyclopropylbenzenesulfonamide
4-(6-amino-5-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'- ***
***
isoquinolin]-6'-yl)pyridin-3-yl)-N-cyclopropylbenzenesulfonamide
4-(6-amino-5-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'- ***
*** isoquinolin]-6'-yl)pyridin-3-yl)-N-cyclopropyl-N-
methylbenzenesulfonamide
(S)-4-(5-amino-6-(3-methyl-5-oxo-2,3,4,5-tetrahydro-1H- *** ***
benzo[e][1,4]diazepin-8-yl)pyrazin-2-yl)-N-cyclopropyl-N-
methylbenzenesulfonamide
(S)-4-(6-amino-5-(3-methyl-5-oxo-2,3,4,5-tetrahydro-1H- *** ***
benzo[e][1,4]diazepin-8-yl)pyridin-3-yl)-N-cyclopropyl-N-
methylbenzenesulfonamide
(S)-4-(6-amino-5-(3-methyl-5-oxo-2,3,4,5-tetrahydro-1H- *** ***
benzo[e][1,4]diazepin-8-yl)pyridin-3-yl)-N-
cyclopropylbenzenesulfonamide
4-(4-(6-amino-5-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-
*** ***
isoquinolin]-6'-yl)pyridin-3-yl)phenyl)tetrahydro-2H-pyran-4-
carbonitrile
4-(6-amino-5-(3'-oxospiro[cyclopropane-1,1'-isoindolin]-6'- *** ***
yl)pyridin-3-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
4-(4-(5-amino-6-(3'-oxospiro[cyclopropane-1,1'-isoindolin]-6'- **
*** yl)pyrazin-2-yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile
4-(6-amino-5-(3'-oxospiro[cyclopropane-1,1'-isoindolin]-6'- *** ***
yl)pyridin-3-yl)-N-cyclopropylbenzenesulfonamide
4-(5-amino-6-(3'-oxospiro[cyclopropane-1,1'-isoindolin]-6'- ** ***
yl)pyrazin-2-yl)-N-cyclopropylbenzenesulfonamide
4-(4-(6-amino-5-(3'-oxospiro[cyclopropane-1,1'-isoindolin]-6'- **
*** yl)pyridin-3-yl)phenyl)tetrahydro-2H-pyran-4-carbonitrile
4-(5-amino-6-(4,4-dideutero-1-oxo-1,2,3,4-tetrahydroisoquinolin-6-
*** *** yl)pyrazin-2-yl)-N-cyclopropylbenzenesulfonamide
5-(3-amino-6-(4-(N-cyclopropyl-N-methylsulfamoyl)phenyl)pyrazin- **
*** 2-yl)-N-methylindoline-1-carboxamide
1-(4-(5-amino-6-(3'-oxospiro[cyclopropane-1,1'-isoindolin]-6'- **
*** yl)pyrazin-2-yl)phenyl)cyclopropanecarbonitrile
6'-(3-amino-6-(4-fluorophenyl)pyrazin-2-yl)-2',3'-dihydro-1'H- *
*** spiro[cyclopropane-1,4'-isoquinolin]-1'-one
4-(5-amino-6-(1'-oxo-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'- **
*** isoquinolin]-6'-yl)pyrazin-2-yl)benzonitrile
4-(5-amino-6-(8-oxo-5,6,7,8-tetrahydro-2,7-naphthyridin-3- *** ***
yl)pyrazin-2-yl)-N-cyclopropylbenzenesulfonamide
4-(5-amino-6-(3,3-dimethyl-1-oxoisoindolin-5-yl)pyrazin-2-yl)-N- **
*** cyclopropylbenzenesulfonamide
4-(6-amino-5-(3,3-dimethyl-1-oxoisoindolin-5-yl)pyridin-3-yl)-N-
*** *** cyclopropyl-N-methylbenzenesulfonamide
4-(5-amino-6-(4-bromo-1-hydroxyisoquinolin-6-yl)pyrazin-2-yl)-N-
*** *** cyclopropyl-N-methylbenzenesulfonamide
4-(6-amino-5-(8-oxo-5,6,7,8-tetrahydro-2,7-naphthyridin-3- ** ***
yl)pyridin-3-yl)benzonitrile
4-(5-amino-6-(8-oxo-5,6,7,8-tetrahydro-2,7-naphthyridin-3- *** ***
yl)pyrazin-2-yl)benzonitrile
4-(5-amino-6-(4,4-dimethyl-1,3-dioxo-1,2,3,4- * *
tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-
cyclopropylbenzenesulfonamide
4-(6-(1-acetyl-3,3-dimethylindolin-5-yl)-5-aminopyrazin-2-yl)-N- **
*** cyclopropyl-N-methylbenzenesulfonamide
6-(2-amino-5-(4-fluorophenyl)pyridin-3-yl)-3,4-dihydro-2,7- ** **
naphthyridin-1(2H)-one
4-(6-amino-5-(8-oxo-5,6,7,8-tetrahydro-2,7-naphthyridin-3- *** ***
yl)pyridin-3-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
4-(5-amino-6-(8-oxo-5,6,7,8-tetrahydro-2,7-naphthyridin-3- *** ***
yl)pyrazin-2-yl)-N-cyclopropyl-N-methylbenzenesulfonamide
6-(3-amino-6-(4-fluorophenyl)pyrazin-2-yl)-3,4-dihydro-2,7- *** ***
naphthyridin-1(2H)-one Table 1 also provides in vitro test results,
which were obtained using the assay described in Example 6.42,
wherein "--" indicates that a measurement was not obtained; * =
IC.sub.50 <0.15 .mu.M; ** = IC.sub.50 <0.05 .mu.M; and *** =
IC.sub.50 <0.01 .mu.M.
6.42. In Vitro Measurement of MST1 Inhibition
[0230] Assays were performed in low-volume 384-well black
proxiplates (Perkin Elmer, PE-Blk-Proxi-6008269). Eight compounds
were first diluted into LDV (Echo) plates using a Multiprobe, at a
starting concentration of 1 mM compound in 100% DMSO. Using the
ECHO, 75 nl compound was pinged into 384-well proxiplates. The
starting concentration of compound in each assay plate is 7.5 uM,
followed by 3-fold dilutions, to give quadruplicate ten-point
concentration curves. A 3.times. solution of MST1 enzyme in kinase
buffer (Invitrogen, PR4940C) was added to wells containing compound
or DMSO controls, followed by a 10 minute preincubation step.
Reactions were initiated by adding 5 .mu.L of a mixture of ATP and
Z'-Lyte S/T Pep 7 (Invitrogen, PV3180) and proceeded at RT for 1
hour. The final concentration of key reagents in the kinase
reactions were 1 .mu.M substrate, 1 nM enzyme, and either 50 .mu.M
ATP or 1 mM ATP. At the end of the kinase reaction, 10 ul of
developing solution (Invitrogen, catalog #PR4876B; development
buffer A diluted 150,000.times. in development buffer B) was added
to each well and incubated at RT for 1 hr. All wells were read on a
Tecan at 400 nm excitation and 460 nm/530 nm emission. Plus and
minus enzyme controls were used to calculate percent inhibition and
IC.sub.50 curves were generated using Excel.
6.43. Cell-Based Assay
[0231] A cell-based assay that monitors autophosphorylation of
intracellular MST1 was also used to characterize compounds of the
invention. In this assay, HEK293F cells were transfected with a
plasmid coding for full-length human MST1. The cells were grown in
cell media (DMEM, 10% FBS, 1.times.GPS) to 80-90% confluency in
24-well issue culture plates. On day one, .about.2.times.10 7 cells
were trypsinized and resuspended in 20 mL cell media minus the GPS.
One mL of this cell suspension was transferred to a Vi-cell sample
cup and cells were counted on a Beckman Coulter Vi-Cell XR. Cells
were then diluted to 3.times.10 5/per mL in DMEM+10% FBS, enough
for 12 mls for one 24-well plate. Each well of a 24-well plate
received 500 .mu.l of this cell suspension for a final
concentration of 1.5.times.10 5 cells per well. Cells were then
incubated overnight at 37.degree. C. and 5% CO.sub.2.
[0232] On day two, each well was transfected with 0.5 .mu.g DNA and
1.5 .mu.l Lipofectamine 2000 (Invitrogen; cat. #11668-019). The DNA
mix is comprised of 5 ng MST1 T183E, 50 ng MEKK1, 445 ng pcDNA3.1,
and 50 ul OPTI-MEM. The Lipofectamine 2000 is comprised of 1.5
.mu.l lipofectamine 2000 and 50 ul OPTI-MEM. The DNA mixture was
first pipetted into 15 ml tubes and incubated at RT for 5 minutes,
followed by addition of the lipofectamine mix and incubation at RT
for 20 minutes. 100 .mu.l of DNA transfection mixture was added to
cells in each well of a 24-well plate, followed by incubation
overnight at 37.degree. C. and 5% CO.sub.2.
[0233] On day 3, compounds were serially diluted with 100% DMSO and
one .mu.L was transferred to wells of a 1 mL deep 96-well plate.
Plates were then transferred to a TC hood and 1 ml of DMEM+0.5% FBS
was added to each well containing 1 .mu.l compound. (0.1% DMSO
final concentration). Next, media/transfection mix was aspirated
from each well of the 24-well plate containing the cells, followed
by addition of 300 .mu.l from each compound dilution (remaining
compound dilutions kept at 4.degree. C. for later use). Plates were
then incubated at 37.degree. C. and 5% CO.sub.2 for 4 hours. One
.mu.l of 225 .mu.M okadaic acid in 100% DMSO was then pipetted into
a new 1 ml deep 96-well plate, followed by addition of 300 .mu.l
compound dilution (stored at 4.degree. C. in the previous step) to
give a final concentration of 0.75 .mu.M okadaic acid. The
media/compound mixture was then aspirated from 24-well plate
containing cells and then replaced with the media/compound/okadaic
acid mixture. Cells were incubated at 37.degree. C. and 5% CO.sub.2
for 2 more hours. Cells were detached by simple pipetting and cell
suspensions from each well were transferred into new 1.5 ml tubes.
Tubes containing media were centrifuged at 1000.times.g for 5
minutes to pellet the cells and supernatants were carefully removed
and discarded. Cell pellets were usually frozen at -80.degree. C.
until the next step.
[0234] On day 4, cells were lysed and prepared for Western Blot
analysis as follows. Cell pellets were thawed on ice. Lysis buffer
consisted of the following components: Tris, pH 7.5, 150 mM NaCl, 1
mM Na.sub.2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate,
1 mM .beta.-glycerophosphate, 1 mM Na.sub.3VO.sub.4, 1 ug/ml
leupeptin, 10 mM EDTA, 2.times. Halt protease & phosphatase
inhibitor cocktail (Thermo Scientific; cat. #1861284). 100 .mu.L of
freshly made lysis buffer was added per sample, followed by
incubation on ice for 15 min, vortexing for 10-15 seconds, and
centrifuging at 12,000.times.g for 30 sec. Supernatant were
carefully removed and mixed with 4.times.LDS Sample Buffer from
Invitrogen (cat. #NP0008; with 50 mM DTT added fresh). Samples were
denatured at 70.degree. C. for 10 minutes on a PCR machine, and 10
.mu.l were loaded per lane on 2.times. Criterion 26-well gels
(Biorad; cat. #345-0034). After SDS-PAGE, samples were transferred
from gel to PVDF membrane, blocked for one hour in TBST+5% milk,
and washed 3.times. for 5-10 min with TBST. One membrane was probed
with rabbit anti-MST1 (1:3000; Millipore, cat. #07-061) diluted in
TBST+5% milk, and another membrane was probed with rabbit
anti-phospho-MST1 (T183; Cell Signaling, cat. #3681) (1:2000)
diluted in TBST+5% BSA, followed by incubate overnight at 4.degree.
C.
[0235] On day 5, Western Blots were washed 3.times. for 5-10
minutes with TBST, probe with anti-rabbit-HRP (1:3000; Biorad, cat.
#170-6515) diluted in TBST+5% milk for 1 hour at RT, and washed
3.times. for 10 minutes with TBST. Blots were developed with ECL
reagent (GE Healthcare; cat. #RPN2132) on a Biorad Versadoc Imagin
System (Model 5000) and volume analysis performed on each band to
obtain density values.
6.44. Pharmacology of
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-N-cy-
clopropyl-N-methylbenzenesulfonamide
[0236] The compound
4-(6-amino-5-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyridin-3-yl)-N-cy-
clopropyl-N-methylbenzenesulfonamide was tested in the EAE disease
model described above. In a first study, the prophylactic effect of
the compound was measured: mice were generally dosed PO, bid, for
22 days, starting on Day (-1) before MOG peptide immunization (in
the 100 mpk group, compound was dosed BID on days -1-9, and QD on
days 10-21.) Four groups of mice (n=10 per group) were tested:
vehicle control; 10 mg/kg, 30 mg/kg, and 100 mg/kg compound.
Results are shown in FIG. 2A, wherein * indicates p<0.05 versus
control.
[0237] A second study examined the therapeutic effect of the
compound. Here, mice were generally dosed PO, BID, for 14 days,
starting on Day 9 after MOG peptide immunization (in the 100 mpk
group, compound was dosed BID on days 9-13, and QD on days 14-23).
Results are shown in FIG. 2B, wherein * indicates p<0.05 versus
control.
[0238] The compound was also studied in a rat EAE model. Here, four
groups of rats (n=10 per group) were administered vehicle control,
10 mg/kg, 25 mg/kg, or 50 mg/kg doses, PO, BID. Dosing began on Day
9 after immunization. As shown in FIG. 3, the compound again
exhibited a dose-dependent reduction in severity of clinical score
(* indicates p<0.05 versus control).
[0239] FIGS. 4A and 4B show results obtained from a study of the
compound in a CIA disease model. In this test, mice were dosed PO,
bid, for 21 days, starting on Day 20 after immunization. Four
groups of mice were used (n=10 per group): vehicle control, 10
mg/kg, 30 mg/kg, and 100 mg/kg. A clear benefit versus control was
observed (* indicates p<0.05 versus control).
[0240] The compound was further studied in a rat CIA disease model,
using four groups of rats (n=10 per group): vehicle control, 10
mg/kg, 25 mg/kg, and 50 mg/kg. The rats were dosed PO, BID (10 and
25 mg/kg groups) or QD (50 mg/kg group) beginning on Day 11 after
immunization. As shown in FIGS. 5A and 5B, the compound again
showed a therapeutic effect. FIG. 5A shows the cumulative arthritis
score as a function of time and dose; FIG. 5B shows the change in
ankle thickness as a function of time and dose (* indicates
p<0.05 versus control). Here, ankle thickness was measured by
volume (water displacement).
[0241] FIG. 6 shows the effect of the compound on liver enzyme and
cytokine response in a Con-A induced hepatitis model. Four groups
of mice were used (n=10 per group): vehicle control, 10 mg/kg, 30
mg/kg, and 100 mg/kg. The mice were dosed 16 h and 1 h before, and
8 h after, the Con-A challenge. A clear effect in TNF-.alpha.,
IL-6, MCP-1, IFN-.gamma., ALT and AST was observed (* indicates
p<0.05 versus control; ** indicates p<0.1 versus
control).
6.45. Pharmacology of
1-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)ph-
enyl)cyclopropanecarbonitrile
[0242] The compound
1-(4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)ph-
enyl)cyclopropanecarbonitrile was studied in a rat EAE model. Here,
five groups of rats (n=10 per group) were administered vehicle
control, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg doses, PO, QD,
starting on Day 10 after immunization. As shown in FIG. 7A, the
compound exhibited a dose-dependent reduction in severity of
clinical score (* indicates p<0.05 versus control). The effect
is also seen in FIG. 7B, which shows the disease development as a
function of test group.
[0243] FIG. 8 shows the effect of the compound on liver enzyme and
cytokine response in a Con-A induced hepatitis model. Five groups
of mice were used (n=10 per group): vehicle control, 1 mg/kg, 3
mg/kg, 10 mg/kg, and 30 mg/kg. The mice were dosed PO 16 h and 1 h
before, and 8 h after, the Con-A challenge. A clear effect in
TNF-.alpha., IL-6, MCP-1, IFN-.gamma., ALT and AST was observed (*
indicates p<0.05 versus control).
6.46. Pharmacology of
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-cy-
clopropyl-N-methylbenzenesulfonamide
[0244] The compound
4-(5-amino-6-(1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazin-2-yl)-N-cy-
clopropyl-N-methylbenzenesulfonamide was studied in a mouse EAE
model. Here, four groups of mice (n=10 per group) were administered
vehicle control, 3 mg/kg, 10 mg/kg, or 30 mg/kg doses BID for 12
days, starting on Day 9 after MOG peptide immunization. FIG. 9A
shows the effect of subcutaneous dosing: a clear dose-dependency in
clinical score was observed (* indicates p<0.05 versus control).
FIG. 9B shows the effect of oral (PO) dosing.
[0245] The effect of the compound in a mouse CIA model was also
observed as a function of dose and method of delivery. FIGS. 10A
and 10B show the effect of the compound on cumulative scores and
change in ankle thickness when administered subcutaneously (*
indicates p<0.05 versus control) to three groups of mice (n=10
per group). The groups were administered vehicle control, 10 mg/kg,
or 30 mg/kg doses BID for three weeks, starting on Day 20 after
collagen immunization. FIGS. 10C and 10D show results obtained when
the compound was administered orally (* indicates p<0.05 versus
control) to four groups of mice (n=10 per group). The groups were
administered vehicle control, 10 mg/kg, 30 mg/kg, or 50 mg/kg doses
BID for three weeks, starting on Day 20 after collagen
immunization.
[0246] All references (e.g., patents and published patent
applications) cited above are incorporated herein by reference.
* * * * *