U.S. patent application number 14/600214 was filed with the patent office on 2015-10-15 for compounds useful as inhibitors of atr kinase.
The applicant listed for this patent is Vertex Pharmaceuticals Incorporated. Invention is credited to Guy Brenchley, Jean-Damien Charrier, Chris Davis, Steven Durrant, Gorka Etxebarria I Jardi, Damien Fraysse, Juan-Miguel Jimenez, David Kay, Ronald Knegtel, Francoise Pierard, Joanne Pinder, David Shaw, Pierre-Henri Storck, John Studley, Heather Twin.
Application Number | 20150291601 14/600214 |
Document ID | / |
Family ID | 49885394 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150291601 |
Kind Code |
A1 |
Brenchley; Guy ; et
al. |
October 15, 2015 |
COMPOUNDS USEFUL AS INHIBITORS OF ATR KINASE
Abstract
The present invention relates to compounds useful as inhibitors
of ATR protein kinase. The invention also relates to
pharmaceutically acceptable compositions comprising the compounds
of this invention; methods of treating of various diseases,
disorders, and conditions using the compounds of this invention;
processes for preparing the compounds of this invention;
intermediates for the preparation of the compounds of this
invention; and methods of using the compounds in in vitro
applications, such as the study of kinases in biological and
pathological phenomena; the study of intracellular signal
transduction pathways mediated by such kinases; and the comparative
evaluation of new kinase inhibitors. The compounds of this
invention have formula I: ##STR00001## or a pharmaceutically
acceptable salt, wherein the variables are as defined herein.
Inventors: |
Brenchley; Guy; (West
Hanney, GB) ; Charrier; Jean-Damien; (Grove, GB)
; Davis; Chris; (Salisbury, GB) ; Durrant;
Steven; (Abingdon, GB) ; Etxebarria I Jardi;
Gorka; (Abingdon, GB) ; Fraysse; Damien;
(Abingdon, GB) ; Jimenez; Juan-Miguel; (Abingdon,
GB) ; Kay; David; (Purton, GB) ; Knegtel;
Ronald; (Abingdon, GB) ; Pierard; Francoise;
(Abingdon, GB) ; Pinder; Joanne; (Didcot, GB)
; Shaw; David; (Oxford, GB) ; Storck;
Pierre-Henri; (Abingdon, GB) ; Studley; John;
(Witney, GB) ; Twin; Heather; (Wantage,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vertex Pharmaceuticals Incorporated |
Boston |
MA |
US |
|
|
Family ID: |
49885394 |
Appl. No.: |
14/600214 |
Filed: |
January 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14098640 |
Dec 6, 2013 |
8957078 |
|
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14600214 |
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61787478 |
Mar 15, 2013 |
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Current U.S.
Class: |
424/649 ;
435/194; 435/375; 514/210.18; 514/210.21; 514/221; 514/233.2;
514/252.02; 514/252.16; 514/259.31; 514/27; 514/393; 514/49;
540/556; 544/117; 544/230; 544/238; 544/281; 600/1 |
Current CPC
Class: |
C07D 453/02 20130101;
A61P 35/00 20180101; A61K 45/06 20130101; C07D 491/107 20130101;
C12Y 207/11001 20130101; A61K 33/24 20130101; A61K 31/36 20130101;
A61N 2005/1087 20130101; A61K 31/5377 20130101; A61P 43/00
20180101; A61N 2005/1089 20130101; A61N 5/1077 20130101; C07D
471/08 20130101; C07D 519/00 20130101; A61K 31/4188 20130101; A61K
31/551 20130101; A61N 2005/109 20130101; A61K 31/7068 20130101;
A61K 31/519 20130101; C07D 487/04 20130101; A61N 5/1028 20130101;
C12N 9/12 20130101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61K 31/519 20060101 A61K031/519; C07D 519/00 20060101
C07D519/00; A61K 31/5377 20060101 A61K031/5377; A61N 5/10 20060101
A61N005/10; A61K 33/24 20060101 A61K033/24; A61K 31/7068 20060101
A61K031/7068; A61K 31/36 20060101 A61K031/36; A61K 31/4188 20060101
A61K031/4188; C12N 9/12 20060101 C12N009/12; A61K 45/06 20060101
A61K045/06; A61K 31/551 20060101 A61K031/551 |
Claims
1. A compound of formula I: ##STR00158## or a pharmaceutically
acceptable salt or prodrug thereof, wherein: R.sup.1 and R.sup.2
are independently selected from H; halo; --C(J.sup.1).sub.2CN;
--CN; W; or M; J.sup.1 is independently selected from H or
C.sub.1-2alkyl; or two occurrences of J.sup.1, together with the
carbon atom to which they are attached, form an optionally
substituted 3-4 membered carbocyclic ring; M is a
C.sub.1-8aliphatic wherein up to three methylene units are
optionally replaced with --O--, --NR--, --C(O)--, or
--S(O).sub.z--, each M is optionally substituted with 0-3
occurrences of R.sup.2a; R.sup.2a is independently selected from
halo; --CF.sub.3; --CN; a C.sub.1-4aliphatic chain wherein up to
two methylene units of the aliphatic chain are optionally replaced
with --O--, --NR--, --C(O)--, or --S(O).sub.z--; or a 3-6 membered
non-aromatic ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; W is independently selected from a 3-7
membered fully saturated, partially unsaturated, or aromatic
monocyclic ring having 0-3 heteroatoms selected from oxygen,
nitrogen or sulfur; or an 7-12 membered fully saturated, partially
unsaturated, or aromatic bicyclic ring having 0-5 heteroatoms
selected from oxygen, nitrogen, or sulfur; wherein W is optionally
substituted with 0-5 occurrences of J.sup.W; J.sup.W is
independently selected from --CN, halo, --CF.sub.3; a
C.sub.1-4aliphatic wherein up to two methylene units are optionally
replaced with --O--, --NR--, --C(O)--, or --S(O).sub.z--; or a 3-6
membered non-aromatic ring having 0-2 heteroatoms selected from
oxygen, nitrogen, or sulfur; two occurrences of J.sup.W on the same
atom, together with atom to which they are joined, form a 3-6
membered ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; or two occurrences of J.sup.W, together with
W, form a 6-10 membered saturated or partially unsaturated bridged
ring system; A is independently selected from: ##STR00159## p is 0,
1, or 2; R.sup.3 is independently selected from -(L).sub.n-Q.sup.1
or T; L and T are each independently a C.sub.1-10aliphatic chain
wherein up to three methylene units of the aliphatic chain are
optionally replaced with --O--, --NR--, --S(O).sub.z--, or
--C(O)--; each L and T is independently substituted with 0-5
occurrences of J.sup.LT; J.sup.LT is independently selected from
halo, --CN, or a C.sub.1-4aliphatic chain wherein up to two
methylene units of the aliphatic chain are optionally replaced with
--O--, --NR--, --C(O)--, or --S(O).sub.z--; n is 0 or 1; Q.sup.1 is
independently selected from a 3-7 membered fully saturated,
partially unsaturated, or aromatic monocyclic ring having 0-3
heteroatoms selected from oxygen, nitrogen or sulfur; or an 7-12
membered fully saturated, partially unsaturated, or aromatic
bicyclic ring having 0-5 heteroatoms selected from oxygen,
nitrogen, or sulfur; wherein Q.sup.1 is independently substituted
with 0-5 occurrences of J.sup.Q; J.sup.Q is independently selected
from halo; --CN; .dbd.O; Q.sup.2; or a C.sub.1-8aliphatic chain
wherein up to three methylene units of the aliphatic chain are
optionally replaced with --O--, --NR--, --C(O)--, or
--S(O).sub.z--; each occurrence of J.sup.Q is optionally
substituted by 0-3 occurrences of J.sup.R; or two occurrences of
J.sup.Q on the same atom, taken together with the atom to which
they are joined, form a 3-6 membered ring having 0-2 heteroatoms
selected from oxygen, nitrogen, or sulfur; wherein the ring formed
by two occurrences of J.sup.Q is optionally substituted with 0-3
occurrences of J.sup.X; or two occurrences of J.sup.Q, together
with Q.sup.1, form a 6-10 membered saturated or partially
unsaturated bridged ring system; Q.sup.2 is independently a 3-7
membered fully saturated, partially unsaturated, or aromatic
monocyclic ring having 0-3 heteroatoms selected from oxygen,
nitrogen, or sulfur; or a 7-12 membered fully saturated, partially
unsaturated, or aromatic bicyclic ring having 0-5 heteroatoms
selected from oxygen, nitrogen, or sulfur; J.sup.R is independently
selected from halo; --CN; .dbd.O; .fwdarw.O; Q.sup.3; or a
C.sub.1-6aliphatic chain wherein up to two methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; each J.sup.R is optionally substituted
with 0-3 occurrences of J.sup.P; or two occurrences of J.sup.R on
the same atom, together with the atom to which they are joined,
form a 3-6 membered ring having 0-2 heteroatoms selected from
oxygen, nitrogen, or sulfur; wherein the ring formed by two
occurrences of J.sup.R is optionally substituted with 0-3
occurrences of J.sup.X; or two occurrences of J.sup.R, together
with Q.sup.2, form a 6-10 membered saturated or partially
unsaturated bridged ring system; Q.sup.3 is a 3-7 membered fully
saturated, partially unsaturated, or aromatic monocyclic ring
having 0-3 heteroatoms selected from oxygen, nitrogen, or sulfur; a
7-12 membered fully saturated, partially unsaturated, or aromatic
bicyclic ring having 0-5 heteroatoms selected from oxygen,
nitrogen, or sulfur; J.sup.X is independently selected from halo or
a C.sub.1-4aliphatic chain wherein up to two methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; or J.sup.P is independently selected
from halo; --CN; .dbd.O; a C.sub.1-6aliphatic chain wherein up to
two methylene units of the aliphatic chain are optionally replaced
with --O--, --NR--, --C(O)--, or --S(O).sub.z--; or a 3-6 membered
non-aromatic ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; each J.sup.P is optionally substituted with
0-3 occurrences of J.sup.M; or two occurrences of J.sup.P on the
same atom, together with the atom to which they are joined, form a
3-6 membered ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; or two occurrences of J.sup.P, together with
Q.sup.3, form a 6-10 membered saturated or partially unsaturated
bridged ring system; R.sup.4 is independently selected from H,
halo, a C.sub.3-4 membered cycloalkyl, 3-4 membered heterocyclyl,
or C.sub.1-4aliphatic chain wherein up to two methylene units of
the aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; J.sup.M is independently selected from
halo or C.sub.1-6aliphatic; z is 0, 1, or 2; and R is independently
selected from H or C.sub.1-4aliphatic.
2.-71. (canceled)
72. The compound of claim 1 independently selected from:
##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164##
##STR00165## ##STR00166## ##STR00167## ##STR00168## ##STR00169##
##STR00170## ##STR00171## ##STR00172## ##STR00173## ##STR00174##
##STR00175##
73. A pharmaceutical composition comprising a compound of claim 1
and a pharmaceutically acceptable carrier.
74. A method for treating cancer in a patient comprising
administering a compound of claim 1 or a pharmaceutically
acceptable derivative thereof.
75. The method of claim 74, further comprising administering to
said patient an additional therapeutic agent independently selected
from a DNA-damaging agent; wherein said additional therapeutic
agent is appropriate for the disease being treated; and said
additional therapeutic agent is administered together with said
compound as a single dosage form or separately from said compound
as part of a multiple dosage form.
76. The method of claim 75, wherein said DNA-damaging agent is
selected chemotherapy or radiation treatment.
77. The method of claim 76, wherein said DNA-damaging agent is
independently selected from ionizing radiation, radiomimetic
neocarzinostatin, a platinating agent, a Topo I inhibitor, a Topo
II inhibitor, an antimetabolite, an alkylating agent, an alkyl
sulphonates, or an antibiotic.
78. (canceled)
79. (canceled)
80. The method of claim 76, wherein said platinating agent is
independently selected from Cisplatin, Oxaliplatin, Carboplatin,
Nedaplatin, Lobaplatin, Triplatin Tetranitrate, Picoplatin,
Satraplatin, ProLindac and Aroplatin; said Topo I inhibitor is
selected from Camptothecin, Topotecan, Irinotecan/SN38, Rubitecan
and Belotecan; said Topo II inhibitor is selected from Etoposide,
Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin,
Amrubicin, Pirarubicin, Valrubicin, Zorubicin and Teniposide; said
antimetabolite is selected from Aminopterin, Methotrexate,
Pemetrexed, Raltitrexed, Pentostatin, Cladribine, Clofarabine,
Fludarabine, Thioguanine, Mercaptopurine, Fluorouracil,
Capecitabine, Tegafur, Carmofur, Floxuridine, Cytarabine,
Gemcitabine, Azacitidine and Hydroxyurea; said alkylating agent is
selected from Mechlorethamine, Cyclophosphamide, Ifosfamide,
Trofosfamide, Chlorambucil, Melphalan, Prednimustine, Bendamustine,
Uramustine, Estramustine, Carmustine, Lomustine, Semustine,
Fotemustine, Nimustine, Ranimustine, Streptozocin, Busulfan,
Mannosulfan, Treosulfan, Carboquone, ThioTEPA, Triaziquone,
Triethylenemelamine, Procarbazine, Dacarbazine, Temozolomide,
Altretamine, Mitobronitol, Actinomycin, Bleomycin, Mitomycin and
Plicamycin.
81. The method of claim 77, wherein said platinating agent is
independently selected from Cisplatin, Oxaliplatin, Carboplatin,
Nedaplatin, or Satraplatin; said Topo I inhibitor is selected from
Camptothecin, Topotecan, irinotecan/SN38, rubitecan; said Topo II
inhibitor is selected from Etoposide; said antimetabolite is
selected from methotrexate, pemetrexed, Thioguanine, Fludarabine,
Cladribine, Cytarabine, gemcitabine, 6-Mercaptopurine, or
5-Fluorouracil; said alkylating agent is selected from nitrogen
mustards, nitrosoureas, triazenes, alkyl sulfonates, Procarbazine,
or aziridines; and said antibiotic is selected from Hydroxyurea,
Anthracyclines, Anthracenediones, or Streptomyces family.
82. The method of claim 77, wherein said DNA-damaging agent is
independently selected from a platinating agent or ionizing
radiation.
83. The method of claim 77, wherein said DNA-damaging agent is
gemcitabine.
84. The method of claim 77, wherein the DNA-damaging agent is
ionizing radiation.
85. The method of claim 77, wherein the DNA-damaging agent is a
platinating agent independently selected from Cisplatin or
Carboplatin.
86. The method of claim 77, wherein the DNA-damaging agent is a
Topo II inhibitor selected from Etoposide.
87. The method of claim 77, wherein the DNA-damaging agent is an
alkylating agent selected from Temozolomide.
88. The method of claim 77, wherein the DNA-damaging agent is
independently selected from one or more of the following:
Cisplatin, Carboplatin, gemcitabine, Etoposide, Temozolomide, or
ionizing radiation.
89. The method of claim 74, wherein said cancer is a solid tumor
selected from the following cancers: Oral: buccal cavity, lip,
tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma,
fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma,
fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma
(squamous cell or epidermoid, undifferentiated small cell,
undifferentiated large cell, adenocarcinoma), alveolar
(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,
chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus
(squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma,
lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas
(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma,
carcinoid tumors, vipoma), small bowel or small intestines
(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,
leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel
or large intestines (adenocarcinoma, tubular adenoma, villous
adenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal;
rectum, Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor
[nephroblastoma], lymphoma), bladder and urethra (squamous cell
carcinoma, transitional cell carcinoma, adenocarcinoma), prostate
(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal
carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial
cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);
Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,
hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma,
biliary passages; Bone: osteogenic sarcoma (osteosarcoma),
fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma,
Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma),
multiple myeloma, malignant giant cell tumor chordoma,
osteochronfroma (osteocartilaginous exostoses), benign chondroma,
chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell
tumors; Nervous system: skull (osteoma, hemangioma, granuloma,
xanthoma, osteitis deformans), meninges (meningioma,
meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,
glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,
oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),
spinal cord neurofibroma, meningioma, glioma, sarcoma);
Gynecological/Female: uterus (endometrial carcinoma), cervix
(cervical carcinoma, pre-tumor cervical dysplasia), ovaries
(ovarian carcinoma [serous cystadenocarcinoma, mucinous
cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell
tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant
teratoma), vulva (squamous cell carcinoma, intraepithelial
carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear
cell carcinoma, squamous cell carcinoma, botryoid sarcoma
(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;
Skin: malignant melanoma, basal cell carcinoma, squamous cell
carcinoma, Karposi's sarcoma, keratoacanthoma, moles dysplastic
nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis, Thyroid
gland: papillary thyroid carcinoma, follicular thyroid carcinoma;
medullary thyroid carcinoma, multiple endocrine neoplasia type 2A,
multiple endocrine neoplasia type 2B, familial medullary thyroid
cancer, pheochromocytoma, paraganglioma; and Adrenal glands:
neuroblastoma.
90. (canceled)
91. The method of claim 88, wherein said cancer is selected from
lung cancer, head and neck cancer, pancreatic cancer, gastric
cancer, or brain cancer.
92. The method of claim 88, wherein said cancer is selected from
non-small cell lung cancer, small cell lung cancer, pancreatic
cancer, biliary tract cancer, head and neck cancer, bladder cancer,
colorectal cancer, glioblastoma, esophageal cancer, breast cancer,
hepatocellular carcinoma, or ovarian cancer.
93. (canceled)
94. A method of treating pancreatic cancer comprising administering
to a patient a compound of a compound of claim 1 in combination
with an additional therapeutic agent selected from Gemcitabine,
radiation therapy, or both Gemcitabine and radiation therapy
together.
95. A method of increasing the sensitivity of pancreatic cancer
cells to a cancer therapy selected from chemotherapy or radiation
therapy by administering to a patient a compound of claim 1.
96. The method of claim 95, wherein the chemotherapy is
gemcitabine.
97. (canceled)
98. (canceled)
99. The method of claim 95, wherein the cancer therapy is
gemcitabine and radiation.
100. A method of inhibiting phosphorylation of Chk1 (Ser 345) in a
pancreatic cancer cell comprising administering a compound of claim
1 in combination with gemcitabine (100 nM) and/or radiation (6
Gy).
101. A method of sensitizing pancreatic cancer cells to
chemoradiation by administering a compound of claim 1 in
combination with chemoradiation.
102. (canceled)
103. A method of radiosensitizing hypoxic pancreatic cancer cells
by administering a compound of claim 1 in combination with
radiation therapy.
104. A method of sensitizing hypoxic pancreatic cancer cells by
administering a compound of claim 1 in combination with
chemotherapy.
105. (canceled)
106. A method of disrupting damage-induced cell cycle checkpoints
by administering a compound of claim 1 in combination with
radiation therapy and/or gemcitabine.
107. A method of inhibiting repair of DNA damage by homologous
recombination in a pancreatic cancer cell by administering a
compound of claim 1 in combination with radiation therapy and/or
gemcitabine.
108. (canceled)
109. (canceled)
110. (canceled)
111. A method of treating non-small cell lung cancer comprising
administering to a patient a compound of claim 1 in combination
with one or more of the following additional therapeutic agents:
Cisplatin or Carboplatin, Etoposide, and ionizing radiation.
112. (canceled)
113. A method of promoting cell death in cancer cells comprising
administering to a patient a compound of claim 1.
114. A method of preventing cell repair from DNA damage comprising
administering to a patient a compound of claim 1.
115. A method of inhibiting ATR in a biological sample comprising
the step of contacting a compound of claim 1 with said biological
sample.
116. (canceled)
117. A method of sensitizing cells to DNA damaging agents
comprising administering to a patient a compound of claim 1.
118.-130. (canceled)
131. A process for preparing a compound of formula I: ##STR00176##
comprising reacting a compound of formula 6: ##STR00177## under
suitable conditions to form an amide bond, wherein: J is H or Cl;
and R.sup.1, R.sup.2, and A are as defined in claim 1.
132. (canceled)
133. A process for preparing a compound of formula I: ##STR00178##
comprising reacting a compound of formula 5: ##STR00179## under
suitable conditions to form an amide bond, wherein R.sup.1,
R.sup.2, and A are as defined in claim 1.
134. (canceled)
135. (canceled)
136. (canceled)
137. (canceled)
138. A process for preparing a compound of formula I: ##STR00180##
comprising reacting a compound of formula 9: ##STR00181## under
suitable condensation conditions to form a pyrimidine ring, wherein
R.sup.1, R.sup.2, and A are as defined in claim 1.
139. (canceled)
140. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
Nonprovisional application Ser. No. 14/098,640, filed Dec. 6, 2013,
which claims the benefit under 35 U.S.C. .sctn.119 of U.S.
Provisional Application No. 61/787,478, filed Mar. 15, 2013.
BACKGROUND OF THE INVENTION
[0002] ATR ("ATM and Rad3 related") kinase is a protein kinase
involved in cellular responses to certain forms of DNA damage
(e.g., double strand breaks and replication stress). ATR kinase
acts with ATM ("ataxia telangiectasia mutated") kinase and many
other proteins to regulate a cell's response to double strand DNA
breaks and replication stress, commonly referred to as the DNA
Damage Response ("DDR"). The DDR stimulates DNA repair, promotes
survival and stalls cell cycle progression by activating cell cycle
checkpoints, which provide time for repair. Without the DDR, cells
are much more sensitive to DNA damage and readily die from DNA
lesions induced by endogenous cellular processes such as DNA
replication or exogenous DNA damaging agents commonly used in
cancer therapy.
[0003] Healthy cells can rely on a host of different proteins for
DNA repair including the DDR kinases ATR and ATM. In some cases
these proteins can compensate for one another by activating
functionally redundant DNA repair processes. On the contrary, many
cancer cells harbour defects in some of their DNA repair processes,
such as ATM signaling, and therefore display a greater reliance on
their remaining intact DNA repair proteins which include ATR.
[0004] In addition, many cancer cells express activated oncogenes
or lack key tumour suppressors, and this can make these cancer
cells prone to dysregulated phases of DNA replication which in turn
cause DNA damage. ATR has been implicated as a critical component
of the DDR in response to disrupted DNA replication. As a result,
these cancer cells are more dependent on ATR activity for survival
than healthy cells. Accordingly, ATR inhibitors may be useful for
cancer treatment, either used alone or in combination with DNA
damaging agents, because they shut down a DNA repair mechanism that
is more important for cellular survival in many cancer cells than
in healthy normal cells.
[0005] In fact, disruption of ATR function (e.g. by gene deletion)
has been shown to promote cancer cell death both in the absence and
presence of DNA damaging agents. This suggests that ATR inhibitors
may be effective both as single agents and as potent sensitizers to
radiotherapy or genotoxic chemotherapy.
[0006] ATR peptide can be expressed and isolated using a variety of
methods known in the literature (see e.g., Unsal-Kacmaz et al, PNAS
99: 10, pp 6673-6678, May 14, 2002; see also Kumagai et al. Cell
124, pp 943-955, Mar. 10, 2006; Unsal-Kacmaz et al. Molecular and
Cellular Biology, February 2004, p 1292-1300; and Hall-Jackson et
al. Oncogene 1999, 18, 6707-6713).
[0007] For all of these reasons, there is a need for the
development of potent and selective ATR inhibitors for the
treatment of cancer, either as single agents or as combination
therapies with radiotherapy or genotoxic chemotherapy.
SUMMARY OF THE INVENTION
[0008] The present invention relates to compounds useful as
inhibitors of ATR protein kinase. The invention also relates to
pharmaceutically acceptable compositions comprising the compounds
of this invention; methods of treating of various diseases,
disorders, and conditions using the compounds of this invention;
processes for preparing the compounds of this invention;
intermediates for the preparation of the compounds of this
invention; and methods of using the compounds in in vitro
applications, such as the study of kinases in biological and
pathological phenomena; the study of intracellular signal
transduction pathways mediated by such kinases; and the comparative
evaluation of new kinase inhibitors.
[0009] The compounds of the invention are very potent ATR
inhibitors. These compounds also show surprising synergy with other
cancer agents, such as cisplatin and gemcitabine, in combination
therapies.
DETAILED DESCRIPTION OF THE INVENTION
[0010] Another aspect of the invention provides a compound of
Formula I:
##STR00002##
or a pharmaceutically acceptable salt or prodrug thereof, wherein:
R.sup.1 and R.sup.2 are independently selected from H; halo;
--C(J.sup.1).sub.2CN; --CN; W; or M; J.sup.1 is independently
selected from H or C.sub.1-2alkyl; or two occurrences of J.sup.1,
together with the carbon atom to which they are attached, form an
optionally substituted 3-4 membered carbocyclic ring; M is a
C.sub.1-8aliphatic wherein up to three methylene units are
optionally replaced with --O--, --NR--, --C(O)--, or
--S(O).sub.z--, each M is optionally substituted with 0-3
occurrences of R.sup.2a; R.sup.2a is independently selected from
halo; --CF.sub.3; --CN; a C.sub.1-4aliphatic chain wherein up to
two methylene units of the aliphatic chain are optionally replaced
with --O--, --NR--, --C(O)--, or --S(O).sub.z--; or a 3-6 membered
non-aromatic ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; W is independently selected from a 3-7
membered fully saturated, partially unsaturated, or aromatic
monocyclic ring having 0-3 heteroatoms selected from oxygen,
nitrogen or sulfur; or an 7-12 membered fully saturated, partially
unsaturated, or aromatic bicyclic ring having 0-5 heteroatoms
selected from oxygen, nitrogen, or sulfur; wherein W is optionally
substituted with 0-5 occurrences of J.sup.W; J.sup.W is
independently selected from --CN, halo, --CF.sub.3; a
C.sub.1-4aliphatic wherein up to two methylene units are optionally
replaced with --O--, --NR--, --C(O)--, or --S(O).sub.z--; or a 3-6
membered non-aromatic ring having 0-2 heteroatoms selected from
oxygen, nitrogen, or sulfur; two occurrences of J.sup.W on the same
atom, together with atom to which they are joined, form a 3-6
membered ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; or two occurrences of J.sup.W, together with
W, form a 6-10 membered saturated or partially unsaturated bridged
ring system; A is independently selected from:
##STR00003##
p is 0, 1, or 2; R.sup.3 is independently selected from
-(L).sub.n-Q.sup.1 or T; L and T are each independently a
C.sub.1-10aliphatic chain wherein up to three methylene units of
the aliphatic chain are optionally replaced with --O--, --NR--,
--S(O).sub.z--, or --C(O)--; each L and T is independently
substituted with 0-5 occurrences of J.sup.LT; J.sup.LT is
independently selected from halo, --CN, or a C.sub.1-4aliphatic
chain wherein up to two methylene units of the aliphatic chain are
optionally replaced with --O--, --NR--, --C(O)--, or
--S(O).sub.z--; n is 0 or 1; Q.sup.1 is independently selected from
a 3-7 membered fully saturated, partially unsaturated, or aromatic
monocyclic ring having 0-3 heteroatoms selected from oxygen,
nitrogen or sulfur; or an 7-12 membered fully saturated, partially
unsaturated, or aromatic bicyclic ring having 0-5 heteroatoms
selected from oxygen, nitrogen, or sulfur; wherein Q.sup.1 is
independently substituted with 0-5 occurrences of J.sup.Q; J.sup.Q
is independently selected from halo; --CN; .dbd.O; Q.sup.2; or a
C.sub.1-8aliphatic chain wherein up to three methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; each occurrence of J.sup.Q is
optionally substituted by 0-3 occurrences of J.sup.R; or two
occurrences of J.sup.Q on the same atom, taken together with the
atom to which they are joined, form a 3-6 membered ring having 0-2
heteroatoms selected from oxygen, nitrogen, or sulfur; wherein the
ring formed by two occurrences of J.sup.Q is optionally substituted
with 0-3 occurrences of J.sup.X; or two occurrences of J.sup.Q,
together with Q.sup.1, form a 6-10 membered saturated or partially
unsaturated bridged ring system; Q.sup.2 is independently a 3-7
membered fully saturated, partially unsaturated, or aromatic
monocyclic ring having 0-3 heteroatoms selected from oxygen,
nitrogen, or sulfur; or a 7-12 membered fully saturated, partially
unsaturated, or aromatic bicyclic ring having 0-5 heteroatoms
selected from oxygen, nitrogen, or sulfur; J.sup.R is independently
selected from halo; --CN; .dbd.O; .fwdarw.O; Q.sup.3; or a
C.sub.1-6aliphatic chain wherein up to two methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; each J.sup.R is optionally substituted
with 0-3 occurrences of J.sup.P; or two occurrences of J.sup.R on
the same atom, together with the atom to which they are joined,
form a 3-6 membered ring having 0-2 heteroatoms selected from
oxygen, nitrogen, or sulfur; wherein the ring formed by two
occurrences of J.sup.R is optionally substituted with 0-3
occurrences of J.sup.X; or two occurrences of J.sup.R, together
with Q.sup.2, form a 6-10 membered saturated or partially
unsaturated bridged ring system; Q.sup.3 is a 3-7 membered fully
saturated, partially unsaturated, or aromatic monocyclic ring
having 0-3 heteroatoms selected from oxygen, nitrogen, or sulfur; a
7-12 membered fully saturated, partially unsaturated, or aromatic
bicyclic ring having 0-5 heteroatoms selected from oxygen,
nitrogen, or sulfur; J.sup.X is independently selected from halo or
a C.sub.1-4aliphatic chain wherein up to two methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; or J.sup.P is independently selected
from halo; --CN; .dbd.O; a C.sub.1-6aliphatic chain wherein up to
two methylene units of the aliphatic chain are optionally replaced
with --O--, --NR--, --C(O)--, or --S(O).sub.z--; or a 3-6 membered
non-aromatic ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; each J.sup.P is optionally substituted with
0-3 occurrences of J.sup.M; or two occurrences of J.sup.P on the
same atom, together with the atom to which they are joined, form a
3-6 membered ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; or two occurrences of J.sup.P, together with
Q.sup.3, form a 6-10 membered saturated or partially unsaturated
bridged ring system; R.sup.4 is independently selected from H,
halo, a C.sub.3-4 membered cycloalkyl, 3-4 membered heterocyclyl,
or C.sub.1-4aliphatic chain wherein up to two methylene units of
the aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; J.sup.M is independently selected from
halo or C.sub.1-6aliphatic; z is 0, 1, or 2; and R is independently
selected from H or C.sub.1-4aliphatic.
[0011] Another aspect of the invention provides a compound of
Formula I:
##STR00004##
or a pharmaceutically acceptable salt or prodrug thereof, wherein:
R.sup.1 is independently selected from H, fluoro, chloro, or
--C(J.sup.1).sub.2CN; J.sup.1 is independently selected from H or
C.sub.1-2alkyl; or two occurrences of J.sup.1, together with the
carbon atom to which they are attached, form an optionally
substituted 3-4 membered carbocyclic ring; R.sup.2 is independently
selected from H; halo; --CN; or a C.sub.1-6aliphatic chain wherein
up to two methylene units of the aliphatic chain are optionally
replaced with --O--, --NR--, --C(O)--, or --S(O).sub.z; each
R.sup.2 is optionally substituted with 0-3 occurrences of R.sup.2a;
R.sup.2a is independently selected from halo, C.sub.1-4alkyl, --CN,
or a 3-6 membered non-aromatic ring having 0-2 heteroatoms selected
from oxygen, nitrogen, or sulfur; A is independently selected
from:
##STR00005##
R.sup.3 is independently selected from -(L).sub.n-Q.sup.1 or T; L
and T are each independently a C.sub.1-10aliphatic chain wherein up
to three methylene units of the aliphatic chain are optionally
replaced with --O--, --NR--, --S(O).sub.z--, or --C(O)--; each L
and T is independently substituted with 0-5 occurrences of
J.sup.LT; J.sup.LT is independently selected from halo, --CN, or a
C.sub.1-4aliphatic chain wherein up to two methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; n is 0 or 1; Q.sup.1 is independently
selected from a 3-7 membered fully saturated, partially
unsaturated, or aromatic monocyclic ring having 0-3 heteroatoms
selected from oxygen, nitrogen or sulfur; or an 7-12 membered fully
saturated, partially unsaturated, or aromatic bicyclic ring having
0-5 heteroatoms selected from oxygen, nitrogen, or sulfur; wherein
Q.sup.1 is independently substituted with 0-5 occurrences of
J.sup.Q; J.sup.Q is independently selected from halo; --CN; .dbd.O;
Q.sup.2; or a C.sub.1-8aliphatic chain wherein up to three
methylene units of the aliphatic chain are optionally replaced with
--O--, --NR--, --C(O)--, or --S(O).sub.z--; each occurrence of
J.sup.Q is optionally substituted by 0-3 occurrences of J.sup.R; or
two occurrences of J.sup.Q on the same atom, taken together with
the atom to which they are joined, form a 3-6 membered ring having
0-2 heteroatoms selected from oxygen, nitrogen, or sulfur; wherein
the ring formed by two occurrences of J.sup.Q is optionally
substituted with 0-3 occurrences of J.sup.X; or two occurrences of
J.sup.Q, together with Q.sup.1, form a 6-10 membered saturated or
partially unsaturated bridged ring system; Q.sup.2 is independently
a 3-7 membered fully saturated, partially unsaturated, or aromatic
monocyclic ring having 0-3 heteroatoms selected from oxygen,
nitrogen, or sulfur; or a 7-12 membered fully saturated, partially
unsaturated, or aromatic bicyclic ring having 0-5 heteroatoms
selected from oxygen, nitrogen, or sulfur; J.sup.R is independently
selected from halo; --CN; .dbd.O; .fwdarw.O; Q.sup.3; or a
C.sub.1-6aliphatic chain wherein up to two methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; each J.sup.R is optionally substituted
with 0-3 occurrences of J.sup.P; or two occurrences of J.sup.R on
the same atom, together with the atom to which they are joined,
form a 3-6 membered ring having 0-2 heteroatoms selected from
oxygen, nitrogen, or sulfur; wherein the ring formed by two
occurrences of J.sup.R is optionally substituted with 0-3
occurrences of J.sup.X; or two occurrences of J.sup.R, together
with Q.sup.2, form a 6-10 membered saturated or partially
unsaturated bridged ring system; Q.sup.3 is a 3-7 membered fully
saturated, partially unsaturated, or aromatic monocyclic ring
having 0-3 heteroatoms selected from oxygen, nitrogen, or sulfur; a
7-12 membered fully saturated, partially unsaturated, or aromatic
bicyclic ring having 0-5 heteroatoms selected from oxygen,
nitrogen, or sulfur; J.sup.X is independently selected from halo or
a C.sub.1-4aliphatic chain wherein up to two methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--,
--C(O)--, or --S(O).sub.z--; or J.sup.P is independently selected
from halo; --CN; .dbd.O; a C.sub.1-6aliphatic chain wherein up to
two methylene units of the aliphatic chain are optionally replaced
with --O--, --NR--, --C(O)--, or --S(O).sub.z--; or a 3-6 membered
non-aromatic ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; or two occurrences of J.sup.P on the same
atom, together with the atom to which they are joined, form a 3-6
membered ring having 0-2 heteroatoms selected from oxygen,
nitrogen, or sulfur; or two occurrences of J.sup.P, together with
Q.sup.3, form a 6-10 membered saturated or partially unsaturated
bridged ring system; R.sup.4 is independently selected from H or
C.sub.1-3aliphatic; z is 0, 1, or 2; and R is independently
selected from H or C.sub.1-4aliphatic.
[0012] For purposes of this application, it will be understood that
when two occurrences of J.sup.Q, together with Q.sup.1, form a
bridged ring system, the two occurrences of J.sup.Q are attached to
separate atoms of Q.sup.1. Additionally, when two occurrences of
J.sup.R, together with Q.sup.2, form a bridged ring system, the two
occurrence of J.sup.R are attached to separate atoms of Q.sup.2.
Moreover, when two occurrences of J.sup.P, together with Q.sup.3,
form a bridged ring system, the two occurrences of J.sup.P are
attached to separate atoms of Q.sup.3. Finally, when two
occurrences of J.sup.W, together with W, form a bridged ring
system, the two occurrences of J.sup.W are attached to separate
atoms of W;
[0013] In one embodiment, the present invention is a compound of
formula I, wherein R.sup.1 is fluoro. In another embodiment, the
present invention is a compound of formula I, wherein R.sup.1 is
--CH.sub.2CN or --CH(C.sub.1-2alkyl)CN. In yet another embodiment,
the present invention is a compound of formula I, wherein R.sup.1
is chloro. In other embodiments, the present invention is a
compound of formula I, wherein R.sup.1 is H.
[0014] In some embodiments, the present invention is a compound of
formula I, wherein R.sup.2 is --CF.sub.3. In another embodiment,
the present invention is a compound of formula I, wherein R.sup.2
is C.sub.1-6aliphatic wherein up to two methylene units are
optionally replaced with --O--, --NR--, --C(O)--, or S. In other
embodiments, the present invention is a compound of formula I,
wherein R.sup.2 is --O(C.sub.1-3alkyl)N(C.sub.1-3alkyl) or
--NR(C.sub.1-3alkyl)N(C.sub.1-3alkyl). In yet another embodiment,
the present invention is a compound of formula I, wherein R.sup.2
is H.
[0015] In some examples, the present invention is a compound of
formula I, wherein A is:
##STR00006##
[0016] In other examples, the present invention is a compound of
formula I, wherein A is:
##STR00007##
[0017] In yet further examples, the present invention is a compound
of formula I, wherein A is:
##STR00008##
[0018] In one or more embodiments, the present invention is a
compound of formula I, wherein R.sup.3 is -(L).sub.n-Q.sup.1. In
another embodiment, the present invention is a compound of formula
I, wherein R.sup.3 is T.
[0019] In some embodiments, the present invention is a compound of
formula I, wherein is 1. In other embodiments, the present
invention is a compound of formula I, wherein n is 0.
[0020] In yet another example, the present invention is a compound
of formula I, wherein L is --O--
[0021] In another aspect, the present invention is a compound of
formula I, wherein Q.sup.1 is independently selected from a 3-7
membered fully saturated, partially unsaturated, or aromatic
monocyclic ring having 0-3 heteroatoms selected from oxygen,
nitrogen, or sulfur. In other aspects, the present invention is a
compound of formula I, wherein Q.sup.1 is a 3-7 membered
heterocyclyl or carbocyclyl. In yet another aspect, the present
invention is a compound of formula I, wherein Q.sup.1 is
independently selected from cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl, pyrrolidinyl, piperidinyl, azepanyl,
pyrazolidinyl, isoxazolidinyl, oxazolidinyl, thiazolidinyl,
imidazolidinyl, piperazinyl, morpholinyl, thiomorpholinyl,
1,3-oxazinanyl, 1,3-thiazinanyl, dihydropyridinyl,
dihydroimidazolyl, 1,3-tetrahydropyrimidinyl, dihydropyrimidinyl,
1,4-diazepanyl, 1,4-oxazepanyl, 1,4-thiazepanyl, and azetidinyl. In
some embodiments, the present invention is a compound of formula I,
wherein Q.sup.1 is independently selected from pyrrolidinyl,
cyclopropyl, cyclohexyl, piperidinyl or piperazinyl.
[0022] In other embodiments, the present invention is a compound of
formula I, wherein Q.sup.1 is a 5-6 membered aryl or heteroaryl. In
yet another embodiment, the present invention is a compound of
formula I, wherein Q.sup.1 is independently selected from phenyl,
pyridinyl, pyrazinyl, pyrimidinyl, tetrahydropyridinyl, pyrrolyl,
imidazolyl, pyrazolyl, 1,2,3-triazolyl, or 1,2,4-triazolyl. In
further embodiments, the present invention is a compound of formula
I, wherein Q.sup.1 is pyridinyl.
[0023] In another example, the present invention is a compound of
formula I, wherein Q.sup.1 is a 7-12 membered fully saturated,
partially unsaturated, or aromatic bicyclic ring having 1-5
heteroatoms selected from oxygen, nitrogen, or sulfur. In some
examples, the present invention is a compound of formula I, wherein
Q.sup.1 is independently selected from
octahydropyrrolo[1,2-a]pyrazinyl,
5,6,7,8-tetrahydroimidazo[1,2-a]pyridinyl,
octahydro-1H-pyrazino[1,2-a]pyrazinyl,
5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl,
2,5-diazabicyclo[4.1.0]heptane, or
octahydropyrazino[2,1-a][1,4]oxazinyl.
[0024] In one or more aspects of the invention, the present
invention is a compound of formula I, wherein J.sup.Q is
C.sub.1-6aliphatic chain wherein up to three methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--, or
--C(O)--. In other aspects, the present invention is a compound of
formula I, wherein J.sup.Q is independently selected from --C(O)--,
C.sub.1-4alkyl, --(C.sub.0-4alkyl)NH.sub.2,
--(C.sub.0-4alkyl)NH(C.sub.1-4alkyl),
--(C.sub.0-4alkyl)N(C.sub.1-4alkyl).sub.2, --(C.sub.0-4alkyl)OH,
--(C.sub.0-4alkyl)O(C.sub.1-4alkyl), --C(O)OH,
--C(O)O(C.sub.1-4alkyl), N(C.sub.1-4alkyl).sub.2,
--C(O)N(C.sub.1-4alkyl).sub.2, or
--(C.sub.1-3alkyl)O(C.sub.1-2alkyl)N(C.sub.1-3alkyl).sub.2. In yet
another aspect, the present invention is a compound of formula I,
wherein J.sup.Q is independently selected from --C(O)--,
C.sub.1-4alkyl, or --(C.sub.0-4alkyl)NH.sub.2.
[0025] In some embodiments, the present invention is a compound of
formula I, wherein J.sup.Q is Q.sup.2. In another embodiment, the
present invention is a compound of formula I, wherein Q.sup.2 is a
3-7 membered fully saturated, partially unsaturated, or aromatic
monocyclic ring having 0-3 heteroatoms selected from oxygen,
sulfur, or nitrogen. In other embodiments, the present invention is
a compound of formula I, wherein Q.sup.2 is independently selected
from selected from cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, oxetanyl, tetrahydropyranyl, tetrahydrofuranyl,
azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl,
thiomorpholinyl, or morpholinyl. In yet another embodiment, the
present invention is a compound of formula I, wherein Q.sup.2 is
oxetanyl, pyrrolidinyl, tetrahydrofuranyl, or
tetrahydropyranyl.
[0026] In one or more examples, the present invention is a compound
of formula I, wherein Q.sup.2 is a 7-12 membered fully saturated,
partially unsaturated, or aromatic bicyclic ring having 0-5
heteroatoms selected from oxygen, nitrogen, or sulfur. In some
examples, the present invention is a compound of formula I, wherein
Q.sup.2 is independently selected from
5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl or
5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl.
[0027] In another aspect, the present invention is a compound of
formula I, wherein two occurrences of J.sup.Q, together with
Q.sup.1, form a bridged ring system. In some aspects, the present
invention is a compound of formula I, wherein two occurrences of
J.sup.Q on the same atom, taken together with the atom to which
they are joined, form a 3-6 membered non-aromatic ring having 0-2
heteroatoms selected from oxygen, nitrogen, or sulfur. In yet
another aspect, the present invention is a compound of formula I,
wherein the ring formed by the two occurrences of J.sup.Q on the
same atom, taken together with the atom to which they are joined,
is selected from oxetanyl, cyclobutyl, or azetidinyl.
[0028] In some embodiments, the present invention is a compound of
formula I, wherein J.sup.R is a 3-6 membered heterocyclyl having
1-3 heteroatoms selected from oxygen, nitrogen, or sulfur. In
another embodiment, the present invention is a compound of formula
I, wherein J.sup.R is independently selected from oxetanyl,
piperidinyl, azetidinyl, piperazinyl, pyrrolidinyl, 1,4-diazepanyl,
or morpholinyl. In other embodiments, the present invention is a
compound of formula I, wherein J.sup.R is a piperazinyl.
[0029] In yet another embodiment, the present invention is a
compound of formula I, wherein J.sup.R is independently selected
from halo, .dbd.O, --OH, C.sub.1-4alkyl,
--(C.sub.0-4alkyl)N(C.sub.1-4alkyl).sub.2, or
--(C.sub.0-4alkyl)O(C.sub.1-4alkyl). In other embodiments, the
present invention is a compound of formula I, wherein two
occurrences of J.sup.R on the same atom, together with the atom to
which they are joined, form a 3-6 membered aromatic or non-aromatic
ring having 0-2 heteroatoms selected from oxygen, nitrogen, or
sulfur.
[0030] In some aspects, the present invention is a compound of
formula I, wherein J.sup.P is halo, --C.sub.1-4alkyl, or a 3-6
membered non-aromatic ring having 0-2 heteroatoms selected from
oxygen, nitrogen, or sulfur. In other aspects, the present
invention is a compound of formula I, wherein J.sup.P is
independently selected from pyrrolidinyl or oxetanyl.
[0031] In another aspect, the present invention is a compound of
formula I, wherein T is independently selected from
--(C.sub.1-4alkyl), --(C.sub.1-4alkyl)N(C.sub.1-4alkyl).sub.2,
--(C.sub.1-3alkyl)O(C.sub.1-2alkyl)N(C.sub.1-3alkyl).sub.2,
--(C.sub.1-4alkyl)OH, --(C.sub.1-4alkyl)NH.sub.2, or
--(C.sub.1-4alkyl)O(C.sub.1-4alkyl). In yet another aspect, the
present invention is a compound of formula I, wherein J.sup.LT is
halo or C.sub.1-3alkyl.
[0032] In still other aspects, the present invention is a compound
of formula I, wherein A is independently selected from:
##STR00009##
[0033] In some embodiments, the present invention is a compound of
formula I, wherein p is 0. In other embodiments, the present
invention is a compound of formula I, wherein p is 1.
[0034] In another embodiment, the present invention is a compound
of formula I, wherein R.sup.4 is independently selected from
C.sup.1-4alkyl or halo. In yet another embodiment, the present
invention is a compound of formula I, wherein R.sup.4 is
independently selected from methyl or fluoro.
[0035] In some embodiments, the present invention is a compound of
formula I, wherein R.sup.3 is -(L).sub.n-Q.sup.1.
[0036] In other embodiments, the present invention is a compound of
formula I, wherein n is 0. In still other embodiments, the present
invention is a compound of formula I, wherein n is 1.
[0037] In one or more aspects, the present invention is a compound
of formula I, wherein L is independently selected from
C.sub.1-4alkyl.
[0038] In another embodiment, Q.sup.1 the present invention is a
compound of formula I, wherein is phenyl. In yet another
embodiment, the present invention is a compound of formula I,
wherein Q.sup.1 is independently selected from a 3-6 membered
carbocyclyl or a 4-6 membered heterocyclyl. In some embodiments,
the present invention is a compound of formula I, wherein Q.sup.1
is independently selected from cyclopropyl, morpholinyl,
pyrrolidinyl, piperidinyl, piperazinyl, tetrahydropyran,
dihydropyran, or tetrahydropyridine. In still other embodiments,
the present invention is a compound of formula I, wherein Q.sup.1
is a 5-6 membered heteroaryl. In other embodiments, the present
invention is a compound of formula I, wherein Q.sup.1 is
independently selected from pyrazolyl, pyridinyl, or
pyrimidinyl.
[0039] In some embodiments, the present invention is a compound of
formula I, wherein J.sup.Q is independently selected from a
C.sub.1-6aliphatic chain wherein up to three methylene units of the
aliphatic chain are optionally replaced with --O--, --NR--, or
--C(O)--. In another embodiment, the present invention is a
compound of formula I, wherein J.sup.Q is independently selected
from --C(O)--, --C(O)C.sub.1-4alkyl or C.sub.1-4alkyl. In other
embodiments, the present invention is a compound of formula I,
wherein J.sup.Q is methyl.
[0040] In another example, the present invention is a compound of
formula I, wherein J.sup.R is independently selected from
piperidinyl or piperazinyl.
[0041] In yet another example, the present invention is a compound
of formula I, wherein J.sup.P is independently selected from
oxetanyl or azetidinyl. In some embodiments, the present invention
is a compound of formula I, wherein R.sup.3 is T. In still other
embodiments, the present invention is a compound of formula I,
wherein T is a C.sub.1-6aliphatic chain wherein up to three
methylene units of the aliphatic chain are optionally replaced with
--O--, --NR--, or --C(O)--. In yet another embodiment, the present
invention is a compound of formula I, wherein T is
--(C.sub.1-3alkyl)O(C.sub.1-3alkyl).
[0042] In other embodiments, the compounds of the present invention
are represented by formula I-A:
##STR00010##
[0043] In some embodiments, the compounds of this invention are as
represented in Table 1:
TABLE-US-00001 TABLE 1 ##STR00011## I-A-1 ##STR00012## I-A-2
##STR00013## I-A-3 ##STR00014## I-A-4 ##STR00015## I-A-5
##STR00016## I-A-6 ##STR00017## I-A-7 ##STR00018## I-A-8
##STR00019## I-A-9 ##STR00020## I-A-10 ##STR00021## I-A-11
##STR00022## I-A-12 ##STR00023## I-A-13 ##STR00024## I-A-14
##STR00025## I-A-15 ##STR00026## I-A-16 ##STR00027## I-A-17
##STR00028## I-A-18 ##STR00029## I-A-19 ##STR00030## I-A-20
[0044] In one or more embodiments, the compounds of the present
invention are represented by formula I-B:
##STR00031##
[0045] In yet another embodiment, the compounds of this invention
are as represented in Table 2:
TABLE-US-00002 TABLE 2 ##STR00032## I-B-1 ##STR00033## I-B-2
##STR00034## I-B-3 ##STR00035## I-B-4 ##STR00036## I-B-5
##STR00037## I-B-6 ##STR00038## I-B-7 ##STR00039## I-B-8
##STR00040## I-B-9 ##STR00041## I-B-10 ##STR00042## I-B-11
##STR00043## I-B-12 ##STR00044## I-B-13 ##STR00045## I-B-14
##STR00046## I-B-15 ##STR00047## I-B-16 ##STR00048## I-B-17
##STR00049## I-B-18 ##STR00050## I-B-19 ##STR00051## I-B-20
##STR00052## I-B-21 ##STR00053## I-B-22 ##STR00054## I-B-23
##STR00055## I-B-24
[0046] In another embodiment, the compounds of the present
invention are represented by formula I-C:
##STR00056##
[0047] In other embodiments, the compounds of this invention are as
represented in Table 3:
TABLE-US-00003 TABLE 3 ##STR00057## I-C-1 ##STR00058## I-C-2
##STR00059## I-C-3 ##STR00060## I-C-4 ##STR00061## I-C-5
##STR00062## I-C-6 ##STR00063## I-C-7 ##STR00064## I-C-8
##STR00065## I-C-9 ##STR00066## I-C-10 ##STR00067## I-C-11
[0048] In another embodiment, the compounds of the present
invention are represented by formula I-D:
##STR00068##
[0049] In other embodiments, the compounds of this invention are as
represented in Table 4:
TABLE-US-00004 TABLE 4 ##STR00069## I-D-1 ##STR00070## I-D-2
##STR00071## I-D-3 ##STR00072## I-D-4 ##STR00073## I-D-5
##STR00074## I-D-6
[0050] In another embodiment, the compounds of the present
invention are represented by formula I-E:
##STR00075##
[0051] In other embodiments, the compounds of this invention are as
represented in Table 5:
TABLE-US-00005 TABLE 5 ##STR00076## I-E-1 ##STR00077## I-E-2
##STR00078## I-E-3 ##STR00079## I-E-4 ##STR00080## I-E-5
##STR00081## I-E-6 ##STR00082## I-E-7 ##STR00083## I-E-8
##STR00084## I-E-9 ##STR00085## I-E-10 ##STR00086## I-E-11
##STR00087## I-E-12 ##STR00088## I-E-13 ##STR00089## I-E-14
##STR00090## I-E-15 ##STR00091## I-E-16 ##STR00092## I-E-17
##STR00093## I-E-18 ##STR00094## I-E-19 ##STR00095## I-E-20
##STR00096## I-E-21
[0052] In another embodiment, the compounds of this invention are
selected from the following:
##STR00097##
[0053] Another aspect of the present invention comprises a process
for preparing a compound of formula I:
##STR00098## [0054] comprising reacting a compound of formula
6:
[0054] ##STR00099## [0055] under suitable conditions to form an
amide bond, wherein J, R.sup.1, R.sup.2, and A are as defined
herein.
[0056] In some examples, the suitable conditions for forming the
amide bond comprises reacting the compound of formula 6 with a
substituted heteroaromatic amine in an aprotic solvent under heat.
In other examples, the aprotic solvent is selected from NMP,
optionally substituted pyridine, or DMF. In another embodiment, the
aprotic solvent is optionally substituted pyridine. In still other
embodiments, the reaction temperature is at least 80.degree. C. In
another embodiment, the reaction temperature is at least
100.degree. C.
[0057] In another embodiment, the process, described above, further
comprises preparing a compound of formula 6:
##STR00100## [0058] by reacting a compound of formula 5:
[0058] ##STR00101## [0059] under suitable conditions to form an
activated ester, wherein J, R.sup.1, and R.sup.2 are as defined
herein.
[0060] In some embodiments, suitable conditions for forming the
activated ester comprises reacting the compound of formula 5 with
an amide coupling agent in the presence of an organic base. In
another embodiment, the organic basis is an aliphatic amine. In
still other embodiments, the organic base is independently selected
from triethylamine or DIPEA. In one or more embodiments, the amide
coupling agent is independently selected from EDCI, TBTU, TCTU,
HATU, T3P, or COMU. In yet another embodiment, the amide coupling
agent is independently selected from TBTU or TCTU. In still other
embodiments, the amide coupling agent is TCTU
[0061] Another aspect of the invention comprises a process for
preparing a compound of formula I:
##STR00102## [0062] comprising reacting a compound of formula
5:
[0062] ##STR00103## [0063] under suitable conditions to form an
amide bond, wherein R.sup.1, R.sup.2, and A are as defined
herein.
[0064] Yet another aspect of the present invention comprises a
process for preparing a compound of formula 5:
##STR00104## [0065] by reacting a compound of formula 4:
[0065] ##STR00105## [0066] under suitable hydrolysis conditions,
wherein R.sup.1 and R.sup.2 are as defined herein.
[0067] In some embodiments, suitable hydrolysis conditions comprise
reacting the compound of formula 4 with a silane in the presence of
a metal catalyst. In other embodiments, the silane is a
phenylsilane. In another embodiment, the metal catalyst is a
palladium catalyst. In yet another embodiment, the palladium
catalyst is Pd(PPh.sub.3).sub.4. In another embodiment, suitable
hydrolytic conditions comprise reacting the compound of formula 4
with 4-methylbenzenesulfinate in the presence of a metal
catalyst.
[0068] In still other embodiments, suitable hydrolytic conditions
comprise reacting the compound of formula 4 with an aqueous alkali.
In some embodiments, the aqueous alkali is selected from LiOH, NaOH
or KOH.
[0069] Another aspect of the present invention comprises a process
for preparing a compound of formula 4:
##STR00106## [0070] by reacting a compound of formula 3:
[0070] ##STR00107## [0071] under suitable condensation conditions
to form a pyrimidine ring.
[0072] In some embodiments, suitable condensation conditions to
form a pyrimidine ring comprise reacting the compound of formula 3
with a 1,3-dielectrophilic species in the presence of a solvent. In
some instances, the condensation reaction is carried out in the
presence of a strong base. In some embodiments, the strong base is
KOH. In another embodiment, the condensation reaction is carried
out in the presence of a weak base. In yet another embodiment, the
weak base is triethylamine. In another embodiment, the
1,3-dielectrophilic species is selected from 1,3-dialdehyde or a
3-(dialkylamino)-prop-2-enal. In still other embodiments, the
solvent is selected from dioxane, DMF, or DMSO in water. In other
embodiments, the 1,3-dielectrophilic species is generated in situ
from a protected 1,3-dielectrophilic species. In other embodiments,
the 1,3-dielectrophilic species is generated in situ from a
protected 1,3-dielectrophilic species. In another embodiment the
1,3-dielectrophilic species is generated from a ketal in the
presence of a sulfonic acid. In some embodiments, the sulfonic acid
is PTSA.
[0073] Another aspect of the present invention comprises a process
for preparing the compound of formula 3:
##STR00108## [0074] by reacting a compound of formula 2:
[0074] ##STR00109## [0075] under suitable condensation conditions
to form a pyrazole ring.
[0076] In some embodiments, suitable condensation conditions to
form a pyrazole ring comprise reacting the compound of formula 2
with a hydrazine or hydrazine hydrate in the presence of an aprotic
solvent under basic conditions. In another embodiment, the aprotic
solvent is DMF. In yet another embodiment, the basic conditions
comprise reacting the compound of formula 2 in the presence of
potassium acetate or sodium acetate.
[0077] Yet another aspect of the present invention comprises a
process for preparing a compound of formula 2:
##STR00110## [0078] by reacting a compound of formula 1:
[0078] ##STR00111## [0079] under suitable anion condensation
conditions.
[0080] In some embodiments, suitable anion condensation conditions
comprise 1) reacting the compound of formula 1 with a base, in the
presence of a solvent, to generate the anion of the compound of
formula 1; and 2) reacting the anion of the compound of formula 1
with trichloroacetonitrile. In still other embodiments, the base is
potassium acetate. In yet another embodiment, the solvent is an
alcohol. In other embodiments, the solvent is isopropylalcohol.
[0081] Another aspect of the invention, comprises a process for
preparing a compound of formula I:
##STR00112## [0082] comprising reacting a compound of formula
9:
[0082] ##STR00113## [0083] under suitable condensation conditions
to form a pyrimidine ring, wherein R.sup.1, R.sup.2, and A are as
defined herein.
[0084] In some embodiments, suitable condensation conditions to
form a pyrimidine ring comprise reacting the compound of formula 9
with a 1,3-dielectrophilic species in the presence of a solvent. In
another embodiment, the 1,3-dielectrophilic species is selected
from 1,3-dialdehyde or a 3-(dialkylamino)-prop-2-enal. In still
other embodiments, the solvent is selected from dioxane, iPrOH in
water, DMF, or DMSO in water. In other embodiments, the
1,3-dielectrophilic species is generated in situ from a protected
1,3-dielectrophilic species. In another embodiment the
1,3-dielectrophilic species is generated from a ketal in the
presence of a sulfonic acid. In yet another embodiment, the
sulfonic acid is PTSA.
[0085] Yet another aspect of the present invention comprises a
process for preparing a compound of formula 9:
##STR00114## [0086] by reacting a compound of formula 8:
[0086] ##STR00115## [0087] under suitable condensation conditions
to form a pyrazole ring.
[0088] In some embodiments, suitable condensation conditions to
form a pyrazole ring comprise 1) reacting the compound of formula 8
with a base, in the presence of a solvent, to generate the anion of
the compound of formula I; 2) reacting the anion with
trichloroacetonitrile; and 3) reacting the product from 2) with a
hydrazine or hydrazine hydrate in the presence of an aprotic
solvent. In another embodiment, the aprotic solvent is NMP or DMF.
In some embodiments, the base is selected from sodium acetate or
potassium acetate.
[0089] Another embodiment comprises a process for preparing a
compound of formula 8:
##STR00116## [0090] by reacting a compound of formula 7
[0090] ##STR00117## [0091] under suitable conditions to form an
amide bond.
[0092] In some examples, the suitable conditions for forming the
amide bond comprises reacting the compound of formula 7 with a
substituted heteroaromatic amine with an amide coupling agent in
the presence of an aprotic solvent and an organic base. In other
examples, the aprotic solvent is selected from NMP, DCM, or DMF. In
another embodiment, the organic base is an aliphatic amine. In
still other embodiments, the organic base is independently selected
from triethylamine or DIPEA. In yet another embodiment, the amide
coupling agent is independently selected from TBTU or TCTU. In
still other embodiments, the reaction temperature is at least
80.degree. C. In another embodiment, the reaction temperature is at
least 100.degree. C.
[0093] Compounds of this invention include those described
generally herein, and are further illustrated by the classes,
subclasses, and species disclosed herein. As used herein, the
following definitions shall apply unless otherwise indicated. For
purposes of this invention, the chemical elements are identified in
accordance with the Periodic Table of the Elements, CAS version,
Handbook of Chemistry and Physics, 75.sup.th Ed. Additionally,
general principles of organic chemistry are described in "Organic
Chemistry", Thomas Sorrell, University Science Books, Sausalito:
1999, and "March's Advanced Organic Chemistry", 5.sup.th Ed., Ed.:
Smith, M. B. and March, J., John Wiley & Sons, New York: 2001,
the entire contents of which are hereby incorporated by
reference.
[0094] As described herein, a specified number range of atoms
includes any integer therein. For example, a group having from 1-4
atoms could have 1, 2, 3, or 4 atoms.
[0095] As described herein, compounds of the invention may
optionally be substituted with one or more substituents, such as
are illustrated generally herein, or as exemplified by particular
classes, subclasses, and species of the invention. It will be
appreciated that the phrase "optionally substituted" is used
interchangeably with the phrase "substituted or unsubstituted." In
general, the term "substituted", whether preceded by the term
"optionally" or not, refers to the replacement of hydrogen radicals
in a given structure with the radical of a specified substituent.
Unless otherwise indicated, an optionally substituted group may
have a substituent at each substitutable position of the group, and
when more than one position in any given structure may be
substituted with more than one substituent selected from a
specified group, the substituent may be either the same or
different at every position. Combinations of substituents
envisioned by this invention are preferably those that result in
the formation of stable or chemically feasible compounds.
[0096] Unless otherwise indicated, a substituent connected by a
bond drawn from the center of a ring means that the substituent can
be bonded to any position in the ring. In example i below, for
instance, J.sup.w can be bonded to any position on the pyridyl
ring. For bicyclic rings, a bond drawn through both rings indicates
that the substituent can be bonded from any position of the
bicyclic ring. In example ii below, for instance, J.sup.w can be
bonded to the 5-membered ring (on the nitrogen atom, for instance),
and to the 6-membered ring.
##STR00118##
[0097] The term "stable", as used herein, refers to compounds that
are not substantially altered when subjected to conditions to allow
for their production, detection, recovery, purification, and use
for one or more of the purposes disclosed herein. In some
embodiments, a stable compound or chemically feasible compound is
one that is not substantially altered when kept at a temperature of
40.degree. C. or less, in the absence of moisture or other
chemically reactive conditions, for at least a week.
[0098] The term "dative bond", as used herein, is defined as the
coordination bond formed upon interaction between molecular
species, one of which serves as a donor and the other as an
acceptor of the electron pair to be shared in the complex
formed.
[0099] The term "aliphatic" or "aliphatic group", as used herein,
means a straight-chain (i.e., unbranched), branched, or cyclic,
substituted or unsubstituted hydrocarbon chain that is completely
saturated or that contains one or more units of unsaturation that
has a single point of attachment to the rest of the molecule.
[0100] Unless otherwise specified, aliphatic groups contain 1-20
aliphatic carbon atoms. In some embodiments, aliphatic groups
contain 1-10 aliphatic carbon atoms. In other embodiments,
aliphatic groups contain 1-8 aliphatic carbon atoms. In still other
embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms,
and in yet other embodiments aliphatic groups contain 1-4 aliphatic
carbon atoms. Aliphatic groups may be linear or branched,
substituted or unsubstituted alkyl, alkenyl, or alkynyl groups.
Specific examples include, but are not limited to, methyl, ethyl,
isopropyl, n-propyl, sec-butyl, vinyl, n-butenyl, ethynyl, and
tert-butyl. Aliphatic groups may also be cyclic, or have a
combination of linear or branched and cyclic groups. Examples of
such types of aliphatic groups include, but are not limited to
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl,
--CH.sub.2-cyclopropyl,
CH.sub.2CH.sub.2CH(CH.sub.3)-cyclohexyl.
[0101] The term "cycloaliphatic" (or "carbocycle" or "carbocyclyl")
refers to a monocyclic C.sub.3-C.sub.8 hydrocarbon or bicyclic
C.sub.8-C.sub.12 hydrocarbon that is completely saturated or that
contains one or more units of unsaturation, but which is not
aromatic, that has a single point of attachment to the rest of the
molecule wherein any individual ring in said bicyclic ring system
has 3-7 members. Examples of cycloaliphatic groups include, but are
not limited to, cycloalkyl and cycloalkenyl groups. Specific
examples include, but are not limited to, cyclohexyl, cyclopropyl,
and cyclobutyl.
[0102] The term "heterocycle", "heterocyclyl", or "heterocyclic" as
used herein means non-aromatic, monocyclic, bicyclic, or tricyclic
ring systems in which one or more ring members are an independently
selected heteroatom. In some embodiments, the "heterocycle",
"heterocyclyl", or "heterocyclic" group has three to fourteen ring
members in which one or more ring members is a heteroatom
independently selected from oxygen, sulfur, nitrogen, or
phosphorus, and each ring in the system contains 3 to 7 ring
members.
[0103] Examples of heterocycles include, but are not limited to,
3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one,
2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl,
3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino,
2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino,
1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl,
1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl,
3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl,
3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl,
5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl,
4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl,
1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl,
5-imidazolidinyl, indolinyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, benzothiolane, benzodithiane, and
1,3-dihydro-imidazol-2-one.
[0104] Cyclic groups, (e.g. cycloaliphatic and heterocycles), can
be linearly fused, bridged, or spirocyclic.
[0105] The term "heteroatom" means one or more of oxygen, sulfur,
nitrogen, phosphorus, or silicon (including, any oxidized form of
nitrogen, sulfur, phosphorus, or silicon; the quaternized form of
any basic nitrogen or; a substitutable nitrogen of a heterocyclic
ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in
pyrrolidinyl) or NR.sup.+ (as in N-substituted pyrrolidinyl)).
[0106] The term "unsaturated", as used herein, means that a moiety
has one or more units of unsaturation. As would be known by one of
skill in the art, unsaturated groups can be partially unsaturated
or fully unsaturated. Examples of partially unsaturated groups
include, but are not limited to, butene, cyclohexene, and
tetrahydropyridine. Fully unsaturated groups can be aromatic,
anti-aromatic, or non-aromatic. Examples of fully unsaturated
groups include, but are not limited to, phenyl, cyclooctatetraene,
pyridyl, thienyl, and 1-methylpyridin-2(1H)-one.
[0107] The term "alkoxy", or "thioalkyl", as used herein, refers to
an alkyl group, as previously defined, attached through an oxygen
("alkoxy") or sulfur ("thioalkyl") atom.
[0108] The terms "haloalkyl", "haloalkenyl", "haloaliphatic", and
"haloalkoxy" mean alkyl, alkenyl or alkoxy, as the case may be,
substituted with one or more halogen atoms. This term includes
perfluorinated alkyl groups, such as --CF.sub.3 and
--CF.sub.2CF.sub.3.
[0109] The terms "halogen", "halo", and "hal" mean F, Cl, Br, or
I.
[0110] The term "aryl" used alone or as part of a larger moiety as
in "arylalkyl", "arylalkoxy", or "aryloxyalkyl", refers to
monocyclic, bicyclic, and tricyclic ring systems having a total of
five to fourteen ring members, wherein at least one ring in the
system is aromatic and wherein each ring in the system contains 3
to 7 ring members. The term "aryl" may be used interchangeably with
the term "aryl ring".
[0111] The term "heteroaryl", used alone or as part of a larger
moiety as in "heteroarylalkyl" or "heteroarylalkoxy", refers to
monocyclic, bicyclic, and tricyclic ring systems having a total of
five to fourteen ring members, wherein at least one ring in the
system is aromatic, at least one ring in the system contains one or
more heteroatoms, and wherein each ring in the system contains 3 to
7 ring members. The term "heteroaryl" may be used interchangeably
with the term "heteroaryl ring" or the term "heteroaromatic".
Examples of heteroaryl rings include, but are not limited to,
2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl,
5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl,
2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g.,
3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl
(e.g., 5-tetrazolyl), triazolyl (e.g., 2-triazolyl and
5-triazolyl), 2-thienyl, 3-thienyl, benzofuryl, benzothiophenyl,
indolyl (e.g., 2-indolyl), pyrazolyl (e.g., 2-pyrazolyl),
isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl,
1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl,
1,3,5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl,
4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl,
3-isoquinolinyl, or 4-isoquinolinyl).
[0112] It shall be understood that the term "heteroaryl" includes
certain types of heteroaryl rings that exist in equilibrium between
two different forms. More specifically, for example, species such
hydropyridine and pyridinone (and likewise hydroxypyrimidine and
pyrimidinone) are meant to be encompassed within the definition of
"heteroaryl."
##STR00119##
[0113] The term "protecting group" and "protective group" as used
herein, are interchangeable and refer to an agent used to
temporarily block one or more desired functional groups in a
compound with multiple reactive sites. In certain embodiments, a
protecting group has one or more, or preferably all, of the
following characteristics: a) is added selectively to a functional
group in good yield to give a protected substrate that is b) stable
to reactions occurring at one or more of the other reactive sites;
and c) is selectively removable in good yield by reagents that do
not attack the regenerated, deprotected functional group. As would
be understood by one skilled in the art, in some cases, the
reagents do not attack other reactive groups in the compound. In
other cases, the reagents may also react with other reactive groups
in the compound. Examples of protecting groups are detailed in
Greene, T. W., Wuts, P. G in "Protective Groups in Organic
Synthesis", Third Edition, John Wiley & Sons, New York: 1999
(and other editions of the book), the entire contents of which are
hereby incorporated by reference. The term "nitrogen protecting
group", as used herein, refers to an agent used to temporarily
block one or more desired nitrogen reactive sites in a
multifunctional compound. Preferred nitrogen protecting groups also
possess the characteristics exemplified for a protecting group
above, and certain exemplary nitrogen protecting groups are also
detailed in Chapter 7 in Greene, T. W., Wuts, P. G in "Protective
Groups in Organic Synthesis", Third Edition, John Wiley & Sons,
New York: 1999, the entire contents of which are hereby
incorporated by reference.
[0114] In some embodiments, a methylene unit of an alkyl or
aliphatic chain is optionally replaced with another atom or group.
Examples of such atoms or groups include, but are not limited to,
nitrogen, oxygen, sulfur, --C(O)--, --C(.dbd.N--CN)--,
--C(.dbd.NR)--, --C(.dbd.NOR)--, --SO--, and --SO.sub.2--. These
atoms or groups can be combined to form larger groups. Examples of
such larger groups include, but are not limited to, --OC(O)--,
--C(O)CO--, --CO.sub.2--, --C(O)NR--, --C(.dbd.N--CN), --NRCO--,
--NRC(O)O--, --SO.sub.2NR--, --NRSO.sub.2--, --NRC(O)NR--,
--OC(O)NR--, and --NRSO.sub.2NR--, wherein R is, for example, H or
C.sub.1-6aliphatic. It should be understood that these groups can
be bonded to the methylene units of the aliphatic chain via single,
double, or triple bonds. An example of an optional replacement
(nitrogen atom in this case) that is bonded to the aliphatic chain
via a double bond would be --CH.sub.2CH.dbd.N--CH.sub.3. In some
cases, especially on the terminal end, an optional replacement can
be bonded to the aliphatic group via a triple bond. One example of
this would be CH.sub.2CH.sub.2CH.sub.2C.ident.N. It should be
understood that in this situation, the terminal nitrogen is not
bonded to another atom.
[0115] It should also be understood that, the term "methylene unit"
can also refer to branched or substituted methylene units. For
example, in an isopropyl moiety [--CH(CH.sub.3).sub.2], a nitrogen
atom (e.g. NR) replacing the first recited "methylene unit" would
result in dimethylamine [--N(CH.sub.3).sub.2]. In instances such as
these, one of skill in the art would understand that the nitrogen
atom will not have any additional atoms bonded to it, and the "R"
from "NR" would be absent in this case.
[0116] Unless otherwise indicated, the optional replacements form a
chemically stable compound. Optional replacements can occur both
within the chain and/or at either end of the chain; i.e. both at
the point of attachment and/or also at the terminal end. Two
optional replacements can also be adjacent to each other within a
chain so long as it results in a chemically stable compound. For
example, a C.sub.3 aliphatic can be optionally replaced by 2
nitrogen atoms to form --C--N.ident.N. The optional replacements
can also completely replace all of the carbon atoms in a chain. For
example, a C.sub.3 aliphatic can be optionally replaced by --NR--,
--C(O)--, and --NR-- to form --NRC(O)NR-- (a urea).
[0117] Unless otherwise indicated, if the replacement occurs at the
terminal end, the replacement atom is bound to a hydrogen atom on
the terminal end. For example, if a methylene unit of
--CH.sub.2CH.sub.2CH.sub.3 were optionally replaced with --O--, the
resulting compound could be --OCH.sub.2CH.sub.3,
--CH.sub.2OCH.sub.3, or --CH.sub.2CH.sub.2OH. In another example,
if a methylene unit of --CH.sub.2CH.sub.2CH.sub.3 was optionally
replaced with --NH--, the resulting compound could be
--NHCH.sub.2CH.sub.3, --CH.sub.2NHCH.sub.3, or
--CH.sub.2CH.sub.2NH.sub.2. It should be understood that if the
terminal atom does not contain any free valence electrons, then a
hydrogen atom is not required at the terminal end (e.g.,
--CH.sub.2CH.sub.2CH.dbd.O or --CH.sub.2CH.sub.2CN).
[0118] Unless otherwise indicated, structures depicted herein are
also meant to include all isomeric (e.g., enantiomeric,
diastereomeric, geometric, conformational, and rotational) forms of
the structure. For example, the R and S configurations for each
asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E)
conformational isomers are included in this invention. As would be
understood to one skilled in the art, a substituent can freely
rotate around any rotatable bonds. For example, a substituent drawn
as
##STR00120##
also represents
##STR00121##
[0119] Therefore, single stereochemical isomers as well as
enantiomeric, diastereomeric, geometric, conformational, and
rotational mixtures of the present compounds are within the scope
of the invention.
[0120] Unless otherwise indicated, all tautomeric forms of the
compounds of the invention are within the scope of the
invention.
[0121] Additionally, unless otherwise indicated, structures
depicted herein are also meant to include compounds that differ
only in the presence of one or more isotopically enriched atoms.
For example, compounds having the present structures except for the
replacement of hydrogen by deuterium or tritium, or the replacement
of a carbon by a .sup.13C- or .sup.14C-enriched carbon are within
the scope of this invention. Such compounds are useful, for
example, as analytical tools or probes in biological assays.
Pharmaceutically Acceptable Salts, Solvates, Chlatrates, Prodrugs
and Other Derivatives
[0122] The compounds described herein can exist in free form, or,
where appropriate, as salts. Those salts that are pharmaceutically
acceptable are of particular interest since they are useful in
administering the compounds described below for medical purposes.
Salts that are not pharmaceutically acceptable are useful in
manufacturing processes, for isolation and purification purposes,
and in some instances, for use in separating stereoisomeric forms
of the compounds of the invention or intermediates thereof
[0123] As used herein, the term "pharmaceutically acceptable salt"
refers to salts of a compound which are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of
humans and lower animals without undue side effects, such as,
toxicity, irritation, allergic response and the like, and are
commensurate with a reasonable benefit/risk ratio.
[0124] Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge et al., describe pharmaceutically
acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66,
1-19, incorporated herein by reference. Pharmaceutically acceptable
salts of the compounds described herein include those derived from
suitable inorganic and organic acids and bases. These salts can be
prepared in situ during the final isolation and purification of the
compounds.
[0125] Where the compound described herein contains a basic group,
or a sufficiently basic bioisostere, acid addition salts can be
prepared by 1) reacting the purified compound in its free-base form
with a suitable organic or inorganic acid and 2) isolating the salt
thus formed. In practice, acid addition salts might be a more
convenient form for use and use of the salt amounts to use of the
free basic form.
[0126] Examples of pharmaceutically acceptable, non-toxic acid
addition salts are salts of an amino group formed with inorganic
acids such as hydrochloric acid, hydrobromic acid, phosphoric acid,
sulfuric acid and perchloric acid or with organic acids such as
acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,
succinic acid or malonic acid or by using other methods used in the
art such as ion exchange. Other pharmaceutically acceptable salts
include adipate, alginate, ascorbate, aspartate, benzenesulfonate,
benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, glycolate, gluconate, glycolate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl
sulfate, malate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate,
palmitate, palmoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, salicylate, stearate,
succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate,
undecanoate, valerate salts, and the like.
[0127] Where the compound described herein contains a carboxy group
or a sufficiently acidic bioisostere, base addition salts can be
prepared by 1) reacting the purified compound in its acid form with
a suitable organic or inorganic base and 2) isolating the salt thus
formed. In practice, use of the base addition salt might be more
convenient and use of the salt form inherently amounts to use of
the free acid form. Salts derived from appropriate bases include
alkali metal (e.g., sodium, lithium, and potassium), alkaline earth
metal (e.g., magnesium and calcium), ammonium and
N.sup.+(C.sub.1-4alkyl).sub.4 salts. This invention also envisions
the quaternization of any basic nitrogen-containing groups of the
compounds disclosed herein. Water or oil-soluble or dispersible
products may be obtained by such quaternization.
[0128] Basic addition salts include pharmaceutically acceptable
metal and amine salts. Suitable metal salts include the sodium,
potassium, calcium, barium, zinc, magnesium, and aluminum. The
sodium and potassium salts are usually preferred. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
Suitable inorganic base addition salts are prepared from metal
bases, which include sodium hydride, sodium hydroxide, potassium
hydroxide, calcium hydroxide, aluminium hydroxide, lithium
hydroxide, magnesium hydroxide, zinc hydroxide and the like.
Suitable amine base addition salts are prepared from amines which
are frequently used in medicinal chemistry because of their low
toxicity and acceptability for medical use Ammonia,
ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine,
choline, N,N'-dibenzylethylenediamine, chloroprocaine,
dietanolamine, procaine, N-benzylphenethylamine, diethylamine,
piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium
hydroxide, triethylamine, dibenzylamine, ephenamine,
dehydroabietylamine, N-ethylpiperidine, benzylamine,
tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, ethylamine, basic amino acids,
dicyclohexylamine and the like are examples of suitable base
addition salts.
[0129] Other acids and bases, while not in themselves
pharmaceutically acceptable, may be employed in the preparation of
salts useful as intermediates in obtaining the compounds described
herein and their pharmaceutically acceptable acid or base addition
salts.
[0130] It should be understood that this invention includes
mixtures/combinations of different pharmaceutically acceptable
salts and also mixtures/combinations of compounds in free form and
pharmaceutically acceptable salts.
[0131] The compounds described herein can also exist as
pharmaceutically acceptable solvates (e.g., hydrates) and
clathrates. As used herein, the term "pharmaceutically acceptable
solvate," is a solvate formed from the association of one or more
pharmaceutically acceptable solvent molecules to one of the
compounds described herein. The term solvate includes hydrates
(e.g., hemihydrate, monohydrate, dihydrate, trihydrate,
tetrahydrate, and the like).
[0132] As used herein, the term "hydrate" means a compound
described herein or a salt thereof that further includes a
stoichiometric or non-stoichiometric amount of water bound by
non-covalent intermolecular forces.
[0133] As used herein, the term "clathrate" means a compound
described herein or a salt thereof in the form of a crystal lattice
that contains spaces (e.g., channels) that have a guest molecule
(e.g., a solvent or water) trapped within.
[0134] In addition to the compounds described herein,
pharmaceutically acceptable derivatives or prodrugs of these
compounds may also be employed in compositions to treat or prevent
the herein identified disorders.
[0135] A "pharmaceutically acceptable derivative or prodrug"
includes any pharmaceutically acceptable ester, salt of an ester,
or other derivative or salt thereof of a compound described herein
which, upon administration to a recipient, is capable of providing,
either directly or indirectly, a compound described herein or an
inhibitorily active metabolite or residue thereof. Particularly
favoured derivatives or prodrugs are those that increase the
bioavailability of the compounds when such compounds are
administered to a patient (e.g., by allowing an orally administered
compound to be more readily absorbed into the blood) or which
enhance delivery of the parent compound to a biological compartment
(e.g., the brain or lymphatic system) relative to the parent
species.
[0136] As used herein and unless otherwise indicated, the term
"prodrug" means a derivative of a compound that can hydrolyze,
oxidize, or otherwise react under biological conditions (in vitro
or in vivo) to provide a compound described herein. Prodrugs may
become active upon such reaction under biological conditions, or
they may have activity in their unreacted forms. Examples of
prodrugs contemplated in this invention include, but are not
limited to, analogs or derivatives of compounds of the invention
that comprise biohydrolyzable moieties such as biohydrolyzable
amides, biohydrolyzable esters, biohydrolyzable carbamates,
biohydrolyzable carbonates, biohydrolyzable ureides, and
biohydrolyzable phosphate analogues. Other examples of prodrugs
include derivatives of compounds described herein that comprise
--NO, --NO.sub.2, --ONO, or --ONO.sub.2 moieties. Prodrugs can
typically be prepared using well-known methods, such as those
described by BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY (1995)
172-178, 949-982 (Manfred E. Wolff ed., 5th ed).
Abbreviations
[0137] The following abbreviations are used: [0138] DMSO dimethyl
sulfoxide [0139] DCM dichloromethane [0140] ATP adenosine
triphosphate [0141] .sup.1HNMR proton nuclear magnetic resonance
[0142] HPLC high performance liquid chromatography [0143] LCMS
liquid chromatography-mass spectrometry [0144] Rt retention time
[0145] RT room temperature [0146] TEA triethylamine [0147] NMP
N-methyl-2-pyrrolidone [0148] TFA trifluoroacetic acid [0149] DMF
dimethylformamide [0150] DIPEA N,N-diisopropylethylamine [0151]
mCPBA meta-chloroperoxybenzoic acid [0152] Bp Boiling point [0153]
THF tetrahydrofuran [0154] HOBT hydroxybenzotriazole [0155] HATU
1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate [0156] T3P Propylphosphonic anhydride
[0157] COMU
1-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino)]-
uroniumhexafluorophosphate [0158] TBTU
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate [0159] TCTU
O-(6-Chloro-1-hydrocibenzotriazol-1-yl)-
-1,1,3,3-tetramethyluronium tetrafluoroborate [0160] EDCI
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
Compound Uses
[0161] One aspect of this invention provides compounds that are
inhibitors of ATR kinase, and thus are useful for treating or
lessening the severity of a disease, condition, or disorder in a
subject or patient where ATR is implicated in the disease,
condition, or disorder.
[0162] As used herein, the terms "subject" and "patient" are used
interchangeably. The terms "subject" and "patient" refer to an
animal, and more specifically a human. In one embodiment, the
subject is a non-human animal such as a rat or dog. In a preferred
embodiment, the subject is a human.
[0163] Another aspect of this invention provides compounds that are
useful for the treatment of diseases, disorders, and conditions
characterized by excessive or abnormal cell proliferation. Such
diseases include a proliferative or hyperproliferative disease.
Examples of proliferative and hyperproliferative diseases include,
without limitation, cancer and myeloproliferative disorders.
[0164] In some embodiments, said compounds are selected from the
group consisting of a compound of formula I. In another aspect,
said compounds are selected from the group consisting of formula
I-A. In yet another aspect of the present invention, said compounds
are selected from the group consisting of formula I-B. In still
other embodiments, said compounds are selected from the group
consisting of formula I-C. In another embodiment, said compounds
are selected from the group consisting of formula I-D. In some
embodiments, said compounds are selected from the group consisting
of formula I-E. The term "cancer" includes, but is not limited to
the following cancers. Oral: buccal cavity, lip, tongue, mouth,
pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma,
rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma,
lipoma and teratoma; Lung: non-small cell, bronchogenic carcinoma
(squamous cell or epidermoid, undifferentiated small cell,
undifferentiated large cell, adenocarcinoma), alveolar
(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,
chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus
(squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma,
lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas
(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma,
carcinoid tumors, vipoma), small bowel or small intestines
(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,
leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel
or large intestines (adenocarcinoma, tubular adenoma, villous
adenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal;
rectum, Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor
[nephroblastoma], lymphoma, leukemia), bladder and urethra
(squamous cell carcinoma, transitional cell carcinoma,
adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis
(seminoma, teratoma, embryonal carcinoma, teratocarcinoma,
choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,
fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma
(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,
angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;
Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant
fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant
lymphoma (reticulum cell sarcoma), multiple myeloma, malignant
giant cell tumor chordoma, osteochronfroma (osteocartilaginous
exostoses), benign chondroma, chondroblastoma, chondromyxofibroma,
osteoid osteoma and giant cell tumors; Nervous system: skull
(osteoma, hemangioma, granuloma, xanthoma, osteitis deformans),
meninges (meningioma, meningiosarcoma, gliomatosis), brain
(astrocytoma, medulloblastoma, glioma, ependymoma, germinoma
[pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma,
retinoblastoma, congenital tumors), spinal cord neurofibroma,
meningioma, glioma, sarcoma); Gynecological/Female: uterus
(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor
cervical dysplasia), ovaries (ovarian carcinoma [serous
cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified
carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell
tumors, dysgerminoma, malignant teratoma), vulva (squamous cell
carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma,
melanoma), vagina (clear cell carcinoma, squamous cell carcinoma,
botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes
(carcinoma), breast; Hematologic: blood (myeloid leukemia [acute
and chronic], acute lymphoblastic leukemia, chronic lymphocytic
leukemia, myeloproliferative diseases, multiple myeloma,
myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's
lymphoma [malignant lymphoma] hairy cell; lymphoid disorders; Skin:
malignant melanoma, basal cell carcinoma, squamous cell carcinoma,
Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma,
angioma, dermatofibroma, keloids, psoriasis, Thyroid gland:
papillary thyroid carcinoma, follicular thyroid carcinoma,
undifferentiated thyroid cancer, medullary thyroid carcinoma,
multiple endocrine neoplasia type 2A, multiple endocrine neoplasia
type 2B, familial medullary thyroid cancer, pheochromocytoma,
paraganglioma; and Adrenal glands: neuroblastoma.
[0165] In some embodiments, the cancer is selected from a cancer of
the lung or the pancreas. In other embodiments, the cancer is
selected from lung cancer, head and neck cancer, pancreatic cancer,
gastric cancer, or brain cancer. In yet other embodiments, the
cancer is selected from non-small cell lung cancer, small cell lung
cancer, pancreatic cancer, biliary tract cancer, head and neck
cancer, bladder cancer, colorectal cancer, glioblastoma, esophageal
cancer, breast cancer, hepatocellular carcinoma, or ovarian
cancer.
[0166] In some embodiments, the cancer is lung cancer. In other
embodiments, the lung cancer is non-small cell lung cancer or small
cell lung cancer. In another embodiment, the cancer is non-small
cell lung cancer. In yet another embodiment, the non-small cell
lung cancer is squamous non-small cell lung cancer.
[0167] Thus, the term "cancerous cell" as provided herein, includes
a cell afflicted by any one of the above-identified conditions. In
some embodiments, the cancer is selected from colorectal, thyroid,
or breast cancer. In other embodiments, the cancer is triple
negative breast cancer.
[0168] The term "myeloproliferative disorders", includes disorders
such as polycythemia vera, thrombocythemia, myeloid metaplasia with
myelofibrosis, hypereosinophilic syndrome, juvenile myelomonocytic
leukemia, systemic mast cell disease, and hematopoietic disorders,
in particular, acute-myelogenous leukemia (AML),
chronic-myelogenous leukemia (CML), acute-promyelocytic leukemia
(APL), and acute lymphocytic leukemia (ALL).
Pharmaceutical Compositions
[0169] The present invention also provides compounds and
compositions that are useful as inhibitors of ATR kinase.
[0170] One aspect of this invention provides pharmaceutically
acceptable compositions that comprise any of the compounds as
described herein, and optionally comprise a pharmaceutically
acceptable carrier, adjuvant or vehicle.
[0171] The pharmaceutically acceptable carrier, adjuvant, or
vehicle, as used herein, includes any and all solvents, diluents,
or other liquid vehicle, dispersion or suspension aids, surface
active agents, isotonic agents, thickening or emulsifying agents,
preservatives, solid binders, lubricants and the like, as suited to
the particular dosage form desired. Remington's Pharmaceutical
Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co.,
Easton, Pa., 1980) discloses various carriers used in formulating
pharmaceutically acceptable compositions and known techniques for
the preparation thereof. Except insofar as any conventional carrier
medium is incompatible with the compounds of the invention, such as
by producing any undesirable biological effect or otherwise
interacting in a deleterious manner with any other component(s) of
the pharmaceutically acceptable composition, its use is
contemplated to be within the scope of this invention.
[0172] Some examples of materials which can serve as
pharmaceutically acceptable carriers include, but are not limited
to, ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, or potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, wool fat, sugars such
as lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil; sesame oil; olive oil; corn oil and soybean
oil; glycols; such a propylene glycol or polyethylene glycol;
esters such as ethyl oleate and ethyl laurate; agar; buffering
agents such as magnesium hydroxide and aluminum hydroxide; alginic
acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol, and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator.
Combination Therapies
[0173] Another aspect of this invention is directed towards a
method of treating cancer in a subject in need thereof, comprising
administration of a compound of this invention or a
pharmaceutically acceptable salt thereof, and an additional
therapeutic agent. In some embodiments, said method comprises the
sequential or co-administration of the compound or a
pharmaceutically acceptable salt thereof, and the additional
therapeutic agent.
[0174] As used herein, the term "in combination" or
"co-administration" can be used interchangeably to refer to the use
of more than one therapy (e.g., one or more therapeutic agents).
The use of the term does not restrict the order in which therapies
(e.g., therapeutic agents) are administered to a subject.
[0175] In some embodiments, said additional therapeutic agent is an
anti-cancer agent. In other embodiments, said additional
therapeutic agent is a DNA-damaging agent. In yet other
embodiments, said additional therapeutic agent is selected from
radiation therapy, chemotherapy, or other agents typically used in
combination with radiation therapy or chemotherapy, such as
radiosensitizers and chemosensitizers. In yet other embodiments,
said additional therapeutic agent is ionizing radiation.
[0176] As would be known by one of skill in the art,
radiosensitizers are agents that can be used in combination with
radiation therapy. Radiosensitizers work in various different ways,
including, but not limited to, making cancer cells more sensitive
to radiation therapy, working in synergy with radiation therapy to
provide an improved synergistic effect, acting additively with
radiation therapy, or protecting surrounding healthy cells from
damage caused by radiation therapy. Likewise chemosensitizers are
agents that can be used in combination with chemotherapy.
Similarly, chemosensitizers work in various different ways,
including, but not limited to, making cancer cells more sensitive
to chemotherapy, working in synergy with chemotherapy to provide an
improved synergistic effect, acting additively to chemotherapy, or
protecting surrounding healthy cells from damage caused by
chemotherapy.
[0177] Examples of DNA-damaging agents that may be used in
combination with compounds of this invention include, but are not
limited to Platinating agents, such as Carboplatin, Nedaplatin,
Satraplatin and other derivatives; Topo I inhibitors, such as
Topotecan, irinotecan/SN38, rubitecan and other derivatives;
Antimetabolites, such as Folic family (Methotrexate, Pemetrexed and
relatives); Purine antagonists and Pyrimidine antagonists
(Thioguanine, Fludarabine, Cladribine, Cytarabine, Gemcitabine,
6-Mercaptopurine, 5-Fluorouracil (5FU) and relatives); Alkylating
agents, such as Nitrogen mustards (Cyclophosphamide, Melphalan,
Chlorambucil, mechlorethamine, Ifosfamide and relatives);
nitrosoureas (eg Carmustine); Triazenes (Dacarbazine,
temozolomide); Alkyl sulphonates (eg Busulfan); Procarbazine and
Aziridines; Antibiotics, such as Hydroxyurea, Anthracyclines
(doxorubicin, daunorubicin, epirubicin and other derivatives);
Anthracenediones (Mitoxantrone and relatives); Streptomyces family
(Bleomycin, Mitomycin C, actinomycin); and Ultraviolet light.
[0178] Other therapies or anticancer agents that may be used in
combination with the inventive agents of the present invention
include surgery, radiotherapy (in but a few examples,
gamma-radiation, neutron beam radiotherapy, electron beam
radiotherapy, proton therapy, brachytherapy, and systemic
radioactive isotopes, to name a few), endocrine therapy, biologic
response modifiers (interferons, interleukins, and tumor necrosis
factor (TNF) to name a few), hyperthermia and cryotherapy, agents
to attenuate any adverse effects (e.g., antiemetics), and other
approved chemotherapeutic drugs, including, but not limited to, the
DNA damaging agents listed herein, spindle poisons (Vinblastine,
Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (Etoposide,
Irinotecan, Topotecan), nitrosoureas (Carmustine, Lomustine),
inorganic ions (Cisplatin, Carboplatin), enzymes (Asparaginase),
and hormones (Tamoxifen, Leuprolide, Flutamide, and Megestrol),
Gleevec.TM., adriamycin, dexamethasone, and cyclophosphamide.
[0179] A compound of the instant invention may also be useful for
treating cancer in combination with any of the following
therapeutic agents: abarelix (Plenaxis Depot.RTM.); aldesleukin
(Prokine.RTM.); Aldesleukin (Proleukin.RTM.); Alemtuzumabb
(Campath.RTM.); alitretinoin (Panretin.RTM.); allopurinol
(Zyloprim.RTM.); altretamine (Hexalen.RTM.); amifostine
(Ethyol.RTM.); anastrozole (Arimidex.RTM.); arsenic trioxide
(Trisenox.RTM.); asparaginase (Elspar.RTM.); azacitidine
(Vidaza.RTM.); bevacuzimab (Avastin.RTM.); bexarotene capsules
(Targretin.RTM.); bexarotene gel (Targretin.RTM.); bleomycin
(Blenoxane.RTM.); bortezomib (Velcade.RTM.); busulfan intravenous
(Busulfex.RTM.); busulfan oral (Myleran.RTM.); calusterone
(Methosarb.RTM.); capecitabine (Xeloda carboplatin
(Paraplatin.RTM.); carmustine (BCNU.RTM., BiCNU.RTM.); carmustine
(Gliadel.RTM.); carmustine with Polifeprosan 20 Implant (Gliadel
Wafer.RTM.); celecoxib (Celebrex.RTM.); cetuximab (Erbitux.RTM.);
chlorambucil (Leukeran.RTM.); cisplatin (Platinol.RTM.); cladribine
(Leustatin.RTM., 2-CdA.RTM.); clofarabine (Clolar.RTM.);
cyclophosphamide (Cytoxan.RTM., Neosar.RTM.); cyclophosphamide
(Cytoxan Injection.RTM.); cyclophosphamide (Cytoxan Tablet.RTM.);
cytarabine (Cytosar-U.RTM.); cytarabine liposomal (DepoCyt.RTM.);
dacarbazine (DTIC-Dome.RTM.); dactinomycin, actinomycin D
(Cosmegen.RTM.); Darbepoetin alfa (Aranesp.RTM.); daunorubicin
liposomal (DanuoXome.RTM.); daunorubicin, daunomycin
(Daunorubicin.RTM.); daunorubicin, daunomycin (Cerubidine.RTM.);
Denileukin diftitox (Ontak.RTM.); dexrazoxane (Zinecard.RTM.);
docetaxel (Taxotere.RTM.); doxorubicin (Adriamycin PFS.RTM.);
doxorubicin (Adriamycin.RTM., Rubex.RTM.); doxorubicin (Adriamycin
PFS Injection.RTM.); doxorubicin liposomal (Doxil.RTM.);
dromostanolone propionate (Dromostanolone.RTM.); dromostanolone
propionate (masterone Injection.RTM.); Elliott's B Solution
(Elliott's B Solution.RTM.); epirubicin (Ellence.RTM.); Epoetin
alfa (Epogen.RTM.); erlotinib (Tarceva.RTM.); estramustine
(Emcyt.RTM.); etoposide phosphate (Etopophos.RTM.); etoposide,
VP-16 (Vepesid.RTM.); exemestane (Aromasin.RTM.); Filgrastim
(Neupogen.RTM.); floxuridine (intraarterial) (FUDR.RTM.);
fludarabine (Fludara.RTM.); fluorouracil, 5-FU (Adrucil.RTM.);
fulvestrant (Faslodex.RTM.); gefitinib (Iressa.RTM.); gemcitabine
(Gemzar.RTM.); gemtuzumab ozogamicin (Mylotarg.RTM.); goserelin
acetate (Zoladex Implant.RTM.); goserelin acetate (Zoladex.RTM.);
histrelin acetate (Histrelin Implant.RTM.); hydroxyurea
(Hydrea.RTM.); Ibritumomab Tiuxetan (Zevalin.RTM.); idarubicin
(Idamycin.RTM.); ifosfamide (IFEX.RTM.); imatinib mesylate
(Gleevec.RTM.); interferon alfa 2a (Roferon A.RTM.); Interferon
alfa-2b (Intron A.RTM.); irinotecan (Camptosar.RTM.); lenalidomide
(Revlimid.RTM.); letrozole (Femara.RTM.); leucovorin
(Wellcovorin.RTM., Leucovorin.RTM.); Leuprolide Acetate
(Eligard.RTM.); levamisole (Ergamisol.RTM.); lomustine, CCNU
(CeeBU.RTM.); meclorethamine, nitrogen mustard (Mustargen.RTM.);
megestrol acetate (Megace.RTM.); melphalan, L-PAM (Alkeran.RTM.);
mercaptopurine, 6-MP (Purmethol.RTM.); mesna (Mesnex.RTM.); mesna
(Mesnex Tabs.RTM.); methotrexate (Methotrexate.RTM.); methoxsalen
(Uvadex.RTM.); mitomycin C (Mutamycin.RTM.); mitotane
(Lysodren.RTM.); mitoxantrone (Novantrone.RTM.); nandrolone
phenpropionate (Durabolin-50.RTM.); nelarabine (Arranon.RTM.);
Nofetumomab (Verluma.RTM.); Oprelvekin (Neumega.RTM.); oxaliplatin
(Eloxatin.RTM.); paclitaxel (Paxene.RTM.); paclitaxel (Taxol.RTM.);
paclitaxel protein-bound particles (Abraxane.RTM.); palifermin
(Kepivance.RTM.); pamidronate (Aredia.RTM.); pegademase (Adagen
(Pegademase Bovine).RTM.); pegaspargase (Oncaspar.RTM.);
Pegfilgrastim (Neulasta.RTM.); pemetrexed disodium (Alimta.RTM.);
pentostatin (Nipent.RTM.); pipobroman (Vercyte.RTM.); plicamycin,
mithramycin (Mithracin.RTM.); porfimer sodium (Photofrin.RTM.);
procarbazine (Matulane.RTM.); quinacrine (Atabrine.RTM.);
Rasburicase (Elitek.RTM.); Rituximab (Rituxan.RTM.); sargramostim
(Leukine.RTM.); Sargramostim (Prokine.RTM.); sorafenib
(Nexavar.RTM.); streptozocin (Zanosar.RTM.); sunitinib maleate
(Sutent.RTM.); talc (Sclerosol.RTM.); tamoxifen (Nolvadex.RTM.);
temozolomide (Temodar.RTM.); teniposide, VM-26 (Vumon.RTM.);
testolactone (Teslac.RTM.); thioguanine, 6-TG (Thioguanine.RTM.);
thiotepa (Thioplex.RTM.); topotecan (Hycamtin.RTM.); toremifene
(Fareston.RTM.); Tositumomab (Bexxar.RTM.); Tositumomab/I-131
tositumomab (Bexxar.RTM.); Trastuzumab (Herceptin.RTM.); tretinoin,
ATRA (Vesanoid.RTM.); Uracil Mustard (Uracil Mustard
Capsules.RTM.); valrubicin (Valstar.RTM.); vinblastine
(Velban.RTM.); vincristine (Oncovin.RTM.); vinorelbine
(Navelbine.RTM.); zoledronate (Zometa.RTM.) and vorinostat
(Zolinza.RTM.).
[0180] For a comprehensive discussion of updated cancer therapies
see, http://www.nci.nih.gov/, a list of the FDA approved oncology
drugs at http://www.fda.gov/cder/cancer/druglistframe.htm, and The
Merck Manual, Seventeenth Ed. 1999, the entire contents of which
are hereby incorporated by reference.
Compositions for Administration into a Subject
[0181] The ATR kinase inhibitors or pharmaceutical salts thereof
may be formulated into pharmaceutical compositions for
administration to animals or humans. These pharmaceutical
compositions, which comprise an amount of the ATR inhibitor
effective to treat or prevent the diseases or conditions described
herein and a pharmaceutically acceptable carrier, are another
embodiment of the present invention.
[0182] The exact amount of compound required for treatment will
vary from subject to subject, depending on the species, age, and
general condition of the subject, the severity of the disorder, the
particular agent, its mode of administration, and the like. The
compounds of the invention are preferably formulated in dosage unit
form for ease of administration and uniformity of dosage. The
expression "dosage unit form" as used herein refers to a physically
discrete unit of agent appropriate for the patient to be treated.
It will be understood, however, that the total daily usage of the
compounds and compositions of the present invention will be decided
by the attending physician within the scope of sound medical
judgment. The specific effective dose level for any particular
patient or organism will depend upon a variety of factors including
the disorder being treated and the severity of the disorder; the
activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific compound employed, and like
factors well known in the medical arts. The term "patient", as used
herein, means an animal, preferably a mammal, and most preferably a
human.
[0183] In some embodiments, these compositions optionally further
comprise one or more additional therapeutic agents. For example,
chemotherapeutic agents or other anti-proliferative agents may be
combined with the compounds of this invention to treat
proliferative diseases and cancer. Examples of known agents with
which these compositions can be combined are listed above under the
"Combination Therapies" section and also throughout the
specification. Some embodiments provide a simultaneous, separate or
sequential use of a combined preparation.
Modes of Administration and Dosage Forms
[0184] The pharmaceutically acceptable compositions of this
invention can be administered to humans and other animals orally,
rectally, parenterally, intracisternally, intravaginally,
intraperitoneally, topically (as by powders, ointments, or drops),
bucally, as an oral or nasal spray, or the like, depending on the
severity of the disorder being treated. In certain embodiments, the
compounds of the invention may be administered orally or
parenterally at dosage levels of about 0.01 mg/kg to about 50 mg/kg
and preferably from about 1 mg/kg to about 25 mg/kg, of subject
body weight per day, one or more times a day, to obtain the desired
therapeutic effect. Alternatively, the dosing schedule of the
compounds of the present invention may vary.
[0185] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active compounds, the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0186] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0187] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0188] In order to prolong the effect of a compound of the present
invention, it is often desirable to slow the absorption of the
compound from subcutaneous or intramuscular injection. This may be
accomplished by the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of
absorption of the compound then depends upon its rate of
dissolution that, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a
parenterally administered compound form is accomplished by
dissolving or suspending the compound in an oil vehicle. Injectable
depot forms are made by forming microencapsule matrices of the
compound in biodegradable polymers such as
polylactide-polyglycolide. Depending upon the ratio of compound to
polymer and the nature of the particular polymer employed, the rate
of compound release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the compound in liposomes or microemulsions that are
compatible with body tissues.
[0189] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
compounds of this invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active compound.
[0190] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0191] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
that can be used include polymeric substances and waxes. Solid
compositions of a similar type may also be employed as fillers in
soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polethylene
glycols and the like.
[0192] The active compounds can also be in microencapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active compound may be admixed with at least one inert diluent such
as sucrose, lactose or starch. Such dosage forms may also comprise,
as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such
a magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions that can be used include polymeric
substances and waxes.
[0193] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, eardrops, and
eye drops are also contemplated as being within the scope of this
invention. Additionally, the present invention contemplates the use
of transdermal patches, which have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
can be made by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel.
[0194] The compositions of the present invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes, but is
not limited to, subcutaneous, intravenous, intramuscular,
intra-articular, intra-synovial, intrasternal, intrathecal,
intrahepatic, intralesional and intracranial injection or infusion
techniques. Preferably, the compositions are administered orally,
intraperitoneally or intravenously.
[0195] Sterile injectable forms of the compositions of this
invention may be aqueous or oleaginous suspension. These
suspensions may be formulated according to techniques known in the
art using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed including
synthetic mono- or di-glycerides. Fatty acids, such as oleic acid
and its glyceride derivatives are useful in the preparation of
injectables, as are natural pharmaceutically-acceptable oils, such
as olive oil or castor oil, especially in their polyoxyethylated
versions. These oil solutions or suspensions may also contain a
long-chain alcohol diluent or dispersant, such as carboxymethyl
cellulose or similar dispersing agents which are commonly used in
the formulation of pharmaceutically acceptable dosage forms
including emulsions and suspensions. Other commonly used
surfactants, such as Tweens, Spans and other emulsifying agents or
bioavailability enhancers which are commonly used in the
manufacture of pharmaceutically acceptable solid, liquid, or other
dosage forms may also be used for the purposes of formulation.
[0196] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, aqueous suspensions or
solutions. In the case of tablets for oral use, carriers commonly
used include, but are not limited to, lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically
added. For oral administration in a capsule form, useful diluents
include lactose and dried cornstarch. When aqueous suspensions are
required for oral use, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening,
flavoring or coloring agents may also be added.
[0197] Alternatively, the pharmaceutical compositions of this
invention may be administered in the form of suppositories for
rectal administration. These can be prepared by mixing the agent
with a suitable non-irritating excipient that is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include, but
are not limited to, cocoa butter, beeswax and polyethylene
glycols.
[0198] The pharmaceutical compositions of this invention may also
be administered topically, especially when the target of treatment
includes areas or organs readily accessible by topical application,
including diseases of the eye, the skin, or the lower intestinal
tract. Suitable topical formulations are readily prepared for each
of these areas or organs.
[0199] Topical application for the lower intestinal tract can be
effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically-transdermal patches may also
be used.
[0200] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, but are not limited to, mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical compositions can be formulated in
a suitable lotion or cream containing the active components
suspended or dissolved in one or more pharmaceutically acceptable
carriers. Suitable carriers include, but are not limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0201] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH adjusted
sterile saline, or, preferably, as solutions in isotonic, pH
adjusted sterile saline, either with or without a preservative such
as benzylalkonium chloride. Alternatively, for ophthalmic uses, the
pharmaceutical compositions may be formulated in an ointment such
as petrolatum.
[0202] The pharmaceutical compositions of this invention may also
be administered by nasal aerosol or inhalation. Such compositions
are prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other conventional solubilizing or dispersing agents.
[0203] The amount of protein kinase inhibitor that may be combined
with the carrier materials to produce a single dosage form will
vary depending upon the host treated, the particular mode of
administration. Preferably, the compositions should be formulated
so that a dosage of between 0.01-100 mg/kg body weight/day of the
inhibitor can be administered to a patient receiving these
compositions. Alternatively, a dosage of between 0.01-50 mg/kg body
weight/dose of the inhibitor can be administered to a patient
receiving these compounds.
[0204] It should also be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician and the severity of the
particular disease being treated. The amount of inhibitor will also
depend upon the particular compound in the composition.
Administering with Another Agent
[0205] Depending upon the particular protein kinase-mediated
conditions to be treated or prevented, additional drugs, which are
normally administered to treat or prevent that condition, may be
administered together with the compounds of this invention.
[0206] Those additional agents may be administered separately, as
part of a multiple dosage regimen, from the protein kinase
inhibitor-containing compound or composition. Alternatively, those
agents may be part of a single dosage form, mixed together with the
protein kinase inhibitor in a single composition.
[0207] Another aspect of this invention is directed towards a
method of treating cancer in a subject in need thereof, comprising
the sequential or co-administration of a compound of this invention
or a pharmaceutically acceptable salt thereof, and an anti-cancer
agent. In some embodiments, said anti-cancer agent is selected from
Platinating agents, such as Cisplatin, Oxaliplatin, Carboplatin,
Nedaplatin, or Satraplatin and other derivatives; Topo I
inhibitors, such as Camptothecin, Topotecan, irinotecan/SN38,
rubitecan and other derivatives; Antimetabolites, such as Folic
family (Methotrexate, Pemetrexed and relatives); Purine family
(Thioguanine, Fludarabine, Cladribine, 6-Mercaptopurine and
relatives); Pyrimidine family (Cytarabine, Gemcitabine,
5-Fluorouracil and relatives); Alkylating agents, such as Nitrogen
mustards (Cyclophosphamide, Melphalan, Chlorambucil,
mechlorethamine, Ifosfamide, and relatives); nitrosoureas (e.g.
Carmustine); Triazenes (Dacarbazine, temozolomide); Alkyl
sulphonates (e.g. Busulfan); Procarbazine and Aziridines;
Antibiotics, such as Hydroxyurea; Anthracyclines (doxorubicin,
daunorubicin, epirubicin and other derivatives); Anthracenediones
(Mitoxantrone and relatives); Streptomyces family (Bleomycin,
Mitomycin C, actinomycin) and Ultraviolet light.
[0208] Another embodiment provides administering a compound of this
invention with an additional therapeutic agent that inhibits or
modulates a base excision repair protein. In some embodiments, the
base excision repair protein is selected from UNG, SMUG1, MBD4,
TDG, OGG1, MYH, NTH1, MPG, NEIL1, NEIL2, NEIL3 (DNA glycosylases);
APE1, APEX2 (AP endonucleases); LIG1, LIG3 (DNA ligases I and III);
XRCC1 (LIG3 accessory); PNK, PNKP (polynucleotide kinase and
phosphatase); PARP1, PARP2 (Poly(ADP-Ribose) Polymerases); PolB,
PolG (polymerases); FEN1 (endonuclease) or Aprataxin. In other
embodiments, the base excision repair protein is selected from
PARP1, PARP2, or PolB. In yet other embodiments, the base excision
repair protein is selected from PARP1 or PARP2. In some
embodiments, the agent is selected from Olaparib (also known as
AZD2281 or KU-0059436), Iniparib (also known as BSI-201 or
SAR240550), Veliparib (also known as ABT-888), Rucaparib (also
known as PF-01367338), CEP-9722, INO-1001, MK-4827, E7016, BMN673,
or AZD2461.
Biological Samples
[0209] As inhibitors of ATR kinase, the compounds and compositions
of this invention are also useful in biological samples. One aspect
of the invention relates to inhibiting ATR kinase activity in a
biological sample, which method comprises contacting said
biological sample with a compound described herein or a composition
comprising said compound. The term "biological sample", as used
herein, means an in vitro or an ex vivo sample, including, without
limitation, cell cultures or extracts thereof; biopsied material
obtained from a mammal or extracts thereof; and blood, saliva,
urine, feces, semen, tears, or other body fluids or extracts
thereof. The term "compounds described herein" includes compounds
of formula I, formula I-A, formula I-B, formula I-C, formula I-D,
and formula I-E.
[0210] Inhibition of ATR kinase activity in a biological sample is
useful for a variety of purposes that are known to one of skill in
the art. Examples of such purposes include, but are not limited to,
blood transfusion, organ-transplantation, and biological specimen
storage.
Study of Protein Kinases
[0211] Another aspect of this invention relates to the study of
protein kinases in biological and pathological phenomena; the study
of intracellular signal transduction pathways mediated by such
protein kinases; and the comparative evaluation of new protein
kinase inhibitors. Examples of such uses include, but are not
limited to, biological assays such as enzyme assays and cell-based
assays.
[0212] The activity of the compounds as protein kinase inhibitors
may be assayed in vitro, in vivo or in a cell line. In vitro assays
include assays that determine inhibition of either the kinase
activity or ATPase activity of the activated kinase. Alternate in
vitro assays quantitate the ability of the inhibitor to bind to the
protein kinase and may be measured either by radiolabelling the
inhibitor prior to binding, isolating the inhibitor/kinase complex
and determining the amount of radiolabel bound, or by running a
competition experiment where new inhibitors are incubated with the
kinase bound to known radioligands. Detailed conditions for
assaying a compound utilized in this invention as an inhibitor of
ATR is set forth in the Examples below.
[0213] Another aspect of the invention provides a method for
modulating enzyme activity by contacting a compound described
herein with ATR kinase.
Methods of Treatment
[0214] In one aspect, the present invention provides a method for
treating or lessening the severity of a disease, condition, or
disorder where ATR kinase is implicated in the disease state. In
another aspect, the present invention provides a method for
treating or lessening the severity of an ATR kinase disease,
condition, or disorder where inhibition of enzymatic activity is
implicated in the treatment of the disease. In another aspect, this
invention provides a method for treating or lessening the severity
of a disease, condition, or disorder with compounds that inhibit
enzymatic activity by binding to the ATR kinase. Another aspect
provides a method for treating or lessening the severity of a
kinase disease, condition, or disorder by inhibiting enzymatic
activity of ATR kinase with an ATR kinase inhibitor.
[0215] One aspect of the invention relates to a method of
inhibiting ATR kinase activity in a patient, which method comprises
administering to the patient a compound described herein, or a
composition comprising said compound. In some embodiments, said
method is used to treat or prevent a condition selected from
proliferative and hyperproliferative diseases, such as cancer.
[0216] Another aspect of this invention provides a method for
treating, preventing, or lessening the severity of proliferative or
hyperproliferative diseases comprising administering an effective
amount of a compound, or a pharmaceutically acceptable composition
comprising a compound, to a subject in need thereof. In some
embodiments, said method is used to treat or prevent cancer. In
some embodiments, said method is used to treat or prevent a type of
cancer with solid tumors. In yet another embodiment, said cancer is
selected from the following cancers: Oral: buccal cavity, lip,
tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma,
fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma,
fibroma, lipoma and teratoma; Lung: non-small cell, bronchogenic
carcinoma (squamous cell or epidermoid, undifferentiated small
cell, undifferentiated large cell, adenocarcinoma), alveolar
(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,
chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus
(squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma,
lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas
(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma,
carcinoid tumors, vipoma), small bowel or small intestines
(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,
leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel
or large intestines (adenocarcinoma, tubular adenoma, villous
adenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal;
rectum, Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor
[nephroblastoma], lymphoma), bladder and urethra (squamous cell
carcinoma, transitional cell carcinoma, adenocarcinoma), prostate
(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal
carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial
cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);
Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,
hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma,
biliary passages; Bone: osteogenic sarcoma (osteosarcoma),
fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma,
Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma),
multiple myeloma, malignant giant cell tumor chordoma,
osteochronfroma (osteocartilaginous exostoses), benign chondroma,
chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell
tumors; Nervous system: skull (osteoma, hemangioma, granuloma,
xanthoma, osteitis deformans), meninges (meningioma,
meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,
glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,
oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),
spinal cord neurofibroma, meningioma, glioma, sarcoma);
Gynecological: uterus (endometrial carcinoma), cervix (cervical
carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian
carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma,
unclassified carcinoma], granulosa-thecal cell tumors,
Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma),
vulva (squamous cell carcinoma, intraepithelial carcinoma,
adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell
carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal
rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Skin:
malignant melanoma, basal cell carcinoma, squamous cell carcinoma,
Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma,
angioma, dermatofibroma, keloids, psoriasis, Thyroid gland:
papillary thyroid carcinoma, follicular thyroid carcinoma;
medullary thyroid carcinoma, multiple endocrine neoplasia type 2A,
multiple endocrine neoplasia type 2B, familial medullary thyroid
cancer, pheochromocytoma, paraganglioma; and Adrenal glands:
neuroblastoma.
[0217] In some embodiments, the cancer is selected from the cancers
described herein. In some embodiments, said cancer is lung cancer,
head and neck cancer, pancreatic cancer, gastric cancer, or brain
cancer. In other embodiments, the cancer is selected from a cancer
of the lung or the pancreas.
[0218] In yet other embodiments, the cancer is selected from
non-small cell lung cancer, small cell lung cancer, pancreatic
cancer, biliary tract cancer, head and neck cancer, bladder cancer,
colorectal cancer, glioblastoma, esophageal cancer, breast cancer,
hepatocellular carcinoma, or ovarian cancer.
[0219] In some embodiments, the lung cancer is small cell lung
cancer and the additional therapeutic agents are cisplatin and
etoposide. In other examples, the lung cancer is non-small cell
lung cancer and the additional therapeutic agents are gemcitabine
and cisplatin. In yet other embodiments, the non-small cell lung
cancer is squamous non-small cell lung cancer. In another
embodiment, the cancer is breast cancer and the additional
therapeutic agent is cisplatin. In other embodiments, the cancer is
triple negative breast cancer.
[0220] In certain embodiments, an "effective amount" of the
compound or pharmaceutically acceptable composition is that amount
effective in order to treat said disease. The compounds and
compositions, according to the method of the present invention, may
be administered using any amount and any route of administration
effective for treating or lessening the severity of said
disease.
[0221] One aspect provides a method for inhibiting ATR in a patient
comprising administering a compound described herein as described
herein. Another embodiment provides a method of treating cancer
comprising administering to a patient a compound described herein,
wherein the variables are as defined herein.
[0222] Some embodiments comprising administering to said patient an
additional therapeutic agent selected from a DNA-damaging agent;
wherein said additional therapeutic agent is appropriate for the
disease being treated; and said additional therapeutic agent is
administered together with said compound as a single dosage form or
separately from said compound as part of a multiple dosage
form.
[0223] In some embodiments, said DNA-damaging agent is selected
from ionizing radiation, radiomimetic neocarzinostatin, a
platinating agent, a Topo I inhibitor, a Topo II inhibitor, an
antimetabolite, an alkylating agent, an alkyl sulphonates, an
antimetabolite, or an antibiotic. In other embodiments, said
DNA-damaging agent is selected from ionizing radiation, a
platinating agent, a Topo I inhibitor, a Topo II inhibitor, or an
antibiotic.
[0224] Examples of Platinating agents include Cisplatin,
Oxaliplatin, Carboplatin, Nedaplatin, Satraplatin and other
derivatives. Other platinating agents include Lobaplatin, and
Triplatin. Other platinating agents include Tetranitrate,
Picoplatin, Satraplatin, ProLindac and Aroplatin.
[0225] Examples of Topo I inhibitor include Camptothecin,
Topotecan, irinotecan/SN38, rubitecan and other derivatives. Other
Topo I inhibitors include Belotecan.
[0226] Examples of Topo II inhibitors include Etoposide,
Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin,
Amrubicin, Pirarubicin, Valrubicin, Zorubicin and Teniposide.
[0227] Examples of Antimetabolites include members of the Folic
family, Purine family (purine antagonists), or Pyrimidine family
(pyrimidine antagonists). Examples of the Folic family include
methotrexate, pemetrexed and relatives; examples of the Purine
family include Thioguanine, Fludarabine, Cladribine,
6-Mercaptopurine, and relatives; examples of the Pyrimidine family
include Cytarabine, gemcitabine, 5-Fluorouracil (5FU) and
relatives.
[0228] Some other specific examples of antimetabolites include
Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Pentostatin,
Cladribine, Clofarabine, Fludarabine, Thioguanine, Mercaptopurine,
Fluorouracil, Capecitabine, Tegafur, Carmofur, Floxuridine,
Cytarabine, Gemcitabine, Azacitidine and Hydroxyurea.
[0229] Examples of alkylating agents include Nitrogen mustards,
Triazenes, alkyl sulphonates, Procarbazine and Aziridines. Examples
of Nitrogen mustards include Cyclophosphamide, Melphalan,
Chlorambucil and relatives; examples of nitrosoureas include
Carmustine; examples of triazenes include Dacarbazine and
temozolomide; examples of alkyl sulphonates include Busulfan.
[0230] Other specific examples of alkylating agents include
Mechlorethamine, Cyclophosphamide, Ifosfamide, Trofosfamide,
Chlorambucil, Melphalan, Prednimustine, Bendamustine, Uramustine,
Estramustine, Carmustine, Lomustine, Semustine, Fotemustine,
Nimustine, Ranimustine, Streptozocin, Busulfan, Mannosulfan,
Treosulfan, Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine,
Procarbazine, Dacarbazine, Temozolomide, Altretamine, Mitobronitol,
Actinomycin, Bleomycin, Mitomycin and Plicamycin.
[0231] Examples of antibiotics include Mitomycin, Hydroxyurea;
Anthracyclines, Anthracenediones, Streptomyces family. Examples of
Anthracyclines include doxorubicin, daunorubicin, epirubicin and
other derivatives; examples of Anthracenediones include
Mitoxantrone and relatives; examples of Streptomyces family inclue
Bleomycin, Mitomycin C, and actinomycin.
[0232] In certain embodiments, said platinating agent is Cisplatin
or Oxaliplatin; said Topo I inhibitor is Camptothecin; said Topo II
inhibitor is Etoposide; and said antibiotic is Mitomycin. In other
embodiments, said platinating agent is selected from Cisplatin,
Oxaliplatin, Carboplatin, Nedaplatin, or Satraplatin; said Topo I
inhibitor is selected from Camptothecin, Topotecan,
irinotecan/SN38, rubitecan; said Topo II inhibitor is selected from
Etoposide; said antimetabolite is selected from a member of the
Folic Family, the Purine Family, or the Pyrimidine Family; said
alkylating agent is selected from nitrogen mustards, nitrosoureas,
triazenes, alkyl sulfonates, Procarbazine, or aziridines; and said
antibiotic is selected from Hydroxyurea, Anthracyclines,
Anthracenediones, or Streptomyces family.
[0233] In some embodiments, the additional therapeutic agent is
ionizing radiation. In other embodiments, the additional
therapeutic agent is Cisplatin or Carboplatin. In yet other
embodiments, the additional therapeutic agent is Etoposide. In yet
other embodiments, the additional therapeutic agent is
Temozolomide.
[0234] In certain embodiments, the additional therapeutic agent is
selected from one or more of the following: Cisplatin, Carboplatin,
gemcitabine, Etoposide, Temozolomide, or ionizing radiation.
[0235] Another embodiment provides methods for treating pancreatic
cancer by administering a compound described herein in combination
with another known pancreatic cancer treatment. One aspect of the
invention includes administering a compound described herein in
combination with gemcitabine. In some embodiments, the pancreatic
cancer comprises one of the following cell lines: PSN-1, MiaPaCa-2
or Panc-1. According to another aspect, the cancer comprises one of
the following primary tumor lines: Panc-M or MRCS.
[0236] Another aspect of the invention includes administering a
compound described herein in combination with radiation therapy.
Yet another aspect provides a method of abolishing
radiation-induced G2/M checkpoint by administering a compound
described herein in combination with radiation treatment.
[0237] Another aspect provides a method of treating pancreatic
cancer by administering to pancreatic cancer cells a compound
described herein in combination with one or more cancer therapies.
In some embodiments, the compound is combined with chemoradiation,
chemotherapy, and/or radiation therapy. As would be understood by
one of skill in the art, chemoradiation refers to a treatment
regime that includes both chemotherapy (such as gemcitabine) and
radiation. In some embodiments, the chemotherapy is
gemcitabine.
[0238] Yet another aspect provides a method of increasing the
sensitivity of pancreatic cancer cells to a cancer therapy selected
from gemcitabine or radiation therapy by administering a compound
described herein in combination with the cancer therapy.
[0239] In some embodiments, the cancer therapy is gemcitabine. In
other embodiments, the cancer therapy is radiation therapy. In yet
another embodiment the cancer therapy is chemoradiation.
[0240] Another aspect provides a method of inhibiting
phosphorylation of Chk1 (Ser 345) in a pancreatic cancer cell
comprising administering a compound described herein after
treatment with gemcitabine (100 nM) and/or radiation (6 Gy) to a
pancreatic cancer cell.
[0241] Another aspect provides method of radiosensitizing hypoxic
PSN-1, MiaPaCa-2 or PancM tumor cells by administering a compound
described herein to the tumor cell in combination with radiation
therapy.
[0242] Yet another aspect provides a method of sensitizing hypoxic
PSN-1, MiaPaCa-2 or PancM tumor cells by administering a compound
described herein to the tumor cell in combination with
gemcitabine.
[0243] Another aspect provides a method of sensitizing PSN-1 and
MiaPaCa-2 tumor cells to chemoradiation by administering a compound
described herein to the tumor cells in combination with
chemoradiation.
[0244] Another aspect provides a method of disrupting
damage-induced cell cycle checkpoints by administering a compound
described herein in combination with radiation therapy to a
pancreatic cancer cell.
[0245] Another aspect provides a method of inhibiting repair of DNA
damage by homologous recombination in a pancreatic cancer cell by
administering a compound described herein in combination with one
or more of the following treatments: chemoradiation, chemotherapy,
and radiation therapy.
[0246] In some embodiments, the chemotherapy is gemcitabine.
[0247] Another aspect provides a method of inhibiting repair of DNA
damage by homologous recombination in a pancreatic cancer cell by
administering a compound described herein in combination with
gemcitabine and radiation therapy.
[0248] In some embodiments, the pancreatic cancer cells are derived
from a pancreatic cell line selected from PSN-1, MiaPaCa-2 or
Panc-1.
[0249] In other embodiments, the pancreatic cancer cells are in a
cancer patient.
[0250] Another aspect of the invention provides a method of
treating non-small cell lung cancer comprising administering to a
patient a compound described herein in combination with one or more
of the following additional therapeutic agents: Cisplatin or
Carboplatin, Etoposide, and ionizing radiation. Some embodiments
comprise administering to a patient a compound described herein in
combination with Cisplatin or Carboplatin, Etoposide, and ionizing
radiation. In some embodiments the combination is Cisplatin,
Etoposide, and ionizing radiation. In other embodiments the
combination is Carboplatin, Etoposide, and ionizing radiation.
[0251] Another embodiment provides a method of promoting cell death
in cancer cells comprising administering to a patient a compound
described herein, or a composition comprising said compound.
[0252] Yet another embodiment provides a method of preventing cell
repair of DNA damage in cancer cells comprising administering to a
patient a compound described herein, or a composition comprising
said compound. Yet another embodiment provides a method of
preventing cell repair caused by of DNA damage in cancer cells
comprising administering to a patient a compound of formula I, or
composition comprising said compound.
[0253] Another embodiment provides a method of sensitizing cells to
DNA damaging agents comprising administering to a patient a
compound described herein, or a composition comprising said
compound.
[0254] In some embodiments, the method is used on a cancer cell
having defects in the ATM signaling cascade. In some embodiments,
said defect is altered expression or activity of one or more of the
following: ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, H2AX,
MCPH1/BRIT1, CTIP, or SMC1. In other embodiments, said defect is
altered expression or activity of one or more of the following:
ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1 or H2AX. According
to another embodiment, the method is used on a cancer, cancer cell,
or cell expressing DNA damaging oncogenes.
[0255] In another embodiment, the cell is a cancer cell expressing
DNA damaging oncogenes. In some embodiments, said cancer cell has
altered expression or activity of one or more of the following:
K-Ras, N-Ras, H-Ras, Raf, Myc, Mos, E2F, Cdc25A, CDC4, CDK2, Cyclin
E, Cyclin A and Rb.
[0256] According to another embodiment, the method is used on a
cancer, cancer cell, or cell has a defect in a protein involved in
base excision repair ("base excision repair protein"). There are
many methods known in the art for determining whether a tumor has a
defect in base excision repair. For example, sequencing of either
the genomic DNA or mRNA products of each base excision repair gene
(e.g., UNG, PARP1, or LIG1) can be performed on a sample of the
tumor to establish whether mutations expected to modulate the
function or expression of the gene product are present (Wang et
al., Cancer Research 52:4824 (1992)). In addition to the mutational
inactivation, tumor cells can modulate a DNA repair gene by
hypermethylating its promoter region, leading to reduced gene
expression. This is most commonly assessed using
methylation-specific polymerase chain reaction (PCR) to quantify
methylation levels on the promoters of base excision repair genes
of interest. Analysis of base excision repair gene promoter
methylation is available commercially
(http://www.sabiosciences.com/dna_methylation_product/HTML/MEAH-421A.html-
).
[0257] Finally, the expression levels of base excision repair genes
can be assessed by directly quantifying levels of the mRNA and
protein products of each gene using standard techniques such as
quantitative reverse transcriptase-coupled polymerase chain
reaction (RT-PCR) and immunhohistochemistry (IHC), respectively
(Shinmura et al., Carcinogenesis 25: 2311 (2004); Shinmura et al.,
Journal of Pathology 225:414 (2011)).
[0258] In some embodiments, the base excision repair protein is
UNG, SMUG1, MBD4, TDG, OGG1, MYH, NTH1, MPG, NEIL1, NEIL2, NEIL3
(DNA glycosylases); APE1, APEX2 (AP endonucleases); LIG1, LIG3 (DNA
ligases I and III); XRCC1 (LIG3 accessory); PNK, PNKP
(polynucleotide kinase and phosphatase); PARP1, PARP2
(Poly(ADP-Ribose) Polymerases); PolB, PolG (polymerases); FEN1
(endonuclease) or Aprataxin.
[0259] In sorme embodiments, the base excision repair protein is
PARP1, PARP2, or PolB. In other embodiments, the base excision
repair protein is PARP1 or PARP2.
[0260] The methods described above (gene sequence, promoter
methylation and mRNA expression) may also be used to characterize
the status (e.g., expression or mutation) of other genes or
proteins of interesting, such DNA-damaging oncogenes expressed by a
tumor or defects in the ATM signaling cascade of a cell.
[0261] Yet another embodiment provides use of a compound described
herein as a radio-sensitizer or a chemo-sensitizer.
[0262] Yet other embodiment provides use of a compound of formula I
as a single agent (monotherapy) for treating cancer. In some
embodiments, the compounds of formula I are used for treating
patients having cancer with a DNA-damage response (DDR) defect. In
other embodiments, said defect is a mutation or loss of ATM, p53,
CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, or H2AX.
Compounds and Compositions for Use
[0263] One embodiment provides a compound or composition as
described herein for use as a radio-sensitizer or a
chemo-sensitizer. Another embodiment provides a compound or
composition as described herein for use as a single agent
(monotherapy) for treating cancer.
[0264] Another embodiment provides a compound or composition as
described herein for treating patients having cancer with a
DNA-damage response (DDR) defect. In some embodiments, said defect
is a mutation or loss of ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1,
MDC1, or H2AX. In other embodiments, said defect is a mutation or
loss of ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, H2AX,
MCPH1/BRIT1, CTIP, or SMC1.
[0265] Another embodiment provides compounds or compositions
described herein for treating cancer. In some embodiments, the
compound or composition is further combined with an additional
therapeutic agent described herein. In some embodiments, the
compound or composition is further combined with a DNA damaging
agent described herein.
[0266] In some embodiments, the cancer has a defect in a pathway
described herein.
Manufacture of Medicaments
[0267] One embodiment provides the use of a compound or composition
described herein for the manufacture of a medicament for use as a
radio-sensitizer or a chemo-sensitizer. Another embodiment provides
the use of a compound or composition described herein for the
manufacture of a medicament for the manufacture of a medicament for
use as a single agent (monotherapy) for treating cancer.
[0268] Yet another embodiment provides the use of a compound or
composition described herein for the manufacture of a medicament
for the manufacture of a medicament for treating patients having
cancer with a DNA-damage response (DDR) defect.
[0269] In some embodiments, said defect is a mutation or loss of
ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, or H2AX. In other
embodiments, said defect is a mutation or loss of ATM, p53, CHK2,
MRE11, RAD50, NBS1, 53BP1, MDC1, H2AX, MCPH1/BRIT1, CTIP, or
SMC1.
[0270] Another embodiment provides the use of a compound or
composition described herein for the manufacture of a medicament
for treating cancer. In some embodiments, the compound or
composition is combined with an additional therapeutic agent, such
as a DNA damaging agent, described herein. In another embodiment,
the cancer has a defect in a pathway described herein.
EXPERIMENTAL MATERIALS AND METHODS
[0271] All commercially available solvents and reagents were used
as received. Microwave reactions were carried out using a CEM
Discovery microwave. Flash chromatography was carried out on an
ISCO.COPYRGT. Combiflash.RTM. Companion.TM. system eluting with a 0
to 100% EtOAc/petroleum ether gradient. Samples were applied
pre-absorbed on silica. Other methods known in the art were also
utilized to perform Flash Chromotography. Where stated,
supercritical fluid chromatography (SFC) was performed on a Berger
Minigram SFC machine. All .sup.1H NMR spectra were recorded using a
Bruker Avance III 500 instrument at 500 MHz. MS samples were
analyzed on a Waters SQD mass spectrometer with electrospray
ionization operating in positive and negative ion mode. Samples
were introduced into the mass spectrometer using chromatography.
All final products had a purity.gtoreq.95%, unless specified
otherwise in the experimental details. HPLC purity was measured on
a Waters Acquity UPLC system with a Waters SQD MS instrument
equipped with a Waters UPLC BEH C8 1.7 .mu.m, 2.1.times.50 mm
column and a Vanguard BEH C8 1.7 .mu.m, 2.1.times.5 mm guard
column.
[0272] As used herein, the term "Rt(min)" refers to the HPLC
retention time, in minutes, associated with the compound. Unless
otherwise indicated, the HPLC methods utilized to obtain the
reported retention times are as described below:
HPLC Method
Instrument: Waters Acquity UPLC-MS;
[0273] Column: Waters UPLC BEH C8 1.7 .mu.m, 2.1.times.50 mm with
Vanguard BEH C8 1.7 .mu.m, 2.1.times.5 mm guard column; Column
temperature: 45.degree. C.; Mobile Phase A: 10 mM ammonium formate
in water:acetonitrile 95:5, pH 9; Mobile Phase B: acetonitrile;
Detection: 210-400 nm;
[0274] Gradient: 0-0.40 min: 2% B, 0.40-4.85 min: 2% B to 98% B,
4.85-4.90 min: 98% B to 2% B, 4.90-5.00 min: hold at 2% B; Flow
rate: 0.6 mL/minute.
EXAMPLES AND SCHEMES
[0275] The compounds of the disclosure may be prepared in light of
the specification using steps generally known to those of ordinary
skill in the art. Those compounds may be analyzed by known methods,
including but not limited to LCMS (liquid chromatography mass
spectrometry) and NMR (nuclear magnetic resonance). The following
generic schemes and examples illustrate how to prepare the
compounds of the present disclosure. The examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in any way.
Scheme 1: General Approach for the Preparation of Compounds of
Formula I
##STR00122##
[0277] Compounds of this invention can be synthesised according to
methods similar to the one depicted in Scheme 1.
[0278] The anion of commercially available allyl cyanoacetate 1 can
react with trichloroacetonitrile to provide intermediate 2. In the
anion condensation step, the anion of commercially available allyl
cyanoacetate 1 can be generated with a base such as potassium
acetate in an appropriate solvent such as an alcohol (e.g.,
isopropylalcohol). The anion then reacts with trichloroacetonitrile
at room temperature.
[0279] In the pyrazole formation step, intermediate 2 is reacted
with hydrazine (or its hydrate) in an aprotic solvent, such as DMF,
to provide the diaminopyrazole 3. The reaction occurs under basic
conditions (eg in the presence of potassium acetate or AcONa) with
heating (e.g., 110.degree. C.) to ensure complete cyclisation.
Intermediate 2 then reacts with hydrazine to form the
diaminopyrazole 3, which can further be condensed with a
dielectrophilic coupling partner to form the pyrimidine 4 a-e.
[0280] In the pyrimidine formation step, intermediate 3 is reacted
with a 1,3-dielectrophilic species (e.g., a 1,3-dialdehyde or a
3-(dialkylamino)-prop-2-enal) in various types of solvents (e.g.,
DMF, or DMSO/water) to furnish the bicyclic cores 4a-e. In some
instances, the reaction is carried out in the presence of a strong
base, e.g., KOH. In other instances, the reaction is carried out in
the presence of a weak base, e.g., triethylamine. When one or two
of the electrophilic centers is protected/masked (eg aldehyde
masked as a ketal), introduction of a sulfonic acid (eg PTSA) may
be required to liberate the reactive functional group.
[0281] Deprotection, e.g., via hydrolysis, of the allyl ester leads
to the carboxylic acids 5 a-e. In the deprotection step, compounds
4a-e are subjected to hydrolytic conditions that are known to those
skilled in the art. For example, treatment of 4 with phenylsilane
or 4-methylbenzenesulfinate in the presence of a catalytic amount
of palladium (e.g., Pd(PPh.sub.3).sub.4) leads to the formation of
the corresponding carboxylic acid 5a-e. Alternatively, compounds
4a-e could be treated with aqueous alkali (e.g., NaOH or KOH) to
produce acids 5a-e.
[0282] In the activated ester formation step, the carboxylic acids
5a-e are reacted with amide coupling agents known to those skilled
in the art. When the coupling agent is chosen appropriately, the
reactions can proceed rapidly (.about.1 h) at room temperature in
the presence of an organic base (e.g., triethylamine, DIPEA) to
provide the activated esters 6. For example, when the amide
coupling agents TBTU [J=H] or TCTU [J=C1 are used, compounds 6 are
obtained readily by filtration of the reaction mixture.
[0283] Formation of the activated esters 6 a-e prior to the amide
bond formation to prepare a compound of formula I is generally
preferred, although a direct conversion of 5 a-e into the compounds
of formula I of this invention is also possible. Alternative
activated esters can also be utilised (isolated or formed in situ)
and will be known to those skilled in the art (e.g, using TCTU,
HATU, T3P, COMU coupling agents).
[0284] In the amide bond formation step, activated esters 6 can
react with a substituted heteroaromatic amine to provide compounds
I of this invention. The reaction conditions for the amide coupling
are generally in a aprotic solvent (e.g., NMP, pyridine, DMF, etc)
with heating (e.g., >90.degree. C.). The heteroaromatic amine
may be further functionalized following amide bond formation.
[0285] Alternatively, the two steps described above can be
combined: carboxylic acids 5a-e can be used as starting points for
the amide bond formation, the activated esters being generated in
situ, using the same amide couplings agents as those described
above. Compounds I of this invention are isolated in a similar
manner to the one described above (specific details are given in
Examples below).
Scheme 2: Alternative Approach for the Preparation of Compounds of
Formula I
##STR00123##
[0287] Alternatively, compounds of the present disclosure can be
prepared according to methods similar to the one depicted in Scheme
2.
[0288] The amide 8 can readily be prepared from commercially
available cyanoacetic acid 7. In the amide bond formation step,
cyanoacetic acid 7 can react with a substituted heteroaromatic
amine to provide compounds 8. The reaction conditions for the amide
coupling are generally in a aprotic solvent (e.g., DCM, NMP, DMF,
etc), in the presence of an organic base such as an aliphatic amine
(e.g., triethylamine or DIPEA) and an amide coupling agent known to
those skilled in the art: for example EDCI, TBTU, COMU, T3P,
etc.
[0289] In the pyrazole formation step, the anion of cyanoamide 8
can be generated with a base (such as potassium or sodium acetate)
in an appropriate solvent such as an alcohol (e.g., ethanol). The
anion then reacts with trichloroacetonitrile at room temperature
(specific details are given in Examples below). The resulting
solid, which can be collected by filtration, is then reacted with
hydrazine (or its hydrate) in an aprotic solvent, such as DMF or
NMP, to provide the diaminopyrazole 9. Intermediate 9 is further
condensed with a dielectrophilic coupling partner to form the
pyrimidine portion of the compounds of formula I of this
invention.
[0290] In the pyrimidine formation step, intermediate 9 is reacted
with a 1,3-dielectrophilic species (e.g., a 1,3-dialdehyde or a
3-(dialkylamino)-prop-2-enal) in various types of solvents (e.g.,
iPrOH/water, DMF, or DMSO/water) to furnish the desired products of
formula I. When one or two of the electrophilic centers is
protected/masked (e.g., aldehyde masked as a ketal), introduction
of a sulfonic acid (e.g., PTSA) is required to liberate the
reactive functional group.
Preparation 1: Allyl 3,5-diamino-1H-pyrazole-4-carboxylate
##STR00124##
[0291] Step 1: allyl 3-amino-4,4,4-trichloro-2-cyanobut-2-enoate
2
[0292] To a solution of KOAc (589.4 g, 6.006 mol) in isopropanol (3
L) was added allyl cyanoacetate (429.4 g, 403.2 mL, 3.432 mol) and
the reaction mixture was cooled to 5.degree. C.
Trichloroacetonitrile (495.5 g, 3.432 mol) was added in 50 mL
portions, maintaining temperature below 15.degree. C. The reaction
mixture was then allowed to warm to 20.degree. C. and stirred for 3
h. Water (.about.4 L) was added to dissolve the inorganic materials
and precipitate out the desired product. The mixture was stirred
for 20 minutes and the solid was isolated by filtration under
vacuum. This solid was filtered, washed with water (2.times.0.5 L)
and dried in a vacuum oven overnight at 40.degree. C. to afford
allyl 3-amino-4,4,4-trichloro-2-cyanobut-2-enoate 2 as an off-white
powder (787 g, 85%)
Step 2: Allyl 3,5-diamino-1H-pyrazole-4-carboxylate 3
[0293] To a suspension of allyl
3-amino-4,4,4-trichloro-2-cyano-but-2-enoate 2 (619 g, 2.297 mol)
and KOAc (676.3 g, 6.891 mol) in DMF (2.476 L) at 0.degree. C. was
slowly added hydrazine hydrate (172.5 g, 167.6 mL, 3.446 mol) over
15 min. The reaction mixture was then stirred at ambient
temperature for 2 h, at which stage .sup.1H NMR shows complete
consumption of the starting material. Reaction mixture was then
heated overnight at 110.degree. C. before being allowed to cool to
ambient and stirred for another 48 h. The mixture was filtered
through a sintered glass funnel to remove the precipitated solid
and the filtrate was evaporated under reduced pressure to give a
thick liquid. DCM (approx 2 L) was added and the mixture filtered
again to remove additional solids that have precipitated. The
filtrate was purified through a 1 kg silica gel plug (gradient of
DCM/MeOH as an eluent) and the solvent was removed to afford an
orange solid which was suspended in acetonitrile and heated at
about 70.degree. C. until all the solid went into solution, at
which point the solution was allowed to cool to ambient
temperature, then to 2.degree. C. The precipitate that formed was
isolated by filtration under vacuum, washed with chilled MeCN
(.about.50 mL) and dried to constant mass in a vacuum oven to
furnish the title compound as an off-white powder (171.2 g,
41%).
Preparation 2a: 1H-benzo[d][1,2,3]triazol-1-yl
2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate
##STR00125##
[0294] Step 1: allyl
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4a
[0295] To a suspension of allyl
3,5-diamino-1H-pyrazole-4-carboxylate 3 (42.72 g, 234.5 mmol) in
DMSO (270.8 mL)/Water (270.8 mL), was added p-TsOH hydrate (46.72
g, 245.6 mmol) and 3-(diisopropylamino)-2-fluoro-prop-2-enal
(described in Tetrahedron Letters, 33(3), 357-60; 1992) (38.69 g,
223.3 mmol). The reaction mixture was heated to 100.degree. C. for
3 h during which time a solid slowly precipitated out of solution.
The orange suspension was allowed to cool down to RT overnight. The
solid was filtered, washed with water and dried under vacuum to
give allyl 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate
4a as a sand solid (45.05 g, 85% yield).
Step 2: 2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid 5a
[0296] To a suspension of allyl
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4a (45 g,
190.5 mmol) in DCM (1.35 L) was added phenylsilane (41.23 g, 46.96
mL, 381.0 mmol), followed by Pd(PPh.sub.3).sub.4 (8.805 g, 7.620
mmol). The reaction was stirred at room temperature for 2 h30 min.
The reaction mixture was filtered and the solid was washed with DCM
to give a light yellow solid (43.2 g). This solid was triturated
further in DCM (225 mL) at RT for 45 min, then filtered and dried
overnight under vacuum to provide
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a as
a light yellow solid (37.77 g, 100% yield).
[0297] In an alternative method, 4-methylbenzenesulfinate
(anhydrous, 1.2 eqv, 22.6 g, 127 mmol) was suspended in dry DMSO
(20 vol, 500 ml). The stirred mixture was warmed to 30.degree. C.
under a nitrogen atmosphere. Upon complete dissolution
Pd(PPh.sub.3).sub.4 (2 mol %, 2.4 g, 2.1 mmol) was added. The
mixture was stirred for 10 min at 25-30.degree. C. after which time
a turbid yellow solution was present. Allyl
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate (25 g,
105.8 mmol) was added portionwise, maintaining the temperature at
25-30.degree. C. Once addition was complete the cloudy solution was
stirred until the reaction was complete by HPLC (2-3 hrs). A heavy
precipitate formed after 15 minutes post addition of the substrate.
The mixture became thicker as the reaction proceeded. The reaction
mixture was diluted with water (125 ml) and 2M HCl (66 ml) was
added slowly, maintaining the temperature at 25-30.degree. C. The
slurry was stirred for 30 minutes, then filtered. The filtration
was slow (2 hrs). The resulting solid was washed with water, then
dried on the sinter. The solid was slurried in DCM (8 vol) for 1
hr. The solid was filtered (rapid filtration) and washed with DCM.
The solid was re-slurried in chloroform (8 vol) for 1 hr. The acid
was filtered and dried on the sinter. It was further dried in a
vacuum oven at 50.degree. C. for 24 hrs. The product was obtained
as an off-white solid (18.6 g, 85%); 1H NMR (500 MHz, DMSO-d6)
.delta. 12.14 (1H, brs), 9.31 (1H, dd), 8.69 (1H, m), 6.47 (2H,
brS); 19F NMR (500 MHz, DMSO-d6) .delta. -153.65; MS (ES+)
197.1.
Step 3: 1H-benzo[d][1,2,3]triazol-1-yl
2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate 6a
[0298] To a suspension of
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a (20
g, 102.0 mmol) in chloroform (300 mL) was added Et.sub.3N (11.35 g,
15.63 mL, 112.2 mmol). The suspension was stirred for .about.5 mins
and then
(benzotriazol-1-yloxy-dimethylamino-methylene)-dimethyl-ammonium
Boron Tetrafluoride was added (32.75 g, 102.0 mmol). The suspension
was heated to 60.degree. C. for 1 h before the thick suspension was
allowed to cool down to RT. The resulting suspension was filtered,
washed with chloroform (200 mL) and dried under vacuum overnight to
afford the title compound 6a as a light yellow powder (32.5 g,
88%).
Preparation 2b:
(6-chlorobenzotriazol-1-yl)-2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3--
carboxylate 6a*
##STR00126##
[0300] In a 2.5 L three-necked flask equipped with stirrer bar,
condenser, nitrogen line and Hanna temperature probe was charged
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a (60
g, 305.9 mmol), chloroform (900.0 mL) and triethylamine (32.44 g,
44.68 mL, 320.6 mmol).
[(6-chlorobenzotriazol-1-yl)oxy-(dimethylamino)methylene]-dimethyl-ammoni-
um (Boron Tetrafluoride Ion (1)) (87.00 g, 244.7 mmol) was added
portionwise over 5 mins (internal dropped from 22.7 to 21.5.degree.
C. on complete addition). Mixture heated at 60.degree. C. (internal
temp) for 2 h, still a cream suspension. Mixture cooled to room
temperature then solid collected by filtration, washed well with
chloroform (until filtrate runs essentially colourless) and dried
by suction to leave product 6a* as a cream solid (82.2 g, 77%
yield). .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 9.55 (dd, 1H),
8.91 (d, 1H), 8.22 (dd, 1H), 8.09 (dd, 1H), 7.57 (dd, 1H) and 6.87
(s, 2H). MS (ES+) 348.1.
[0301] In an alternative method,
2-Amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5a (30
g, 153 mmol) was slurried in acetonitrile (540 ml). Triethylamine
(22.5 ml, 153 mmol) was added, followed by
[(6-chlorobenzotriazol-lyl)oxy-(dimethylamino)methylene]-dimethylammonium
tetrafluoroborate (TCTU, 54.4 g, 153 mmol). The mixture was stirred
at room temperature for 2 hrs. The product was isolated by
filtration--the filter cake was washed with acetonitrile
(2.times.60 ml) (49.3 g, 93%); .sup.1H NMR (500 MHz, DMSO-d.sub.6)
.delta. 9.55 (dd, 1H), 8.91 (d, 1H), 8.22 (dd, 1H), 8.09 (dd, 1H),
7.57 (dd, 1H) and 6.87 (s, 2H); 19F NMR (500 MHz, DMSO-d6) .delta.
-150.1; MS (ES+) 348.1.
Preparation 3: 1H-benzo[d][1,2,3]triazol-1-yl
2-amino-6-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate
##STR00127##
[0302] Step 1: allyl
2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4b
[0303] To a suspension of allyl
3,5-diamino-1H-pyrazole-4-carboxylate 3 (1 g, 5.489 mmol) in DMF (5
mL) was added
(Z)-2-chloro-3-dimethylamino-prop-2-enylidene]-dimethyl-ammonium
hexafluorophosphate (1.683 g, 5.489 mmol), followed by
triethylamine (722.1 mg, 994.6 .mu.L, 7.136 mmol). The reaction
mixture was heated to 60.degree. C. for 4 h during which time a
solid slowly precipitated out of solution. The brown suspension was
allowed to cool down to RT. The solid was filtered, washed with
water and dried under vacuum to give allyl
2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4b as a
brown solid (1.092 g, 72% yield).
Step 2: 2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid 5b
[0304] To a suspension of allyl
2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4b (1 g,
3.96 mmol) in DCM (15 mL) was added phenylsilane (856.6 mg, 0.9756
mL, 7.916 mmol), followed by Pd(PPh.sub.3).sub.4 (182.9 mg, 0.1583
mmol). The reaction was stirred at room temperature for 7 h. The
reaction mixture was filtered and the solid was washed with DCM to
give a light yellow solid (43.2 g). This solid was triturated
further in DCM (225 mL) at RT for 45 min, then filtered and dried
overnight under vacuum to provide
2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 5b as
a yellow solid (791 m, 94% yield).
Step 3: 1H-benzo[d][1,2,3]triazol-1-yl
2-amino-6-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate 6b
[0305] To a solution of
2-amino-6-chloro-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid 2.5b
(1.51 g, 7.103 mmol) in chloroform (15.1 mL) was added TBTU boron
tetrafluoride (2.737 g, 8.524 mmol) and TEA (862.5 mg, 1.188 mL,
8.524 mmol). The reaction mixture was stirred at 50.degree. C. for
one hour. The resulting suspension was filtered, and the solid
triturated in ethyl acetate to afford the title compound 6b as a
yellow solid (2.05 g, 88%).
Scheme 3--Preparation of Butanenitrile Intermediates
##STR00128##
[0306] Step 1: 3-(dimethoxymethyl)-4,4-dimethoxybutanenitrile
11
[0307] 2-(dimethoxymethyl)-3,3-dimethoxy-propan-1-ol 10 (Journal of
the American Chemical Society (1973), 95(26), 8741) (92 g, 473.7
mmol) was dissolved in dry THF (920 mL) and the mixture was cooled
down with an ice bath. Triethylamine (143.8 g, 198.1 mL, 1.421 mol)
was added at once, followed by dropwise addition of methane
sulfonyl chloride (59.69 g, 40.33 mL, 521.1 mmol), over 1 h and
keeping the internal temperature below 5.degree. C. The reaction
mixture was stirred for 1 h and then allowed to warm to room
temperature. The mixture was diluted with ethyl acetate (920 mL)
and water (920 mL). The layers were separated and the organic layer
was isolated, washed with a saturated solution of NaHCO.sub.3, then
brine. The organics were dried over MgSO.sub.4, filtered and
evaporated to give
[2-(dimethoxymethyl)-3,3-dimethoxypropyl]methanesulfonate as an
orange oil (125.31 g, 97%) which was used directly without further
purification.
[0308] Tetraethylammonium cyanide (142.3 g, 910.8 mmol) was added
portionwise over 10 minutes to a solution of
[2-(dimethoxymethyl)-3,3-dimethoxypropyl]methanesulfonate (124 g,
455.4 mmol) in MeCN (1.24 L). The reaction mixture was stirred at
room temperature for 72 h, then portioned between ethyl acetate
(1.24 L) and water (1.24 L). The layers were separated and the
organic layer was isolated, washed with brine. The organics were
dried over MgSO.sub.4, filtered and evaporated to give
3-(dimethoxymethyl)-4,4-dimethoxybutanenitrile 11 as a dark brown
oil (86.1 g).
Step 2: 3-(dimethoxymethyl)-4,4-dimethoxy-2-methylbutanenitrile 12a
and 3-(dimethoxymethyl)-4,4-dimethoxy-2,2-dimethylbutanenitrile
13a
[0309] To a solution of
3-(dimethoxymethyl)-4,4-dimethoxy-butanenitrile 11 (250 mg, 1.205
mmol) in THF (3 mL) at -75.degree. C. was added a solution of
iodomethane (513.1 mg, 225.0 .mu.L, 3.615 mmol) in THF (1 ml). A
THF solution of (bis(trimethylsilyl)amino)sodium (1.808 mL of 2M,
3.615 mmol) was then added, keeping the temperature below
-60.degree. C. After addition, the reaction mixture was stirred at
-75.degree. C. for 2 hrs and then slowly quenched with aq. sat.
NH.sub.4Cl solution (5 ml). The mixture diluted with water and
ether and layers separated. The organic layer was washed with
brine, dried (Na.sub.2SO.sub.4) and concentrated in vacuo to afford
a yellow oil which was purified by chromatography on silica gel,
eluting with a petroleum ether:EtOAc gradient of 100:0 to 80:20.
Solvents were concentrated in vacuo to afford a clear oil (194 mg).
NMR proved this oil to be a mixture of 80% mono methyl compound 12a
with and 20% bis methyl compound 13a. This mixture was used
directly in subsequent steps.
Step 3: 3-(dimethoxymethyl)-2-ethyl-4,4-dimethoxybutanenitrile 12b
and 3-(dimethoxymethyl)-2-diethyl-4,4-dimethoxybutanenitrile
13b
[0310] When ethyl iodide was used instead of methyl iodide in a
similar procedure to Scheme 3, step 2, above, a mixture of
monosubsituted compound 12b and disubstituted compound 13b was
isolated and used directly in subsequent steps.
Preparation 4: 1H-benzo[d][1,2,3]triazol-1-yl
2-amino-6-(cyanomethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
##STR00129##
[0311] Step 1: allyl
2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate
4c
[0312] To a suspension of allyl
3,5-diamino-1H-pyrazole-4-carboxylate 3 (63.49 g, 348.5 mmol) in a
mixture of DMSO (340 mL) and water (340 mL), was added
3-(dimethoxymethyl)-4,4-dimethoxy-butanenitrile (85 g, 418.2 mmol),
followed by para-toluene Sulfonic acid hydrate (1) (11.27 g, 59.24
mmol). The reaction mixture was heated to 85.degree. C. and stirred
overnight. The reaction mixture was cooled with an ice bath. The
mixture was diluted with EtOAc (680 mL) and a saturated aqueous
solution of NaHCO.sub.3 (1.36 L). The precipitate was filtered and
rinsed with water, then with a mixture of water and EtOAc. The
brown solid was dried under vacuum to give allyl
2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4c
as a brown solid (55.94 g, 62% yield).
Step 2:
2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
5c
[0313] To a suspension of allyl
2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate 4c
(10.2 g, 39.65 mmol) in DCM (350 mL) was added phenylsilane (8.581
g, 9.773 mL, 79.3 mmol), followed by Pd(PPh.sub.3).sub.4 (1.5 g,
1.298 mmol). The reaction was stirred at room temperature for 2 h.
The reaction mixture was filtered and the solid was washed with DCM
and dried under vacuum to provide
2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
5c as a yellow solid (8.61 g, 100% yield).
Step 3: 1H-benzo[d][1,2,3]triazol-1-yl
2-amino-6-(cyanomethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
6c
[0314] To a solution of
2-amino-6-(cyanomethyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
5c (5.11 g, 23.53 mmol) in DCM (51 mL) was added TBTU boron
tetrafluoride (9.067 g, 28.24 mmol) and TEA (2.858 g, 3.937 mL,
28.24 mmol). The reaction mixture was stirred at room temperature
for one hour. The resulting suspension was filtered, and the solid
triturated in hot chloroform to afford the title compound 6c as a
beige solid (6.59 g, 84%).
Preparation 5: 1H-benzo[d][1,2,3]triazol-1-yl
2-amino-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
##STR00130##
[0315] Step 1: allyl
2-amino-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
4d
[0316] 4-methylbenzenesulfonic acid monohydrate (500.1 mg, 467.4
.mu.L, 2.629 mmol) was added to a solution of
4-ethoxy-1,1,1-trifluoro-but-3-en-2-one (200.97 g, 1.195 mol) and
ethylene glycol (77.27 g, 69.42 mL, 1.245 mol) in toluene (502.5
mL). The reaction mixture was heated to reflux for 1 h30 min. (oil
bath). Then, the reflux was continued with simultaneous
distillation of ethanol/toluene. The Dean-Stark was removed and the
flask was connected to a distillation apparatus.
3-(1,3-dioxolan-2-yl)-1,1,1-trifluoro-propan-2-one was distilled
using a KNF pump (Bp 81.degree. C.) as a light yellow oil (148 g,
67% yield).
[0317] 3-(1,3-Dioxolan-2-yl)-1,1,1-trifluoro-propan-2-one (55.58 g,
301.9 mmol) and allyl 3,5-diamino-1H-pyrazole-4-carboxylate (55 g,
301.9 mmol) were dissolved in 1,4-dioxane (330 mL). KOH (1.694 g,
30.19 mmol) was added and the yellow suspension was stirred at room
temperature for 4 h30 min. The reaction mixture was heated at
90.degree. C. for 16 h. The mixture was cooled down to room
temperature. The reaction mixture was concentrated onto silica and
purified by column chromatography (CombiFlash Companion XL, 1.5 kg
column, 0.5 to 40% EtOAc in DCM) to afford allyl
2-amino-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
4d as a bright yellow solid (68 g, 79% yield).
Step 2:
2-amino-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid 5d
[0318] allyl
2-amino-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
4d (67.45 g, 235.7 mmol) in dioxane (670 mL)/water (670 mL) was
treated with lithium hydroxide (16.93 g, 707.1 mmol). The resulting
light brown suspension was stirred at 70.degree. C. for 1 h45 min.
The bright yellow suspension was cooled down to ambient temperature
and concentrated in vacuo. The crude mixture was cooled down in a
water bath containing ice (T.degree..about.10.degree. C.) and the
pH was adjusted to .about.3 with 2M HCl. The mixture was stirred
for 2 h and the yellow solid was filtered, washed with water and
dried on the sinter to give 63.6 g of a wet yellow solid. The solid
was dried overnight in a vacuum oven at 40.degree. C. (KNF pump) to
afford
2-amino-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid 5d as a yellow solid (55.2 g, 95% yield).
Step 3: 1H-benzo[d][1,2,3]triazol-1-yl
2-amino-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate
6d
[0319] Triethylamine (994.9 mg, 1.370 mL, 9.832 mmol) was added to
a suspension of
2-amino-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid 5d (2.2 g, 8.938 mmol) in chloroform (22 mL). TBTU (2.870 g,
8.938 mmol) was added to the solution and the reaction mixture was
heated at 50.degree. C. for 20 min, during which time a yellow
precipitate formed. The reaction mixture was cooled to ambient
temperature. The precipitate was isolated by filtration and washed
with chloroform to leave the product (benzotriazol-1-yl
2-amino-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylate)
6d as an off white solid (2.8 g, 86% yield).
Preparation 6: 1H-benzo[d][1,2,3]triazol-1-yl
2-amino-5-(2-(dimethylamino)ethoxy)pyrazolo[1,5-a]pyrimidine-3-carboxylat-
e
##STR00131##
[0320] Step 1: allyl
2-amino-5-(2-(dimethylamino)ethoxy)pyrazolo[1,5-a]pyrimidine-3-carboxylat-
e 4e
[0321] To a suspension of allyl
3,5-diamino-1H-pyrazole-4-carboxylate 3 (1.0 g, 5.4 mmol) and
Cs.sub.2CO.sub.3 (2.5 g, 7.6 mmol) in EtOH (10 mL) was added methyl
prop-2-ynoate (553.0 mg, 586.0 .mu.l, 6.5 mmol), dropwise over 15
minutes and the reaction mixture was stirred overnight at room
temperature. The deep orange mixture was filtered. The filtrate was
cooled on an ice-bath and ether (100 mL) was slowly added with
stirring. The mixture was stirred at 0.degree. C. for 20 minutes by
which time a yellow solid had precipitated out of solution. The
solid was filtered, further triturated in ether (20 mL) and
filtered. The yellow solid was dried to afford allyl
2-amino-5-oxo-4,5-dihydropyrazolo[1,5-a]pyrimidine-3-carboxylate
(700 mg, 54% yield).
[0322] To a mixture of allyl
2-amino-5-oxo-4H-pyrazolo[1,5-a]pyrimidine-3-carboxylate (1 g,
4.270 mmol) in MeCN (15 mL) was added
2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (975.1 mg, 957.9
.mu.L, 6.405 mmol) and
benzotriazol-1-yloxy(tripyrrolidin-1-yl)phosphonium (Phosphorus
Hexafluoride Ion) (2.666 g, 5.124 mmol). After 5 minutes,
2-dimethylaminoethanol (3.806 g, 4.291 mL, 42.70 mmol) and cesium
carbonate (5 g) were added and the mixture was stirred at room
temperature for 30 minutes, followed by heating to 50.degree. C.
for 30 minutes. After cooling to room temperature the mixture was
filtered and the solid was rinsed with acetonitrile. The filtrate
was concentrated in vacuo to an orange oil and purified by column
chromatography using 4% MeOH/DCM as eluant. Solvent evaporations in
vacuo afforded allyl
2-amino-5-(2-(dimethylamino)ethoxy)pyrazolo[1,5-a]pyrimidine-3-carboxylat-
e as a light orange solid. (450 mg, 35% yield).
Step 2:
2-amino-5-(2-(dimethylamino)ethoxy)pyrazolo[1,5-a]pyrimidine-3-car-
boxylic acid 5e
[0323] To a solution of phenylsilane (478.5 mg, 550.0 .mu.L, 4.422
mmol) and allyl
2-amino-5-[2-(dimethylamino)ethoxy]pyrazolo[1,5-a]pyrimidine-3--
carboxylate (900 mg, 2.948 mmol) in DCM (5 mL) was added palladium
triphenylphosphane (170.3 mg, 0.1474 mmol). The reaction mixture
was stirred at room temperature for 3 h. Ether was slowly added to
the mixture and a white solid precipitated. This solid was filtered
off, washed with small amount of diethyl ether and dried to afford
pure
2-amino-5-(2-(dimethylamino)ethoxy)pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid as off-white solid. (530 mg, 67% yield).
Step 3: 3a,7a-dihydro-1H-benzo[d][1,2,3]triazol-1-yl
2-amino-5-(2-(dimethylamino)ethoxy)pyrazolo[1,5-a]pyrimidine-3-carboxylat-
e 6e
[0324] To a suspension of
2-amino-5-[2-(dimethylamino)ethoxy]pyrazolo[1,5-a]pyrimidine-3-carboxylic
acid (80 mg, 0.3016 mmol) in NMP (1 mL) was added Et.sub.3N (37.24
mg, 51.29 .mu.L, 0.3680 mmol). To the mixture was added TBTU (Boron
Tetrafluoride Ion (1)) (109.0 mg, 0.3396 mmol) portionwise over a 5
minutes. The mixture was allowed to stir at room temperature for 20
minutes before being partitioned between ethylacetate and water.
The aqueous layer was extracted twice with ethylacetate and the
combined organic layers were washed with brine, dried (MgSO.sub.4),
and concentrated in vacuo to afford the product
3a,7a-dihydro-1H-benzo[d][1,2,3]triazol-1-yl
2-amino-5-(2-(dimethylamino)ethoxy)pyrazolo[1,5-a]pyrimidine-3-carboxylat-
e as an oil (92 mg, 80% yield).
Example 1
(R)-2-amino-6-fluoro-N-(4-((1-methylpiperidin-3-yl)oxy)pyrimidin-5-yl)pyra-
zolo[1,5-a]pyrimidine-3-carboxamide
##STR00132##
[0326] A solution of benzotriazol-1-yl
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 6a (125
mg, 0.3990 mmol) and
4-[[(3R)-1-methyl-3-piperidyl]oxy]pyrimidin-5-amine 7a (83.10 mg,
0.3990 mmol) in NMP (2 mL) was heated at 100.degree. C. overnight.
The reaction mixture was cooled to RT, passed through a SCX
cartridge, eluting with 2M ammonia in methanol. The product
fractions were combined, concentrated in vacuo and purified by
HPLC. The title product I-A-1 was isolated (60 mg., 38.9%)
[0327] The following compounds were prepared according to a
methodology similar to the one described in Example 1, starting
from the appropriate activated acid 6 a-e and aromatic amine:
[0328]
(S)-2-amino-6-fluoro-N-(4-((1-methylpiperidin-3-yl)oxy)pyrimidin-5-yl)pyr-
azolo[1,5-a]pyrimidine-3-carboxamide I-A-2; [0329]
(R)-2-amino-6-fluoro-N-(4-(quinuclidin-3-yloxy)pyrimidin-5-yl)pyrazolo[1,-
5-a]pyrimidine-3-carboxamide I-A-3; [0330]
(S)-2-amino-6-fluoro-N-(4-(quinuclidin-3-yloxy)pyrimidin-5-yl)pyrazolo[1,-
5-a]pyrimidine-3-carboxamide I-A-4; [0331]
2-amino-6-fluoro-N-(4-((1-methylpiperidin-4-yl)oxy)pyrimidin-5-yl)pyrazol-
o[1,5-a]pyrimidine-3-carboxamide I-A-5; [0332]
2-amino-6-chloro-N-(4-((1-methylpiperidin-4-yl)oxy)pyrimidin-5-yl)pyrazol-
o[1,5-a]pyrimidine-3-carboxamide I-A-6; [0333]
2-amino-N-(4-((1-methylpiperidin-4-yl)oxy)pyrimidin-5-yl)-5-(trifluoromet-
hyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-A-7; [0334]
2-amino-6-fluoro-N-(4-((1-(oxetan-3-yl)piperidin-4-yl)oxy)pyrimidin-5-yl)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide I-A-8; [0335]
2-amino-6-chloro-N-(4-((1-(oxetan-3-yl)piperidin-4-yl)oxy)pyrimidin-5-yl)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide I-A-9; [0336]
(R)-2-amino-6-fluoro-N-(4-((1-methylpyrrolidin-3-yl)oxy)pyrimidin-5-yl)py-
razolo[1,5-a]pyrimidine-3-carboxamide I-A-10; [0337]
(S)-2-amino-6-fluoro-N-(4-((1-methylpyrrolidin-3-yl)oxy)pyrimidin-5-yl)py-
razolo[1,5-a]pyrimidine-3-carboxamide I-A-11; [0338]
2-amino-N-(4-((1r,4r)-4-(dimethylamino)cyclohexyl)oxy)pyrimidin-5-yl)-6-f-
luoropyrazolo[1,5-a]pyrimidine-3-carboxamide I-A-12; [0339]
2-amino-N-(4-((1r,4r)-4-(dimethylamino)cyclohexyl)oxy)pyrimidin-5-yl)-5-(-
trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-A-13;
[0340]
2-amino-6-fluoro-N-(4-isopropoxypyrimidin-5-yl)pyrazolo[1,5-a]pyrimidine--
3-carboxamide I-A-14; [0341]
2-amino-5-(2-(dimethylamino)ethoxy)-N-(4-isopropoxypyrimidin-5-yl)pyrazol-
o[1,5-a]pyrimidine-3-carboxamide I-A-15; [0342]
2-amino-5-(2-(dimethylamino)ethoxy)-N-(4-methoxypyrimidin-5-yl)pyrazolo[1-
,5-a]pyrimidine-3-carboxamide I-A-16; [0343]
2-amino-6-(cyanomethyl)-N-(4-methoxypyrimidin-5-yl)pyrazolo[1,5-a]pyrimid-
ine-3-carboxamide I-A-17; [0344]
2-amino-6-fluoro-N-(4-(4-(4-methylpiperazine-1-carbonyl)piperidin-1-yl)py-
rimidin-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-A-19; [0345]
2-amino-6-fluoro-N-(4-(4-methylpiperazin-1-yl)pyrimidin-5-yl)pyrazolo[1,5-
-a]pyrimidine-3-carboxamide I-A-20; [0346]
2-amino-6-fluoro-N-(5-(4-methylpiperazin-1-yl)pyridazin-4-yl)pyrazolo[1,5-
-a]pyrimidine-3-carboxamide I-B-1; [0347]
2-amino-6-fluoro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridazin-4-yl)pyraz-
olo[1,5-a]pyrimidine-3-carboxamide I-B-2; [0348]
2-amino-6-fluoro-N-(5-(4-(tetrahydro-2H-pyran-4-yl)piperazin-1-yl)pyridaz-
in-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-3; [0349]
2-amino-6-fluoro-N-(5-(2-methylpyridin-3-yl)pyridazin-4-yl)pyrazolo[1,5-a-
]pyrimidine-3-carboxamide I-B-4; [0350]
2-amino-6-fluoro-N-(5-(4-(4-methylpiperazine-1-carbonyl)piperidin-1-yl)py-
ridazin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-5; [0351]
2-amino-N-(5-(4-((3,3-difluoropyrrolidin-1-yl)methyl)piperidin-1-yl)pyrid-
azin-4-yl)-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-6;
[0352]
2-amino-6-fluoro-N-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)pyridazin-
-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-7; [0353]
2-amino-6-fluoro-N-(5-(4-(morpholinomethyl)piperidin-1-yl)pyridazin-4-yl)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-8; [0354]
2-amino-6-fluoro-N-(5-(piperidin-1-yl)pyridazin-4-yl)pyrazolo[1,5-a]pyrim-
idine-3-carboxamide I-B-9; [0355]
2-amino-N-(5-(4-(4-(tert-butyl)piperazine-1-carbonyl)piperidin-1-yl)pyrid-
azin-4-yl)-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-10;
[0356]
2-amino-6-fluoro-N-(5-(4-((4-methylpiperazin-1-yl)methyl)piperidin-1-yl)p-
yridazin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-11;
[0357]
2-amino-6-fluoro-N-(5-morpholinopyridazin-4-yl)pyrazolo[1,5-a]pyrimidine--
3-carboxamide I-B-12; [0358]
2-amino-6-fluoro-N-(5-(4-(4-methylpiperazine-1-carbonyl)phenyl)pyridazin--
4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-13; [0359]
2-amino-6-fluoro-N-(6-methyl-5-(4-(4-methylpiperazine-1-carbonyl)piperidi-
n-1-yl)pyridazin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
I-B-14; [0360]
2-amino-6-fluoro-N-(5-(1-methyl-1H-imidazol-5-yl)pyridazin-4-yl)py-
razolo[1,5-a]pyrimidine-3-carboxamide I-B-15; [0361]
2-amino-6-fluoro-N-(5-(pyrrolidin-1-yl)pyridazin-4-yl)pyrazolo[1,5-a]pyri-
midine-3-carboxamide I-B-16; [0362]
2-amino-6-fluoro-N-(5-(o-tolyl)pyridazin-4-yl)pyrazolo[1,5-a]pyrimidine-3-
-carboxamide I-B-17; [0363]
2-amino-N-(5-cyclopropylpyridazin-4-yl)-6-fluoropyrazolo[1,5-a]pyrimidine-
-3-carboxamide I-B-18; [0364]
N-(5-(2-oxa-7-azaspiro[3.5]nonan-7-yl)pyridazin-4-yl)-2-amino-6-fluoropyr-
azolo[1,5-a]pyrimidine-3-carboxamide I-B-19; [0365]
2-amino-6-fluoro-N-(5-(4-fluoropiperidin-1-yl)pyridazin-4-yl)pyrazolo[1,5-
-a]pyrimidine-3-carboxamide I-B-20; [0366]
2-amino-N-(5-(4-(3,3-difluoroazetidine-1-carbonyl)piperidin-1-yl)pyridazi-
n-4-yl)-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-21;
[0367]
2-amino-6-fluoro-N-(5-(4-(3-fluoroazetidine-1-carbonyl)piperidin-1-yl)pyr-
idazin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-22; [0368]
2-amino-6-fluoro-N-(5-(4-(methylsulfonyl)piperidin-1-yl)pyridazin-4-yl)py-
razolo[1,5-a]pyrimidine-3-carboxamide I-B-23; [0369]
2-amino-6-fluoro-N-(5-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyridazin-
-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-B-24; [0370]
2-amino-6-fluoro-N-(3-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-4-yl)pyrazol-
o[1,5-a]pyrimidine-3-carboxamide I-C-1; [0371]
2-amino-6-fluoro-N-(3-(4-methylpiperazin-1-yl)pyridin-4-yl)pyrazolo[1,5-a-
]pyrimidine-3-carboxamide I-C-2; [0372]
2-amino-6-fluoro-N-(3-(4-(4-methylpiperazine-1-carbonyl)piperidin-1-yl)py-
ridin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide I-C-3; [0373]
2-amino-N-(3-methoxypyridin-4-yl)-5-(trifluoromethyl)pyrazolo[1,5-a]pyrim-
idine-3-carboxamide I-C-4; [0374]
2-amino-6-fluoro-N-(2'-methyl-[3,3'-bipyridin]-4-yl)pyrazolo[1,5-a]pyrimi-
dine-3-carboxamide I-C-5; [0375]
2-amino-6-fluoro-N-(3'-methyl-[3,4'-bipyridin]-4-yl)pyrazolo[1,5-a]pyrimi-
dine-3-carboxamide I-C-6; [0376]
2-amino-6-fluoro-N-(3-(4-(methylsulfonyl)piperidin-1-yl)pyridin-4-yl)pyra-
zolo[1,5-a]pyrimidine-3-carboxamide I-C-10; and [0377]
2-amino-6-fluoro-N-(3-(pyrrolidin-1-yl)pyridin-4-yl)pyrazolo[1,5-a]pyrimi-
dine-3-carboxamide I-C-11.
Example 2
N-(4-((4s,6r)-1-azaspiro[3.3]heptan-6-yloxy)pyrimidin-5-yl)-2-amino-6-fluo-
ropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00133##
[0379] A solution of (4s,6r)-tert-butyl
6-((5-aminopyrimidin-4-yl)oxy)-1-azaspiro[3.3]heptane-1-carboxylate
(prepared according to a procedure similar to Example 1) (242 mg,
0.7899 mmol) and benzotriazol-1-yl
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate (247.4 mg,
0.7899 mmol) in NMP (2.5 mL) was heated at 100.degree. C. for 72 h.
The reaction mixture was filtered through a SCX cartridge, eluting
with a 2M NH.sub.3 in MeOH. The appropriate fractions were
concentrated in vacuo and the residue was dissolved in DCM (10 mL).
TFA (2 mL, 26 mmol) was added and the mixture was stirred at RT for
2 h, then concentrated in vacuo. The residue was purified using
preparative HPLC (32 mg, 6.4%).
Example 3
2-amino-6-fluoro-N-(3-(4-(4-(oxetan-3-yl)piperazine-1-carbonyl)piperidin-1-
-yl)pyridin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00134##
[0380] Step 1:
1-(4-(2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamido)pyridin-3-y-
l)piperidine-4-carboxylic acid
[0381] To a suspension of tert-butyl
1-[4-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-3-pyr-
idyl]piperidine-4-carboxylate trifluoroacetic acetate (prepared
according to a methodology similar to the one described in Example
1) (101 mg, 0.1773 mmol) in DCM (5 mL) was added TFA (1 mL, 12.98
mmol). The mixture was stirred at room temperature for 20 hours.
The solvent was removed in vacuo and the residue azeotroped with
DCM (.times.2) and diethyl ether (.times.2) to give title compound
as a beige solid (98% Yield, di-TFA salt). .sup.1H NMR (500 MHz,
DMSO) .delta. 11.10 (s, 1H), 9.58 (dd, 1H), 8.91 (d, 1H), 8.86 (d,
1H), 8.68 (s, 1H), 8.56 (d, 1H), 6.92 (s, 2H), 3.10 (d, 2H),
2.99-2.82 (m, 2H), 2.53-2.50 (m, 1H), 2.15-1.93 (m, 4H); .sup.19F
NMR (471 MHz, DMSO) .delta. -74.10, -151.83; LC-MS ES+: 400.0.
Step 2:
2-amino-6-fluoro-N-(3-(4-(4-(oxetan-3-yl)piperazine-1-carbonyl)pip-
eridin-1-yl)pyridin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
I-C-9
[0382] A mixture of
1-[4-[(2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carbonyl)amino]-3-pyr-
idyl]piperidine-4-carboxylic acid (ditrifluoroacetate) (36 mg,
0.05738 mmol), 1-(oxetan-3-yl)piperazine (20.41 mg, 0.1435 mmol),
Et.sub.3N (23.22 mg, 31.98 uL, 0.2295 mmol) and TBTU
tetrafluoroborate (27.64 mg, 0.08607 mmol) in DMF (1 mL) was
stirred at ambient temperature for 66 hours. The crude reaction
mixtures was purified directly by reverse phase preparative HPLC
(Waters Sunfire C18, 10 .mu.M, 100 .ANG. column) to give the title
compound as a beige solid. (17 mg, 39%/TFA salt). .sup.1H NMR (500
MHz, DMSO) .delta. 11.16 (s, 1H), 9.57 (dd, 1H), 9.17 (d, 1H), 8.92
(d, 1H), 8.65 (s, 1H), 8.57 (d, 1H), 6.92 (s, 2H), 4.69 (d, 4H),
4.06 (br s, 2H), 3.80 (br s, 4H), 3.12 (dd, 2H), 3.01-2.80 (m, 6H),
2.15 (qd, 2H), 1.82 (d, 2H); .sup.19F NMR (471 MHz, DMSO) .delta.
-73.97, -151.62; LC-MS ES+: 524.2, ES-522.2.
[0383] The following compounds were prepared according to a
methodology similar to the one described in Example 3, starting
from the appropriate aromatic amine: [0384]
N-(3-(4-(1,4-diazabicyclo[3.2.2]nonane-4-carbonyl)piperidin-1-yl)pyridin--
4-yl)-2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamide
I-C-7; and [0385]
2-amino-6-fluoro-N-(3-(4-(4-(pyrrolidin-1-yl)piperidine-1-carbonyl-
)piperidin-1-yl)pyridin-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
I-C-8.
[0386] The synthesis of the novel intermediates below was required
for the preparation of some of the compounds described in this
patent application.
Preparation 7:
(R)-4-((1-methylpiperidin-3-yl)oxy)pyrimidin-5-amine
##STR00135##
[0387] Step 1:
(R)-4-chloro-6-((1-methylpiperidin-3-yl)oxy)pyrimidin-5-amine
[0388] Sodium t-butoxide (586.0 mg, 6.098 mmol) was added to a
solution of 4,6-dichloropyrimidin-5-amine (500 mg, 3.049 mmol) and
(3R)-1-methylpiperidin-3-ol (351.2 mg, 3.049 mmol) in THF (25.00
mL) at RT. The reaction mixture was heated at 70.degree. C.
overnight. The reaction mixture was quenched with 1 mL water and
concentrated in vacuo. The residue was extracted with DCM and the
combined organic extract were dried and concentrated in vacuo to
afford (R)-4-chloro-6-(1-methylpiperidin-3-yl)oxy)pyrimidin-5-amine
which was used in next step without further purification.
Step 2: (R)-4-((1-methylpiperidin-3-yl)oxy)pyrimidin-5-amine 7a
[0389]
(R)-4-chloro-6-((1-methylpiperidin-3-yl)oxy)pyrimidin-5-amine was
dissolved in MeOH (15.0 mL) and Pd/C 10% (324.5 mg, 0.3049 mmol)
was added and the reaction mixture stirred under a hydrogen
atmosphere for 2 h. The reaction vessel was evacuated and flushed
with nitrogen (3.times.), filtered through a celite pad washing
with methanol followed by ethyl acetate. The combined filtrates
were concentrated in vacuo to leave the title product 7a as a
colourless oil (631 mg, 99%).
[0390] The following amines were prepared according to a
methodology similar to the one described in Preparation 7: [0391]
(S)-6-((1-methylpiperidin-3-yl)oxy)pyrimidin-5-amine 7b; [0392]
(R)-6-(quinuclidin-3-yloxy)pyrimidin-5-amine 7c; [0393]
(S)-6-(quinuclidin-3-yloxy)pyrimidin-5-amine 7d; [0394]
4-((1-methylpiperidin-4-yl)oxy)pyrimidin-5-amine 7e; [0395]
4-((1-(oxetan-3-yl)piperidin-4-yl)oxy)pyrimidin-5-amine 7f; [0396]
(4s,6r)-tert-butyl
6-((5-aminopyrimidin-4-yl)oxy)-1-azaspiro[3.3]heptane-1-carboxylate
7g; [0397] (R)-4-((1-methylpyrrolidin-3-yl)oxy)pyrimidin-5-amine
7h; [0398] (S)-4-((1-methylpyrrolidin-3-yl)oxy)pyrimidin-5-amine
7i; [0399]
4-(((1r,4r)-4-(dimethylamino)cyclohexyl)oxy)pyrimidin-5-amine 7j;
[0400] 4-isopropoxypyrimidin-5-amine 7k; and [0401]
(1-(5-aminopyrimidin-4-yl)piperidin-4-yl)(4-methylpiperazin-1-yl)methanon-
e 7p; and [0402] 4-(4-methylpiperazin-1-yl)pyrimidin-5-amine
7gg.
Preparation 8.1: 3-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-4-amine
7l
##STR00136##
[0403] Step 1:
1-(4-nitro-1-oxido-pyridin-1-ium-3-yl)-4-(oxetan-3-yl)piperazine
[0404] A mixture of 3-bromo-4-nitro-1-oxido-pyridin-1-ium (500 mg,
2.283 mmol) and 1-(oxetan-3-yl)piperazine (649.3 mg, 4.566 mmol) in
EtOH (10 mL) were heated at reflux for 17 hours. The reaction was
cooled to ambient temperature and the solvent removed in vacuo. The
residue was passed through a 25 g SCX-2 cartridge (pre-wetted with
MeOH), eluting with 2M NH.sub.3 in MeOH/DCM. The solvents were
removed in vacuo and the residue purified by column chromatography
(ISCO Companion, 40 g column, eluting with 0 to 10% MeOH/DCM,
loaded in DCM) to give
1-(4-nitro-1-oxido-pyridin-1-ium-3-yl)-4-(oxetan-3-yl)piperazine as
an orange solid (553 mg, 86% Yield).
Step 2: 3-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-4-amine 7l
[0405]
1-(4-nitro-1-oxido-pyridin-1-ium-3-yl)-4-(oxetan-3-yl)piperazine
(553 mg, 1.973 mmol) in MeOH (15 mL)/EtOAc (25 mL) was passed
through the H-cube apparatus to hydrogenate over raney nickel at
20.degree. C. and full H.sub.2 mode, flow rate 1 ml/min. The
solvent was removed in vacuo to give the title compound 7l as an
orange solid (462 mg, 100% Yield).
[0406] The following amines were prepared according to a
methodology similar to the one described in Preparation 8.1: [0407]
3-(4-methylpiperazin-1-yl)pyridin-4-amine 7m; [0408]
(1-(4-aminopyridin-3-yl)piperidin-4-yl)(1-methylpiperidin-4-yl)methanone
7n; [0409] tert-butyl
1-(4-aminopyridin-3-yl)piperidine-4-carboxylate 7w; [0410]
3-(4-(methylsulfonyl)piperidin-1-yl)pyridin-4-amine 7hh; and [0411]
3-(pyrrolidin-1-yl)pyridin-4-amine 7ii.
Preparation 8.2: 2'-methyl-[3,3'-bipyridin]-4-amine
##STR00137##
[0413] A mixture of (4-amino-3-pyridyl)boronic acid hydrochloric
acid (100 mg, 0.5734 mmol), 3-bromo-2-methyl-pyridine (108.5 mg,
0.64 mmol), NaHCO.sub.3 (860 .mu.L of 2 M, 1.72 mmol) and palladium
triphenylphosphane (66.26 mg, 0.05734 mmol) in dioxane (4 mL) was
heated at 105.degree. C. for 12 h. The mixture was then cooled to
room temperature and partitioned between DCM and water. The organic
layer was filtered through a SCX column, eluting with a 2M solution
of NH.sub.3 in MeOH. The eluate was concentrated in vacuo to give
the title compound 7q (60 mg, 56% Yield).
[0414] The following amines were prepared according to a
methodology similar to the one described in Preparation 8.2: [0415]
3'-methyl-[3,4'-bipyridin]-4-amine 7r.
Preparation 9.1: 5-(4-methylpiperazin-1-yl)pyridazin-4-amine
##STR00138##
[0416] Step 1:
3,4-dichloro-5-(4-methylpiperazin-1-yl)pyridazine
[0417] 3,4,5-Trichloropyridazine (3 g, 16.36 mmol) was dissolved in
dry NMP (18 mL) and cooled in an ice-bath. DIPEA (2.326 g, 3.135
mL, 18 mmol) was added, followed, dropwise, by 1-methylpiperazine
(1.721 g, 1.906 mL, 17.18 mmol). The resulting mixture was stirred
at RT overnight. The reaction mixture was concentrated under
reduced pressure to give an brown solid which was partitioned
between 10% MeOH in DCM and saturated NaHCO.sub.3. The aqueous
layer was extracted with further 10% MeOH in DCM (5.times.20 mL)
and the combined organics were dried over Na.sub.2SO.sub.4,
filtered and concentrated under reduced pressure to give a brown
oil which was purified by column chromatography (7.5% MeOH in DCM,
.about.300 mL silica, loaded in DCM) to provide
3,4-dichloro-5-(4-methylpiperazin-1-yl)pyridazine as a light yellow
solid (2.36 g, 58% Yield).
Step 2:
[4-chloro-5-(4-methylpiperazin-1-yl)pyridazin-3-yl]hydrazine
[0418] 3,4-Dichloro-5-(4-methylpiperazin-1-yl)pyridazine (500 mg,
2.023 mmol) was placed in a microwave tube and hydrazine
monohydrate (1.772 g, 1.717 mL, 35.40 mmol) was added. The
resulting suspension was stirred at 100.degree. C. for .about.10
mins. The brown reaction mixture was allowed to cool to RT,
whereupon solid started to precipitate. The suspension was
sonicated and the suspended solid was collected by filtration. This
material was dissolved in water, basified with saturated
NaHCO.sub.3 and partitioned with 10% MeOH in DCM. The aqueous layer
was extracted with further 10% MeOH in DCM (6.times.10 mL) and the
combined organics were dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure to give a yellow solid. This
material was sonicated in ether and the suspended solid was
collected by filtration to give
[4-chloro-5-(4-methylpiperazin-1-yl)pyridazin-3-yl]hydrazine as an
ochre powder (155.9 mg, 32% Yield).
Step 3: 4-chloro-5-(4-methylpiperazin-1-yl)pyridazine
[0419] [4-Chloro-5-(4-methylpiperazin-1-yl)pyridazin-3-yl]hydrazine
(145 mg, 0.5974 mmol) and copper sulfate pentahydrate (522.1 mg,
2.091 mmol) were dissolved in water (9 mL) and stirred at
95.degree. C. for .about.30 minutes. The brown suspension was
allowed to cool and 15 wt % NaOH (2 mL) was added. The resulting
suspension was heated to 95.degree. C. for .about.5 mins, allowed
to cool to RT and filtered through a pre-wetted (10% MeOH in DCM)
celite cartridge (10 g). The cartridge was washed with 10% MeOH in
DCM and the filtrate was partitioned with water. The aqueous layer
was extracted with further 10% MeOH in DCM (3.times.10 mL) and the
combined organics were dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography (7.5% MeOH/1% NH 4 OH in DCM, .about.75 mL
silica) to give 4-chloro-5-(4-methylpiperazin-1-yl)pyridazine as an
off-white solid (100.6 mg, 79% Yield).
Step 4: 5-(4-methylpiperazin-1-yl)pyridazin-4-amine 7o
[0420] 4-chloro-5-(4-methylpiperazin-1-yl)pyridazine (720 mg, 3.385
mmol), tert-butyl carbamate (1.982 g, 16.92 mmol), sodium
tert-butoxide (1.659 g, 17.26 mmol), BrettPhos pre-catalyst (269.7
mg, 0.3385 mmol) and BrettPhos (181.7 mg, 0.3385 mmol) were placed
in a Schlenk tube and degassed by vacuum/nitrogen cycles
(.times.5). Dry toluene (14.4 mL) was added and the resulting
mixture was placed into a pre-heated block at 100.degree. C. The
mixture was stirred at 100.degree. C. overnight. The reaction
mixture was partitioned between 10% MeOH in DCM and saturated
NH.sub.4Cl. The aqueous layer was extracted with further 10% MeOH
in DCM (3.times.10 mL) and the combined organics were dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure
to give an orange gum which was purified by column chromatography
(7.5% MeOH/1% NH.sub.4OH in DCM, .about.100 mL silica) to give an
orange foam (618.8 mg, 62% Yield). The material was dissolved in
DCM (5 mL) and cooled in an ice-bath. TFA (5 mL) was added slowly
and the resultant solution was stirred at 0.degree. C. for
.about.20 mins and at RT for .about.90 mins. The reaction mixture
was concentrated under reduced pressure. The residue was dissolved
in MeOH and added to a pre-wetted (MeOH) SCX-2 cartridge (25 g),
eluting with 2M NH.sub.3 in MeOH. The light yellow eluate was
concentrated under reduced pressure to give a light yellow gum
which was purified by column chromatography (9% MeOH/1% NH.sub.4OH
in DCM, .about.100 mL silica) to give
5-(4-methylpiperazin-1-yl)pyridazin-4-amine 7o as a light yellow
solid (286.3 mg, 44% Yield).
[0421] The following amines were prepared according to a
methodology similar to the one described in Preparation 9.1: [0422]
5-(4-(oxetan-3-yl)piperazin-1-yl)pyridazin-4-amine 7s; [0423]
5-(4-(tetrahydro-2H-pyran-4-yl)piperazin-1-yl)pyridazin-4-amine 7t;
[0424]
(1-(5-aminopyridazin-4-yl)piperidin-4-yl)(4-methylpiperazin-1-yl)m-
ethanone 7u; [0425]
(1-(5-aminopyridazin-4-yl)piperidin-4-yl)(4-(tert-butyl)piperazin-1-yl)me-
thanone 7x; [0426]
(1-(5-amino-3-methylpyridazin-4-yl)piperidin-4-yl)(4-methylpiperazin-1-yl-
)methanone 7y; [0427] 5-(pyrrolidin-1-yl)pyridazin-4-amine 7jj;
[0428] 5-(2-oxa-7-azaspiro[3.5]nonan-7-yl)pyridazin-4-amine 7kk;
[0429] 5-(4-fluoropiperidin-1-yl)pyridazin-4-amine 7ll; [0430]
(1-(5-aminopyridazin-4-yl)piperidin-4-yl)(3,3-difluoroazetidin-1-yl)metha-
none 7 mm; [0431]
(1-(5-aminopyridazin-4-yl)piperidin-4-yl)(3-fluoroazetidin-1-yl)methanone
Inn; [0432] 5-(4-(methylsulfonyl)piperidin-1-yl)pyridazin-4-amine
7oo; and [0433]
5-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyridazin-4-amine
7pp.
Preparation 9.2: 5-(2-methylpyridin-3-yl)pyridazin-4-amine
##STR00139##
[0435] A mixture of 5-chloropyridazin-4-amine (50 mg, 0.386 mmol),
(2-methyl-3-pyridyl)boronic acid (63.43 mg, 0.463 mmol), palladium
triphenylphosphane (22.3 mg, 0.0193 mmol) and Na.sub.2CO.sub.3 (386
.mu.L of 2M, 0.772 mmol) in dioxane (2 mL) was heated at
140.degree. C. for 1 h. The mixture was then cooled to room
temperature and partitioned between DCM and water. The organic
layer was filtered through a SCX column, eluting with a 2M solution
of NH.sub.3 in MeOH. The eluate was concentrated in vacuo to give
the title compound 7v (72 mg, 100% Yield).
[0436] The following amines were prepared according to a
methodology similar to the one described in Preparation 9.2: [0437]
(4-(5-aminopyridazin-4-yl)phenyl)(4-methylpiperazin-1-yl)methanone
7z; [0438]
5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)pyridazin-4-amine 7aa;
[0439] 5-(1-methyl-1H-imidazol-5-yl)pyridazin-4-amine 7qq; [0440]
5-(o-tolyl)pyridazin-4-amine 7rr; and [0441]
5-cyclopropylpyridazin-4-amine 7ss.
Preparation 9.3:
5-(4-((3,3-difluoropyrrolidin-1-yl)methyl)piperidin-1-yl)pyridazin-4-amin-
e
##STR00140##
[0442] Step 1: tert-butyl
4-((3,3-difluoropyrrolidin-1-yl)methyl)piperidine-1-carboxylate
[0443] A mixture of 3,3-difluoropyrrolidine hydrochloride (965 mg,
6.722 mmol), tert-butyl 4-formylpiperidine-1-carboxylate (1.720 g,
8.066 mmol), DIPEA (955.6 mg, 1.288 mL, 7.394 mmol) and crushed 4A
MS (965 mg) in DCE (30 mL) were stirred at ambient temperature for
3 hours. NaBH(OAc).sub.3 (2.848 g, 13.44 mmol) was added and the
reaction stirred at ambient temperature for a further 16 hours. The
mixture was filter through Celite (washing with DCM) and the
filtrate concentrated in vacuo. The residue was purified by column
chromatography (ISCO Companion, 80 g column, eluting with 0 to 10%
MeOH/DCM, loaded in DCM) to give the sub-title product as a
colourless oil that was used directly into next step without
further purification. LC-MS ES+: 305.1.
Step 2: 4-((3,3-difluoropyrrolidin-1-yl)methyl)piperidine
[0444] TFA (766.5 mg, 517.9 .mu.L, 6.722 mmol) was added to a
stirred solution of tert-butyl
4-[(3,3-difluoropyrrolidin-1-yl)methyl]piperidine-1-carboxylate
(2.046 g, 6.722 mmol) in DCM (15 mL) and the reaction stirred at
ambient temperature for 66 hours. The solvent was removed in vacuo
and the residue azeotroped with DCM (.times.2) and ether
(.times.2). The residue was passed through a 50 g SCX-2 cartridge
and washed with MeOH/DCM mixtures. The product was eluted by
washing the cartridge with 2M NH.sub.3 in MeOH/DCM mixtures. The
solvent was removed in vacuo to give the sub-title compound as a
pale yellow solid (1.15 g, 84% Yield). .sup.1H NMR (500 MHz, DMSO)
.delta. 2.89 (dt, 2H), 2.82 (t, 2H), 2.64 (t, 2H), 2.42 (td, 2H),
2.32-2.09 (m, 4H), 1.67-1.58 (m, 2H), 1.52-1.44 (m, 1H), 0.95 (dtd,
2H); .sup.19F NMR (471 MHz, DMSO) .delta. -90.80; LC-MS ES+:
205.1.
Step 3:
5-(4-((3,3-difluoropyrrolidin-1-yl)methyl)piperidin-1-yl)pyridazin-
-4-amine 7bb
[0445] A mixture of 5-chloropyridazin-4-amine (50 mg, 0.3860 mmol),
4-[(3,3-difluoropyrrolidin-1-yl)methyl]piperidine (197.1 mg, 0.9650
mmol) in NMP (10 mL) were heated under microwave conditions at
170.degree. C. for 7 hours. The reaction was passed through a 10 g
SCX-2 cartridge and washed with MeOH/DCM mixtures. The product was
eluted by washing the cartridge with 2M NH.sub.3 in MeOH/DCM
mixtures. The solvent was removed in vacuo and the residue was
purified by column chromatography (ISCO Companion, 12 g column,
eluting with 0 to 10% MeOH/DCM, loaded in DCM) to give the
sub-title product as a beige solid (27 mg, 23% Yield). .sup.1H NMR
(500 MHz, DMSO) .delta. 8.46 (d, 1H), 8.41 (s, 1H), 5.85 (s, 2H),
3.20 (d, 2H), 2.87 (t, 2H), 2.69 (t, 2H), 2.59 (td, 2H), 2.34 (d,
2H), 2.24 (tt, 2H), 1.83-1.74 (m, 2H), 1.65-1.53 (m, 1H), 1.43-1.30
(m, 2H); .sup.19F NMR (471 MHz, DMSO) .delta. -90.81; LC-MS ES+:
298.1, ES-: 296.1.
[0446] The following amines were prepared according to a
methodology similar to the one described in Preparation 9.3: [0447]
5-morpholinopyridazin-4-amine 7cc; [0448]
5-(4-((4-methylpiperazin-1-yl)methyl)piperidin-1-yl)pyridazin-4-amine
7dd; [0449] 5-(piperidin-1-yl)pyridazin-4-amine 7ee; and [0450]
5-(4-(morpholinomethyl)piperidin-1-yl)pyridazin-4-amine 7ff.
Preparation 10:
1-(tetrahydro-2H-pyran-4-yl)-1H-imidazol-5-amine
[0451] Tetrahydropyran-4-amine (451 mg, 4.459 mmol) was added to a
stirred solution of ethyl N-(cyanomethyl)formimidate (500 mg, 4.459
mmol) in DCM (5 mL) and the reaction was heated at reflux for 1
hour. The reaction was cooled to ambient temperature then passed
through a 25 g SCX-2 cartridge and washed with MeOH/DCM mixtures.
The product was eluted by washing the cartridge with 2M NH.sub.3 in
MeOH/DCM mixtures. The solvent was removed in vacuo and the residue
tritruated from DCM/Et.sub.2O and the resultant precipitate
isolated by filtration to give the sub-title compound as a grey
solid (123 mg, 17% Yield). MS (ES+) 168.1.
[0452] The following aminoimidazoles intermediates were synthesized
according to Preparation 10: [0453]
1-cyclopropyl-1H-imidazol-5-amine:
[0453] ##STR00141## [0454]
1-(pyridin-3-yl)-1H-imidazol-5-amine:
##STR00142##
[0454] and [0455] 1-phenyl-1H-imidazol-5-amine:
##STR00143##
[0455] Example 4
2-amino-6-fluoro-N-(1-(tetrahydro-2H-pyran-4-yl)-1H-imidazol-5-yl)pyrazolo-
[1,5-a]pyrimidine-3-carboxamide (Compound I-D-4)
##STR00144##
[0457] (6-chlorobenzotriazol-1-yl)
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate (120.1 mg,
0.3454 mmol) and 3-tetrahydropyran-4-ylimidazol-4-amine (75 mg,
0.314 mmol) (prepared according to a procedure similar to
Preparation 10) were suspended in NMP (1 mL) and stirred at
100.degree. C. for 19 hours. The reaction was cooled to ambient
temperature and purified by passing the crude reaction mixture
through a 10 g SCX-2 cartridge (pre-washed with MeOH). The
cartridge was washed with DCM/MeOH mixtures then the product eluted
with 2M NH.sub.3 in MeOH/DCM mixtures. The solvent was removed in
vacuo and the material was purified by fractionlynx. The clean
fractions were freeze-dried to give title compound
2-amino-6-fluoro-N-(1-(tetrahydro-2H-pyran-4-yl)-1H-imidazol-5-yl)pyrazol-
o[1,5-a]pyrimidine-3-carboxamide as a beige solid (21.1 mg, 19%
Yield). MS (ES+) 346.1.
[0458] The following compounds were successfully prepared using a
procedure similar to Example 4: [0459]
2-amino-N-(1-benzyl-1H-imidazol-5-yl)-6-(cyanomethyl)pyrazolo[1,5-a]pyrim-
idine-3-carboxamide (Compound I-D-1); [0460]
2-amino-6-fluoro-N-(1-phenyl-1H-imidazol-5-yl)pyrazolo[1,5-a]pyrimidine-3-
-carboxamide (Compound I-D-2); [0461]
2-amino-N-(1-cyclopropyl-1H-imidazol-5-yl)-6-fluoropyrazolo[1,5-a]pyrimid-
ine-3-carboxamide (Compound I-D-3); and [0462]
2-amino-6-fluoro-N-(1-(pyridin-3-yl)-1H-imidazol-5-yl)pyrazolo[1,5-a]pyri-
midine-3-carboxamide (Compound I-D-5).
Example 5
2-amino-6-fluoro-N-(2-methyl-1-phenyl-1H-imidazol-5-yl)pyrazolo[1,5-a]pyri-
midine-3-carboxamide (Compound I-D-6)
##STR00145##
[0463] Step 1: benzyl
(2-methyl-1-phenyl-1H-imidazol-5-yl)carbamate
[0464] To a suspension of 2-methyl-3-phenyl-imidazole-4-carboxylic
acid (Hydrochloric Acid (1)) (500 mg, 2.095 mmol) in dioxane (6 mL)
was sequentially added dppa (634.1 mg, 0.4966 mL, 2.304 mmol) and
Et.sub.3N (466.4 mg, 0.6424 mL, 4.609 mmol). The mixture was heated
at 90.degree. C. for 5 mins before benzyl alcohol (0.651 mL, 6.291
mmol) was added. The reaction mixture was heated at 90.degree. C.
for 1 h, then partitioned between water and EtOAc. The combined
organics were washed with brine, dried (MgSO.sub.4) and
concentrated in vacuo to give a brown oil. The crude product was
purified by chromatography on silica gel eluting with 1% MeOH in
EtOAc (0.1% NH.sub.4OH) to give benzyl
(2-methyl-1-phenyl-1H-imidazol-5-yl)carbamate as a yellow oil. (75
mg, 9.5%). MS (ES+) 309.3.
Step 2: 2-methyl-1-phenyl-1H-imidazol-5-amine
[0465] To a mixture of benzyl
N-(2-methyl-3-phenyl-imidazol-4-yl)carbamate (510 mg, 1.659 mmol)
and Pd on C, wet, Degussa (176.6 mg, 0.1659 mmol) was added
methanol (10 mL). The reaction was hydrogenated (balloon pressure)
for 2 h before the catalyst was filtered off and the filtrate
concentrated in vacuo yielding
2-methyl-1-phenyl-1H-imidazol-5-amine as a yellow oil that is
immediately used in next step without further purification. MS
(ES+) 174.4.
Step 3: 2-cyano-N-(2-methyl-1-phenyl-1H-imidazol-5-yl)acetamide
[0466] To a solution of 2-methyl-1-phenyl-1H-imidazol-5-amine (250
mg, 1.44 mmol) in DCM (7.5 mL) was sequentially added DIPEA (1.006
mL, 5.776 mmol) and cyanoacetic acid (184.2 mg, 2.165 mmol). The
mixture was cooled on an ice bath then
3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine;
hydrochloride (415.0 mg, 2.165 mmol) was added. The reaction
mixture was stirred at 0.degree. C. for 30 mins and then allowed to
warm to room temperature and stirred overnight. The reaction
mixture was partitioned between DCM and water. Combined organic
extract was dried (MgSO.sub.4) and concentrated in vacuo to give a
redish oil which was purified by chromatography on silica gel,
eluting with 5% MeOH in DCM (0.5% NH.sub.4OH) to give
2-cyano-N-(2-methyl-1-phenyl-1H-imidazol-5-yl)acetamide as a redish
oil/solid. (105 mg, 32.3%). MS (ES+) 242.1.
Step 4:
3-amino-4,4,4-trichloro-2-cyano-N-(2-methyl-1-phenyl-1H-imidazol-5-
-yl)but-2-enamide
[0467] To a solution of
2-cyano-N-(2-methyl-3-phenyl-imidazol-4-yl)acetamide (100 mg,
0.4162 mmol) in Ethanol (1.5 mL) was sequentially added sodium
acetate (68.28 mg, 0.8324 mmol), 2,2,2-trichloroacetonitrile (0.051
mL, 0.5016 mmol) and the reaction was stirred at room temperature
for 4 h. A further 10 mg of sodium acetate and 10 uL
2,2,2-trichloroacetonitrile was sequentially added and the mixture
was stirred for a further 2 h at RT. The reaction was concentrated
in vacuo and the residue was partitioned between water and EtOAc.
Combined organic extract was washed with brine, dried (MgSO.sub.4)
and concentrated in vacuo to give
3-amino-4,4,4-trichloro-2-cyano-N-(2-methyl-1-phenyl-1H-imidazol-5-yl)but-
-2-enamide as a redish oil. (153 mg, 79%). MS (ES+) 384.0.
Step
5:3,5-diamino-N-(2-methyl-1-phenyl-1H-imidazol-5-yl)-1H-pyrazole-4-ca-
rboxamide
[0468] To a solution of
3-amino-4,4,4-trichloro-2-cyano-N-(2-methyl-3-phenyl-imidazol-4-yl)but-2--
enamide (150 mg, 0.39 mmol) in NMP (1.5 mL) was added hydrazine
hydrate (0.032 mL, 1.02 mmol) and the mixture heated to 85.degree.
C. for 3 h. The reaction was allowed to cool to RT and concentrated
in vacuo to give
3,5-diamino-N-(2-methyl-1-phenyl-1H-imidazol-5-yl)-1H-pyrazole-4-carboxam-
ide as an orange oil. (116 mg, 100%). MS (ES+) 298.2.
Step 6:
2-amino-6-fluoro-N-(2-methyl-1-phenyl-1H-imidazol-5-yl)pyrazolo[1,-
5-a]pyrimidine-3-carboxamide
[0469] To a mixture of
3,5-diamino-N-(2-methyl-3-phenyl-imidazol-4-yl)-1H-pyrazole-4-carboxamide
(116 mg, 0.3902 mmol), 3-(diisopropylamino)-2-fluoro-prop-2-enal
(67.59 mg, 0.3902 mmol) in isopropanol (0.58 mL) and water (0.58
mL) was added acetic acid (0.221 mL, 3.904 mmol) and the solution
was heated to 88.degree. C. for 2.5 h. The reaction mixture was
partitioned between EtOAc and saturated sodium carbonate solution.
Combined organic extract was dried (MgSO.sub.4), and concentrated
in vacuo to give as a redish oil that was purified by fractionlynx.
Clean fractions were freeze-dried to give
2-amino-6-fluoro-N-(2-methyl-1-phenyl-1H-imidazol-5-yl)pyrazolo[1,5--
a]pyrimidine-3-carboxamide as a yellow solid. (16.4 mg, 8.5%). MS
(ES+) 352.2.
Preparation 11:
2-amino-N-(4-bromo-3-methylisothiazol-5-yl)-6-fluoropyrazolo[1,5-a]pyrimi-
dine-3-carboxamide
##STR00146##
[0471] NaH (210.3 mg, 5.259 mmol) was added to a solution of
4-bromo-3-methyl-isothiazol-5-amine (533 mg, 2.761 mmol) in dry NMP
(21.95 mL) at RT. The solution was stirred at RT for 5 min before
(6-chlorobenzotriazol-1-yl)
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate 6a* (914.4
mg, 2.630 mmol) was added. After 10 min, the reaction mixture was
treated with water (120 mL) and stirred for 15 mins. The pale brown
solid was filtered off and dried in vacuo to afford
4-bromo-3-methylisothiazol-5-yl
2-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate. (801 mg,
82%). MS (ES+) 370.5.
Preparation 12: 3-methyl-4-(pyridin-3-yl)isothiazol-5-amine
##STR00147##
[0473] 3-pyridylboronic acid (127.3 mg, 1.036 mmol),
4-bromo-3-methyl-isothiazol-5-amine (100 mg, 0.518 mmol),
Na.sub.2CO.sub.3 (777 .mu.L of 2 M, 1.554 mmol), palladium
triphenylphosphane (29.93 mg, 0.0259 mmol) in dioxane (6 mL) was
heated at 110.degree. C. in a microwave for 30 min. The mixture was
concentrated in vacuo and the residue was loaded on a SCX column,
washed with DCM/MeOH mixtures before the product was eluted with a
2M solution of ammonia in MeOH. The eluent was concentrated in
vacuo to yield 3-methyl-4-(pyridin-3-yl)isothiazol-5-amine as a
pale yellow oil that was used in next step without further
purification. MS (ES+) 192.0.
[0474] The following 3-aminoisothiazole intermediates were prepared
using preparation 12: [0475]
3-methyl-4-(2-methylpyridin-3-yl)isothiazol-5-amine:
[0475] ##STR00148## [0476]
4-cyclopropyl-3-methylisothiazol-5-amine:
[0476] ##STR00149## [0477]
3-methyl-4-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)isothiazol-5-amine:
[0477] ##STR00150## [0478]
4-(3,6-dihydro-2H-pyran-4-yl)-3-methylisothiazol-5-amine:
##STR00151##
[0478] and [0479] 3-methyl-4-phenylisothiazol-5-amine:
##STR00152##
[0479] Preparation 13:
3-methyl-4-(pyrrolidin-1-yl)isothiazol-5-amine
##STR00153##
[0480] Step 1: methyl
2-((3-methyl-4-nitroisothiazol-5-yl)carbamoyl)benzoate
[0481] A mixture of 5-bromo-3-methyl-4-nitro-isothiazole (200 mg,
0.8967 mmol), (1,3-dioxoisoindolin-2-yl)potassium (174.4 mg, 0.9415
mmol) in dry DMF (2 mL) was stirred at RT overnight. The mixture
was quenched by the addition of methanol, stirred at RT for 1 h,
then the reaction mixture was concentrated in vacuo. The residue
was triturated in a small volume of dry MeOH. The precipitate was
collected by filtration and dried in vacuo, yielding methyl
2-((3-methyl-4-nitroisothiazol-5-yl)carbamoyl)benzoate as a pale
yellow solid. (165 mg, 57%) MS (ES+) 322.1.
Step 2: methyl
2-((4-amino-3-methylisothiazol-5-yl)carbamoyl)benzoate
[0482] Methyl
2-((3-methyl-4-nitroisothiazol-5-yl)carbamoyl)benzoate was
suspended in MeOH (30 mL), Pd/C 10% (477.1 mg, 4.483 mmol) was
added and the mixture was hydrogenated at RT under 1 atm of
hydrogen (balloon pressure) for 2 h. The catalyst was filtered off
and the mixture was concentrated in vacuo, yielding methyl
2-((4-amino-3-methylisothiazol-5-yl)carbamoyl)benzoate. (150 mg,
100%). MS (ES+) 292.1.
Step 3:
2-(3-methyl-4-(pyrrolidin-1-yl)isothiazol-5-yl)isoindoline-1,3-dio-
ne
[0483] DIEA (332.7 mg, 448.4 .mu.L, 2.574 mmol), 1,4-dibromobutane
(555.8 mg, 307.4 .mu.L, 2.574 mmol) was added to a solution of
methyl 2-[4-amino-3-methyl-isothiazol-5-yl)carbamoyl]benzoate (150
mg, 0.5149 mmol) in DMF (4 mL) and the mixture was stirred at
130.degree. C. for 40 min in a microwave. The reaction mixture was
concentrated in vacuo to yield
2-(3-methyl-4-(pyrrolidin-1-yl)isothiazol-5-yl)isoindoline-1,3-dion-
e that was used in next step without further purification. MS (ES+)
314.1.
Step 4: 3-methyl-4-(pyrrolidin-1-yl)isothiazol-5-amine
[0484] Hydrazine hydrate (25.56 mg, 25.03 .mu.L, 0.5106 mmol) was
added to a solution of
2-(3-methyl-4-pyrrolidin-1-yl-isothiazol-5-yl)isoindoline-1,3-dione
(160 mg, 0.5106 mmol) in EtOH (5 mL) and the mixture was stirred at
RT for 10 min. The mixture was heated for 15 min at 120.degree. C.
in a microwave, then was concentrated in vacuo. The residue was
purified by fractionlynx. The clean fractions were concentrated in
vacuo and dried by azeotropic distillation with toluene (.times.2)
to yield 3-methyl-4-(pyrrolidin-1-yl)isothiazol-5-amine as pale
yellow solid. (61.7 mg, 59.9% over two steps). MS (ES+) 184.1.
Example 6
2-amino-6-fluoro-N-(3-methyl-4-(1-methyl-1H-imidazol-5-yl)isothiazol-5-yl)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-6)
##STR00154##
[0486]
2-amino-N-(4-bromo-3-methyl-isothiazol-5-yl)-6-fluoro-pyrazolo[1,5--
a]pyrimidine-3-carboxamide (20 mg, 0.05388 mmol),
tributyl-(3-methylimidazol-4-yl)stannane (40.01 mg, 0.1078 mmol)
dichloropalladium triphenylphosphane (7.566 mg, 0.01078 mmol) in
DMF (2 mL) was degassed for 5 min, then heated at 110.degree. C.
After 2 h, the reaction mixture was loaded on a SCX column, washed
with DCM/MeOH mixtures. The product was eluted with a 2M solution
of ammonia in MeOH and the eluent was concentrated in vacuo. The
residue was purified by fractionlynx to yield
2-amino-6-fluoro-N-(3-methyl-4-(1-methyl-1H-imidazol-5-yl)isothiazol-5-yl-
)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a pale yellow solid.
(8.09 mg, 37.4%). MS (ES+) 373.1.
Example 7
2-amino-6-fluoro-N-(3-methyl-4-(morpholinomethyl)isothiazol-5-yl)pyrazolo[-
1,5-a]pyrimidine-3-carboxamide (compound I-E-15)
##STR00155##
[0488]
2-amino-N-(4-bromo-3-methyl-isothiazol-5-yl)-6-fluoro-pyrazolo[1,5--
a]pyrimidine-3-carboxamide (50 mg, 0.1078 mmol),
trifluoro(morpholinomethyl)boranuide (Potassium Ion (1)) (29.01 mg,
0.1401 mmol), dicesium carbonate (105.4 mg, 0.3234 mmol),
palladium(+2) cation diacetate (4.84 mg, 0.02156 mmol),
dicyclohexyl-[2-(2,4,6-triisopropylphenyl)phenyl]phosphane (20.56
mg, 0.04312 mmol) in dioxane (4 mL)-water (0.5 mL) was heated at
90.degree. C. for 5 h. The reaction mixture was concentrated in
vacuo and the residue was purified by fractionlynx. The clean
fractions were freeze dried, yielding
2-amino-6-fluoro-N-(3-methyl-4-(morpholinomethyl)isothiazol-5-yl)pyrazolo-
[1,5-a]pyrimidine-3-carboxamide as a pale yellow solid. (21 mg,
32%). MS (ES+) 392.2.
Example 8
2-amino-6-fluoro-N-(3-methyl-4-(pyrrolidin-1-yl)isothiazol-5-yl)pyrazolo[1-
,5-a]pyrimidine-3-carboxamide (compound I-E-21)
##STR00156##
[0490] A mixture of (6-chlorobenzotriazol-1-yl)
2-amino-6-fluoro-pyrazolo[1,5-a]pyrimidine-3-carboxylate (140.4 mg,
0.4038 mmol), 3-methyl-4-(pyrrolidin-1-yl)isothiazol-5-amine (37
mg, 0.2019 mmol) (synthesized according to preparation 13) in
pyridine (3 mL) was heated at 105.degree. C. for 24 h. The mixture
was concentrated in vacuo and the residue was purified by
fractionlynx. The clean fractions were combined and freeze dried to
yield
2-amino-6-fluoro-N-(3-methyl-4-(pyrrolidin-1-yl)isothiazol-5-yl)pyrazolo[-
1,5-a]pyrimidine-3-carboxamide as a pale yellow solid. (25 mg,
26%). MS (ES+) 362.1.
[0491] The following compounds were successfully prepared using a
procedure similar to Example 6 or Example 7: [0492]
N-(4-(1-acetyl-1,2,3,6-tetrahydropyridin-4-yl)-3-methylisothiazol-5-yl)-2-
-amino-6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxamide (compound
I-E-9); [0493]
2-amino-6-fluoro-N-(3-methyl-4-(pyrimidin-5-yl)isothiazol-5-yl)pyr-
azolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-11); [0494]
2-amino-6-fluoro-N-(3-methyl-4-(1-methyl-1H-pyrazol-4-yl)isothiazol-5-yl)-
pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-12); [0495]
2-amino-6-fluoro-N-(3-methyl-4-(2-methylpyridin-4-yl)isothiazol-5-yl)pyra-
zolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-13); [0496]
2-amino-6-fluoro-N-(3-methyl-4-(pyridin-4-yl)isothiazol-5-yl)pyrazolo[1,5-
-a]pyrimidine-3-carboxamide (compound I-E-14); [0497]
2-amino-6-fluoro-N-(3-methyl-4-(morpholinomethyl)isothiazol-5-yl)pyrazolo-
[1,5-a]pyrimidine-3-carboxamide (compound I-E-15); [0498]
2-amino-N-(4-(1,3-dimethyl-1H-pyrazol-5-yl)-3-methylisothiazol-5-yl)-6-fl-
uoropyrazolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-16);
[0499]
2-amino-6-fluoro-N-(3-methyl-4-(pyrrolidin-1-ylmethyl)isothiazol-5-yl)pyr-
azolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-17); [0500]
2-amino-N-(4-(1,3-dimethyl-1H-pyrazol-4-yl)-3-methylisothiazol-5-yl)-6-fl-
uoropyrazolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-18);
[0501]
2-amino-6-fluoro-N-(3-methyl-4-((4-methylpiperazin-1-yl)methyl)isothiazol-
-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-19);
and [0502]
2-amino-6-fluoro-N-(4-(methoxymethyl)-3-methylisothiazol-5-yl)pyra-
zolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-20).
[0503] The following compounds were successfully prepared using a
procedure similar to Example 8: [0504]
2-amino-6-fluoro-N-(3-methyl-4-(2-methylpyridin-3-yl)isothiazol-5-yl)pyra-
zolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-1); [0505]
2-amino-N-(4-cyclopropyl-3-methylisothiazol-5-yl)-6-fluoropyrazolo[1,5-a]-
pyrimidine-3-carboxamide (compound I-E-2); [0506]
2-amino-6-fluoro-N-(3-methyl-4-(pyridin-3-yl)isothiazol-5-yl)pyrazolo[1,5-
-a]pyrimidine-3-carboxamide (compound I-E-3); [0507]
2-amino-N-(4-(3,6-dihydro-2H-pyran-4-yl)-3-methylisothiazol-5-yl)-6-fluor-
opyrazolo[1,5-a]pyrimidine-3-carboxamide (compound I-E-4); [0508]
2-amino-6-fluoro-N-(3-methyl-4-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)i-
sothiazol-5-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound
I-E-5); [0509]
2-amino-6-fluoro-N-(3-methyl-4-phenylisothiazol-5-yl)pyrazolo[1,5--
a]pyrimidine-3-carboxamide (compound I-E-7); and [0510]
2-amino-6-fluoro-N-(3-methyl-4-(pyrrolidin-1-yl)isothiazol-5-yl)pyrazolo[-
1,5-a]pyrimidine-3-carboxamide (compound I-E-21).
Example 9
2-amino-6-fluoro-N-(3-methyl-4-(1H-pyrazol-1-yl)isothiazol-5-yl)pyrazolo[1-
,5-a]pyrimidine-3-carboxamide (compound I-E-10)
##STR00157##
[0512] A mixture of
2-amino-N-(4-bromo-3-methyl-isothiazol-5-yl)-6-fluoro-pyrazolo[1,5-a]pyri-
midine-3-carboxamide (20 mg, 0.05388 mmol), 1H-pyrazole (7.339 mg,
0.1078 mmol), Cut (21 mg, 0.1103 mmol), Cs.sub.2CO.sub.3 (71 mg,
0.2179 mmol) in DMF (2 mL) was degassed with nitrogen and heated at
140.degree. C. in a microwave for 1 h. The insoluble material was
filtered off and the filtrate was purified by fractionlynx. The
clean fractions were freeze-dried yielding
2-amino-6-fluoro-N-(3-methyl-4-(1H-pyrazol-1-yl)isothiazol-5-yl)pyrazolo[-
1,5-a]pyrimidine-3-carboxamide as a pale yellow solid. (0.63 mg,
1.9%). MS (ES+) 359.1.
Compound Analytical Data
TABLE-US-00006 [0513] Cmpds of Formula I LCMS LCMS and I-A ES+ (Rt
min) HNMR I-A-1 387.3 2.18 .sup.1H NMR (500 MHz, DMSO-d6) .delta.
1.67 (2H, m), 2.01 (2H, m), 2.26 (3H, m), 2.52 (br s, 3H), 2.92
(1H, m), 5.26 (1H, m), 6.74 (2H, br s), 8.49 (1H, s), 8.74 (1H, m),
9.50 (2H, m) and 9.99 (1H, br s). I-A-2 387.3 2.17 .sup.1H NMR (500
MHz, DMSO-d6) .delta. 1.61-.171 (2H, m), 1.93 (1H, m), 2.02 (1H,
m), 2.29 (1H, m), 2.40 (1H, m), 2.47 (1H, m), 2.85 (1H, m),
5.21-5.25 (1H, m), 6.74 (2H, br s), 8.48 (1H, m), 8.73 (1H, d),
9.50 (2H, m) and 10.01 (1H, s) ppm. I-A-3 399.0 2.01 .sup.1H NMR
(500 MHz, DMSO-d6) .delta. 9.92 (s, 1H), 9.81 (s, 1jH), 9.55 (s,
1H), 9.52 (dd, J = 4.8, 2.5 Hz, 1H), 8.74 (dd, J = 2.5, 0.5 Hz,
1H), 8.50 (s, 1H), 6.78 (s, 2H), 5.50-5.42 (m, 1H), 3.90 (m, 1H),
3.53-3.25 (m, 5H), 2.59 (s, 1H), 2.47-2.37 (m, 1H), 2.08-1.93 (m,
3H). I-A-4 399.0 2.02 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 9.87
(s, 1H), 9.82 (s, 1H), 9.55 (s, 1H), 9.52 (dd, J = 4.8, 2.5 Hz,
1H), 8.79-8.72 (m, 1H), 8.51 (s, 1H), 6.78 (s, 2H), 5.45 (d, J =
9.1 Hz, 1H), 3.90 (m, 1H), 3.50-3.26 (m, 5H), 2.43 (d, J = 11.4 Hz,
1H), 2.07-1.94 (m, 3H). I-A-5 387.0 2.00 .sup.1H NMR (500 MHz,
DMSO-d6 @ 375K) .delta. 2.25-2.34 (5H, m), 3.44 (3H, m), 5.47 (1H,
m), 8.50 (1H, s), 8.73 (1H, m), 9.34 (1H, m), 9.49 (1H, s) and 9.78
(1H, br s) ppm. I-A-6 403.1 2.08 .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 9.90 (s, 1H), 9.50 (s, 1H), 9.45 (d, J = 2.2 Hz, 1H), 8.60
(d, J = 2.2 Hz, 1H), 8.48 (s, 1H), 6.80 (s, 2H), 5.21 (s, 1H), 2.74
(br s, 2H), 2.32 (br s, 2H), 2.24 (s, 3H), 2.08 (br s, 2H), 1.91
(br s, 2H). I-A-7 437.2 2.30 .sup.1H NMR (500 MHz, DMSO-d6) .delta.
9.49 (s, 1H), 9.35 (s, 1H), 9.29 (d, J = 6.9 Hz, 1H), 8.52 (s, 1H),
7.53 (d, J = 6.9 Hz, 1H), 7.04 (s, 2H), 5.24 (br s, 1H), 2.79 (br
s, 2H), 2.21 (s, 3H), 2.14-1.98 (m, 4H), 1.91-1.70 (m, 2H). I-A-8
429.2 1.90 .sup.1H NMR (500 MHz, DMSO-d6 @ 360K) .delta. 2.21-2.38
(4H, m), 3.22-3.30 (4H, m), 4.35 (1H, m), 4.79 (4H, m), 5.45 (1H,
m), 8.50 (1H, s), 8.72 (1H, m), 9.38 (1H, m), 9.49 (1H, s) and 9.81
(1H, br s) ppm. I-A-9 445.1 1.99 .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 9.88 (s, 1H), 9.50 (s, 1H), 9.45 (d, J = 2.2 Hz, 1H), 8.60
(d, J = 2.2 Hz, 1H), 8.49 (s, 1H), 6.80 (s, 2H), 5.25 (dp, J =
11.8, 4.0 Hz, 1H), 4.56 (t, J = 6.5 Hz, 2H), 4.47 (t, J = 6.1 Hz,
2H), 3.45 (p, J = 6.4 Hz, 1H), 2.68-2.61 (m, 2H), 2.25 (t, J = 9.5
Hz, 2H), 2.15-2.06 (m, 2H), 1.90 (dtd, J = 11.7, 8.1, 3.3 Hz, 2H).
I-A-10 373.0 1.97 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.25 (s,
1H), 9.91 (d, J = 44.6 Hz, 1H), 9.52 (s, 2H), 8.90 (d, J = 2.4 Hz,
1H), 8.53 (s, 1H), 6.76 (s, 2H), 5.72 (d, J = 4.4 Hz, 1H),
4.18-3.76 (m, 3H), 3.46-3.18 (m, 2H), 2.94 (dd, J = 26.9, 3.9 Hz,
3H). I-A-11 373.0 1.97 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.35
(s, 1H), 9.91 (d, J = 44.9 Hz, 1H), 9.52 (d, J = 2.5 Hz, 2H), 8.89
(d, J = 2.5 Hz, 1H), 8.53 (s, 1H), 6.75 (s, 1H), 5.71 (d, J = 6.2
Hz, 1H), 3.92 (s, 4H), 3.62-3.18 (m, 2H), 3.01-2.84 (m, 3H). I-A-12
415.0 2.16 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.08 (s, 1H),
9.74 (s, 1H), 9.52 (dd, J = 4.8, 2.5 Hz, 1H), 9.48 (s, 1H), 8.84
(d, J = 2.5 Hz, 1H), 8.49 (s, 1H), 6.74 (s, 2H), 5.15 (m, 1H),
3.42-3.32 (m, 1H), 2.80 (d, J = 4.9 Hz, 6H), 2.35 (d, J = 8.9 Hz,
2H), 2.12 (s, 2H), 1.80-1.58 (m, 4H). I-A-13 465.0 2.44 .sup.1H NMR
(500 MHz, DMSO-d6) .delta. 9.48 (s, 1H), 9.35 (s, 1H), 9.31-9.24
(m, 1H), 8.50 (s, 1H), 7.52 (d, J = 6.9 Hz, 1H), 7.03 (s, 2H), 5.24
(td, J = 10.9, 5.4 Hz, 1H), 2.20 (s, 6H), 2.18 (s, 1H), 2.15 (m,
2H), 1.93-1.85 (m, 2H), 1.54 (td, J = 12.7, 12.0, 3.3 Hz, 2H), 1.38
(qd, J = 13.3, 3.2 Hz, 2H). I-A-14 332.1 2.39 .sup.1H NMR (500 MHz,
DMSO-d6) .delta. 1.47-1.49 (6H, d), 5.41-5.43 (1H, m), 8.50 (1H,
s), 8.82-8.83 (1H, d), 9.47 (1H, s), 9.50-9.51 (1H, dd), 10.06 (1H,
s). I-A-15 401.2 2.23 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 1.4
(6H, d), 2.88-2.92 (6H, m), 3.6-3.63 (2H, m), 4.8-4.82 (2H, m), 6.5
(2H, brs), 6.6 (1H, d), 8.55 (1H, s), 8.82 (1H, d), 9.06 (1H, s),
9.22 (1H, s), 9.62 (1H, brs). I-A-16 373.2 1.92 .sup.1H NMR (500
MHz, DMSO-d6) .delta. 2.82-2.87 (6H, m), 3.6-3.63 (2H, m), 4.12
(3H, s), 4.8-4.83 (2H, m), 6.5 (2H, brs), 6.6 (1H, d), 8.55 (1H,
s), 8.82 (1H, d), 9.52 (1H, s), 9.62 (1H, brs) I-A-17 325.1 1.79 --
I-A-18 385.0 1.83 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.05 (s,
1H), 9.55 (dd, J = 4.8, 2.5 Hz, 1H), 9.48 (s, 1H), 9.07 (s, 2H),
8.80 (d, J = 2.5 Hz, 1H), 8.49 (s, 1H), 6.76 (s, 2H), 5.21 (p, J =
6.8 Hz, 1H), 3.90-3.78 (m, 2H), 3.14-3.06 (m, 2H), 2.90-2.82 (m,
2H), 2.65 (t, J = 8.2 Hz, 2H). I-A-19 483.4 1.77 .sup.1H NMR (500
MHz, DMSO-d6) .delta. 9.90 (s, 1H), 9.55-9.49 (m, 2H), 9.13 (s,
1H), 8.85 (d, J = 2.5 Hz, 1H), 8.63 (d, J = 0.6 Hz, 1H), 6.76 (s,
2H), 4.51 (s, 1H), 3.95 (d, J = 12.8 Hz, 2H), 3.10-2.95 (m, 5H),
2.93 (s, 2H), 2.83 (s, 3H), 2.58 (s, 1H), 1.90 (s, 2H), 1.76 (dd, J
= 13.2, 3.8 Hz, 2H). I-A-20 372.2 1.69 .sup.1H NMR (500 MHz,
DMSO-d6) 9.82 (s, 1H), 9.53 (dd, J = 4.8, 2.5 Hz, 1H), 9.39 (s,
1H), 9.25 (s, 1H), 9.00 (dd, J = 2.6, 0.6 Hz, 1H), 8.64 (s, 1H),
6.76 (s, 2H), 4.01 (d, J = 9.9 Hz, 2H), 3.37-3.10 (m, 5H), 2.94 (s,
3H).
TABLE-US-00007 Cmpds of Formula I LCMS LCMS and I-B ES+ (Rt min)
HNMR I-B-1 372.1 1.63 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.11
(d, J = 0.7 Hz, 1H), 9.92 (s, 1H), 9.52 (dd, J = 4.8, 2.5 Hz, 1H),
9.03 (d, J = 0.8 Hz, 1H), 8.83 (d, J = 2.5 Hz, 1H), 6.83 (s, 2H),
3.08 (t, J = 4.8 Hz, 4H), 2.60 (t, J = 4.8 Hz, 4H), 2.32 (s, 3H).
I-B-2 414.2 1.55 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 12.38 (s,
1H), 10.16 (s, 1H), 9.95 (s, 1H), 9.58 (dd, J = 4.6, 2.5 Hz, 1H),
9.22 (s, 1H), 9.08 (d, J = 2.7 Hz, 1H), 6.88 (s, 2H), 4.92 (t, J =
6.7 Hz, 2H), 4.75 (t, J = 7.2 Hz, 2H), 4.66 (s, 1H), 3.62-3.55 (m,
4H), 3.18 (s, 4H). I-B-3 442.2 1.73 .sup.1H NMR (500 MHz,
Methanol-d4) .delta. 10.27 (d, J = 0.7 Hz, 1H), 9.13 (dd, J = 4.3,
2.5 Hz, 1H), 9.04 (d, J = 0.7 Hz, 1H), 8.84 (dd, J = 2.6, 0.6 Hz,
1H), 4.15 (dd, J = 11.6, 4.5 Hz, 2H), 3.58-3.44 (m, 10H), 2.18 (d,
J = 11.6 Hz, 2H), 1.81 (tt, J = 13.3, 6.7 Hz, 2H), 1.39-1.27 (m,
1H). I-B-4 365.0 1.68 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.47
(s, 1H), 9.61 (s, 1H), 9.44 (dd, J = 4.7, 2.5 Hz, 1H), 9.11 (s,
1H), 8.83 (dd, J = 5.0, 1.8 Hz, 1H), 7.98 (d, J = 2.6 Hz, 1H), 7.59
(s, 1H), 6.60 (bs, 3H), 2.32 (s, 3H). I-B-5 483.2 1.78 .sup.1H NMR
(500 MHz, DMSO-d6) .delta. 1.75 (2H, m), 2.09-2.14 (2H, m), 2.21
(3H, s), 2.29 (2H, m), 2.37 (2H, m), 2.91-2.96 (3H, m), 3.17 (2H,
m), 3.57 (4H, m), 6.84 (2H, br s), 9.01 (1H, s), 9.04 (1H, d), 9.52
(1H, dd), 10.24 (1H, s) and 10.27 (1H, br s) ppm. I-B-6 476.1 2.56
.sup.1H NMR (500 MHz, Methanol-d4) .delta. 10.14 (s, 1H), 9.14 (dd,
J = 4.3, 2.5 Hz, 1H), 9.06 (s, 1H), 8.79 (d, J = 2.4 Hz, 1H),
3.87-3.72 (m, 4H), 3.69-3.48 (m, 2H), 3.26-2.98 (m, 4H), 2.63 (ddd,
J = 21.3, 14.6, 7.9 Hz, 2H), 2.11 (ddt, J = 18.4, 7.4, 3.7 Hz, 1H),
2.04 (d, J = 12.7 Hz, 2H), 1.70 (qd, J = 12.7, 12.2, 3.8 Hz, 2H).
I-B-7 462.1 2.03 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.42 (d, J
= 0.9 Hz, 1H), 10.07 (s, 1H), 9.47 (dd, J = 4.8, 2.5 Hz, 1H), 9.06
(d, J = 0.9 Hz, 1H), 8.17 (d, J = 2.5 Hz, 1H), 7.65 (s, 4H), 6.82
(s, 2H), 3.85 (br s, 2H), 3.43 (br s, 2H), 3.06 (br s, 4H), 2.81
(s, 3H), 2.47 (br s, 4H). I-B-8 456.2 2.06 .sup.1H NMR (500 MHz,
DMSO-d6) .delta. 10.14 (s, 1H), 9.91 (s, 1H), 9.56 (dd, J = 4.8,
2.5 Hz, 1H), 9.32 (s, 1H), 9.06 (s, 1H), 8.91 (d, J = 2.5 Hz, 1H),
6.87 (s, 2H), 4.01 (d, J = 12.9 Hz, 2H), 3.73 (t, J = 12.3 Hz, 2H),
3.52 (d, J = 12.7 Hz, 2H), 3.38 (d, J = 11.6 Hz, 2H), 3.31-3.22 (m,
2H), 3.18-3.07 (m, 2H), 2.97-2.88 (m, 2H), 2.06 (s, 1H), 1.92 (d, J
= 12.3 Hz, 2H), 1.57 (dd, J = 23.7, 11.3 Hz, 2H). I-B-9 357.0 2.28
.sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.01 (s, 1H), 9.96 (s, 1H),
9.54 (dd, J = 4.7, 2.5 Hz, 1H), 9.09 (s, 1H), 8.91 (d, J = 2.5 Hz,
1H), 6.84 (s, 2H), 3.22 (t, J = 5.4 Hz, 4H), 1.82-1.74 (m, 4H),
1.66 (q, J = 5.9 Hz, 2H). I-B-10 525.2 2.18 .sup.1H NMR (500 MHz,
DMSO-d6) .delta. 10.29-10.22 (m, 2H), 10.15 (s, 1H), 9.55 (dd, J =
4.8, 2.5 Hz, 1H), 9.07-8.98 (m, 2H), 6.85 (s, 2H), 4.64 (d, J =
13.6 Hz, 1H), 4.28 (d, J = 14.5 Hz, 1H), 3.58 (ddd, J = 37.3, 20.9,
8.3 Hz, 4H), 3.22 (d, J = 11.2 Hz, 1H), 3.10 (dt, J = 24.6, 12.0
Hz, 2H), 2.95 (tdd, J = 11.8, 9.4, 3.4 Hz, 4H), 2.12 (dd, J = 23.3,
12.2 Hz, 2H), 1.83 (t, J = 16.7 Hz, 2H), 1.37 (s, 10H). I-B-11
469.1 1.94 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.04 (s, 1H),
9.91 (s, 1H), 9.56 (dd, J = 4.7, 2.5 Hz, 1H), 9.10 (s, 1H), 8.87
(d, J = 2.5 Hz, 1H), 6.86 (s, 2H), 4.89 (br s, 4H), 3.53 (br d,
4H), 3.53-3.16 (br m, 2H), 2.97 (t, J = 11.4 Hz, 2H), 2.81 (s, 3H),
2.74 (br s, 2H), 1.89 (d, J = 12.2 Hz, 3H), 1.49 (q, J = 12.6 Hz,
2H). I-B-12 359.0 1.68 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.10
(s, 1H), 10.00 (s, 1H), 9.54 (dd, J = 4.7, 2.5 Hz, 1H), 9.12 (d, J
= 0.6 Hz, 1H), 9.02-8.92 (m, 1H), 6.84 (s, 2H), 3.94-3.80 (m, 4H),
3.28-3.15 (m, 4H). I-B-13 476.0 1.84 .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 10.42 (d, J = 0.9 Hz, 1H), 9.83 (s, 1H), 9.28 (dd, J = 4.7,
2.5 Hz, 1H), 9.02 (d, J = 0.9 Hz, 1H), 8.31 (dd, J = 2.5, 0.5 Hz,
1H), 7.76-7.65 (m, 4H), 3.81 (s, 4H), 3.26 (s, 4H), 2.83 (s, 3H).
I-B-14 497.2 1.9 .sup.1H NMR (500 MHz, DMSO-d6) d 1.79 (2H, m),
2.13 (2H, m), 2.77 (3H, s), 2.85 (3H, s), 3.00-3.06 (6H, m), 3.29
(2H, m), 3.49 (2H, m), 4.34 (2H, m), 4.60 (2H, m), 6.92 (2H, br s),
9.27 (1H, s), 9.55 (1H, m), 10.07 (1H, br s), 10.35 (1H, s) and
10.95 (1H, s) ppm. I-B-15 354.1 1.42 .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 10.44 (d, J = 1.0 Hz, 1H), 9.96 (s, 1H), 9.47 (dd, J = 4.8,
2.5 Hz, 1H), 9.09 (d, J = 1.0 Hz, 1H), 8.34 (d, J = 2.5 Hz, 1H),
8.10 (s, 1H), 7.32 (d, J = 1.1 Hz, 1H), 6.80 (s, 2H), 3.54 (s, 3H)
I-B-16 343.1 1.72 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 1.84-1.92
(4H, m), 3.62-3.7 (4H, m), 6.75 (2H, s), 8.76 (1H, s), 8.83 (1H,
d), 9.08 (1H, s), 9.49-9.53 (2H, m) I-B-17 364.1 2.36 .sup.1H NMR
(500 MHz, DMSO-d6) .delta. 10.44 (d, 1H), 9.61 (s, 1H), 9.41 (dd,
1H), 9.00 (d, 1H), 7.96 (d, 1H), 7.74-7.19 (m, 4H), 6.77 (s, 2H),
2.10 (s, 3H). I-B-18 314.1 1.92 .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 10.59 (s, 1H), 10.29 (d, 1H), 9.56 (dd, 1H), 9.08 (s, 1H),
8.97 (dd, 1H), 6.87 (s, 2H), 2.05-2.01 (m, 1H), 1.33-1.29 (m, 2H),
0.97-0.94 (m, 2H). I-B-19 399.0 1.66 .sup.1H NMR (500 MHz,
Methanol-d4) .delta. 9.06 (dd, J = 4.4, 2.6 Hz, 1H), 8.97 (s, 1H),
8.71-8.66 (m, 2H), 3.98-3.83 (m, 2H), 3.80-3.74 (m, 2H), 3.74-3.66
(m, 4H), 2.12-1.93 (m, 4H). I-B-20 375.0 2.02 .sup.1H NMR (500 MHz,
DMSO-d6) .delta. 2.00-2.12 (2H, m), 2.10-2.22 (2H, m), 3.15-3.20
(2H, m), 3.22-3.27 (2H, m), 4.97-5.00 (0.5H, m), 5.05-5.10 (0.5H,
m), 6.83 (2H, brs), 8.90 (1H, d), 9.15 (1H, s), 9.52-9.55 (1H, m),
10.03 (1H, s), 10.12 (1H, brs) I-B-21 476.1 2.02 .sup.1H NMR (500
MHz, DMSO-d6) .delta. 10.28 (s, 1H), 10.24 (d, J = 0.7 Hz, 1H),
9.53 (dd, J = 4.7, 2.5 Hz, 1H), 9.05 (dd, J = 2.5, 0.6 Hz, 1H),
9.04 (d, J = 0.8 Hz, 1H), 6.84 (s, 2H), 4.75 (t, J = 12.4 Hz, 2H),
4.37 (t, J = 12.6 Hz, 2H), 3.29 (d, J = 0.9 Hz, 1H), 3.21-3.15 (m,
2H), 2.89 (td, J = 12.0, 2.4 Hz, 2H), 2.14-2.01 (m, 2H), 1.89-1.82
(m, 2H). I-B-22 458.1 1.82 .sup.1H NMR (500 MHz, DMSO-d6) .delta.
10.29 (s, 1H), 10.24 (d, J = 0.9 Hz, 1H), 9.52 (dd, J = 4.7, 2.5
Hz, 1H), 9.10 (d, J = 2.5 Hz, 1H), 9.03 (d, J = 0.8 Hz, 1H), 6.83
(s, 2H), 5.44 (dtt, J = 57.3, 6.2, 3.1 Hz, 1H), 4.64-4.52 (m, 1H),
4.40-4.20 (m, 2H), 4.03-3.91 (m, 1H), 3.30 (d, J = 6.9 Hz, 1H),
3.17 (d, J = 11.2 Hz, 2H), 2.98-2.85 (m, 2H), 2.15-2.00 (m, 2H),
1.80 (t, J = 14.4 Hz, 2H). I-B-23 435.0 1.66 .sup.1H NMR (500 MHz,
DMSO-d6) .delta. 2.03-2.12 (2H, m), 2.18-2.25 (2H, m), 2.94-3.02
(2H, m), 3.08 (3H, s), 3.42-3.49 (3H, m), 6.88 (2H, brs), 8.82 (1H,
d), 9.17 (1H, s), 9.54-9.57 (1H, m), 10.18 (1H, s), 10.22 (1H, brs)
I-B-24 455.1 1.65 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.04 (s,
1H), 9.86 (s, 1H), 9.56 (dd, J = 4.7, 2.5 Hz, 1H), 9.09 (s, 1H),
8.84 (d, J = 3.1 Hz, 1H), 6.86 (s, 2H), 4.76 (s, 4H), 3.66-2.93 (m,
9H), 2.81 (s, 3H), 2.05 (d, J = 10.3 Hz, 2H), 1.81 (t, J = 12.1 Hz,
2H).
TABLE-US-00008 Cmpds of Formula I LCMS LCMS and I-C ES+ (Rt min)
HNMR I-C-1 413.2 1.91 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.41
(s, 1H), 9.49 (dd, J = 4.8, 2.5 Hz, 1H), 8.80 (d, J = 2.5 Hz, 1H),
8.48 (d, 1H), 8.41 (d, J = 5.5 Hz, 1H), 8.27 (d, J = 5.3 Hz, 1H),
6.80 (s, 2H), 4.62 (t, J = 6.5 Hz, 2H), 4.49 (t, J = 6.1 Hz, 2H),
3.66 (p, J = 6.3 Hz, 1H), 2.99 (t, J = 4.7 Hz, 4H), 2.54 (s, 4H).
I-C-2 371.2 2.04 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.48 (s,
1H), 10.03 (s, 1H), 9.58 (dd, J = 4.7, 2.5 Hz, 1H), 9.08 (d, J =
2.5 Hz, 1H), 8.73 (d, J = 6.1 Hz, 1H), 8.66 (s, 1H), 8.51 (d, J =
6.2 Hz, 1H), 6.89 (s, 2H), 3.66 (d, J = 8.0 Hz, 2H), 3.41 (d, J =
15.4 Hz, 2H), 3.35-3.22 (m, 2H), 3.22-3.11 (m, 2H), 3.02 (s, 3H).
I-C-3 482.2 2.20 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 11.04 (s,
1H), 9.78 (s, 1H), 9.56 (dd, J = 4.7, 2.5 Hz, 1H), 9.13 (dd, J =
2.5, 0.7 Hz, 1H), 8.80 (s, 1H), 8.58 (s, 1H), 8.49 (d, J = 6.2 Hz,
1H), 6.90 (s, 2H), 4.60 & 4.29 (2 .times. s, 1H), 3.49 (s, 2H),
3.11 (d, J = 6.8 Hz, 2H), 3.06-2.89 (m, 4H), 2.85 (s, 3H),
2.56-2.48 (m, 4H), 2.15 (s, 2H), 1.83 (d, J = 14.3 Hz, 2H). I-C-4
353.0 2.61 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 9.81 (s, 1H),
9.26 (dd, J = 6.9, 0.7 Hz, 1H), 8.19 (dd, J = 9.1, 0.7 Hz, 1H),
8.10 (dd, J = 3.1, 0.7 Hz, 1H), 7.53-7.44 (m, 2H), 6.98 (s, 2H),
3.83 (s, 3H). I-C-5 364.0 1.95 .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 9.45 (s, 1H), 9.40 (dd, J = 4.8, 2.5 Hz, 1H), 8.76 (dd, J =
4.9, 1.8 Hz, 1H), 8.59-8.51 (m, 2H), 8.36 (d, J = 0.6 Hz, 1H), 7.93
(d, J = 2.5 Hz, 1H), 7.74 (dd, J = 7.6, 1.8 Hz, 1H), 7.48 (ddd, J =
7.7, 4.9, 0.7 Hz, 1H), 6.72 (s, 2H), 2.27 (s, 3H). I-C-6 364.0 2.01
.sup.1H NMR (500 MHz, DMSO-d6) .delta. 9.40 (dd, J = 4.8, 2.5 Hz,
1H), 9.36 (s, 1H), 8.76-8.73 (m, 1H), 8.64 (dd, J = 4.7, 0.8 Hz,
1H), 8.56 (s, 2H), 8.36 (s, 1H), 7.88 (d, J = 2.5 Hz, 1H), 7.36 (d,
J = 4.9 Hz, 1H), 6.72 (s, 2H), 2.09 (s, 3H). I-C-7 508.1 2.17
.sup.1H NMR (500 MHz, DMSO-d6) .delta. 11.15 (s, 0.4H), 11.09 (s,
0.6H), 10.22 (s, 1H), 9.57 (dd, J = 4.7, 2.5 Hz, 1H), 9.32 (d, J =
2.5 Hz, 0.4H), 9.18-9.01 (m, 0.6H), 8.85 (s, 1H), 8.61 (s, 1H),
8.53 (d, J = 6.3 Hz, 1H), 6.90 (s, 2H), 4.75 (s, 0.6H), 4.56 (s,
0.4H), 3.98 (dt, J = 11.0, 5.5 Hz, 2H), 3.54-3.35 (m, 6H),
3.24-3.07 (m, 2H), 2.94 (q, J = 12.1, 11.7 Hz, 3H), 2.32-1.97 (m,
6H), 1.84 (d, J = 12.4 Hz, 2H). I-C-8 536.2 2.28 .sup.1H NMR (500
MHz, DMSO-d6) .delta. 11.10 (s, 1H), 9.60 (s, 1H), 9.57 (dd, J =
4.7, 2.5 Hz, 1H), 9.19 (d, J = 2.5 Hz, 1H), 8.85 (d, J = 5.1 Hz,
1H), 8.60 (s, 1H), 8.52 (d, J = 6.4 Hz, 1H), 6.90 (s, 2H), 4.62 (d,
J = 13.3 Hz, 1H), 4.21 (d, J = 13.6 Hz, 1H), 3.54 (s, 2H), 3.41 (s,
2H), 3.12 (t, J = 11.3 Hz, 6H), 3.05-2.86 (m, 3H), 2.16 (dd, J =
25.6, 11.5 Hz, 3H), 2.03 (t, J = 7.4 Hz, 2H), 1.96-1.83 (m, 2H),
1.79 (d, J = 13.0 Hz, 2H), 1.61-1.38 (m, 2H). I-C-9 524.2 2.04
.sup.1H NMR (500 MHz, DMSO-d6) .delta. 11.16 (s, 1H), 9.57 (dd, J =
4.7, 2.5 Hz, 1H), 9.17 (d, J = 2.4 Hz, 1H), 8.92 (d, J = 6.6 Hz,
1H), 8.65 (s, 1H), 8.57 (d, J = 6.5 Hz, 1H), 6.92 (s, 2H), 4.69 (d,
J = 6.8 Hz, 4H), 4.06 (br s, 2H), 3.80 (br s, 4H), 3.12 (dd, J =
11.2, 4.0 Hz, 2H), 3.01-2.80 (m, 6H), 2.15 (qd, J = 12.8, 3.9 Hz,
2H), 1.82 (d, J = 11.3 Hz, 2H). I-C-10 434.0 2.0 .sup.1H NMR (500
MHz, DMSO-d6) .delta. 10.95 (s, 1H), 9.58-9.55 (m, 1H), 8.96-8.94
(m, 1H), 8.88 (d, 1H), 8.68 (s, 1H), 8.53 (d, 1H), 6.84 (2H, brs),
3.44-3.38 (m, 1H), 3.28-3.24 (m, 1H), 3.12 (s, 3H), 2.97-2.90 (m,
2H), 2.28-2.20 (m, 2H), 2.15-2.07 (m, 2H) I-C-11 342.1 2.37 .sup.1H
NMR (500 MHz, DMSO-d6) .delta. 2.03-2.12 (4H, m), 3.02-3.10 (4H,
m), 6.90 (2H, s), 8.47 (1H, d), 8.57 (1H, s), 8.83 (1H, d), 8.87
(1H, d), 9.55-9.58 (2H, m), m 10.68 (1H, s)
TABLE-US-00009 Cmpds of Formula I LCMS LCMS and I-D ES+ (Rt min)
HNMR I-D-1 373.1 1.91 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 9.60
(s, 1H), 9.07 (d, J = 2.0 Hz, 1H), 9.01 (br s, 1H), 8.57 (d, J =
2.0 Hz, 1H), 7.68 (s, 1H), 7.42-7.33 (m, 5H), 6.67 (s, 2H), 5.48
(s, 2H), 4.14 (s, 2H). I-D-2 338.0 2.04 .sup.1H NMR (500 MHz,
DMSO-d6) .delta. 9.50 (s, 1H), 9.41 (dd, J = 4.9, 2.5 Hz, 1H), 8.48
(d, J = 2.5 Hz, 1H), 7.78 (d, J = 1.2 Hz, 1H), 7.66-7.59 (m, 2H),
7.58-7.52 (m, 3H), 7.19 (dd, J = 1.1, 0.6 Hz, 1H), 6.62 (s, 2H).
I-D-3 302.1 1.69 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 9.92 (s,
1H), 9.48 (dd, 1H), 8.89 (dd, 1H), 7.45 (d, 1H), 7.03 (d, 1H), 6.67
(s, 2H), 3.28-3.24 (m, 1H), 1.20-1.16 (m, 2H), 1.03-1.00 (m, 2H).
I-D-4 346.1 1.51 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 9.48 (dd,
1H), 9.29 (s, 1H), 8.83 (dd, 1H), 7.75 (s, 1H), 6.94 (s, 1H), 6.64
(s, 2H), 4.16-4.09 (m, 1H), 4.00-3.96 (m, 2H), 3.47-3.42 (m, 2H),
1.99-1.92 (m, 4H). I-D-5 339.1 1.45 .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 9.43 (dd, 1H), 9.39 (s, 1H), 8.78 (dd, 1H), 8.69 (dd, 1H),
8.56 (d, 1H), 8.05 (ddd, 1H), 7.92 (d, 1H), 7.64 (ddd, 1H), 7.17
(t, 1H), 6.59 (s, 2H). I-D-6 352.0 2.08 .sup.1H NMR (500 MHz,
DMSO-d6) d 9.57 (1H, s), 9.43 (1H, dd), 8.25 (1H, s), 7.79-7.78
(3H, m), 7.73-7.71 (3H, m), 6.65 (2H, brs), 2.37 (3H, s).
Compound Analytical Data
TABLE-US-00010 [0514] Cmpds of Formula I LCMS LCMS and I-E ES+ (Rt
min) HNMR I-E-1 384.0 2.17 .sup.1H NMR (500 MHz, DMSO-d6) .delta.
10.30 (s, 1H), 9.47 (dd, J = 4.7, 2.5 Hz, 1H), 8.81 (dd, J = 5.2,
1.7 Hz, 1H), 8.28 (d, J = 2.5 Hz, 1H), 8.08 (d, J = 7.7 Hz, 1H),
7.72 (dd, J = 7.5, 5.2 Hz, 1H), 6.74 (s, 2H), 2.39 (s, 3H), 2.21
(s, 3H). I-E-2 333.0 2.51 .sup.1H NMR (500 MHz, DMSO-d6) .delta.
10.89 (s, 1H), 9.53 (dd, J = 4.8, 2.5 Hz, 1H), 8.98 (d, J = 2.5 Hz,
1H), 2.39 (s, 3H), 1.70 (tt, J = 8.2, 5.4 Hz, 1H), 1.24-1.10 (m,
2H), 0.69-0.54 (m, 2H). I-E-3 370.0 2.13 .sup.1H NMR (500 MHz,
DMSO-d6) .delta. 10.75 (s, 1H), 9.49 (dd, J = 4.7, 2.5 Hz, 1H),
8.82 (dd, J = 2.3, 0.9 Hz, 1H), 8.80 (dd, J = 5.0, 1.6 Hz, 1H),
8.42 (d, J = 2.5 Hz, 1H), 8.16 (dt, J = 7.8, 1.9 Hz, 1H), 7.83
(ddd, J = 7.9, 5.0, 0.9 Hz, 1H), 2.34 (s, 3H). I-E-4 375.0 2.28
.sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.67 (s, 1H), 9.51 (dd, J =
4.7, 2.5 Hz, 1H), 8.84 (d, J = 2.5 Hz, 1H), 6.03-5.93 (m, 1H), 4.38
(q, J = 2.7 Hz, 2H), 3.94 (t, J = 5.3 Hz, 2H), 2.30 (s, 5H). I-E-5
388.0 2.24 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.55 (s, 1H),
10.19 (s, 1H), 9.48 (dd, J = 4.7, 2.5 Hz, 1H), 8.98 (dd, J = 2.5,
0.6 Hz, 1H), 6.82-6.44 (m, 2H), 6.02-5.87 (m, 1H), 4.24-3.29 (m,
6H), 3.06 (s, 3H), 2.33 (s, 3H). I-E-6 373.0 1.81 .sup.1H NMR (500
MHz, DMSO-d6) .delta. 10.50 (s, 1H), 9.50 (dd, J = 4.7, 2.5 Hz,
1H), 8.50 (d, J = 2.5 Hz, 1H), 8.29 (s, 1H), 7.31 (d, J = 1.2 Hz,
1H), 6.76 (s, 2H), 3.52 (s, 3H), 2.25 (s, 3H). I-E-7 369.1 2.74
.sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.75 (s, 1H), 9.47 (dd, J =
4.8, 2.5 Hz, 1H), 8.40 (d, J = 2.5 Hz, 1H), 7.76-7.59 (m, 2H),
7.61-7.38 (m, 3H), 2.33 (s, 3H) I-E-8 373.1 2.11 .sup.1H NMR (500
MHz, DMSO-d6) .delta. 10.53 (s, 1H), 9.49 (dd, J = 4.8, 2.5 Hz,
1H), 8.45 (d, J = 2.5 Hz, 1H), 7.76 (d, J = 1.9 Hz, 1H), 6.76 (s,
2H), 6.52 (d, J = 1.9 Hz, 1H), 3.66 (s, 3H), 2.25 (s, 3H). I-E-9
416.1 1.98 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.64 (s, 1H),
10.60 (s, 0H), 9.52 (dt, J = 4.8, 2.6 Hz, 1H), 8.89-8.78 (m, 1H),
6.75 (s, 2H), 6.00-5.74 (m, 1H), 4.28 (q, J = 2.9 Hz, 2H), 3.77
(dt, J = 10.8, 5.5 Hz, 2H), 2.30 (d, J = 5.9 Hz, 3H), 2.14 (d, J =
4.5 Hz, 3H). I-E-10 359.1 2.19 .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 11.46 (s, 1H), 9.48 (dd, J = 4.8, 2.5 Hz, 1H), 8.77 (d, J =
2.5 Hz, 1H), 8.25 (dd, J = 2.4, 0.6 Hz, 1H), 8.12 (dd, J = 1.9, 0.6
Hz, 1H), 6.76 (s, 2H), 6.69 (dd, J = 2.4, 1.9 Hz, 1H), 2.41 (s,
3H). I-E-11 370.0 1.85 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.76
(s, 1H), 9.49 (dd, J = 4.7, 2.5 Hz, 1H), 9.37 (s, 1H), 9.06 (s,
2H), 8.36 (d, J = 2.5 Hz, 1H), 6.76 (s, 2H), 6.52 (s, 1H), 2.34 (s,
3H). I-E-12 373.1 1.97 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.66
(s, 1H), 9.51 (dd, J = 4.8, 2.5 Hz, 1H), 8.62 (d, J = 2.5 Hz, 1H),
8.12-8.02 (m, 1H), 7.75 (d, J = 0.8 Hz, 1H), 6.76 (s, 2H), 4.04 (s,
3H), 2.35 (s, 3H). I-E-13 384.1 2.19 .sup.1H NMR (500 MHz, DMSO-d6)
.delta. 10.86 (s, 1H), 9.51 (dd, J = 4.7, 2.5 Hz, 1H), 8.90 (d, J =
5.6 Hz, 1H), 8.60 (d, J = 2.5 Hz, 1H), 7.95-7.57 (m, 2H), 6.79 (s,
2H), 2.72 (s, 3H), 2.40 (s, 3H). I-E-14 370.1 2.07 .sup.1H NMR (500
MHz, DMSO-d6) .delta. 10.86 (s, 1H), 9.50 (dd, J = 4.8, 2.5 Hz,
1H), 8.98-8.85 (m, 2H), 8.59 (d, J = 2.5 Hz, 1H), 7.77-7.65 (m,
2H), 6.87-6.67 (m, 2H), 2.38 (s, 3H). I-E-15 392.0 2.19 .sup.1H NMR
(500 MHz, DMSO-d6) .delta. 11.04 (s, 1H), 9.42-9.32 (m, 1H), 8.79
(s, 1H), 3.84 (s, 2H), 3.67 (s, 4H), 2.73 (s, 4H), 2.37 (s, 3H).
I-E-16 387.1 2.23 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.50 (s,
1H), 9.49 (dd, J = 4.8, 2.5 Hz, 1H), 8.42 (d, J = 2.5 Hz, 1H), 6.76
(s, 2H), 6.28 (d, J = 0.6 Hz, 1H), 3.57 (s, 3H), 2.33 (s, 3H), 2.24
(s, 3H). I-E-17 376.0 2.67 .sup.1H NMR (500 MHz, DMSO-d6) .delta.
10.85 (s, 1H), 9.90 (s, 1H), 9.57 (dd, J = 5.0, 2.5 Hz, 1H),
9.09-8.81 (m, 1H), 6.80 (s, 1H), 4.54 (d, J = 5.0 Hz, 1H), 3.63 (s,
1H), 3.29 (d, J = 15.4 Hz, 1H), 2.47 (s, 2H), 2.15 (s, 1H), 1.90
(d, J = 6.2 Hz, 1H). I-E-18 387.2 2.02 .sup.1H NMR (500 MHz,
DMSO-d6) .delta. 10.37 (s, 1H), 9.48 (dd, J = 4.8, 2.5 Hz, 1H),
8.48 (d, J = 2.5 Hz, 1H), 7.83 (s, 1H), 6.75 (s, 2H), 3.95 (s, 3H),
2.22 (s, 3H), 2.04 (s, 3H). I-E-19 405.3 2.07 .sup.1H NMR (500 MHz,
DMSO-d6) .delta. 11.05 (s, 1H), 9.55 (dd, J = 4.7, 2.5 Hz, 1H),
9.43 (s, 1H), 8.90-8.85 (m, 1H), 6.79 (s, 2H), 3.70 (s, 2H), 3.41
(d, J = 12.5 Hz, 2H), 3.05 (d, J = 12.8 Hz, 2H), 2.94 (d, J = 10.0
Hz, 2H), 2.75 (s, 3H), 2.44 (t, J = 11.5 Hz, 2H), 2.34 (s, 3H).
I-E-20 337.1 2.1 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 11.19 (s,
1H), 9.51 (dd, J = 4.8, 2.5 Hz, 1H), 8.91 (dd, J = 2.6, 0.5 Hz,
1H), 6.75 (s, 2H), 4.62 (s, 2H), 3.54 (s, 3H), 2.30 (s, 3H). I-E-21
362.1 2.74 .sup.1H NMR (500 MHz, DMSO-d6) .delta. 10.67 (s, 0H),
9.51 (dd, J = 4.8, 2.5 Hz, 0H), 8.92-8.80 (m, 0H), 3.26-3.07 (m,
1H), 2.33 (s, 1H), 2.12-1.99 (m, 1H).
Example 10
Cellular ATR Inhibition Assay
[0515] Compounds can be screened for their ability to inhibit
intracellular ATR using an immunofluorescence microscopy assay to
detect phosphorylation of the ATR substrate histone H2AX in
hydroxyurea treated cells. HT29 cells are plated at 14,000 cells
per well in 96-well black imaging plates (BD 353219) in McCoy's 5A
media (Sigma M8403) supplemented with 10% foetal bovine serum (JRH
Biosciences 12003), Penicillin/Streptomycin solution diluted 1:100
(Sigma P7539), and 2 mM L-glumtamine (Sigma G7513), and allowed to
adhere overnight at 37.degree. C. in 5% CO.sub.2. Compounds are
then added to the cell media from a final concentration of 25 .mu.M
in 3-fold serial dilutions and the cells are incubated at
37.degree. C. in 5% CO.sub.2. After 15 min, hydroxyurea (Sigma
H8627) is added to a final concentration of 2 mM.
[0516] After 45 min of treatment with hydroxyurea, the cells are
washed in PBS, fixed for 10 min in 4% formaldehyde diluted in PBS
(Polysciences Inc 18814), washed in 0.2% Tween-20 in PBS (wash
buffer), and permeabilised for 10 min in 0.5% Triton X-100 in PBS,
all at room temperature. The cells are then washed once in wash
buffer and blocked for 30 min at room temperature in 10% goat serum
(Sigma G9023) diluted in wash buffer (block buffer). To detect H2AX
phosphorylation levels, the cells are then incubated for 1 h at
room temperature in primary antibody (mouse monoclonal
anti-phosphorylated histone H2AX Ser139 antibody; Upstate 05-636)
diluted 1:250 in block buffer. The cells are then washed five times
in wash buffer before incubation for 1 h at room temperature in the
dark in a mixture of secondary antibody (goat anti-mouse Alexa
Fluor 488 conjugated antibody; Invitrogen A11029) and Hoechst stain
(Invitrogen H3570); diluted 1:500 and 1:5000, respectively, in wash
buffer. The cells are then washed five times in wash buffer and
finally 100 ul PBS is added to each well before imaging.
[0517] Cells are imaged for Alexa Fluor 488 and Hoechst intensity
using the BD Pathway 855 Bioimager and Attovision software (BD
Biosciences, Version 1.6/855) to quantify phosphorylated H2AX
Ser139 and DNA staining, respectively. The percentage of
phosphorylated H2AX-positive nuclei in a montage of 9 images at
20.times. magnification is then calculated for each well using BD
Image Data Explorer software (BD Biosciences Version 2.2.15).
Phosphorylated H2AX-positive nuclei are defined as Hoechst-positive
regions of interest containing Alexa Fluor 488 intensity at
1.75-fold the average Alexa Fluor 488 intensity in cells not
treated with hydroxyurea. The percentage of H2AX positive nuclei is
finally plotted against concentration for each compound and IC50s
for intracellular ATR inhibition are determined using Prism
software (GraphPad Prism version 3.0cx for Macintosh, GraphPad
Software, San Diego Calif., USA).
[0518] The compounds described herein can also be tested according
to other methods known in the art (see Sarkaria et al, "Inhibition
of ATM and ATR Kinase Activities by the Radiosensitizing Agent,
Caffeine: Cancer Research 59: 4375-5382 (1999); Hickson et al,
"Identification and Characterization of a Novel and Specific
Inhibitor of the Ataxia-Telangiectasia Mutated Kinase ATM" Cancer
Research 64: 9152-9159 (2004); Kim et al, "Substrate Specificities
and Identification of Putative Substrates of ATM Kinase Family
Members" The Journal of Biological Chemistry, 274(53): 37538-37543
(1999); and Chiang et al, "Determination of the catalytic
activities of mTOR and other members of the
phosphoinositide-3-kinase-related kinase family" Methods Mol. Biol.
281:125-41 (2004)).
Example 11
ATR Inhibition Assay
[0519] Compounds were screened for their ability to inhibit ATR
kinase using a radioactive-phosphate incorporation assay. Assays
were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM
MgCl.sub.2 and 1 mM DTT. Final substrate concentrations were 10
.mu.M [.gamma.-33P]ATP (3 mCi 33P ATP/mmol ATP, Perkin Elmer) and
800 .mu.M target peptide (ASELPASQPQPFSAKKK).
[0520] Assays were carried out at 25.degree. C. in the presence of
5 nM full-length ATR. An assay stock buffer solution was prepared
containing all of the reagents listed above, with the exception of
ATP and the test compound of interest. 13.5 .mu.L of the stock
solution was placed in a 96 well plate followed by addition of 2
.mu.L of DMSO stock containing serial dilutions of the test
compound (typically starting from a final concentration of 15 .mu.M
with 3-fold serial dilutions) in duplicate (final DMSO
concentration 7%). The plate was pre-incubated for 10 minutes at
25.degree. C. and the reaction initiated by addition of 15 .mu.L
[.gamma.-33P]ATP (final concentration 10 .mu.M).
[0521] The reaction was stopped after 24 hours by the addition of
30 .mu.L 0.1M phosphoric acid containing 2 mM ATP. A multiscreen
phosphocellulose filter 96-well plate (Millipore, Cat no.
MAPHNOB50) was pretreated with 100 .mu.L 0.2M phosphoric acid prior
to the addition of 45 .mu.L of the stopped assay mixture. The plate
was washed with 5.times.200 .mu.L 0.2M phosphoric acid. After
drying, 100 .mu.L Optiphase `SuperMix` liquid scintillation
cocktail (Perkin Elmer) was added to the well prior to
scintillation counting (1450 Microbeta Liquid Scintillation
Counter, Wallac).
[0522] After removing mean background values for all of the data
points, Ki(app) data were calculated from non-linear regression
analysis of the initial rate data using the Prism software package
(GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, San
Diego Calif., USA).
[0523] Table 6, below, shows the ATR Inhibition Ki values of
compounds of the disclosure. Compounds with a Ki value of <0.01
.mu.M are marked with "+++." Compounds with a Ki value>0.01
.mu.M but <1 .mu.M are marked with "++." Compounds with a Ki
value>1 .mu.M but <5 .mu.M are marked with "+."
TABLE-US-00011 TABLE 6 Cmpd. # ATR Ki I-A-1 + II-A-2 ++ I-A-3 ++
I-A-4 ++ I-A-5 ++ I-A-6 + I-A-7 -- I-A-8 ++ I-A-9 + I-A-10 ++
I-A-11 + I-A-12 ++ I-A-13 ++ I-A-14 ++ I-A-15 + I-A-16 + I-A-17 ++
I-A-18 + I-A-19 + I-A-20 + I-B-1 ++ I-B-2 ++ I-B-3 ++ I-B-4 ++
I-B-5 ++ I-B-6 ++ I-B-7 ++ I-B-8 ++ I-B-9 ++ I-B-10 ++ I-B-11 ++
I-B-12 ++ I-B-13 ++ I-B-14 ++ I-B-15 ++ I-B-16 + I-B-17 -- I-B-18
-- I-B-19 -- I-B-20 ++ I-B-21 ++ I-B-22 +++ I-B-23 ++ I-B-24 ++
I-C-1 ++ I-C-2 ++ I-C-3 ++ I-C-4 ++ I-C-5 ++ I-C-6 ++ I-C-7 ++
I-C-8 ++ I-C-9 ++ I-C-10 +++ I-C-11 ++ I-D-1 +++ I-D-2 ++ I-D-3 ++
I-D-4 ++ I-D-5 ++ I-D-6 ++ I-E-1 +++ I-E-2 ++ I-E-3 ++ I-E-4 +++
I-E-5 ++ I-E-6 ++ I-E-7 +++ I-E-8 ++ I-E-9 ++ I-E-10 +++ I-E-11 ++
I-E-12 ++ I-E-13 ++ I-E-14 ++ I-E-15 ++ I-E-16 ++ I-E-17 +++ I-E-18
++ I-E-19 +++ I-E-20 ++ I-E-21 ++
Example 12
Cisplatin Sensitization Assay
[0524] Compounds can be screened for their ability to sensitize
HCT116 colorectal cancer cells to Cisplatin using a 96 h cell
viability (MTS) assay. HCT116 cells, which possess a defect in ATM
signaling to Cisplatin (see, Kim et al.; Oncogene 21:3864 (2002);
see also, Takemura et al.; JBC 281:30814 (2006)) are plated at 470
cells per well in 96-well polystyrene plates (Costar 3596) in 150
.mu.l of McCoy's 5A media (Sigma M8403) supplemented with 10%
foetal bovine serum (JRH Biosciences 12003),
Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539), and 2
mM L-glumtamine (Sigma G7513), and allowed to adhere overnight at
37.degree. C. in 5% CO.sub.2. Compounds and Cisplatin are then both
added simultaneously to the cell media in 2-fold serial dilutions
from a top final concentration of 10 .mu.M as a full matrix of
concentrations in a final cell volume of 200 .mu.l, and the cells
are then incubated at 37.degree. C. in 5% CO.sub.2. After 96 h, 40
.mu.l of MTS reagent (Promega G358a) is added to each well and the
cells are incubated for 1 h at 37.degree. C. in 5% CO.sub.2.
Finally, absorbance is measured at 490 nm using a SpectraMax Plus
384 reader (Molecular Devices) and the concentration of compound
required to reduce the IC50 of Cisplatin alone by at least 3-fold
(to 1 decimal place) can be reported.
[0525] Table 7, below, shows the Cisplatin sensitization values of
compounds of the disclosure. Compounds with a Cisplatin
sensitization value of <0.02 .mu.M are marked with "+++."
Compounds with a Cisplatin sensitization value>0.02 .mu.M but
<0.2 .mu.M are marked with "++." Compounds with a Cisplatin
sensitization value>0.2 .mu.M but <5 .mu.M are marked with
"+."
TABLE-US-00012 TABLE 7 Cisplatin Sensitization Cmpd. # Assay I-A-1
+ I-A-2 + I-A-3 ++ I-A-4 ++ I-A-5 ++ I-A-6 ++ I-A-7 -- I-A-8 +++
I-A-9 + I-A-10 + I-A-11 ++ I-A-12 ++ I-A-13 ++ I-A-14 ++ I-A-15 +
I-A-16 + I-A-17 ++ I-A-18 + I-A-19 + I-A-20 + I-B-1 ++ I-B-2 ++
I-B-3 ++ I-B-4 +++ I-B-5 ++ I-B-6 +++ I-B-7 +++ I-B-8 ++ I-B-9 +++
I-B-10 ++ I-B-11 ++ I-B-12 ++ I-B-13 ++ I-B-14 +++ I-B-15 ++ I-B-16
-- I-B-17 -- I-B-18 -- I-B-19 -- I-B-20 +++ I-B-21 +++ I-B-22 +++
I-B-23 ++ I-B-24 -- I-C-1 ++ I-C-2 ++ I-C-3 ++ I-C-4 -- I-C-5 ++
I-C-6 ++ I-C-7 +++ I-C-8 +++ I-C-9 ++ I-C-10 +++ I-C-11 ++ I-D-1
+++ I-D-2 +++ I-D-3 +++ I-D-4 ++ I-D-5 ++ I-D-6 +++ I-E-1 +++ I-E-2
+++ I-E-3 +++ I-E-4 +++ I-E-5 +++ I-E-6 +++ I-E-7 +++ I-E-8 +++
I-E-9 +++ I-E-10 +++ I-E-11 ++ I-E-12 ++ I-E-13 +++ I-E-14 +++
I-E-15 +++ I-E-16 +++ I-E-17 +++ I-E-18 +++ I-E-19 +++ I-E-20 +++
I-E-21 ++
Example 13
Single Agent HCT116 Activity
[0526] Compounds can be screened for single agent activity against
HCT116 colorectal cancer cells using a 96 h cell viability (MTS)
assay. HCT116 are plated at 470 cells per well in 96-well
polystyrene plates (Costar 3596) in 150 .mu.l of McCoy's 5A media
(Sigma M8403) supplemented with 10% foetal bovine serum (JRH
Biosciences 12003), Penicillin/Streptomycin solution diluted 1:100
(Sigma P7539), and 2 mM L-glumtamine (Sigma G7513), and allowed to
adhere overnight at 37.degree. C. in 5% CO.sub.2. Compounds are
then added to the cell media in 2-fold serial dilutions from a top
final concentration of 10 .mu.M as a full matrix of concentrations
in a final cell volume of 200 .mu.l, and the cells are then
incubated at 37.degree. C. in 5% CO.sub.2. After 96 h, 40 .mu.l of
MTS reagent (Promega G358a) is added to each well and the cells are
incubated for 1 h at 37.degree. C. in 5% CO.sub.2. Finally,
absorbance is measured at 490 nm using a SpectraMax Plus 384 reader
(Molecular Devices) and IC50 values can be calculated.
Example 14
ATR-Complex Inhibition Assay
[0527] Compounds were screened for their ability to inhibit ATR
kinase, in the presence of partner proteins ATRIP, CLK2 and TopBP1,
using a radioactive-phosphate incorporation assay. Assays were
carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM
MgCl.sub.2 and 1 mM DTT. Final substrate concentrations were 10
.mu.M [g-33P]ATP (3.5 .mu.Ci 33P ATP/nmol ATP, Perkin Elmer,
Massachusetts, USA) and 800 .mu.M target peptide
(ASELPASQPQPFSAKKK, Isca Biochemicals, Cambridgeshire, UK).
[0528] Assays were carried out at 25.degree. C. in the presence of
4 nM full-length ATR, 40 nM full-length ATRIP, 40 nM full-length
CLK2 and 600 nM TopBP1(A891-S1105). An enzyme stock buffer solution
was prepared containing all of the reagents listed above, with the
exception of target peptide, ATP and the test compound of interest.
This enzyme stock was pre-incubated for 30 minutes at 25.degree. C.
8.5 .mu.L of the enzyme stock solution was placed in a 96-well
plate followed by addition of 5 .mu.l of target peptide and 2 .mu.L
of DMSO stock containing serial dilutions of the test compound
(typically starting from a final concentration of 1.5 .mu.M with
2.5-fold serial dilutions) in duplicate (final DMSO concentration
7%). The plate was pre-incubated for 10 minutes at 25.degree. C.
and the reaction initiated by addition of 15 .mu.L [g-33P]ATP
(final concentration 10 .mu.M).
[0529] The reaction was stopped after 20 hours by the addition of
30 .mu.L 0.3 M phosphoric acid containing 2 mM ATP. A
phosphocellulose filter 96-well plate (Multiscreen HTS MAPHNOB50,
Merck-Millipore, Massachusetts, USA) was pretreated with 100 .mu.L
0.1 M phosphoric acid prior to the addition of 45 .mu.L of the
stopped assay mixture. The plate was washed with 5.times.200 .mu.L
0.1 M phosphoric acid. After drying, 50 .mu.L Optiphase `SuperMix`
liquid scintillation cocktail (Perkin Elmer, Massachusetts, USA)
was added to the well prior to scintillation counting (Wallac 1450
Microbeta Liquid Scintillation Counter, Perkin Elmer,
Massachusetts, USA).
[0530] After removing mean background values for all of the data
points, Ki(app) data were calculated from non-linear regression
analysis of the initial rate data using the Prism software package
(GraphPad Prism version 6.0c for Macintosh, GraphPad Software Inc.,
San Diego, USA).
[0531] Table 8, below, shows the ATR Inhibition Ki values of
compounds of the disclosure. Compounds with a Ki value of <0.01
.mu.M are marked with "+++." Compounds with a Ki value>0.01
.mu.M but <1 .mu.M are marked with "++." Compounds with a Ki
value>1 .mu.M but <5 .mu.M are marked with "+."
TABLE-US-00013 TABLE 8 Cmpd. # ATR Ki I-B-13 +++ I-C-1 +++
[0532] While we have described a number of embodiments of this
invention, it is apparent that our basic examples may be altered to
provide other embodiments that utilize the compounds, methods, and
processes of this invention. Therefore, it will be appreciated that
the scope of this invention is to be defined by the appended claims
rather than by the specific embodiments that have been represented
by way of example herein.
Sequence CWU 1
1
1117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ala Ser Glu Leu Pro Ala Ser Gln Pro Gln Pro Phe
Ser Ala Lys Lys 1 5 10 15 Lys
* * * * *
References