U.S. patent application number 12/551623 was filed with the patent office on 2010-04-29 for compounds useful for inhibiting chk1.
Invention is credited to Laurence Edward Burgess, Adam Wade Cook, Scott Douglas Cowen, John Joseph Gaudino, Kathleen S. Keegan, Edward A. Kesicki.
Application Number | 20100105683 12/551623 |
Document ID | / |
Family ID | 23042649 |
Filed Date | 2010-04-29 |
United States Patent
Application |
20100105683 |
Kind Code |
A1 |
Keegan; Kathleen S. ; et
al. |
April 29, 2010 |
COMPOUNDS USEFUL FOR INHIBITING CHK1
Abstract
Aryl- and heteroaryl-substituted urea compounds useful in the
treatment of diseases and conditions related to DNA damage or
lesions in DNA replication are disclosed. Methods of making the
compounds, and their use as therapeutic agents, for example, in
treating cancer and other diseases characterized by defects in DNA
replication, chromosome segregation, or cell division also are
disclosed.
Inventors: |
Keegan; Kathleen S.; (Mercer
Island, WA) ; Kesicki; Edward A.; (Bothell, WA)
; Gaudino; John Joseph; (Longmont, CO) ; Cook;
Adam Wade; (Longmont, CO) ; Cowen; Scott Douglas;
(Longmont, CO) ; Burgess; Laurence Edward;
(Boulder, CO) |
Correspondence
Address: |
ELI LILLY & COMPANY
PATENT DIVISION, P.O. BOX 6288
INDIANAPOLIS
IN
46206-6288
US
|
Family ID: |
23042649 |
Appl. No.: |
12/551623 |
Filed: |
September 1, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11115993 |
Apr 27, 2005 |
7608618 |
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12551623 |
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10087715 |
Mar 1, 2002 |
7067506 |
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11115993 |
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60273124 |
Mar 2, 2001 |
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Current U.S.
Class: |
514/235.8 ;
514/252.13; 514/255.05; 514/255.06 |
Current CPC
Class: |
C07D 401/12 20130101;
C07D 239/42 20130101; C07D 453/02 20130101; C07D 241/20 20130101;
C07D 213/40 20130101; C07D 251/22 20130101; A61K 31/498 20130101;
A61P 29/02 20180101; C07D 213/77 20130101; C07D 257/06 20130101;
C07D 233/56 20130101; A61K 2300/00 20130101; C07D 241/44 20130101;
C07D 213/75 20130101; C07D 253/07 20130101; C07D 401/14 20130101;
A61K 31/498 20130101; C07D 409/12 20130101; C07D 409/14 20130101;
A61P 35/02 20180101; C07D 239/47 20130101; C07D 241/12 20130101;
C07D 249/08 20130101; C07D 403/12 20130101; C07D 215/34 20130101;
C07D 213/30 20130101; A61K 45/06 20130101; C07D 231/40 20130101;
C07D 231/12 20130101; C07D 277/48 20130101; C07D 403/06 20130101;
C07D 405/12 20130101; A61K 31/4965 20130101; A61K 31/4965 20130101;
A61K 2300/00 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/235.8 ;
514/255.06; 514/255.05; 514/252.13 |
International
Class: |
A61K 31/5377 20060101
A61K031/5377; A61K 31/4965 20060101 A61K031/4965; A61K 31/497
20060101 A61K031/497; A61K 31/496 20060101 A61K031/496; A61P 35/00
20060101 A61P035/00 |
Claims
1-30. (canceled)
31. A method of treating a cancer selected from the group
consisting of a colorectal cancer, a breast cancer, a leukemia, a
melanoma, a renal cell carcinoma, an ovarian cancer, a lung
carcinoma, a cervical cancer, a colon cancer, and a prostate cancer
which comprises administering to a patient in need thereof a
therapeutically effective amount of a compound having a formula
##STR00467## wherein: Y' is O or S; W' is ##STR00468## optionally
substituted with from one to four substituents selected from the
group consisting of C.sub.1-6alkyl, aryl, N(R.sup.7).sub.2,
OR.sup.7, N.sub.3, CN, C(O)R.sup.7, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.2).sub.2, halo, and ##STR00469## Z' is
selected from the group consisting of: ##STR00470## wherein Q' is
OR.sup.7; J' is selected from the group consisting of CR.sup.8,
NR.sup.8, O, and S; K' is selected from the group consisting of
CR.sup.9, NR.sup.9, O, and S; L' is selected from the group
consisting of CR.sup.10, NR.sup.10, O, and S; M' is selected from
the group consisting of CR.sup.11, NR.sup.11, O, and S; and
provided when Z' is a six-membered ring J', K', L', and M' cannot
be O or S; wherein: R.sup.7 is
C.sub.1-3alkyleneC.sub.3-8heterocycloalkyl; R.sup.8, R.sup.9, and
R.sup.10 are each independently selected from the group consisting
of hydrogen, halo, optionally substituted C.sub.1-6alkyl,
C.sub.2-6alkenyl, OCF.sub.3, NO.sub.2, CN, NC, N(R.sup.7).sub.2,
OR.sup.7, CO.sub.2R.sup.7, C(O)N(R.sup.7).sub.2, C(O)R.sup.7,
N(R.sup.13)C(O)R.sup.7, N(R.sup.13)C(O)OR.sup.7,
N(R.sup.7)C(O)OR.sup.7, N(R.sup.7)C(O)C.sub.1-3alkyleneC(O)R.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneC(O)OR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneOR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.7, CF.sub.3,
C.sub.1-3alkyleneN(R.sup.12)SO.sub.2aryl,
C.sub.1-3alkyleneN(R.sup.12)SO.sub.2heteroaryl,
C.sub.1-3alkyleneOC.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.12)C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.12)C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneN(R.sup.12)C(O)R.sup.7,
C.sub.1-3alkyleneN(R.sup.12)C(O)C.sub.1-3alkyleneOR.sup.2,
C.sub.1-3alkyleneN(R.sup.12)C(O)aryl,
C.sub.1-3alkyleneN(R.sup.12)C(O)C.sub.1-3alkyleneN(R.sup.12).sub.2,
C.sub.1-3alkyleneN(R.sup.12)C(O)heteroaryl,
C.sub.1-3alkyleneOR.sup.7, and SR.sup.7, wherein R.sup.7 is as
defined above; R.sup.11 is selected from the group consisting of
hydrogen, optionally substituted C.sub.1-6alkyl, and halo; R.sup.12
is selected from the group consisting of hydrogen, C.sub.1-6alkyl,
cycloalkyl, aryl, heteroaryl, C.sub.1-3alkylenearyl, and
SO.sub.2C.sub.1-6alkyl, or two R.sup.12 groups are taken together
to form an optionally substituted 3- to 6-membered ring; and
R.sup.13 is hydrogen; or a pharmaceutically acceptable salt
thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application Ser. No. 60/273,124, filed Mar. 2, 2001.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to compounds useful for
inhibiting enzymes that maintain and repair the integrity of
genetic material. More particularly, the present invention relates
to a series of aryl- and heteroaryl-substituted urea compounds,
methods of making the compounds, and their use as therapeutic
agents, for example, in treating cancer and other diseases
characterized by defects in deoxyribonucleic acid (DNA)
replication, chromosome segregation, or cell division.
BACKGROUND OF THE INVENTION
[0003] An important and significant goal in healthcare is to
discover and make available safer and more effective drugs for the
treatment of cancer. Most chemotherapeutic agents act by disrupting
DNA metabolism, DNA synthesis, DNA transcription, or microtubule
spindle function, or by perturbing chromosomal structural integrity
by introducing DNA lesions. These processes affect both normal and
tumor cells. The maintenance of DNA integrity is essential to cell
viability in normal cells, therefore, anticancer drugs have the
lowest therapeutic index of any drug class.
[0004] An individual cell creates an exact copy of its chromosomes,
and then segregates each copy into two cells by a process called
mitosis. The mitotic cycle can be divided into three major events:
DNA replication, chromosome segregation, and cell division. Cells
have sensing mechanisms to maintain the order of these steps with
respect to one another and to ensure that each step is executed
with high fidelity. The sensing mechanisms for these processes are
referred to as "checkpoints" in L. H. Hartwell et al., Science,
Nov. 3, 1989, 246(4930):629-34.
[0005] Cell cycle checkpoints have been reported to comprise at
least three distinct classes of polypeptides. Each class of
polypeptides acts sequentially in response to cell cycle signals or
defects in chromosomal mechanisms (Carr, (1996) Science,
271:314-315). One family of proteins detects or senses DNA damage
or abnormalities in the cell cycle. These sensors include
Ataxia-Telangiectasia Mutated (Atm) and Ataxia-Telangiectasia
Rad-related (Atr) (Keegan et al., (1996) Genes Dev., 10:2423-2437).
Another class of polypeptides amplify and transmit the signal
detected by the detector and is exemplified by Rad53 (Allen et al.
(1994) Genes Dev., 8:2416-2488) and Chk1. In addition, cell cycle
effectors, such as p53, mediate a cellular response, including, for
example, arrest of mitosis and/or meiosis and apoptosis.
[0006] DNA damage can be induced by drugs, radiation, or can be
spontaneously generated during the course of normal metabolism. DNA
damage checkpoints ensure that cells with unrepaired DNA lesions do
not progress into the DNA synthesis phase or mitosis until
chromosomal lesions have been removed. Cell cycle arrest can
enhance the opportunity for DNA repair and increase the fidelity of
cell division. DNA damage can be recognized throughout the cell
cycle. Checkpoints ensure that the growth of cells is arrested at
multiple cell cycle phases. As a result, multiple cell cycle
signaling pathways may result during sensitization of cells to DNA
damaging agents.
[0007] Much of the current understanding of the function of cell
cycle checkpoints has been derived from the study of tumor-derived
cell lines. In many cases, tumor cells have lost key cell cycle
checkpoints (Hartwell et al., Science, Dec. 16, 1994; 266(5192):
1821-8). It has been reported that a key step in the evolution of
cells to a neoplastic state is the acquisition of mutations that
inactivate cell cycle checkpoint pathways, such as p53. (Weinberg,
R. A. (1995) Cell 81:323-330; Levine, A. J. (1997) Cell 88:
3234-331). Loss of these cell cycle checkpoints results in the
inappropriate cycling of tumor cells in response to DNA damaging
agents. When faced with cellular stresses, such as DNA damage, and
cell cycle events with decreased fidelity, tumor cells have
difficulty altering the kinetics of cell cycle progression.
Therefore, inhibition and disruption of additional DNA damage
checkpoint pathways may further sensitize tumor cells to anticancer
treatments, such as radiation and chemotherapy.
[0008] Noncancerous tissue, which has intact cell cycle
checkpoints, typically is insulated from temporary disruption of a
single checkpoint pathway. Tumor cells, however, have defects in
pathways controlling cell cycle progression such that the
perturbation of additional checkpoints, for example, the DNA damage
checkpoint, renders them particularly sensitive to DNA damaging
agents. For example, tumor cells that contain mutant p53 are
defective both in the G1 DNA damage checkpoint and in the ability
to maintain the G2 DNA damage checkpoint. (Bunz et al., Science,
Nov. 20, 1998; 282(5393): 1497-501; Levine). Checkpoint inhibitors
that target initiation of the G2 checkpoint or the S phase
checkpoint are expected to further cripple the ability of these
tumor cells to repair DNA damage and, therefore, selectively kill
them over normal cells. Therefore, checkpoint inhibitors are
expected to enhance the therapeutic index, which is a measure of
the probability of tumor control relative to the probability of
toxicity to normal tissue, of both radiation and systemic
chemotherapy.
[0009] The ability of checkpoint inhibitors to enhance the
therapeutic index may be dependent upon tumor type. Tumors with
cell cycle defects complementary to the DNA damage checkpoint
pathways may be most sensitive to inhibitor drug treatment. In
contrast, DNA-PK inhibitors, another distinct class of potential
therapeutic agents, are expected to sensitize tumors independently
of cell type. A systematic approach of applying checkpoint
inhibitors and DNA-PK inhibitors also may be effective in the
treatment of metastatic diseases that radiation therapy cannot
target.
[0010] The checkpoint proteins Atm and Atr are hypothesized to
initiate a signal transduction pathway leading to cell cycle arrest
in the presence of DNA damage or any block to DNA replication. Atm
has been shown to play a role in a DNA damage checkpoint in
response to ionizing radiation (IR). Patients lacking functional
Atm develop the disease Ataxia-Telangiectasia (A-T). Symptoms of
A-T include extreme sensitivity to ionizing radiation (IR),
cerebellar degeneration, oculotaneous telangiectasias, gonadal
deficiencies, immunodeficiencies and increased risk of cancer
(Shiloh, Eur. J. Hum. Genet 1995; 3(2):116-38). Fibroblasts derived
from these patients are thought to have defects in G1, S, and G2
checkpoints and are defective in their response to IR (Kastan et
al., Cell, Nov. 13, 1992; 71(4): 587-97; Scott et al., Int. J.
Radiat. Biol., December, 1994; 66(6 Suppl): S157-63; and Beamish et
al., J. Biol. Chem. Aug. 26, 1993; 271(34):20486-93). Therefore,
Atm may sense double-strand DNA damage caused by IR and
radiomimetic drugs, and signal the cell cycle to arrest, such that
damage can be repaired.
[0011] Atr is a checkpoint protein stimulated by agents that cause
double strand DNA breaks, single strand DNA breaks, and agents that
block DNA radiation. Overexpression of Atr in muscle cells on
iso-chromosome 3q results in a block to differentiation, abnormal
centrosome numbers, chromosome instability, and abolishes the G1
arrest in response to IR (Smith et al., Nat. Genet., May 1998;
19(1): 39-46). Overexpression of a kinase inactive, dominant
negative mutant of Atr sensitizes cells to IR, ultraviolet light
(UV), MMS, and cisplatin (Cliby et al., EMBO J. Jan. 2, 1998,
17(1):159-69 and Wright et al., Proc. Nat'l Acad. Sci. U.S.A., Jun.
23, 1998; 95(13):7445-50). Cells containing overexpressed, mutant
strain Atr also fail to arrest in G2 in response to IR. In
addition, Atr is associated with chromosomes in meiotic cells where
DNA breaks and abnormal DNA structures persist as a result of the
process of meiotic recombination (Keegan et al., Genes Dev. Oct. 1,
1996; 10(19): 433-37). Atr, like Atm, also senses DNA damage and
agents that block DNA replication, as well as initiates a cell
cycle arrest at G2 and S for DNA repair.
[0012] Chk1 is hypothesized to lie downstream from protein kinases
Atm and/or Atr in the DNA damage checkpoint signal transduction
pathway. (See, Sanchez et al., Science, 1997; 277:1497-1501; U.S.
Pat. No. 6,218,109.) In mammalian cells, Chk1 is phosphorylated in
response to agents that cause DNA damage including IR, UV, and
hydroxyurea (Sanchez et al., 1997; Lui et al., Genes Dev. 2000;
14:1448-1459). The phosphorylation and activation of Chk1 in
mammalian cells is dependent on Atm (Chen et al., 1999) and Atr
(Lui et al., 2000). In the yeast S. pombe, Chk1 also appears to be
involved in the response to IR and blocks to replication (Boddy et
al., 1998; Walworth et al., 1993). Furthermore, Chk1 has been shown
to phosphorylate both weel (O'Connell et al., EMBO J. 1997;
16:545-554) and Pds1 (Sanchez et al., Science 1999; 286:1166-1171)
gene products known to be important in cell cycle control. These
studies demonstrate that mammalian Chk1 plays a role in both the
Atm-dependent DNA damage checkpoint leading to arrest at S phase.
However, a role for Chk1 in the S phase replication checkpoint in
mammalian cells has yet to be elucidated. Interestingly, Chk1
knockout mice are embryonically lethal, thereby suggesting a role
for Chk1 in a developing organism in addition to its role in DNA
damage checkpoints.
[0013] Chk1 may invoke a G2 arrest by phosphorylating and
inactivating Cdc25C, the dual specificity phosphatase that normally
dephosphorylates cyclin B/cdc2 as cells progress into mitosis
(Fernery et al., Science, Sep. 5, 1997; 277(5331): 1495-7; Sanchez
et al.; Matsuoka et al.; and Blasina et al., Curr. Biol., Jan. 14,
1999; 9(1):1-10). This mechanism of regulation of Cdc2 activity
stimulates cell cycle arrest to prevent cells from entering mitosis
in the presence of DNA damage or unreplicated DNA.
SUMMARY OF THE INVENTION
[0014] The present invention is directed to potent and selective
chemosensitizing agents useful in the treatment of diseases and
conditions related to DNA damage or lesions in DNA replication. The
present compounds are inhibitors of the checkpoint kinase Chk1. In
particular, aryl- and heteroaryl-substituted urea compounds have
demonstrated significant activity for inhibiting Chk1.
[0015] In one aspect, the present invention is directed to a method
of inhibiting checkpoint kinase Chk1 comprising the step of
administering a compound of formula (I), or a composition
containing the same, to an individual. Compounds of formula (I)
have a structural formula:
##STR00001##
wherein:
[0016] X.sup.1 is null, --O--, --S--, --CH.sub.2--, or
--N(R.sup.1)--;
[0017] X.sup.2 is --O--, --S--, or --N(R.sup.1)--;
[0018] Y is O or S; or .dbd.Y represents two hydrogen atoms
attached to a common
[0019] W is selected from the group consisting of heteroaryl, aryl,
heterocycloalkyl, cycloalkyl, and C.sub.1-3 alkyl substituted with
a heteroaryl or aryl group; and
[0020] Z is selected from the group consisting of hydro, aryl, and
heteroaryl;
[0021] wherein said aryl groups of W and Z are optionally
substituted with one to four substituents represented by R.sup.2,
said heteroaryl groups of W and Z are optionally substituted with
one to four substituents represented by R.sup.5, and said
heterocycloalkyl and cycloalkyl groups of W are optionally
substituted with one to two substituents represented by
R.sup.6;
[0022] R.sup.1 is selected from the group consisting of hydrogen,
C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl, and aryl;
[0023] R.sup.2 is selected from the group consisting of halogen,
C.sub.1-6alkyl, C.sub.2-6alkenyl, OCF.sub.3, NO.sub.2, CN, NC,
N(R.sup.3).sub.2, OR.sup.3, CO.sub.2R.sup.3, C(O)N(R.sup.3).sub.2,
C(O)R.sup.3, N(R.sup.1)COR.sup.3, N(R.sup.1)C(O)OR.sup.3,
N(R.sup.3)C(O)OR.sup.3,
N(R.sup.3)C(O)--C.sub.1-3--alkylene-C(O)R.sup.3,
N(R.sup.3)C(O)--C.sub.1-3alkylene-C(O)OR.sup.3,
N(R.sup.3)C(O)--C.sub.1-3alkylene-OR.sup.3,
N(R.sup.3)C(O)--C.sub.1-3alkylene-NHC(O)OR.sup.3,
N(R.sup.3)C(O)--C.sub.1-3alkylene-SO.sub.2NR.sup.3,
C.sub.1-3alkylene-OR.sup.3, and SR.sup.3,
[0024] R.sup.3 is selected from the group consisting of hydro,
C.sub.1-6alkyl, C.sub.2-6alkenyl, cycloalkyl, aryl, heteroaryl,
SO.sub.2R.sup.4, C.sub.1-6alkyl substituted with one or more of
halo, hydroxy, aryl, heteroaryl, heterocycloalkyl,
N(R.sup.4).sub.2, and SO.sub.2R.sup.4, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneC.sub.3-8heterocycloalkyl,
C.sub.1-3alkyleneSO.sub.2aryl, optionally substituted
C.sub.1-3alkyleneN(R.sup.4).sub.2, OCF.sub.3,
C.sub.1-3alkyleneN(R.sup.4).sub.3.sup.+, C.sub.3-8heterocycloalkyl,
and CH(C.sub.1-3alkyleneN(R.sup.4).sub.2).sub.2, or two R.sup.3
groups are taken together to form an optionally substituted 3- to
6-membered aliphatic ring;
[0025] R.sup.4 is selected from the group consisting of hydro,
C.sub.1-6alkyl, cycloalkyl, aryl, heteroaryl,
C.sub.1-3alkylenearyl, and SO.sub.2C.sub.1-6alkyl, or two R.sup.4
groups are taken together to form an optionally substituted 3- to
6-membered ring;
[0026] R.sup.5 is selected from the group consisting of
C.sub.1-6alkyl, aryl, N(R.sup.3).sub.2, OR.sup.3, halo, N.sub.3,
CN, C.sub.1-3alkylenearyl, C.sub.1-3alkyleneN(R.sup.3).sub.2,
C(O)R.sup.3, and
##STR00002##
[0027] R.sup.6 is selected from the group consisting of halo and
C.sub.1-6alkyl,
[0028] and pharmaceutically acceptable salts or solvates
thereof.
[0029] In another aspect, the present invention is directed to
aryl- and heteroaryl-disubstituted urea compounds having a
structural formula (II)
##STR00003##
[0030] wherein Y' is O or S;
[0031] W' is selected from the group consisting of
##STR00004##
optionally substituted with from one to four substituents selected
from the group consisting of C.sub.1-6alkyl, aryl,
N(R.sup.7).sub.2, OR.sup.7, N.sub.3, CN, C(O)R.sup.7,
C.sub.1-3alkylenearyl, C.sub.1-3alkyleneN(R.sup.12).sub.2,
##STR00005##
[0032] Z' is selected from the group consisting of:
##STR00006##
wherein:
[0033] Q' is selected from the group consisting of hydrogen,
OR.sup.7, SR.sup.7, and N(R.sup.7).sub.2;
[0034] J' is selected from the group consisting of C--R.sup.8,
N--R.sup.8, O, and S;
[0035] K' is selected from the group consisting of C--R.sup.9,
N--R.sup.9, O, and S;
[0036] L' is selected from the group consisting of C--R.sup. ,
N--R.sup.10, O, and S;
[0037] M' is selected from the group consisting of C--R.sup.11,
N--R.sup.11, O, and S;
wherein:
[0038] R.sup.7, independently, is selected from the group
consisting of hydro, C.sub.1-6alkyl, C.sub.2-6alkenyl, cycloalkyl,
aryl, heteroaryl, SO.sub.2R.sup.12, C.sub.1-6alkyl substituted with
one or more of halo, hydroxy, aryl, heteroaryl, heterocycloalkyl,
N(R.sup.12).sub.2, and SO.sub.2R.sup.12, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneC.sub.3-8heterocycloalkyl,
C.sub.1-3alkyleneSO.sub.2aryl, optionally substituted
C.sub.1-3alkyleneN(R.sup.12).sub.2, OCF.sub.3,
C.sub.1-3alkyleneN(R.sup.12).sub.3.sup.+,
C.sub.3-8heterocycloalkyl, and
CH(C.sub.1-3alkyleneN(R.sup.12).sub.2).sub.2, or two R.sup.7 groups
are taken together to form an optionally substituted 3- to
6-membered aliphatic ring;
[0039] R.sup.8, R.sup.9, and R.sup.10 are each independently
selected from the group consisting of null, hydro, halo, optionally
substituted C.sub.1-6 alkyl, C.sub.2-6alkenyl, OCF.sub.3, NO.sub.2,
CN, NC, N(R.sup.7).sub.2, OR.sup.7, CO.sub.2R.sup.7,
C(O)N(R.sup.7).sub.2, C(O)R.sup.7, N(R.sup.13)COR.sup.7,
N(R.sup.13)C(O)OR.sup.7, N(R.sup.7)C(O)OR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneC(O)R.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkylene C(O)OR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneOR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.7, CF.sub.3,
C.sub.1-3alkyleneN(R.sup.12)SO.sub.2aryl,
C.sub.1-3alkyleneN(R.sup.12)SO.sub.2heteroaryl,
c.sub.1-3alkyleneOC.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.12)C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.12)C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneN(R.sup.12)C(O)R.sup.7,
C.sub.1-3alkyleneN(R.sup.12)C(O)C.sub.1-3alkyleneOR.sup.2,
C.sub.1-3alkyleneN(R.sup.12)C(O)aryl,
C.sub.1-3alkyleneN(R.sup.12)C(O)C.sub.1-3alkyleneN(R.sup.12).sub.2,
C.sub.1-3alkyleneN(R.sup.12)C(O)heteroaryl,
C.sub.1-3alkyleneOR.sup.7, and SR.sup.7, wherein R.sup.7 is as
defined above;
[0040] R.sup.11 is selected from the group consisting of null,
hydro, C.sub.1-6alkyl, and halo;
[0041] R.sup.12 is selected from the group consisting of hydro,
C.sub.1-6alkyl, cycloalkyl, aryl, and SO.sub.2C.sub.1-6alkyl, or
two R.sup.12 groups are taken together to form a 3- to 6-membered
ring; and
[0042] R.sup.13 is selected from the group consisting of hydrogen,
C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl, and aryl;
[0043] provided that when Q' is hydrogen or OCH.sub.3, at least one
of R.sup.8, R.sup.9, and R.sup.10 is not selected from hydrogen,
CH.sub.3, OCH.sub.3, or halo,
[0044] and pharmaceutically acceptable salts or solvates
thereof.
[0045] Another aspect of the present invention relates to
carbamido-substituted heteroaryl groups having the structural
formula (Ill)
##STR00007##
[0046] wherein W''' is selected from the group consisting of
heteroaryl, aryl, heterocycloalkyl, cycloalkyl, and C.sub.1-3 alkyl
substituted with a heteroaryl or aryl group;
[0047] wherein said aryl groups are optionally substituted with one
to four substituents represented by R.sup.14, said heteroaryl
groups are optionally substituted with one to four substituents
represented by R.sup.18, and said heterocycloalkyl and cycloalkyl
groups are optionally substituted with one to two substituents
represented by R.sup.19;
[0048] R.sup.14 is selected from the group consisting of halo,
C.sub.1-6alkyl, C.sub.2-6alkenyl, OCF.sub.3, NO.sub.2, CN, NC,
N(R.sup.16).sub.2, OR.sup.16, CO.sub.2R.sup.16,
C(O)N(R.sup.16).sub.2, C(O)R.sup.16, N(R.sup.15)COR.sup.16,
N(R.sup.15)C(O)OR.sup.16, N(R.sup.16)C(O)OR.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneC(O)R.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneC(O)OR.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneOR.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.16,
C.sub.1-3alkyleneOR.sup.16, and SR.sup.16;
[0049] R.sup.15 is selected from the group consisting of hydro,
C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl, and aryl;
[0050] R.sup.16 is selected from the group consisting of hydro,
C.sub.1-6alkyl, C.sub.2-6alkenyl, cycloalkyl, aryl, heteroaryl,
SO.sub.2R.sup.17, C.sub.1-6alkyl substituted with one or more of
halo, hydroxy, aryl, heteroaryl, heterocycloalkyl,
N(R.sup.17).sub.2, and SO.sub.2R.sup.17, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneC.sub.3-8heterocycloalkyl,
C.sub.1-3alkyleneSO.sub.2aryl, optionally substituted
C.sub.1-3alkyleneN(R.sup.17).sub.2, OCF.sub.3,
C.sub.1-3alkyleneN(R.sup.17).sub.3.sup.+,
C.sub.3-8heterocycloalkyl,
CH(C.sub.1-3alkyleneN(R.sup.17).sub.2).sub.2;
[0051] or two R.sup.16 groups are taken together to form an
optionally substituted 3- to 6-membered aliphatic ring.
[0052] R.sup.17 is selected from the group consisting of hydro,
C.sub.1-6alkyl, cycloalkyl, aryl, and SO.sub.2C.sub.1-6alkyl, or
two R.sup.17 groups are taken together to form an optionally
substituted 3- to 6-membered ring;
[0053] R.sup.18 is selected from the group consisting of
C.sub.1-6alkyl, aryl, N(R.sup.15).sub.2, OR.sup.15, and halo;
and
[0054] R.sup.19 is selected from the group consisting of halo and
C.sub.1-6alkyl.
[0055] The present invention also is directed to pharmaceutical
compositions containing one or more compounds of structural formula
(II), to use of the compounds and compositions containing the
compounds in therapeutic treatment of a disease or disorder, and to
methods of preparing the compounds and intermediates involved in
the synthesis of the compounds of structural formula (II).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0056] Radiation and most chemotherapeutic agents are
therapeutically beneficial because they take advantage of
inappropriate tumor cell proliferation. Cellular processes, such as
DNA damage repair and cell cycle checkpoints, protect tumor cells
from the toxic effects of physical and chemical agents. Treatments
that modulate the underlying molecular mechanisms of cell cycle
progression and resistance to DNA damage can potentiate tumor cell
killing and enhance the therapeutic index of existing
therapies.
[0057] Most chemotherapeutic agents act by disrupting DNA
metabolism. Because these processes are shared by both normal and
tumor cells, and because the maintenance of DNA integrity is
essential to cell viability, anticancer drugs have the lowest
therapeutic index of any drug class. By identifying and inhibiting
cellular processes that tumor cells rely upon, the effectiveness of
radiation and chemotherapy treatment regimens can be enhanced.
[0058] The interruption of the DNA damage checkpoint protein
function provides a novel means of killing tumor cells relative to
normal cells. For example, Chk1 ensures that cells with unrepaired
DNA lesions caused by certain drugs or radiation do not progress
through DNA synthesis phase or mitosis until chromosomal lesions
have been removed. Accordingly, a tumor cell treated with a Chk1
inhibitor in combination with a DNA damaging agent can kill using
lower amounts of DNA damaging agent than tumor cells treated with
the DNA damaging agent alone.
[0059] Failure of cell cycle checkpoints in normal cells
predisposes an individual to, or directly causes, many disease
states, such as cancer, ataxia telangiectdsia, embryo
abnormalities, and various immunological defects associated with
aberrant B and T cell development. The latter are associated with
the pathological states of lupus, arthritis, and autoimmune
diseases. Intense research efforts, therefore, have focused on
identifying cell cycle checkpoints and the proteins essential for
the function of the checkpoints.
[0060] Noncancerous tissue having intact cell checkpoints typically
is insulated from temporary disruption of a single checkpoint
pathway, such as the Chk1 pathway. Tumor cells, however, have
multiple defects in pathways controlling cell cycle progression
such that perturbation of the DNA damage checkpoint can render
cells particularly sensitive to DNA damaging agents. Therefore,
checkpoint inhibitors are expected to enhance the therapeutic
index, which is a measure of the probability of tumor control
relative to the probability of toxicity to normal tissue to
radiation and systemic chemotherapy. In contrast, other classes of
inhibitors may not be amenable to combination chemotherapy because
both normal and tumor tissue may be similarly sensitized.
[0061] One aspect of the present invention is directed to a method
of inhibiting Chk1, comprising the step of administering a
therapeutically effective amount of a compound of formula (I), or a
composition containing the same, to an individual. Compounds of
formula (I) have a structural formula
##STR00008##
[0062] wherein X is null, --O--, --S--, --CH.sub.2--, or
--N(R.sup.1)--;
[0063] X.sup.2 is --O--, --S--, or --N(R.sup.1)--;
[0064] Y is O or S; or .dbd.Y represents two hydrogen atoms
attached to a common carbon atom;
[0065] W is selected from the group consisting of heteroaryl, aryl,
heterocycloalkyl, cycloalkyl, and C.sub.1-3 alkyl substituted with
a heteroaryl or aryl group; and
[0066] Z is selected from the group consisting of hydrogen, aryl,
and heteroaryl;
[0067] wherein said aryl groups of W and Z are optionally
substituted with one to four substituents represented by R.sup.2,
said heteroaryl groups of W and Z are optionally substituted with
one to four substituents represented by R.sup.5, and said
heterocycloalkyl and cycloalkyl groups of W are optionally
substituted with one to two substituents represented by
R.sup.6;
[0068] R.sup.1 is selected from the group consisting of hydrogen,
C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl, and aryl;
[0069] R.sup.2 is selected from the group consisting of halogen,
C.sub.1-6alkyl, C.sub.2-6alkenyl, OCF.sub.3, NO.sub.2, CN, NC,
N(R.sup.3).sub.2, OR.sup.3, CO.sub.2R.sup.3, C(O)N(R.sup.3).sub.2,
C(O)R.sup.3, N(R.sup.1)COR.sup.3, N(R.sup.1)C(O)OR.sup.3,
N(R.sup.3)C(O)OR.sup.3, N(R.sup.3)C(O)C.sub.1-3alkyleneC(O)R.sup.3,
N(R.sup.3)C(O)C.sub.1-3alkylene-C(O)OR.sup.3,
N(R.sup.3)C(O)C.sub.1-3alkyleneOR.sup.3,
N(R.sup.3)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.3,
N(R.sup.3)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.3,
C.sub.1-3alkyleneOR.sup.3, and SR.sup.3;
[0070] R.sup.3 is selected from the group consisting of hydro,
C.sub.1-6alkyl, C.sub.2-6alkenyl, cycloalkyl, aryl, heteroaryl,
SO.sub.2R.sup.4, C.sub.1-6alkyl substituted with one or more of
halo, hydroxy, aryl, heteroaryl, heterocycloalkyl,
N(R.sup.4).sub.2, and SO.sub.2R.sup.4, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneC.sub.3-8heterocycloalkyl,
C.sub.1-3alkyleneSO.sub.2aryl, optionally substituted
C.sub.1-3alkyleneN(R.sup.4).sub.2, OCF.sub.3,
C.sub.1-3alkyleneN(R.sup.4).sub.3.sup.+, C.sub.3-8heterocycloalkyl
and CH(C.sub.1-3alkyleneN(R.sup.4).sub.2).sub.2, or two R.sup.3
groups are taken together to form an optionally substituted 3- to
6-membered aliphatic ring;
[0071] R.sup.4 is selected from the group consisting of hydro,
C.sub.1-6alkyl, cycloalkyl, aryl, heteroaryl,
C.sub.1-3alkylenearyl, and SO.sub.2C.sub.1-6alkyl, or two R.sup.4
groups are taken together to form an optionally substituted 3- to
6-membered ring;
[0072] R.sup.5 is selected from the group consisting of
C.sub.1-6alkyl, aryl, N(R.sup.3).sub.2, OR.sup.3, halo, N.sub.3,
CN, C.sub.1-3alkylenearyl, C.sub.1-3alkyleneN(R.sup.3).sub.2,
C(O)R.sup.3, and
##STR00009##
[0073] R.sup.6 is selected from the group consisting of halo and
C.sub.1-6alkyl,
[0074] and pharmaceutically acceptable salts or solvates
thereof.
[0075] Preferred compounds used in the method are those wherein
X.sup.1 and X.sup.2 are --N(H)--;
[0076] Y is O or S;
[0077] W is heteroaryl containing at least two heteroatoms selected
from the group consisting of N, O, and S, said ring is optionally
substituted with from one to four substituents selected from the
group consisting of C.sub.1-6alkyl, aryl, N(R.sup.3).sub.2,
OR.sup.3, and halo, wherein R.sup.3 is as previously defined;
[0078] Z is selected from the group consisting of:
##STR00010##
wherein
[0079] Q is selected from the group consisting of hydrogen,
OR.sup.3, SR.sup.3, and N(R.sup.3).sub.2;
[0080] J is selected from the group consisting of CR.sup.20,
NR.sup.20, O, and S;
[0081] K is selected from the group consisting of CR.sup.21,
NR.sup.21, O, and S;
[0082] L is selected from the group consisting of CR.sup.22,
NR.sup.22, O, and S;
[0083] M is selected from the group consisting of CR.sup.23,
NR.sup.23, O, and S;
wherein:
[0084] R.sup.20, R.sup.21, and R.sup.22 are each independently
selected from the group consisting of null, hydro, halo,
C.sub.1-6oalkyl, C.sub.2-6alkenyl, OCF.sub.3, NO.sub.2, CN, NC,
N(R.sup.25).sub.2, OR.sup.25, CO.sub.2R.sup.25,
C(O)N(R.sup.25).sub.2, C(O)R.sup.25, N(R.sup.24)COR.sup.25,
N(R.sup.24)C(O)OR.sup.25, N(R.sup.25)C(O)OR.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneC(O)R.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneC(O)OR.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneOR.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.25, CF.sub.3,
C.sub.1-3alkyleneN(R.sup.25)SO.sub.2aryl,
C.sub.1-3alkyleneN(R.sup.25)SO.sub.2heteroaryl,
C.sub.1-3alkyleneOC.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.25)C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.25)C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneN(R.sup.25)C(O)R.sup.7,
C.sub.1-3alkyleneN(R.sup.25)C(O)C.sub.1-3alkyleneOR.sup.2,
C.sub.1-3alkyleneN(R.sup.25)C(O)aryl,
C.sub.1-3alkyleneN(R.sup.25)C(O)C.sub.1-3-alkyleneN(R.sup.25).sub.2,
C.sub.1-3alkyleneN(R.sup.25)C(O)heteroaryl,
C.sub.1-3alkyleneOR.sup.25, and SR.sup.25;
[0085] R.sup.23 is selected from the group consisting of null,
hydro, C.sub.1-6alkyl, and halo;
[0086] R.sup.24 is selected from the group consisting of hydro,
C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl, and aryl;
[0087] R.sup.25 is selected from the group consisting of hydro,
C.sub.1-6alkyl, C.sub.2-6alkenyl, cycloalkyl, aryl, heteroaryl,
SO.sub.2R.sup.26, and C.sub.1-6alkyl substituted with halo,
hydroxy, aryl, heteroaryl, heterocycloalkyl, N(R.sup.26).sub.2, or
SO.sub.2R.sup.26; and
[0088] R.sup.26 is selected from the group consisting of hydro,
C.sub.1-6alkyl, cycloalkyl, aryl, and SO.sub.2C.sub.1-6alkyl, or
two R.sup.26 groups are taken together to form an optionally
substituted 3- to 6-membered ring.
[0089] More preferred compounds of the method are those of
structural formula (I) wherein W is selected from the group
consisting of pyridazinyl, pyrimidinyl, pyrazinyl, and triazinyl,
optionally substituted with from one to four substituents selected
from the group consisting of C.sub.1-6alkyl, aryl,
N(R.sup.3).sub.2, OR.sup.3, N.sub.3, CN, C(O)R.sup.7,
C.sub.1-3alkylenearyl, C.sub.1-3alkyleneN(R.sup.4),
##STR00011##
and halo, wherein R.sup.3, X.sup.1, X.sup.2, Y, and Z are as
defined for formula (I).
[0090] Additional preferred compounds of formula (I) are those
wherein W is selected from the group consisting of pyridazine,
pyrimidine, pyrazine, and triazine, optionally substituted with
from one to four substituents selected from the group consisting of
optionally substituted C.sub.1-6alkyl, aryl, N(R.sup.3).sub.2,
OR.sup.3, C.sub.1-3alkylenearyl, C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneC.sub.3-8heterocycloalkyl,
C.sub.1-3alkyleneSO.sub.2aryl, optionally substituted
C.sub.1-3alkyleneN(R.sup.4).sub.2, OCF.sub.3,
C.sub.1-3alkyleneN(R.sup.4).sub.3.sup.+, C.sub.3-8heterocycloalkyl,
CH(C.sub.1-3alkyleneN(R.sup.4).sub.2).sub.2, and halo; X.sup.1 and
X.sup.2 are --N(H)--; Y is O or S; and Z is selected from the group
consisting of:
##STR00012##
wherein R.sup.3, Q, J, K, L, and M are as previously defined.
[0091] Compounds preferred for use in the method also include those
of formula (I) wherein J is selected from the group consisting of
CR.sup.20 and NR.sup.20, wherein R.sup.20 is null, hydro,
C.sub.1-6alkyl, and halo;
[0092] K is selected from the group consisting of CR.sup.21 and
NR.sup.21;
[0093] L is selected from the group consisting of CR.sup.22 and
NR.sup.22; and
[0094] one of R.sup.21 and R.sup.22 is hydro and the other is a
substituent selected from the group consisting of CO.sub.2R.sup.25,
C(O)N(R.sup.25).sub.2, C(O)R.sup.25, N(R.sup.24)COR.sup.25,
N(R.sup.24)C(O)OR.sup.25, N(R.sup.25)C(O)OR.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneC(O)R.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneC(O)OR.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneOR.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.25,
N(R.sup.25)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.25,
C.sub.1-3alkyleneOR.sup.25, and SR .sup.25, wherein R.sup.24,
R.sup.25, W, X.sup.1, X.sup.2, Y and M are as previously
defined.
[0095] Compounds useful in the method also include structures of
formula (I) wherein X.sup.1 is null, X.sup.2 is --N(H)--, Y is O, Z
is hydro, and W is as previously defined.
[0096] The method of inhibiting Chk1 also can be used to sensitize
a tumor cell to a chemotherapeutic agent. As such, the present
invention also is directed to a method of sensitizing a tumor cell
to a chemotherapeutic agent comprising administering a compound of
formula (I), or a salt, solvate, or derivative thereof, or a
composition comprising the same, to an individual.
[0097] In another aspect, the present invention is directed to
aryl- and heteroaryl-disubstituted urea compounds having a
structural formula (II)
##STR00013##
[0098] wherein Y' is O or S;
[0099] W' is selected from the group consisting of
##STR00014##
optionally substituted with from one to four substituents selected
from the group consisting of C.sub.1-6oalkyl, aryl,
N(R.sup.7).sub.2, OR.sup.7, N.sub.3, CN, C(O)R.sup.7,
C.sub.1-3alkylenearyl C.sub.1-3alkyleneN(R.sup.12).sub.2,
##STR00015##
[0100] Z' is selected from the group consisting of:
##STR00016##
wherein
[0101] Q' is selected from the group consisting of hydrogen,
OR.sup.7, SR.sup.7, and N(R.sup.7).sub.2;
[0102] J' is selected from the group consisting of C--R.sup.8,
N--R.sup.8, O, and S;
[0103] K' is selected from the group consisting of C--R.sup.9,
N--R.sup.9, O, and S;
[0104] L.varies. is selected from the group consisting of
C--R.sup.10, N--R.sup.10, O, and S;
[0105] M' is selected from the group consisting of C--R.sup.11,
N--R.sup.11, O, and S;
wherein:
[0106] R.sup.7, independently, is selected from the group
consisting of hydro, C.sub.1-6alkyl, C.sub.2-6alkenyl, cycloalkyl,
aryl, heteroaryl, SO.sub.2R.sup.12, C.sub.1-6alkyl substituted with
one or more of halo, hydroxy, aryl, heteroaryl, heterocycloalkyl,
N(R.sup.12).sub.2, and SO.sub.2R.sup.12, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneC.sub.3-8heterocycloalkyl,
C.sub.1-3alkyleneSO.sub.2aryl, optionally substituted
C.sub.1-3alkyleneN(R.sup.12).sub.2, OCF.sub.3,
C.sub.1-3alkyleneN(R.sup.12).sub.3.sup.+,
C.sub.3-8heterocycloalkyl, and
CH(C.sub.1-3alkyleneN(R.sup.12).sub.2).sub.2, or two R.sup.7 groups
are taken together to form an optionally substituted 3- to
6-membered aliphatic ring;
[0107] R.sup.8, R.sup.9, and R.sup.10 are each independently
selected from the group consisting of null, hydro, halo, optionally
substituted C.sub.1-6alkyl, C.sub.2-6alkenyl, OCF.sub.3, NO.sub.2,
CN, NC, N(R.sup.7).sub.2, OR.sup.7, CO.sub.2R.sup.7,
C(O)N(R.sup.7).sub.2, C(O)R.sup.7, N(R.sup.13)COR.sup.7,
N(R.sup.13)C(O)OR.sup.7, N(R.sup.7)C(O)OR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneC(O)R.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkylene(C(O)R.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneOR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.7,
N(R)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.7, CF.sub.3,
C.sub.1-3alkyleneN(R.sup.12)SO.sub.2aryl,
C.sub.1-3alkyleneN(R.sup.12)SO.sub.2heteroaryl,
C.sub.1-3alkyleneOC.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.12)C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.12)C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneN(R.sup.12)C(O)R.sup.7,
C.sub.1-3alkyleneN(R.sup.12)C(O)C.sub.1-3alkyleneOR.sup.2,
C.sub.1-3alkyleneN(R.sup.12)C(O)aryl,
C.sub.1-3alkyleneN(R.sup.12)C(O)C.sub.1-3alkyleneN(R.sup.12).sub.2,
C.sub.1-3alkyleneN(R.sup.12)C(O)heteroaryl,
C.sub.1-3alkyleneOR.sup.7, and SR.sup.7, wherein R.sup.7 is as
defined above;
[0108] R.sup.11 is selected from the group consisting of null,
hydro, optionally substituted C.sub.1-6alkyl, and halo;
[0109] R.sup.12 is selected from the group consisting of hydro,
C.sub.1-6alkyl, cycloalkyl, aryl, heteroaryl,
C.sub.1-3alkylenearyl, and SO.sub.2C.sub.1-6alkyl, or two R.sup.12
groups are taken together to form an optionally substituted 3- to
6-membered ring; and
[0110] R.sup.13 is selected from the group consisting of hydro,
C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl, and aryl;
[0111] provided that when Q' is hydro or OCH.sub.3, at least one of
R.sup.8, R.sup.9, and R.sup.10 is different from hydro, CH.sub.3,
OCH.sub.3, and halo,
[0112] and pharmaceutically acceptable salts or solvates
thereof.
[0113] Preferred compounds of structural formula (II) are those
wherein W' is selected from the group consisting of
##STR00017##
optionally substituted with one to four substituents selected from
the group consisting of C.sub.1-6alkyl, optionally substituted
aryl, N(R.sup.7).sub.2, CF.sub.3, C(O)R.sup.7, N.sub.3, CN,
C.sub.1-3alkylenearyl, C.sub.1-3alkyleneN(R.sup.12).sub.2,
OR.sup.7, halo,
##STR00018##
wherein R.sup.7, Y and Z are as previously defined.
[0114] More preferred compounds of formula (II) are those
wherein:
[0115] J' is selected from the group consisting of CR.sup.8 and
NR.sup.8, wherein R.sup.8 is null, hydro, C.sub.1-6alkyl, and
halo;
[0116] K' is selected from the group consisting of CR.sup.9 and
NR.sup.9;
[0117] L' is selected from the group consisting of CR.sup.10 and
NR.sup.10; and
[0118] one of R.sup.9 and R.sup.10 is hydro and the other is a
substituent selected from the group consisting of CO.sub.2R.sup.7,
C(O)N(R.sup.7).sub.2, C(O)R.sup.7, N(R.sup.13)COR.sup.7,
N(R.sup.13)C(O)OR.sup.7, N(R.sup.7)C(O)OR.sup.7,
N(R.sup.7C(O)C.sub.1-3alkyleneC(O)R.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneC(O)OR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneOR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.7,
N(R.sup.7)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.7,
C.sub.1-3alkyleneOR.sup.7, CF.sub.3,
C.sub.1-3alkyleneN(R.sup.12)SO.sub.2aryl,
C.sub.1-3alkyleneN(R.sup.12)SO.sub.2heteroaryl,
C.sub.1-3alkyleneOC.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.12)C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneN(R.sup.12)C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneN(R.sup.12)C(O)R.sup.7,
C.sub.1-3alkyleneN(R.sup.12)C(O)C.sub.1-3alkyleneOR.sup.2,
C.sub.1-3alkyleneN(R.sup.12)C(O)aryl,
C.sub.1-3alkyleneN(R.sup.12)C(O)C.sub.1-3alkyleneN(R.sup.12).sub.2,
C.sub.1-3alkyleneN(R.sup.12)C(O)heteroaryl, and SR.sup.7, wherein
R.sup.7, R.sup.13, W', Y', and M' are as previously defined.
[0119] Yet another aspect of the invention relates to compounds,
and compositions containing compounds, of structural formula
(III)
##STR00019##
[0120] wherein W''' is selected from the group consisting of
heteroaryl, aryl, heterocycloalkyl, cycloalkyl, and C.sub.1-3 alkyl
substituted with a heteroaryl or aryl group;
[0121] wherein said aryl groups are optionally substituted with one
to four substituents represented by R.sup.14, said heteroaryl
groups are optionally substituted with one to four substituents
represented by R.sup.18, and said heterocycloalkyl and cycloalkyl
groups are optionally substituted with one to two substituents
represented by R.sup.19;
[0122] R.sup.14 is selected from the group consisting of halo,
C.sub.1-6alkyl, C.sub.2-6alkenyl, OCF.sub.3, NO.sub.2, CN, NC,
N(R.sup.16).sub.2, OR.sup.16, CO.sub.2R.sup.16,
C(O)N(R.sup.16).sub.2, C(O)R.sup.16, N(R.sup.15)COR.sup.16,
N(R.sup.15)C(O)OR.sup.16, N(R.sup.16)C(O)OR.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneC(O)R.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneC(O)OR.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneOR.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.16,
N(R.sup.16)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.16,
C.sub.1-3alkyleneOR.sup.26, and SR.sup.16;
[0123] R.sup.15 is selected from the group consisting of hydro,
C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl, and aryl;
[0124] R.sup.16 is selected from the group consisting of hydro,
C.sub.1-6alkyl, C.sub.2-6alkenyl, cycloalkyl, aryl, heteroaryl,
SO.sub.2R.sup.17, and C.sub.1-6alkyl substituted with one or more
of halo, hydroxy, aryl, heteroaryl, heterocycloalkyl,
N(R.sup.17).sub.2, and SO.sub.2R.sup.17, C.sub.1-3alkylenearyl,
C.sub.1-3alkyleneheteroaryl,
C.sub.1-3alkyleneC.sub.3-8heterocycloalkyl,
C.sub.1-3alkyleneSO.sub.2aryl, optionally substituted
C.sub.1-3alkyleneN(R.sup.17).sub.2, OCF.sub.3,
C.sub.1-3alkyleneN(R.sup.17).sub.3.sup.+,
C.sub.3-8heterocycloalkyl,
CH(C.sub.1-3alkyleneN(R.sup.17).sub.2).sub.2, or two R.sup.16
groups are taken together to form a 3- to 6-membered aliphatic
ring.
[0125] R.sup.17 is selected from the group consisting of hydro,
C.sub.1-6alkyl, cycloalkyl, aryl, and SO.sub.2C.sub.1-6alkyl, or
two R.sup.17 groups are taken together to form an optionally
substituted 3- to 6-membered ring;
[0126] R.sup.18 is selected from the group consisting of
C.sub.1-6alkyl, aryl, N(R.sup.15).sub.2, OR.sup.15, and halo;
and
[0127] R.sup.19 is selected from the group consisting of halo and
C.sub.1-6alkyl.
[0128] As used herein, the term "alkyl" includes straight chained
and branched hydrocarbon groups containing the indicated number of
carbon atoms, typically methyl, ethyl, and straight-chain and
branched propyl and butyl groups. Unless otherwise indicated, the
hydrocarbon group can contain up to 20 carbon atoms. The term
"alkyl" includes "bridged alkyl," i.e., a C.sub.6-C.sub.16 bicyclic
or polycyclic hydrocarbon group, for example, norbornyl, adamantyl,
bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or
deca hydronaphthyl. Alkyl groups can be substituted, for example,
with hydroxy (OH), halo, aryl, heteroaryl, cycloalkyl,
heterocycloalkyl, amino (N(R.sup.a).sub.2), and sulfonyl
(SO.sub.2R.sup.a), wherein R.sup.a is selected from the group
consisting of hydro, C.sub.1-6alkyl, cycloalkyl, aryl, and
SO.sub.2C.sub.1-6alkyl, or two R.sup.a groups are taken together to
form an optionally substituted 3- to 6-membered ring.
[0129] The term "cycloalkyl" is defined as a cyclic
C.sub.3-8hydrocarbon group, e.g., cyclopropyl, cyclobutyl,
cyclohexyl, and cyclopentyl. "Heterocycloalkyl" is defined
similarly as cycloalkyl, and includes bicyclic and polycyclic
groups, except the ring contains one to three heteroatoms selected
from the group consisting of oxygen, nitrogen, and sulfur.
Cycloalkyl and heterocycloalkyl groups can be saturated or
partially unsaturated ring systems substituted with, for example,
one to three groups, independently selected from C.sub.1-4alkyl,
C.sub.1-3alkyleneOH, C(.dbd.O)NH.sub.2, NH.sub.2, oxo (.dbd.O),
aryl, trifluoroethanoyl, and OH. Heterocycloalkyl groups optionally
are further N-substituted with C.sub.1-3alkylenearyl or
C.sub.1-3alkyleneheteroaryl.
[0130] The term "alkenyl" is defined identically as "alkyl," except
the substituent contains a carbon-carbon double bond.
[0131] The term "alkynyl" is defined identically as "alkyl," except
the substituent contains a carbon-carbon triple bond.
[0132] The term "alkylene" refers to an alkyl group having a
substituent. For example, the term "C.sub.1-3alkyleneC(O)OR" refers
to an alkyl group containing one to three carbon atoms substituted
with a --C(O)OR group. The alkylene group is optionally substituted
with one or more of aryl, heteroaryl, and OR.sup.7, wherein R.sup.7
is defined hereafter.
[0133] The term "halo" or "halogen" is defined herein to include
fluorine, bromine, chlorine, and iodine.
[0134] The term "aryl," alone or in combination, is defined herein
as a monocyclic or polycyclic aromatic group, preferably a
monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl.
Unless otherwise indicated, an "aryl" group can be unsubstituted or
`substituted, for example, with one or more, and in particular one
to four, halo, C.sub.1-6alkyl, C.sub.2-6alkenyl, OCF.sub.3,
NO.sub.2, CN, NC, N(R.sup.a).sub.2, OR.sup.b, CO.sub.2R.sup.b,
C(O)N(R.sup.b).sub.2, C(O)R.sup.b, N(R.sup.a)COR.sup.b,
N(R.sup.a)C(O)OR.sup.b, N(R.sup.a)C(O)OR.sup.b,
N(R.sup.a)C(O)C.sub.1-3alkyleneC(O)R.sup.b,
N(R.sup.b)C(O)C.sub.1-3alkyleneC(O)OR.sup.b,
N(R.sup.b)C(O)C.sub.1-3alkyleneOR.sup.b,
N(R.sup.b)C(O)C.sub.1-3alkyleneNHC(O)OR.sup.b,
N(R.sup.b)C(O)C.sub.1-3alkyleneSO.sub.2NR.sup.b,
C.sub.1-3alkyleneOR.sup.b, and SR.sup.b, wherein R.sup.b is
selected from the group consisting of hydro, C.sub.1-6alkyl,
C.sub.2-6alkenyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
SO.sub.2R.sup.a, and C.sub.1-6alkyl substituted with halo, hydroxy,
aryl, heteroaryl, heterocycloalkyl, N(R.sup.a).sub.2, or
SO.sub.2R.sup.a, and R.sup.a, as previously defined. Exemplary aryl
groups include phenyl, naphthyl, tetrahydronaphthyl,
2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-methylphenyl,
4-methoxyphenyl, 3-trifluoromethyl phenyl, 4-nitrophenyl,
2-methoxyphenyl, 2,4-methoxychlorophenyl, and the like. The terms
"arylC.sub.1-3alkyl" and "heteroarylC.sub.1-3alkyl" are defined as
an aryl or heteroaryl group having a C.sub.1-3alkyl
substituent.
[0135] The term "heteroaryl" is defined herein as a monocyclic or
bicyclic ring system containing one or two aromatic rings and
containing at least one nitrogen, oxygen, or sulfur atom in an
aromatic ring, and that can be unsubstituted or substituted, for
example, with one or more, and in particular one to four,
substituents, for example, hydrogen, C.sub.1-6alkyl,
C.sub.1-6alkoxy, aryl, N(R.sup.a).sub.2, OR.sup.b, and halo,
wherein R.sup.a and R.sup.b are as previously defined. Examples of
heteroaryl groups include, but are not limited to, thienyl, furyl,
pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl,
isothiazolyl, isoxazolyl, imidizolyl, benzothiazolyl, pyrazinyl,
pyrimidinyl, thiazolyl, and thiadiazolyl.
[0136] The term "hydroxy" is defined as --OH.
[0137] The term "3- to 6-membered ring" as used herein refers to
carbocyclic and heterocyclic aliphatic or aromatic groups,
including, but not limited to, morpholinyl, piperidinyl, phenyl,
thiophenyl, furyl, pyrrolyl, imidazolyl, pyrimidinyl, and
pyridinyl, optionally substituted with one or more, and in
particular one to three, groups exemplified above for "aryl"
groups.
[0138] The carbon atom content of hydrocarbon-containing moieties
is indicated by a subscript designating the minimum and maximum
number of carbon atoms in the moiety, e.g., "C.sub.1-6alkyl" refers
to an alkyl group having one to six carbon atoms, inclusive.
[0139] In the structures herein, for a bond lacking a substituent,
the substituent is methyl, for example,
##STR00020##
[0140] When no substituent is indicated as attached to a carbon
atom on a ring, it is understood that the carbon atom contains the
appropriate number of hydrogen atoms. In addition, when no
substituent is indicated as attached to a carbonyl group or a
nitrogen atom, for example, the substituent is understood to be
hydrogen, e.g.,
##STR00021##
[0141] The abbreviation "Me" is methyl. The abbreviation CO and
C(O) is carbonyl (C.dbd.(O)).
[0142] The notation N(R.sup.x).sub.2, wherein x represents an alpha
or numeric character, such as for example R.sup.a, R.sup.b,
R.sup.4, R.sup.12, and the like, is used to denote two R.sup.x
groups attached to a common nitrogen atom. When used in such
notation, the R.sup.x group can be the same or different, and is
selected from the group as defined by the R.sup.x group.
[0143] The present invention also is directed to pharmaceutical
compositions containing one or more compounds of structural formula
(II) and (III), to use of the compounds and compositions containing
the compounds in therapeutic treatment of a disease or disorder,
and to methods of preparing the compounds and intermediates
involved in the synthesis of the compounds of structural formula
(II) and (III).
[0144] Compounds useful for the method of the present invention
have demonstrated activity in inhibiting Chk1 in vitro. Compounds
of the present invention have demonstrated selectivity for Chk1 as
against other protein kinases including Cdc2, Chk2, Atr, DNA-PK,
PKA, and CaM KII.
[0145] Compounds of the present invention can be used to potentiate
the therapeutic effects of radiation and/or chemotherapeutics used
in the treatment of cancers and other cell proliferation disorders
in humans or animals. For example, compounds of the invention can
be used to enhance treatment of tumors that are customarily treated
with an antimetabolite, e.g., methotrexate or 5-fluorouracil
(5-FU). The method of the present invention comprises
administration of a Chk1 inhibitor compound in combination with a
chemotherapeutic agent that can effect single- or double-strand DNA
breaks or that can block DNA replication or cell proliferation.
Alternatively, the method of the present invention comprises
administration of a Chk1 inhibitor compound in combination with
therapies that include use of an antibody, e.g., herceptin, that
has activity in inhibiting the proliferation of cancer cells.
Accordingly, cancers such as colorectal cancers, head and neck
cancers, pancreatic cancers, breast cancers, gastric cancers,
bladder cancers, vulvar cancers, leukemias, lymphomas, melanomas,
renal cell carcinomas, ovarian cancers, brain tumors,
osteosarcomas, and lung carcinomas, are susceptible to enhanced
treatment in combination with the Chk1 inhibitors of the
invention.
[0146] Tumors or neoplasms include growths of tissue cells wherein
multiplication of cells is uncontrolled and progressive. Some such
growths are benign, but others are termed "malignant," and can lead
to death of the organism. Malignant neoplasms, or "cancers," are
distinguished from benign growths in that, in addition to
exhibiting aggressive cellular proliferation, can invade
surrounding tissues and metastasize. Moreover, malignant neoplasms
are characterized by showing a greater loss of differentiation
(greater "dedifferentiation") and organization relative to one
another and surrounding tissues. This property is called
"anaplasia."
[0147] Neoplasms treatable by the present invention also include
solid tumors, i.e., carcinomas and sarcomas. Carcinomas include
malignant neoplasms derived from epithelial cells which infiltrate
(i.e., invade) surrounding tissues and give rise to metastases.
Adenocarcinomas are carcinomas derived from glandular tissue, or
from tissues that form recognizable glandular structures. Another
broad category of cancers includes sarcomas, which are tumors whose
cells are embedded in a fibrillar or homogeneous substance, like
embryonic connective tissue. The invention also enables treatment
of cancers of the myeloid or lymphoid systems, including leukemias,
lymphomas, and other cancers that typically are not present as a
tumor mass, but are distributed in the vascular or lymphoreticular
systems.
[0148] Chk1 activity is associated with various forms of cancer in,
for example, adult and pediatric oncology, growth of solid
tumors/malignancies, myxoid and round cell carcinoma, locally
advanced tumors, metastatic cancer, human soft tissue sarcomas,
including Ewing's sarcoma, cancer metastases, including lymphatic
metastases, squamous cell carcinoma, particularly of the head and
neck, esophageal squamous cell carcinoma, oral carcinoma, blood
cell malignancies, including multiple myeloma, leukemias, including
acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic
lymphocytic leukemia, chronic myelocytic leukemia, and hairy cell
leukemia, effusion lymphomas (body cavity based lymphomas), thymic
lymphoma lung cancer (including small cell carcinoma, cutaneous T
cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer
of the adrenal cortex, ACTH-producing tumors, nonsmall cell
cancers, breast cancer, including small cell carcinoma and ductal
carcinoma), gastrointestinal cancers (including stomach cancer,
colon cancer, colorectal cancer, and polyps associated with
colorectal neoplasia), pancreatic cancer, liver cancer, urological
cancers (including bladder cancer, such as primary superficial
bladder tumors, invasive transitional cell carcinoma of the
bladder, and muscle-invasive bladder cancer), prostate cancer,
malignancies of the female genital tract (including ovarian
carcinoma, primary peritoneal epithelial neoplasms, cervical
carcinoma, uterine endometrial cancers, vaginal cancer, cancer of
the vulva, uterine cancer and solid tumors in the ovarian
follicle), malignancies of the male genital tract (including
testicular cancer and penile cancer), kidney cancer (including
renal cell carcinoma, brain cancer (including intrinsic brain
tumors, neuroblastoma, astrocytic brain tumors, gliomas, and
metastatic tumor cell invasion in the central nervous system), bone
cancers (including osteomas and osteosarcomas), skin cancers
(including malignant melanoma, tumor progression of human skin
keratinocytes, and squamous cell cancer), thyroid cancer,
retinoblastoma, neuroblastoma, peritoneal effusion, malignant
pleural effusion, mesothelioma, Wilms's tumors, gall bladder
cancer, trophoblastic neoplasms, hemangiopericytoma, and Kaposi's
sarcoma.
[0149] Compounds of the present invention also can potentiate the
efficacy of drugs in the treatment of inflammatory diseases.
Examples of diseases that can benefit from combination therapy with
compounds suitable for the method of the present invention are
rheumatoid arthritis, psoriasis, vitiligo, Wegener's
granulomatosis, and systemic lupus erythematosus (SLE). Treatment
of arthritis, Wegener's granulomatosis, and SLE often involves the
use of immunosuppressive therapies, such as ionizing radiation,
methotrexate, and cyclophosphamide. Such treatments typically
induce, either directly or indirectly, DNA damage. Inhibition of
Chk1 activity within the offending immune cells render the cells
more sensitive to control by these standard treatments. Psoriasis
and vitiligo commonly are treated with ultraviolet radiation (UV)
in combination with psoralen. The present DNA damaging agents
induce the killing effect of UV and psoralen, and increase the
therapeutic index of this treatment regimen. In general, compounds
useful in methods of the present invention potentiate control of
inflammatory disease cells when in combination with currently used
immunosuppressive drugs.
[0150] The present invention includes all possible stereoisomers
and geometric isomers of compounds of the present method and of
structural formulae (I), (II), and (III). The present invention
includes not only racemic compounds, but optically active isomers
as well. When a compound of structural formulae (I), (II), or (III)
is desired as a single enantiomer, it can be obtained either by
resolution of the final product or by stereospecific synthesis from
either isomerically pure starting material or use of a chiral
auxiliary reagent, for example, see Z. Ma et al., Tetrahedron:
Asymmetry, 8(6), pages 883-888 (1997). Resolution of the final
product, an intermediate, or a starting material can be achieved by
any suitable method known in the art. Additionally, in situations
where tautomers of the compounds of structural formulae (I), (II),
and (III) are possible, the present invention is intended to
include all tautomeric forms of the compounds. As demonstrated
hereafter, specific stereoisomers can exhibit an exceptional
ability to inhibit Chk1 in combination with chemo- or radiotherapy
with diminshed adverse effects typically associated with
chemotherapeutic or radiotherapeutic treatments.
[0151] Prodrugs of compositions of structural formulae (I), (II),
and (III) also can be used as the compound and in the method of the
present invention. It is well established that a prodrug approach,
wherein a compound is derivatized into a form suitable for
formulation and/or administration, and then is released as a drug
in vivo, has been successfully employed to transiently (e.g.,
bioreversibly) alter the physicochemical properties of the compound
(see, H. Bundgaard, Ed., Design of Prodrugs, Elsevier, Amsterdam,
(1985); R. B. Silverman, The Organic Chemistry of Drug Design and
Drug Action, Academic Press, San Diego, chapter 8, (1992); K. M.
Hillgren et al., Med. Res. Rev., 15, 83 (1995)).
[0152] Compounds of the present invention can contain several
functional groups. The introduced functional groups, if desired or
necessary, then can be modified to provide a prodrug for dose of
formulation and/or administration. Suitable prodrugs include, for
example, acid derivatives, like amides, esters, and the like. It
also is appreciated by those skilled in the art that N-oxides can
be used as a prodrug.
[0153] As used herein, the term pharmaceutically acceptable salts
refers compounds of structural formula (I), (II), and (III) which
contain acidic moieties and form salts with suitable cations.
Suitable pharmaceutically acceptable cations include alkali metal
(e.g., sodium or potassium) and alkaline earth metal (e.g., calcium
or magnesium) cations. The pharmaceutically acceptable salts of the
compounds of structural formula (I), (II), and (III), which contain
a basic center, are acid addition salts formed with
pharmaceutically acceptable acids. Examples include the
hydrochloride, hydrobromide, sulfate or bisulfate, phosphate or
hydrogen phosphate, acetate, benzoate, succinate, fumarate,
maleate, lactate, citrate, tartrate, gluconate, methanesulfonate,
benzene sulphonate, and p-toluenesulphonate salts. In light of the
foregoing, any reference to compounds of the present invention
appearing herein is intended to include compounds of structural
formula (I), (II), and (III), as well as pharmaceutically
acceptable salts and solvates thereof.
[0154] The compounds of the present invention can be
therapeutically administered as the neat chemical, but it is
preferable to administer compounds of structural formula (I), (II),
and (III) as a pharmaceutical composition or formulation.
Accordingly, the present invention further provides pharmaceutical
formulations comprising a compound of structural formula (I), (II),
and/or (III), or pharmaceutically acceptable salts thereof,
together with one or more pharmaceutically acceptable carriers and,
optionally, other therapeutic and/or prophylactic ingredients. The
carriers are "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not deleterious to the
recipient thereof.
[0155] Inhibition of the checkpoint kinase typically is measured
using a dose-response assay in which a sensitive assay system is
contacted with a compound of interest over a range of
concentrations, including concentrations at which no or minimal
effect is observed, through higher concentrations at which partial
effect is observed, to saturating concentrations at which a maximum
effect is observed. Theoretically, such assays of the dose-response
effect of inhibitor compounds can be described as a sigmoidal curve
expressing a degree of inhibition as a function of concentration.
The curve also theoretically passes through a point at which the
concentration is sufficient to reduce activity of the checkpoint
enzyme to a level that is 50% that of the difference between
minimal and maximal enzyme activity in the assay. This
concentration is defined as the Inhibitory Concentration (50%) or
IC.sub.50 value. Determination of IC.sub.50 values preferably are
made using conventional biochemical (acellular) assay techniques or
cell-based assay techniques.
[0156] Comparisons of the efficacy of inhibitors often are provided
with reference to comparative IC.sub.50 values, wherein a higher
IC.sub.50 indicates that the test compound is less potent, and a
lower IC.sub.50 indicates that the compound is more potent, than a
reference compound. Compounds useful in the method of the present
invention demonstrate an IC.sub.50 value of at least 0.1 nM when
measured using the dose-response assay. Preferred compounds
demonstrate an IC.sub.50 value of less than 10 .quadrature.M. More
preferred compounds demonstrate an IC.sub.50 value of less than 500
nM. Still more preferred compounds of the present invention
demonstrate an IC.sub.50 value of less than 250 nM, less than 100
nM, or less than 50 nM.
[0157] Compounds and pharmaceutical compositions suitable for use
in the present invention include those wherein the active
ingredient is administered in an effective amount to achieve its
intended purpose. More specifically, a "therapeutically effective
amount" means an amount effective to inhibit development of, or to
alleviate the existing symptoms of, the subject being treated.
Determination of the effective amount is well within the capability
of those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0158] A "therapeutically effective dose" refers to that amount of
the compound that results in achieving the desired effect. Toxicity
and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD.sub.50 (the dose lethal to
50% of the population) and the ED.sub.50 (the dose therapeutically
effective in 50% of the population). The dose ratio between toxic
and therapeutic effects is the therapeutic index, which is
expressed as the ratio of LD.sub.50 to ED.sub.50. Compounds that
exhibit high therapeutic indices (i.e., a toxic dose that is
substantially higher than the effective dose) are preferred. The
data obtained can be used in formulating a dosage range for use in
humans. The dosage of such compounds preferably lies within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed, and the route of
administration utilized.
[0159] The exact formulation, route of administration, and dosage
is chosen by the individual physician in view of the patient's
condition. Dosage amount and interval can be adjusted individually
to provide plasma levels of the active compound that are sufficient
to maintain desired therapeutic effects.
[0160] Pharmaceutical compositions of the invention can be
formulated to include one or more cytokines, lymphokines, growth
factors, or other hematopoietic factors which can reduce negative
side effects that may arise from, or be associated with,
administration of the pharmaceutical composition alone. Cytokines,
lymphokines, growth factors, or other hematopoietic factors
particularly useful in pharmaceutical compositions of the invention
include, but are not limited to, M-CSF, GM-CSF, TNF, IL-1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNF, G-CSF, Meg-CSF,
GM-CSF, thrombopoietin, stem cell factor, erythropoietin,
angiopoietins, including Ang-1, Ang-2, Ang-4, Ang-Y, and/or the
human angiopoietin-like polypeptide, vascular endothelial growth
factor (VEGF), angiogenin, bone morphogenic protein-1 (BMP-1),
BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10,
BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP receptor IA, BMP
receptor IB, brain derived neurotrophic factor, ciliary neutrophic
factor, ciliary neutrophic factor receptor cytokine-induced
neutrophil chemotactic factor 1, cytokine-induced neutrophil
chemotactic factor 2, cytokine-induced neutrophil chemotactic
factor 2, endothelial cell growth factor, endothelin 1, epidermal
growth factor, epithelial-derived neutrophil attractant, fibroblast
growth factor (FGF) 4, FGF 5, FGF 6, FGF 7, FGF 8, FGF 8b, FGF 8c,
FGF 9, FGF 10, FGF acidic, FGF basic, glial cell line-derived
neutrophic factor receptor 1, glial cell line-derived neutrophic
factor receptor 2, growth related protein, growth related protein,
growth related protein, growth related protein, heparin binding
epidermal growth factor, hepatocyte growth factor, hepatocyte
growth factor receptor, insulin-like growth factor I, insulin-like
growth factor receptor, insulin-like growth factor II, insulin-like
growth factor binding protein, keratinocyte growth factor, leukemia
inhibitory factor, leukemia inhibitory factor receptor, nerve
growth factor nerve growth factor receptor, neurotrophin-3,
neurotrophin-4, placenta growth factor, placenta growth factor 2,
platelet-derived endothelial cell growth factor, platelet derived
growth factor, platelet derived growth factor A chain, platelet
derived growth factor AA, platelet derived growth factor AB,
platelet derived growth factor B chain, platelet derived growth
factor BB, platelet derived growth factor receptor, platelet
derived growth factor receptor, pre-B cell growth stimulating
factor, stem cell factor, stem cell factor receptor, transforming
growth factor (TGF), TGF, TGF 1, TGF 1.2, TGF 2, TGF 3, TGF 5,
latent TGF 1, TGF, binding protein I, TGF binding protein II, TGF
binding protein III, tumor necrosis factor receptor type I, tumor
necrosis factor receptor type II, urokinase-type plasminogen
activator receptor, vascular endothelial growth factor, and
chimeric proteins and biologically or immunologically active
fragments thereof.
[0161] The compounds useful according to the invention may be
conjugated or linked to auxiliary moieties that promote any
property of the compounds that may be beneficial in methods of
therapeutic use. Such conjugates can enhance delivery of the
compounds to a particular anatomical site or region of interest
(e.g., a tumor), enable sustained therapeutic concentrations of the
compounds in target cells, alter pharmacokinetic and
pharmacodynamic properties of the compounds, and/or improve the
therapeutic index or safety profile of the compounds. Suitable
auxiliary moieties include, for example, amino acids,
aligopeptides, or polypeptides, e.g., antibodies such as monoclonal
antibodies and other engineered antibodies; and natural or
synthetic ligands to receptors in target cells or tissues. Other
suitable auxiliaries include fatty acid or lipid moieties, to
promote biodistribution or uptake of the compound by target cells
(see, e.g., Bradley et al., Clin. Cancer Res. (2001) 7:3229.
[0162] The therapeutic index of compositions comprising one or more
compounds of the invention can be enhanced by conjugation of the
compound(s) with antitumor antibodies as previously described (for
example, Pietersz and McKinzie, Immunol. Rev. (1992) 129:57; Trail
et al., Science (1993) 261:212; Rowlinson-Busza and Epenetos, Curr.
Opin. Oncol. 1992; 4:1142). Tumor directed delivery of compounds of
the invention would enhance the therapeutic benefit by minimizing
potential nonspecific toxicities which can result from radiation
treatment or chemotherapy. In another aspect, Chk1 inhibitors and
radioisotopes or chemotherapeutic agents can be conjugated to the
same antibody molecule. Alternatively, Chk1 inhibitor-conjugated
tumor specific antibodies can be administered before, during, or
after administration of chemotherapeutic-conjugated antitumor
antibody or radioimmunotherapy.
[0163] Compounds of the present invention can enhance the
therapeutic benefit of radiation and chemotherapy treatment,
including induction chemotherapy, primary (neoadjuvant)
chemotherapy, and both adjuvant radiation therapy and adjuvant
chemotherapy. In addition, radiation and chemotherapy are
frequently indicated as adjuvants to surgery in the treatment of
cancer. The goal of radiation and chemotherapy in the adjuvant
setting is to reduce the risk of recurrence and enhance
disease-free survival when the primary tumor has been controlled.
Chemotherapy is utilized as a treatment adjuvant for colon, lung,
and breast cancer, frequently when the disease is metastatic.
Adjuvant radiation therapy is indicated in several diseases
including colon, lung, and breast cancers as described above. For
example, radiation frequently is used both pre- and post-surgery as
components of the treatment strategy for rectal carcinoma.
Compounds for the present invention are therefore particularly
useful following surgery in the treatment of cancer in combination
with radio- and/or chemotherapy.
[0164] A compound of the present invention also can radiosensitize
a cell. The term "radiosensitize," as used herein, is defined as a
molecule, preferably a low molecular weight molecule, administered
to human or other animal in a therapeutically effective amount to
increase the sensitivity of the cells to be radiosensitized to
electromagnetic radiation and/or to promote the treatment of
diseases that are treatable with electromagnetic radiation.
Diseases that are treatable with electromagnetic radiation include
neoplastic diseases, benign and malignant tumors, and cancerous
cells.
[0165] Electromagnetic radiation treatment of other diseases not
listed herein also is contemplated by the present invention. The
terms "electromagnetic radiation" and "radiation" as used herein
include, but are not limited to, radiation having the wavelength of
10.sup.-20 to 100 meters. Preferred embodiments of the present
invention employ the electromagnetic radiation of: gamma-radiation
(10.sup.-20 to 10.sup.-13 m), X-ray radiation (10.sup.-12 to
10.sup.-9 m), ultraviolet light (10 nm to 400 nm), visible light
(400 nm to 700 nm), infrared radiation (700 nm to 1.0 mm), and
microwave radiation (1 mm to 30 cm).
[0166] Many cancer treatment protocols currently employ
radiosensitizers activated by electromagnetic radiation, e.g.,
X-rays. Examples of X-ray-activated radiosensitizers include, but
are not limited to, the following: metronidazole, misonidazole,
desmethylmisonidazole, pimonidazole, etanidazole, nimorazole,
mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide,
5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),
bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea,
cisplatin, and therapeutically effective analogs and derivatives of
the same.
[0167] Photodynamic therapy (PDT) of cancers employs visible light
as the radiation activator of the sensitizing agent. Examples of
photodynamic radiosensitizers include the following, but are not
limited to: hematoporphyrin derivatives, PHOTOFRIN.RTM.,
benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2),
pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines,
phthalocyanines, zinc phthalocyanine, and therapeutically effective
analogs and derivatives of the same.
[0168] Radiosensitizers can be administered in conjunction with a
therapeutically effective amount of one or more compounds in
addition to the Chk1 inhibitor, such compounds including, but not
limited to, compounds that promote the incorporation of
radiosensitizers to the target cells, compounds that control the
flow of therapeutics, nutrients, and/or oxygen to the target cells,
chemotherapeutic agents that act on the tumor with or without
additional radiation, or other therapeutically effective compounds
for treating cancer or other disease. Examples of additional
therapeutic agents that can be used in conjunction with
radiosensitizers include, but are not limited to, 5-fluorouracil
(5-FU), leucovorin, oxygen, carbogen, red cell transfusions,
perfluorocarbons (e.g., FLUOSOLW.RTM.-DA), 2,3-DPG, BW12C, calcium
channel blockers, pentoxifylline, antiangiogenesis compounds,
hydralazine, and L-BSO.
[0169] Chemotherapeutic agents that can be used include, but are
not limited to, alkylating agents, antimetabolites, hormones and
antagonists thereof, radioisotopes, antibodies, as well as natural
products, and combinations thereof. For example, an inhibitor
compound of the present invention can be administered with
antibiotics, such as doxorubicin and other anthracycline analogs,
nitrogen mustards, such as cyclophosphamide, pyrimidine analogs
such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its
natural and synthetic derivatives, and the like. As another
example, in the case of mixed tumors, such as adenocarcinoma of the
breast, where the tumors include gonadotropin-dependent and
gonadotropin-independent cells, the compound can be administered in
conjunction with leuprolide or goserelin (synthetic peptide analogs
of LH-RH). Other antineoplastic protocols include the use of an
inhibitor compound with another treatment modality, e.g., surgery
or radiation, also referred to herein as "adjunct anti-neoplastic
modalities." Examples of chemotherapeutic agents useful for the
method of the present invention are listed in the following
table.
TABLE-US-00001 Alkylating agents Nitrogen mustards mechlorethamine
cyclophosphamide ifosfamide melphalan chlorambucil Nitrosoureas
carmustine (BCNU) lomustine (CCNU) semustine (methyl-CCNU)
Ethylenimine/Methylmelamine thriethylenemelamine (TEM) triethylene
thiophosphoramide (thiotepa) hexamethylmelamine (HMM, altretamine)
Alkyl sulfonates busulfan Triazines dacarbazine (DTIC)
Antimetabolites Folic Acid analogs methotrexate trimetrexate
Pyrimidine analogs 5-fluorouracil fluorodeoxyuridine gemcitabine
cytosine arabinoside (AraC, cytarabine) 5-azacytidine
2,2'-difluorodeoxycytidine Purine analogs 6-mercaptopurine
6-thioguanine azathioprine 2'-deoxycoformycin (pentostatin)
erythrohydroxynonyladenine (EHNA) fludarabine phosphate
2-chlorodeoxyadenosine (cladribine, 2-CdA) Type I Topoisomerase
Inhibitors camptothecin topotecan irinotecan Natural products
Antimitotic drugs paclitaxel Vinca alkaloids vinblastine (VLB)
vincristine vinorelbine Taxotere .RTM. (docetaxel) estramustine
estramustine phosphate Epipodophylotoxins etoposide teniposide
Antibiotics actimomycin D daunomycin (rubidomycin) doxorubicin
(adriamycin) mitoxantroneidarubicin bleomycinsplicamycin
(mithramycin) mitomycinC dactinomycin Enzymes L-asparaginase
Biological response modifiers interferon-alpha IL-2 G-CSF GM-CSF
Differentiation Agents retinoic acid derivatives Radiosensitizers
metronidazole misonidazole desmethylmisonidazole pimonidazole
etanidazole nimorazole RSU 1069 EO9 RB 6145 SR4233 nicotinamide
5-bromodeozyuridine 5-iododeoxyuridine bromodeoxycytidine
Miscellaneous agents Platinium coordination complexes cisplatin
carboplatin Anthracenedione mitoxantrone Substituted urea
hydroxyurea Methylhydrazine derivatives N-methylhydrazine (MIH)
procarbazine Adrenocortical suppressant mitotane (o,p'-DDD)
ainoglutethimide Cytokines interferon.(,.,) interleukin-2 Hormones
and antagonists Adrenocorticosteroids/antagonists prednisone and
equivalents dexamethasone ainoglutethimide Progestins
hydroxyprogesterone caproate medroxyprogesterone acetate megestrol
acetate Estrogens diethylstilbestrol ethynyl estradiol/equivalents
Antiestrogen tamoxifen Androgens testosterone propionate
fluoxymesterone/equivalents Antiandrogens flutamide
gonadotropin-releasing hormone analogs leuprolide Nonsteroidal
antiandrogens flutamide Photosensitizers hematoporphyrin
derivatives Photofrin .RTM. benzoporphyrin derivatives Npe6 tin
etioporphyrin (SnET2) pheoboride-a bacteriochlorophyll-a
naphthalocyanines phthalocyanines zinc phthalocyanines
[0170] Examples of chemotherapeutic agents that are particularly
useful in conjunction with radiosensitizers include, for example,
adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin,
doxorubicin, interferon (alpha, beta, gamma), interleukin 2,
irinotecan, docetaxel, paclitaxel, topotecan, and therapeutically
effective analogs and derivatives of the same.
[0171] As appreciated by persons skilled in the art, reference
herein to treatment extends to prophylaxis, as well as to treatment
of established diseases or symptoms. It is further appreciated that
the amount of a compound of the invention required for use in
treatment varies with the nature of the condition being treated,
and with the age and the condition of the patient, and is
ultimately determined by the attendant physician or veterinarian.
In general, however, doses employed for adult human treatment
typically are in the range of 0.001 mg/kg to about 100 mg/kg per
day. The desired dose can be conveniently administered in a single
dose, or as multiple doses administered at appropriate intervals,
for example as two, three, four or more subdoses per day. In
practice, the physician determines the actual dosing regimen most
suitable for an individual patient, and the dosage varies with the
age, weight, and response of the particular patient. The above
dosages are exemplary of the average case, but there can be
individual instances in which higher or lower dosages are merited,
and such are within the scope of the present invention.
[0172] Formulations of the present invention can be administered in
a standard manner for the treatment of the indicated diseases, such
as orally, parenterally, transmucosally (e.g., sublingually or via
buccal administration), topically, transdermally, rectally, via
inhalation (e.g., nasal or deep lung inhalation). Parenteral
administration includes, but is not limited to intravenous,
intraarterial, intraperitoneal, subcutaneous, intramuscular,
intrathecal, and intraarticular. Parenteral administration also can
be accomplished using a high pressure technique, like
POWDERJECT.TM..
[0173] For oral administration, including buccal administration,
the composition can be in the form of tablets or lozenges
formulated in conventional manner. For example, tablets and
capsules for oral administration can contain conventional
excipients such as binding agents (for example, syrup, acacia,
gelatin, sorbitol, tragacanth, mucilage of starch, or
polyvinylpyrrolidone), fillers (for example, lactose, sugar,
microcrystalline cellulose, maize-starch, calcium phosphate, or
sorbitol), lubricants (for example, magnesium stearate, stearic
acid, talc, polyethylene glycol or silica), disintegrants (for
example, potato starch or sodium starch glycolate), or wetting
agents (for example, sodium lauryl sulfate). The tablets can be
coated according to methods well known in the art.
[0174] Alternatively, the compounds of the present invention can be
incorporated into oral liquid preparations such as aqueous or oily
suspensions, solutions, emulsions, syrups, or elixirs, for example.
Moreover, formulations containing these compounds can be presented
as a dry product for constitution with water or other suitable
vehicle before use. Such liquid preparations can contain
conventional additives, for example suspending agents, such as
sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin,
hydroxyethylcellulose, hydroxypropylmethylcellulose,
carboxymethylcellulose, aluminum stearate gel, and hydrogenated
edible fats; emulsifying agents, such as lecithin, sorbitan
monooleate, or acacia; nonaqueous vehicles (which can include
edible oils), such as almond oil, fractionated coconut oil, oily
esters, propylene glycol, and ethyl alcohol; and preservatives,
such as methyl or propyl p-hydroxybenzoate and sorbic acid.
[0175] Such preparations also can be formulated as suppositories,
e.g., containing conventional suppository bases, such as cocoa
butter or other glycerides. Compositions for inhalation typically
can be provided in the form of a solution, suspension, or emulsion
that can be administered as a dry powder or in the form of an
aerosol using a conventional propellant, such as
dichlorodifluoromethane or trichlorofluoromethane. Typical topical
and transdermal formulations comprise conventional aqueous or
nonaqueous vehicles, such as eye drops, creams, ointments, lotions,
and pastes, or are in the form of a medicated plaster, patch, or
membrane.
[0176] Additionally, compositions of the present invention can be
formulated for parenteral administration by injection or continuous
infusion. Formulations for injection can be in the form of
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and can contain formulation agents, such as suspending,
stabilizing, and/or dispersing agents. Alternatively, the active
ingredient can be in powder form for constitution with a suitable
vehicle (e.g., sterile, pyrogen-free water) before use.
[0177] A composition in accordance with the present invention also
can be formulated as a depot preparation. Such long acting
formulations can be administered by implantation (for example,
subcutaneously or intramuscularly) or by intramuscular injection.
Accordingly, the compounds of the invention can be formulated with
suitable polymeric or hydrophobic materials (e.g., an emulsion in
an acceptable oil), ion exchange resins, or as sparingly soluble
derivatives (e.g., a sparingly soluble salt).
[0178] For veterinary use, a compound of formula (I), (II), or
(III), or a nontoxic salt thereof, is administered as a suitably
acceptable formulation in accordance with normal veterinary
practice. The veterinarian can readily determine the dosing regimen
and route of administration that is most appropriate for a
particular animal.
[0179] Thus, the invention provides a pharmaceutical composition
comprising a compound of the formula (I), (II), or (III), together
with a pharmaceutically acceptable diluent or carrier therefor.
Also provided is a process of preparing a pharmaceutical
composition comprising a compound of formula (I), (II), or (III)
comprising mixing a compound of formula (I), (II), or (III),
together with a pharmaceutically acceptable diluent or carrier
therefor.
[0180] Specific, nonlimiting examples of compounds of structural
formula (I), (II), and (III) are provided below, the synthesis of
which were performed in accordance with the procedures set forth
below.
[0181] For ease of understanding, a compound having a particular
structure is identified by the corresponding compound number
provided in the following tables summarizing some of the compounds
useful in the method. For example, the structure identified as
Compound 1 is a compound of structural formula (IV), wherein
R.sup.27 is hydrogen and R.sup.28 is
--C(O)NH(CH.sub.2).sub.2(2-N-methylpyrrolidyl).
[0182] Compounds suitable in the method include, but are not
limited to:
TABLE-US-00002 (IV) ##STR00022## Compound No. R.sup.27 R.sup.28 1 H
##STR00023## 2 H ##STR00024## 3 H ##STR00025## 4 H ##STR00026## 5 H
##STR00027## 6 H ##STR00028## 7 H ##STR00029## 8 H ##STR00030## 9 H
##STR00031## 10 ##STR00032## H 11 ##STR00033## H 12 CH.sub.3 H 13
##STR00034## H 14 NH.sub.2 H 15 ##STR00035## H 16 ##STR00036## H 17
##STR00037## H 18 ##STR00038## H 19 H ##STR00039## 20 ##STR00040##
H 21 Cl ##STR00041## 22 ##STR00042## H 23 Cl H 24 H ##STR00043## 25
H ##STR00044## 26 ##STR00045## H 27 H ##STR00046## 28 H
##STR00047## 29 H Cl 30 ##STR00048## H 31 ##STR00049## H 32 H
NH.sub.2 33 H ##STR00050## 34 H ##STR00051## 35 H ##STR00052## 36
##STR00053## H 37 ##STR00054## H 38 H H 39 H ##STR00055## 40 H
##STR00056## 41 H ##STR00057## 42 ##STR00058## H 43 H ##STR00059##
44 H ##STR00060## 45 ##STR00061## H 46 H ##STR00062## 47 H
##STR00063## 48 ##STR00064## H 49 ##STR00065## H 50 ##STR00066## H
51 ##STR00067## H 52 H ##STR00068## 53 H ##STR00069## 54
##STR00070## H 55 ##STR00071## H 56 ##STR00072## H 57 H
##STR00073## 58 H ##STR00074## 59 ##STR00075## H 60 H ##STR00076##
61 H ##STR00077## 62 ##STR00078## H 63 ##STR00079## H 64
##STR00080## H 65 H ##STR00081## 66 H ##STR00082## 67 H
##STR00083## 68 H ##STR00084## 69 H ##STR00085## 70 H ##STR00086##
71 H ##STR00087## 72 H ##STR00088## 73 H ##STR00089## 74 H
##STR00090## 75 H ##STR00091## 76 H ##STR00092## 77 ##STR00093## H
78 ##STR00094## H 79 ##STR00095## H 80 ##STR00096## H 81
##STR00097## H 82 ##STR00098## H 83 ##STR00099## H 84 ##STR00100##
H 85 ##STR00101## H 86 ##STR00102## H 87 ##STR00103## H 88
##STR00104## H
Hetero Ring Substitutions:
TABLE-US-00003 [0183] (V) ##STR00105## Compound No. R.sup.29 89
##STR00106## 90 ##STR00107## 91 ##STR00108## 92 ##STR00109## 93
##STR00110## 94 ##STR00111## 95 ##STR00112## 96 ##STR00113## 97
##STR00114## 98 ##STR00115## 99 ##STR00116## 100 ##STR00117## 101
##STR00118## 102 ##STR00119## 103 ##STR00120## 104 ##STR00121## 105
##STR00122## 106 ##STR00123## 107 ##STR00124##
Thiourea Compounds:
TABLE-US-00004 [0184] (VI) ##STR00125## Compound No. R.sup.30
R.sup.31 108 ##STR00126## H 109 ##STR00127## H 110 ##STR00128## H
111 ##STR00129## H 112 ##STR00130## Cl 113 ##STR00131## H 114
##STR00132## Cl
[0185] Miscellaneous Class:
TABLE-US-00005 (VII) ##STR00133## Compound No. R.sup.32 Ar.sup.1
Ar.sup.2 115 --N(H)-- ##STR00134## ##STR00135## 116 --N(H)--
##STR00136## ##STR00137## 117 --N(H)-- ##STR00138## ##STR00139##
118 --N(CH.sub.3)-- ##STR00140## ##STR00141## 119 --N(H)--
##STR00142## ##STR00143## 120 --N(H)-- ##STR00144## ##STR00145##
121 --N(H)-- ##STR00146## ##STR00147## 122 --N(H)-- ##STR00148##
##STR00149## 123 --N(H)-- ##STR00150## ##STR00151## 124 --N(H)--
##STR00152## ##STR00153## 125 --N(H)-- ##STR00154## ##STR00155##
126 --N(H)-- ##STR00156## ##STR00157## 127 --N(H)-- ##STR00158##
##STR00159## 128 --O-- ##STR00160## ##STR00161## 129 --N(H)--
##STR00162## ##STR00163## 130 --N(H)-- ##STR00164## ##STR00165##
131 --N(H)-- ##STR00166## ##STR00167## 132 null ##STR00168##
##STR00169## 133 --N(H)-- ##STR00170## ##STR00171## 134 --N(H)--
##STR00172## ##STR00173## 135 --O-- ##STR00174## ##STR00175## 136
--N(H)-- ##STR00176## ##STR00177## 137 --N(H)-- ##STR00178##
##STR00179## 138 --N(H)-- ##STR00180## ##STR00181## 139 --N(H)--
##STR00182## ##STR00183## 140 --O-- ##STR00184## ##STR00185## 141
--O-- ##STR00186## ##STR00187## 142 --N(H)-- ##STR00188##
##STR00189## 143 --O-- ##STR00190## ##STR00191## 144 --N(H)--
##STR00192## ##STR00193## 145 --N(H)-- ##STR00194## ##STR00195##
146 --N(H)-- ##STR00196## ##STR00197## 147 --N(H)-- ##STR00198##
##STR00199## 148 --N(H)-- ##STR00200## ##STR00201## 149 --O--
##STR00202## ##STR00203## 150 --N(H)-- ##STR00204## ##STR00205##
151 --N(H)-- ##STR00206## ##STR00207## 152 --N(H)-- ##STR00208##
##STR00209## 153 --N(H)-- ##STR00210## ##STR00211## 154 --N(H)--
##STR00212## ##STR00213## 155 --N(H)-- ##STR00214## ##STR00215##
156 --N(H)-- ##STR00216## ##STR00217## 157 --N(H)-- ##STR00218##
##STR00219## 158 --N(H)-- ##STR00220## ##STR00221## 159 --N(H)--
##STR00222## ##STR00223## 160 --N(H)-- ##STR00224## ##STR00225##
161 --N(H)-- ##STR00226## ##STR00227## 162 --N(H)-- ##STR00228##
##STR00229## 163 --N(H)-- ##STR00230## ##STR00231## 164 --N(H)--
##STR00232## ##STR00233## 165 --N(H)-- ##STR00234##
##STR00235##
[0186] Some preferred compounds include Compound Nos. 2, 4, 6, 12,
72, 76, 83, 84, 88, 89, and 90.
[0187] Generally, compounds of structural formulae (I), (II), and
(III), including those of formulae (IV), (V), (VI), and (VII), can
be prepared according to the following synthetic scheme. In the
scheme described below, it is understood in the art that protecting
groups can be employed where necessary in accordance with general
principles of synthetic chemistry. These protecting groups are
removed in the final steps of the synthesis under basic, acidic, or
hydrogenolytic conditions which are readily apparent to those
skilled in the art. By employing appropriate manipulation and
protection of any chemical functionalities, synthesis of compounds
of structural formulae (I), (II), and (III) not specifically set
forth herein can be accomplished by methods analogous to the
schemes set forth below.
[0188] Unless otherwise noted, all starting materials were obtained
from commercial suppliers and used without further purification.
All reactions and chromatography fractions were analyzed by
thin-layer chromatography on 250-mm silica gel plates, visualized
with UV (ultraviolet) light and I.sub.2 (iodine) stain. Flash
column chromatography was carried out using Biotage 40M silica gel
(230-400 mesh). Products and intermediates were purified by flash
chromatography or reverse-phase HPLC.
[0189] As illustrated below, the compounds of general structural
formulae (I) and (II) can be prepared by the following general
synthetic schemes.
##STR00236##
[0190] In general, an aryl amine represented by the formula
Ar--NH.sub.2 is reacted with about 0.75-1.25 molar equivalent of
4-nitrophenyl chloroformate. The reaction preferably is performed
under an inert atmosphere, for example, nitrogen (N.sub.2), and
typically is maintained at low temperature (about 0.degree. C.).
The resulting product is treated with about 0.75-1.25 molar
equivalent of a heteroaryl amine represented by the formula
HetAr--NH.sub.2, preferably under an inert atmosphere at room
temperature (about 25.degree. C.), to afford a crude aryl
pyrazine-disubstituted urea compound.
[0191] A more particular illustration for the preparation compounds
of standard formulae (I) and (II) can include, for example, the
following General Scheme 2.
Step (1): TMS Diazomethane Esterification
[0192] To a cooled (about 0.degree. C.), stirred solution of
4-amino-3-methoxybenzoic acid (5.0 g; 30 mmol) in dry methanol (150
mL) was added trimethylsilyl diazomethane (60 mL of 2.0 M solution
in hexanes, 120 mmol) slowly over 1 hour. After stirring for 4
hours, the reaction was concentrated at reduced pressure, dissolved
in ethyl acetate (200 mL), washed with 10% aqueous sodium carbonate
and brine, then dried (MgSO.sub.4), filtered, and concentrated in
vacuo to
##STR00237##
provide the desired ester as an off-white solid (94% yield). Step
(2): p-Nitrophenyl Carbamate Procedure
[0193] To a stirred, cooled (about 0.degree. C.) solution of
methyl-3-amino-4-methoxy benzoate (5.0 g; 27.6 mmol) in dry
dichloromethane (175 mL) was added pyridine (2.34 mL; 29 mmol)
followed by 4-nitrophenyl chloroformate (5.8 g; 29 mmol) under a
nitrogen (N.sub.2) atmosphere. After stirring for 8 hours, the
reaction was washed with 2N aqueous hydrochloric acid (2.times.200
mL), saturated aqueous sodium bicarbonate (2.times.200 mL), and
brine (200 mL), then dried (MgSO.sub.4), and filtered. The filtered
solution was diluted with ethyl acetate and hexanes (about 800 mL)
until a precipitate formed. The solid was collected on a Buchner
funnel with suction, and air dried to provide the desired carbamate
as a white solid (70% yield).
Step (3): Carbamate Coupling Procedure
[0194] To a stirred solution of
4-methoxy-3-(4-nitro-phenoxycarbonylamino)-benzoic acid methyl
ester (30 g; 8.7 mmol) in dry N-methyl pyrrolidine (50 mL) was
added the amino pyrazine (0.84 g; 8.8 mmol) under a N.sub.2
atmosphere at room temperature. The reaction mixture was heated to
80.degree. C. for 6 hours, then allowed to cool to room
temperature. Dilution with ethyl acetate (200 mL) and water (200
mL) provided the desired urea as a white solid (54% yield).
Step (4): Lithium Hydroxide Hydrolysis Procedure
[0195] To a stirred solution of
4-methoxy-4-(3-pyrazin-2-yl-ureido)-benzoic acid methyl ester (1.0
g; 3.3 mmol) in methanol (35 mL) was added aqueous lithium
hydroxide (5 mL of a 2N solution; 10 mmol) at room temperature. The
reaction was heated to 67.degree. C. for 15 hours, then allowed to
cool to room temperature. The reaction then was diluted with water
(100 mL), and washed with ethyl acetate (2.times.100 mL). The pH of
the aqueous layer was adjusted to pH 5.2 with 2N aqueous
hydrochloric acid, and the resulting precipitate was collected on a
Buchner funnel with suction and air-dried to provide the desired
acid as a white solid.
Step (5): HBTU Coupling Procedure
[0196] To a stirred solution of the acid (30 mg; 0.11 mmol) in dry
N-methyl pyrrolidinone (2 mL) was added
O-benzotrazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HBTU; 45 mg; 0.12 mmol), 4-(2-aminioethyl)-morpholine (15.7 L;
0.12 mmol) and diisopropyl ethyl amine (34 L, 0.2 mmol) at room
temperature under a nitrogen atmosphere. The resulting solution was
stirred 5 hours, then diluted with ethyl acetate (30 mL) and 10%
aqueous sodium carbonate (30 mL). After stirring vigorously at room
temperature for 15 minutes, the precipitate was collected on a
Buchner funnel with suction and air-dried to provide the desired
amide as a white solid (59% yield).
[0197] The following compounds were prepared using the general
procedure described accompanying General Scheme 2, but substituting
the R group below for the R group shown in General Scheme 2:
TABLE-US-00006 Compound No. R group Characterization 22
##STR00238## .sup.1H NMR (400 MHz, D.sub.6 DMSO) d 10.15 (br s,
1H), 8.90 (s, 1H), 8.69 (d, 1H), 8.53 (t, 1H), 8.27 (t, 1H), 8.18
(d, 1H), 7.53 (dd, 1H), 3.96 (br s, 2H), 3.93 (s, 3H), 3.67-3.53
(m, 6H), 3.29 (t, 2H), 3.10 (t, 2H). LRMS (esi, positive) m/e 401.2
(M + 1). 11 ##STR00239## .sup.1H NMR (400 MHz, D.sub.6 DMSO) d
10.14 (br s, 1H), 8.91 (s, 1H), 8.66 (d, 1H), 8.27 (dd, 1H), 8.18
(d, 1H), 7.51 (dd, 1H), 7.03 (d, 1H), 3.91 (s, 3H), 3.53 (br s,
1H), 3.33 (d, 2H), 3.21 (br s, 1H), 3.02 (br s, 1H), 2.78 (d, 2H),
2.48 (s, 3H), 2.31 (br s, 1H), 2.13 (br s, 1H), 1.99-1.82 (m, 2H).
LRMS (esi, positive) m/e 399.2 (M + 1). 25 ##STR00240## .sup.1H NMR
(400 MHz, D.sub.6 DMSO) d 10.20 (br s, 1H), 8.87 (s, 1H), 8.31-
8.26 (M, 2H), 8.21 (br s, 1H), 7.52- 7.44 (m, 2H), 3.95 (s, 3H),
3.72 (t, 2H), 3.56 (t, 3H). 28 ##STR00241## .sup.1H NMR (400 MHz,
D.sub.6 DMSO) d 10.22 (br s, 1H), 8.90 (br s, 1H), 8.64 (t, 1H),
8.31-8.28 (m, 2H), 8.19 (d, 1H), 7.51-7.47 (m, 2H), 3.97 (br s,
2H), 3.93 (s, 3H), 3.69-3.52 (m, 6H), 3.30 (t, 2H), 3.11 (t, 2H).
LRMS (esi, positive) m/e 401.1 (M + 1). 1 ##STR00242## .sup.1H NMR
(400 MHz, D.sub.6 DMSO) d 10.20 (br s, 1H), 8.88 (br s, 1H), 8.40
(t, 1H), 8.32 (t, 1H), 8.23 (dd, 2H), 7.47 (d, 1H), 7.43 (dd, 1H),
3.94 (s, 3H), 2.91 (c, 2H), 2.19 (s, 3H), 2.05-1.98 (m, 2H),
1.93-1.80 (m, 2H), 1.60 (c, 1H), 1.43 (c, 1H). LRMS (esi, positive)
m/e 399.0 (M + 1). 35 ##STR00243## .sup.1H NMR (400 MHz, D.sub.6
DMSO) d 10.21 (br s, 1H), 8.88 (br s, 1H), 8.48 (br s, 1H), 8.32
(t, 1H), 8.26 (s, 1H), 8.23 (d, 1H), 7.50 (br s, 1H), 7.46 (dd,
1H), 3.95 (s, 3H), 3.36 (c, 2H), 3.10 (t, 2H), 2.83 (s, 3H). LRMS
(esi, positive) m/e 409.0 (M + 1). 44 ##STR00244## .sup.1H NMR (400
MHz, D.sub.6 DMSO) d 11.47 (br s, 1H), 8.48 (br s, 1H), 8.41 (s,
1H), 8.35 (d, 1H), 8.25-8.23 (M, 2H), 7.06-7.02 (m, 2H), 3.98 (s,
3H), 3.38 (br s, 2H), 3.04 (br s, 3H), 1.21 (br s, 3H). LRMS (esi,
positive) m/e 330.0 (M + 1).
##STR00245##
Isocyanate Procedure:
[0198] To a stirred solution of 2-methoxy-5-methyl-phenylisocyanate
(43 mL; 0.3 mmol) in dry dichloroethane (0.4 mL) was added
2-aminoquinoxaline (43.5 mg; 0.3 mmol) in a reaction vial under a
nitrogen atmosphere. The vial was capped and heated to 80.degree.
C. overnight (14 hours). The reaction mixture then was filtered,
and the residue washed with dichloromethane to provide the desired
urea as a white solid (91% yield).
[0199] The following compounds were prepared using the procedure
described accompanying General Scheme 3, but substituting the Ar
group in the table below for the Ar group in General Scheme 3:
TABLE-US-00007 Compound No. Ar group Characterization 90
##STR00246## .sup.1H NMR (400 MHz, D.sub.6 DMSO) d 11.65 (br s,
1H), 10.58 (br s, 1H), 10.58 (br s, 1H), 8.80 (s, 1H), 8.15 (d,
1H), 7.98 (d 1H), 7.84-7.80 (m, 2H), 7.65 (c, 1H), 6.98 (d, 1H),
6.98 (d, 1H), 6.84 (dd, 1H), 3.99 (s, 3H), 2.25 (s, 3H). LRMS (esi,
positive) m/e 309.1 (M + 1). 98 ##STR00247## .sup.1H NMR (400 MHz,
D.sub.6 DMSO) d 9.61 (s, 1H), 8.06 (d, 1H), 7.35 (d, 1H), 6.88 (dd,
1H), 8.87 (d, 1H), 5.42 (d, 1H), 3.89 (s, 3H), 2.33 (s, 3H). LRMS
(esi, positive) m/e 259.0 (M + 1). 97 ##STR00248## .sup.1H NMR (400
MHz, D.sub.6 DMSO) d 11.29 (s, 1H), 10.64 (s, 1H), 9.05 (d, 1H),
8.70 (d, 1H), 8.05 (d, 1H), 6.92 (d, 1H), 6.81 (c, 1H), 3.85 (s,
3H), 2.24 (s, 3H). LRMS (esi, positive) m/e 259.0 (M + 1). 91
##STR00249## .sup.1H NMR (400 MHz, D.sub.6 CDCl.sub.3) d 11.12 (br
s, 1H), 8.61 (br s, 1H), 8.28 (s, 1H), 8.24 (s, 1H), 8.14 (d, 1H),
8.04-8.01 (m, 2H), 7.55-7.50 (m, 3H), 6.86 (dd, 1H), 6.76 (d, 1H),
3.52 (s, 3H), 2.35 (s, 3H). LRMS (esi, positive) m/e 335.2 (M + 1).
12 ##STR00250## .sup.1H NMR (300 Mhz, d.sub.6-DMSO) .delta.: 10.10
(s, 1H), 10.00 (br s, 1H), 8.90 (s, 1H), 8.32 (s, 1H), 8.23 (s,
1H), 8.04 (s, 1H), 7.93 (d, 1H), 6.81 (d, 1H), 3.87 (s, 3H), 2.28
(s, 3H). .sup.13C NMR (75 Mhz, d.sub.6-DMSO) .delta.: 151.5, 149.3,
146.0, 140.9, 137.2, 135.2, 129.2, 127.7, 122.7, 119.5, 110.8,
55.9, 20.5
Examples
Example 1
Preparation of Compound 115
##STR00251##
[0200] 1-[2-(1,1-difluoromethoxy)-phenyl]-3-pyrazin-2-yl-urea
[0201] 2-(Difluoromethoxy)phenylisocyanate (1.0 g, 5.4 mmol) and
aminopyrazine (0.51 g, 5.4 mmol) were reacted for 6 hours in
refluxing dimethoxyethane (20 mL). The reaction mixture was cooled
to room temperature to precipitate the product, which was collected
by filtration, washed with ethyl acetate, and dried in vacuo (765
mg, 50%). .sup.1H NMR (300 Mhz, d.sub.6-DMSO): .delta. 10.49 (br s,
1H), 10.26 (s, 1H), 8.83 (s, 1H), 8.35-8.24 (m, 3H), 7.53-7.00
(m,4H).
Example 2
Preparation of Compound 165
##STR00252##
[0202] 1-(2-methylsulfanylphenyl)-3-pyrazin-2-yl urea
[0203] 2-(Methylthiophenyl)-isocyanate (1.0 g, 6.1 mmol) and
aminopyrazine (0.58 g, 6.1 mmol) were reacted for 16 hours in
refluxing dimethoxyethane (40 mL). The product precipitated from
the cooled reaction mixture and was collected by filtration, washed
with dimethoxyethane, and dried in vacuo (715 mg, 45%). .sup.1H NMR
(300 MHz, d.sub.6-DMSO) .delta.: 10.35(br s, 1H), 10.29 (s, 1H),
8.84 (s, 1H), 8.33 (s, 1H), 8.27 (s, 1H), 8.09 (d, 1H), 7.45 (d,
1H), 7.29, (t, 1H), 7.10 (t, 1H), 2.43 (s, 3H). .sup.13C NMR (75
Mhz, d.sub.6-DMSO) .delta.: 151.8, 149.2, 140.5, 137.5, 137.3,
135.2, 130.1, 127.1, 126.9, 123.7, 121.4, 16.5.
Example 3
Preparation of Compound 159
##STR00253##
[0204] 1-(2-methoxy-5-nitrophenyl)-3-pyrazin-2-yl-urea
[0205] A mixture of 2-methoxy-5-nitrophenyl isocyanate (5.0 g, 25
mmol) and aminopyrazine (2.5 g, 26 mmol) in tetrahydrofuran (THF,
250 mL) was stirred at reflux for 24 hours. The product was
precipitated from the cooled reaction mixture and was collected by
filtration, washed with ethyl acetate, and dried in vacuo (4.3 g,
57%). .sup.1H NMR (300 MHz, d.sub.6-DMSO) (mixture of rotamers)
.delta.: 10.38 (br, s, 1H), 10.27 (s, 1H), 9.39, 8.88 (2 singlets,
1H), 9.10(d, 1H), 8.33 (s, 1H), 8.26 (d, 1H), 7.98-8.25 (m, 1H),
7.97-7.84 (m, 1H), 4.05, 4.03 (2 singlets, 77:28 ratio, 3H).
Example 4
Preparation of Compound 14
##STR00254##
[0206] 1-(5-amino-2-methoxyphenyl)-3-pyrazin-2-yl-urea
[0207] A solution of (2-methoxy-5-nitrophenyl)-3-pyrazin-2-yl-urea
(Compound 159, Example 3) (16.9 g, 55 mmol) in dimethylformamide
(DMF, 320 mL) was shaken under H.sub.2 in the presence of palladium
on carbon (Pd/C) catalyst (1.6 g, 10% Pd) at 80.degree. C. for 12
h. A second portion of catalyst was added (1.6 g) and shaking was
continued for an additional 8 h at the same temperature. The
solution was filtered through a pad of celite using an additional
200 mL of DMF. The filtrate was concentrated in vacuo and the
residue was triturated with methanol (100 mL). The solid was
collected, stirred in boiling methanol, and solids present (1.8 g)
were filtered off and discarded. The filtrate was cooled at
4.degree. C. overnight. Solids (1.4 g) were removed by filtration
and the filtrate was concentrated in vacuo to a tan solid (2.6 g).
The crude solid product was triturated with THF (200 mL), collected
by filtration, and dried in vacuo to afford the product as a tan
solid (1.85 g, 13%). .sup.1H NMR (300 Mhz, d.sub.6-DMSO) .delta.:
10.10 (s, 1H), 9.94 (br s, 1H), 8.89 (s, 1H), 8.32 (s, 1H), 8.22
(s, 1H), 7.58 (s, 1H), 7.75 (d, 1H), 6.21 (d, 1H), 4.70 (s, 2H),
3.76 (s, 3H). .sup.13C NMR (75 Mhz, d.sub.6-DMSO) .delta.: 151.4,
149.4, 142.6, 140.9, 139.8, 137.2, 135.2, 128.7, 112.8, 107.7,
106.0, 56.8.
Example 5
Preparation of Compound 48
##STR00255##
[0208] N-[4-methoxy-3-(3-pyrazin-2-yl-ureido)phenyl]-succinamic
acid
[0209] A solution of
1-(5-amino-2-methoxyphenyl)-3-pyrazin-2-yl-urea (Compound 14,
Example 4) (260 mg, 1 mmol) and succinic anhydride (131 mg, 1.3
mmol) in dry pyridine (10 mL) was stirred 16 h at room temperature.
The resulting solid was collected by filtration and triturated with
chloroform, and dried in vacuo to afford the off-White product (175
mg, 50%). .sup.1H NMR (300 Mhz, d.sub.6-DMSO) .delta.: 10.15 (s,
1H), 10.05 (s, 1H), 9.87 (s, 1H), 8.90 (s, 1H), 8.33 (s, 2H), 8.24
(s, 1H), 7.42 (ddd, J=8.8, 2.2 Hz, 1H), 6.96 (d, J=8.8 Hz, 1H),
3.87 (s, 3H), 2.54 (br s, 4H).
Example 6
Preparation of Compound 36
##STR00256##
[0210] (S)-1-(2,2,2-trifluoroethanoyl)pyrrolidine-2-carboxylic acid
[4-methoxy-3-(3-pyrazin-2-yl-ureido)phenyl]-amide
[0211] A solution of 1-(5-amino-methoxyphenyl)-3-pyrazin-2-yl-urea
(Compound 14, Example 4) (105 mg, 0.4 mmol) in dry pyridine (2 mL)
at 0.degree. C. was treated with a solution of
N-trifluoroacetyl-(S)-prolyl chloride (0.1 M in dichloromethane,
4.5 mL, 0.45 mmol) and stirred 2 h at room temperature. The
reaction was quenched with 1 N HCl (50 mL) and extracted with ethyl
acetate (3.times.50 mL). The combined organic layers were washed
with 1 N HCl (2.times.20 mL), water (20 mL), brine (20 mL), dried
over sodium sulfate, and concentrated in vacuo to a beige solid (60
mg). Recrystallization from acetonitrile yielded the final solid
product (30 mg, 17%). .sup.1H NMR (300 Mhz, d.sub.6-DMSO) .delta.:
(10.16-10.06, m, 3H), 8.90 (s, 1H), 8.36-8.30 (m, 2H), 8.25 (d,
J=2.6 Hz, 1H), 7.42 (dd, J=8.8, 2.6 Hz, 1H), 6.98 (d, J=8.8 Hz,
1H), 4.57 (dd, J=8.5, 4.4 Hz, 1H), 3.88 (s, 3H), 3.73 (t, J=6.5 Hz,
1H), 2.27-2.21 (m, 1H), 2.06-1.90 (m, 3H). LRMS (ESI positive) m/e
453.1 (M+1).
Example 7
Preparation of Compound 16
##STR00257##
[0212] (S)-pyrrolidine-2-carboxylic acid
[4-methoxy-3-(3-pyrazin-2-yl-ureido)-plienyl]-amide
[0213] A suspension of
(S)-1-(2,2,2-trifluoroethanoyl)-pyrrolidine-2-carboxylic acid
[4-methoxy-3-(3-pyrazin-2-yl-ureido)-phenyl]-amide (Compound 36,
Example 6) (22 mg, 0.05 mmol) in a mixture of methanol (MeOH, 5 mL)
and water (about 0.25 mL) was treated with KOH (100 mg, large
excess). Within 10 minutes, all ingredients were in solution. The
reaction mixture was treated with water (20 mL) and extracted with
ethyl acetate (2.times.20 mL). The organic layers were combined and
washed with water (10 mL) and brine (10 mL), dried with sodium
sulfate, and concentrated to a tan solid (13 mg, 75%). .sup.1H NMR
(300 MHz, d.sub.6-DMSO) .delta.: 10.13 (s, 1H), 10.03 (s, 1H), 9.85
(s, 1H), 8.90 (s, 1H), 8.38-8.28 (m, 2H), 8.25 (d, J=2.6 Hz, 1H),
7.41 (dd, J=8.8, 2.6 Hz), 6.98 (d, J=8.8 Hz, 1H), 3.88 (s, 3H, 3.69
(dd, J=8.7, 5.6 Hz, 1H), 2.94-2.87 (m, 2H), 2.10-1.97 (m, 1H),
1.81-1.60 (m, 3H). LRMS (ESI, positive) m/e 357.1 (M+1).
Example 8
Preparation of Compound 42
##STR00258##
[0214]
N-[4-methoxy-3-(3-pyrazin-2-yl-ureido)phenyl]-methanesulfonamide
[0215] A solution of
1-(5-amino-2-methoxyphenyl)-3-pyrazin-2-yl-urea (Compound 14,
Example 4) (260 mg, 1 mmol) in dry pyridine (15 mL) was treated
with methanesulfonyl chloride (0.08 mL, 1 mmol) and stirred 16 h at
room temperature. The reaction mixture was concentrated in vacuo
and the solid residue was triturated with ethanol, collected by
filtration, and dried in vacuo to afford the product (205 mg, 61%).
.sup.1H NMR (300 Mhz, d.sub.6-DMSO) .delta.: 10.16 (s, 1H), 10.07
(s, 1H), 9.40 (s, 1H), 8.92 (s, 1H), 8.35 (s, 1H), 8.27 (s, 1H),
8.19 (d, J=2.2 Hz, 1H), 7.04 (d, J=8.8 Hz, 1H), 6.91 (dd, J=8.7,
2.4 Hz, 1H), 3.91 (s, 3H), 2.91 (s, 3H).
Example 9
Preparation of Compound 65
##STR00259##
[0216] 1-(2-methoxy-4-nitrophenyl)-3-pyrazin-2-yl-urea
[0217] A mixture of 2-methoxy-4-nitrophenyl isocyanate (15.0 g, 77
mmol) and aminopyrazine (7.35 g, 77 mmol) in THF (600 mL) was
stirred at reflux for 24 hours. The product precipitated from the
cooled reaction mixture and was collected by filtration, washed
with ethyl acetate, triturated with hot ethanol, and dried in vacuo
(16.3 g, 73%). .sup.1H NMR (300 Mhz, d.sub.6DMSO) .delta.: 10.50
(br s, 1H), 10.42 (s, 1H), 8.94 (s, 1H), 8.48 (d, 1H), 8.39 (s,
1H), 8.32 (d, 1H), 7.95 (dd, J=9.1, 2.4 Hz, 1H), 7.84(d, J=2.4 Hz,
1H), 4.08 (s, 3H).
Example 10
Preparation of Compound 32
##STR00260##
[0218] 1-(4-amino-2-methoxyphenyl)-3-pyrazin-2-yl-urea
[0219] A solution of (2-methoxy-4-nitrophenyl)-3-pyrazin-2-yl-urea
(Compound 65, Example 9) (7.9 g, 27 mmol) in DMF (300 mL) was
shaken under H.sub.2 in the presence of Pd/C catalyst (1.6 g, 10%
Pd) at 110.degree. C. for 4 h. The mixture was filtered through a
pad of celite using an additional 200 mL of DMF. The filtrate was
concentrated in vacuo and the residue was recrystallized from
ethanol (with a hot filtration step) to yield the light gray
product (2.9 g, 41%). .sup.1H NMR (300 MHz, d.sub.6-DMSO),: 9.83
(s, 1H), 9.50 (s, 1H), 8.86 (s, 1H), 8.28 (s, 1H), 8.19 (d, J=2.5
Hz, 1H), 7.64 (d, J=8.5 Hz, 1H), 6.31 (d, J=2.0 Hz, 1H), 6.13 (dd,
J=8.5, 2.0 Hz, 1H), 4.92 (s, 2H), 3.79 (s, 3H). .sup.13C NMR (75
Mhz, d.sub.6-DMSO) .delta.: 151.6, 150.0, 149.6, 145.2, 140.9,
136.9, 135.1, 121.6, 116.8, 105.5, 98.0, 55.4.
Example 11
Preparation of Compound 3
##STR00261##
[0220]
C-dimethylamino-N-[3-methoxy-4-(3-pyrazin-2-yl-ureido)phenyl]-aceta-
mide
[0221] A solution of N,N-dimethylglycine (124 mg, 1.2 mmol) and
triethylamine (0.33 mL, 2.4 mmol) in dry acetonitrile (5 mL) at
0.degree. C. was treated dropwise with isobutyl chloroformate (0.16
mL, 1.2 mmol) and stirred 15 min. This mixture was treated dropwise
with a solution of 1-(4-amino-2-methoxyphenyl)-3-pyrazin-2-yl-urea
(Compound 32, Example 10) (100 mg, 0.4 mmol) in dimethyl sulfoxide
(DMSO, 1 mL). The reaction mixture was stirred at room temperature
for 3 h, quenched with water (20 mL), and extracted with ethyl
acetate (2.times.15 mL). The combined organic layers were washed
with water (10 mL) and brine (10 mL), dried over sodium sulfate,
and concentrated in vacuo. The residue was dissolved in DMSO (1 mL)
and purified by HPLC (YMC 20.times.50 mm C18 CombiPrep column, 20
mL/min, 2-50% CH.sub.3CN/water in 6 min, all solvents contained
0.05% trifluoroacetic acid (TFA), 0.35 mL injections, detector at
254 nm, detector path length 0.2 mm). Fractions containing the
product were concentrated in vacuo to afford the product as the
trifluoroacetate (TFA) salt (24 mg, 17%).
Example 12
Preparation of Compound 8
##STR00262##
[0222]
3-chloro-N-[3-methoxy-4-(3-pyrazin-2-yl-ureido)phenyl]-propionamide
[0223] A solution of
1-(4-amino-2-methoxyphenyl)-3-pyrazin-2-yl-urea (Compound 32,
Example 10) (259 mg, 1 mmol) in pyridine (3 mL) at 0.degree. C. was
treated with chloroacetyl chloride (0.29 mL, 3 mmol). The
suspension was warmed at 80.degree. C. until most solids dissolved,
the reaction mixture was cooled to room temperature and the product
was precipitated with ether (10 mL). This crude product was used
without purification for further reactions, but a portion (30 mg)
was purified by HPLC (Luna 10.times.250 mm C18 column, 4.7 mL/min,
2-80% CH.sub.3CN/water in 15 min, all solvents contained 0.05% TFA,
0.25 mL injections, detector at 254 nm, detector path length 0.3
mm). Fractions containing the product were concentrated in vacuo to
afford the product. .sup.1H NMR (300 Mhz, d.sub.6-DMSO) .delta.:
10.00 (s, 2H), 9.94 (s, 1H), 8.87 (s, 1H), 8.32 (dd, J=2.5, 1.5 Hz,
1H), 8.23 (d, J=2.6 Hz, 1H), 8.05 (d, J=8.7 Hz, 1H), 7.49 (d, J=2.1
Hz, 1H), 7.07 (dd, J=8.7, 2.1 Hz, 1H), 3.90-3.80 (m, 5H), 2.80 (t,
J=6.2 Hz, 2H). LRMS (ESI, positive) m/e 350, 352 (M+1).
Example 13
Preparation of Compound 4
##STR00263##
[0224]
(cyclohexyl-methyl-amino)-N-[3-methoxy-4-(3-pyrazin-2-yl-ureido)phe-
nyl]-propionamide
[0225] A mixture of
3-chloro-N-[3-methoxy-4-(3-pyrazin-2-yl-ureido)phenyl]-propionamide
(Compound 8, Example 12) and N-cyclohexyl-methylamine (0.5 mL,
large excess) was warmed at 80.degree. C. for 1 h and cooled to
room temperature. Crude product was precipitated from ether (10
mL), collected by filtration, and dissolved in DMSO (0.5 mL).
Aliquots (about 0.25 mL) were purified by HPLC (Luna 10.times.250
mm C18 column, 4.7 mL/min, 2-80% CH.sub.3CN/water in 15 min, all
solvents contained 0.05% trifluoroacetic acid, detector at 254 nm,
detector path length 0.3 mm). Fractions containing the product were
concentrated in vacuo to afford the product as the TFA salt (4.7
mg, 11%). .sup.1H NMR (300 Mhz, d.sub.6-DMSO) .delta.: 10.18 (s,
1H), 10.07 (s, 1H), 9.98 (s, 1H), 9.04 (br s, 1H), 8.88 (s, 1H),
8.33 (dd, J=2.6, 1.5 Hz, 1H), 8.24 (d, J=2.6 Hz, 1H), 8.08 (d,
J=8.7 Hz, 1H), 7.43(d, J=2.1 Hz, 1H), 7.09(dd, J=8.7, 2.1 Hz, 1H),
3.90-3.83 (m, 1H), 3.30-3.16 (m, 2H), 2.81 (t, J=6.7 Hz, 1H), 2.74
(d, J=5.0 Hz, 2H), 2.03-1.89 (m, 2H), 1.89-1.75 (m, 2H), 1.70-1.53
(m, 1H), 1.46-1.10(m, 5H). LRMS (ESU, positive) m/e 427.2
(M+1).
Example 14
Preparation of Compound 2
##STR00264##
[0226]
3-cyclopentylamino-N-[3-methoxy-4-(3-pyrazin-2-yl-ureido)phenyl]-pr-
opionamide
[0227] A mixture of
3-chloro-N-[3-methoxy-4-(3-pyrazin-2-yl-ureido)phenyl]-propionamide
(Compound 8, Example 12) and cyclopentylamine (0.5 mL, large
excess) was warmed at 80.degree. C. for 1 h and cooled to room
temperature. The product was precipitated from ether (10 mL),
collected by filtration, washed with ether, and dried in vacuo (24
mg, 60%). .sup.1H NMR (300 Mhz, d.sub.6-DMSO) .delta.: 10.14 (s,
1H), 10.03 (s, 1H), 9.93 (s, 1H), 8.87 (d, J=1.1 Hz, 1H), 8.31 (dd,
J=2.6, 1.5 Hz. 1H), 8.23 (d, J=2.7 Hz, 1H), 8.03(d, J=8.7 Hz, 1H),
7.47(d, J=2.1 Hz, 1H), 7.04(dd, J=8.7, 2.1 Hz, 1H), 3.87 (s, 3H),
3.05 (quintet, J=6.3 Hz, 1H), 2.80 (t, J=6.6 Hz, 2H), 2.44 (t,
J=6.6 Hz, 2H), 1.80-1.67 (m, 2H), 1.65-1.56 (m, 2H), 1.53-142 (m,
2H), 1.37-1.27 (m, 2H). LRMS (ESI, positive) m/e 399.1 (M+1).
[0228] Compound 166:
##STR00265##
3-Methoxy-4-(3-pyrazin-2-yl-ureido)-benzoic acid
[0229] Step 1: Methyl-3-amino-4-methoxy benzoate. To a cooled
(about 0.degree. C.), stirred solution of 4-amino-3-methoxybenzoic
acid (5.0 g, 30 mmol) in dry methanol (150 mL) was added
trimethylsilyldiazomethane (60 mL of 2M solution in hexanes, 120
mmol) slowly over 1 hour. After stirring for 4 hours, the reaction
was concentrated at reduced pressure, dissolved in ethyl acetate
(200 mL), washed with 10% aqueous sodium carbonate and brine, then
dried (MgSO.sub.4), filtered, and concentrated in vacuo to provide
the desired ester as an off-white solid (94% yield).
[0230] Step 2: 4-Methoxy-3-(4-nitro-phenoxycarbonylamino)-benzoic
acid methyl ester. To a stirred, cooled (about 0.degree. C.)
solution of methyl-3-amino-4-methoxy benzoate (5.0 g, 27.6 mmol) in
dry dichloromethane (90 mL) was added pyridine (2.34 mL, 29 mmol)
followed by 4-nitophenyl chloroformate (5.8 g, 29 mmol) under a
nitrogen atmosphere. After stirring for 1 hour, the reaction was
diluted to 200 mL with dichloromethane and washed with 2N aqueous
hydrochloric acid (2.times.200 mL), saturated aqueous sodium
bicarbonate (2.times.200 mL), and brine (200 mL), then dried
(MgSO.sub.4), and filtered. The filtered solution was concentrated
to a white solid corresponding to the desired carbamate (98%
yield).
[0231] Step 3: 3-Methoxy-4-(3-pyrazin-2-yl-ureido)-benzoic acid
methyl ester. To a stirred solution of
4-methoxy-3-(4-nitro-phenoxycarbonylamino)-benzoic acid methyl
ester (10.64 g, 30.7 mmol) in dry N-methyl pyrrolidinone (31 mL) at
room temperature under nitrogen was added aminopyrazine (2.92 g,
30.7 mmol) and the reaction was warmed to 85.degree. C. After 6
hours, the reaction was cooled to room temperature and triturated
with ethyl acetate (200 mL). The precipitate formed was filtered
off, rinsed with ethyl acetate and dried to give the urea as a tan
solid (66% yield).
[0232] Step 4: 3-Methoxy-4-(3-pyrazin-2-yl-ureido)-benzoic acid. To
a stirred suspension of 3-methoxy-4-(3-pyrazin-2-yl-ureido)-benzoic
acid methyl ester (6.07 g, 20 mmol) in 200 mL 3:1 MeOH:H.sub.20 at
room temperature under nitrogen was added lithium hydroxide
monohydrate (8.4 g, 200 mmol) and the reaction heated to 65.degree.
C. overnight. The reaction was then cooled to room temperature and
most of the methanol removed by rotary evaporation. The remaining
suspension was neutralized to pH about 4 with concentrated HCl. The
formed precipitate was isolated by filtration and rinsing with
H.sub.20 and then drying under high vacuum to give the desired acid
as a white solid (5.34 g, 93%).
[0233] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.89 (br s, 1H),
8.34 (s, 1H), 8.24 (s, 1H), 8.16 (d, 1H), 7.56 (s, 1H), 7.50 (d,
1H), 3.91 (s, 3H)
[0234] Compound 167:
##STR00266##
N-Butyl-3-methoxy-4-(3-pyrazin-2-yl-ureido)-benzamide
[0235] To a stirred suspension of Compound 1xx (32 mg, 0.11 mmol)
in 1 mL of NMP at room temperature in a capped reaction vial was
added HBTU (0.4 M in NMP, 300 .mu.L, 0.12 mmol) and the suspension
stirred for 15 minutes. N-Butyl amine (0.4 M in NMP, 300 .mu.L,
0.12 mmol) was then added followed by DIEA (38 .mu.L, 0.22 mmol).
The reaction was stirred at room temperature overnight and was then
diluted with EtOAc (20 mL) and 10% Na.sub.2CO.sub.3 (20 mL) and
stirred rapidly for 5 minutes. A precipitate formed which was
isolated by filtration and rinsing with H.sub.2O and EtOAc. After
air drying, the amide was isolated as an off-white solid (12.2 mg,
32%).
[0236] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (br s, 1H),
8.37 (br s, 2H), 8.23 (d, 1H), 8.21 (s, 1H), 7.53 (s, 1H), 7.47 (d,
1H), 3.97 (s, 3H), 3.23 (q, 2H), 1.52 (m, 2H), 1.35 (m, 2H), 0.92
(t, 3H)
[0237] LRMS (apci, positive) m/e 344.1 (M+1)
##STR00267##
N-Benzyl-3-methoxy-4-(3-pyrazin-2-yl-ureido)-benzamide
[0238] Prepared according to the procedure of Compound 167 using
benzyl amine (39% yield).
[0239] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.96 (t, 1H),
8.91 (s, 1H), 8.56 (s, 1H), 8.50 (s, 1H), 8.44 (m, 1H), 7.58 (s,
1H), 7.56 (d, 1H), 7.35 (m, 4H), 7.23 (m, 1H), 4.46 (d, 2H), 3.97,
(s, 3H)
[0240] LRMS (apci, positive) m/e 378.1 (M+1)
[0241] Compound 169:
##STR00268##
3-Methoxy-N-phenethyl-4-(3-pyrazin-2-yl-ureido)-benzamide
[0242] Prepared according to the procedure of Compound 167 using
phenethyl amine (49% yield).
[0243] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (br s, 1H),
8.51 (t, 1H), 8.36 (br s, 1H), 8.25 (s, 1H), 8.22 (d, 1H), 7.52 (s,
1H), 7.45 (d, 1H), 7.36-7.20 (m, 5H), 3.98 (s, 3H), 3.46 (m, 2H),
2.84 (dd, 2H)
[0244] LRMS (apci, positive) m/e 392.1 (M+1)
[0245] Compound 170:
##STR00269##
3-Methoxy-N-(3-phenyl-propyl)-4-(3-pyrazin-2-yl-ureido)-benzamide
[0246] Prepared according to the procedure of Compound 167 using
phenpropyl amine (71% yield).
[0247] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (br s, 1H),
8.40 (t, 1H), 8.36 (br s, 1H), 8.25 (d, 1H), 8.23 (s, 1H), 7.52 (s,
1H), 7.46 (d, 1H), 7.32-7.18 (m, 5H), 3.97 (s, 3H), 3.25 (m, 2H),
2.61 (m, 2H), 1.82 (m, 2H)
[0248] LRMS (apci, positive) m/e 406.1 (M+1)
[0249] Compound 171:
##STR00270##
N-(2-Benzenesulfonyl-ethyl)-3-methoxy-4-(3-pyrazin-2-yl-ureido)-benzamide
[0250] Prepared according to the procedure of Compound 167 using
2-benzenesulfonyl-ethylamine (57% yield).
[0251] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (br s, 1H),
8.43 (br m, 1H), 8.35 (br s, 1H), 8.25 (s, 1H), 8.22 (d, 1H), 7.96
(s, 1H), 7.93 (s, 1H), 7.72 (m, 1H), 7.63 (m, 2H), 7.38 (s, 1H),
7.33 (d, 1H), 3.95 (s, 3H), 3.59 (m, 2H), 3.55 (m, 2H)
[0252] LMRS (apci, positive) m/e 456.0 (M+1)
[0253] Compound 172:
##STR00271##
N-(4-Iodo-benzyl)-3-methoxy-4-(3-pyrazin-2-yl-ureido)-benzamide
[0254] Prepared according to the procedure of Compound 167 using
4-iodo benzyl amine (66% yield).
[0255] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.97 (t, 1H),
8.90 (s, 1H), 8.36 (s, 1H), 8.26 (d, 1H), 8.24 (s, 1H), 7.69 (d,
2H), 7.56 (m, 2H), 7.16 (d, 2H), 4.41 (d, 2H), 3.97 (s, 3H)
[0256] LRMS (apci, positive) m/e 504.0 (M+1)
[0257] Compound 173:
##STR00272##
3-Methoxy-4-(3-pyrazin-2-yl-ureido-N-(2-pyridin-2-yl-ethyl)-benzamide
[0258] Prepared according to the procedure of Compound 167 using
2-pyridin-2-yl-ethylamine (57% yield).
[0259] .sup.1-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (br s, 1H),
8.51 (br m, 2H), 8.35 (s, 1H), 8.22 (m, 2H), 7.71 (t, 1H), 7.48 (s,
1H), 7.43 (d, 1H), 7.26 (d, 1H), 7.21 (m, 1H), 3.97 (s, 3H), 3.60
(m, 2H), 3.00 (dd, 2H)
[0260] LRMS (esi, positive) m/e 393.3 (M+1)
[0261] Compound 174:
##STR00273##
3-Methoxy-4-(3-pyrazin-2-yl-ureido)-N-(2-pyridin-4-yl-ethyl)-benzamide
[0262] Prepared according to the procedure of Compound 167 using
2-pyridin-4-yl-ethylamine (45% yield).
[0263] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.93 (s, 1H),
8.63 (d, 2H), 8.51 (t, 1H), 8.37 (s, 1H), 8.27 (s, 1H), 8.24 (d,
1H), 7.60 (d, 2H), 7.43 (m, 2H), 3.97 (s, 3H), 3.58 (m, 2H), 3.01
(m, 2H)
[0264] LRMS (esi, positive) m/e 393.1 (M+1)
[0265] Compound 175:
##STR00274##
N-(1H-Benzoimidazol-2-ylmethyl)-3-methoxy-4-(3-pyrazin-2-yl-ureido)-benza-
mide
[0266] Prepared according to the procedure of Compound 167 using
C-(1H-benzoimidazol-2-yl)-methylamine (53% yield).
[0267] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (s, 1H),
8.35 (s, 1H), 8.32 (d, 1H), 8.22 (s, 1H), 7.61 (m, 3H), 7.47 (m,
2H), 7.12 (m, 2H), 4.66 (s, 2H), 3.98 (s, 3H)
[0268] LRMS (esi, positive) m/e 418.2 (M+1)
[0269] Compound 176:
##STR00275##
N-[2-(1H-Indol-3-yl)-ethyl]-3-methoxy-4-(3-pyrazin-2-yl-ureido)-benzamide
[0270] Prepared according to the procedure of Compound 167 using
tryptamine (74% yield).
[0271] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (s, 1H),
8.55 (br t, 1H), 8.26 (s, 1H), 8.24 (d, 1H), 8.23 (s, 1H), 7.60 (d,
1H), 7.53 (s, 1H), 7.50 (d, 1H), 7.26 (d, 1H), 7.19 (s, 1H), 7.05
(dd, 1H), 6.98 (dd, 1H), 3.97 (s, 3H), 3.56 (m, 2H), 2.96 (m,
2H)
[0272] LRMS (esi, positive) m/e 431.2 (M+1)
[0273] Compound 177:
##STR00276##
3-Methoxy-N-[3-(methyl-phenyl-amino)-propyl]-4-(3-pyrazin-2-yl-ureido)-be-
nzamide
[0274] Prepared according to the procedure of Compound 167 using
N1-methyl-N1-phenyl-propane-1,3-diamine (68% yield).
[0275] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.89 (s, 1H),
8.41 (br s, 1H), 8.36 (s, 1H), 8.23 (d, 1H), 8.22 (s, 1H), 7.52 (s,
1H), 7.49 (d, 1H), 7.16 (m, 2H), 6.70 (d, 2H), 6.59 (dd, 1H), 3.96
(s, 3H), 3.38 (m, 2H), 3.30 (m, 2H), 2.87 (s, 3H), 1.77 (m, 2H)
[0276] LRMS (esi, positive) m/e 435.2 (M+1)
[0277] Compound 178:
##STR00277##
N-(1-Benzyl-pyrrolidin-3-yl)-3-methoxy-4-(3-pyrazin-2-yl-ureido)-benzamid-
e
[0278] Prepared according to the procedure of Compound 167 using
3-amino-1-benzyl pyrrolidine (62% yield).
[0279] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.88 (br s, 1H),
8.36 (br m, 2H), 8.24 (d, 1H), 8.21 (s, 1H), 7.51 (s, 1H), 7.50 (d,
1H), 7.32 (m, 4H), 7.22 (m, 1H), 4.39 (br m, 1H), 3.96 (s, 3H),
3.59 (s, 2H), 2.79 (m, 1H), 2.62 (m, 1H), 2.40 (m, 1H), 2.16 (m,
1H), 1.81 (m, 2H)
[0280] LRMS (esi, positive) m/e 447.2 (M+1)
[0281] Compound 179;
##STR00278##
N-(3-(R)-1-Benzyl-pyrrolidin-3-yl)-3-methoxy-4-(3-pyrazin-2-yl-ureido)-be-
nzamide
[0282] Prepared according to the procedure of Compound 167 using
3-(R)-amino-1-benzyl pyrrolidine (57% yield).
[0283] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.83 (br s, 1H),
8.36-8.24 (m, 3H), 8.15 (m, 1H), 7.48 (m, 2H), 7.32 (m, 4H), 7.22
(m, 1H), 4.37 (m, 1H), 3.96 (s, 3H), 3.59 (s, 2H), 2.78 (m, 1H),
2.63 (m, 1H), 2.41 (m, 1H), 2.17 (m, 1H), 1.80 (m, 2H)
[0284] LRMS (esi, positive) m/e 447.1 (M+1)
[0285] Compound 180:
##STR00279##
N-(3-(S)-1-Benzyl-pyrrolidin-3-yl)-3-methoxy-4-(3-pyrazin-2-yl-ureido)-be-
nzamide
[0286] Prepared according to the procedure of Compound 167 using
3-(S)-amino-1-benzyl pyrrolidine (57% yield).
[0287] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.85 (s, 1H),
8.34 (br s, 1H), 8.30 (s, 1H), 8.25 (d, 1H), 8.19 (s, 1H), 7.50 (m,
2H), 7.32 (m, 4H), 7.22 (m, 1H), 4.39 (m, 1H), 3.97 (s, 3H), 3.59
(s, 2H), 2.79 (m, 1H), 2.62 (m, 1H), 2.41 (m, 1H), 2.16 (m, 1H),
1.81 (m, 2H)
[0288] LRMS (esi, positive) m/e 447.1 (M+1)
[0289] Compound 181:
##STR00280##
N-(2-Dimethylamino-ethyl)-3-methoxy-N-methyl-4-(3-pyrazin-2-yl-ureido)-be-
nazmide
[0290] Prepared according to the procedure of Compound 167 using
N,N,N'-trimethyl-ethane-1,2-diamine (57% yield).
[0291] .sup.1H-NMR (400 MHz, D.sub.2O) .delta. 8.17 (s, 1H), 8.05
(s, 1H), 7.94 (s, 1H), 7.76 (d, 1H), 6.92 (m, 2H), 3.79 (m, 2H),
3.75 (s, 3H), 3.36 (m, 2H), 2.97 (s, 3H), 2.88 (s, 6H)
[0292] LRMS (esi, positive) m/e 373.2 (M+1)
[0293] Compound 182:
##STR00281##
3-Methoxy-N-(3-methylamino-propyl)-4-(3-pyrazin-2-yl-ureido)-benzamide
[0294] Prepared according to the procedure of Compound 167 using
N1-methyl-propane-1,3-diamine (25% yield).
[0295] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.93 (s, 1H),
8.38-8.25 (m, 4H), 7.59 (d, 1H), 7.52 (m, 1H), 3.98 (s, 3H), 3.9
(m, 2H), 2.92 (m, 2H), 2.50 (s, 3H), 1.82 (m, 2H)
[0296] LRMS (esi, positive) m/e 359.1 (M+1)
[0297] Compound 183:
##STR00282##
N-(3-Dimethylamino-propyl)-3-methoxy-4-(3-pyrazin-2-yl-ureido)-benzamide
[0298] Prepared according to the procedure of Compound 167 using
N,N-dimethyl propyldiamine (81% yield).
[0299] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.93 (s, 1H),
8.56 (t, 1H), 8.37 (s, 1H), 8.25 (d, 1H), 8.23 (s, 1H), 7.52 (s,
1H), 7.50 (d, 1H), 4.10 (m, 2H), 3.97 (s, 3H), 3.35 (s, 6H), 3.05
(m, 2H), 1.84 (m, 2H)
[0300] LRMS (esi, positive) m/e 373.1 (M+1)
[0301] Compound 184:
##STR00283##
N-(3-Dimethylamino-propyl)-3-methoxy-N-methyl-4-(3-pyrazin-2-yl-ureido)-b-
enazmide
[0302] Prepared according to the procedure of Compound 167 using
N,N,N'-trimethyl propyldiamine (88% yield).
[0303] .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD) .delta. 8.60
(s, 1H), 8.33 (d, 1H), 8.23 (s, 1H), 8.19 (s, 1H), 7.01 (m, 2H),
3.99 (s, 3H), 3.83 (s, 3H), 3.59 (m, 2H), 2.78 (m, 2H), 2.59 (s,
6H), 2.22 (m, 2H)
[0304] LRMS (esi, positive) m/e 387.1 (M+1)
[0305] Compound 185:
##STR00284##
3-Methoxy-N-(3-morpholin-4-yl-propyl)-4-(3-pyrazin-2-yl-ureido)-benzamide
[0306] Prepared according to the procedure of Compound 167 using
3-morpholin-4-yl-propylamine (53% yield).
[0307] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (s, 1H),
8.57 (t, 1H), 8.36 (s, 1H), 8.28 (d, 1H), 8.24 (s, 1H), 7.52 (s,
1H), 7.50 (d, 1H), 3.96 (s, 3H), 3.61 (m, 2H), 3.42 (m, 2H), 3.32
(m, 4H), 3.10 (m, 4H), 1.90 (m, 2H)
[0308] LRMS (esi, positive) m/e 415.1 (M+1)
[0309] Compound 186:
##STR00285##
3-Methoxy-N-[3-(4-methyl-piperazin-1-yl)-propyl]-4-(3-pyrazin-2-yl-ureido-
)-benzamide
[0310] Prepared according to the procedure of Compound 167 using
3-(4-methyl-piperazin-1-yl)-propylamine (63% yield).
[0311] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (s, 1H),
8.41 (m, 1H), 8.36 (s, 1H), 8.24 (m, 2H), 7.52 (s, 1H), 7.49 (d,
1H), 3.97 (s, 3H), 3.31 (m, 11H), 2.70 (m, 2H), 2.41 (m, 2H), 1.72
(m, 2H)
[0312] LRMS (esi, positive) m/e 428.1 (M+1)
[0313] Compound 187:
##STR00286##
{2-[3-Methoxy-4-(3-pyrazin-2-yl-ureido)-benzoylamino]-etyl}-trimethyl-amm-
onium chloride
[0314] Prepared according to the procedure of Compound 167 using
2-N,N,N-trimethylammonium ethylamine (46% yield).
[0315] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (s, 1H),
8.77 (m, 1H), 8.36 (s, 1H), 8.30 (d, 1H), 8.24 (s, 1H), 7.53 (s,
1H), 7.51 (d, 1H), 3.97 (s, 3H), 3.70 (m, 2H), 3.50 (m, 2H), 3.15
(s, 9H)
[0316] LRMS (esi, positive) m/e 373.1 (M+)
[0317] Compound 188:
##STR00287##
4-Methoxy-3-(3-pyrazin-2-yl-ureido)-benzoic acid
[0318] Step 1: 4-Methoxy-3-(4-nitro-phenoxycarbonylamino)-benzoic
acid methyl ester. To a stirred, cooled (0.degree. C.) solution of
methyl-3-amino-4-methoxy benzoate (5.0 g; 27.6 mmol) in methylene
chloride (100 mL) was added pyridine (2.34 mL; 29 mmol) followed by
4 nitrophenyl chloroformate (5.8 g; 29 mmol). After stirring for 8
hours, the reaction was diluted with methylene chloride (100 mL),
washed with 1N hydrochloric acid (2.times.125 mL), 10% aqueous
sodium carbonate (2.times.125 mL), brine (1.times.125 mL), then
dried MgSO.sub.4), and filtered. The filtered material was
concentrated under reduced pressure. The residue was taken up in
ethyl acetate (100 mL) followed by hexanes (700 mL). A precipitate
formed which was filtered to yield an off white solid (80%
yield).
[0319] Step 2: 4-Methoxy-3-(3-pyrazin-2-yl-ureido)-benzoic acid
methyl ester. To a stirred solution of the carbamate piece (1.0 g;
2.9 mmol) in N-methyl pyrrolidinone (5 mL) was added amino pyrazine
(285 mg; 3.0 mmol). The reaction was heated to 85.degree. C. and
stirred for 12 hours. The reaction was allowed to cool to room
temperature, then diluted with ethyl acetate (50 mL) and water (50
mL). A precipitate formed which was filtered and dried under
reduced pressure to yield an off white solid (55% yield).
[0320] Step 3: 4-Methoxy-3-(3-pyrazin-2-yl-ureido)-benzoic acid. To
a stirred solution of 4-methoxy-3-(3-pyrazin-2-yl-ureido)-benzoic
acid methyl ester (1.0 g; 3.3 mmol) in methanol (25 mL) was added
lithium hydroxide (5 mL of a 2M aqueous solution). The reaction was
heated to 60.degree. C. and stirred for 12 hours. The reaction was
allowed to cool to room temperature and the pH was adjusted to 5.5
with hydrochloric acid (1N). A precipitate formed which was
filtered and dried under reduced pressure to yield an off white
solid (58% yield).
[0321] Compound 189:
##STR00288##
N-Butyl-4-methoxy-3-(3-pyrazin-2-yl-ureido)-benzamide
[0322] To a stirred solution of
4-Methoxy-3-(3-pyrazin-2-yl-ureido)-benzoic acid (32 mg; 0.11 mmol)
in N-methyl pyrrolidinone (1 mL) was added
O-benzotrazol-1-yl-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HBTU; 45 mg; 0.12 mmol), butyl amine (12
.mu.L, 0.12 mmol), and diisopropylethylamine (35 .mu.t; 0.20 mmol).
The reaction was stirred at room temperature for 12 hours. The
reaction was diluted with ethyl acetate (20 mL) and 10% aqueous
sodium carbonate (20 mL) and stirred for 5 minutes. A precipitate
formed which was filtered and dried under reduced pressure to yield
an off white solid (49% yield).
[0323] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (s, 1H),
8.63 (s, 1H), 8.35 (s, 1H), 8.23 (m, 1H), 8.21 (s, 1H), 7.52 (d,
1H), 7.06 (d, 1H), 3.95 (s, 3H), 3.22 (m, 2H), 1.50 (m, 2H), 1.33
(m, 2H), 0.90 (t, 3H)
[0324] LRMS (apci, positive) m/e 344.1 (M+1)
[0325] Compound 190:
##STR00289##
N-Benzyl-4-methoxy-3-(3-pyrazin-2-yl-ureido)-benzamide
[0326] Prepared according to the procedure of Compound 189 using
benzyl amine (70% yield).
[0327] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (s, 1H),
8.90 (br s, 1H), 8.73 (s, 1H), 8.33 (s, 1H), 8.23 (s, 1H), 7.60 (d,
1H), 7.33 (m, 4H), 7.23 (m, 1H), 7.12 (d, 1H), 4.44 (s, 2H), 3.96
(s, 3H)
[0328] LRMS (apci positive) m/e 378.1 (M+1)
[0329] Compound 191:
##STR00290##
4-Methoxy-N-phenethyl-3-(3-pyrazin-2-yl-ureido)-benzamide
[0330] Prepared according to the procedure of Compound 189 using
phenethyl amine (68% yield).
[0331] `H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (s, 1H), 8.64
(s, 1H), 8.40 (m, 1H), 8.35 (s, 1H), 8.23 (s, 1H), 7.52 (d, 1H),
7.33-7.18 (m, 5H), 7.08 (d, 1H), 3.96 (s, 3H), 3.44 (m, 2H), 2.82
(m, 2H)
[0332] LRMS (apci positive) m/e 392.1 (M+1)
[0333] Compound 192:
##STR00291##
4-Methoxy-N-(3-phenyl-propyl)-3-(3-pyrazin-2-yl-ureido)-benzamide
[0334] Prepared according to the procedure of Compound 189 using
phenpropyl amine (65% yield).
[0335] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (s, 1H),
8.64 (s, 1H), 8.35 (s, 1H), 8.23 (s, 1H), 7.56 (d, 1H), 7.32-7.22
(m, 5H), 7.18 (m, 1H), 7.10 (d, 111), 3.97 (s, 3H), 3.24 (m, 2H),
2.62 (dd, 2H), 1.82 (m, 2H)
[0336] LRMS (apci positive) m/e 406.1 (M+1)
[0337] Compound 193:
##STR00292##
N-(2-Benzenesulfonyl-ethyl)-4-methoxy-3-(3-pyrazin-2-yl-ureido)-benzamide
[0338] Prepared according to the procedure of Compound 189 using
2-benzenesulfonyl-ethylamine (42% yield).
[0339] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (s, 1H),
8.58 (s, 1H), 8.38 (br s, 1H), 8.32 (s, 1H), 8.23 (s, 1H), 7.94 (d,
2H), 7.74 (m, 1H), 7.65 (d, 2H), 7.38 (d, 1H), 7.05 (d, 1H), 3.95
(s, 3H), 3.57 (m, 2H), 3.51 (m, 2H)
[0340] LRMS (apci positive) m/e 456.0 (M+1)
[0341] Compound 194:
##STR00293##
4-Methoxy-3-(3-pyrazin-2-yl-ureido)-N-(2-pyridin-2-yl-ethyl)-benzamide
[0342] Prepared according to the procedure of Compound 189 using
2-pyridin-2-yl-ethylamine (16% yield).
[0343] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (s, 1H),
8.64 (s, 1H), 8.52 (d, 1H), 8.41 (m, 1H), 8.32 (s, 1H), 8.21 (s,
1H), 7.72 (m, 1H), 7.50 (d, 1H), 7.28 (d, 1H), 7.22 (m, 1H), 7.08
(d, 1H), 3.96 (s; 3H), 3.59 (m, 2H), 2.98 (m, 2H)
[0344] LRMS (esi positive) m/e 415.2 (M+1)
[0345] Compound 195:
##STR00294##
4-Methoxy-3-(3-pyrazin-2-yl-ureido)-N-(2-pyridin-4-yl-ethyl)-benzamide
[0346] Prepared according to the procedure of Compound 189 using
2-pyridin-4-yl-ethylamine (41% yield).
[0347] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.91 (s, 1H),
8.63 (s, 1H), 8.46 (d, 2H), 8.40 (m, 1H), 8.35 (s, 1H), 8.24 (s,
1H), 7.47 (d, 1H), 7.26 (d, 2H), 7.08 (d, 1H), 3.96 (s, 3H), 3.50
(m, 2H), 2.84 (m, 2H)
[0348] LRMS (esi positive) m/e 415.2 (M+1)
[0349] Compound 196:
##STR00295##
N-(1H-Benzoimidazol-2-ylmethyl)-4-methoxy-3-(3-pyrazin-2-yl-ureido)-benza-
mide
[0350] Prepared according to the procedure of Compound 189 using
C-(1H-benzoimidazol-2-yl)-methylamine (26% yield).
[0351] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.99 (t, 1H),
8.91 (s, 1H), 8.77 (s, 1H), 8.36 (s, 1H), 8.23 (s, 1H), 7.66 (d,
1H), 7.56 (d, 1H), 7.44 (d, 1H), 7.15 (m, 3H), 4.65 (d, 2H), 3.97
(s, 3H)
[0352] LRMS (esi positive) m/e 418.1 (M+1)
[0353] Compound 197:
##STR00296##
N-[2-(1H-Indol-3-yl)-ethyl]-4-methoxy-3-(3-pyrazin-2-yl-ureido)-bezamide
[0354] Prepared according to the procedure of Compound 189 using
tryptamine (51% yield).
[0355] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (s, 1H),
8.69 (s, 1H), 8.43 (t, 1H), 8.33 (s, 1H), 8.24 (s, 1H), 7.60 (d,
1H), 7.56 (d, 1H), 7.35 (d, 1H), 7.17 (s, 1H), 7.11 (d, 1H), 7.07
(m, 1H), 6.97 (m, 1H), 3.96 (s, 3H), 3.54 (m, 2H), 2.95 (m,
2H).
[0356] LRMS (esi positive) m/e 431.1 (M+1)
[0357] Compound 198:
##STR00297##
4-Methoxy-N-[3-(methyl-phenyl-amino)-propyl]-3-(3-pyrazin-2-yl-ureido)-be-
nzamide
[0358] Prepared according to the procedure of Compound 189 using
N-methyl-N-phenyl propyldiamine (81% yield).
[0359] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.92 (s, 1H),
8.64 (s, 1H), 8.37 (m, 1H), 8.35 (s, 1H), 8.23 (s, 1H), 7.55 (d,
1H), 7.14 (m, 3H), 6.70 (d, 2H), 6.58 (t, 1H), 3.96 (s, 3H), 3.37
(m, 2H), 3.25 (m, 2H), 2.86 (s, 3H), 1.77 (m, 2H).
[0360] LRMS (esi positive) m/e 435.2 (M+1)
[0361] Compound 199:
##STR00298##
N-(1-Benzyl-pyrrolidin-3-yl)-4-methoxy-3-(3-pyrazin-2-yl-ureido)-benzamid-
e
[0362] Prepared according to the procedure of Compound 189 using
3-amino-1-benzyl pyrrolidine (48% yield).
[0363] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (s, 1H),
8.62 (s, 1H), 8.36 (s, 1H), 8.30 (d, 1H), 8.22 (s, 1H), 7.56 (d,
1H), 7.32 (m, 4H), 7.22 (m, 1H), 7.15 (d, 1H), 4.37 (m, 1H), 3.96
(s, 3H), 3.59 (s, 23H), 2.79 (m, 1H), 2.60 (m, 1H), 2.39 (m, 1H),
2.14 (m, 1H), 1.80 (m, 2H).
[0364] LRMS (esi positive) m/e 447.2 (M+1)
[0365] Compound 200:
##STR00299##
N-(2-Dimethylamino-ethyl)-4-methoxy-N-methyl-3-(3-pyrazin-2-yl-ureido)-be-
nzamide
[0366] Prepared according to the procedure of Compound 189 using
N,N,N'-trimethylethyldiamine (93% yield).
[0367] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 9.39 (br s, 1H),
8.91 (s, 1H), 8.35 (s, 1H), 8.33 (d, 1H), 8.23 (s, 1H), 7.16-7.09
(m, 2H), 3.96 (s, 3H), 3.76 (m, 2H), 3.37 (m, 2H), 2.99 (s, 3H),
2.85 (br s, 6H).
[0368] LRMS (esi, positive) m/e 373.2 (M+1)
[0369] Compound 201:
##STR00300##
4-Methoxy-N-(3-methylamino-propyl)-3-(3-pyrazin-2-yl-ureido)-benzamide
[0370] Prepared according to the procedure of Compound 189 using
N-methyl propyldiamine (15% yield).
[0371] .sup.1H-NMR (400 MHz, D.sub.2O) .delta. 7.98 (m, 2H), 7.91
(s, 1H), 7.82 (s, 1H), 7.17 (d, 1H), 6.73 (d, 1H), 3.73 (s, 3H),
3.29 (m, 2H), 2.98 (m, 2H), 2.61 (s, 3H), 1.88 (m, 2H).
[0372] LRMS (esi, positive) m/e 359.2 (M+1)
[0373] Compound 202:
##STR00301##
N-(3-Dimethylamino-propyl)-4-methoxy-3-(3-pyrazin-2-yl-ureido)-benzamide
[0374] Prepared according to the procedure of Compound 189 using
N,N-dimethyl propyldiamine (51% yield).
[0375] .sup.1H-NMR (400 MHz, D.sub.2O) .delta. 7.98 (s, 1H), 7.96
(s, 2H), 7.81 (s, 1H), 7.16 (d, 1H), 6.72 (d, 1H), 3.73 (s, 3H),
3.29 (m, 2H), 3.09 (m, 2H), 2.80 (s, 6H), 1.93 (m, 2H).
[0376] LRMS (esi, positive) m/e 373.2 (M+1)
[0377] Compound 203:
##STR00302##
N-(3-Dimethylamino-propyl)-4-methoxy-N-methyl-3-(3-pyrazin-2-yl-ureido)-b-
enzamide
[0378] Prepared according to the procedure of Compound 189 using
N,N,N'-trimethyl propyldiamine (60% yield).
[0379] .sup.1H-NMR (400 MHz, D.sub.2O) .delta. 8.37 (s, 1H), 8.19
(s, 1H), 8.04 (s, 1H), 7.80 (d, 1H), 7.13 (m, 1H), 7.01 (m, 1H),
3.82 (s, 3H), 3.51 (m, 2H), 3.11 (m, 2H), 2.96 (s, 3H), 2.80 (s,
6H), 2.01 (m, 2H).
[0380] LRMS (esi, positive) m/e 387.1 (M+1)
[0381] Compound 204:
##STR00303##
4-Methoxy-N-[3-(4-methyl-piperazin-1-yl)-propyl]-3-(3-pyrazin-2-yl-ureido-
)-benzamide
[0382] Prepared according to the procedure of Compound 189 using
3-(4-methyl-piperazin-1-yl)-propylamine (57% yield).
[0383] .sup.1H-NMR (400 MHz, D.sub.2O) .delta. 8.18 (s, 1H), 8.06
(s, 1H), 8.04 (s, 1H), 7.96 (s, 1H), 7.32 (d, 1H), 6.85 (d, 1H),
3.79 (s, 3H), 3.48 (br s, 8H), 3.36 (m, 2H), 3.17 (m, 2H), 2.83 (s,
3H), 1.96 (m, 2H)
[0384] LRMS (esi, positive) m/e 428.2 (M+1)
[0385] Compound 205:
##STR00304##
{2-[4-Methoxy-3-(3-pyrazin-2-yl-ureido)-benzoylamino]-ethyl}-trimethyl-am-
monium chloride
[0386] Prepared according to the procedure of Compound 189 using
2-trimethylammonium ethyl amine (63% yield).
[0387] .sup.1H-NMR (400 MHz, D.sub.2O) .delta. 8.17 (s, 1H), 8.05
(s, 1H), 8.03 (s, 1H), 7.93 (s, 1H), 7.32 (d, 1H), 6.84 (d, 1H),
3.80 (s, 3H), 3.76 (m, 2H), 3.44 (m, 2H), 3.11 (s, 9H).
[0388] LRMS (esi, positive) m/e 373.0 (M+)
[0389] Compound 206:
##STR00305##
4-Methoxy-N-(3-morpholin-4-yl-propyl)-3-(3-pyrazin-2-yl-ureido)-benzamide
[0390] Prepared according to the procedure of Compound 189 using
3-morpholin-4-yl-propylamine (69% yield).
[0391] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.91 (s, 1H),
8.63 (s, 1H), 8.36 (s, 1H), 8.35 (m, 1H), 8.23 (s, 1H), 7.54 (d,
1H), 7.10 (d, 1H), 3.96 (s, 3H), 3.57 (m, 4H), 3.26 (m, 2H), 2.34
(m, 4H), 2.32 (m, 2H), 1.66 (m, 2H).
[0392] LRMS (esi, positive) m/e 415.2 (M+1)
[0393] Compound 207:
##STR00306##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic acid
[0394] Step 1: (5-Methyl-pyrazin-2-yl)-carbamic acid tert-butyl
ester. To a stirred solution of 5-methyl pyrazine carboxylic acid
(13.8 g, 100 mmol) in 300 mL of toluene at room temperature under
nitrogen was added triethyl amine (14 mL, 100 mmol) followed by
diphenyl phosphoryl azide (21 6 mL, 100 mmol). After 30 min. at
room temperature, 2-methyl-2-propanol (19 mL, 200 mmol) was added
and the solution immersed in a 90.degree. C. oil bath. After 2
hours, the reaction was cooled to RT, diluted to 600 mL with EtOAc,
and washed 3.times.60 mL with 10% Na.sub.2CO.sub.3 and 1.times.600
mL with saturated NaCl. The organics were dried (MgSO.sub.4),
filtered and concentrated to a yellow solid (17.5 g, 83%).
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 9.16 (s, 1H), 8.05 (s,
1H), 7.56 (br s, 1H), 2.50 (s, 3H), 1.55 (s, 9H).
[0395] Step 2: 5-Methyl-2-aminopyrazine. To a stirred solution of
(5-methyl-pyrazin-2-yl)-carbamic acid tert-butyl ester (2.1 g, 10
mmol) in 30 mL CH.sub.2Cl.sub.2 at 0.degree. C. under nitrogen was
added trifluoroacetic acid (30 mL). The solution was allowed to
warm to RT overnight. The solution was rotary evaporated to remove
TFA and the residue was redissolved in 200 mL CH.sub.2Cl.sub.2 and
stirred with 100 mL 10% Na.sub.2CO.sub.3. The organics were
isolated with the aqueous solution extracted 3.times.100 mL with
CH.sub.2Cl.sub.2. The organics were combined, dried (MgSO.sub.4),
filtered and concentrated to an orange solid (1 g, 92%).
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.46 (s, 1H), 7.70 (s,
1H), 2.49 (s, 3H).
[0396] Step 3:
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic acid methyl
ester. To a stirred solution of
3-methoxy-4-(4-nitro-phenoxycarbonylamino)-benzoic acid methyl
ester (11.7 g, 33.8 mmol) in 34 mL NMP at room temperature under
nitrogen was added 5-methyl-2-aminopyrazine (3.69 g, 33.8 mmol) and
the reaction was immersed in an 85.degree. C. oil bath. After 6
hours the reaction was allowed to cool to room temperature and a
precipitate formed. EtOAc (200 mL) was added and the precipitate
was isolated by filtration (4.7 g, 44%). .sup.1H-NMR (400 MHz,
d.sub.6-DMSO) .delta. 8.79 (br s, 1H), 8.36 (d, 1H), 8.23 (s, 1H),
7.60 (d, 1H), 7.52 (s, 1H), 3.98 (s, 3H), 3.81 (s, 3H), 2.42 (s,
3H).
[0397] Step 4:
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic acid. To a
stirred suspension of
3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic acid methyl
ester (7.15 g, 22.6 mmol) in 3:1 MeOH:H.sub.2O (226 mL) at RT under
N.sub.2 was added lithium hydroxide monohydrate (9.5 g, 226 mmol)
as a solid and the mixture heated to 65.degree. C. After reaching
temperature, the suspension gradually became a bright yellow
solution. After about 4 hours a precipitate formed but the reaction
was continued overnight. After cooling to RT, MeOH was removed by
rotovap and the aqueous suspension diluted with 100 mL H.sub.2O and
neutralized to pH=5 with concentrated HCl. As pH=5 was approached,
the suspension turned from yellow to white. The suspension was then
filtered through paper on a large ceramic funnel. The filtration
went very slowly, taking several hours. The filter cake was washed
twice with H.sub.2O. When most of the H.sub.2O was removed, the
residue was dried under high vacuum in a dessicator overnight to
give the free acid as a white solid (6 g, 88%). .sup.1H-NMR (400
MHz, d.sub.6-DMSO) .delta. 8.79 (br s, 1H), 8.36 (d, 1H), 8.22 (s,
1H), 7.57 (d, 1H), 7.51 (s, 1H), 3.97 (s, 3H), 2.42 (s, 3H).
[0398] Compound 208:
##STR00307##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(2-pyridin-2-yl-ethyl)-b-
enzamide
[0399] To a stirred solution of
3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic acid (30 mg,
0.1 mmol) in 1 ML NPM at RT in a capped reaction vial was added
HBTU (42 mg, 0.11 mmol). The suspension was stirred for 15 minutes
and then treated with 2-ethylaminopyridine (13.2 0.11 mmol)
followed by Hunigs Base (35 .mu.L, 0.2 mmol). After stirring
overnight, NMP was removed by bulb to bulb transfer at 70.degree.
C. under high vacuum and the residue stirred with a mixture of
CH.sub.2Cl.sub.2 (10 mL) and 10% Na.sub.2CO.sub.3 (10 mL) until
complete dissolution occurred. The organics were isolated, dried
(MgSO.sub.4), filtered and concentrated. The residue was triturated
with EtOAc to produce a solid which was isolated by filtration (71%
yield).
[0400] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (s, 1H),
8.58 (d, 1H), 8.50 (t, 1H), 8.23 (d, 1H), 8.21 (s, 1H), 7.82 (t,
1H), 7.48-7.31 (m, 4H), 3.97 (s, 3H), 3.62 (m, 2H), 3.04 (m, 2H),
2.42 (s, 3H).
[0401] LRMS (esi, positive) m/e 407.1 (M+1)
[0402] Compound 209:
##STR00308##
N-(1-Benzyl-piperidin-4-yl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido-
]-benzamide
[0403] Prepared according to the method of Compound 208 using
4-amino-1-benzyl piperidine except the crude product was purified
by chromatography on a Biotage 12S column eluting with 92.5/7.5
Ch.sub.2Cl.sub.2/MeOH. (61% yield).
[0404] .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD) .delta. 8.44
(br s, 1H), 8.32 (d, 1H), 8.09 (s, 1H), 7.47 (s, 1H), 7.38-7.28 (m,
6H), 7.09 (d, 1H), 4.00 (m, 1H), 3.99 (s, 3H), 3.60 (s, 2H), 2.98
(m, 2H), 2.52 (s, 3H), 2.25 (m, 2H), 2.00 (m, 2H), 1.64 (m,
2H).
[0405] LRMS (esi, positive) m/e 475.2 (M+1)
[0406] Compound 210:
##STR00309##
N-(3-Dimethylamino-propyl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-
-benzamide
[0407] Prepared according to the method of Compound 208 using
N,N-dimethyl propyldiamine (70% yield).
[0408] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (br s, 1H),
8.55 (t, 1H), 8.26 (d, 1H), 8.22 (s, 1H), 7.52 (s, 1H), 7.50(d,
1H), 3.98 (s, 3H), 3.32 (m, 2H), 3.07 (m, 2H), 2.77 (s, 6H), 2.41
(s, 3H), 1.89 (m, 2H).
[0409] LRMS (esi, positive) m/e 387.1 (M+1)
[0410] Compound 211:
##STR00310##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-morpholin-4-yl-propyl-
)-benzamide
[0411] Prepared according to the method of Compound 208 using
3-morpholin-4-yl-propylamine (79% yield).
[0412] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (br s, 1H),
8.57 (t, 1H), 8.25 (d, 1H), 8.21 (s, 1H), 7.51 (s, 1H), 7.50 (d,
1H), 3.97 (s, 3H), 3.62 (m, 2H), 3.31 (m, 8H), 3.12 (m, 2H), 2.41
(s, 3H), 1.92 (m, 2H).
[0413] LRMS (esi, positive) m/e 429.1 (M+1)
[0414] Compound 212:
##STR00311##
N-(2-Dimethylamino-2-phenyl-ethyl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-
-ureido]-benzamide
[0415] Prepared according to the method of Compound 208 using
N-(2-dimethylamino)-2-phenyl ethyl amine except the crude product
was purified by chromatography on a Biotage 12S column eluting with
92.5/7.5 CH.sub.2CH.sub.2/MeOH. (12% yield).
[0416] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.05 (s, 1H),
8.88 (s, 1H), 8.20 (m, 3H), 7.41-7.19 (m, 7H), 3.96 (s, 3H), 3.78
(m, 1H), 3.70 (m, 1H), 3.57 (m, 1H), 3.38 (m, 1H), 2.50 (s, 6H),
2.40 (s, 3H)
[0417] LRMS (esi, positive) m/e 448.9 (M+1)
[0418] Compound 213:
##STR00312##
N-(2-Dimethylamino-1-phenyl-ethyl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-
-ureido]-benzamide
[0419] Prepared according to the method of Compound 208 using
N-(2-dimethylamino)-1-phenyl ethyl amine except the crude product
was purified by chromatography on a Biotage 12S column eluting with
92.5/7.5 CH.sub.2Cl.sub.2/MeOH. (27% yield).
[0420] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 11.61 (br s,
1H), 9.71 (br s, 1H), 8.42 (d, 1H), 8.36 (s, 1H), 8.09 (s, 1H),
7.52-7.21 (m, 7H), 5.05 (br s, 1H), 3.93 (s, 3H), 2.80 (m, 1H),
2.52 (s, 3H), 2.37 (s, 6H), 1.79 (br s, 2H)
[0421] LRMS (esi, positive) m/e 449.0 (M+1)
[0422] Compound 214:
##STR00313##
N-(1-Aza-bicyclo[2,2,2]octo-3-yl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)--
ureido]-benzamide
[0423] Prepared according to the method of Compound 208 using
1-aza-bicyclo[2,2,2]oct-3-ylamine (27% yield).
[0424] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.17 (br s,
1H), 8.90 (br s, 1H), 8.24 (d, 1H), 8.23 (s, 1H), 8.17 (d, 1H),
7.53 (d, 1H), 7.50 (s, 1H), 3.98 (s, 3H), 3.95 (m, 1H0, 3.10 (m,
1H), 2.90 (m, 1H), 2.64 (m, 4H), 2.42 (s, 3H), 1.87 (m, 1H), 1.80
(m, 1H), 1.59 (m, 2H), 1.31 (m, 1H)
[0425] LRMS (esi, positive) m/e 411.0 (M+1)
[0426] Compound 215:
##STR00314##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-R-1-pyridin-2-ylmethy-
l-pyrrolidin-3-yl)-benzamide
[0427] Step 1: (3-R-1-Pyridin-2-ylmethyl-pyrrolidin-3-yl)-carbamic
acid tert-butyl ester. To a stirred solution of
(R)-boc-3-aminopyrrolidine (372.5 mg, 2 mmol) in dichloroethane (6
mL) at room temperature under nitrogen was added
pyridine-2-carboxaldehyde (190 .mu.L, 2 mmol) followed by sodium
triacetoxyborohydride (593 mg, 2.8 mmol). The reaction was stirred
at room temperature overnight and was then quenched by addition of
saturated NaHCO.sub.3 (6 mL) with stirring for 15 minutes. The
reaction was then partitioned between CH.sub.2Cl.sub.2 (25 mL) and
10% Na.sub.2CO.sub.3 (25 mL). The organics were isolated, dried
(MgSO.sub.4), filtered and concentrated to the pure product (526
mg, 95%).
[0428] Step 2: 3-R-1-Pyridin-2-ylmethyl-pyrrolidin-3-ylamine
dihydrochloride. A stirred solution of
(3-R-1-pyridin-2-ylmethyl-pyrrolidin-3-yl)-carbamic acid tert-butyl
ester (277 mg, 1 mmol) in 10 mL 4N HCl in dioxane at room
temperature in a capped flask was reacted overnight. The reaction
was concentrated by rotary evaporation and high vacuum to give the
di HCl salt (250 mg, quantitative).
[0429] Step 3: Prepared according to the procedure of Compound 208
except 3-R-1-pyridin-2-ylmethyl-pyrrolidin-3-ylamine
dihydrochloride salt was mixed with excess DIEA (70 .mu.L, 0.4
mmol) in 500 .mu.L NMP to form a solution which was added to the
acid/HBTU mixture. The crude product was purified by chromatography
on a Biotage 12S column eluting with 9/1 CH.sub.2Cl.sub.2/MeOH (60%
yield).
[0430] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.57 (s, 1H), 8.51
(d, 1H), 8.39 (d, 1H), 8.17 (s, 1H), 8.09 (s, 1H), 7.92 (s, 1H),
7.64 (d, 1H), 7.50 (s, 1H), 7.30 (d, 1H), 6.70 (d, 1H), 4.69 (m,
1H), 4.00 (s, 3H), 3.66 (dd, 2H), 2.98 (m, 1H), 2.80 (m, 1H), 2.70
(m, 1H), 2.53 (s, 3H), 2.39 (m, 3H), 1.76 (m, 1H).
[0431] LRMS (esi, positive) m/e 462.3 (M+1)
[0432] Compound 216:
##STR00315##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-R-1-methyl-pyrrolidin-
-3-yl)-benzamide
[0433] Prepared according to the procedure of Compound 215 using
3-(R)-amino-1-methyl pyrrolidine (29% yield).
[0434] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.78 (br s, 1H),
8.36 (d, 1H), 8.22 (d, 1H), 8.20 (s, 1H), 7.52 (s, 1H), 7.50 (d,
1H), 4.39 (m, 1H), 3.96 (s, 3H), 2.64 (m, 1H), 2.61 (m, 1H), 2.43
(s, 3H), 2.39 (m, 2H), 2.24 (s, 3H), 2.17 (m, 1H), 1.76 (m,
1H).
[0435] LRMS (esi, positive) m/e 385.3 (M+1)
[0436] Compound 217:
##STR00316##
N-(3-R-1-Benzyl-pyrrolidin-3-yl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-u-
reido]-benzamide
[0437] Prepared according to the method of Compound 215 using
3-(R)-amino-1-benzyl pyrrolidine except the crude product was
purified by chromatography on a Biotage 12S column eluting with
92.5/7.5 CH.sub.2Cl.sub.2/MeOH. (54% yield).
[0438] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 9.09 (br s, 1H),
8.43 (d, 1H), 8.09 (s, 1H), 8.02 (s, 1H), 7.57 (m, 1H), 7.53 (s,
1H), 7.46 (d, 1H), 7.33-7.22 (m, 4H), 4.79 (m, 1H), 4.00 (s, 3H),
3.78 (dd, 2H), 3.13 (m, 2H), 2.76 (m, 1H), 2.55 (s, 3H), 2.44 (m,
1H), 1.79 (m, 1H)
[0439] LRMS (esi, positive) m/e 461.1 (M+1)
[0440] Compound 218:
##STR00317##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-R-1-pyridin-4-ylmethy-
l-pyrrolidin-3-yl)-benzamide
[0441] Prepared according to the procedure of Compound 215 using
1-(R)-Pyridin-4-ylmethyl-pyrrolidin-3-ylamine (60% yield).
[0442] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.56 (d, 2H), 8.41
(d, 1H), 8.21 (d, 1H), 8.20 (s, 1H), 8.08 (s, 1H), 7.52 (s, 1H),
7.32 (d, 1H), 7.25 (d, 2H), 6.71 (d, 1H), 4.73 (m, 1H), 4.00 (s,
3H), 3.66 (dd, 2H), 2.97 (m, 1H), 2.82 (m, 1H), 2.71 (m, 1H), 2.54
(s, 3H), 2.39 (m, 2H), 1.78 (m, 1H).
[0443] LRMS (esi, positive) m/e 462.3 (M+1)
[0444] Compound 219:
##STR00318##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-R-1-thiophen-2-ylmeth-
yl-pyrrolidin-3-yl)-benzamide
[0445] Prepared according to the procedure of Compound 215 using
(R)-1-thiophen-2-ylmethyl-pyrrolidin-3-ylamine (60% yield).
[0446] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.96 (br s, 1H),
8.43 (d, 1H), 8.09 (d, 2H), 7.57 (s, 1H), 7.50 (d, 1H), 7.10 (d,
1H), 6.91 (m, 2H), 4.80 (m, 1H), 4.00 (s, 3H), 3.96 (dd, 2H), 3.17
(m, 1H), 3.08 (m, 1H), 2.71 (m, 1H), 2.54 (s, 3H), 2.42 (m, 2H),
1.80 (m, 1H).
[0447] LRMS (esi, positive) m/e 467.2 (M+1)
[0448] Compound 220:
##STR00319##
N-(3-R-1-Cyclohexylmethyl-pyrrolidin-3-yl)-3-methoxy-4-[3-(5-methyl-pyraz-
in-2-yl)-ureido]-benzamide
[0449] Prepared according to the procedure of Compound 215 using
1-cyclohexylmethyl-pyrrolidin-3-R-ylamine (71% yield).
[0450] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.89 (br s, 1H),
8.32 (d, 1H), 8.23 (d, 1H), 8.21 (s, 1H), 7.51 (s, 1H), 7.49 (d,
1H), 4.38 (m, 1H), 3.96 (s, 3H), 2.76 (m, 1H), 2.56 (m, 1H), 2.42
(s, 3H), 2.38 (m, 1H), 2.25-2.06 (m, 3H), 1.77 (m, 3H), 1.61 (m,
3H), 1.40 (m, 1H), 1.19 (m, 3H), 0.83 (m, 2H)
[0451] LRMS (esi, positive) m/e 467.3 (M+1)
[0452] Compound 221:
##STR00320##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-R-1-methyl-pyrrolidin-
-3-yl)-benzamide
[0453] Prepared according to the procedure of Compound 215 using
1-methyl-pyrrolidin-3-R-ylamine (51% yield).
[0454] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.78 (br s, 1H),
8.35 (d, 1H), 8.22 (d, 1H), 8.21 (s, 1H), 7.52 (s, 1H), 7.49 (d,
1H), 4.39 (m, 1H), 3.96 (s, 3H), 2.66 (m, 1H), 2.61 (m, 1H), 2.43
(s, 3H), 2.39 (m, 2H), 2.24 (s, 3H), 2.17 (m, 1H), 1.75 (m, 1H)
[0455] LRMS (esi, positive) m/e 385.4 (M+1)
[0456] Compound 222:
##STR00321##
N-(3-S-1-Benzyl-pyrrolidin-3-yl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-u-
reido]-benzamide
[0457] Prepared according to the method of Compound 215 using
1-Benzyl-pyrrolidin-3-S-ylamine except the crude product was
purified by chromatography on a Biotage 12S column eluting with
92.5/7.5 CH.sub.2Cl.sub.2/MeOH. (54% yield).
[0458] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.81 (br s, 1H),
8.43 (d, 1H), 8.06 (d, 2H), 7.53 (s, 1H), 7.42 (d, 1H), 7.35-7.20
(m, 5H), 4.78 (m, 1H), 3.99 (s, 3H), 3.78 (dd, 2H), 3.15 (m, 1H),
3.04 (m, 1H), 2.77 (m, 1H), 2.54 (s, 3H), 2.44 (m, 1H), 1.79 (m,
2H).
[0459] LRMS (esi, positive) m/e 461.1 (M+1)
[0460] Compound 223:
##STR00322##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-S-1-pyridin-2-ylmethy-
l-pyrrolidin-3-yl)-benzamide
[0461] Prepared according to the procedure of Compound 215 using
1-pyridin-2-ylmethyl-pyrrolidin-3-S-ylamine (60% yield).
[0462] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.56 (d, 1H), 8.38
(s, 1H), 8.32 (d, 1H), 8.06 (s, 1H), 7.68 (t, 1H), 7.51 (s, 1H),
7.40 (d, 1H), 7.35 (d, 1H), 7.21 (m, 1H), 4.72 (m, 1H), 4.00 (s,
3H), 3.79 (dd, 2H), 3.40 (m, 1H), 3.03 (m, 1H), 2.86 (m, 1H), 2.64
(m, 1H), 2.53 (s, 3H), 2.35 (m, 1H), 1.78 (m, 2H).
[0463] LRMS (esi, positive) m/e 462.1 (M+1)
[0464] Compound 224:
##STR00323##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-S-1-pyridin-3-ylmethy-
l-pyrrolidin-3-yl)-benzamide
[0465] Prepared according to the procedure of Compound 215 using
1-pyridin-3-ylmethyl-pyrrolidin-3-S-ylamine (60% yield).
[0466] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.56 (s, 1H), 8.51
(m, 1H), 8.39 (d, 1H), 8.18 (s, 1H), 8.08 (s, 2H), 7.64 (d, 1H),
7.50 (s, 1H), 7.33 (d, 1H), 6.77 (d, 1H), 4.72 (m, 1H), 4.00 (s,
3H), 3.66 (dd, 2H), 2.97 (m, 1H), 2.82 (m, 1H), 2.71 (m, 1H), 2.54
(s, 3H), 2.40 (m, 2H), 1.76 (m, 2H).
[0467] LRMS (esi, positive) m/e 462.3 (M+1)
[0468] Compound 225:
##STR00324##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-S-1-pyridin-4-ylmethy-
l-pyrrolidin-3-yl)-benzamide
[0469] Prepared according to the procedure of Compound 215 using
1-pyridin-4-ylmethyl-pyrrolidin-3-S-ylamine (60% yield).
[0470] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.56 (d, 2H), 8.41
(d, 1H), 8.24 (s, 1H), 8.20 (s, 1H), 8.08 (s, 1H), 7.53 (s, 1H),
7.32 (d, 1H), 7.24 (d, 2H), 6.75 (d, 1H), 4.72 (m, 1H), 4.00 (s,
3H), 3.67 (dd, 2H), 2.98 (m, 1H), 2.82 (m, 1H), 2.71 (m, 1H), 2.54
(s, 3H), 2.40 (m, 2H), 1.79 (m, 2H).
[0471] LRMS (esi, positive) m/e 462.3 (M+1)
[0472] Compound 226:
##STR00325##
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(3-S-1-thiophen-2-ylmeth-
yl-pyrrolidin-3-yl)-benzamide
[0473] Prepared according to the procedure of Compound 215 using
1-thiophen-2-ylmethyl-pyrrolidin-3-S-ylamine (55% yield).
[0474] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.71 (br s, 1H),
8.42 (d, 1H), 8.11 (d, 2H), 7.55 (s, 1H), 7.44 (d, 1H), 7.39 (d,
1H), 7.20 (d, 1H), 6.91 (m, 2H), 4.78 (m, 1H), 4.00 (s, 3H), 3.94
(dd, 2H), 3.17 (m, 1H), 3.05 (m, 1H), 2.69 (m, 1H), 2.53 (s, 3H),
2.42 (m, 2H), 1.80 (m, 2H).
[0475] LRMS (esi, positive) m/e 467.2 (M+1)
[0476] Compound 227:
##STR00326##
N-(3-S-1-Cyclohexylmethyl-pyrrolidin-3-yl-3-methoxy-4-[3-(5-methyl-pyrazi-
n-2-yl)-ureido]-benzamide
[0477] Prepared according to the procedure of Compound 215 using
1-cyclohexylmethyl-pyrrolidin-3-S-ylamine (60% yield).
[0478] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.78 (s, 1H),
8.31 (d, 1H), 8.23 (d, 1H), 8.20 (s, 1H), 7.51 (s, 1H), 7.48 (d,
1H), 4.37 (m, 1H), 3.96 (s, 3H), 2.76 (m, 1H), 2.56 (m, 1H), 2.42
(s, 3H), 2.38 (m, 1H), 2.25-2.06 (m, 3H), 1.77 (m, 3H), 1.61 (m,
3H), 1.40 (m, 1H), 1.19 (m, 3H);0.83 (m, 2H).
[0479] LRMS (esi, positive) m/e 467.3 (M+1)
[0480] Compound 228:
##STR00327##
N-(3-S-1-Benzyl-pyrrolidin-3-yl)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-3-t-
rifluoromethoxy-benzamide
[0481] Step 1: 4-Amino-3-trifluoromethoxy-benzoic acid methyl
ester. To a stirred solution of 4-amino-3-trifluoromethoxy-benzoic
acid (1.2 g, 5.4 mmol) in 16 mL of 4:1 THF:MeOH at 0.degree. C. was
added TMS-diazomethane (2M solution in hexane, 6 mL, 12 mmol)
dropwise and conversion was monitored by TLC in 2/3 EtOAc/hexane.
When complete, the reaction was concentrated to a white solid
corresponding to the methyl ester (1.27 g, quantitative).
[0482] Step 2:
4-(4-Nitro-phenoxycarbonylamino)-3-trifluoromethoxy-benzoic acid
methyl ester. To a stirred solution of 4-amino-3-trifluoromethoxy
benzoic acid methyl ester (1.38 gm, 5.9 mmol) in 18 mL
CH.sub.2Cl.sub.2 at 0.degree. C. under nitrogen was added pyridine
(521 .mu.L, 6.4 mmol) followed by p-nitrophenylchloroformate (1.18
gm, 5.9 mmol). After 4 hours at 0.degree. C. the reaction was
diluted to 60 mL with CH.sub.2Cl.sub.2 and washed 2.times.60 mL
with 2N HCl, 1.times.60 mL with H.sub.2O and 1.times.60 mL with
saturated NaCl. The organics were dried (MgSO.sub.4), filtered and
concentrated to a white solid corresponding to the carbamate (2.1
gm, 89%).
[0483] Step 3:
4-[3-(5-Methyl-pyrazin-2-yl)-ureido]-3-trifluoromethoxy-benzoic
acid methyl ester. To a stirred solution of
4-(4-nitro-phenoxycarbonylamino)-3-trifluoromethoxy-benzoic acid
methyl ester (400 mg, 1 mmol) in 1 mL of NMP in a capped reaction
vial at room temperature was added 2-amino-5-methyl-pyrazine (109
mg, 1 mmol) and the solution heated to 90.degree. C. for 6 hours.
After cooling to room temperature, the solution was diluted to 30
mL with EtOAc and washed 4.times.30 mL with 10% NaHCO.sub.3 to
remove the phenol by-product and 1.times.30 mL with saturated NaCl.
The organics were dried (MgSO.sub.4), filtered and concentrated.
Trituration and filtration with EtOAc gave a beige solid
corresponding to the urea ester (136 mg, 37%).
[0484] Step 4:
4-[3-(5-Methyl-pyrazin-2-yl)-ureido]-3-trifluoromethoxy-benzoic
acid. To a stirred suspension of
4-(3-(5-methyl-pyrazin-2-yl)-ureido]-3-trifluoromethoxy-benzoic
acid methyl ester (136 mg, 0.37 mmol) in 4 mL 3:1 MeOH:H.sub.2O
under nitrogen was added lithium hydroxide monohydrate (154 mg, 3.7
mmol), and the reaction heated to 65.degree. C. The reaction turned
yellow/green and eventually became a solution. After stirring
overnight; the reaction was cooled to RT and partially concentrated
by rotovap to remove most of the MeOH. The residual suspension was
neutralized with 2N HCl until pH=6. The reaction formed a
flocculent precipitate which was filtered off with H2O and dried
overnight under high vacuum to give the acid as a white solid (102
mg, 78%).
[0485] Step 5: To a stirred suspension of
4-[3-(5-methyl-pyrazin-2-yl)-ureido]-3-trifluoromethoxy-benzoic
acid (102 mg, 0.29 mmol) in 2.9 mL NMP in a capped reaction vial at
room temperature was added HBTU (109 mg, 0.29 mmol). After 15
minutes 3-S-1-Benzyl-pyrrolidin-3-ylamine dihydrochloride (prepared
analogously to 1-Pyridin-2-ylmethyl-pyrrolidin-3-ylamine used in
the synthesis of Compound 215) (73 mg, 0.29 mmol) was added
followed by DIEA (200 .mu.L, 1.2 mmol). The reaction was stirred
overnight and NMP was then removed under high vacuum at 70.degree.
C. The residue was partitioned between 30 mL CH.sub.2Cl.sub.2 and
30 mL 10% Na.sub.2CO.sub.3. The organics were isolated and washed
1.times.30 mL with saturated NaCl, dried (MgSO.sub.4), filtered and
concentrated to a yellow foam corresponding to the desired amide
(103 mg, 70%).
[0486] .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD) .delta. 8.56
(d, 1H), 8.37 (br s, 1H), 8.08 (s, 1H), 7.84 (s, 1H), 7.66 (d, 1H),
7.40-7.28 (m, 6H), 4.73 (m, 1H), 3.68 (dd, 2H), 2.77 (dd, 2H), 2.53
(s, 3H), 2.43 (m, 1H), 2.37 (dd, 2H), 1.78 (m, 1H).
[0487] LRMS (esi, positive) m/e 514.9 (M+1)
[0488] Compound 229:
##STR00328##
N-(1-Benzyl-piperidin-4-ylmethyl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)--
ureido]-benzamide
[0489] Step 1: N-benzylisonipecotamide. To a suspension of
isonipecotamide (12.3 g, 96 mmol) in 200 mL of dichloromethane, was
added, benzaldehyde (10.6 g, 100 mmol) and sodium
triacetoxyborohydride (29.7 gm, 140 mmol) and the mixture was
stirred at room temperature for 5 days. The thick white mixture was
diluted with 100 mL water and extracted with EtOAc (2.times.20 mL).
The aqueous phase was basified with 1N NaOH to pH>12. The
resulting white precipitate was collected by suction filtration.
The white solid was subsequently taken up in 50 mL of EtOAc, and
was washed with 20 mL of brine, then dried (MgSO.sub.4), filtered
and concentrated to give 10.84 g (50%) of the desired product.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.85 (m, 5H), 5.45 (s,
1H), 5.34 (s, 1H), 3.5 (s, 2H), 2.93 (d, J=10.96 Hz, 2H), 2.16 (t,
J=12.13 Hz, 1H), 2.01 (t, J=11.74 Hz, 2H), 1.87 (d, J=12.52 Hz,
2H), 1.76 (q, J=12.52 Hz, 2H).
[0490] Step 2: 4-Aminomethyl-1-benzyl piperdine. To a solution of
N-benzylisonipecotamide (7.34 g, 34 mmol) in 60 mL of anhydrous
THF, was added, LiAlH.sub.4 (1.9 g, 51 mmol) and the mixture was
stirred at room temperature for 10 minutes followed by heating to
reflux for 3 h. The reaction was quenched by addition of 100 mL
sat'd. sodium potassium tartrate, and was extracted with EtOAc
(3.times.50 mL). The combined extracts were washed with 20 mL water
and 20 mL brine then dried over MgSO.sub.4 filtered and
concentrated to give the desired product. .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 7.13 (m, 5H), 3.42 (s, 2H), 2.91 (m, 2H), 2.59
(m, 2H), 1.97 (m, 2H), 1.62 (m, 2H), 2.21 (m, 5H).
[0491] Step 3:
N-(1-Benzyl-piperidin-4-ylmethyl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)--
ureido]-benzamide. Prepared from 4-aminomethyl-1-benzyl piperdine,
according to the procedure of Compound 208:
[0492] .sup.1H-NMR (400 MHz, d6-DMSO) .delta. 10.1 (s, 2H), 8.81
(s, 1H), 8.48 (s, 1H), 8.26 (m, 2H), 7.52 (m, 7H), 3.97 (s, 5H),
3.33 (s, 3H), 3.18 (s, 3H), 2.43 (s, 3H), 1.75 (s, 3H), 1.44 (s,
2H). MS APCI-pos, M+1=489.1.
[0493] Compound 230:
##STR00329##
N-[3-S-1-(4-Fluoro-benzyl)-pyrrolidin-3-yl]-3-methoxy-4-[3-(5-methyl-pyra-
zin-2-yl)-ureido]-benzamide
[0494] Step 1:
(3S)-3-(tert-Butoxycarbonylamino)-1-(4-fluoro-benzyl)-pyrrolidine.
To a solution of (3S)-(+)-3-(tert-butoxycarbonylamino)pyrrolidine
(410 mg, 2.20 mmol), in 22 mL EtOH, was added, (288 .mu.L, 2.31
mmol) of 4-fluorobenzyl bromide, and (788 mg, 2.42 mmol) of finely
powdered cesium carbonate. The stirred reaction mixture was heated
at 80.degree. C., under nitrogen for 3 h, after which time, TLC
indicated the reaction was complete. The reaction was then
concentrated in vacuo to about 5 mL, and was then diluted with 30
mL of EtOAc, and washed with 20 mL of 5% NH.sub.4OH. The aqueous
fraction was extracted with diethyl ether (3.times.20 mL). The
combined organics were washed with 20 mL of brine, dried
(MgSO.sub.4), filtered and concentrated. The crude white solid was
triturated with 1:1 ether-hexane to give 501 mg (77%) of the
desired product as a white solid. .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 7.2-7.3 (m, 2H), 6.9-7.1 (m, 2H), 4.85 (br s, 1H,
exchanges), 4.18 (br s, 1H), 3.55 (s, 2H), 2.65 (br m, 1H), 2.58
(m, 1H), 2.50 (m, 1H), 2.2-2.4 (m, 2H), 1.5-1.7 (m, 1H), 1.4 (s,
9H).
[0495] Step 2: (3S)-3-Amino-1-(4-fluoro-benzyl)-pyrrolidine. A
solution of
(3S)-3-(tert-butoxycarbonylamino)-1-(4-fluoro-benzyl)-pyrrolidine
(400 mg, 1.36 mmol) was stirred in 15 mL of formic acid at room
temperature. After 3 h, the clear colorless solution was
concentrated to dryness and the residue was taken-up in 20 mL of
EtOAc and washed with 10 mL of 5% NH.sub.4OH, followed by 10 mL of
brine. The solution was then dried over MgSO.sub.4, filtered and
concentrated to give 240 mg (91%) of product, as a yellow oil.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.28 (m, 2H), 6.99 (m,
2H), 3.56 (d, J=6.2 Hz, 2H), 3.47-3.53 (m, 1H), 2.67-2.71 (m, 2H),
2.42-2.48 (m, 1H), 2.28-2.31 (m, 1H), 2.21-2.28 (m, 1H), 1.59 (s,
2H, NH.sub.2), 1.43-1.52 (m, 1H).
[0496] Step 3:
N-[(3S)-1-(4-Fluoro-benzyl)-pyrrolidin-3-yl]-3-methoxy-4-[3-(5-methyl-pyr-
azin-2-yl)-ureido]-benzamide. Prepared from
(3S)-3-amino-1-(4-fluoro-benzyl)-pyrrolidine, according to the
procedure of Compound 208. .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 10.8 (s, 2H), 8.8 (s, 1H), 8.38 (s, 1H), 8.27 (s, 1H), 8.24
(s, 2H), 7.51 (s, 1H), 7.49 (s, 1H), 7.37 (s, 2H), 7.15 (m, 2H),
4.4 (br s, 1H), 3.98 (s, 3H), 3.6 (br s, 2H), 3.06 (br.s, 1H), 2.65
(br.s, 1H), 2.81 (br.s, 1H), 2.43 (s, 3H), 2.16 (s, 1H), 1.83 (s,
1H), MS apc:-pos M+1=479.2.
[0497] Compound 231:
##STR00330##
3-Methoxy-4-[3-(5-methyl-pyrazin-2yl)-ureido]-benzoic acid
3-S-1-benzyl-pyrrolidin-3-yl ester
[0498] Step 1: 3-Methoxy-4-nitro-benzoyl chloride. Thionyl chloride
3.7 mL, (50 mmol) was added drop wise, at room temperature, to a
stirred solution of 3-methoxy-4-nitro-benzoic acid (1.0 g, 5.07
mmol) in 15 mL of dioxane under a nitrogen atmosphere. After the
addition was complete, the reaction was allowed to stir at room
temperature for 1 hr. The reaction flask was then fitted with a
distillation head, and excess thionyl chloride and about 1/2 of the
solvent was removed by distillation in a 120.degree. C. oil bath.
The remaining solvent was removed by rotary evaporation, and the
residue was taken up in 20 mL toluene and again about 1/2 of the
solvent was removed by distillation. The remaining solution was
then cooled to 0.degree. C., and the white solid which
precipitated, was collected by suction filtration, and rinsed with
1-1 Et.sub.2O-hexane. .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.
7.87 (d, J=7.83 Hz, 1H), 7.82 (d, J=7.83 Hz, 1H), 7.777 (s, 1H),
4.04 (s, 3H).
[0499] Step 2: (3S)-1-Benzyl-pyrrolidin-3-ol. A solution of
(3S)-3-hydroxypyrrolidine (2.0 g, 25 mmol) in 50 mL
CH.sub.2Cl.sub.2, under a nitrogen atmosphere, was cooled to
0.degree. C. and benzaldehyde (2.92 g, 27.5 mmol) was added,
followed by 1 g of powdered 4 A molecular sieves. Sodium
triacetoxyborohydride (7.4 gm, 35 mmol) was added in several
portions over 30 min, and the reaction was allowed to stir at room
temperature for 18 h. The reaction was again cooled to 0.degree. C.
and quenched by addition of 10 mL methanol, followed by 5 mL of 1N
HCl. The molecular sieve solids were removed by filtration through
a glass fiber paper, and the filtrate was extracted with 20 mL
diethyl ether. The organic phase was discarded, and the aqueous
phase was first basified by addition of conc. ammonium hydroxide,
and then extracted with EtOAc (3.times.20 mL). The combined
extracts were washed with 20 mL brine, dried (MgSO.sub.4),
filtered, and concentrated in vacuo to furnish 3.2 g (73%) of clear
yellow oil, which required no further purification. .sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 7.2-7.4 (m, 5H), 4.33 (m, 1H), 3.63
(s, 2H), 2.83-3.89 (m, 1H), 2.67 (d, J=9.9 Hz, 1H), 2.53-2.55 (m,
1H), 2.23-2.34 (m, 1H), 2.14-2.20 (m, 2H), 1.70-1.77 (m, 1H).
[0500] Step 3: 3-Methoxy-4-nitro-benzoic acid
(3S)-1-benzyl-pyrrolidin-3-yl ester. A solution of
3-methoxy-4-nitro-benzoyl chloride (608 mg, 2.82 mmol) in 10 mL of
CH.sub.2Cl.sub.2 was added to stirred solution of 500 mg (2.82
mmol) of (3S)-1-benzyl-pyrrolidin-3-ol and 1 mL of pyridine in 15
mL CH.sub.2Cl.sub.2 at room temperature. After stirring for 18 h,
the reaction mixture was diluted with 20 mL of saturated aqueous
NaHCO.sub.3 and extracted with CH.sub.2Cl.sub.2 (2.times.10 mL).
The combined extracts were washed with 20 mL of brine, dried over
MgSO.sub.4, filtered, and concentrated in vacuo to furnish 780 mg
(71%) of the desired ester as a yellow oil, which solidified after
drying under high vacuum. The resulting solid was further purified
by trituration with 1:1 ether-hexane. .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 7.83 (d, J=7.83 Hz, 1H), 7.74 (s, 1H), 7.68 (d,
J=7.83 Hz, 1H), 7.2-7.4 (m, 5H), 5.44 (br.m, 1H), 4.02 (s, 2H),
3.69 (dd, J=30 Hz, J=13 Hz, 1 h), 2.8-3.0 (m, 2H), 2.5-2.6 (m, 1H),
2.3-2.45 (m, 1H), 2.0-2.1 (m, 1H).
[0501] Step 4: 4-Amino-3-methoxy-benzoic acid
(3S)-1-benzyl-pyrrolidin-3-yl ester. The aniline was prepared by
nickel boride reduction of 3-methoxy-4-nitro-benzoic acid
(3S)-1-benzyl-pyrrolidin-3-yl ester analogously to the preparation
of 4-[(Benzyl-methyl-amino)-methyl]-2-methoxy-phenylamine used in
the synthesis of Compound 340. .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 7.54 (d, J=8.21 Hz, 1H), 7.44 (s, 1H), 7.2-7.4 (m, 5H),
6.65 (d, J=8.21 Hz, 1H), 5.38 (br.m, 1H), 4.22 (s, 1H), 3.90 (s,
3H), 3.69 (q, J=24.63 Hz, 1H), 3.0 (m, 1H), 2.7-2.9 (m, 2H),
2.5-2.6 (m, 1H), 2.3-2.4 (m, 1H), 1.9-2.1 (m, 1H). MS apci-pos
M+1=327.2.
[0502] Step 5:
3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic acid
(3S)-1-benzyl-pyrrolidin-3-yl ester. Prepared from
4-amino-3-methoxy-benzoic acid (3S)-1-benzyl-pyrrolidin-3-yl ester
according to the procedure of Compound 208: .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 10.15 (s, 2H), 8.8 (s, 1H), 8.35 (d, J=8.61 Hz,
1H), 8.25 (s, 1H), 7.58 (d, J=8.61 Hz, 1H), 7.5 (s, 1H), 7.30-7.35
(m, 4H), 7.26-7.24 (m, 1H), 5.28 (s, 1H), 3.99 (s, 3H), 3.62 (s,
2H), 2.82 (m, 1H), 2.74 (m, 1H), 2.67 (m, 1H), 2.44 (s, 3H), 2.28
(m, 1H), 1.91 (m, 1H). MS apci-pos M+1=462.2.
[0503] Compound 232:
##STR00331##
N-(3-S-1-Benzyl-pyrrolidin-3-yl)-3-methoxy-4-[3-(5-trifluoromethyl-pyrazi-
n-2-yl)-ureido]-benzamide
[0504] Step 1:
3-Methoxy-4-[3-(5-trifluoromethyl-pyrazin-2-yl)-ureido]-benzoic
acid methyl ester. To a stirred solution of
5-trifluoromethylaminopyrazine (326 mg, 2 mmol) at room temperature
in NMP (2 mL) in a capped reaction vial was added
4-methoxy-3-(4-nitrophenylcarbonylamino)-benzoic acid methyl ester
(692 mg, 2 mmol) and the solution was heated to 85.degree. C. for 6
hours. The reaction was cooled to RT and triturated with EtOAc (5
mL) and the tan solid that formed was isolated by filtration and
rinsing with EtOAc (213 mg, 28%).
[0505] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.78 (s, 1H),
10.16 (br s, 1H), 9.07 s, 1H), 8.84 (s, 1H), 8.37 (d, 1H), 7.63 (d,
1H), 7.56 (s, 1H), 4.01 (s, 3H), 3.83 (s, 3H).
[0506] Step 2:
3-Methoxy-4-[3-(5-trifluoromethyl-pyrazin-2-yl)-ureido]-benzoic
acid. To a stirred solution of
3-methoxy-4-[3-(5-trifluoromethyl-pyrazin-2-yl)-ureido]-benzoic
acid methyl ester (213 mg, 0.575 mmol) in 5.75 mL 3:1 MeOH:H.sub.2O
at room temperature under nitrogen was added lithium hydroxide
monohydrate (240 mg, 5.8 mmol) and the reaction warmed to
65.degree. C. After reaching temperature, the suspension gradually
became a bright yellow solution. After about 4 hours a precipitate
formed but the reaction was continued overnight. After cooling to
RT, MeOH was removed by rotovap and the aqueous suspension
neutralized to pH=5 with concentrated HCl. As pH=5 was approached,
the suspension turned from yellow to white. The suspension was then
filtered through paper on a ceramic funnel. When most of the
H.sub.2O was removed the residue was dryed under high vacuum in a
dessicator overnight 133 mg, 55%).
[0507] Step 3: To a stirred solution of
3-methoxy-4-[3-(5-trifluoromethyl-pyrazin-2-yl)-ureido]-benzoic
acid (35 mg, 0.1 mmol) in 0.5 mL NMP in a capped reaction vial at
RT was added HBTU (41 mg, 0.11 mmol) and the activation stirred for
15 min. 1-Benzyl-pyrrolidin-3-ylamine dihydrochloride (prepared
analogously to 1-Pyridin-2-ylmethyl-pyrrolidin-3-ylamine used in
the synthesis of Compound 215) (crude 0.1 mmol) was then added as a
solution in 0.5 mL NMP with DIEA (69 .mu.L, 0.4 mmol). After
stirring overnight at RT, NMP was removed by distillation under
high vacuum at 70.degree. C. The residue was dissolved in 25 mL
CH.sub.2Cl.sub.2 with a small amount of MeOH and washed 2.times.25
mL with 10% Na.sub.2CO.sub.3. The organics were dried (MgSO.sub.4),
filtered and concentrated to a residue that was chromatographed on
a biotage 12S column with 5/95 MeOH/CH.sub.2Cl.sub.2. This material
was concentrated to dryness and triturated/filtered with Et.sub.2O
to give pure product as a white solid (9.5 mg, 19%).
[0508] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.53 (s, 1H),
9.86 (br s, 1H), 8.57 (s, 1H), 8.40 (d, 1H), 8.12 (s, 1H), 7.56 (s,
1H), 7.46 (m, 2H), 7.25 (m, 4H), 4.79 (m, 1H), 4.01 (s, 3H), 3.73
(dd, 2H), 3.22 (m, 2H), 2.76 (m, 1H), 2.47 (m, 2H), 1.76 (m,
1H).
[0509] LRMS (esi, positive) m/e 515.1 (M+1)
[0510] Compound 233:
##STR00332##
5-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(2-pyridin-2-yl-ethyl)-2-
-trifluoromethyl-benzamide
[0511] Step 1: Mixture of
5-Fluoro-4-nitro-2-trifluoromethyl-benzoic acid and
5-Fluoro-3-nitro-2-trifluoromethyl-benzoic acid. To a stirred
solution of 3-fluoro-6-trifuoromethylbenzoic acid (3.58 gm, 17.2
mmol) in 17 mL concentrated H.sub.2SO.sub.4 at 0.degree. C. was
carefully added 70% HNO.sub.3 (17 mL) dropwise. The reaction was
heated to 100.degree. C. overnight and was then cooled to room
temperature and poured into 250 mL of H.sub.2O/ice with stirring.
EtOAc (250 mL) was added and the mixture stirred. The layers were
separated and the organics washed 1.times.250 mL with H.sub.2O and
1.times.250 mL with saturated NaCl. The organics were dried
MgSO.sub.4), filtered and concentrated to an oil which solidified
upon standing. The solid was a 1:1 mixture of regioisomers and was
carried on crude.
[0512] Step 2: Mixture of
5-Fluoro-4-nitro-2-trifluoromethyl-benzoic acid methyl ester and
5-Fluoro-3-nitro-2-trifluoromethyl-benzoic acid methyl ester. To a
stirred solution of the nitro acids (crude, 17.2 mmol) in 60 mL of
4:1 THF:MeOH at 0.degree. C. was added 2M TMS-diazomethane in
hexane dropwise until a yellow color persisted. After 30 minutes
the reaction was concentrated to a crude oil and used directly in
the next step.
[0513] Step 3: Mixture of
5-Methoxy-4-nitro-2-trifluoromethyl-benzoic acid methyl ester and
5-Methoxy-3-nitro-2-trifluoromethyl-benzoic acid methyl ester. To a
stirred solution of the fluoro nitro esters (crude, 17.2 mmol) in
22 mL MeOH at room temperature was added 150 mL of 0.5 M sodium
methoxide in MeOH. The red solution was stirred for 30 minutes and
then partitioned between CH.sub.2Cl.sub.2 (500 mL) and H.sub.2O
(500 mL). The organics were washed 2.times.500 mL with H.sub.2O,
dried (MgSO.sub.4), filtered and concentrated to a white solid.
[0514] Step 4: 5-Methoxy-4-amino-2-trifluoromethyl-benzoic acid
methyl ester. To a stirred solution of the methoxy nitro esters
(crude, 17.2 mmol) in 172 mL MeOH at room temperature purged with
nitrogen was carefully added 10% Pd on C (1 gm). The suspension was
put through a vacuum/purge cycle three times with hydrogen gas and
then held under 1 atmosphere of hydrogen. After stirring overnight
the suspension was filtered through GF/F filter paper and
concentrated to a clear oil. This material was loaded directly onto
a Biotage 40M column with CH.sub.2Cl.sub.2 and eluted first with
9/1 hexane/EtOAc to elute the undesired hi Rf regioisomer and then
with 85/15 hexane/EtOAc to elute the desired lower Rf regioisomer.
After concentration, the lower Rf isomer solidified to a clear
solid (1.75 gm, 41%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.
7.33 (s, 1H), 6.98 (s, 1H), 3.92 (s, 3H), 3.85 (s, 3H)
[0515] Step 5:
5-Methoxy-4-(4-nitro-phenoxycarbonylamino)-2-trifluoromethyl-benzoic
acid methyl ester. To a stirred solution of
5-methoxy-4-amino-2-trifluoromethyol-benzoic acid methyl ester (552
mg, 2.22 mmol) in 6.6 mL CH.sub.2Cl.sub.2 at 0.degree. C. under
nitrogen was added pyridine (180 .mu.L, 2.22 mmol) followed by
p-nitrophenyl chloroformate (448 mg, 2.22 mmol) as a solid. After
stirring for 1 hour, the reaction was diluted to 30 mL with
CH.sub.2Cl.sub.2 and washed 2.times.30 mL with 2N HCl and
1.times.30 mL with H.sub.2O. The organics were dried (MgSO.sub.4),
filtered and concentrated to the p-nitrophenyl carbamate which was
isolated as a white foam (878 mg, 96%). .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 8.57 (br s, 1H), 8.30 (d, 2H), 7.77 (br s, 1H),
7.40 (d, 2H), 7.37 (s, 1H), 4.03 (s, 3H), 3.94 (s, 3H).
[0516] Step 6:
5-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-2-trifluoromethyl-benzoic
acid methyl ester. To a stirred solution of
5-methoxy-4-(4-nitro-phenoxycarbonylamino)-2-trifluoromethyl-benzoic
acid methyl ester (878 mg, 2.1 mmol) in 4.2 mL NMP at room
temperature under nitrogen was added 2-amino-5-methylpyrazine (232
mg, 2.1 mmol) and the reaction heated to 85.degree. C. After six
hours the reaction was cooled to room temperature and a precipitate
formed. The precipitate was triturated with EtOAc (25 mL) and the
urea isolated by filtration as a tan solid (470 mg, 58%).
[0517] Step 7:
5-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-2-trifluoromethyl-benzoic
acid. To a stirred suspension of
5-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-2-trifluoromethyl-benzoic
acid methyl ester (257 mg, 0.67 mmol) in 6.7 mL 3:1 MeOH:H.sub.2O
at room temperature under nitrogen was added lithium hydroxide
monohydrate (281 mg, 6.7 mmol) and the mixture was heated to
85.degree. C. After stirring overnight the reaction was cooled to
room temperature, MeOH was removed by rotovap and the residual
suspension was neutralized with concentrated HCl to pH of
approximately 5. The suspension was filtered and rinsed with
H.sub.2O and the filter cake dried under high vacuum to give the
acid as a white solid (161 mg, 61%). .sup.1H-NMR (400 MHz,
d.sub.6-DMSO) .delta. 8.90 (br s, 1H), 8.63 (s, 1H), 8.22 (s, 1H),
7.38 (s, 1H), 4.00 (s, 3H), 2.42 (s, 3H). LRMS (esi, negative) m/e
369.0 (M-1)
[0518] Step 8:
5-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(2-pyridine-2-yl-ethyl)--
2-trifluoromethyl-benzamide. To a stirred solution of
5-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-2-trifluoromethyl-benzoic
acid (37 mg, 0.1 mmol) in 1 mL NMP at room temperature in a capped
reaction vial was added HBTU (42 mg, 0.11 mmol) and the suspension
stirred for 15 minutes. 2-aminoethylpyridine (13 .mu.L, 0.11 mmol)
was added followed by DIEA (35 .mu.L, 0.2 mmol) and the reaction
was stirred overnight. NMP was then removed by bulb to bulb
transfer under high vacuum at 70.degree. C. and the residue
triturated and filtered with EtOAc to give the desired amide as a
tan solid (32 mg, 68%).
[0519] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.90 (br s, 1H),
8.66 (d, 1H), 8.61 (s, 1H), 8.52 (t, 1H), 8.23 (s, 1H), 8.10 (br m,
1H), 7.61 (br d, 1H), 7.57 (br m, 1H), 7.07 (s, 1H), 4.00 (s, 3H),
3.62 (m, 2H), 3.11 (m, 2H), 2.42 (s, 3H)
[0520] LRMS (esi, positive) m/e 475.1 (M+1).
[0521] Compound 234:
##STR00333##
3-(3-Dimethylamino-propoxy)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic
acid
[0522] Step 1: 3-(3-Dimethylamino-propoxy)-4-nitro-benzoic acid
methyl ester. To a stirred, cooled (about 0.degree. C.) solution of
3-hydroxy-4-nitro benzoic acid methyl ester (394 mg, 2.0 mmol),
triphenyl phosphine (525 mg, 2.0 mmol), and
3-(dimethylamino)-propanol (237 .mu.L, 2.0 mmol) in dry
tertrahydrofuran (5 mL) was added diisopropyl azodicarboxylate (394
.mu.L, 2.0 mmol in 1 mL of tetrahydrofuran). After stirring for 12
hours, the reaction was diluted with hydrochloric acid (30 mL of a
1M solution) and extracted with ethyl acetate (2.times.50 mL). The
aqueous solution was basified with 10% aqueous sodium carbonate (50
mL) and the product was extracted with ethyl acetate (3.times.100
mL). The ethyl acetate was washed with brine (1.times.30 mL), then
dried MgSO.sub.4) and filtered. The filtered solution was
concentrated under reduced pressure to provide the desired crude
product (86% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.81
(d, 1H), 7.78 (s, 1H), 7.65 (d, 1H), 4.23 (t, 2H), 3.91 (s, 3H),
2.44 (t, 2H), 2.22 (s, 6H), 2.05 (m, 2H).
[0523] Step 2: 4-Amino-3-(3-dimethylamino-propoxy)-benzoic acid
methyl ester. To a stirred, cooled (about 0.degree. C.) solution of
3-(3-dimethylamino-propoxy)-4-nitro-benzoic acid methyl ester (282
mg, 1.0 mmol) in methanol (2 mL) and saturated aqueous ammonium
chloride (1 mL) was added zinc dust (2.0 mmol). After stirring for
12 hours, the reaction was diluted with 30 mL of ethyl acetate and
washed with 30 mL of 10% aqueous sodium carbonate (2.times.30 mL),
brine (1.times.30 mL), then dried MgSO.sub.4) and filtered. The
filtered solution was concentrated under reduced pressure to yield
the desired product as a tan solid (88% yield). .sup.1H-NMR (400
MHz, CDCl.sub.3) .delta. 7.52 (d, 1H), 7.48 (s, 1H), 7.25 (s, 1H),
6.65 (d, 1H), 4.29 (br s, 1H), 4.09 (t, 2H), 3.85 (s, 3H), 2.49 (t,
2H), 2.25 (s, 6H), 2.00 (m, 2H).
[0524] Step 3:
3-(3-Dimethylamino-propoxy)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic
acid methyl ester. To a stirred, cooled (about 0.degree. C.)
solution of 4-amino-3-(3-dimethylamino-propoxy)-benzoic acid methyl
ester (252 mg, 1.0 mmol) in toluene (3.0 mL) was added
triethylamine (139 .mu.L, 1.0 mmol) and triphosgene (98 mg, 0.33
mmol). After stirring for 30 minutes, 5-methyl-2-amino pyrazine
(109 mg, 1.0 mmol) was added and the reaction was heated to 65
degrees C. The reaction was allowed to cool to room temperature,
then diluted with ethyl acetate (50 mL) and water (50 mL). A
precipitate formed which was filtered and dried under reduced
pressure to yield the desired material as a white solid (40%
yield). .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.62 (br s,
1H), 8.41 (d, 1H), 8.19 (s, 1H), 7.59 (d, 1H), 7.49 (s, 1H), 4.15
(t, 2H), 3.81 (s, 3H), 2.42 (m, 5H), 2.18 (s, 6H), 2.01 (m,
2H).
[0525] Step 4:
3-(3-Dimethylamino-propoxy)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic
acid. To a stirred solution of
3-(3-dimethylamino-propoxy)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzoic
acid methyl ester (1.0 gm; 3.3 mmol) in a methanol (25 mL) was
added lithium hydroxide (5 mL of a 2M aqueous solution). The
reaction was heated to 60 degrees and stirred for 12 hours. The
reaction was allowed to cool to room temperature and the pH was
adjusted to 5.5 with 1N hydrochloric acid. A precipitate formed
which was filtered and dried under reduced pressure to yield the
desired product as a tan solid (52% yield). .sup.1H-NMR (400 MHz,
d.sub.6-DMSO) .delta. 8.62 (br s, 1H), 8.39 (d, 1H), 8.21 (s, 1H),
7.59 (d, 1H), 7.43 (s, 1H), 4.15 (t, 2H), 2.42 (m, 5H), 2.18 (s,
6H), 2.01 (m, 2H).
[0526] Compound 235:
##STR00334##
4-[3-(5-Methyl-pyrazin-2-yl)-ureido]-3-(pyridine-3-ylmethoxy)-benzoic
acid
[0527] Step 1: 4-Nitro-3-(pyridine-3-ylmethoxy)-benzoic acid methyl
ester. To a stirred, cooled (about 0.degree. C.) solution of
3-hydroxy-4-nitro benzoic acid methyl ester (394 mg, 2.0 mmol),
triphenyl phosphine (525 mg, 2.0 mmol), and 3-pyridylcarbinol (194
.mu.L, 2.0 mmol) in dry tertrahydrofuran (5 mL) was added
diisopropyl azodicarboxylate (394 .mu.L, 2.0 mmol in 1 mL of
tetrahydrofuran). After stirring for 12 hours, the reaction was
diluted with hydrochloric acid (30 mL of a 1M solution) and
extracted with ethyl acetate (2.times.50 mL). The aqueous solution
was basified with 10% aqueous sodium carbonate (50 mL) and the
product was extracted with ethyl acetate (3.times.100 mL). The
ethyl acetate was washed with brine (1.times.30 mL), then dried
(MgSO.sub.4) and filtered. The filtered solution was concentrated
under reduced pressure to provide the desired crude product as an
off white solid (76% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 8.71 (s, 1H), 8.62 (d, 1H), 7.88 (m, 3H), 7.79 (d, 1H),
7.39 (M, 1H), 5.31 (s, 2H), 3.98 (s, 3H).
[0528] Step 2: 4-Amino-3-(pyridin-3-ylmethoxy)-benzoic acid methyl
ester. To a stirred, cooled (about 0.degree. C.) solution of
4-nitro-3-(pyridin-3-ylmethoxy)-benzoic acid methyl ester (288 mg,
1.0 mmol) in methanol (2 mL) and saturated aqueous ammonium
chloride (1 mL) was added zinc dust (131 mg, 2.0 mmol). After
stirring for 12 hours, the reaction was diluted with 30 mL of ethyl
acetate and washed with 30 mL of 10% aqueous sodium carbonate
(2.times.30 mL), brine (1.times.30 mL), then dried MgSO.sub.4) and
filtered. The filtered solution was concentrated under reduced
pressure to yield the desired product as a yellow solid (97%
yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.78 (s, 1H),
8.61 (d, 1H), 7.79 (d, 1H), 7.59 (m, 2H), 7.39 (m, 1H), 6.71 (d,
1H), 5.18 (s, 2H), 4.28 (br s, 2H), 3.85 (s, 3H).
[0529] Step 3:
4-[3-(5-Methyl-pyrazin-2-yl)-ureido]-3-(pyridin-3-ylmethoxy)-benzoic
acid methyl ester. To a stirred, cooled (about 0.degree. C.)
solution of 4-amino-3-(pyridin-3-ylmethoxy)-benzoic acid methyl
ester (258 mg, 1.0 mmol) in toluene (3.0 mL) was added
triethylamine (139 .mu.L, 1.0 mmol) and triphosgene (98 mg, 0.33
mmol). After stirring for 30 minutes, 5-methyl-2-amino pyrazine
(109 mg, 1.0 mmol) was added and the reaction was heated to 65
degrees C. The reaction was allowed to cool to room temperature,
then diluted with ethyl acetate (50 mL) and water (50 mL). A
precipitate formed which was filtered and dried under reduced
pressure to yield the desired material as a white solid (47%
yield). .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.29 (s, 1H),
8.79 (s, 1H), 8.68 (d, 1H), 8.59 (br s, 1H), 8.48 (d, 1H), 7.70 (s,
1H), 7.62 (d, 1H), 7.51 (m, 1H), 5.32 (s, 2H), 3.88 (s, 3H), 2.32
(s, 3H).
[0530] Step 4:
4-[3-(5-Methyl-pyrazin-2-yl)-ureido]-3-(pyridine-3-ylmethoxy)-benzoic
acid. To a stirred solution of
4-[3-(5-methyl-pyrazin-2-yl)-ureido]-3-(pyridine-3-ylmethoxy)-benzoic
acid methyl ester (1.3 gm, 3.3 mmol) in methanol (25 mL) was added
lithium hydroxide (5 mL of a 2M aqueous solution). The reaction was
heated to 60 degrees and stirred for 12 hours. The reaction was
allowed to cool to room temperature and the pH was adjusted to 5.5
with 1N hydrochloric acid. A precipitate formed which was filtered
and dried under reduced pressure to yield the desired product as a
tan solid (90% yield). .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta.
10.29 (s, 1H), 8.79 (s, 1H), 8.68 (d, 1H), 8.59 (br s, 1H), 8.48
(d, 1H), 7.70 (s, 1H), 7.62 (d, 1H), 7.51 (m, 1H), 5.32 (s, 2H),
2.32 (s, 3H).
[0531] Compound 236:
##STR00335##
3-(3-Dimethylamino-propoxy)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzamid-
e
[0532] Prepared according to the procedure of Compound 208 using
ammonia (33% yield).
[0533] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.18 (s, 1H),
8.56 (br s, 1H), 8.20 (d, 1H), 8.11 (s, 1H), 7.43 (d, 1H), 7.40 (s,
1H), 4.03 (m, 2H), 2.40 (m, 2H), 2.33 (s, 3H), 2.05 (s, 6H), 1.91
(m, 2H).
[0534] LRLCMS (esi, positive) m/e 374.2 (M+1)
[0535] Compound 237:
##STR00336##
3-(3-Dimethylamino-propoxy)-N,N-dimethyl-4-[3-(5-methyl-pyrazin-2-yl)-ure-
ido]-benzamide
[0536] Prepared according to the procedure of Compound 208 using
dimethylamine (97% yield).
[0537] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.30 (br s, 1H),
8.39 (d, 1H), 8.29 (s, 1H), 8.20 (s, 1H), 7.78 (br s, 1H), 7.07 (s,
1H), 7.04 (d, 1H), 4.16 (t, 2H), 2.55 (s, 3H), 2.53 (m, 2H), 2.26
(s, 6H), 2.10 (m, 2H).
[0538] LRMS (esi, positive) m/e 401.1 (M+1)
[0539] Compound 238:
##STR00337##
N-Benzyl-3-(3-dimethylamino-propoxy)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-
-benzamide
[0540] Prepared according to the procedure of Compound 208 using
benzylamine (65% yield).
[0541] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.43 (d, 1H), 8.26
(s, 1H), 8.20 (s, 1H), 7.91 (br s, 1H), 7.56 (s, 1H), 7.38 (m, 4H),
7.31 (m, 1H), 6.41 (m, 1H), 4.64 (d, 2H), 4.20 (t, 2H), 2.53 (s,
3H), 2.52 (m, 2H), 2.24 (s, 6H), 2.14 (m, 2H).
[0542] LRMS (esi, positive) m/e 463.2 (M+1)
[0543] Compound 239:
##STR00338##
N-Benzyl-3-(3-dimethylamino-propoxy)-N-methyl-4-[3-(5-methyl-pyrazin-2-yl-
)-ureido]-benzamide
[0544] Prepared according to the procedure of Compound 208 using
N-methyl benzylamine (68% yield).
[0545] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.37 (br s, 1H),
8.25 (s, 1H), 8.18 (s, 1H), 7.37 (m, 4H), 7.31 (m, 1H), 7.18 (m,
2H), 4.67 (br m, 2H), 4.07 (br m, 2H), 2.99 (br s, 3H), 2.53 (s,
3H), 2.50 (br m, 2H), 2.24 (s, 6H), 2.06 (br m, 2H).
[0546] LRMS (esi, positive) m/e 477.2 (M+1)
[0547] Compound 240:
##STR00339##
3-(3-Dimethylamino-propoxy)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-(2-mor-
pholin-4-yl-ethyl)-benzamide
[0548] Prepared according to the procedure of Compound 208 using
2-morpholin-4-yl-ethylamine (69% yield).
[0549] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.44 (d, 1H), 8.31
(s, 1H), 8.21 (s, 1H), 7.90 (br s, 1H), 7.55 (s, 1H), 7.24 (d, 1H),
6.76 (m, 1H), 4.20 (t, 2H), 3.76 (m, 4H), 3.56 (m, 2H), 2.61 (m,
2H), 2.53 (m, 7H), 2.24 (s, 6H), 2.13 (m, 2H).
[0550] LRMS (esi, positive) m/e 486.2 (M+1)
[0551] Compound 241:
##STR00340##
3-(3-Dimethylamino-propoxy)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-N-[2-(1--
methyl-pyrrolidin-2-yl)-ethyl]-benzamide
[0552] Prepared according to the procedure of Compound 208 using
2-(1-methyl-pyrrolidin-2-ylamine (68% yield).
[0553] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.35 (br s, 1H),
8.42 (s, 1H), 8.40 (s, 1H), 8.24 (s, 1H), 8.20 (s, 1H), 7.57 (s,
1H), 7.46 (br s, 1H), 7.20 (m, 1H), 4.19 (m, 2H), 3.76 (m, 1H),
3.44 (m, 1H), 3.15 (m, 1H), 2.54 (s, 3H), 2.42 (m, 1H), 2.37 (s,
3H), 2.25 (s, 6H), 2.22 (m, 1H), 2.13 (m, 2H), 1.90 (m, 2H), 1.77
(m, 2H).
[0554] LRMS (esi, positive) m/e 484.3 (M+1)
[0555] Compound 242:
##STR00341##
N-(2-dimethylamino-ethyl)-3-(3-dimethylamino-propoxy)-4-[3-(5-methyl-pyra-
zin-2-yl)-ureido]-benzamide
[0556] Prepared according to the procedure of Compound 208 using
2-dimethylamino-ethylamine (37% yield).
[0557] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.38 (br s, 1H),
8.42 (d, 1H), 8.30 (s, 1H), 8.20 (s, 1H), 7.96 (br s, 1H), 7.53 (s,
1H), 7.27 (d, 1H), 6.79 (m, 1H), 4.20 (m, 2H), 3.53 (m, 2H), 2.52
(m, 2H), 2.52 (s, 3H), 2.26 (m, 2H), 2.24 (s, 6H), 2.22 (s, 6H),
2.12 (m, 2H).
[0558] LRMS (esi, positive) m/e 444.2 (M+1).
[0559] Compound 243:
##STR00342##
N-(3-S-1-Benzyl-pyrrolidin-3-yl)-3-(3-dimethylamino-propoxy)-4-[3-(5-meth-
yl-pyrazin-2-yl)-ureido]-benzamide
[0560] Prepared according to the procedure of Compound 215 using
1-benzyl-pyrrolidin-3-S-ylamine (20% yield)
[0561] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.61 (brs, 1H),
8.45 (d, 1H), 8.20 (s, 1H), 8.16 (m, 1H), 7.51 (s, 1H), 7.39 (d,
1H), 7.31 (m, 4H), 7.16 (m, 1H), 4.72 (m, 1H), 4.18 (m, 2H), 3.72
(dd, 2H), 3.04 (m, 1H), 2.96 (m, 1H), 2.69 (m, 1H), 2.53 (m, 2H),
2.51 (s, 3H), 2.40 (m, 2H), 2.25 (s, 6H), 2.11 (m, 2H), 1.76 (m,
1H).
[0562] LRMS (esi, positive) m/e 532.2 (M+1)
[0563] Compound 244:
##STR00343##
4-[3-(5-Methyl-pyrazin-2-yl)-ureido]-3-(pyridine-3-ylmethoxy)-benzamide
[0564] Prepared according to the procedure of Compound 208 using
ammonia (99% yield).
[0565] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.27 (s, 1H),
8.82 (s, 1H), 8.66 (d, 1H), 8.60 (br s, 1H), 8.29 (d, 1H), 8.03 (m,
1H), 8.00 (s, 1H), 7.76 (s, 1H), 7.56 (d, 1H), 7.50 (m, 1H), 7.37
(br m, 1H), 7.26 (br s, 1H), 5.32 (s, 2H), 2.32 (s, 3H).
[0566] LRMS (apci, positive) m/e 379.1 (M+1)
[0567] Compound 245:
##STR00344##
N-Methyl-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-3-(pyridine-3-ylmethoxy)-be-
nzamide
[0568] Prepared according to the procedure of Compound 208 using
methylamine (99% yield).
[0569] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.25 (s, 1H),
8.80 (s, 1H), 8.60 (m, 3H), 8.26 (d, 1H), 8.03 (d, 1H), 7.78 (s,
1H), 7.52 (m, 2H), 7.37 (m, 1H), 5.34 (s, 2H), 2.79 (d, 3H), 2.36
(s, 3H).
[0570] LRMS (apci, positive) m/e 393.2 (M+1)
[0571] Compound 246:
##STR00345##
N,N-Dimethyl-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-3-(pyridine-3-ylmethoxy-
)-benzamide
[0572] Prepared according to the procedure of Compound 208 using
dimethylamine (88% yield).
[0573] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.16 (s, 1H),
8.80 (s, 1H), 8.66 (d, 1H), 8.58 (s, 1H), 8.28 (d, 1H), 7.96 (d,
1H), 7.50 (m, 1H), 7.37 (br s, 1H), 7.25 (s, 1H), 7.03 (d, 1H),
5.30 (s, 2H), 2.95 (s, 6H), 2.33 (s, 3H).
[0574] LRMS (apci, positive) m/e 407.4 (M+1)
[0575] Compound 247:
##STR00346##
N-Benzyl-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-3-(pyridine-3-ylmethoxy)-be-
nzamide
[0576] Prepared according to the procedure of Compound 208 using
benzylamine (41% yield).
[0577] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.23 (s, 1H),
9.05 (t, 1H), 8.82 (s, 1H), 8.67 (d, 1H), 8.58 (br s, 1H), 8.33 (d,
1H), 8.00 (d, 1H), 7.78 (s, 1H), 7.59 (d, 1H), 7.50 (m, 1H), 7.33
(m, 4H), 7.25 (m, 1H), 5.32 (s, 2H), 4.50 (d, 2H), 2.32 (s,
3H).
[0578] LRMS (apci, positive) m/e 469.1 (M+1)
[0579] Compound 248:
##STR00347##
N-Benzyl-N-methyl-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-3-(pyridin-3-ylmet-
hoxy)-benzamide
[0580] Prepared according to the procedure of Compound 208 using
N-methyl benzylamine (71% yield).
[0581] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.16 (s, 1H),
8.76 (br s, 1H), 8.64 (d, 1H), 8.57 (m, 1H), 8.28 (br m, 1H), 7.95
(br m, 1H), 7.48 (m, 1H), 7.40-7.23 (br m, 2H), 7.06 (m, 1H), 5.23
(br m, 2H), 4.62 (br m, 2H), 2.88 (s, 3H), 2.33 (s, 3H).
[0582] LRMS (apci, positive) m/e 483.3 (M+1)
[0583] Compound 249:
##STR00348##
4-[3-(5-Methyl-pyrazin-2-yl)-ureido]-N-(2-morpholin-4-yl-ethyl)-3-(pyridi-
n-3-ylmethoxy)-benzamide
[0584] Prepared according to the procedure of Compound 208 using
2-morphlin-4-yl-ethylamine (99% yield).
[0585] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.27 (s, 1H),
8.81 (s, 1H), 8.66 (d, 1H), 8.61 (br s, 1H), 8.52 (t, 1H), 8.29 (d,
1H), 8.03 (d, 1H), 7.76 (s, 1H), 7.52 (m, 2H), 7.36 (m, 1H), 5.33
(s, 2H), 3.56 (m, 4H), 3.40 (m, 2H), 2.48 (m, 2H), 2.43 (m, 4H),
2.34 (s, 3H).
[0586] LRMS (apci, positive) m/e 492.4 (M+1)
[0587] Compound 250:
##STR00349##
4-[3-(5-Methyl-pyrazin-2-yl)-ureido]-N-[2-(1-methyl-pyrrolidin-2-yl)-ethy-
l]-3-(pyridin-3-ylmethoxy)-benzamide
[0588] Prepared according to the procedure of Compound 208 using
2-(1-methyl-pyrrolidin-2-yl)-ethylamine (99% yield).
[0589] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.21 (s, 1H),
8.81 (s, 1H), 8.64 (d, 1H), 8.58 (br s, 1H), 8.49 (m, 1H), 8.33 (d,
1H), 8.00 (d, 1H), 7.70 (s, 1H), 7.52 (m, 2H), 7.34 (m, 1H), 5.33
(s, 2H), 3.28 (m, 2H), 2.95 (m, 1H), 2.36 (s, 3H), 2.21 (s, 3H),
2.04 (m, 2H), 1.90 (m, 2H), 1.62 (m, 2H), 1.44 (m, 2H).
[0590] LRMS (apci, positive) m/e 490.3 (M+1)
[0591] Compound 251:
##STR00350##
N-(2-dimethylamino-ethyl)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-3-(pyridin-
e-3-ylmethoxy)-benzamide
[0592] Prepared according to the procedure of Compound 208 using
2-dimethylamino-ethylamine (99% yield).
[0593] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.24 (s, 1H),
8.82 (s, 1H), 8.66 (d, 1H), 8.60 (br s, 1H), 8.43 (t, 1H), 8.30 (d,
1H), 8.02 (d, 1H), 7.74 (s, 1H), 7.52 (m, 2H), 7.35 (br m, 1H),
5.32 (s, 2H), 3.36 (m, 2H), 2.39 (m, 2H), 2.33 (s, 3H), 2.18 (s,
6H).
[0594] LRMS (apci, positive) m/e 450.2 (M+1)
[0595] Compound 252:
##STR00351##
N-(3-S-1-Benzyl-pyrrolidin-3-yl)-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-3-(-
pyridine-3-ylmethoxy)-benzamide
[0596] Prepared according to the procedure of Compound 215 using
1-benzyl-pyrrolidin-3-S-ylamine (99% yield).
[0597] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.26 (s, 1H),
8.82 (s, 1H), 8.66 (d, 1H), 8.60 (br s, 1H), 8.47 (d, 1H), 8.28 (d,
1H), 8.02 (d, 1H), 7.74 (s, 1H), 7.55 (d, 1H), 7.51 (m, 1H), 7.32
(m, 4H), 7.24 (m, 1H), 5.31 (s, 2H), 4.40 (m, 1H), 3.60 (s, 2H),
2.80 (m, 1H), 2.64 (m, 1H), 2.45 (m, 2H), 2.34 (s, 3H), 2.15 (m,
1H), 1.85 (m, 1H).
[0598] LRMS (apci, positive) m/e 538.2 (M+1)
[0599] Compound 253:
##STR00352##
1-[2-(3-Dimethylamino-propoxy)-5-methyl-phenyl]-3-pyrazin-2-yl-urea
[0600] Step 1: (2-Hydroxy-5-methyl-phenyl)-carbamic acid tert-butyl
ester. To a stirred solution of 2-amino-4-methyl-phenol (6.15 gm;
50 mmol) in dioxane (100 mL) was added carbonic acid di-tert-butyl
ester (9.8 gm; 45 mmol) followed by sodium bicarbonate (12.6 gm;
150 mmol in 75 mL of H.sub.2O). After stirring for 8 hours, the
reaction was diluted with 100 mL of ethyl acetate and washed with
1N aqueous hydrochloric acid (2.times.100 mL), saturated aqueous
sodium bicarbonate (1.times.100 mL), and brine (100 mL), then dried
(MgSO.sub.4) and filtered. The filtered solution was concentrated
under reduced pressure to provide the desired
(2-Hydroxy-5-methyl-phenyl)-carbamic acid tert-butyl ester as a
brown solid (95% yield).
[0601] Step 2:
[2-(3-Dimethylamino-propoxy)-5-methyl-phenyl]-carbamic acid
tert-butyl ester. To a stirred, cooled (about 0.degree. C.)
solution of (2-hydroxy-5-methyl-phenyl)-carbamic acid tert-butyl
ester (447 mg, 2.0 mmol), triphenyl phosphine (525 mg, 2.0 mmol),
and 3-(dimethylamino)-1-propanol (237 .mu.L, 2.0 mmol) in dry
tetrahydrofuran (5 mL) was added diisopropyl azodicarboxylate (394
.mu.L, 2.0 mmol) in 1 mL of tetrahydrofuran). After stirring for 12
hours, the reaction was diluted with 30 mL of ethyl acetate and
washed with 30 mL of 10% aqueous sodium carbonate (2.times.30 mL),
brine (1.times.30 mL), then dried (MgSO.sub.4) and filtered. The
filtered solution was concentrated under reduced pressure to
provide the desired crude product.
[0602] Step 3: 2-(3-Dimethylamino-propoxy)-5-methyl-phenylamine. To
a stirred solution of
[2-(3-dimethylamino-propoxy)-5-methyl-phenyl]-carbamic acid
tert-butyl ester (617 mg, 2.0 mmol) in 5 mL of dioxane was added
hydrochloric acid (2 mL; 4N in dioxane). After stirring for 12
hours, the reaction was diluted with 20 mL of 1N hydrochloric acid
and washed with ethyl acetate (2.times.30 mL). The aqueous layer
was basified with 10% aqueous sodium carbonate (50 mL) and
extracted with ethyl acetate (3.times.50 mL). The ethyl acetate was
washed with brine (1.times.30 mL), then dried (MgSO.sub.4), and
filtered. The filtered solution was concentrated under reduced to
yield the corresponding aniline.
[0603] Step 4:
1-[2-(3-Dimethylamino-propoxy)-5-methyl-phenyl]-3-pyrazin-2-yl-urea.
To a stirred, cooled (about 0.degree. C.) solution of
2-(3-dimethylamino-propoxy)-5-methyl-phenylamine (208 mg, 1.0 mmol)
in toluene (3.0 mL) was added triethylamine (140 .mu.L, 1.0 mmol)
and triphosgene (98 mg, 0.33 mmol). After stirring for 30 minutes,
amino pyrazine (95 mg, 1.0 mmol) was added and the reaction was
heated to 65 degrees C. After stirring for 4 hours, the reaction
was cooled to room temperature and stirred for 8 hours. The
precipitate that formed was filtered, rinsed with toluene
(2.times.1 mL), and dried under reduced pressure (35% yield).
[0604] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.45 (s, 1H), 8.39
(br s, 1H), 8.22 (s, 1H), 8.21 (d, 1H), 8.19 (d, 1H), 4.09 (t, 2H),
2.55 (t, 2H), 2.36 (s, 3H), 2.26 (s, 6H), 2.05 (m, 2H).
[0605] LRMS (esi, positive) m/e 330.10 (M+1).
[0606] Compound 254:
##STR00353##
1-[2-(2-Dimethylamino-ethoxy)-5-methyl-phenyl]-3-pyrazin-2-yl-urea
[0607] Prepared according to the method of Compound 253 using
N,N-dimethyl ethanolamine (36% yield).
[0608] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.79 (br s, 1H),
8.25 (s, 1H), 8.22 (s, 1H), 8.05 (s, 1H), 6.95 (d, 1H), 6.80 (d,
1H), 4.15 (t, 2H), 2.55 (s, 3H), 2.31 (t, 2H), 2.26 (s, 6H).
[0609] LRMS (esi, positive) m/e 316.21 (M+1)
[0610] Compound 255:
##STR00354##
1-[5-Methyl-2-(pyridine-3-ylmethoxy)-phenyl]-3-pyrazin-2-yl-urea
[0611] Prepared according to the method of Compound 253 using
3-hydroxymethyl pyridine (10% yield).
[0612] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.82 (s, 1H), 8.65
(d, 1H), 8.35 (s, 1H), 8.30 (s, 1H), 8.21 (s, 2H), 8.05 (s, 1H),
7.81 (m, 2H), 7.35 (m, 1H), 6.9 (dd, 2H), 5.15 (s, 2H), 2.39 (s,
3H).
[0613] LRMS (esi, positive) m/e 336.21 (M+1)
[0614] Compound 256:
##STR00355##
1-{5-Methyl-2-[3-(2-oxo-pyrrolidin-1-yl)-propoxyl]-phenyl}-3-pyrazin-2-yl-
-urea
[0615] Prepared according to the method of Compound 253 using
(3-(2-oxo-pyrrolidin-1-yl)-propanol (10% yield).
[0616] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.79 (s, 1H),
8.29 (s, 1H), 8.25 (s, 1H), 8.05 (s, 1H), 6.92 (d, 1H), 6.79 (d,
1H), 3.99 (t, 2H), 3.38 (m, 4H), 2.22 (s, 3H), 2.20 (t, 2H), 2.00
(t, 2H), 1.91 (t, 2H).
[0617] LRMS (esi, positive) m/e 392.2 (M+Na).
[0618] Compound 257:
##STR00356##
1-[5-Methyl-2-(2-morpholin-4-yl-ethoxy)-phenyl]-3-pyrazin-2-yl-urea
[0619] Prepared according to the method of Compound 253 using
2-morpholin-4-yl-ethanol (39% yield).
[0620] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.79 (s, 1H),
8.29 (d, 1H), 8.25 (d, 1H), 8.05 (s, 1H), 6.95 (d, 1H), 6.79 (d,
1H), 4.19 (t, 2H), 3.59 (m, 4H), 2.80 (t, 2H), 2.49 (m, 4H), 2.22
(s, 3H).
[0621] LRMS (esi, positive) m/e 358.2 (M+1)
[0622] Compound 258:
##STR00357##
1-[5-Methyl-2-(3-morpholin-4-yl-propoxy)-phenyl]-3-(5-methyl-pyrazin-2-yl-
)-urea
[0623] Prepared according to the method of Compound 253 using
3-morpholin-4-yl-propanol (8% yield).
[0624] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 9.01 (s, 1H),
8.62 (br s, 1H), 8.22 (s, 1H), 8.19 (s, 1H), 8.05 (s, 1H), 6.91 (d,
1H), 6.79 (d, 1H), 4.05 (t, 2H), 3.59 (m, 4H), 2.48 (s, 3H), 2.45
(t, 2H), 2.35 (m, 4H), 2.21 (s, 3H), 2.00 (t, 2H).
[0625] LRMS (esi, positive) m/e 386.31 (M+1)
[0626] Compound 259:
##STR00358##
1-[2-(3-Dimethylamino-propoxy)-5-methyl-phenyl]-3-(5-methyl-pyrazin-2-yl)-
-urea
[0627] Prepared according to the method of Compound 253
3-dimethylamino-propanol (40% yield).
[0628] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.37 (s, 1H), 8.19
(s, 1H), 8.18 (s, 1H), 8.09 (br s, 1H), 6.80 (dd, 2H), 4.05 (t,
2H), 2.55 (t, 2H), 2.54 (s, 3H), 2.36 (s, 3H), 2.26 (s, 6H), 2.05
(m, 2H).
[0629] LRMS (esi, positive) m/e 344.20 (M+1)
[0630] Compound 260:
##STR00359##
1-[5-Methyl-2-(2-morpholin-4-yl-ethoxy)-phenyl]-3-(5-methyl-pyrazin-2-yl)-
-urea
[0631] Prepared according to the method of Compound 253 using
2-morpholin-4-yl-ethanol (10% yield).
[0632] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 10.79 (br s, 1H),
8.82 (s, 1H), 8.59 (s, 1H), 8.19 (s, 1H), 8.05 (s, 1H), 6.81 (dd,
2H), 4.20 (t, 2H), 3.75 (m, 4H), 2.91 (t, 2H), 2.61 (m, 4H), 2.55
(s, 3H), 2.35 (s, 3H).
[0633] LRMS (esi, positive) m/e 372.1 (M+1)
[0634] Compound 261:
##STR00360##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(pyridin-2-ylmethoxy)-phenyl]-ure-
a
[0635] Prepared according to the method of Compound 253 using
2-hydroxymethyl pyridine (21% yield).
[0636] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.61 (d, 1H), 8.29
(s, 1H), 8.11 (s, 1H), 7.61 (t, 1H), 7.31 (d, 1H), 7.18 (t, 1H),
6.88 (d, 1H), 6.75 (d, 1H), 5.18 (s, 2H), 2.30 (s, 3H).
[0637] LRMS (esi, positive) m/e 372.2 (M+Na).
[0638] Compound 262:
##STR00361##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(pyridin-4-ylmethoxy)-phenyl]-ure-
a
[0639] Prepared according to the method of Compound 253 using
4-hydroxymethyl pyridine (18% yield).
[0640] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.84 (s, 1H),
8.55 (d, 2H), 7.91 (s, 1H), 7.47 (d, 2H), 6.88 (d, 1H), 6.72 (d,
1H), 5.28 (s, 2H), 2.22 (s, 3H).
[0641] LRMS (esi, positive) m/e 350.21 (M+1)
[0642] Compound 263:
##STR00362##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(pyridin-3-ylmethoxy)-phenyl]-ure-
a
[0643] Prepared according to the method of Compound 253 using
3-hydroxymethyl pyridine (10% yield).
[0644] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.82 (s, 1H), 8.68
(m, 2H), 8.25 (s, 1H), 8.20 (s, 1H), 7.84 (d, 1H), 7.38 (m, 1H),
6.99 (s, 1H), 6.88 (d, 1H), 6.80 (d, 1H), 5.10 (s, 2H), 2.38 (s,
3H), 2.35 (s, 3H).
[0645] LRMS (esi, positive) m/e 350.21 (M+1)
[0646] Compound 264:
##STR00363##
1-[2-(2-Dimethylamino-ethoxy)-5-methyl-phenyl]-3-(5-methyl-pyrazin-2-yl)--
urea
[0647] Prepared according to the method of Compound 253 using
N,N-dimethyl ethanolamine (11% yield).
[0648] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.69 (s, 1H),
8.18 (s, 1H), 8.03 (s, 1H), 6.95 (d, 1H), 6.79 (d, 1H), 4.11 (t,
2H), 2.72 (t, 2H), 2.43 (s, 3H), 2.25 (s, 3H), 2.22 (s, 6H).
[0649] LRMS (esi, positive) m/e 330.20 (M+1)
[0650] Compound 265:
##STR00364##
1-(5-Methyl-pyrazin-2-yl)-3-[2-(pyridin-3-ylmethoxy)-5-trifluoromethyl-ph-
enyl]-urea
[0651] Prepared according to the method of Compound 253
3-hydroxymethyl pyridine and 2-hydroxy-5-trifluoromethyl aniline
(40% yield).
[0652] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.81 (s, 1H),
8.69 (s, 1H), 8.65 (s, 1H), 8.59 (br s, 1H), 8.01, (d, 1H), 7.45
(t, 1H), 7.3 (br s, 1H), 5.39 (s, 2H), 2.35 (s, 3H).
[0653] LRMS (esi, positive) m/e 404.10 (M+1)
[0654] Compound 266:
##STR00365##
1-[5-Methyl-2-(6-methyl-pyridin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazin--
2-yl)-urea
[0655] Step 1: (6-Methyl-pyridin-3-yl)-methanol. To a stirred,
cooled (0.degree. C.) solution of 6-methyl nicotinic acid (5.0
mmol) in tetrahydrofuran (10 mL) was added lithium aluminum hydride
(20 mmol; 20 mL of 1M solution in tetrahydrofuran) dropwise. The
reaction was stirred for 4 hours, treated sequentially with 1 mL of
H.sub.2O, 1 mL of 15% aqueous sodium hydroxide, and 3 mL of
H.sub.2O. The reaction was filtered and washed with tetrahydrofuran
(3.times.50 mL). The filtrate was concentrated under reduced
pressure to yield the alcohol as a clear viscous oil.
[0656] Steps 2-3: Mitsunobu reaction and aniline deprotection
according to the method of Compound 253.
[0657] Step 4: To a stirred solution of
5-methyl-pyrazine-2-carboxylic acid (138 mg, 1.0 mmol) in toluene
(3.0 mL) was added diphenylphosphoryl azide (216 .mu.L, 1.0 mmol)
and triethylamine (140 .mu.L, 1.0 mmol). The reaction was placed
under nitrogen and heated to 90 degrees C. for 15 minutes. The
temperature was reduced to 65.degree. C. and
5-methyl-2-(6-methyl-pyridin-3-ylmethoxy)-phenylamine (228 mg, 1.0
mmol) was added. The reaction was stirred at that temperature for 4
hours and then stirred at room temperature for 8 hours. The
precipitate that formed during the reaction was filtered, rinsed
with toluene (2.times.1 mL), and dried under reduced pressure (36%
yield).
[0658] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.45 (br s, 1H),
8.65 (s, 1H), 8.29 (s, 1H), 8.22 (s, 1H), 8.20 (s, 1H), 7.70 (d,
1H), 7.19 (d, 1H), 6.9 (m, 3H), 5.05 (s, 2H), 2.65 (s, 3H), 2.4 (s,
3H), 2.35 (s, 3H).
[0659] LRMS (esi, positive) 364.16 (M+1).
[0660] Compound 267:
##STR00366##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(2-pyridin-2-yl-ethoxy)-phenyl]-u-
rea
[0661] Steps 1-2: Mitsunobu reaction using 2-(2-pyridyl)-ethanol
and aniline deprotection according to the method of Compound
253.
[0662] Step 3: Urea formation according to the method of Compound
266 using 5-methyl-2-(2-pyridin-2-yl-ethoxy)-phenylamine (37%
yield).
[0663] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 10.70 (br s, 1H),
8.65 (d, 1H), 8.45 (br s, 1H), 8.21 (s, 1H), 7.85 (s, 1H), 7.59 (t,
1H), 7.29 (t, 1H), 6.80 (dd, 2H), 4.49 (t, 2H), 3.39 (t, 2H), 2.49
(s, 3H), 2.39 (s, 3H).
[0664] LRMS (esi, positive) m/e 364.14 (M+1)
[0665] Compound 268:
##STR00367##
1-(5-Methyl-pyrazin-2-yl)-3-]5-methyl-2-(3-pyridin-2-yl-propoxy)-phenyl]--
urea
[0666] Steps 1-2: Mitsunobu reaction using 3-(2-pyridyl)-propanol
and aniline deprotection according to the method of Compound
253.
[0667] Step 3: Urea formation according to the method of Compound
266 using 5-methyl-2-(3-pyridin-2-yl-propoxy)-phenylamine (5%
yield).
[0668] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 10.89 (br s, 1H),
8.59 (d, 1H), 8.49 (s, 1H), 8.45 (br s, 1H), 8.22 (s, 1H), 8.15 (s,
1H), 7.59 (t, 1H), 7.15 (d, 2H), 6.88 (dd, 2H), 4.05 (t, 2H), 3.10
(t, 2H), 2.45 (s, 3H), 2.40 (t, 2H), 2.35 (s, 3H).
[0669] LRMS (esi, positive) m/e 378.10 (M+1)
[0670] Compound 269:
##STR00368##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(3-pyridin-4-yl-propoxy)-phenyl]--
urea
[0671] Steps 1-2: Mitsunobu reaction using 3-(4-pyridyl)-propanol
and aniline deprotection according to the method of Compound
253.
[0672] Step 3: Urea formation according to the method of Compound
266 using 5-methyl-2-(4-pyridin-2-yl-propoxy)-phenylamine (28%
yield).
[0673] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.15 (br s, 1H),
8.51 (d, 2H), 8.25 (s, 1H), 8.20 (s, 1H), 7.79 (s, 1H), 7.75 (s,
1H), 7.15 (d, 2H), 6.82 (d, 1H), 6.75 (d, 1H), 4.05 (t, 2H), 2.91
(t, 2H), 2.40 (s, 3H), 2.36 (s, 3H), 2.25 (m, 2H).
[0674] LRMS (esi, positive) m/e 378.16 (M+1)
[0675] Compound 270:
##STR00369##
1-{2-[2-(Benzyl-methyl-amino)-ethoxy]-5-methyl-phenyl}-3-(5-methyl-pyrazi-
n-2-yl)-urea
[0676] Steps 1-2: Mitsunobu reaction using N-methyl-N-benzyl
ethanolamine and aniline deprotection according to the method of
Compound 253.
[0677] Step 3: Urea formation according to the method of Compound
266 using 2-[2-(benzyl-methyl-amino)-ethoxy]-5-methyl-phenylamine
(17% yield).
[0678] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 10.70 (br s, 1H),
8.49 (s, 1H), 8.20 (s, 1H), 7.95 (s, 1H), 7.85 (s, 1H), 7.32 (m,
5H), 6.85 (s, 2H), 4.15 (t, 2H), 3.62 (s, 2H), 2.92 (t, 2H), 2.49
(s, 3H), 2.33 (s, 3H), 2.29 (s, 3H).
[0679] LRMS (esi, positive) m/e 406.01 (M+1)
[0680] Compound 271:
##STR00370##
1-[5-Methyl-2-(1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazi-
n-2-yl)-urea
[0681] Steps 1-2: Mitsunobu reaction using 3-hydroxymethyl-1-methyl
piperidine and aniline deprotection according to the method of
Compound 253.
[0682] Step 3: Urea formation according to the method of Compound
266 using 5-methyl-2-(1-methyl-piperidin-3-ylmethoxy)-phenylamine
(16% yield).
[0683] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.25 (br s, 1H),
8.45 (br s, 1H), 8.35 (s, 1H), 8.22 (s, 2H), 6.80 (d, 1H), 6.74 (d,
1H), 3.80 (m, 2H), 3.15 (br d, 1H), 2.80 (br d, 1H), 2.51 (s, 3H),
2.35 (s, 3H), 2.30 (m, 1H), 2.22 (s, 3H), 1.50-2.00 (m, 6H),
1.00-1.25 (m, 2H).
[0684] LRMS (esi, positive) m/e 370.01 (M+1)
[0685] Compound 272:
##STR00371##
1-{2-[2-(4-Dimethylamino-phenyl)-ethoxy]-5-methyl-phenyl}-3-(5-methyl-pyr-
azin-2-yl)-urea
[0686] Steps 1-2: Mitsunobu reaction using
2-(4-dimethylamino-phenyl)-ethanol and aniline deprotection
according to the method of Compound 253.
[0687] Step 3: Urea formation according to the method of Compound
266 using
2-[2-(4-dimethylamino-phenyl)-ethoxy]-5-methyl-phenylamine (10%
yield).
[0688] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.15 (br s, 1H),
8.35 (s, 1H), 8.25 (s, 1H), 7.80 (m, 2H), 7.25 (m, 2H), 6.82 (s,
2H), 6.75 (d, 2H), 4.25 (t, 2H), 3.20 (t, 2H), 2.99 (s, 6H), 2.55
(s, 3H), 2.41 (s, 3H).
[0689] LRMS (esi, positive) m/e 405.90 (M+1)
[0690] Compound 273:
##STR00372##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(3-pyridin-3-yl-propoxy)-phenyl]--
urea
[0691] Steps 1-2: Mitsunobu reaction using 3-(3-pyridyl)-propanol
and aniline deprotection according to the method of Compound
253.
[0692] Step 3: Urea formation according to the method of Compound
266 using 5-methyl-2-(3-pyridin-3-yl-propoxy)-phenylamine (16%
yield).
[0693] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 10.95 (br s, 1H),
8.51 (m, 2H), 8.35 (s, 1H), 8.25 (s, 1H), 8.15 (s, 1H), 7.80 (s,
1H), 7.50 (d, 1H), 7.21 (t, 1H), 6.79 (d, 1H), 6.75 (d, 1H), 4.09
(t, 2H), 2.90 (t, 2H), 2.45 (s, 3H), 2.35 (s, 3H), 2.25 (m,
2H).
[0694] LRMS (esi, positive) m/e 377.91 (M+1)
[0695] Compound 274:
##STR00373##
1-[2-(2-Dimethylamino-1-dimethylaminomethyl-ethoxy)-5-methyl-phenyl]-3-(5-
-methyl-pyrazin-2-yl)-urea
[0696] Steps 1-2: Mitsunobu reaction using
1,3-bis-dimethylamino-propan-2-ol and aniline deprotection
according to the method of Compound 253.
[0697] Step 3: Urea formation according to the method of Compound
266 using
2-(2-amino-4-methyl-phenoxy)-N,N,N',N'-tetramethyl-propane-1,3-diam-
ine (4% yield).
[0698] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 9.69 (br s, 1H),
8.95 (br s, 1H), 8.19 (s, 1H), 8.05 (s, 1H), 6.80 (dd, 2H), 4.19
(m, 1H), 4.09 (m, 1H), 3.05 (m, 1H), 2.65 (m, 2H), 2.50 (s, 3H),
2.45 (s, 6H), 2.38 (s, 6H), 2.35 (s, 3H).
[0699] LRMS (esi, positive) m/e 386.92 (M+1)
[0700] Compound 275:
##STR00374##
1-[5-Methyl-2-(2-S-1-methyl-pyrrolidin-2-ylmethoxy)-phenyl]-3-(5-methyl-p-
yrazin-2-yl)-urea
[0701] Steps 1-2: Mitsunobu reaction using
1-methyl-pyrrolidin-2-S-ylmethanol and aniline deprotection
according to the method of Compound 253.
[0702] Step 3: Urea formation according to the method of Compound
266 using
5-methyl-2-(1-methyl-pyrrolidin-2-S-ylmethoxy)-phenylamine (12%
yield).
[0703] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.10 (s, 1H),
9.85 (br s, 1H), 9.65 (br s, 1H), 8.78 (s, 1H), 8.25 (s, 1H), 6.90
(s, 1H), 7.02 (d, 1H), 6.85 (d, 1H), 4.33 (br s, 2H), 3.88 (m, 1H),
3.59 (m, 1H), 3.19 (m, 1H), 2.99 (d, 2H), 2.70-2.85 (m, 2H), 2.45
(s, 3H), 2.29 (s, 3H), 1.80-2.10 (m, 3H).
[0704] LRMS (esi, positive) m/e 355.91 (M+1)
[0705] Compound 276:
##STR00375##
1-[2-(2-S-1-Benzyl-pyrrolidin-2-ylmethoxy)-5-methyl-phenyl]-3-(5-methyl-p-
yrazin-2-yl)-urea
[0706] Steps 1-2: Mitsunobu reaction using
1-benzyl-pyrrolidin-2-S-ylmethanol and aniline deprotection
according to the method of Compound 253.
[0707] Step 3: Urea formation according to the method of Compound
266 using
5-methyl-2-(1-benzyl-pyrrolidin-2-S-ylmethoxy)-phenylamine (3%
yield).
[0708] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 9.95 (s, 1H),
9.90 (br s, 1H), 9.59 (br s, 1H), 8.69 (s, 1H), 8.10 (s, 1H), 7.89
(s, 1H), 7.25-7.50 (m, 6H), 6.96 (d, 1H), 6.90 (d, 1H), 4.75 (d,
2H), 4.33 (m, 4H), 4.10 (m, 2H), 2.40 (s, 3H), 2.25 (s, 3H),
1.80-2.10 (m, 3H), 1.10-1.30 (m, 2H).
[0709] LRMS (esi, positive) m/e 432.31 (M+1).
[0710] Compound 277:
##STR00376##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(2-pyrrolidin-1-yl-ethoxy)-phenyl-
]-urea
[0711] Prepared according to the procedure for Compound 266, using
2-pyrrolidin-1-yl-ethanol. (28% yield).
[0712] .sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.01 (s, 1H),
9.85 (br s, 1H), 9.72 (br s, 1H), 8.75 (s, 1H), 8.25 (s, 1H), 8.01
(s, 1H), 7.01 (d, 1H), 6.80 (d, 1H), 4.40 (t, 2H), 3.62 (m, 4H),
3.21 (m, 2H), 2.40 (s, 3H), 2.25 (s, 3H), 2.00 (m, 2H), 1.88 (m,
2H).
[0713] LRMS (esi, positive) m/e 356.2 (M+1).
[0714] Compound 278:
##STR00377##
1-{5-Methyl-2-[2-(1-methyl-pyrrolidin-2-yl)-ethoxy]-phenyl}-3-(5-methyl-p-
yrazin-2-yl)-urea
[0715] Prepared according to the procedure for Compound 266, using
2-(1-methyl-pyrrolidin-2-yl)-ethanol (32% yield). .sup.1H-NMR (400
MHz, d.sub.6-DMSO) .delta. 10.01 (s, 1H), 9.85 (br.s, 1H), 9.72 (br
s, 1H), 8.75 (s, 1H), 8.25 (s, 1H), 8.01 (s, 1H), 7.01 (d, 1H),
6.80 (d, 1H), 4.20 (m, 3H), 3.00-4.00 (m, 11H), 2.80 (d, 2H), 2.40
(s, 3H), 2.25 (s, 3H).
[0716] LRMS (esi, positive) m/e 370.2 (M+1)
[0717] Compound 279:
##STR00378##
1-[2-(3H-Imidazol-4-ylmethoxy)-5-methyl-phenyl]-3-(5-methyl-pyrazin-2-yl)-
-urea
[0718] Prepared according to the procedure for Compound 266, using
(3H-imidazol-4-yl)-methanol. (24% yield). .sup.1H-NMR (400 MHz,
d.sub.6-DMSO) .delta. 8.51 (br s, 1H), 8.01 (s, 1H), 7.69 (s, 1H),
7.20-7.50 (m, 2H), 7.10 (d, 1H), 6.75 (d, 1H), 4.99 (s, 2H), 2.40
(s, 3H), 2.25 (s, 3H). LRMS (esi, positive) m/e 339.1 (M+1).
[0719] Compound 280:
##STR00379##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(2-pyridin-3-yl-ethoxy)-phenyl]-u-
rea
[0720] Prepared according to the procedure for Compound 266, using
2-pyridin-3-yl-ethanol. (16% yield). .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 10.98 (br s, 1H), 8.65 (s, 1H), 8.49 (d, 1H),
8.35 (s, 2H), 8.20 (s, 1H), 7.75 (s, 1H), 7.65 (d, 1H), 7.19 (m,
1H), 6.82 (dd, 2H), 4.31 (t, 2H), 3.21 (t, 2H), 2.48 (s, 3H), 2.35
(s, 3H). LRMS (esi, positive) m/e 364.2 (M+1)
[0721] Compound 281:
##STR00380##
1-[5-Methyl-2-(3-piperidin-1-yl-propoxy)-phenyl]-3-(5-methyl-pyrazin-2-yl-
)-urea
[0722] Prepared according to the procedure for Compound 266 using
3-piperidin-1-yl-propan-1-ol (33% yield). .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 11.21 (br s, 1H), 8.35 (s, 1H), 8.25 (s, 2H),
8.15 (s, 1H), 6.80 (dd, 2H), 4.15 (t, 2H), 2.53 (t, 2H), 2.52 (s,
3H), 2.45 (m, 4H), 2.39 (s, 3H), 2.10 (m, 2H), 1.61 (m, 4H), 1.45
(m, 2H). LRMS (esi, positive) m/e 384.2 (M+1)
[0723] Compound 282:
##STR00381##
1-[5-Methyl-2-(1-methyl-piperidin-4-yloxy)-phenyl]-3-(5-methyl-pyrazin-2--
yl)-urea
[0724] Prepared according to the procedure for Compound 266, using
1-methyl-piperidin-4-ol (4% yield). .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 11.22 (br s, 1H), 8.43 (s, 1H), 8.25 (s, 1H),
8.20 (s, 1H), 8.10 (s, 1H), 6.81 (dd, 2H), 4.25 (m, 1H), 2.8 (m,
2H), 2.59 (s, 3H), 2.39 (s, 3H), 2.36 (s, 3H), 2.19 (m, 4H), 1.90
(m, 2H). LRMS (esi, positive) m/e 355.9 (M+1).
[0725] Compound 283:
##STR00382##
1-[2-(1-Benzyl-piperidin-4-yloxy)-5-methyl-phenyl]-3-(5-methyl-pyrazin-2--
yl)-urea
[0726] Prepared according to the procedure for Compound 266, using
1-benzyl-piperidin-4-ol (1% yield). LRMS (esi, positive) m/e 432.0
(M+1).
[0727] Compound 284:
##STR00383##
1-[5-Methyl-2-(3-(S)-1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl--
pyrazin-2-yl)-urea
[0728] Step 1: (1-Methyl-piperidin-3-(S)-yl)-methanol. To a
stirred, cooled solution of (S)-(+)-N-boc nipeocotic acid (5.0
mmol) in tetrahydrofuran (10 mL) lithium aluminum hydride (20 mL,
20 mmol, 1M in tetrahydrofuran) was added dropwise. The reaction
was refluxed for 12 hours and then cooled to 0.degree. C. The
reaction was quenched with 1 mL of H.sub.2O, 1 mL of 15% aqueous
sodium hydroxide, 3 mL of H.sub.2O. The reaction was filtered and
the filter cake was washed with tetrahydrofuran (3.times.50 mL).
The filtrate was concentrated under reduced pressure to yield a
clear viscous oil.
[0729] Steps 2-3:
5-Methyl-2-(1-methyl-piperidin-3-(S)-ylmethoxy)-phenylamine.
Mitsunobu reaction and aniline deprotection according to procedure
for Compound 253.
[0730] Step 4: Prepared according to the procedure for Compound 266
(33% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.25 (br s,
1H), 8.45 (br s, 1H), 8.35 (s, 1H), 8.22 (s, 2H), 6.80 (d, 1H),
6.74 (d, 1H), 3.80 (m, 2H), 3.15 (br d, 1H), 2.80 (br d, 1H), 2.51
(s, 3H), 2.35 (s, 3H), 2.30 (m, 1H), 2.22 (s, 3H); 1.50-2.00 (m,
6H), 1.00-1.25 (m, 2H). LRMS (esi, positive) m/e 370.0 (M+1)
[0731] Compound 285:
##STR00384##
1-[5-Methyl-2-(3-(R)-1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl--
pyrazin-2-yl)-urea
[0732] Steps 1-3:
5-Methyl-2-(1-methyl-piperidin-3-(R)-ylmethoxy)-phenylamine
according to the method for Compound 284, using (R)-(+)-N-boc
nipecotic acid.
[0733] Step 4: 5-Methylpyrazine-2-carboxylic azide (1.2 eq.)
dissolve in anhydrous toluene (0.1M concentration) was heated to
90.degree. C. After 20 minutes N.sub.2 evolution had subsided, and
the caramel colored reaction mixture was cooled to 60.degree. C.
before adding the aniline prepared above as a solution in toluene
(1 eq.). After stirring for 4 hours at 60.degree. C., the reaction
mixture was cooled to room temperature overnight. A precipitate
formed which was isolated by filtration (49% yield). .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 11.25 (br s, 1H), 8.45 (br s, 1H),
8.35 (s, 1H), 8.22 (s, 2H), 6.80 (d, 1H), 6.74 (d, 1H), 3.80 (m,
21-1), 3.15 (br d, 1H), 2.80 (br d, 1H), 2.51 (s, 3H), 2.35 (s,
3H), 2.30 (m, 1H), 2.22 (s, 3H), 1.50-2.00 (m, 6H), 1.00-1.25 (m,
2H). LRMS (esi, positive) m/e 370.0 (M+1).
[0734] Compound 286:
##STR00385##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(1-pyridin-3-yl-ethoxy)-phenyl]-u-
rea
[0735] Step 1: 1-Pyridin-3-yl-ethanol. To a stirred, cooled
(-78.degree. C.) solution of pyridine-3-carbaldehyde (15 mmol) in
tetrahydrofuran (40 mL), methyl magnesium bromide (5 mL, 15 mmol,
3M in diethyl ether) was added. After stirring for 2 hours, the
reaction was quenched with saturated aqueous ammonium chloride (5
mL). The pH was adjusted to 5.0 with aqueous sodium carbonate and
the product was extracted with ethyl acetate (3.times.100 mL). The
ethyl acetate was washed with brine (1.times.100 mL), dried
(MgSO.sub.4), and filtered. The filtered material was concentrated
under reduced pressure to a yield a yellow oil.
[0736] Steps 2-3. 5-Methyl-2-(1-pyridin-3-yl-ethoxy)-phenylamine.
Mitsunobu reaction and aniline deprotection according to the method
for Compound 253.
[0737] Step 4: Urea formation was conducted according to the
procedure for Compound 285 (17% yield). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 11.49 (br s, 1H), 8.81 (s, 1H), 8.73 (s, 1H),
8.52 (d, 1H), 8.35 (s, 1H), 8.25 (s, 1H), 8.18 (s, 1H), 7.80 (s,
1H), 7.72 (d, 1H), 7.20 (t, 1H), 6.70 (d, 1H), 6.65 (d, 1H), 5.49
(q, 1H), 2.50 (s, 3H), 2.30 (s, 3H), 1.75 (d, 3H).
[0738] LRMS (esi, positive) m/e 363.8 (M+1).
[0739] Compound 287:
##STR00386##
1-[5-Methyl-2-(1-methyl-piperidin-2-ylmethoxy)-phenyl]-3-(5-methyl-pyrazi-
n-2-yl)-urea
[0740] Steps 1-2:
5-Methyl-2-(1-methyl-piperidin-2-ylmethoxy)-phenylamine. Mitsunobu
reaction and aniline deprotection according to the method for
compound 253.
[0741] Step 3: Urea formation was conducted according to the
procedure for compound 285 (11% yield). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 10.90 (br s, 1H), 8.45 (s, 1H), 8.21 (s, 1H),
8.18 (s, 1H), 7.80 (s, 1H), 6.80 (s, 2H), 4.51 (m, 1H), 2.58-100
(m, 4H), 2.52 (s, 3H), 2.46 (s, 3H), 2.35 (s, 3H), 1.50-2.25 (m,
7H). LRMS (esi, positive) m/e 369.9 (M+1)
[0742] Compound 288:
##STR00387##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(tetrahydro-furan-2-ylmethoxy)-ph-
enyl]-urea
[0743] Steps 1-2:
5-Methyl-2-(tetrahydro-furan-2-ylmethoxy)-phenylamine. Mitsunobu
reaction and aniline deprotection according to the method for
compound 253.
[0744] Step 3: Urea formation was conducted according to the
procedure for compound 285 (12% yield). .sup.1H NMR (400 Mz,
CDCl.sub.3) .delta. 11.25 (br s, 1H), 8.49 (s, 1H), 8.40 (s, 1H),
8.22 (s, 1H), 8.18 (s, 1H), 6.8 (s, 2H), 4.42 (m, 1H), 3.80-4.10
(m, 4H), 2.52 (s, 3H), 2.35 (s, 3H), 1.20-2.20 (m, 4H). LRMS (esi,
positive) m/e 342.9 (M+1).
[0745] Compound 289:
##STR00388##
1-[5-Methyl-2-(1-methyl-piperidin-4-ylmethoxy)-phenyl]-3-(5-methyl-pyrazi-
n-2-yl)-urea
[0746] Step 1: (1-Methyl-piperidin-4-yl)-methanol. To a stirred,
cooled solution of 1-methyl-piperidine-4-carboxylic acid (5.0 mmol)
in tetrahydrofuran (10 mL) lithium aluminium hydride (20 mL, 20
mmol, 1M tetrahydrofuran) was added dropwise. The reaction was
stirred for 4 hours, quenched with 1 mL of H.sub.2O, 1 mL of 15%
aqueous sodium hydroxide, and 3 mL of H.sub.2O. The reaction was
filtered and washed with tetrahydrofuran (3.times.50 mL). The
filtrate was concentrated under reduced pressure to yield a clear
viscous oil.
[0747] Steps 2-3:
5-Methyl-2-(1-methyl-piperidin-4-ylmethoxy)-phenylamine. Mitsunobu
and aniline deprotection according to the procedure for compound
253.
[0748] Step 4: Urea formation according to the procedure for
compound 285 (54% yield).
[0749] .sup.1H NMR (400 Mz, CDCl.sub.3) .delta. 11.18 (br s, 1H),
8.62 (br s, 1H), 8.38 (br s, 1H), 8.25 (s, 1H), 8.18 (s, 1H), 6.82
(d, 1H), 6.78 (d, 1H), 3.85 (d, 2H), 2.90 (br d, 2H), 2.51 (s, 3H),
2.35 (m, 6H), 1.50-2.10 (m, 7H).
[0750] LRMS (esi, positive) m/e 369.2 (M+1).
[0751] Compound 290:
##STR00389##
1-[5-Methyl-2-(1-methyl-piperidin-3-yloxy)-phenyl]-3-(5-methyl-pyrazin-2--
yl)-urea
[0752] Steps 1-2:
5-Methyl-2-(1-methyl-piperidin-3-yloxy)-phenylamine. Mitsunobu
reaction and aniline deprotection according to the method for
compound 253.
[0753] Step 3: Urea formation according to the procedure for
compound 285 (3% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.
10.75 (br s, 1H), 8.59 (br s, 1H), 8.18 (s, 1H), 8.05 (s, 1H), 7.62
(s, 1H), 6.90 (d, 1H), 6.80 (d, 1H), 4.40 (m, 1H), 2.58 (s, 3H),
2.39 (s, 6H), 1.60-2.80 (m, 8H).
[0754] LRMS (ESI, positive) m/e 356.1 (M+1).
[0755] Compound 291:
##STR00390##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(1,2,3,4-tetrahydro-quinolin-3-yl-
methoxy)-phenyl]-urea
[0756] Step 1: Quinoline-3-carboxylic acid methyl ester. To a
stirred solution of quinoline-3-carboxylic acid (346 mg, 2 mmol)
dissolved in 4:1 THF:MeOH (6 mL) at 0.degree. C. was added
TMS-diazomethane (2M in hexane) portionwise until a diazomethane
yellow color persisted. The reaction was concentrated to the give
the methyl ester as a tan solid (244 mg, 65%). .sup.1H NMR (400 Mz,
CDCl.sub.3) .delta. 9.44 (s, 1H), 8.85 (s, 1H), 8.17 (d, 1H), 7.96
(d, 1H), 7.84 (dd, 1H), 7.62 (dd, 1H), 4.02 (s, 3H).
[0757] Step 2: 1,2,3,4-Tetrahydro-quinoline-3-carboxylic acid
methyl ester and 1-Ethyl-1,2,3,4-tetrahydroquinoline-3-carboxylic
acid methyl ester. To a stirred solution of the
quinoline-3-carboxylic acid methyl ester (244 mg, 1.3 mmol) in
glacial acetic acid (13 mL) at room temperature was added
NaBH.sub.4 (345 mg, 9.1 mmol) portionwise (vigorous reaction).
After complete addition the reaction was dark yellow. After
stirring for 3 hours the color had become pale yellow. The reaction
mixture was poured into 50 mL of H.sub.2O and 50 mL of
CH.sub.2Cl.sub.2 and stirred rapidly for 15 min. The layers were
separated and the organics concentrated to a yellow oil. TLC in
15/85 EtOAc/hexane showed complete consumption of starting material
and two new lower rf spots. The compound was chromatographed using
a Biotage 12M column (loaded with CH.sub.2Cl.sub.2) and eluted with
15/85 EtOAc/hexane. The higher rf spot corresponds the N-ethylated
product (123 mg, 43%). The lower rf spot corresponds to the desired
tetrahydroquinoline-3-carboxylic acid methyl ester (93 mg, 37%).
N-ethyl derivative: .sup.1H NMR (400 Mz, CDCl.sub.3) .delta. 7.08
(dd, 1H). 6.99 (d, 1H), 6.60 (m, 2H), 3.73 (s, 3H), 3.42 (m, 3H),
3.27 (m, 1H), 2.98 (m, 3H), 1.14 (t, 3H), N--H derivative: .sup.1H
NMR (400 Mz, CDCl.sub.3) .delta. 6.98 (m, 2H), 6.62 (dd, 1H), 6.47
(d, 1H), 3.71 (s, 3H), 3.52 (m, 1H), 3.34 (m, 1H), 3.00 (m, 2H),
2.90 (m, 1H).
[0758] Step 3: (1,2,3,4-Tetrahydro-quinolin-3-yl)-methanol. To a
stirred solution of the 1,2,3,4-Tetrahydro-quinoline-3-carboxylic
acid methyl ester (93 mg, 0.49 mmol) in 1.5 mL of Et.sub.2O at
0.degree. C. under nitrogen was added LiAlH.sub.4 (1M in Et.sub.2O)
dropwise with vigorous gas evolution and a white precipitate
formation. After 30 min., the reaction was carefully quenched with
15% NaOH (3 mL) and 3 mL of Et.sub.2O was added and the mixture
stirred rapidly at RT for 15 min. The layers were separated and the
aqueous layer extracted (1.times.10 mL) with Et.sub.2O. The
organics were combined dried (MgSO.sub.4), filtered and
concentrated to the alcohol (64 mg, 80%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 6.97 (m, 2H), 6.62 (dd, 1H), 6.47 (d, 1H), 3.66
(m, 1H), 3.58 (m, 1H), 3.40 (m, 1H), 3.08 (m, 1H), 2.82 (m, 1H),
2.53 (m, 1H), 2.18 (m, 1H).
[0759] Step 4:
[5-Methyl-2-(1,2,3,4-tetrahydro-quinolin-3-ylmethoxy)-phenyl]-carbamic
acid tert-butyl ester. To a stirred solution of
2-N-Boc-amino-4-methylphenol (88 mg, 0.39 mmol),
(1,2,3,4-Tetrahydro-quinolin-3-yl)-methanol (64 mg, 0.39 mmol) and
triphenylphosphine (103 mg, 0.39 mmol) in 850 .mu.L of THF at
0.degree. C. under nitrogen was added a solution of DIAD (77 .mu.L,
0.39 mmol) in 850 .mu.L THF. The reaction was allowed to warm to RT
overnight, was concentrated and loaded directly onto a Biotage 12M
column with CH.sub.2CL.sub.2 and eluted with 96/4 hexane/EtOAc. The
product was isolated as a yellow oil (129 mg, 89%). .sup.1H-NMR 400
(MHz, CDCl.sub.3) .delta. 7.92 (br s, 1H), 7.06 (br s, 1H), 6.99
(m, 2H), 6.81 (m, 1H), 6.73 (s, 2H), 6.65 (dd, 1H), 6.51 (d, 1H),
3.96 (m, 2H), 3.37 (ddd, 2H), 2.79 (ddd, 2H), 2.56 (m, 1H), 2.28
(s, 3H), 1.54 (s, 9H).
[0760] Step 5:
3-(2-tert-Butoxycarbonylamino-4-methyl-phenoxymethyl)-3,4-dihydro-2H-quin-
oline-1-carboxylic acid benzyl ester. To a stirred solution of
[5-Methyl-2-(1,2,3,4-tetrahydro-quinolin-3-ylmethoxy)-phenyl]-carbamic
acid tert-butyl ester. To a stirred solution of
2-N-Boc-amino-4-methylphenol (129 mg, 0.35 mmol) in
CH.sub.2Cl.sub.2 (1.5 mL) at 0.degree. C. under nitrogen was added
DIEA 61 .mu.L, 0.35 mmol) followed by benzyl chloroformate (50
.mu.L, 0.35 mmol) and DMAP (4 mg, 0.035 mmol). After 24 hours, the
reaction was diluted to 30 mL with CH.sub.2Cl.sub.2 and washed
(2.times.30 mL) with 2N HCl and (2.times.30 mL) with saturated
NaHCO.sub.3. The organics were dried (MgSO.sub.4), filtered and
concentrated to a brown oil, which appeared to be a mixture of
product and starting material.
[0761] Step 6:
3-(2-Amino-4-methyl-phenoxymethyl)-3,4-dihydro-2H-quinoline-1-carboxylic
acid benzyl ester. A solution of the crude
(3-(2-tert-Butoxycarbonylamino-4-methyl-phenoxymethyl)-3,4-dihydro-2H-qui-
noline-1-carboxylic acid benzyl ester (crude, 0.35 mmol) in 4N HCl
in dioxane (2 mL) at room temperature was stirred under a drying
tube overnight. The suspension was concentrated by rotovap, diluted
to 30 mL with CH.sub.2Cl.sub.2 and shaken with 10% Na.sub.2CO.sub.3
(30 mL). The organics were isolated, dried (MgSO.sub.4), filtered
and concentrated to a brown oil corresponding to the crude aniline,
which was used without purification in the urea forming
reaction.
[0762] Step 7:
3-{4-Methyl-2-[3-(5-methyl-pyrazin-2-yl)-ureido]-phenoxymethyl}-3,4-dihyd-
ro-2H-quinoline-1-caroboxylic acid benzyl ester. A 0.5 M solution
of the 5-methyl-pyrazine-2-carboxylic acid azide (204 .mu.L) was
diluted with toluene (408 .mu.L) in a septum capped reaction vial
under nitrogen and with stirring, immersed in a 90.degree. C. oil
bath. After about 20 minutes nitrogen gas evolution had stopped so
the reaction was allowed to cool to RT and was treated with a
solution of the crude
3-(2-Amino-4-methyl-phenoxymethyl)-3,4-dihydro-2H-quinoline-1-carboxylic
acid benzyl ester (ca. 0.101 mmol) in toluene (620 .mu.L). The
mixture was stirred at 65.degree. C. for 2 hours. The reaction was
cooled to rt overnight and a precipitate formed. The precipitate
was filtered off with toluene and appeared to be a mixture of
Cbz-protected and deprotected product.
[0763] Step 8:
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(1,2,3,4-tetrahydro-quinolin-3-yl-
methoxy)-phenyl]-urea. A stirred suspension of the crude
(3-{4-Methyl-2-[3-(5-methyl-pyrazin-2-yl)-ureido]-phenoxymethyl}-3,4-dihy-
dro-2H-quinoline-1-carboxylic acid benzyl ester (6.6 mg, 12
.mu.mol) was heated in 5 mL EtOAc on a heat gun until in solution.
The clear solution was cooled to rt and treated with triethylamine
(3.4 .mu.L, 24 .mu.mol) followed by Pearlman's catalyst (20%
palladium hydroxide on carbon, 9 mg). The mixture was put through a
vacuum/purge cycle three times with hydrogen gas and then held
under 1 atmosphere hydrogen pressure for 1 hour. The reaction was
filtered through GF/F filter paper with EtOAc and concentrated to a
white solid which corresponds to the desired product (4.7 mg,
100%). .sup.1H-NMR (400 MHz, CDCl.sub.3/CD.sub.3OD) .delta. 8.23
(s, 1H), 8.18 (s, 1H), 7.96 (s, 1H), 7.04 (t, 1H), 6.97 (d, 1H),
6.81 (m, 2H), 6.67 (t, 1H), 6.58 (d, 1H), 4.04 (m, 2H), 3.57 (d,
1H), 3.26 (t, 1H), 2.92 (ddd, 2H), 2.64 (m, 1H), 2.34 (s, 3H), 2.31
(s, 3H). LRMS (APCI, positive) m/3 404.2 (M+1).
[0764] Compound 292:
##STR00391##
1-[2-1-Ethyl-1,2,3,4-tetrahydro-quinolin-3-ylmethoxy)-5-methyl-phenyl]-3--
(5-methyl-pyrazin-2-yl)-urea
[0765] Step 1: (1-Ethyl-1,2,3,4-tetrahydro-quinolin-3-yl)-methanol.
To a stirred solution of
1-Ethyl-1,2,3,4-tetrahydro-quinoline-3-carboxylic acid methyl ester
(123 mg, 0.56 mmol) in 1.5 mL of Et.sub.2O at 0.degree. C. under
nitrogen. LAH (1M in Et.sub.2O) was added dropwise with vigorous
gas evolution and a white precipitate formation. After 30 min., TLC
in 3/7 EtOAc/hexane showed complete loss of s.m. and appearance of
a clean lower rf spot. The reaction was carefully quenched with 15%
NaOH (3 mL) and 3 mL of Et.sub.2O was added and the mixture stirred
rapidly at RT for 15 min. The layers were separated and the aqueous
layer extracted 1.times.10 mL with Et.sub.2O. The organics were
combined, dried (MgSO.sub.4), filtered and concentrated to a clear
oil corresponding to the desired alcohol (105 mg, 95%).
[0766] Step 2:
[2-(1-Ethyl-1,2,3,4-tetrahydro-quinolin-3-ylmethoxy)-5-methyl-phenyl]-car-
bamic acid tert-butyl ester. To a stirred solution of
(1-Ethyl-1,2,3,4-tetrahydro-quinolin-3-yl)-methanol (105 mg, 0.55
mmol, prepared in step 2, compound 126xx),
2-N-Boc-amino-4-methylphenol (123 mg, 0.55 mmol), and
triphenylphosphine (144 mg, 0.55 mmol) in 850 .mu.L of THF at
0.degree. C. under nitrogen was added a solution of DIAD (108
.mu.L, 0.55 mmol) in 850 .mu.L THF. The reaction was allowed to
warm to RT overnight, was concentrated and loaded directly onto a
Biotage 12M column with CH.sub.2Cl.sub.2 and eluted with 96/4
hexane/EtOAc to give the desired alkylated phenol as a white foam
(40 mg, 18%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.92 (br s,
1H), 7.08 (dd, 1H), 7.02 (m, 1H), 6.99 (d, 1H), 6.75 (s, 2H), 6.62
(d, 1H), 6.59 (dd, 1H), 3.98 (m, 2H), 3.39 (m, 4H), 3.20 (m, 1H),
2.79 (ddd, 2H), 2.58 (m, 1H), 2.28 (s, 3H), 1.56 (s, 9H), 1.16 (t,
3H).
[0767] Step 3:
2-(1-Ethyl-1,2,3,4-tetrahydro-quinolin-3-ylmethoxy)-5-methyl-phenylamine.
A solution of
[2-(1-Ethyl-1,2,3,4-tetrahydro-quinolin-3-ylmethoxy)-5-methyl-phenyl]-car-
bamic acid tert-butyl ester (40 mg, 0.1 mmol) was stirred in 4N HCl
in dioxane (2 mL) at room temperature under a drying tube
overnight. The suspension was concentrated by rotovap, diluted to
30 mL with CH.sub.2Cl.sub.2 and shaken with 10% Na.sub.2CO.sub.3
(30 mL). The organics were isolated, dried (MgSO.sub.4), filtered
and concentrated to a brown oil, which was used without
purification in the following reaction.
[0768] Step 4:
1-[2-(1-Ethyl-1,2,3,4-tetrahydro-quinolin-3-ylmethoxy)-5-methyl-phenyl]-3-
-(5-methyl-pyrazin-2-yl)-urea. A 0.5 M solution of the acyl azide
(182 .mu.L) was diluted with 364 .mu.L toluene in a septum capped
reaction vial under nitrogen and with stirring, immersed in a
90.degree. C. oil bath. After about 20 minutes N.sub.2 gas
evolution had stopped so the reaction was allowed to cool to rt and
was treated with a solution of
2-(1-ethyl-1,2,3,4-tetrahydro-quinolin-3-ylmethoxy)-5-methyl-phenylamine
(27 mg, 0.91 mmol) in 550 .mu.L of toluene. The mixture was stirred
at 65.degree. C. for 2 hours. The reaction was cooled to rt
overnight and a precipitate formed. The precipitate was filtered
off with toluene and the desired urea isolated as a tan solid (7
mg, 18%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.34 (br s,
1H), 8.25 (s, 1H), 8.21 (s, 1H), 8.11 (br s,1H), 7.83 (s, 1H), 7.25
(m, 1H), 7.18 (d, 1H), 7.12 (t, 1H), 6.97 (d, 1H), 6.80 (m, 2H),
6.70 (d, 1H), 6.61 (t, 1H), 4.05 (m, 2H), 3.52 (m, 2H), 3.2 (t,
1H), 3.17 (m, 1H), 2.91 (ddd, 2H), 2.69 (m, 1H), 2.36 (s, 3H), 2.27
(s, 3H), 1.04 (t, 3H). LRMS (APCI, Positive) m/e 431.9 (M+1).
[0769] Compound 293:
##STR00392##
1-[5-Methyl-2-(1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-quinoxalin-2-yl--
urea
[0770] Step 1: Quinoxaline-2-carbonyl azide. A stirred solution
quinoxaline-2-carboxylic acid (348 mg, 2 mmol) in THF (6 mL) at rt
under nitrogen was treated with diisopropylethylamine (365 .mu.L,
2.1 mmol) followed by diphenylphosphoryl azide (410 .mu.L, 1.9
mmol). After stirring overnight the reaction was diluted to 60 mL
with Et.sub.2O and washed 2.times.60 mL with sat. NaCl. There was
an insoluble brown oil, which was drained off with the aqueous
layer and assumed to be a diphenyl phosphate impurity. The organics
were dried (MgSO.sub.4), filtered and concentrated to a tan solid,
which corresponds to the acyl azide (350 mg, 92%). .sup.1H-NMR (400
MHz, CDCl.sub.3) .delta. 9.58 (s, 1H), 8.32 (d, 1H), 8.21 (d, 1H),
7.94 (m, 2H).
[0771] Step 2:
1-[5-Methyl-2-(1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-quinoxalin-2-yl--
urea. A solution of quinoxaline-2-carbonyl azide (66 mg, 0.33 mmol)
in toluene (1.7 mL) was stirred under nitrogen and immersed in a
90.degree. C. heating bath. After 20 min. the reaction was cooled
to 65.degree. C. and solid
5-methyl-2-(1-methyl-piperidin-3-ylmethoxy)-phenylamine (70 mg, 0.3
mmol) was added. The reaction was stirred at 65.degree. C. for 4
hours and then allowed to cool to rt overnight. The resulting
precipitate was collected by filtration and washed with toluene (62
mg, 51%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.58 (br s,
1H), 9.27 (br s, 1H), 8.63 (s, 1H), 8.22 (s, 1H), 8.03 (d, 1H),
7.91 (d, 1H), 7.76 (t, 1H), 7.61 (t, 1H), 6.86 (m, 2H), 4.03 (m,
2H), 2.91 (d, 1H), 2.61 (d, 1H), 2.37 (s, 3H), 2.25 (m, 1H), 2.09
(s, 1H), 1.81 (m, 3H), 1.57 (m, 2H), 1.05 (m, 1H). LRMS (APCI,
Positive) m/e 405.9 (M+1).
[0772] Compound 294:
##STR00393##
1-[5-Methyl-2-(pyridin-3-ylmethoxy)-phenyl]-3-quinoxalin-2-yl-urea
[0773] Step 1:
115-Methyl-2-(pyridin-3-ylmethoxy)-phenyl]-3-quinoxalin-2-yl-urea.
A stirred solution of quinoxaline-2-carbonyl azide (92 mg, 0.46
mmol, prepared as above) in 1.5 mL toluene under nitrogen was
immersed in a 90.degree. C. heating bath. After 20 min. the
reaction was cooled to 65.degree. C. and treated with solid
5-methyl-2-(pyridin-3-ylmethoxy)-phenylamine (90 mg, 0.42 mmol).
The reaction was stirred at 65.degree. C. for 4 hours and then
allowed to cool to rt overnight. The resulting precipitate was
collected by filtration. The crude product was chromatographed on a
Biotage 12M column with 2/3 EtOAc/hexane to give pure urea as a tan
solid (20 mg, 12%). .sup.1H-NMR (400 MHz, CDCl.sub.3), .delta.
11.99 (br s, 1H), 9.64 (br s, 1H), 8.76 (s, 1H), 8.61 (s, 1H), 8.48
(d, 1H), 8.24 (s, 1H), 7.98 (d, 1H), 7.79 (d, 1H), 7.55 (t,
1H),7.42 (t, 1H), 7.17 (d, 1H), 7.12 (m, 1H), 6.93 (m, 2H), 5.26
(s, 2H), 2.41 (s, 3H). LRMS (APCI, Positive) m/e 385.9 (M+1).
[0774] Compound 295:
##STR00394##
[0775] Step 1 Mitsunobo Procedure:
[0776] 1-[2-(4-Methyl-2-nitro-phenoxy)-ethyl]aziridine. A solution
of 2-nitro-4-methylphenol (505 mg, 3.3 mmol, 1.1 eq.) and
2-aziridin-1-yl-ethanol (3.0 mmol, 1 eq.) in 10 mL THF was stirred
at 0.degree. C. Triphenylphosphine (0.87 g, 3.30 mmol, 1.1 eq.) and
diisopropylazodicarboxylate, (0.67 g, 3.30 mmol, 1.1 eq.) were
added, and the solution was allowed to warm to temperature. After
18 h, the reaction mixture was diluted with 100 mL EtOAc, and was
washed with water (3.times.20 mL). The organic phase was washed
again with 1 N HCl (3.times.20 ml). The aqueous layer was basified
with 3 N NaOH to pH>12 and extracted with EtOAc, (3.times.50 mL)
to give crude product. The final product was purified by flash
chromatography eluting with 5-10% MeOH in dichloromethane.
.sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 7.66 (s, 1H), 7.32 (d;
J=8.61 Hz, 1H), 7.01 (d, J=8.61 Hz, 1H), 4.26 (t, J=5.09 Hz, 2H),
2.67 (t, J=5.48 Hz, 2H), 2.34 (s, 3H), 1.79 (m, 2H), 1.34 (m,
2H).
[0777] Step 2 Nitro Reduction.
[0778] 2-(2-Aziridin-1-yl-ethoxy)-5-methyl-phenylamine. A solution
of 3-nitro-4-alkoxy toluene (1.0 mmol) in 20 mL EtOH was
hydrogenated at 2 atm over 300 mg of 10% Pd on carbon for 30
minutes. The catalyst was removed by filtration through a glass
fiber filter and the filtrate was concentrated to give the desired
product, which was used directly without further purification.
[0779] Step 3 Urea Formation.
[0780]
1-[2-(2-Aziridin-1-yl-ethoxy)-5-methyl-phenyl}-3-(5-methyl-pyrazin--
2-yl)-urea. A solution of 5-methylpyrazine-2-carboxylic azide (196
mg, 1.2 mmol, 1.2 eq.) in 20 mL anhydrous toluene was heated to
90.degree. C. After 20 minutes N.sub.2 evoluation had subsided, and
the reaction mixture was cooled to 60.degree. C. before adding
aniline (1.0 mmol, 1 eq.) as a solution in 2 mL toluene. After
stirring for 4 h at 60.degree. C. The reaction mixture was then
partitioned between 50 mL EtOAc and sat. NaHCO.sub.3. The organic
phase was washed with brine, dried over MgSO.sub.4, filtered and
concentrated. The residue was purified by flash chromatography,
eluting with 5% MeOH in dichloromethane. .sup.1H-NMR (400 MHz,
d.sub.6-DMSO): .delta. 10.80 (s, 1H), 8.64 (s, 1H), 8.53 (s, 1H),
8.15 (s, 1H), 8.07 (s, 1H), 6.82 (m, 2H), 4.2 (m, 2H), 2.7 (m, 2H),
2.5 (s, 3H), 2.32 (s, 3H), 1.89 (s, 2H), 1.30 (m, 2H). MS APCI-Pos,
M/e 328.0 (M+1).
[0781] Compound 296:
##STR00395##
1-[2-(3-Dimethylamino-benzyloxy)-5-methyl-phenyl]-3-(5-methyl-pyrazin-2-y-
l)-urea
[0782] Prepared from (3-dimethylamino-phenyl)-methanol, as
described above for compound 295. .sup.1H-NMR (400 MHz,
CDCl.sub.3): .delta. 11.69 (s, 1H), 8.26 (s, 1H), 8.11 (s, 1H),
7.93 (s, 1H), 7.26 (m, 1H), 6.87 (m, 6H), 5.01 (s, 2H), 2.93 (s,
6H), 2.35 (s, 6H). MS APCI-Pos, M/e 391.9 (M+1).
[0783] Compound 297:
##STR00396##
1-[2-(1-Isopropyl-pyrrolidin-3-yloxy)-5-methyl-phenyl]-3-(5-methyl-pyrazi-
n-2-yl)-urea
[0784] Prepared from 3-hydroxy-1-isopropyl-pyrrolidine, as
described above for compound 295. .sup.1H-NMR (400 MHz,
d.sub.6-DMSO): .delta. 10.08 (s, 2H), 8.65 (s, 1H), 8.14 (s, 1H),
8.03 (s, 1H), 6.81 (d, J=7.83 Hz, 1H), 6.74 (d, J=8.61 Hz, 1H),
4.85 (s, 1H), 2.91 (m, 1H), 2.75 (m, 2H), 2.48 (m, 1H), 2.4 (s,
3H), 2.36 (m, 1H), 2.27 (m, 1H), 2.21 (s, 3H), 1.85 (m, 1H), 1.01
(m, 6H). MS APCI-Pos, M/e 369.9 (M+1).
[0785] Compound 298:
##STR00397##
1-[5-Methyl-2-(1-methyl-pyrrolidin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyraz-
in-2-yl)-urea
[0786] Prepared from (1-methyl-pyrrolidin-3-yl)-methanol as
described above for compound 295. .sup.1H-NMR (400 MHz,
d.sub.6-DMSO): .delta. 10.13 (s, 2H), 8.67 (s, 1H), 8.15 (s, 1H),
8.06 (s, 1H), 6.81 (d, J=8.61 Hz, 1H), 6.75 (d, J=7.83 Hz, 1H),
4.88 (s, 1H), 2.75 (m, 4H), 2.5 (m, 2H), 2.43 (s, 3H), 2.3 (s, 3H),
2.24 (s, 3H). MS APCI-Pos, M/e 341.9 (M+1).
[0787] Compound 299:
##STR00398##
[0788] Step 1: 3-Hydroxymethyl-piperidine-1-carboxylic acid
tert-butyl ester. To a stirred solution of 3-hydroxymethyl
piperdine (403 mg, 3.5 mmol, 1 eq.) in 20 mL of CH.sub.2Cl.sub.2
and 5 mL of sat'd NaHCO.sub.3, 0.degree. C. was added di-tert-butyl
dicarbonate (803 mg, 3.68 mmol, 1.05 eq.) in several portions.
After stirring at 0.degree. C. for 2 h, the solution was diluted
with 10 mL of water and was extracted with 2.times.20 mL
CH.sub.2CL.sub.2 combined extracts were washed with water, then
brine and were dried over MgSO.sub.4, filtered and concentrated to
give the Boc protected amine which was used in the next step.
[0789] Steps 2-4:
3-{4-Methyl-2-[3-(5-methyl-pyrazin-2-yl)-ureido]-phenoxymethyl}-piperidin-
e-1-carboxylic acid tert-butyl ester was prepared from
3-hydroxymethyl-piperidine-1-carboxylic acid tert-butyl ester, as
described above for compound 295. It was purified by flash
chromatography eluting with 5% MeOH in CH.sub.2Cl.sub.2.
[0790] Step 5:
1-[5-Methyl-2-(piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazin-2-yl)-u-
rea. Removal of the Boc group was accomplished by treatment of a
0.degree. C. solution of the protected derivative (180 mg, 0.395
mmol) in 15 mL CH.sub.2Cl.sub.2 with 2 mL of TFA. After stirring
for 18 h at room temperature, the reaction was concentrated in
vacuo, and the residue was taken up in 20 mL of EtOAc and was
washed with 10 mL of NaHCO.sub.3. The aqueous phase was extracted
with EtOAc (2.times.30 mL), and the combined extracts were washed
with 20 mL of brine, dried over MgSO.sub.4, filtered and
concentrated to give 128 mg (91%) of the desired amine. .sup.1H-NMR
(400 MHz, d.sub.6-DMSO): .delta. 10.16 (s, 1H), 10.09 (s, 1H), 8.53
(s, 1H), 8.11 (s, 1H), 7.97 (s, 1H), 6.8 (d, J=7.8 Hz, 1H), 6.69
(d, J=8.6 Hz, 1H), 3.77 (s, 2H), 3.09 (m, 1H), 2.82 (m, 1H), 2.34
(m, 2H), 2.3 (s, 3H), 2.27 (m, 1H), 2.15 (s, 3H), 1.92 (m, 1H),
1.75 (m, 1H), 1.53 (m, 1H), 1.37 (m, 1H), 1.11 (m, 1H). MS
APCI-Pos, M/e 356.0 (M+1).
[0791] Compound 300:
##STR00399##
1-[5-Fluoro-2-(pyridin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazin-2-yl)-ure-
a
[0792] Step 1: 3-(4-Fluoro-2-nitro-phenoxymethyl)pyridine. To a
stirred, cooled (about 0.degree. C.) solution of
1,4-Difluoro-2-nitro-benzene (3.0 mmol) and 3-pyridylcarbinol (3.1
mmol) in tetrahydrofuran (8 mL) was added lithium
bis(trimethylsilyl)amide (3.2 mmol; 3.2 mL of a 1.0 M solution in
tetrahydrofuran). After stirring for 12 hours, the reaction was
diluted with 30 mL of ethyl acetate and washed with 30 mL of 10%
aqueous sodium carbonate (2.times.30 mL), brine (1.times.30 mL),
then dried (MgSO.sub.4) and filtered. The filtered solution was
concentrated under reduced pressure to provide the desired crude
product.
[0793] Step 2: 5-Fluoro-2-(pyridin-3-ylmethoxy)-phenylamine. To a
stirred, cooled (about 0.degree. C.) solution of a
4-Fluoro-2-nitro-phenoxymethyl)-pyridine (1.0 mmol) in methanol (2
mL) and saturated aqueous ammonium chloride (1 mL) was added zinc
dust (2.0 mmol). After stirring for 12 hours, the reaction was
diluted with 30 mL of ethyl acetate and washed with 30 mL of 10%
aqueous sodium carbonate (2.times.30 mL), brine (1.times.30 mL),
then dried (MgSO.sub.4) and filtered. The filtered solution was
concentrated under reduced pressure to provide the desired crude
product.
[0794] Step 3: Urea formation according to method for compound 295
(23% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 11.62 (br s,
1H), 8.80 (s, 1H), 8.75 (d, 1H), 8.43 (br s, 1H), 8.25 (d, 1H),
8.15 (s, 1H), 7.85 (d, 1H), 7.43 (m, 1H), 6.95 (m, 2H), 6.68 (m,
1H), 5.15 (s, 2H), 2.43 (s, 3H). LRMS (ESI, Positive) m/e 354.10
(M+1).
[0795] Compound 301:
##STR00400##
1-[5-Fluoro-2-(1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazi-
n-2-yl)-urea
[0796] Steps 1-2: According to procedure for compound 300, using
1,4-difluoro-2-nitrobenzene and 1-methyl-3-hydroxymethyl
piperidine.
[0797] Step 3: Urea formation according to method for compound 285
(62% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.22 (m,
3H), 7.21 (m, 2H), 6.78 (m, 2H), 3.85 (m, 2H), 3.21 (m, 1H), 2.85
(m, 1H), 2.52 (s, 3H), 2.39 (s, 3H), 1.50-2.30 (m, 8H). LRMS (ESI,
Positive) m/e 374.21 (M+1).
[0798] Compound 302:
##STR00401##
1-[5-Fluoro-2-(1-methyl-piperidin-4-yloxy)-phenyl]-3-(5-methyl-pyrazin-2--
yl)-urea
[0799] Steps 1-2: According to procedure for compound 300, using
1,4-difluoro-2-nitrobenzene and 1-methyl-4-hydroxypiperidine.
[0800] Step 3: Urea formation according to method for compound 295
(78% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 11.49 (br s,
1H), 8.89 (br s, 1H), 8.35 (s, 1H), 8.22 (d, 1H), 8.10 (s, 1H),
6.80 (m, 1H), 6.70 (m, 1 h), 4.25 (m, 1H), 2.90 (m, 2H), 2.55 (s,
3H), 2.38 (s, 3H), 2.35 (s, 3H), 1.80-2.30 (m, 6H). LRMS (ESI,
Positive) m/e 359.91 (M+1).
[0801] Compound 303:
##STR00402##
1-[4-Fluoro-2-(pyridin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazin-2-yl)-ure-
a
[0802] Step 1: 3-(5-Fluoro-2-nitro-phenoxymethyl)-pyridine. To a
stirred, cooled (about 0.degree. C.) solution of
2-nitro-5-fluoro-phenol (2.0 mmol), triphenyl phosphine (2.0 mmol)
and 3-hydroxymethylpyridine (2.0 mmol) in dry tertrahydrofuran (5
mL) was added diisopropyl azodicarboxylate (2.0 mmol in 1 mL of
tetrahydrofuran). After stirring for 12 hours, the reaction was
diluted with 30 mL of ethyl acetate and washed with 30 mL of 10%
aqueous sodium carbonate (2.times.30 mL), brine (1.times.30 mL),
then dried (MgSO.sub.4) and filtered. The filtered solution was
concentrated under reduced pressure to provide the desired crude
product.
[0803] Step 2: 4-Fluoro-2-(pyridin-3-ylmethoxy)-phenylamine. Nitro
reduction according to the method for compound 300.
[0804] Step 3: Urea formation according to method for compound 295
(60% yield).
[0805] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 11.41 (br s, 1H),
8.85 (s, 1H), 8.75 (d, 1H), 8.40 (t, 1H), 8.18 (s, 1H), 7.88 (s,
1H), 7.80 (d, 1H), 7.43 (t, 1H), 7.00 (s, 1H), 6.80 (m, 2H), 5.12
(s, 2H), 2.43 (s, 3H). LRMS (ESI, Positive) m/e 354.21 (M+1).
[0806] Compound 304:
##STR00403##
1-[4-Fluoro-2-(1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazi-
n-2-yl)-urea
[0807] Prepared according to the methods for compound 303, using
2-nitro-5-fluorophenol and 1-methyl-3-hydroxymethyl piperidine.
[0808] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.50 (br s, 1H),
8.19 (m, 2H), 6.65 (m, 2H), 3.85 (m, 2H), 3.60 (s, 3H), 2.80-3.20
(m, 2H), 2.54 (s, 3H), 2.39 (s, 3H), 1.60-2.10 (m, 5H). LRMS (ESI,
Positive) m/e 373.95 (M+1).
[0809] Compound 305:
##STR00404##
1-[4-Fluoro-2-(1-methyl-piperidin-4-yloxy)-phenyl]-3-(5-methyl-pyrazin-2--
yl)-urea
[0810] Prepared according to the methods for compound 303 using
2-nitro-5-fluorophenol and 1-methyl-4-hydroxypiperidine.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.35 (br s, 1H), 9.49
(s, 1H), 8.35 (m, 2H), 8.05 (s 1H), 6.65 (m, 2H), 4.35 (m, 1H),
2.90 (m, 2H), 2.54 (s, 3H), 2.35 (s, 3H), 1.80-2.30 (m, 6H).
[0811] LRMS (ESI, Positive) m/e 359.93 (M+1).
[0812] Compound 306:
##STR00405##
1-[3,4-Difluoro-2-(pyridin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazin-2-yl)-
-urea
[0813] Step 1: 3-(2,3-Difluoro-6-nitro-phenoxymethyl)-pyridine. To
a stirred, cooled (about 0.degree. C.) solution of
2,3-difluoro-6-nitrophenol (2.0 mmol), triphenyl phosphine (2.0
mmol), and 3-hydroxymethylpyridine (2.0 mmol) in dry
tertrahydrofuran (5 mL) was added diisopropyl azodicarboxylate (2.0
mmol in 1 mL of tetrahydrofuran). After stirring for 12 hours, the
reaction as diluted with 30 mL of ethyl acetate and washed with 30
mL of 10% aqueous sodium carbonate (2.times.30 mL), brine
(1.times.30 mL), then dried (MgSO.sub.4) and filtered. The filtered
solution was concentrated under reduced pressure to provide the
desired crude product.
[0814] Step 2: 3,4-Difluoro-2-(pyridin-3-ylmethoxy)-phenylamine.
Nitro reduction according to the method for compound 300.
[0815] Step 3: Urea formation according to method for compound 295
(20% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.49 (br s,
1H), 8.89 (s, 1H), 8.85 (s, 1H), 8.65 (d, 1H), 8.25 (s, 1H), 8.10
(m, 1H), 7.88 (d, 1H), 7.35 (t, 1H), 7.18 (s, 1H), 6.98 (m, 1H),
5.25 (s, 2H), 2.52 (s, 3H). LRMS (ESI, Positive) m/e 372.10
(M+1).
[0816] Compound 307:
##STR00406##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(pyridin-4-yloxy)-phenyl]-urea
[0817] Step 1: 5-Methyl-2-(pyridin-4-yloxy)-phenylamine. To a
stirred solution of 2-amino-4-methyl phenol (616 mg; 5.0 mmol) and
4-chloro pyridine (625 mg; 5.5 mmol) in dimethyl sulfoxide (5 mL)
was added sodium hydroxide (600 mg; 15.0 mmol, in 1 mL of water).
The reaction was heated to 100.degree. C. and stirred for 12 hours.
The reaction was cooled to room temperature, diluted with 50 mL of
ethyl acetate and washed with aqueous 10% sodium carbonate
(1.times.50 mL), and brine (50 mL), then dried (MgSO.sub.4) and
filtered. The crude product was purified using the biotage 40M
cartridge eluting with methylene chloride:methanol:ammonia (90:8:2)
to yield a light yellow oil (10% yield).
[0818] Step 2: Urea formation according to method for compound 295
(36% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.41 (br s,
1H), 8.52 (m, 3H), 8.33 (s, 1H), 8.22 (s, 1H), 7.61 (s, 1H),
6.80-7.00 (m, 4H), 2.49 (s, 3H), 2.45 (s, 3H). LRMS (ESI, Positive)
m/e 335.91 (M+1).
[0819] Compound 308:
##STR00407##
1-(5-Methyl-pyrazin-2-yL)-3-[5-methyl-2-(pyridin-3-yloxy)-phenyl]-urea
[0820] Step 1: 3-(4-Methyl-2-nitro-phenoxy)-pyridine. To a stirred
solution of 1-chloro-4-methyl-2-nitrobenzene (686 mg; 4.0 mmol) and
pyridin-3-ol (418 mg; 4.40 mmol) in dimethylforamide (5 mL) was
added potassium carbonate (1.22 g, 8.80 mmol). The reaction was
heated to 50.degree. C. and stirred for 12 hours. The reaction was
cooled to room temperature, diluted with 50 mL of ethyl acetate and
washed with aqueous 10% sodium carbonate (1.times.50 mL), and brine
(50 mL), then dried (MgSO.sub.4) and filtered. The crude product
was purified using the biotage 40M cartridge eluting with hexanes
and ethyl acetate (1:1) to yield a light yellow oil (27%
yield).
[0821] Step 2: 5-Methyl-2-(pyridin-3-yloxy)-phenylamine. To a
stirred, cooled (about 0.degree. C.) solution of
3-(4-Methyl-2-nitro-phenoxy)-pyridine (1.0 mmol) in methanol (2 mL)
and saturated aqueous ammonium chloride (1 mL) was added zinc dust
(2.0 mmol). After stirring for 12 hours, the reaction was diluted
with 30 mL of ethyl acetate and washed with 30 mL of 10% aqueous
sodium carbonate (2.times.30 mL), brine 1.times.30 mL), then dried
(MgSO.sub.4) and filtered. The filtered solution was concentrated
under reduced pressure to yield a brown oil (95% yield).
[0822] Step-3: Urea formation according to method for compound 295
(45% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.49 (br s,
1H), 8.55 (s, 1H), 8.39 (d, 1H), 8.35 (s, 1H), 8.15 (s, 1H), 8.05
(br s, 1H), 7.21 (m, 2H), 6.92 (m, 2H), 2.49 (s, 3H), 2.45 (s,
3H).
[0823] LRMS (ESI, Positive) m/e 335.91 (M+1).
[0824] Compound 309:
##STR00408##
1-(5-Methyl-pyrazin-2-yl)-3-[5-methyl-2-(pyridin-2-yloxy)-phenyl]-urea
[0825] Step 1: 2-(4-Methyl-2-nitro-phenoxy)-pyridine. To a stirred
solution of 1-chloro-4-methyl-2-nitrobenzene (686 mg; 4.0 mmol) and
pyridin-2-ol (418 mg; 4.40 mmol) in dimethylforamide (5 mL) was
added potassium carbonate (1.22 g, 8.80 mmol). The reaction was
heated to 50.degree. C. and stirred for 12 hours. The reaction was
cooled to room temperature, diluted with 50 mL of ethyl acetate and
washed with aqueous 10% sodium carbonate (1.times.50 mL), and brine
(50 mL), then dried (MgSO.sub.4) and filtered. The crude product
was purified using the biotage 40M cartridge eluting with hexanes
and ethyl acetate (1:1) to yield a light yellow oil (11%
yield).
[0826] Step 2: 5-Methyl-2-(pyridin-2-yloxy)-phenylamine. To a
stirred, cooled (about 0.degree. C.) solution of
2-(4-Methyl-2-nitro-phenoxy)-pyridine (1.0 mmol) in methanol (2 mL)
and saturated aqueous ammonium chloride (1 mL) was added zinc dust
(2.0 mmol). After stirring for 12 hours, the reaction was diluted
with 30 mL of ethyl acetate and washed with 30 mL of 10% aqueous
sodium carbonate (2.times.30 mL), brine (1.times.30 mL), then dried
(MgSO.sub.4) and filtered. The filtered solution was concentrated
under reduced pressure to yield a white foam (77% yield).
[0827] Step 3: Urea formation according to method for compound 295
(43% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.51 (br s,
1H), 8.42 (br s, 1H), 8.00 (s, 1H), 7.80 (s, 1H), 7.51 (t, 1H),
7.29 (d, 1H), 7.05 (d, 1H), 6.95 (d, 1H), 6.75 (d, 1H), 6.35 (t,
1H), 2.49 (s, 3H), 2.45 (s, 3H). LRMS (ESI, Positive) m/e 335.91
(M+1).
[0828] Substituted Aminopyrazine Ureas. General Procedure:
[0829] To a 0.3 M stirred solution of the aminopyrazine derivative
(1 equiv.) in dichloroethane at room temperature under nitrogen was
added 2-methoxy-5-methylphenylisocyanate (1 equiv.). The reaction
was warmed to 80.degree. C. overnight and then cooled to room
temperature. In most cases, the product precipitated and was
isolated by filtration. Alternatively, the product could be
isolated by silica gel chromatography using EtOAc/hexane or
CH.sub.2Cl.sub.2/MeOH as eluant.
[0830] Compound 310:
##STR00409##
3-(2-Methoxy-5-methyl-phenyl)-1-methyla-1-pyrazin-2-yl-urea
[0831] Step 1: 2-methylaminopyrazine. To a stirred solution of 2M
methylamine in 1 mL of methanol, at room temperature, was added
2-chloropyrazine. The reaction was sealed and heated to 60.degree.
C. for 24 hours. The reaction was concentrated to a mixture of
starting material and the desired 2-methylaminopyrazine in a 1:2
ratio. The material was used crude in a urea forming reaction.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 7.97 (d, 1H), 7.85 (s,
1H), 7.73 (d, 1H), 2.96 (s, 3H).
[0832] Step 2: To a 0.3 M stirred solution of 2-methylaminopyrazine
(1 equiv.) in dichloroethane at room temperature under nitrogen was
added 2-methoxy-5-methylphenylisocyanate (1 equiv.). The reaction
was warmed to 80.degree. C. overnight and then cooled to room
temperature. In most cases, the product precipitated and was
isolated by filtration. Alternatively the product could be isolated
by silica gel chromatography using EtOAc/hexane or
CH.sub.2Cl.sub.2/MeOH as eluant. .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 8.57 (s, 1H), 8.32 (s, 1H), 8.31 (s, 1H), 8.17 (s, 1H),
6.80 (m, 2H), 3.90 (s, 3H), 3.56 (S, 3H), 2.32 (s, 3H). LRMS (ESI,
Positive) m/e 273.2 (M+1).
[0833] Compound 311:
##STR00410##
1-(2-Methoxy-5-methyl-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea
[0834] Prepared according to general procedure described for
compound 310, using 2-amino-4-methylpyrazine. .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 11.12 (br s, 1H), 8.28 (s, 1H), 8.17 (s, 1H),
8.09 (s, 1H), 6.81 (m, 2H), 3.91 (s, 3H), 2.54 (s, 3H), 2.35 (s,
3H). LRMS (ESI, Positive) m/e 273.2 (M+1).
[0835] Compound 312:
##STR00411##
1-(5,6-Dimethyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0836] Step 1: 2-Amino-5,6-dimethylpyrazine. Glycine amidine
dihydrobromide (620 mg, 2.64 mmol) was stirred in 6 mL of MeOH at
-30.degree. C. (acetonitrile/CO.sub.2 bath) in a capped flask.
Butanedione (232 .mu.L, 2.64 mmol) was stirred separately in 6 ML
H.sub.2O with sodium acetate (700 mg) until homogeneous. The
diketone was added to the amidine solution by pipet followed by 2.5
mL of 3.6 M NaOH. The yellow solution was allowed to warm slowly to
RT and was then stirred overnight. MeOH was removed by rotovap and
the aqueous solution extracted 3.times.30 mL with EtOAc. The
combined organic extracts were dried (MgSO.sub.4), filtered and
concentrated to a yellow solid which contained some impurities. The
solid was triturated with EtOAc/Et.sub.2O and filtered to give pure
compound (55 mg, 17%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta.
7.76 (s, 1H), 4.25 (br s, 2H), 2.40 (s, 3H), 2.37 (s, 3H).
[0837] Step 2: Prepared according to general procedure described
for compound 310, using 2-amino-5,6-dimethylpyrazine. .sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 11.43 (br s, 1H), 8.23 (s, 1H), 8.00
(s, 1H), 7.64 (br s, 1H), 6.81 (m, 2H), 3.95 (s, 3H), 2.59 (s, 3H),
2.52 (s, 3H), 2.35 (s, 3H). LRMS (APCI, Positive) m/e 287.1
(M+1).
[0838] Compound 313:
##STR00412##
1-(2-Methoxy-5-methyl-phenyl)-3-(5-trifluoromethyl-pyrazin-2-yl)-urea
[0839] Step 1: 2-amino-5-trifluoromethylpyrazine. Prepared
according to the method of Miesel, J. U.S. Pat. No. 4,293,552
(1981).
[0840] Step 2: Prepared according to general procedure described
for compound 310, using 2-amino-5-trifluoromethylpyrazine.
.sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.59 (s, 1H), 9.78 (br
s, 1H), 9.06 (s, 1H), 8.80 (s, 1H), 8.00 (s, 1H), 6.96 (d, 1H),
6.81 (d, 1H), 3.87 (s, 3H), 2.22 (s, 3H). LRMS (ESI, Positive) m/e
327.1 (M+1).
[0841] Compound 314:
##STR00413##
1-(5,6-Diphenyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0842] Step 1: 2-Hydroxy-5,6-diphenylpyrazine. To a stirred
suspension of glycinamide hydrochloride (1.1 gm, 10 mmol) in 20 mL
MeOH at 0.degree. C. was added 20% NaOH (10 mL, 50 mmol). A clear
solution formed and was treated slowly portionwise with benzil (2.1
gm, 109 mmol) as a solid. The yellow solution was stirred at
0.degree. C. for 2 hours and then neutralized to approximately pH=7
with concentrated HCl. The bright yellow color disappeared and a
tan precipitate formed. The material was isolated by filtration
with MeOH and triturated with EtOAc to give
2-hydroxy-5,6-diphenylpyrazine (2 gm, 80%). .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 8.24 (s, 1H), 7.42-7.31 (m, 4H), 7.39-7.21 (m,
6H).
[0843] Step 2: 2-Chloro-5,6-diphenylpyrazine. A stirred solution of
2-Hydroxy-5,6-diphenylpyrazine (430 mg, 1.7 mmol) in 5.2 mL
POCl.sub.3 in a capped reaction vial was heated to 100.degree. C.
for 4 hours. The orange solution was cooled to room temperature and
stirred rapidly in a mixture of CH.sub.2Cl.sub.2 (100 mL) and ice
cold 10% Na.sub.2CO.sub.3 (100 mL) for 15 minutes. The organic
layer was isolated and washed 2.times.100 mL with 10%
Na.sub.2CO.sub.3. The organics were isolated, dried (MgSO.sub.4),
filtered and concentrated to the chloropyrazine, which existed as
white solid (450 mg, quanitative). .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 8.59 (s, 1H), 7.45-7.39 (m, 4H), 7.36-7.24 (m,
6H).
[0844] Step 3: 2-Azido-5,6-diphenylpyrazine. To a stirred solution
of 2-Chloro-5,6-diphenylpyrazine (45 mg, 0.17 mmol) in 500 .mu.L
DMF at room temperature under nitrogen was added sodium azide (11
mg, 0.17 mmol) and the reaction was warmed to 100.degree. C. After
stirring overnight, the reaction was cooled to room temperature,
diluted with EtOAc (30 mL) and washed 4.times.30 mL with H.sub.2O
and 1.times.30 mL with saturated NaCl. The organics were isolated,
dried (MgSO.sub.4), filtered and concentrated to the
2-azidopyrazine, which exists as a yellow solid (45 mg,
quantitative). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 9.73 (s,
1H), 7.58-7.42 (m, 6H), 7.36-7.23 (m, 4H).
[0845] Step 4: 2-Amino-5,6-diphenylpyrazine. To a stirred solution
of 2-Azido-5,6-diphenylpyrazine (45 mg, 0.17 mmol) in 50 mL EtOAc
at room temperature was added triethylamine (100 .mu.l) followed by
Pearlman's Catalyst (50 mg). The suspension was put through a
vacuum/purge cycle three times with hydrogen gas and then held
under 1 atmosphere of hydrogen for 2 hours. The suspension was then
filtered through GF/F filter paper with EtOAc and concentrated. The
crude product was eluted through a Biotage 12S column with 1/1
EtOAc/hexane to give pure product, as a clear oil (25 mg, 59%).
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.04 (s, 1H), 7.42-7.20
(m, 10H), 4.62 (br s, 2H).
[0846] Step 5: Prepared according to general procedure described
for compound 310, using 2-amino-5,6-diphenylpyrazine. .sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 8.34 (s, 1H), 8.13 (s, 1H), 7.80 (s,
1H), 7.46 (d, 2H), 7.37-7.23 (m, 10H), 6.81 (d, 1H), 6.66 (d, 1H),
3.17 (s, 3H), 2.33 (s,3H).
[0847] Compound 315:
##STR00414##
1-[3-Benzyl-5-(4-methoxy-phenyl)-pyrazin-2-yl]-3-(2-methoxy-5-methyl-phen-
yl)-urea
[0848] Prepared according to general procedure described for
compound 310, using 2-amino-3-benzyl-4-(4-methoxyphenyl)pyrazine.
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.51 (s, 1H), 8.11 (s,
1H), 7.96 (d, 2H), 7.34 (m, 5H), 7.03 (d, 2H), 6.80 (m, 2H), 4.28
(s, 2H), 3.95 (s, 3H), 3.90 (s, 3H), 2.30 (s, 3H). LRMS (ESI,
Positive) m/e 477.2 (M+1).
[0849] Compound 316:
##STR00415##
1-(6-Azido-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0850] Step 1: Tetrazolo[1,5-a]pyrazin-5-ylamine. Prepared
according to the method of Shaw, J. T., et al., J. Heterocyclic
Chem. 1980, 17, 11.
[0851] Step 2: Prepared using p-nitrophenyl carbamate general
procedure described for compound 166 (step 2) using
Tetrazolo[1,5-a]pyrazin-5-ylamine. .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 9.72 (br s, 1H), 8.23 (s, 1H), 7.97 (s, 1H),
7.87 (s, 1H), 7.80 (br s, 1H), 6.88 (d, 1H), 6.80 (d, 1H), 3.84 (s,
3H), 2.36 (s, 3H). LRMS (ESI, Positive) m/e 300.0 (M+1).
[0852] Compound 317:
##STR00416##
1-(6-Amino-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0853] To a stirred solution of
1-(6-azido-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea (8 mg,
27 .mu.mol) 95% EtOH (2 mL) at room temperature was added
concentrated NH.sub.4OH (10 .mu.L) and 10% Pd on C (25 mg). The
suspension was put through a vacuum/purge cycle three times with
hydrogen gas and then held under 50 psi of hydrogen pressure and
shaken on a Parr Shaker. After 2 hours the vacuum/purge cycle was
repeated and the reaction held under hydrogen for another 2 hours.
The suspension was then filtered through GF/F filter paper with
EtOH and concentrated to a yellow film (3 mg, 41%). .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.19 (s, 1H), 7.61 (s, 1H), 7.56 (s,
1H), 6.83 (s, 2H), 3.95 (s, 3H), 2.33 (s, 3H). LRMS (ESI, Positive)
m/e 274.2 (M+1).
[0854] Compound 318:
##STR00417##
1-(6-Chloro-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0855] To a stirred solution of 2-amino-6-chloropyrazine (130 mg, 1
mmol) in 3 mL THF at 0.degree. C. under nitrogen was added methyl
magnesium iodide (3M in Et.sub.2O, 330 .mu.L, 1 mmol) to give a
yellow suspension that was stirred at 0.degree. C. for 15 minutes.
The suspension was treated with the isocyanate neat (147 .mu.L, 1
mmol) and allowed to warm to room temperature overnight. The
reaction was partitioned between EtOAc (30 mL) and 10%
Na.sub.2CO.sub.3 (30 mL). The organics were isolated and washed
1.times.30 mL with 10% Na.sub.2CO.sub.3 and 1.times.30 mL with
saturated NaCl. The organics were dried (MgSO.sub.4), filtered and
concentrated to a crude residue that was triturated with EtOAc to
give, after filtration, the urea product as a white solid (27 mg,
9%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.26 (s, 1H), 8.23
(s, 1H), 8.17 (s, 1H), 8.09 (br s, 1H), 6.84 (d, 1H), 6.81 (d, 1H),
3.96 (s, 3H), 2.35 (s, 3H). LRMS (ESI, Positive) m/e 293.0
(M+1).
[0856] Compound 319:
##STR00418##
1-(5-Bromo-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0857] Step 1: 2-amino-5-bromopyrazine. To a stirred, cooled
(0.degree. C.) solution of amino pyrazine (5.0 g, 52.6 mmol) in
methylene chloride (200 mL) was added N-bromosuccinimide (9.39 g,
52.8 mmol). After stirring for 24 hours, the reaction was washed
with aqueous 10% sodium carbonate (3.times.50 mL), water (50 mL),
then dried (MgSO.sub.4) and filtered. The filtered material was
concentrated under reduced pressure, taken up in minimal ethyl
acetate (5 mL) followed by hexanes (200 mL). Yellow crystals formed
which were filtered and dried. (56% yield).
[0858] Step 2: Prepared according to general procedure described
for compound 310, using 2-amino-4-bromopyrazine. .sup.1H-NMR (400
MHz, CDCl.sub.3/CD.sub.3OD) .delta. 8.55 (s, 1H), 8.32 (s, 1H),
8.03 (s, 1H), 6.81 (m, 2H), 3.92 (s, 3H), 2.34 (s, 3H).
[0859] Compound 320:
##STR00419##
1-(3,5-Dibromo-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0860] Prepared according to general procedure described for
compound 310, using 2-amino-4,6-dibromopyrazine. .sup.1H-NMR (400
MHz, CDCl.sub.3) .delta. 7.98 (s, 1H), 7.13 (s, 1H), 6.79 (m, 2H),
3.83 (s, 3H), 2.32 (s, 3H).
[0861] Compound 321:
##STR00420##
1-[5-(1,3-Dioxo-1,3-dihydro-isoindol-2-ylmethyl)-pyrazin-2-yl]-3-(2-metho-
xy-5-methyl-phenyl)-urea
[0862] Step 1: (5-Bromomethyl-pyrazin-2-yl)-carbamic acid
tert-butyl ester. To a stirred solution of 2-Boc-amino-5-methyl
pyrazine (1.34 gm, 6.4 mmol), in 20 mL CCl.sub.4 at room
temperature under nitrogen was added N-bromosuccinimide (1.14 gm,
6.4 mmol) followed by benzoyl peroxide (125 mg). The solution was
irradiated with a 100 watt flood lamp, which caused the reaction to
reflux vigorously. After 2 hours, the reaction was cooled to room
temperature, diluted to 125 mL with CH.sub.2Cl.sub.2 and washed
1.times.125 mL with 10% sodium bisulfate solution and 1.times.125
mL with saturated NaCl. The organics were dried (MgSO.sub.4),
filtered and concentrated to a brown oil, which was directly loaded
onto a Biotage 40S column with CH.sub.2Cl.sub.2 and eluted with
15/85 EtOAc/hexane to give the desired benzylic bromide as a yellow
solid (954 mg, 51%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 9.22
(s, 1H), 8.29 (s, 1H), 7.37 (br s, 1H), 4.54 (s, 2H), 1.55 (s,
9H).
[0863] Step 2:
[5-(1,3-Dioxo-1,3-dihydro-isoindol-2-ylmethyl)-pyrazin-2-yl]-carbamic
acid tert-butyl ester. To a stirred solution of phthalimide (971
mg, 6.6 mmol) and powdered K.sub.2CO.sub.3 (1.37 gm, 9.9 mmol) in
acetonitrile (9.9 mL) at room temperature under nitrogen was added
the bromide (954 mg, 3.3 mmol) as a solid. The suspension was
heated to 65.degree. C. for 4 hours. After cooling to room
temperature the reaction was partitioned between EtOAc (60 mL) and
H.sub.2O (60 mL). The organics were isolated and washed 2.times.50
mL with H.sub.2O and 1.times.50 mL with saturated NaCl. The
organics were dried (MgSO.sub.4), filtered and concentrated. The
crude product was triturated with CH.sub.2Cl.sub.2 and filtered to
remove solid excess phthalimide and the filtrated partially
concentrated and loaded directly onto a Biotage 40S column and
eluted with 3/7 EtOAc/hexane to give the desired phthalimide as a
white solid (495 mg, 42%). .sup.1H-NMR (400 MHz, CDCl.sub.3)
.delta. 9.17 (s, 1H), 8.23 (s, 1H), 7.85 (m, 2H), 7.71 (m, 2H),
7.39 (br s, 1H), 4.97 (s, 2H), 1.52 (s, 9H).
[0864] Step 3: 2-(5-Amino-pyrazin-2-ylmethyl)-isoindole-1,3-dione.
To a stirred solution of the phthalimide (495 mg, 1.4 mmol) in 7 mL
CH.sub.2Cl.sub.2 at room temperature in a capped flask was added
trifluoroacetic acid (7 mL). After stirring overnight, the reaction
was concentrated to remove excess trifluoroacetic acid and was then
dissolved in 200 mL 10/1 CH.sub.2Cl.sub.2/MeOH, stirred rapidly,
and treated with a solution of 10% Na.sub.2CO.sub.3 (200 mL). The
organics were isolated dried (MgSO.sub.4), filtered and
concentrated to give the free aminopyrazine as a yellow solid (260
mg, 73%). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.25 (s, 1H),
7.85 (m, 2H), 7.77 (s, 1H), 7.74 (m, 2H), 4.83 (s, 2H).
[0865] Step 4: Prepared according to general procedure described
for compound 310 using
2-(5-Amino-pyrazin-2-ylmethyl)-isoindole-1,3-dione. .sup.1H-NMR
(400 MHz, CDCl.sub.3) .delta. 8.38 (s, 1H), 8.29 (s, 1H), 8.03 (s,
1H), 7.86 (m, 2H), 7.76 (m, 2H), 6.81 (m, 2H), 4.98 (s, 2H), 3.91
(s, 3H), 2.31 (s, 3H). LRMS (ESI, Positive) m/e 418.1 (M+1).
[0866] Compound 322:
##STR00421##
1-(5-Aminomethyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0867] To a stirred solution of
1-[5-(1,3-dioxo-1,3-dihydro-isoindol-2-ylmethyl)-pyrazin-2-yl]-3-(2-metho-
xy-5-methyl-phenyl)-urea (16 mg, 38 .mu.mol) in 380 .mu.L 95% EtOH
and 100 .mu.L DMF at room temperature in a capped reaction vial was
added hydrazine monohydrate (3.8 .mu.L, 76 .mu.mol). After stirring
overnight at room temperature, a white precipitate formed. The
precipitate was filtered off, dried and triturated with EtOAc to
remove phthalimide based impurities to provide the product as a
white solid (7.9 mg, 72%). .sup.1H-NMR (400 MHz, d.sub.6-DMSO)
.delta. 9.54 (s, 1H), 8.80 (s, 1H), 8.46 (s, 1H), 8.02 (s, 1H),
6.93 (d, 1H), 6.79 (d, 1H), 4.51 (d, 2H), 4.39 (br s, 2H), 3.89 (s,
3H), 2.23 (s, 3H). LRLCMS (ESI, Positive) m/e 288.2 (M+1).
[0868] Compound 323:
##STR00422##
1-(2-Methoxy-5-methyl-phenyl)-3-(6-methoxy-pyrazin-2-yl)-urea
[0869] Step 1: 2-amino-6-methoxypyrazine. To a stirred solution of
methanol (89 .mu.L; 2.2 mmol) in dioxane (1 mL) was added sodium
hydride (53 mg; 2.2 mmol). After stirring for 30 minutes,
2-amino-6-chloropyrazine (258 mg; 2.0 mmol) was added and the
reaction was heated to 90.degree. C. After stirring for 12 hours,
the reaction was cooled to room temperature, diluted with 30 mL of
ethyl acetate and washed with aqueous 10% sodium carbonate
(1.times.30 mL), and brine (30 mL), then dried (MgSO.sub.4) and
filtered. The crude product was purified using the Biotage 12i
cartridge eluting with hexane and ethyl acetate (3:1) to yield a
white solid (11% yield).
[0870] Step 2: Prepared according to general procedure described
for compound 310 using 2-amino-6-methoxypyrazine. (8% yield).
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.15 (s, 1H), 8.05 (br s,
1H), 7.92 (s, 1H), 7.82 (s, 1H), 6.89 (d, 1H), 6.80 (d, 1H), 4.05
(s, 3H), 3.81 (s, 3H), 2.38 (s, 3H). LRMS (ESI, Positive) m/e
289.10 (M+1).
[0871] Compound 324:
##STR00423##
1-(6-Benzyloxy-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0872] Step 1: 2-amino-6-benzyloxypyrazine. To a stirred solution
of benzyl alcohol (432 .mu.L; 4.0 mmol) in dioxane (2 mL) was added
sodium hydride (96 mg; 4.0 mmol). After stirring for 30 minutes,
2-amino-6-chloropyrazine (258 mg; 2.0 mmol) was added and the
reaction was heated to 90.degree. C. After stirring for 12 hours,
the reaction was cooled to room temperature, diluted with 30 mL of
ethyl acetate and washed with aqueous 10% sodium carbonate
(1.times.30 mL), and brine (30 mL), then dried (MgSO.sub.4) and
filtered. The crude product was purified using the biotage 12i
cartridge eluting with hexane and ethyl acetate (3:1) to yield a
white solid (33% yield).
[0873] Step 2: Prepared according to general procedure described
for compound 310 using 2-amino-6-benzyloxypyrazine. (34% yield).
.sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 9.99 (s, 1H), 9.18 (s,
1H), 8.62 (s, 1H), 7.99 (s, 1H), 7.95 (s, 1H), 7.52 (d, 2H), 7.41
(m, 3H), 6.92 (d, 1H), 6.80 (d, 1H), 5.39 (s, 2H), 3.80 (s, 3H),
2.21 (s, 3H). LRMS (ESI, Positive) m/e 365.10 (M+1).
[0874] Compound 325:
##STR00424##
1-(2-Methoxy-5-methyl-phenyl)-3-(5-methoxy-pyrazin-2-yl)-urea
[0875] To a stirred solution of
1-(5-bromo-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea (47 mg;
0.14 mmol) in N-methyl pyrrolidinone (300 .mu.L) was added sodium
methoxide (0.5 mmol). The reaction was heated to 100.degree. C.
After stirring for 12 hours, the reaction was cooled to room
temperature, diluted with 30 mL of ethyl acetate and washed with
aqueous 10% sodium carbonate (1.times.30 mL), brine (30 mL), then
dried (MgSO.sub.4) and filtered. The crude product was purified
using a 0.5 mm prep plate eluting with hexane and ethyl acetate
(1:1) to yield a yellow solid (13% yield). .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 8.12 (s, 1H), 7.99 (s, 1H), 7.91 (s, 1H), 6.80
(dd, 2H), 3.95 (s, 3H), 3.89 (s, 3H), 2.38 (s, 3H). LRMS (ESI,
Positive) m/e 289.10 (M+1).
[0876] Compound 326:
##STR00425##
1-(5-Ethynyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0877] Step 1: 2-Amino-5-alkynylpyrazine. To a stirred solution of
5-bromo-2-aminopyrazine (432 mg; 2.5 mmol),
Pd(Ph.sub.3P).sub.2Cl.sub.2 (91 mg; 0.13 mmol), CuI (1.2 g, 6.5
mmol) in triethylamine (8 mL) was added TMS-acetylene. The reaction
was stirred at 60.degree. C. for 12 hours. The reaction was cooled
to room temperature, diluted with 30 mL of ethyl acetate and washed
with aqueous 10% sodium carbonate (1.times.30 mL), brine (30 mL),
then dried (MgSO.sub.4) and filtered. The crude product was diluted
in 1 mL of methanol and sodium hydroxide (10 mL of a 1N aqueous
solution). After stirring for 12 hours the reaction was diluted
with 30 mL of ethyl acetate and washed with aqueous 10% sodium
carbonate (1.times.30 mL), brine (30 mL), then dried MgSO.sub.4)
and filtered. The crude product was purified using a biotage 12L
column eluting with methylene chloride and methanol (98:2) to yield
and off white solid (40% yield).
[0878] Step 2: Prepared according to general procedure described
for compound 310 using 2-Amino-5-alkynylpyrazine. (20% yield).
.sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 10.25 (s, 1H), 9.80 (br
s, 1H), 8.90 (s, 1H), 8.50 (s, 1H), 8.00 (s, 1H), 6.95 (d, 1H),
6.82 (d, 1H), 4.42 (s, 1H), 3.82 (s, 3H), 2.22 (s, 3H). LRMS (ESI,
Positive) m/e 283.10 (M+1).
[0879] Compound 327:
##STR00426##
1-(5-Ethyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0880] Step 1: 2-Amino-5-ethylpyrazine. To a stirred solution of
5-ethynyl-2-aminopyrazine (18 mg; 0.151 mmol), in ethyl acetate
(500 .mu.L) was added triethylamine (63 .mu.L; 0.45 mmol) and
Pd(OH).sub.2 (0.01 mmol; 20% wt on carbon). The reaction was placed
under a hydrogen atmosphere at 45 psi and shook for 6 hours. The
reaction was filtered and concentrated under reduced pressure to
yield an off white solid (84% yield).
[0881] Step 2: Prepared according to the general procedure
described for compound 310 using 2-Amino-5-ethylpyrazine.
(27%yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.65 (s, 1H),
8.38 (s, 1H), 8.18 (s, 1H), 8.08 (s, 1H), 6.80 (dd, 2H), 3.92 (s,
3H), 2.81 (q, 2H), 2.39 (s, 3H), 1.39 (t, 3H). LRMS (ESI, Positive)
m/e 287.21 (M+1).
[0882] Compound 328:
##STR00427##
1-(5-Cyano-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0883] Step 1: 2-amino-5-cyanopyrazine. To a stirred solution of
5-bromo-2-aminopyrazine (1.0 g, 5.8 mmol), CuI (2.76 g, 14.5 mmol),
18-crown-6 (121 mg; 0.46 mmol), potassium cyanide (943 mg; 14.5
mmol) in dimethylformamide (20 mL) was added Pd(PPh.sub.3).sub.4
(196 mg; 0.17 mmol). After stirring at room temperature for 20
minutes the reaction was placed in an oil bath at 155.degree. C.
for 2 hours. The reaction was allowed to cool to room temperature
and then poured into chloroform (300 mL). A precipitate formed that
was filtered and triturated with hexanes to yield an off white
solid (60% yield).
[0884] Step 2: Prepared according to general procedure described
for compound 310 using 2-amino-5-cyanopyrazine. (30% yield).
.sup.1H-NMR (400 MHz, d.sub.6-DMSO) .delta. 8.89 (s, 1H), 8.79 (s,
1H), 8.05 (s, 1H), 6.91 (d, 1H), 6.80 (d, 1H), 3.85 (s, 3H), 2.22
(s, 3H). LRMS (ESI, Positive) m/e 283.91 (M+1).
[0885] Compound 329:
##STR00428##
1-(5-Benzoyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0886] Step 1: 5-Benzoyl-pyrazine-2-carboxylic acid. To a stirred,
cooled (0.degree. C.) solution of 2-pyrazine carboxylic acid (3.0
g, 24.2 mmol) and benzaldehyde (7.4 mL; 73 mmol) in a 50% aqueous
solution of sulfuric acid (40 mL) and 25 mL of acetic acid was
added FeSO.sub.4 7 H.sub.2O (20.3 g, 73 mmol dissolved in 50 mL of
water) and t-butyl peroxide (9.2 mL; 73 mmol) simultaneously. After
stirring for 1 hour, the reaction was treated with 200 mL of water.
A precipitate formed which was filtered and washed with methylene
chloride (3.times.100 mL) to yield a tan solid (36% yield).
[0887] Step 2:
1-(5-Benzoyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea. To a
stirred solution of 5-Benzoyl-pyrazine-2-carboxylic acid (912 mg;
4.0 mmol) and triethylamine (584 .mu.L; 4.0 mmol) in toluene (12
mL) was added diphenyl phosphoryl azide (860 .mu.L; 8.0 mmol). The
reaction was stirred for 30 minutes followed by the addition of
t-butanol (764 .mu.L; 8.0 mmol). The reaction was heated to
90.degree. C. and stirred for 3 hours. The reaction was cooled to
room temperature, diluted with 30 mL of ethyl acetate and washed
with aqueous 10% sodium carbonate (1.times.30 mL), brine (30 mL),
then dried (MgSO.sub.4) and filtered. The material was purified
using a biotage 40M cartridge eluting with hexane and ethyl acetate
(1:1) to yield an off white solid (14% yield). .sup.1H-NMR (400
MHz, CDCl.sub.3) .delta. 9.41 (s, 1H), 9.01 (s, 1H), 8.45 (s, 1H),
8.18 (s, 1H), 8.08 (d, 2H), 7.61 (t, 1H), 7.52 (t, 2H), 6.90 (d,
1H), 6.82 (d, 1H), 3.92 (s, 3H), 2.39 (s, 3H). LRMS (ESI, Positive)
m/e 363.21(M+1).
[0888] Compound 330:
##STR00429##
1-[5-(Hydroxy-phenyl-methyl)-pyrazin-2-yl]-3-(2-methoxy-5-methyl-phenyl)--
urea
[0889] To a stirred solution of
1-(5-benzoyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea (22
mg; 0.061 mmol) in methanol (1 mL) was added sodium borohydride (10
mg; 0.3 mmol). After stirring for 12 hours, the reaction diluted
with 30 mL of ethyl acetate and washed with aqueous 10% sodium
carbonate (1.times.30 mL), brine (30 mL), then dried (MgSO.sub.4)
and filtered. The filtered material was concentrated under reduced
pressure to yield a white solid (91% yield). .sup.1H-NMR (400 MHz,
CDCl.sub.3) .delta. 8.48 (br s, 1H), 8.39 (s, 1H),8.22 (s, 1H),
8.12 (s, 1H), 7.25-7.45 (m, 5H), 6.89 (d, 1H), 6.80 (d, 1H), 5.85
(d, 1H), 3.88 (s, 3H), 2.37 (s, 3H). LRMS (ESI, Positive) m/e
365.24 (M+1).
[0890] Compound 331:
##STR00430##
1-(2-Methoxy-5-methyl-phenyl)-3-(6-phenyl-pyrazin-2-yl)-urea
[0891] Suzuki Procedure
[0892] Step 1: To a stirred solution of 2-amino-6-chloro pyrazine
(400 mg; 3.1 mmol) and phenyl boronic acid (415 mg; 3.4 mmol) in
dioxane (6 mL) and ethanol (3 mL) was added cesium carbonate (2.28
g, 7.0 mmol in 3 mL of water) followed by Pd(PPh.sub.3).sub.4 (185
mg; 0.16 mmol). The reaction was heated to 75.degree. C. and
stirred for 12 hours. The reaction was cooled to room temperature,
diluted with 50 mL of ethyl acetate and washed with aqueous 10%
sodium carbonate (1.times.50 mL), brine (50 mL), then dried
(MgSO.sub.4) and filtered. The material was purified using a
biotage 40M cartridge eluting with ethyl acetate to yield an off
white solid (84% yield).
[0893] Step 2: Prepared according to general procedure described
for compound 310 using 2-amino-6-phenylpyrazine. (33% yield).
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 11.11 (br s, 1H), 8.612
(s, 1H), 8.29 (s, 1H), 8.24 (s, 1H), 8.14 (s, 1H), 8.03 (m, 2H),
7.51 (m, 3H), 6.87 (d, 1H), 6.77 (d, 1H). LRMS (ESI, Positive) m/e
355.6 (M+1).
[0894] Compound 332:
##STR00431##
1-(3-Bromo-5-phenyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0895] Step 1: 2-amino-3-bromo-5-phenylpyrazine. To a stirred
solution of 3,5-dibromo-2-aminopyrazine (200 mg; 0.79 mmol) and
phenyl boronic acid (106 mg; 0.87 mmol) in dioxane (4 mL) and
ethanol (2 mL) was added cesium carbonate (571 mg; 1.75 mmol in 2
mL of water) followed by Pd(PPh.sub.3).sub.4 (46 mg; 0.04 mmol).
The reaction was heated to 75.degree. C. and stirred for 12 hours.
The reaction was cooled to room temperature, diluted with 50 mL of
ethyl acetate and washed with aqueous 10% sodium carbonate
(1.times.50 mL), brine (50 mL), then dried (MgSO.sub.4) and
filtered. The material was purified using a biotage 12L cartridge
eluting with hexanes and ethyl acetate (3:1) to yield and off white
solid (88% yield).
[0896] Step 2: Prepared according to the general procedure
described for compound 310 using 2-amino-3-bromo-5-phenylpyrazine.
(18% yield). .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.36 (s,
1H), 8.09 (s, 1H), 7.63 (d, 2H), 7.59 (m, 3H), 6.85 (dd, 2H), 3.92
(s, 3H), 2.39 (s, 3H).
[0897] LRMS (ESI, Positive) m/e 413.2-415.2 (M+1).
[0898] Compound 333:
##STR00432##
1-(2-Methoxy-5-methyl-phenyl)-3-(5-phenyl-pyrazin-2-yl)-urea
[0899] Step 1: 2-amino-5-phenylpyrazine. To a stirred solution of
3-bromo-5-phenyl-2-amino pyrazine (80 mg; 0.32 mmol), in ethyl
acetate (1 mL) was added triethylamine (139 .mu.L; 1.0 mmol) and
Pd(OH).sub.2 (10 mg; 20% wt on carbon). The reaction was placed
under a hydrogen atmosphere at 45 psi and shook for 6 hours. The
reaction was filtered and concentrated under reduced pressure. The
product was purified using a biotage 12L eluting with ethyl acetate
to yield an off white solid (75% yield).
[0900] Step 2: Prepared according to general procedure described
for compound 310 using 2-amino-5-phenylpyrazine. (25% yield).
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.36 (s, 1H), 8.22 (s,
1H), 8.18 (s, 1H), 7.63 (m, 2H), 7.59 (m, 3H), 7.28 (br s, 1H),
6.82 (m, 2H), 3.92 (s, 3H), 2.33 (s, 3H). LRMS (ESI, Positive) m/e
335.21 (M+1).
[0901] Compound 334:
##STR00433##
1-(2-Methoxy-5-methyl-phenyl)-3-quinoxalin-2-yl-urea
[0902] Step 1: To 2-chloroquinoxaline (1.0 g, 6 mmol) was added
ammonia in methanol (8 mL of a 2M solution). The reaction was
sealed in a vial, heated to 80.degree. C., and stirred for 12
hours. The reaction was cooled to room temperature and concentrated
under reduced pressure. The residue was taken up in methylene
chloride and filtered. Hexane was added until a precipitate formed
which was filtered and found to be the desired product (5%
yield).
[0903] Step 2: Prepared according to general procedure described
for compound 310 using Quinoxalin-2-ylamine. (26% yield).
.sup.1H-NMR (400 MHz, D.sub.6-DMSO) .delta. 11.63 (br s, 1H), 10.59
(br s, 1H), 8.80 (s, 1H), 8.15 (s, 1H), 7.97 (d, 1H), 7.81 (m, 2H),
7.64 (m, 1H), 6.98 (d, 1H), 6.82 (d, 1H), 3.97 (s, 3H), 2.23
(s,3H). LRMS (ESI, Positive) m/e 309.4 (M+1).
[0904] Compound 335:
##STR00434##
1-(3,6-Dimethyl-pyrazin-2-yl)-3-(2-methoxy-5-methyl-phenyl)-urea
[0905] Step 1: 2-Azido-3,6-dimethylpyrazine. To a stirred solution
of 2-chloro-3,5-dimethyl pryazine (1.0 mL; 8.3 mmol) in
dimethylformamide (10 mL) was added sodium azide (539 mg; 8.3
mmol). The reaction was heated to 100.degree. C. and stirred for 12
hours. The reaction was cooled to room temperature, diluted with 50
mL of ethyl acetate and washed with aqueous 10% sodium carbonate
(1.times.50 mL), brine (50 mL), then dried (MgSO.sub.4) and
filtered. The material was purified using a biotage 12L cartridge
eluting with hexanes and ethyl acetate (3:1) to yield an off white
solid (42% yield).
[0906] Step 2: 2-Amino-3,6-dimethylpyrazine. To a stirred solution
of 3-azido-2,5-dimethyl pyrazine (100 mg; 0.66 mmol) in methanol
(800 .mu.L) was added 12N HCl (100 .mu.L) and tin chloride
dehydrate (149 mg; 0.66 mmol). The reaction was heated to
60.degree. C. and stirred for 12 hours. The reaction was cooled to
room temperature, diluted with 50 mL of ethyl acetate and washed
with aqueous 10% sodium carbonate (1.times.50 mL), brine (50 mL),
then dried (MgSO.sub.4) and filtered. The material was purified
using a biotage 12i cartridge eluting with ethyl acetate to yield
an off white solid (38% yield).
[0907] Step 3: Prepared according to general procedure described
for compound 310 using 2-Amino-3,6-dimethylpyrazine. (15% yield).
.sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. 8.22 (br s, 1H), 8.01 (s,
1H), 6.82 (m, 2H), 3.92 (s, 3H), 2.59 (s, 3H), 2.55 (s, 3H), 2.38
(s, 3H).
[0908] LRMS (ESI, Positive) m/e 287.20 (M+1).
[0909] Compound 336:
##STR00435##
[0910] Step 1: 2-Methoxy-4-methoxymethyl-1-nitro-benzene. To a 250
mL round bottomed flask containing 5.4 g (39 mmol) of
3-methoxy-4-nitrobenzyl alcohol in 30 mL THF and 30 mL DMF was
added, 38 g (117 mmol, 3 eq.) of finely powdered cesium carbonate
followed by 24 mL (390 mmol, 10 eq.) iodomethane. The mixture was
stirred at room temperature for 18 h, and was then partitioned
between 100 mL water and 100 mL diethyl ether. The aqueous phase
was extracted with ether (2.times.100 mL), and the combined organic
extracts were washed with brine (2.times.50 mL), dried over
MgSO.sub.4, filtered through a short plug of silica, and
concentrated. The residue was purified by flash chromatography,
eluting with 1:1 EtOAc-Hexane, to give 6.76 g (88%) of the methyl
ester as a yellow oil. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.
7.84, (d, J=8.2 Hz, 1H), 7.10 (s, 1H), 6.94 (d, J=9.1 Hz, 1H), 4.51
(s, 2H), 3.98 (s, 3H), 3.44 (s, 3H).
[0911] Step 2: 2-Methoxy-4-methoxymethyl-phenylamine. In a 250 mL
Parr apparatus, 2.1 g (10.6 mmol) of
2-methoxy-4-methoxymethyl-1-nitro-benzene, in 40 mL of ethanol was
hydrogenated at 2 atm over 300 mg of 10% Pd on carbon for 2.5 h.
The catalyst was removed by filtration through a glass fiber filter
and the filtrate was concentrated to give 1.61 g (91%) of product
as a light yellow oil. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta.
6.80 (s, 1H), 6.74 (d, J=7.8 Hz, 1H), 6.67 (d, J=7.8 Hz), 4.35 (s,
2H), 3.86 (s, 3H), 3.78 (br d, 2H), 3.35 (s, 3H). MS ESI-pos,
M+1=168.1
[0912] Step 3:
1-(2-Methoxy-4-methoxymethyl-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea.
General diphenylphosphoryl azide coupling method: To a solution of
5-methylpyrzine-2-carboxylic acid (365 mg, 2.64 mmol) in 20 mL of
anhydrous toluene, was added, diisopropylethylamine (483 .mu.L,
2.77 mmol) and the mixture was stirred at room temperature until
the solid dissolved. Then diphenylphosphoryl azide was added and
the solution was heated to 90.degree. C. After 20 minutes N.sub.2
evolution had subsided, and the caramel colored reaction mixture
was cooled to 60.degree. C. before adding
2-methoxy-4-methoxymethylaniline as a solution in 4 mL toluene.
After stirring for 6 hr at 60.degree. C., the mixture was cooled to
room temperature and diluted with 20 mL of 5% NH.sub.4OH, and
extracted with EtOAc (3.times.50 mL). The combined extracts were
washed with 20 mL water and 20 mL brine then dried over MgSO.sub.4,
filtered and concentrated. The brown residue was purified by flash
chromatography (eluting with 5% MeOH in CH.sub.2Cl.sub.2) to give
219 mg (27%) of the desired product. .sup.1H-NMR (400 MHz,
CDCl.sub.3): .delta. 11.36 (s, 1H), 9.47 (s, 1H), 8.40 (s, 1H),
8.31 (s, 1H), 8.08 (s, 1H), 6.95 (s, 1H), 6.94 (d, J=7.8 Hz, 1H),
4.45 (s, 2H), 3.97 (s, 3H), 3.39 (s, 3H), 2.52 (s, 3H). MS ESI-pos
M+1=303.2.
[0913] Compound 337:
##STR00436##
1-(4-Benzyloxymethyl-2-methoxy-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea
[0914] Step 1: 4-Benzyloxymethyl-2-methoxy-1-nitro-benzene. To a
stirred suspension of finely powdered cesium carbonate (8.0 g, 24.5
mmol) was added 3-methoxy-4-nitrobenzyl alcohol (1.5 g, 8.18 mmol),
followed by benzyl bromide (2 mL, 16.4 mmol). After stirring at
room temperature for 18 h, the suspension diluted with 100 mL
diethyl ether, and washed with 3.times.50 mL water, then 50 mL
brine. The organic phase was dried over MgSO.sub.4, filtered
through a plug of silica, and concentrated. The resulting orange
oil was purified by flash chromatography, eluting with 2:1
hexane-EtOAc, to give 1.87 g (84%) of benzyl ether. .sup.1H-NMR
(400 MHz, CDCl.sub.3): .delta. 7.85 (d, J=8.2 Hz, 1H), 7.3-7.4 (m,
5H), 7.26 (s, 1H), 6.97 (d, J=8.2 Hz, 1H), 4.61 (s, 2 h), 4.59 (s,
2H), 3.96 (s, 3H).
[0915] Step 2: 4-Benzyloxymethyl-2-methoxy-phenylamine. A solution
of 4-nitro-3-methoxybenzylbenzyl ether (2.2 g, 8.1 mmol) and
ammonium acetate (2.46 g, 32 mmol, 4 eq.) in 30 mL MeOH was stirred
at 0.degree. C. and 1.3 g (20 mmol, 2.5 eq.) of zinc dust was added
in several portions. After 1 h, the reaction mixture was
partitioned between 40 mL water and 40 mL ethyl acetate. The
organic phase was dried over MgSO.sub.4 and concentrated. The
residue was used directly in next step without further
purification. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 7.35 (m,
5H0, 6.82 (s, 1H), 6.77 (d, J=6.3 Hz, 1H), 6.67 (d, J=7.8 Hz, 1H),
4.51 (s, 2H), 4.45 (s, 2H), 3.85 (s, 3H), 3.79 (s, 2H). MS ESI-pos,
M+1=244.2.
[0916] Step 3:
1-(4-Benzyloxymethyl-2-methoxy-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea.
Prepared according to the general diphenylphosphoryl azide coupling
method described above for compound 336. .sup.1H-NMR (400 MHz,
CDCl.sub.3): .delta. 11.36 (s, 1H), 9.23 (s, 1H), 8.39 (s, 1H),
8.32 (d, J=7.8 Hz, 1H), 8.09 (s, 1H), 7.38 (m, 5H), 6.97 (s, 2H),
4.56 (s, 4H), 3.96 (s, 3H), 2.53 (s, 3H). MS APCI-pos,
M+1=379.3.
[0917] Compound 338:
##STR00437##
1-{4-[(Benzyl-methyl-amino)-methyl]-2-methoxy-phenyl}-3-(5-methyl-pyrazin-
-2-yl)-urea
[0918] Step 1: 3-Methoxy-4-nitro-benzyl bromide. To a 250 mL round
bottomed flask containing 10 g (54.6 mmol) of
4-nitro-3-methoxybenzyl alcohol in 30 mL THF was added, 36 g (109
mmol, 2 eq.) of carbon tetrabromide followed by 15.9 g (60 mmol,
1.1 eq.) triphenylphosphine at 0.degree. C. The mixture was stirred
at 0.degree. C. for 3 hours. Upon removal of the solvent, the
residue was purified by flash chromatography, eluting with 10:90
EtOAc-Hexane, to give 11 g (82%) of the product as a yellow solid.
.sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 7.86 (s, 1H), 7.84 (m,
1H), 7.12 (s, 1H), 7.07 (m, 1H), 4.47 (s, 2H), 4.00 (s, 3H).
[0919] Step 2: N-Benzyl-N-(3-methoxy-4-nitro-benzyl)-amine. To a
150 mL round bottomed flask containing 1.97 g (8.0 mmol) of
3-methoxy-4-nitro-benzyl bromide in 20 mL THF was added 2.4 g (24
mmol, 3 eq.) of triethylamine followed by 2.5 g (24 mmol, 3 eq.) of
benzylamine. The mixture was stirred at room temperature for 2 h,
and was then partitioned between 50 mL ethyl acetate and brine. The
organic phase was dried over MgSO.sub.4 and concentrated. The
residue was purified by flash chromatography, eluting with 1-4%
MeOH in dichloromethane to give 1.6 g (73%) of the benzyl amine as
a yellow oil. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 7.84 (d,
J=8.61 Hz, 1H), 7.34 (m, 5H), 7.16 (s, 1H), 6.99 (d, J=8.61 Hz,
1H), 3.97 (s, 3H), 3.86 (s, 2H), 3.81 (s, 2H).
[0920] Step 3: Benzyl-(3-methoxy-4-nitro-benzyl)-carbamic acid
tert-butyl ester. To a 150 mL round bottomed flask containing 0.92
g (3.4 mmol, 1 eq.) of N-benzyl-N-(3-methoxy-4-nitro-benzyl)-amine
in 2 mL dichloromethane was added Boc anhydride (0.74 g, 3.4 mmol,
1 eq.) then stirred at room temperature for 18 h. The reaction
mixture was then partitioned between 40 mL water and 40 mL ethyl
acetate. The organic phase was dried over MgSO.sub.4 and
concentrated. No further purification was necessary. .sup.1H-NMR
(400 MHz, CDCl.sub.3): .delta. .sup.1H-NMR (400 MHz, CDCl.sub.3):
.delta. 7.81 (d, J=8.61 Hz, 1H), 7.2-7.3 (m, 6H), 6.93 (m, 1H),
6.82 (s, 1H), 4.39 (m, 4H), 3.88 (s, 3H), 1.53 (s, 9H).
[0921] Steps 4-6:
1-{4-[(Benzyl-methyl-amino)-methyl]-2-methoxy-phenyl}-3-(5-methyl-pyrazin-
-2-yl)-urea. Benzyl-(3-methoxy-4-nitro-benzyl)-carbamic acid
tert-butyl ester was reduced to the corresponding aniline according
to the general hydrogenation procedure detailed above for compound
336. The crude aniline was used in the coupling step as follows: A
solution of 5-methylpyrazine-2-carboxylic acid (34.5 mg, 0.25 mmol)
and added triethylamine (28 mg, 0.275 mmol) in 5 mL of anhydrous
toluene was stirred at room temperature until the solid dissolved.
Diphenylphosphoryl azide (62 mg, 0.225 mmol) was added and the
solution heated to 90.degree. C. for 20 min. The reaction pot was
then transferred to a 60.degree. C. oil bath, and the aniline (0.25
mmol) was added as a solution in 2 mL toluene. After stirring for
4.5 hr at 60.degree. C., the mixture was cooled to room
temperature, diluted with EtOAc, washed with sat'd NaHCO.sub.3,
then brine. The organic phase was dried over MgSO.sub.4, filtered
and concentrated. The resulting residue was purified by preparative
TLC, eluting with 5% MeOH in CH.sub.2Cl.sub.2, to give the desired
urea. The Boc group was removed by treatment of the Boc protected
amine in 15 mL of CH.sub.2Cl.sub.2 with 3 mL TFA and stirring at
room temperature for 3 h. The mixture was diluted with EtOAc (50
mL), washed with 20 mL of sat'd NaHCO.sub.3 followed by 20 mL of
brine. The organic phase was then dried over MgSO.sub.4, filtered
and concentrated to give the free amine. .sup.1H-NMR (400 MHz,
d.sub.6-DMSO): .delta. 11.35 (s, 1H), 9.68 (s, 1H), 8.41 (s, 1H),
8.28 (d, J=8.21 Hz, 1H), 8.06 (s, 1H), 7.36 (s, 6H), 6.96 (s, 1H),
6.94 (d, J=8.21 Hz, 1H), 3.95 (s, 3H), 3.84 (s, 2H), 3.81 (s, 2H),
2.52 (s, 3H), 2.13 (s, 1H). MS APCI-pos, M+1=377.9.
[0922] Compound 339:
##STR00438##
1-(2-Methoxy-4-methylaminomethyl-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea
[0923] Step 1+2: (3-Methoxy-4-nitro-benzyl)-methyl-carbamic acid
tert-butyl ester. In a fashion similar to that described above for
the analogous benzyl derivative compound 338,
3-methoxy-4-nitro-benzyl bromide was alkylated with methylamine,
and the resulting secondary amine was protected as the Boc
derivative. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 7.84 (d,
J=8.61 Hz, 1H), 6.98 (s, 1H), 6.87 (d, J=8.61 Hz, 1H), 4.46 (s,
2H), 3.95 (s, 7H), 2.85 (s, 3H), 1.53 (s, 9H).
[0924] Step 3: (4-amino-3-methoxy-benzyl)-methyl-carbamic acid
tert-butyl ester. In a 250 mL Parr apparatus, 0.98 g (3.5 mmol) of
(3-methoxy-4-nitro-benzyl)-methyl-carbamic acid tert-butyl ester in
40 mL of ethanol was hydrogenated at 2 atm over 300 mg of 10% Pd on
carbon for 15 minutes. The catalyst was removed by filtration
through a glass fiber filter and the filtrate was concentrated to
give a crude product as light yellow oil. .sup.1H-NMR (400 MHz,
CDCl.sub.3): .delta. 6.69 (m, 3H), 3.95 (s, 2H), 3.84 (s, 3H), 2.8
(m, 2H), 2.75 (s, 3H), 1.51 (s, 9H).
[0925] Step 4+5:
1-(2-Methoxy-4-methylaminomethyl-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea.
A solution of (4-amino-3-methoxy-benzyl)-methyl-carbamic acid
tert-butyl ester was converted to the urea according to the general
diphenylphosphoryl azide coupling method detailed for compound 336.
The Boc group was removed as described for above compound 338.
.sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.91 (s, 2H), 8.78 (s,
1H), 8.21 (s, 1H), 8.07 (d, J=8.61 Hz, 1H), 7.01 (s, 1H), 6.85 (d,
J=8.61 Hz, 1H), 3.89 (s, 3H), 3.59 (s, 1H), 2.42 (s, 3H), 2.26 (s,
3H). MS APCI-Pos, M+1=301.8.
[0926] Compound 340:
##STR00439##
1-{4-[(Benzyl-methyl-amino)-methyl]-2-methoxy-phenyl}-3-(5-methyl-pyrazin-
-2-yl)-urea
[0927] Step 1: N-Benzyl-N-(3-methoxy-4-nitro-benzyl)-methyl-amine.
A solution of N-methyl-benzyl amine was akylated with
3-methoxy-4-nitro-benzyl bromide as described above for compound
338. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 7.83 (d, J=8.6 Hz,
1H), 7.35 (s, 4H), 7.26 (s, 1H), 7.17 (s, 1H), 7.01 (d, J=7.04 Hz,
1H), 3.97 (s, 3H), 3.55 (s, 4H), 2.22 (s, 3H).
[0928] Step 2:
4-[(Benzyl-methyl-amino)-methyl]-2-methoxy-phenylamine. Nickel
boride Reduction General Method: To a stirred solution of nickel
chloride hexahydrate (820 mg, 3.45 mmol) in 12 mL EtOH and 3 mL
THF, at 0.degree. C., NaBH.sub.4 (130 mg, 3.45 mmol) was added. The
resulting black suspension was stirred at 0.degree. C. while
N-benzyl-(3-methoxy-4-nitro-benzyl)-methyl-amine was added as a
solution in 5 mL THF. After several minutes, 260 mg of NaBH.sub.4
was added in several portions over 10 minutes, and the reaction
mixture was subsequently allowed to warm to room temperature. After
2 h, TLC indicated complete conversion to a new, more polar
product. At this point, 1.5 mL of 5% NH.sub.4OH was added and the
reaction was stirred for about 10 min, until the black solids
achieved a granular consistency. The reaction was filtered through
as glass fiber filter, rinsing with THF. The clear colorless
filtrate was concentrated to about 1/4 volume, diluted with 30 mL
water and extracted with EtOAc (3.times.30 mL). The combined
extracts were washed with brine, dried over MgSO.sub.4 and filtered
through a short plug of silica and concentrated in vacuo to afford
575 mg (65%) of the desired product as an off-white solid.
.sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 7.2-7.4 (m, 5H), 7.85
(s, 1H), 6.85 (s, 1H), 6.74 (d, J=8.2 Hz, 1H), 6.66 (d, J=8.2 Hz,
1H), 3.87 (s, 3H), 3.73 (br s, 2H), 3.49 (s, 2H), 3.45 (s, 2H),
2.18 (s, 3H). MS (APCI-pos)M+1=256.9.
[0929] Step 3:
1-{4-[(Benzyl-methyl-amino)-methyl]-2-methoxy-phenyl}-3-(5-methyl-pyrazin-
-2-yl)-urea. In a 50 mL round bottom flask,
5-methylpyrazine-2-carboxylic acid (250 mg, 1.8 mmol) and
diisopropylethylamine (330 .mu.L, 1.9 mmol) in 20 mL toluene, was
stirred under a nitrogen atmosphere until the acid dissolved.
Diphenylphosphoryl azide (523 mg, 1.9 mmol) was added and the
solution was heated to 90.degree. C. After 20 minutes, nitrogen
evolution had subsided, and the solution had darkened to a caramel
color. The reaction was cooled to 65.degree. C., and
4-[(benzyl-methyl-amino)-methyl]-2-methoxy-phenylamine (486 mg, 1.9
mmol) was added as a solution in 5 mL toluene. The reaction was
allowed to stir at 65.degree. C. for 6 h, and was then cooled to
room temperature, diluted with 30 mL EtOAc, and washed with 15 mL
5% NH.sub.4OH. The aqueous phase was extracted with EtOAc
(2.times.20 mL) and the combined organics were washed with 30 mL
brine, dried over MgSO.sub.4, filtered and concentrated. The
resulting residue was purified by flash chromatography to give 285
mg (40%) of the desired product, which was further purified by
trituration with diethyl ether. Mp=142-143.degree. C. .sup.1H-NMR
(400 MHz, CDCl.sub.3): .delta. 11.33 (s, 1H), 9.51 (s, 1H), 8.41
(s, 1H), 8.27 (d, J=8.61 Hz, 1H), 8.08 (s, 1H), 7.2-7.4 (m, 5H),
6.96 (d, J=7.83 Hz, 1H), 3.97 (s, 3H), 3.53 (s, 4H), 2.53 (s, 3H),
2.22 (s, 3H). .sup.13C-NMR (400 MHz, CDCl.sub.3): .delta. 153.70,
149.03, 147.48, 147.4, 145.99, 138.73, 137.38, 134.81, 129.19,
128.42, 127.16, 121.89, 119.81, 111.05, 104.49, 94.98, 87.22,
61.81, 56.37, 42.5. MS (APCI-pos) M+1=392.0.
[0930] Compound 341:
##STR00440##
1-(4-Dimethylaminomethyl-2-methoxy-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea
[0931] Step 1: (3-Methoxy-4-nitro-benzyl)-dimethyl-amine. According
to the method described above for compound 338,
3-methoxy-4-nitro-benzyl bromide was treated with dimethyl amine to
give the desired product. .sup.1H-NMR (400 MHz, CDCl.sub.3):
.delta. 7.86 (d, J=7:8 Hz, 1H), 7.3 (s, 1H), 6.98 (d, J=8.6 Hz,
1H), 4.01 (s, 3H), 3.5 (s, 2H), 2.3 (s, 6H).
[0932] Step 2: 4-Dimethylaminomethyl-2-methoxy-phenylamine.
According to the nickel-boride method described for compound 340
above, (3-methoxy-4-nitro-benzyl)-dimethyl-amine, was reduced to
the corresponding aniline and was used in the next step without
characterization.
[0933] Step 3:
1-(4-Dimethylaminomethyl-2-methoxy-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea-
. According to the diphenylphosphoryl azide method described above
for compound 336, 4-dimethylaminomethyl-2-methoxy-phenylamine was
converted to the (5-methyl-pyrazin-2-yl)-urea. The crude product
was purified by preparative TLC, eluting with 5% MeOH in
CH.sub.2Cl.sub.2. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta.
11.21 (s, 1H), 8.95 (s, 1H), 8.39 (s, 1H), 8.21 (d, J=7.83 Hz, 1H),
8.04 (s, 1H), 7.01 (s, 1H), 6.89 (d, J=7.83 Hz, 1H), 3.98 (s, 3H),
3.92 (s, 2H), 2.26 (s, 3H), 2.24 (s, 6H). MS APCI-Pos,
M+1=316.0.
[0934] Compound 342:
##STR00441##
1-(4-Aminomethyl-2-methoxy-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea
[0935] Step 1: 2-(3-Methoxy-4-nitro-benzyl)-isoindole-1,3-dione. To
a 250 mL round bottomed flask containing 10 g (55 mmol) of
4-nitro-3-methoxybenzyl alcohol in 150 mL THF was added,
diethylazodiacarboxylate (8.03 g, 54.6 mmol, 1 eq.) and
triphenylphosphine (15.0 g, 57.3 mmol) followed by 11.6 g of
phthalimide (57.3 mmol) at 0.degree. C. Reaction was then allowed
to gradually warm to room temperature over night. A white
precipitate formed and was collected by suction filtration.
Recrystallization from acetonitrile gave 13.8 g (81%) of the
desired product. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 7.79
(m, 5H), 7.19 (s, 1H), 7.08 (d, J=8.80 Hz, 1H), 4.91 (s, 2H), 3.87
(s, 3H).
[0936] Step 2: 2-(4-Amino-3-methoxy-benzyl)-isoindole-1,3-dione. In
a 500 mL Parr vessel, a partially dissolved suspension of
2-(3-methoxy-4-nitro-benzyl)-isoidole-1,3-dione (1.50 g, 4.80 mmol)
in 100 mL EtOH and 30 mL THF was hydrogentated over 250 mg 10%
Pd--C at 2.5 atm for 1 h. The catalyst was removed by filtration
through a glass fiber filter, and the clear light yellow filtrate
was concentrated in vacuo. The product was washed with 30 mL of
diethyl ether and collected by suction filtration to give 1.28 g
(95%) of the aniline as fine, light green needles. .sup.1H-NMR (400
MHz, CDCl.sub.3): .delta. 7.82 (m, 2H), 7.81 (m, 2H), 6.93 (s, 1H),
6.9 (d, J=7.8 Hz, 1H), 6.63 (d, J=7.8 Hz, 1H), 4.74 (s, 2H), 3.84
(s, 3H).
[0937] Step 3: Acyl Azide coupling General Method
1-[4-(1,3-Dioxo-1,3-dihydro-isoindol-2-ylmethyl)-2-methoxy-phenyl]-3-(5-m-
ethyl-pyrazin-2-yl)-urea. In a 50 mL round bottom flask, a solution
of 5-methyl-pyrazine-2-carbonyl azide (510 mg, 3.15 mmol) in 15 mL
of anhydrous toluene was stirred under a nitrogen atmosphere. The
reaction flask was immersed in a 90.degree. C. oil bath, and as the
internal temperature approached 90.degree. C., N.sub.2 release was
evident and the solution started to darken. After 20 min,
effervescence had subsided, and the solution had darkened to a
caramel color. The reaction flask was moved to a 65.degree. C.
bath, and 2-(4-amino-3-methoxy-benzyl)-isoindole-1,3-dione (884 mg,
3.15 mmol) suspended in toluene (5 mL) was added. The reaction was
stirred at 65.degree. C. for 6 h, then cooled to room temperature.
The product precipitated from solution after cooling to room
temperature, and was collected by suction filtration.
[0938] In cases where the product does not precipitate from the
reaction mixture, the following work-up is applied: After cooling
to room temperature, the brown solution is diluted with 5% aq.
NH.sub.4OH and the extracted with EtOAc (3.times.). The combined
extracts are washed with brine, dried over MgSO.sub.4, filtered and
concentrated. The residue is then purified by flash chromatography
in an appropriate solvent system.
[0939] Step 4:
1-(4-Aminomethyl-2-mkethoxy-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea.
In a 100 mL round bottom flask, a suspension of
1-[4-(1,3-dioxo-1,3-dihydro-isoindol-2-ylmethyl)-2-methoxy-phenyl]-3-(5-m-
ethyl-pyrazin-2-yl)-urea (620 mg, 1.48 mmol) in 22 mL of EtOH,
under a nitrogen atmosphere, was warmed, with stirring to
70.degree. C. Hydrazine monohydrate (1.4 mL) was added, and the
reaction was stirred at 70.degree. C. After 10 minutes, the
reaction had become a completely homogenous caramel colored
solution. After several minutes more, product started to
precipitate from the solution. After 20 min, the reaction was
cooled to room temperature, and the white solid product was
collected by suction filtration. The crude product, which contained
some phthalhydrazide by-product, was taken up in 80 mL of EtOAc,
and washed with water (3.times.20 mL). The washings were back
extracted with 30 mL EtOAc, and the combined organics were washed
with brine, then dried over MgSO.sub.4, filtered and concentrated
in vacuo to afford 400 mg (94%) of the desired amine.
Mp=168-169.degree. C. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta.
9.88 (br s, 2H), 8.78 (s, 1H), 8.21 (s, 1H), 8.05 (d, J=8.2 Hz,
1H), 7.05 (s, 1H), 6.85 (d, J=8.1 Hz, 1H), 3.89 (s, 3H), 3.68 (s,
2H), 2.42 (s, 3H), 1.80 (br s, 2H, NH.sub.2). MS (APCI-pos)M-17
(--NH.sub.3)=270.1 apci-neg M-1=285.8.
Alkyl Derivatives of Compound 342
[0940] Compound 343:
##STR00442##
[0941] A solution of
1(4-aminomethyl-2-methoxy-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea
(0.25 mmol, 1.0 eq.) and 1 mL trimethylorthoformate in 4 mL MeOH
was stirred at room temperature, then thiophene-2-carboxaldehyde
(2.5 mmol, 10 eq.) was added and the mixture was heated at
80.degree. C. After 18 h the reaction was cooled to room
temperature and was concentrated in vacuo. The resulting imine was
taken up in 5 mL of anhydrous MeOH and stirred at 0.degree. C.
Sodium borohydride (0.75 mmol, 3 eq.) was added, and the reaction
was stirred at 0.degree. C. for 30 minutes and was then diluted
with 2 mL of water and partitioned between 50 mL EtOAc and 30 mL
sat. NaHCO.sub.3. The organic phase was washed with brine, dried
over MgSO.sub.4, filtered and concentrated. If needed, the product
was purified by flash chromatography, eluting with an appropriate
MeOH--CH.sub.2Cl.sub.2 mixture.
[0942]
1-(2-Methoxy-4-{[(thiophen-2-ylmethyl)-amino]-methyl}-phenyl)-3-(5--
methyl-pyrazin-2-yl)-urea. .sup.1H-NMR (400 MHz, d.sub.6-DMSO):
.delta. 10.01 (s, 2H), 8.79 (s, 1H), 8.23 (s, 1H), 8.17 (d, J=8.61
Hz, 1H), 7.59 (d, J=6.26 Hz, 1H), 7.33 (s, 1H), 7.15 (m, 2H), 7.05
(s, 1H), 6.97 (d, J=8.61 Hz, 1H), 4.02 (s, 2H), 3.91 (s, 3H), 3.88
(s, 1H), 3.78 (s, 1H), 2.43 (s, 3H). MS APCI-Pos no detectable
molecular ion.
[0943] Compound 344:
##STR00443##
[0944] Prepared according to the general procedure described for
compound 343 using thiophene-3-carboxaldehyde.
[0945]
1-(2-Methoxy-4-{[(thiophen-3-ylmethyl)-amino]-methyl}-phenyl)-3-(5--
methyl-pyrazin-2-yl)-urea. .sup.1H-NMR (400 MHz, d.sub.6-DMSO):
.delta. 9.92 (s, 2H), 8.78 (s, 1H), 8.21 (s, 1H), 8.07 (d, J=7.81
Hz, 1H), 7.47 (s, 1H), 7.3 (s, 1H), 7.11 (d, J=4.88 Hz, 1H), 7.04
(s, 1H), 6.87 (d, J=7.8 Hz, 1H), 3.9 (s, 3H), 3.67 (s, 2H), 3.65
(s, 2H), 2.42 (s, 3H). MS APCI-Neg, M-1=382.0.
[0946] Compound 345:
##STR00444##
[0947] Prepared according to the general procedure described for
compound 343 using furfural.
[0948]
1-(4-{[(Furan-2-ylmethyl)-amino]-methyl}-2-methoxy-phenyl)-3-(5-met-
hyl-pyrazin-2-yl)-urea. .sup.1H-NMR (400 MHz, MeOD): .delta. 8.57
(s, 1H), 8.2 (s, 1H), 8.1 (d, J=8.61 Hz, 1H), 7.46 (s, 1H), 7.03
(s, 1H), 6.88 (d, J=8.61 Hz, 1H), 6.36 (s, 1H), 6.27 (s, 1H), 3.96
(s, 3H), 3.74 (s, 2H), 3.71 (s, 2H), 2.48 (s, 3H). MS APCI-Neg,
M-1=366.0.
[0949] Compound 346:
##STR00445##
[0950] Prepared according to the general procedure described for
compound 343 using furan-3-carboxaldehyde.
[0951]
1-(4-{[(Furan-3-ylmethyl)-amino]-methyl}-2-methoxy-phenyl)-3-(5-met-
hyl-pyrazin-2-yl)-urea. .sup.1H-NMR (400 MHz, MeOD): .delta. 8.58
(s, 1H), 8.21 (s, 1H), 8.13 (d, J=7.83 Hz, 1H), 7.49 (s, 1H), 7.05
(s, 1H), 6.91 (d, J=7.04 Hz, 1H), 6.5 (s, 1H), 3.97 (s, 3H), 3.78
(s, 2H), 3.69 (s, 2H), 2.49 (s, 3H). MS APCI-Neg, M-1=366.0.
[0952] Compound 347:
##STR00446##
[0953] Prepared according to the general procedure described for
compound 343 using 2-methoxybenzaldehyde.
[0954]
1-{2-Methoxy-4-[(2-methoxy-benzlamino)-methyl]-phenyl}-3-(5-methyl--
pyrazin-2-yl)-urea. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 11.3
(s, 1H), 9.97 (s, 1H), 8.44 (s, 1H), 8.22 (d, J=7.83 Hz, 1H), 7.98
(s, 1H), 7.28 (m, 2H), 7.06 (s, 1H), 6.88 (m, 3H), 4.08 (s, 1H),
3.93 (s, 5H), 3.81 (s, 5H), 2.5 (s, 3H). MS APCI-Pos,
M+1=407.8.
[0955] Compound 348:
##STR00447##
[0956] Prepared according to the general procedure described for
compound 343 using 3-methoxybenzaldehyde.
[0957]
1-{2-Methoxy-4-[(3-methoxy-benzylamino)-methyl]-phenyl}-3-(5-methyl-
-pyrazin-2-yl)-urea. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta.
8.57 (s, 1H), 8.20 (s, 1H), 8.11 (d, J=8.61 Hz, 1H), 7.22 (t,
J=7.83 Hz, 1H), 7.04 (s, 1H), 6.91 (m, 3H), 6.83 (d, J=8.61 Hz,
1H), 3.96 (s, 3H), 3.79 (s, 3H), 3.75 (s, 4H), 2.48 (s, 3H). MS
APCI-Neg, M-1=406.0.
[0958] Compound 349:
##STR00448##
[0959] Prepared according to the general procedure for compound 343
using 4-methoxybenzaldehyde.
[0960]
1-{2-Methoxy-4-[(4-methoxy-benzylamino)-methyl]-phenyl}-3-(5-methyl-
-pyrazin-2-yl)-urea. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta.
9.91 (s, 2H), 8.78 (s, 1H), 8.21 (s, 1H), 8.07 (d, J=7.81 Hz, 1H),
7.25 (d, J=8.78 Hz, 2H), 7.03 (s, 1H), 6.88 (m, 2H), 3.89 (s, 3H),
3.73 (s, 3H), 3.62 (s, 2H), 3.6 (s, 2H), 2.42 (s, 3H). MS APCI-Pos,
M+1=407.9.
Acyl Derivatives
Compound 350:
##STR00449##
[0962] A solution of
1-(4-aminomethyl-2-methoxy-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea
(100 mg, 0.35 mmol) in 30 mL THF and 15 mL aqueous NaHCO.sub.3 was
treated with 3.7 mmol (1.05 eq.) of acetyl chloride. The biphasic
reaction mixture was vigorously stirred at room temperature and
after 2 h was diluted with 10 mL water and extracted with EtOAc
(3.times.20 mL). The combined extracts were washed with brine,
dried over MgSO.sub.4 and filtered through a short plug of silica.
The filtrate was concentrated and the resulting residue was
triturated with diethyl ether. If required, further purification
was accomplished by flash chromatography, eluting with an
appropriate methanol-CH.sub.2Cl.sub.2 solvent system.
[0963]
N-{3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-acetamide-
. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.92 (s, 2H), 8.75
(s, 1H), 8.28 (t, J=5.87 Hz, 1H), 8.19 (s, 1H), 8.07 (d, J=8.61 Hz,
1H), 6.92 (s, 1H), 6.78 (d, J=8.61 Hz, 1H), 4.19 (s, 1H), 4.18 (s,
1H), 3.87 (s, 3H), 2.4 (s, 3H), 1.85 (s, 3H). MS ESI-pos,
M+1=330.2.
[0964] Compound 351:
##STR00450##
[0965] Prepared according to the general procedure described for
compound 350 using methoxyacetyl chloride.
[0966]
2-Methoxy-N-{3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-
-acetamide. .sup.1H-NMR (400 MHz, D.sub.6-DMSO): .delta. 9.51 (s,
2H), 8.34 (s, 1H), 7.89 (t, J=6.26 Hz, 1H), 7.78 (s, 1H), 7.65 (d,
J=7.83 Hz, 1H), 6.54 (s, 1H), 6.38 (d, J=7.83 Hz, 1H), 4.2 (s, 1H),
4.19 (s, 1H), 3.82 (s, 3H), 3.79 (s, 2H), 3.29 (s, 3H), 2.35 (s,
3H). MS APCI-Pos, M+1=359.9.
[0967] Compound 352:
##STR00451##
[0968] Prepared according to the general procedure described for
compound 350 using dimethylamino-acetyl chloride.
[0969]
2-Dimethylamino-N-{3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-b-
enzyl}-acetamide. .sup.1H-NMR (400 MHz, MeOD): .delta. 8.56 (s,
1H), 8.18 (s, 1H), 8.09 (d, J=7.83 Hz, 1H), 6.97 (s, 1H), 6.86 (d,
J=7.83 Hz, 1H), 4.37 (s, 2H), 3.94 (s, 3H), 3.03 (s, 2H), 2.47 (s,
3H), 2.3 (s, 6H). MS APCI-Neg, M-1=370.9.
[0970] Compound 353:
##STR00452##
[0971] Prepared according to the general procedure described for
compound 350 using 2-(2-pyridyl)-acetyl chloride.
[0972]
N-{3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-2-pyridin-
-2-yl-acetamide. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.94
(s, 2H), 8.77 (s, 1H), 8.63 (t, J=5.87 Hz, 1H), 8.49 (s, 1H), 8.44
(d, J=6.26 Hz, 1H), 8.22 (s, 1H), 8.08 (d, J=7.83 Hz, 1H), 7.7 (d,
J=10.17 Hz, 1H), 7.35 (q, J=5.74 Hz, 1H), 6.87 (s, 1H), 6.79 (d,
J=9.39 Hz, 1H), 4.25 (s, 1H), 4.24 (s, 1H), 3.82 (s, 3H), 3.53 (s,
2H), 2.42 (s, 3H). MS APCI-Pos M+1=407.1.
[0973] Compound 354:
##STR00453##
[0974] Prepared according to the general procedure described for
compound 350 using 2-(4-methoxyphenyl)-acetyl chloride.
[0975]
N-{3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-2-(4-meth-
oxy-phenyl)-acetamide. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta.
9.94 (s, 2H), 8.77 (s, 1H), 8.54 (t, J=4.7 Hz, 1H), 8.21 (s, 1H),
8.08 (d, J=7.83 Hz, 2H), 7.89 (m, 1H), 7.22 (t, J=7.83 Hz, 1H),
6.89 (s, 3H), 4.25 (s, 1H), 4.23 (s, 1H), 3.8 (s, 3H), 3.73 (s,
2H), 3.45 (s, 2H), 2.42 (s, 3H). MS (APCI-Pos) M+1=436.2.
[0976] Compound 355:
##STR00454##
[0977] Prepared according to the general procedure described for
compound 350 using benzoyl chloride.
[0978]
N-{3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-benzamide-
. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.95 (s, 2H), 9.02
(t, J=5.48 Hz, 1H), 8.77 (s, 1H), 8.22 (s, 1H), 8.1 (d, J=8.61 Hz,
1H), 7.9 (d, J=7.83 Hz, 2H), 7.48 (m, 3H), 7.04 (s, 1H), 6.88 (d,
J=10.17 Hz, 1H), 4.46 (s, 1H), 4.44 (s, 1H), 3.89 (s, 3H), 2.42 (s,
3H). MS APCI-Pos, M+1=391.9.
[0979] Compound 356:
##STR00455##
[0980] Prepared according to the general procedure described for
compound 350 using pyridine-2-carbonyl chloride.
[0981] Pyridine-2-carboxylic acid
3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzylamide.
.sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.94 (s, 2H), 9.29 (t,
J=6.26 Hz, 1H), 8.77 (m, 1H), 8.65 (m, 1H), 8.21 (s, 1H), 8.05 (m,
3H), 7.61 (m, 1H), 7.07 (s, 1H), 6.89 (d, J=8.61 Hz, 1H), 4.47 (s,
1H), 4.45 (s, 1H), 3.88 (s, 1H), 2.42 (s, 3H). MS APCI-Pos no
detectable molecular ion.
[0982] Compound 357:
##STR00456##
[0983] Prepared according to the general procedure described for
compound 350 using nicotinoyl chloride.
[0984]
N-{3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-nicotinam-
ide. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 10 (s, 2H), 9.26
(t, J=5.87 Hz, 1H), 9.11 (s, 1H), 8.82 (s, 1H), 8.77 (d, J=4.7 Hz,
1H), 8.27 (s, 2H), 8.16 (d, J=7.83 Hz, 1H), 7.58 (m, 1H), 7.09 (s,
1H), 6.95 (d, J=8.61 Hz, 1H), 4.53 (s, 1H), 4.51 (s, 1H), 3.95 (s,
3H), 2.47 (s, 3H). MS APCI-Pos, M-1=397.9.
[0985] Compound 358:
##STR00457##
[0986] Prepared according to the general procedure described for
compound 350 using isonicotinoyl chloride.
[0987]
N-{3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-isonicoti-
namide. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.92 (s, 2H),
9.27 (t, J=5.87 Hz, 1H), 8.74 (s, 1H), 8.71 (d, J=6.3 Hz, 2H), 8.18
(s, 1H), 8.08 (d, J=8.6 Hz, 1H), 7.77 (d, J=6.3 Hz, 2H), 7 (s, 1H),
6.86 (d, J=8.6 Hz, 1H), 4.44 (s, 1H), 4.43 (s, 1H), 3.86 (s, 3H),
2.39 (s, 3H). MS APCI-pos no detectable molecular ion.
[0988] Compound 359:
##STR00458##
[0989] Prepared according to the general procedure described for
compound 350 using thiophene-2-carbonyl chloride.
[0990] Thiophene-2-carboxylic acid
3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzylamide.
.sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.96 (s, 2H), 9.03 (t,
J=6.26 Hz, 1H), 8.78 (s, 1H), 8.22 (s, 1H), 8.11 (d, J=7.83 Hz,
1H), 7.82 (d, J=4.7 Hz, 1H), 7.77 (d, J=3.91 Hz, 1H), 7.16 (d,
J=3.91 Hz, 1H), 7.03 (s, 1H), 6.88 (d, J=10.17 Hz, 1H), 4.43 (s,
1H), 4.42 (s, 1H), 3.89 (s, 3H), 2.42 (s, 3H). MS APCI-pos,
M-1=397.9.
[0991] Compound 360:
##STR00459##
[0992] Prepared according to the general procedure described for
compound 350 using 4-dimethylamino-2-carbonyl chloride.
[0993]
3-Dimethylamino-N-{3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-b-
enzamide. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.94 (s,
2H), 8.9 (t, J=5.87 Hz, 1H), 8.77 (s, 1H), 8.21 (s, 1H), 8.09 (d,
J=8.61 Hz, 1H), 7.26 (t, J=7.83 Hz, 1H), 7.2 (s, 2H), 7.02 (s, 1H),
6.87 (d, J=8.61 Hz, 2H), 4.44 (s, 1H), 4.42 (s, 1H), 3.88 (s, 3H),
2.93 (s, 6H), 2.42 (s, 3H). MS APCI-pos, M+1=434.9.
[0994] Compound 361:
##STR00460##
[0995] Prepared according to the general procedure described for
compound 350 using
1-phenyl-4-trifluoromethyl-1H-pyrazole-3-carbonyl chloride.
[0996] 1-Phenyl-4-trifluoromethyl-1H-pyrazole-3-carboxylic acid
3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzylamide.
.sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.96 (s, 2H), 9.09 (t,
J=6.26 Hz, 1H), 8.78 (s, 1H), 8.22 (s, 1H), 8.19 (s, 1H), 8.12 (d,
J=7.83 Hz, 1H), 7.59 (m, 6H), 7.02 (s, 1H), 6.89 (d, J=8.61 Hz,
1H), 4.43 (s, 1H), 4.42 (s, 1H), 3.9 (s, 3H), 2.42 (s, 3H). MS
TIC-pos M+1=526.2.
Sulfonylated Derivatives
[0997] Compound 362:
##STR00461##
[0998] A solution of
1-(4-aminomethyl-2-methoxy-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea
(0.25 mmol, 1.0 eq.), 4-dimethylaminopyridine (5 mg),
diisopropylethylamine (36 mg, 0.275 mmol, 1.1 eq.) in THF (5 mL)
was prepared and thiophene-2-sulfonyl chloride (0.275 mmol, 1.1
eq.) was added. The mixture was stirred at room temperature for 24
h. The reaction mixture was partitioned between EtOAc (75 mL) and
sat. NaHCO.sub.3. After separation of the layers, the organic layer
was washed with water, saturated brine, dried over MgSO.sub.4,
filtered and concentrated. The residue was purified by flash
chromatography eluting with 5% MeoH in dichloromethane and
trituration with ether to give pure products.
[0999] Thiophene-2-sulfonic acid
3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzylamide.
.sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.96 (s, 2H), 8.77 (s,
1H), 8.33 (t, J=7.04 Hz, 1H), 8.31 (s, 1H), 8.07 (d, J=7.83 Hz,
1H), 7.93 (d, J=4.7 Hz, 1H), 7.59 (s, 1H), 7.18 (t, J=3.91 Hz, 1H),
6.9 (s, 1H), 6.8 (t, J=7.04 Hz, 1H), 4.05 (s, 1H), 4.03 (s, 1H),
3.85 (s, 3H), 2.51 (s, 3H). MS APCI-Pos, M+1=433.9.
[1000] Compound 363:
##STR00462##
[1001] Prepared according to the general procedure described for
compound 362 using benzenesulfonyl chloride.
[1002]
N-{3-Methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-benzenesu-
lfonamide. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.95 (s,
2H), 8.77 (s, 1H), 8.21 (s, 1H), 8.13 (t, J=6.26 Hz, 1H), 8.05 (d,
J=8.61 Hz, 1H), 7.81 (d, J=7.04 Hz, 2H), 7.58 (m, 3H), 6.85 (s,
1H), 6.77 (d, J=8.61 Hz, 1H), 3.96 (s, 1H), 3.95 (s, 1H), 3.81 (s,
3H), 2.42 (s, 3H). MS APCI-Pos, M+1=428.2.
[1003] Compound 364:
##STR00463##
[1004] Prepared according to the general procedure described for
compound 362 using 2-trifluoromethoxybenzenesulfonyl chloride.
[1005]
N-{3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-2-trifluo-
romethoxybenzenesulfonamide. .sup.1H-NMR (400 MHz, d.sub.6-DMSO):
.delta. 9.94 (s, 2H), 8.77 (s, 1H), 8.38 (s, 1H), 8.21 (s, 1H),
8.02 (d, J=8.61 Hz, 1H), 7.89 (m, 1H), 7.72 (t, J=7.83 Hz, 1H),
7.51 (d, J=7.83 Hz, 2H), 6.88 (s, 1H), 6.75 (d, J=10.17 Hz, 1H),
4.11 (s, 2H), 3.82 (s, 3H), 2.42 (s, 3H). MS APCI-Pos,
M+1=512.0.
[1006] Compound 365:
##STR00464##
[1007] Prepared according to the general procedure described for
compound 362 using 3-methoxybenzenesulfonyl chloride.
[1008]
3-Methoxy-N-{3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-
-benzenesulfonamide. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta.
9.92 (s, 2H), 8.73 (s, 1H), 8.17 (s, 1H), 8.08 (s, 1H), 8.01 (d,
J=7.83 Hz, 1H), 7.45 (t, J=7.83 Hz, 1H), 7.34 (d, J=7.83 Hz, 1H),
7.24 (s, 1H), 7.14 (m, 1H), 6.81 (s, 1H), 6.74 (d, J=7.83 Hz, 1H),
3.93 (s, 2H), 3.77 (s, 3H), 3.76 (s,3H), 2.38 (s, 3H). MS APCI-Pos,
M+1=458.0.
[1009] Compound 366:
##STR00465##
[1010] Prepared according to the general procedure described for
compound 362 using 4-methoxybenzenesulfonyl chloride.
[1011]
4-Methoxy-N-{3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzyl}-
-benzenesulfonamide. .sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta.
9.93 (s, 2H), 8.74 (s, 1H), 8.18 (s, 1H), 8.02 (d, J=8.61 Hz, 1H),
7.92 (s, 1H), 7.69 (d, J=8.61 Hz, 2H), 7.05 (d, J=9.39 Hz, 2H),
6.81 (s, 1H), 6.74 (d, J=7.83 Hz, 1H), 3.88 (s, 2H), 3.79 (s, 6H),
2.39 (s, 3H). MS APCI-Pos, M+1=458.2.
[1012] Compound 367:
##STR00466##
[1013] Prepared according to the general procedure described for
compound 362 using pyridine-2-sulfonyl chloride.
[1014] Pyridine-2-sulfonic acid
3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzylamide.
.sup.1H-NMR (400 MHz, d.sub.6-DMSO): .delta. 9.91 (s, 2H), 8.74 (s,
1H), 8.68 (d, J=4.7 Hz, 1H), 8.17 (s, 1H), 7.99 (m, 2H), 7.86 (d,
J=7.83 Hz, 1H), 7.6 (s, 1H), 6.86 (s, 1H), 6.74 (d, J=8.61 Hz, 1H),
4.1 (s, 2H), 3.80 (s, 3H), 3.32 (s, 1H), 2.38 (s, 3H). MS APCI-Pos,
M+1=428.9.
[1015] Additional preferred compounds of the present invention
include [1016]
N-(2-dimethylamino-1-phenyl-ethyl)-3-methoxy-4-[3-(5-methyl-pyrazi-
n-2-yl)-ureido]-benzamine; [1017]
N-(1-aza-bicyclo[2.2.2]oct-3-yl)-3-methoxy-4-[3-(5-methyl-pyrazin-2-yl)-u-
reido]-benzamide; [1018]
N-(3-R-1-cyclohexylmethyl-pyrrolidin-3-yl)-3-methoxy-4-[3-(5-methyl-pyraz-
in-2-yl)-ureido]-benzamide; [1019]
1-[2-(2-dimethylamino-ethoxy)-5-methyl-phenyl]-3-pyrazin-2-yl-urea;
[1020]
1-[2-(3-dimethylamino-propoxy)-5-methyl-phenyl]-3-(5-methyl-pyrazi-
n-2-yl)-urea; [1021]
1-(5-methyl-pyrazin-2-yl)-3-[5-methyl-2-(pyridin-3-ylmethoxy)-phenyl]-ure-
a; [1022]
1-[2-(2-dimethylamino-1-dimethylaminomethyl-ethoxy)-5-methyl-phe-
nyl]-3-(5-methyl-pyrazin-2-yl)-urea; [1023]
1-[5-methyl-2-(2-S-1-methyl-pyrrolidin-2-ylmethoxy)-phenyl]-3-(5-methyl-p-
yrazin-2-yl)-urea; [1024]
1-{5-methyl-2-[2-(1-methyl-pyrrolidin-2-yl)-ethoxy]-phenyl}-3-(5-methyl-p-
yrazin-2-yl)-urea; [1025]
1-{5-methyl-2-(1-methyl-piperidin-4-yloxy)-phenyl]-3-(5-methyl-pyrazin-2--
yl)-urea; [1026]
1-[5-methyl-2-(3-(S)-1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl--
pyrazin-2-yl)-urea; [1027]
1-[5-methyl-2-(3-(R)-1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl--
pyrazin-2-yl)-urea; [1028]
1-[5-methyl-2-(1-methyl-piperidin-2-ylmethoxy)-phenyl]-3-(5-methyl-pyrazi-
n-2-yl)-urea; [1029]
1-[5-methyl-2-(1-methyl-piperidin-3-yloxy)-phenyl]-3-(5-methyl-pyrazin-2--
yl)-urea; [1030]
1-[5-methyl-2-(1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-quinoxalin-2-yl--
urea; [1031]
1-[5-methyl-2-(piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazin-2-yl)-u-
rea; [1032]
1-[5-fluoro-2-(1-methyl-piperidin-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazi-
n-2-yl)-urea; [1033]
1-[5-fluoro-2-(1-methyl-piperidin-4-yloxy)-phenyl]-3-(5-methyl-pyrazin-2--
yl)-urea; [1034]
1-[4-fluoro-2-(1-methyl-piperidin-4-yloxy)-phenyl]-3-(5-methyl-pyrazin-2--
yl)-urea; [1035]
1-(2-methoxy-4-methylaminomethyl-phenyl)-3-(5-methyl-pyrazin-2-yl)-urea;
[1036]
1-(4-{[(furan-3-ylmethyl)-amino]-methyl}-2-methoxy-phenyl)-3-(5-me-
thyl-pyrazin-2-yl)-urea; and [1037]
1-{2-methoxy-4-[(4-methoxy-benzylamino)-methyl]-phenyl}-3-(5-methyl-pyraz-
in-2-yl)-urea.
Example 15
Identification of Chk1 Inhibitors
[1038] The human Chk1 cDNA was identified and cloned as described
previously in International Application No. PCT/US98/18558, filed
Sep. 4, 1998. A FLAG.RTM. tag was inserted in frame with the amino
terminus of the full-length Chk1. The 5' primer contains an EcoRI
site, a Kozak sequence, and also encodes a FLAG.RTM. tag for
affinity purification using the M2 Antibody (Sigma, Saint Louis,
Ill.). The 3' primer contains a SalI site. The PCR-amplified
fragment was cloned into pCI-Neo as an EcoRI-SalI fragment
(Invitrogen, Carlsbad, Calif.), then subcloned as an EcoRI-NotI
fragment into pFastBacI (Gibco-BRL, Bethesda, Md.). Recombinant
baculovirus was prepared as described in the Gibco-BRL Bac-to-Bac
manual and used to infect Sf-9 cells grown in CCM3 medium (HyClone
Laboratories, Logan, Utah) for expression of FLAG.RTM.-tagged Chk1
protein.
[1039] FLAG.RTM.-tagged Chk1 was purified from frozen pellets of
baculovirus-infected SF9 cells. Frozen cell pellets were mixed with
an equal volume of 2.times. lysis buffer containing 100 mM Tris-HCl
pH 7.5, 200 mM NaCl, 50 mM B-glycerophosphate, 25 mM NaF, 4 mM
MgCl.sub.2, 0.5 mM EGTA, 0.2% TWEEN.RTM.-20, 2 mM sodium vanadate,
2 mM DTT, and a cocktail of protease inhibitors (Complete mini,
Boehringer Mannheim 2000 catalog #1836170). Cells were then dounced
20 times with the loose pestle of a dounce homogenizer and
centrifuged at 48,400.times.g for 1 hour. The M2 affinity was
prewashed with 10 column volumes of 50 mM glycine pH 3.5 followed
by 20 mM Tris pH 7.5, 150 mM NaCl alternating three times and
ending with a Tris NaCl wash. The column was then washed with 25
column volumes of 20 mM Tris pH 7.5, 150 mM NaCl, 0.1%
TWEEN.RTM.-20, 1 mM EGTA, 1 mM EDTA and 1.times. complete mini
protease tablets. The cleared lysate was then bound to M2 affinity
resin in batch at 4.degree. C. for 4 hours. The mixture of resin
and lysate was then poured into a column and the flow through
collected. The resin was washed with 10 column volumes of 20 mM
Tris pH 7.5, 150 mM NaCl, and 3 mM N-octyl glucoside.
FLAG.RTM.-tagged Chk1 was then eluted from the column with 6 column
volumes of cold 20 mM Tris pH 7.5, 150 mM NaCl, 3 mM N-octyl
glucoside containing 0.5 mg/mL FLAG.RTM. peptide (Sigma, 2000
Catalog # F-3290). Three fractions were collected an analyzed for
the presence of FLAG-tagged Chk1.
[1040] The protein kinase was used to developed an assay for Chk1
kinase activity that includes 100 ng purified FLAG.RTM.-Chk1 (150
pmol of ATP/min), 20 .mu.m Cdc25C peptide
(H-leu-tyr-arg-ser-pro-ser-met-pro-glu-asn-leu-asn-arg-arg-arg-arg-OH)
(SEQ ID NO: 1), 400 .mu.m ATP, 2 .mu.Ci [.sup.32P]-gATP, 20 mM
Hepes pH 7.2, 5 mM MgCl.sub.2, 0.1% NP40 and 1 mM DTT. This assay
was used to screen approximately 100,000 small molecule inhibitors.
Reactions were initiated by the addition of ATP-containing reaction
mix and carried out at room temperature for 10 min. Reactions were
stopped by the addition of phosphoric acid (150 mM final
concentration) and transferred to phosphocellulose discs. The
phosphocellulose discs were washed five times with 150 mM
phosphoric acid and air-dried. Scintillation fluid was added and
discs were counted in a Wallac scintillation counter. The screen
identified a number of Chk1 inhibitors having IC.sub.50 values in
the range of 1 to 100 .mu.M.
Example 16
Chk1 Kinase Inhibitors are Selective
[1041] Chk1 inhibitors of the invention were tested for selectivity
as against one or more other protein kinases, i.e., DNA-PK, Cdc2,
Casein Kinase I (CKI), Chk2, p38 MAP kinase, Protein Kinase A
(PKA), and calcium-calmodulin protein kinase II (CaM KII). Assay
procedures for all of these kinases except Chk2 have been
previously described in the literature, including U.S. provisional
patent application 60/229,899, filed Sep. 1, 2000, and U.S. patent
application Ser. No. 08/184,605, filed Jan. 21, 1994, both of which
are herein incorporated by reference. Activity of the compounds
against Chk2 was assayed as follows: 128 ng of purified His-tagged
Chk2 was incubated with up to 100 mM Chk1 inhibitor in the presence
of 4 mM ATP, 1 mCi [32P]g-ATP, 20 mM Hepes pH 7.5, 5 mM MgCl.sub.2,
and 0.25% NP40 for 20 minutes at room temperature. Reactions were
stopped with a final concentration of 150 mM phosphoric acid, and
5/8 of the reaction mixture was transferred to phosphocellulose
discs. The discs were washed five times with 150 mM phosphoric
acid, and air-dried. Scintillant was added and radioactivity was
counted using a Wallac beta counter. p38 MAP kinase, PKA, CaM KII,
and Cdc2 were purchased from New England Biolabs, and assays were
performed according to the manufacturer's instructions using 4-50
.mu.M ATP and testing Chk1 inhibitor concentrations as high as 100
.mu.M. All inhibitors tested were showed at least 5-fold
selectivity for Chk1 over the other enzymes.
Example 17
Chk1 Inhibitors Block Chk1 Function in Cells
[1042] Chk 1 is activated in response to ionizing radiation and
certain chemical DNA damaging agents. In the presence of DNA
damage, Chk1 is activated and causes a cell cycle arrest. In
mammalian cells, the best-characterized cell cycle arrest invoked
by Chk1 is a G2 arrest. Activation of Chk 1 by DNA damage results
in the phosphorylation and inactivation of Cdc25C, the dual
specificity phosphatase that normally dephosphorylates cyclin
B/cdc2 as cells progress into mitosis (Funari et al., Science, Sep.
5, 1997; 277(5331)1495-7; Sanchez et al.; Matsuoka et al.; and
Blasina et al.). This negative regulation of Cdc2 activity causes
cell cycle arrest in order to prevent cells from entering mitosis
in the presence of DNA damage or unreplicated DNA. Inhibition of
Chk1, therefore, allows cells to progress through the cell cycle in
the presence of DNA damage of unreplicated DNA.
[1043] To establish that the Chk1 inhibitors prevented Chk1
function in cells, inhibitors were tested in molecular cell-based
assays. Since mammalian Chk1 has been shown to phosphorylate Cdc25C
in vitro, suggesting that it negatively regulates cyclin B/cdc2 in
response to DNA damage, the ability of the Chk1 inhibitors to
enhance the activity of CyclinB/cdc2 was analyzed. The experiment
was designed as follows: HeLa cells were irradiated with 800 rads
and incubated for 7 hours at 37.degree. C. Because these cells are
functionally p53 negative, they arrest exclusively in G2. Then,
nocodazole was added to a concentration of 0.5 .mu.g/mL and
incubated for 15 hours at 37.degree. C. The addition of nocodazole
was designed to trap any cells that progressed through the G2
arrest into M. Finally, a Chk1 inhibitor was added for 8 hours, the
cells harvested, lysed and immunoprecipitated equal amounts of
protein with an antibody to Cyclin B1 (New England Biolabs) as
suggested by the manufacturer. IPs then were analyzed for
CyclinB-associated cdc2 kinase activity by assaying histone H1
kinase activity (Yu et al., J Biol Chem. Dec. 11,
1998;273(50):33455-64). The results demonstrated that Compound 29
overrides the IR-induced inactivation of Cyclin B/Cdc2.
[1044] In addition, whether the Chk1 inhibitors abrogate the
IR-induced G2 DNA damage checkpoint as assayed by mitotic index
experiments was tested. HeLa cells (approximately 1.times.10.sup.6)
were treated as described above. Cells were harvested by
centrifugation, washed once with PBS, then resuspended in 2.5 mL 75
mM KCl and centrifuged again. The cells then were fixed in 3 mL of
freshly prepared cold, acetic acid: methanol (1:3) and incubated on
ice for 20 minutes. Cells were pelleted, fix solution aspirated and
resuspended in 0.5 mL of PBS. Mitotic spreads were prepared by
pipeting 100 .mu.L of the fixed cells onto a glass microscope slide
and flooding the sample with 1 ml of fix solution. Slides were then
air dried, stained with Wrights stain (Sigma) for 1 minutes,
followed by one wash in water and one wash in 50% methanol. The
presence of condensed chromosomes and lack of nuclear envelope
identified mitotic cells. Both Compounds 12 and 29 showed an
increase in the number of mitotic cells in the presence of
irradiation demonstrating abrogation of the IR-induced G2
arrest.
[1045] Abrogation of the IR-induced G2 checkpoint allows the cells
to continue through the cell cycle, presumably in the presence of
DNA damage, as demonstrated by analysis of DNA content by FACS
profile. 293T cells were treated with 800 rads of ionizing
radiation and increasing concentrations (up to 80 mM) of some of
the Chk1 inhibitors. The cells then were harvested and fixed with 5
mL of cold 70% ethanol at -20.degree. C. overnight. The cells then
were pelleted by centifugation at 1000.times.g for 10 minutes, and
stained with 1 mL of solution containing 50 mg/mL propidium iodide
and 250 mg/mL RNase for 30 minutes at room temperature. Stained
cells were then analyzed by FACS on FL2 using a Becton-Dickinson
apparatus. These experiments demonstrated that, while the cells
treated with radiation and vehicle alone remained arrested in G2,
the Chk1 inhibitor treated cells were distributed in G1 and S
phase. These data, taken together with the data above, suggest that
the Chk1 inhibitors allow cells to continue cycling in the presence
of ionizing radiation.
Example 18
Chk1 Inhibitors Enhance Killing of Cells by Cancer Treatments
[1046] To test the hypothesis that inhibition of Chk1 potentiates
the killing effect of DNA-damaging agents, cells were incubated in
the presence of selective Chk1 inhibitors and either irradiation or
chemical DNA-damaging agents. Cells plated at a density of
1000-2000 per well in 96-well microtitre plates were grown in RMPI
1640 containing 10% FBS, 100 U/mL penicillin and 100 .mu.g/mL
streptomycin for 18 hours at 37.degree. C. in a humidified
incubator with 5% CO.sub.2. Cells tested included HeLa, ACHN,
786-0, HCT116, SW620, HT29, Colo205, SK-MEL-5, SK-MEL-28, A549,
H322, OVCAR-3, SK-OV-3, MDA-MB-231, MCF-7, PC-3, HL-60, K562, and
MOLT4. All cell line designations refer to human cell lines and
refer to the following:
TABLE-US-00008 HeLa cervical adenocarcinoma ACHN renal
adenocarcinoma 786-0 renal adenocarcinoma HCT116 colon carcinoma
SW620 colon carcinoma, lymph node metastasis HT-29 colonrectal
adenocarcinoma Colo205 colon adenocarcinoma SK-MEL-5 melanoma
SK-MEL-28 malignant melanoma A549 lung carcinoma H322
broncholoalveolar carcinoma OVCAR-3 ovarian adenocarcinoma SK-OV-3
ovarian adenocarcinoma MDA-MB- breast adenocarcinoma 231 MCF-7
breast adenocarcinoma PC-3 prostate adenocarcinoma, from metastasis
to bone HL-60 acute promyelocytic leukemia K562 chronic myelogenous
leukemia MOLT4 acute lymphoblastic leukemia; T lymphoblast
[1047] Cells were treated with media containing chemotherapeutic
drugs alone or chemotherapeutic drugs and Compounds 12 and 29.
Cells were incubated for approximately 5 days before growth was
measured by determination of levels of .sup.3H-thymidine uptake.
Chemotherapeutic drugs included etoposide, doxorubicin, cisplatin,
chlorambucil, 5-fluorouracil (5-FU). The drug concentration
necessary to inhibit cell growth to 90% of untreated control cells
was defined as the GL.sub.90. At concentrations less than 100
.mu.M, Compounds 12 and 29 enhanced the killing of 5-FU from 2- to
10-fold.
[1048] Compounds 2 and 12 were tested with additional
antimetabolites, including methotrexate, hydroxyurea,
2-chloroadenosine, fludarabine, azacytidine, and gemcitibine for an
ability to enhance killing of the agents. These Chk1 inhibitors
were found to enhance the killing of cells to hydroxyurea,
fludarabine, 5-azacytidine, and methotrexate suggesting that the
combination of inhibition of Chk1 and blocking of DNA synthesis
leads to increased cell death by these agents.
[1049] In addition, the ability of the Chk1 inhibitor to enhance
killing by irradiation was tested. In HeLa cells, Compounds 12 and
29 were found to enhance killing by irradiation 2-3 fold.
Example 19
Animal Tumor Models
[1050] To test the ability of the Chk1 inhibitors to enhance the
killing of tumors by 5-FU in mice, xenograft tumor models using
colon tumor cell lines were established. Colo205 and HT29 cells
(human colon carcinoma) were used to propagate xenograft tumors in
6-8 week old female thymic Balb/c (nu/nu) mice. Mice were
maintained in a laminar airflow cabinet under pathogen-free
conditions and fed sterile food and water ad libitum. Cell lines
were grown to subconfluence in RPMI 1640 media supplemented with
10% FBS, 100 U/mL penicillin, 100 .mu.g/mL streptomycin, and 1.5 mM
L-glutamine in a 5% CO.sub.2 humidified environment. Single cell
suspensions were prepared in CMF-PBS, and cell concentration
adjusted to 1.times.10.sup.8 cells/mL. Mice were inoculated
subcutaneously (s.c). on the right flank or right leg with a total
of 1.times.10.sup.7 cells (100 .mu.L).
[1051] Mice were randomized (5 mice/group) into four treatment
groups and used when tumors reached a weight of 75-100 mg (usually
7-11 days post-inoculation). Tumors were measured with vernier
calipers and tumor weights were estimated using the empirically
derived formula: tumor weight (mg)=tumor length (mm).times.tumor
width (mm).sup.2/3.3. Treatment consisted of i) 100 .mu.L
intraperitoneal (i.p). injection of 5-FU at 50 mg/kg, 100 mg/kg, or
150 mg/kg. A dose-dependent delay in tumor growth was observed in
the mice treated with 5-FU. Tumor size was monitored every other
day for the duration of the experiment.
[1052] Obviously, many modifications and variations of the
invention as hereinbefore set forth can be made without departing
from the spirit and scope thereof, and, therefore, only such
limitations should be imposed as are indicated by the appended
claims.
* * * * *