TRICYCLIC HETEROARENES, PHARMACEUTICAL COMPOSITIONS CONTAINING THE SAME, AND METHODS OF USING THE SAME
20230142913 · 2023-05-11
Inventors
Cpc classification
C07D221/16
CHEMISTRY; METALLURGY
C07D519/00
CHEMISTRY; METALLURGY
International classification
C07D221/16
CHEMISTRY; METALLURGY
Abstract
Disclosed are compounds and pharmaceutically acceptable salts thereof that may be used in the treatment of subjects in need thereof. The compounds disclosed herein may be inhibitors of tyrosine and threonine-specific cdc2-inhibitory kinase (Myt1). Also disclosed are pharmaceutical compositions containing the compounds or pharmaceutically acceptable salts thereof and methods of their preparation and use.
Claims
1. A compound of formula (IA): ##STR00224## or a pharmaceutically acceptable salt thereof, wherein each is a single or double bond; one, two, or three X groups are N, and the remaining X groups are C; each Y is independently N or C; each Z is independently N or CH; R.sup.1 is OH, and R.sup.3 is hydrogen, optionally substituted C.sub.1-6 alkyl, halogen, or optionally substituted C.sub.3-8 cycloalkyl; or R.sup.1 and R.sup.3 combine to form —CR.sup.9═N—NH—; each R.sup.2 is independently absent, hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, optionally substituted C.sub.3-8 cycloalkyl, optionally substituted C.sub.3-8 cycloalkenyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.2-9 heterocyclyl C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.1-9 heteroaryl C.sub.1-6 alkyl, halogen, cyano, —N(R.sup.7).sub.2, —OR.sup.7, —C(O)N(R.sup.8).sub.2, —SO.sub.2N(R.sup.8).sub.2, —SO.sub.2R.sup.7A, or -Q-R.sup.7B; and R.sup.2A and R.sup.2B together with the atoms to which they are attached, combine to form ring A; or R.sup.2 and R.sup.2A, together with the atoms to which they are attached, combine to form ring A, and R.sup.2B is absent or hydrogen; R.sup.4 is hydrogen, optionally substituted C.sub.1-6 alkyl, halogen, or optionally substituted C.sub.3-8 cycloalkyl; R.sup.5 is hydrogen, halogen, or —N(R.sup.7).sub.2; R.sup.6 is —C(O)NH(R.sup.8), —C(O)R.sup.7A, or —SO.sub.2R.sup.7A; each R.sup.7 is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl C.sub.1-6 alkyl, optionally substituted C.sub.3-8 cycloalkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.1-9 heteroaryl C.sub.1-6 alkyl, or —SO.sub.2R.sup.7A; or two R.sup.7 groups, together with the atom to which both are attached, combine to form an optionally substituted C.sub.2-9 heterocyclyl; each R.sup.7A is independently optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.6-10 aryl; each R.sup.7B is independently hydroxyl, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.1-9 heteroaryl, —N(R.sup.7).sub.2, —C(O)N(R.sup.8).sub.2, —SO.sub.2N(R.sup.8).sub.2, —SO.sub.2R.sup.7A, or optionally substituted alkoxy; each R.sup.8 is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkoxyalkyl, optionally substituted C.sub.6-10 aryl C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.1-9 heteroaryl; or two R.sup.8, together with the atom to which they are attached, combine to form an optionally substituted C.sub.2-9 heterocyclyl; R.sup.9 is hydrogen or halogen; ring A is a 5- or 6-membered carbocyclic ring or a 5- or 6-membered heterocyclic ring, wherein A is optionally substituted with 1, 2, 3, or 4 non-hydrogen R.sup.2 groups; and Q is optionally substituted C.sub.1-6 alkylene, optionally substituted C.sub.2-6 alkenylene, optionally substituted C.sub.2-6 alkynylene, optionally substituted C.sub.3-8 cycloalkylene, optionally substituted C.sub.3-8 cycloalkenylene optionally substituted C.sub.6-10 arylene, optionally substituted C.sub.2-9 heterocyclylene, or optionally substituted C.sub.1-9 heteroarylene; wherein each R.sup.2 is absent, if attached to Y that is N.
2. A compound of formula (I): ##STR00225## or a pharmaceutically acceptable salt thereof, wherein each is a single or double bond; A is a 5- or 6-membered carbocyclic ring or a 5- or 6-membered heterocyclic ring, wherein A is optionally substituted with 1, 2, 3, or 4 non-hydrogen R.sup.2 groups; one, two, or three X groups are N, and the remaining X groups are C; each Y is independently N or C; each Z is independently N or CH; R.sup.1 is OH, and R.sup.3 is hydrogen, optionally substituted C.sub.1-6 alkyl, halogen, or optionally substituted C.sub.3-8 cycloalkyl; or R.sup.1 and R.sup.3 combine to form —CR.sup.9═N—NH—; each R.sup.2 is independently absent, hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, optionally substituted C.sub.3-8 cycloalkyl, optionally substituted C.sub.3-8 cycloalkenyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.2-9 heterocyclyl C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.1-9 heteroaryl C.sub.1-6 alkyl, halogen, cyano, —N(R.sup.7).sub.2, —OR.sup.7, —C(O)N(R.sup.8).sub.2, —SO.sub.2N(R.sup.8).sub.2, —SO.sub.2R.sup.7A, or -Q-R.sup.7B; R.sup.4 is hydrogen, optionally substituted C.sub.1-6 alkyl, halogen, or optionally substituted C.sub.3-8 cycloalkyl; R.sup.5 is hydrogen, halogen, or —N(R.sup.7).sub.2; R.sup.6 is —C(O)NH(R.sup.8), —C(O)R.sup.7A, or —SO.sub.2R.sup.7A; each R.sup.7 is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl C.sub.1-6 alkyl, optionally substituted C.sub.3-8 cycloalkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.1-9 heteroaryl C.sub.1-6 alkyl, or —SO.sub.2R.sup.7A; or two R.sup.7 groups, together with the atom to which both are attached, combine to form an optionally substituted C.sub.2-9 heterocyclyl; each R.sup.7A is independently optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.6-10 aryl; each R.sup.7B is independently hydroxyl, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.1-9 heteroaryl, —N(R.sup.7).sub.2, —C(O)N(R.sup.8).sub.2, —SO.sub.2N(R.sup.8).sub.2, —SO.sub.2R.sup.7A, or optionally substituted alkoxy; each R.sup.8 is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkoxyalkyl, optionally substituted C.sub.6-10 aryl C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.1-9 heteroaryl; or two R.sup.8, together with the atom to which they are attached, combine to form an optionally substituted C.sub.2-9 heterocyclyl; R.sup.9 is hydrogen or halogen; and Q is optionally substituted C.sub.1-6 alkylene, optionally substituted C.sub.2-6 alkenylene, optionally substituted C.sub.2-6 alkynylene, optionally substituted C.sub.3-8 cycloalkylene, optionally substituted C.sub.3-8 cycloalkenylene optionally substituted C.sub.6-10 arylene, optionally substituted C.sub.2-9 heterocyclylene, or optionally substituted C.sub.1-9 heteroarylene; wherein each R.sup.2 is absent, if attached to Y that is N.
3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein one X group is N, and the remaining X groups are C.
4. (canceled)
5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein all Y groups are C, or one or two Y groups are N and the remaining Y groups are C.
6. (canceled)
7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (II): ##STR00226##
8. The compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein the compound is enriched for the atropisomer of formula (II-i): ##STR00227##
9-34. (canceled)
35. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (III): ##STR00228##
36. The compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein ring A is a 5- or 6-membered heteroaryl ring or 5- or 6-membered carbocyclic ring that is optionally substituted with 1, 2, 3, or 4 non-hydrogen R.sup.2 groups.
37. (canceled)
38. The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein Z proximal to R.sup.1 or R.sup.4 is N.
39-41. (canceled)
42. The compound of any one of claim 36, or a pharmaceutically acceptable salt thereof, wherein R.sup.3 is optionally substituted C.sub.1-6 alkyl or halogen.
43-44. (canceled)
45. The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein R.sup.1 and R.sup.3 combine to form —CR.sup.9═N—NH—.
46-47. (canceled)
48. The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein R.sup.4 is optionally substituted C.sub.1-6 alkyl or halogen.
49. (canceled)
50. The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein zero, one, two, three, or four R.sup.2 groups are independently halogen, optionally substituted C.sub.1-6 alkyl, methyl, 3-hydroxypropyl, 2-hydroxyprop-2-yl, methoxycarbonyl, hydroxycarbonyl, aminocarbonyl, N,N-dimethylaminocarbonyl, or N-ethylaminocarbonyl, 2-propenyl, optionally substituted heteroaryl, 1-methylpyrazolyl, pyrazolyl, pyridyl, C.sub.3-8 cycloalkenyl, cyclopentenyl, bromine, chlorine, fluorine, cyano, -Q-R.sup.7B, cyclopropylethynyl, pyrazinylethynyl, 3-(N-morpholinyl)propynyl, 3-hydroxypropynyl, C.sub.6-10 aryl, phenyl, cyanophenyl, [N-(2-hydroxyethyl)-N′-piperazinyl]phenyl, hydrogen, cyano, N-(2-hydroxyethyl)-N′-piperazinyl, or —C(O)N(R.sup.8).sub.2, C.sub.2-9 heterocyclyl, or dihydropyranyl, wherein Q is one of optionally substituted C.sub.2-6 alkynylene, optionally substituted C.sub.6-10 arylene, or phenylene, and R.sup.7B is hydrogen, cyano, or N-(2-hydroxyethyl)-N′-piperazinyl.
51-88. (canceled)
89. A pharmaceutical composition comprising the compound of claim 36, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
90-92. (canceled)
93. A method of inducing cell death in a cancer cell overexpressing CCNE1 or an FBCW7-mutated cancer cell, the method comprising contacting the cell with an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.
94. The method of claim 93, wherein the cancer cell is a uterine cancer, ovarian cancer, breast cancer, stomach cancer, esophageal cancer, lung cancer, or endometrial cancer cell.
95-98. (canceled)
99. The method of claim 94, wherein the cell is in a subject.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0266]
[0267]
[0268]
[0269]
[0270]
[0271]
[0272]
[0273]
[0274]
[0275]
[0276]
[0277]
[0278]
DETAILED DESCRIPTION
[0279] In general, the invention provides compounds, pharmaceutical compositions containing the same, methods of preparing the compounds, and methods of use. Compounds of the invention may be Myt1 inhibitors. These compounds may be used to inhibit Myt1 in a cell, e.g., a cell in a subject (e.g., a cell overexpressing CCNE1 or having an inactivating mutation in the FBXW7 gene). The subject may be in need of a treatment for a disease or condition, e.g., a disease or condition having a symptom of cell hyperproliferation, e.g., a cancer. The Myt1 inhibitory activity of the compounds disclosed herein is useful for treating a subject in need of a treatment for cancer.
[0280] Myt1 is a cell cycle regulating kinase localized predominantly in the endoplasmic reticulum and golgi complex. It is part of the Wee family of kinases that includes Wee1 and Wee1b. It is involved in the negative regulation of the CDK1-Cyclin B complex which promotes the progression of cells from G2-phase into the mitotic phase (M-phase) of the cell cycle. During DNA damage, Myt1 drives the phosphorylation on CDK1 (both Tyr15 and Thr14 of CDK1) which maintains the kinase complex in an inactive state in G2 as part of the G2 checkpoint response along with Wee1 (which mediates only Tyr15 phosphorylation) and prevents entry into mitosis until the damage has been repaired. Additionally, it has been proposed that Myt1 directly interacts with CDK1 complexes in the cytoplasm and prevents their nuclear translocation thus inhibiting cell cycle progression.
[0281] Myt1 has been implicated as a potentially important cancer target as it is essential in many cancer cells. Overexpression of Myt1 has been observed in various cancers including hepatocellular carcinoma as well as clear-cell renal-cell carcinoma. Myt1 downregulation has a minor role in unperturbed cells but has a more prominent role in cells exposed to DNA damage. Additionally, cells that exhibit high levels of replication stress in addition to defective G1 checkpoint regulation may be particularly sensitive to loss of Myt1 function, as these cells will be prone to entering mitosis prematurely with compromised genomic material leading to mitotic catastrophe.
[0282] Inhibitors of Myt1, a regulator of G2-M transition, may be particularly useful in the treatment of tumors harboring CCNE1-amplification or FBXW7 loss-of-function mutations using a synthetic lethal therapeutic strategy.
[0283] Cyclin E1 (encoded by the CCNE1 gene) is involved in the G1 to S phase cell cycle transition. In late G1 phase of the cell cycle, it complexes with cyclin-dependent kinase 2 (CDK2) to promote E2F transcription factor activation and entry into S-phase. Cyclin E1 levels are tightly regulated during normal cell cycles, accumulating at the G1/S transition and being completely degraded by the end of S phase. The cell cycle-dependent proteasomal degradation of Cyclin E1 is mediated by the SCF.sup.FBW7 ubiquitin ligase complex. Once activated in late G1, the Cyclin E1/CDK2 complex promotes the transition into S phase through phosphorylation and inactivation of RB1 and subsequent release of E2F transcription factors. S phase is promoted by E2F-mediated transcription of numerous genes involved in DNA replication including the pre-replication complex subunits ORC1, CDC6, CDT1, and the MCM helicase factors.
[0284] CCNE1 is frequently amplified and/or over-expressed in human cancers (
[0285] Defective cell cycle-regulated proteolysis of Cyclin E1 by the SCF.sup.FBW7 ubiquitin ligase complex is another mechanism of CCNE1 over-expression observed in tumors. The F-box protein gene, FBXW7, is frequently mutated in several cancer types including endometrial, colorectal, and gastric, ranging in frequency from 5-35% (
[0286] Cyclin E1 over-expression and/or FBXW7 loss-of-function is thought to drive tumorigenesis by inducing genome instability (e.g., increased origin firing, defective nucleotide pools, transcription-replication conflicts, and/or fork instability). Over-expression of Cyclin E1 has been shown to induce replication stress characterized by slowed or stalled replication forks and loss-of-heterozygosity at fragile sites. The primary mechanism by which Cyclin E1 over-expression causes replication stress is increased origin firing in early S-phase followed by depletion of replication factors including nucleotide pools. The decrease in overall replication proteins and nucleotides decreases fork progression and causes stalling and subsequent collapse or reversal.
[0287] The compound of the invention may be, e.g., a compound of formula (IA):
##STR00031##
or a pharmaceutically acceptable salt thereof,
wherein
[0288] each is a single or double bond;
[0289] one, two, or three X groups are N, and the remaining X groups are C;
[0290] each Y is independently N or C;
[0291] each Z is independently N or CH;
[0292] R.sup.1 is OH, and R.sup.3 is hydrogen, optionally substituted C.sub.1-6 alkyl, halogen, or optionally substituted C.sub.3-8 cycloalkyl; or R.sup.1 and R.sup.3 combine to form —CR.sup.9═N—NH—;
[0293] each R.sup.2 is independently absent, hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, optionally substituted C.sub.3-8 cycloalkyl, optionally substituted C.sub.3-8 cycloalkenyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.2-9 heterocyclyl C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.1-9 heteroaryl C.sub.1-6 alkyl, halogen, cyano, —N(R.sup.7).sub.2, —OR.sup.7, —C(O)N(R.sup.8).sub.2, —SO.sub.2N(R.sup.8).sub.2, —SO.sub.2R.sup.7A, or -Q-R.sup.1B; and R.sup.2A and R.sup.2B, together with the atoms to which they are attached, combine to form ring A; or R.sup.2 and R.sup.2A, together with the atoms to which they are attached, combine to form ring A, and R.sup.2B is absent or hydrogen;
[0294] R.sup.4 is hydrogen, optionally substituted C.sub.1-6 alkyl, halogen, or optionally substituted C.sub.3-8 cycloalkyl;
[0295] R.sup.5 is hydrogen, halogen, or —N(R.sup.7).sub.2;
[0296] R.sup.6 is —C(O)NH(R.sup.8), —C(O)R.sup.7A, or —SO.sub.2R.sup.7A;
[0297] each R.sup.7 is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl C.sub.1-6 alkyl, optionally substituted C.sub.3-8 cycloalkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.1-9 heteroaryl C.sub.1-6 alkyl, or —SO.sub.2R.sup.7A; or two R.sup.7 groups, together with the atom to which both are attached, combine to form an optionally substituted C.sub.2-9 heterocyclyl;
[0298] each R.sup.7A is independently optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.6-10 aryl;
[0299] each R.sup.7B is independently hydroxyl, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.1-9 heteroaryl, —N(R.sup.7).sub.2, —C(O)N(R.sup.8).sub.2, —SO.sub.2N(R.sup.8).sub.2, —SO.sub.2R.sup.7A, or optionally substituted alkoxy;
[0300] each R.sup.8 is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkoxyalkyl, optionally substituted C.sub.6-10 aryl C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.1-9 heteroaryl; or two R.sup.8, together with the atom to which they are attached, combine to form an optionally substituted C.sub.2-9 heterocyclyl;
[0301] R.sup.9 is hydrogen or halogen;
[0302] ring A is a 5- or 6-membered carbocyclic ring or a 5- or 6-membered heterocyclic ring, wherein A is optionally substituted with 1, 2, 3, or 4 non-hydrogen R.sup.2 groups; and
[0303] Q is optionally substituted C.sub.1-6 alkylene, optionally substituted C.sub.2-6 alkenylene, optionally substituted C.sub.2-6 alkynylene, optionally substituted C.sub.3-8 cycloalkylene, optionally substituted C.sub.3-8 cycloalkenylene optionally substituted C.sub.6-10 arylene, optionally substituted C.sub.2-9 heterocyclylene, or optionally substituted C.sub.1-9 heteroarylene;
[0304] wherein each R.sup.2 is absent, if attached to Y that is N.
[0305] In some embodiments, the compound is a compound of formula (I):
##STR00032##
or a pharmaceutically acceptable salt thereof,
where
[0306] each is a single or double bond;
[0307] A is a 5- or 6-membered carbocyclic ring or a 5- or 6-membered heterocyclic ring, where A is optionally substituted with 1, 2, 3, or 4 non-hydrogen R.sup.2 groups;
[0308] one, two, or three X groups are N, and the remaining X groups are C;
[0309] each Y is independently N or C;
[0310] each Z is independently N or CH;
[0311] R.sup.1 is OH, and R.sup.3 is hydrogen, optionally substituted C.sub.1-6 alkyl, halogen, or optionally substituted C.sub.3-8 cycloalkyl; or R.sup.1 and R.sup.3 combine to form —CR.sup.9═N—NH—;
[0312] each R.sup.2 is independently absent, hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkenyl, optionally substituted C.sub.2-6 alkynyl, optionally substituted C.sub.3-8 cycloalkyl, optionally substituted C.sub.3-8 cycloalkenyl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.2-9 heterocyclyl C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.1-9 heteroaryl C.sub.1-6 alkyl, halogen, cyano, —N(R.sup.7).sub.2, —OR.sup.7, —C(O)N(R.sup.8).sub.2, —SO.sub.2N(R.sup.8).sub.2, —SO.sub.2R.sup.7A, or -Q-R.sup.7B;
[0313] R.sup.4 is hydrogen, optionally substituted C.sub.1-6 alkyl, halogen, or optionally substituted C.sub.3-8 cycloalkyl;
[0314] R.sup.5 is hydrogen, halogen, or —N(R.sup.7).sub.2;
[0315] R.sup.6 is —C(O)NH(R.sup.8), —C(O)R.sup.7A, or —SO.sub.2R.sup.7A;
[0316] each R.sup.7 is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl C.sub.1-6 alkyl, optionally substituted C.sub.3-8 cycloalkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.1-9 heteroaryl C.sub.1-6 alkyl, or —SO.sub.2R.sup.7A; or two R.sup.7 groups, together with the atom to which both are attached, combine to form an optionally substituted C.sub.2-9 heterocyclyl;
[0317] each R.sup.7A is independently optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.6-10 aryl;
[0318] each R.sup.7B is independently hydroxyl, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.2-9 heterocyclyl, optionally substituted C.sub.1-9 heteroaryl, —N(R.sup.7).sub.2, —C(O)N(R.sup.8).sub.2, —SO.sub.2N(R.sup.8).sub.2, —SO.sub.2R.sup.7A, or optionally substituted alkoxy;
[0319] each R.sup.8 is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.2-6 alkoxyalkyl, optionally substituted C.sub.6-10 aryl C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.1-9 heteroaryl; or two R.sup.8, together with the atom to which they are attached, combine to form an optionally substituted C.sub.2-9 heterocyclyl;
[0320] R.sup.9 is hydrogen or halogen; and
[0321] Q is optionally substituted C.sub.1-6 alkylene, optionally substituted C.sub.2-6 alkenylene, optionally substituted C.sub.2-6 alkynylene, optionally substituted C.sub.3-8 cycloalkylene, optionally substituted C.sub.3-8 cycloalkenylene optionally substituted C.sub.6-10 arylene, optionally substituted C.sub.2-9 heterocyclylene, or optionally substituted C.sub.1-9 heteroarylene;
[0322] where each R.sup.2 is absent, if attached to Y that is N.
[0323] The compound of the invention may be, e.g., a compound listed in Table 1 below or a pharmaceutically acceptable salt thereof.
TABLE-US-00001 TABLE 1
[0324] The invention includes (where possible) individual diastereomers, enantiomers, epimers, and atropisomers of the compounds disclosed herein, and mixtures of diastereomers and/or enantiomers thereof including racemic mixtures. Although the specific stereochemistries disclosed herein are preferred, other stereoisomers, including diastereomers, enantiomers, epimers, atropisomers, and mixtures of these may also have utility in treating Myt1-mediated diseases. Inactive or less active diastereoisomers and enantiomers may be useful, e.g., for scientific studies relating to the receptor and the mechanism of activation.
[0325] It is understood that certain molecules can exist in multiple tautomeric forms. This invention includes all tautomers even though only one tautomer may be indicated in the examples.
[0326] The invention also includes pharmaceutically acceptable salts of the compounds, and pharmaceutical compositions comprising the compounds and a pharmaceutically acceptable carrier. The compounds are especially useful, e.g., in certain kinds of cancer and for slowing the progression of cancer once it has developed in a patient.
[0327] The compounds disclosed herein may be used in pharmaceutical compositions comprising (a) the compound(s) or pharmaceutically acceptable salts thereof, and (b) a pharmaceutically acceptable carrier. The compounds may be used in pharmaceutical compositions that include one or more other active pharmaceutical ingredients. The compounds may also be used in pharmaceutical compositions in which the compound disclosed herein or a pharmaceutically acceptable salt thereof is the only active ingredient.
[0328] Optical Isomers—Diastereomers—Geometric Isomers—Tautomers
[0329] Compounds disclosed herein may contain, e.g., one or more stereogenic centers and can occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, and mixtures of diastereomers and/or enantiomers. The invention includes all such isomeric forms of the compounds disclosed herein. It is intended that all possible stereoisomers (e.g., enantiomers and/or diastereomers) in mixtures and as pure or partially purified compounds are included within the scope of this invention (i.e., all possible combinations of the stereogenic centers as pure compounds or in mixtures).
[0330] Some of the compounds described herein may contain bonds with hindered rotation such that two separate rotomers, or atropisomers, may be separated and found to have different biological activity which may be advantageous. It is intended that all of the possible atropisomers are included within the scope of this invention.
[0331] Some of the compounds described herein may contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
[0332] Some of the compounds described herein may exist with different points of attachment of hydrogen, referred to as tautomers. An example is a ketone and its enol form, known as keto-enol tautomers. The individual tautomers as well as mixtures thereof are encompassed by the invention.
[0333] Compounds disclosed herein having one or more asymmetric centers may be separated into diastereoisomers, enantiomers, and the like by methods well known in the art.
[0334] Alternatively, enantiomers and other compounds with chiral centers may be synthesized by stereospecific synthesis using optically pure starting materials and/or reagents of known configuration.
[0335] Metabolites—Prodrugs
[0336] The invention includes therapeutically active metabolites, where the metabolites themselves fall within the scope of the claims. The invention also includes prodrugs, which are compounds that are converted to the claimed compounds as they are being administered to a patient or after they have been administered to a patient. The claimed chemical structures of this application in some cases may themselves be prodrugs.
[0337] Isotopically Enriched Derivatives
[0338] The invention includes molecules which have been isotopically enriched at one or more position within the molecule. Thus, compounds enriched for deuterium fall within the scope of the claims.
Methods of Preparing a Compound of the Invention
[0339] Compounds of the present invention may be prepared using reactions and techniques known in the art and those described herein. One of skill in the art will appreciate that methods of preparing compounds of the invention described herein are non-limiting and that steps within the methods may be interchangeable without affecting the structure of the end product.
Method A
[0340] Compounds of the present invention may be prepared as shown in Scheme A and described herein. One chloro of a 2,3-dichloropyrazine can be substituted by malononitrile under S.sub.NAr or palladium-mediated conditions. The remaining chloro can be substituted with an aromatic amine under S.sub.NAr or palladium-mediated conditions to afford an aminopyrrole. Depending on the nature of the arylamine, a protecting group may be required to be in place prior to this reaction. Hydrolysis of the nitrile can be done under acidic or basic conditions to give compounds of the present invention. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. Fused pyrrolopyrazine core may bear substituent(s) which can be substituted and/or derivatized at any step in the synthesis including at the end. In some cases, a single or major regioisomer is obtained. In other cases, a mixture of regioisomers is separated. In some instances, the regiochemistry is known, in some other instances, a single regioisomer is depicted with two possible configurations. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00143##
Method B
[0341] Compounds of the present invention may be prepared as shown in Scheme B and described herein. One chloro of a 2,3-dichloropyrazine can be substituted by an aromatic amine under S.sub.NAr or palladium-mediated conditions. The remaining chloro can be substituted with malononitrile under S.sub.NAr or palladium-mediated conditions to afford an aminopyrrole. Depending on the nature of the arylamine, a protecting group may be required to be in place prior to this reaction. Hydrolysis of the nitrile can be done under acidic or basic conditions to give compounds of the present invention. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. Fused pyrrolopyrazine core may bear substituent(s) which can be substituted and/or derivatized at any step in the synthesis including at the end. In some cases, a single or major regioisomer is obtained. In other cases, a mixture of regioisomers is separated. In some instances, the regiochemistry is known, in some other instances, a single regioisomer is depicted with two possible configurations. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00144##
Method C
[0342] Compounds of the present invention may be prepared as shown in Scheme C and described herein. Commercially available 3,5-dibromo-6-chloro-pyrazin-2-amine may be condensed with a substituted isothiocyanate to provide a fused thiazolopyrazine. The bromo can be substituted with the anion of malononitrile under palladium catalyzed conditions and the chloro can be substituted with an arylamine to afford the fused pyrrolothiazolopyrazine described herein. Hydrolysis of the nitrile can be done under acidic or basic conditions to give compounds of the present invention. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. The R substituent may be removed or modified at any step in the synthesis including at the end. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00145##
Method D
[0343] Compounds of the present invention may be prepared as shown in Scheme D and described herein. The Bromo of commercially available 3-bromo-2-chloropyridyl ring system can be selectively substituted with an arylamine under palladium catalyzed conditions. The remaining chloro can be substituted with the anion of malononitrile under S.sub.NAr conditions, which cyclizes to a substituted pyrrolopyridyl tricyclic ring system. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base, and/or fluoride to give compounds of the present invention. This deprotection step may be realized before hydrolysis of the nitrile which can be done under acidic or basic conditions to give compounds of the present invention. Fused pyrrolopyridyl tricyclic core may bear substituent(s) which can be substituted and/or derivatized at any step in the synthesis including at the end. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00146##
Method E
[0344] Compounds of the present invention may be prepared as shown in Scheme E and described herein. The 2-halogen atom of 2,3-dihalogenopyrido bicycle can be selectively substituted with an arylamine under SNAr or palladium catalyzed conditions. The remaining halogen can be substituted with malononitrile under palladium-catalyzed conditions to provide the aminopyrrole. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. This deprotection step may be realized before hydrolysis of the nitrile which can be done under acidic or basic conditions to give compounds of the present invention. Fused pyrrolopyridyl tricyclic core may bear substituent(s) which can be substituted and/or derivatized at any step in the synthesis including at the end. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00147##
Method F
[0345] Compounds of the present invention may be prepared as shown in Scheme F and described herein. Using a procedure similar to method B, one chloro of a fused 2,3-dichloropyrazine can be substituted by an aromatic amine under S.sub.NAr or palladium-mediated conditions. The remaining chloro can be substituted with a 2-cyanoacetamide under S.sub.NAr or palladium-mediated conditions to afford an aminopyrrole. Depending on the nature of the arylamine, a protecting group may be required to be in place prior to this reaction. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. Fused pyrrolopyrazine core may bear substituent(s) which can be substituted and/or derivatized at any step in the synthesis including at the end. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00148##
Method G
[0346] Compounds of the present invention may be prepared as shown in Scheme G and described herein. The amino of an aminopyrrole described herein can be substituted by a proton or a chlorine under diazotization conditions. The nitrile can be hydrolyzed to the carboxamide under acidic or basic conditions to give compound of the present invention. Alternatively, the nitrile may be hydrolyzed prior to substitution of the amino group. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. A similar protocol may be applied to the other fused bicycles from the methods to substitute the amino group by a proton or chlorine. Depending on the nature of the N-aryl group, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00149##
Method H
[0347] Compounds of the present invention may be prepared as shown in Scheme H and described herein. The halogen or triflate of a fused tricyclic system constructed via one of the methods described herein and bearing a halogen or triflate substituent at any of the 4 positions on the fused aryl ring may be substituted with a cyano group. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00150##
Method I
[0348] Compounds of the present invention may be prepared as shown in Scheme I and described herein. The halogen or triflate of a fused tricyclic system constructed via one of the methods described herein and bearing a halogen or triflate at any of the 4 positions on the fused aryl ring may be substituted with an alkynyl group by a Sonogashira-type metal mediated coupling reaction with a substituted alkyne. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. The alkyne may optionally be reduced to produce the corresponding substituted alkyl. In other cases, the alkyne substituent may bear a protecting group which may be removed concomitantly or in a separate step. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00151##
Method J
[0349] Compounds of the present invention may be prepared as shown in Scheme J and described herein. The halogen or triflate of a fused tricyclic system constructed via one of the methods described herein and bearing a halogen or triflate at any of the 4 positions on the fused aryl ring may be substituted by a palladium mediated Suzuki-type coupling with a boronic acid or boronate ester. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. In other cases, the new substituent may bear a protecting group which may be removed concomitantly or in a separate step. The new substituent may also contain an insaturation which may optionally be reduced. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00152##
Method K
[0350] Compounds of the present invention may be prepared as shown in Scheme K and described herein. The ester of a fused tricyclic system constructed via one of the methods described herein and bearing a carboxylate at any of the 4 positions on the fused phenyl ring may be hydrolyzed to the corresponding acid and coupled with an amine under amide forming conditions. Depending on the nature of the arylamine, a protecting group may be required to be in place prior to this reaction. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. In other cases, the amine substituent may bear a protecting group which may be removed concomitantly or in a separate step. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00153##
Method L
[0351] Compounds of the present invention may be prepared as shown in Scheme L and described herein. The ester of a fused tricyclic system bearing a carboxylate at any of the 4 positions on the fused phenyl ring may react with a Grignard reagent to provide the corresponding tertiary alcohol. Depending on the nature of the arylamine, a protecting group may be required to be in place prior to this reaction. In the case where the arylamine group bears a protecting group, a deprotection step(s) may be required using acid, base and/or fluoride to give compounds of the present invention. The tertiary alcohol functional group may be dehydrated to the corresponding alkene during the deprotection steps. Depending on the nature of the arylamine, an atropisomeric mixture may be obtained. In such cases it may be necessary to isolate the atropisomer of interest to give compounds of the present invention. Alternatively, an atropisomerically enriched (e.g., pure) intermediate can be isolated and may be derivatized further to give compounds of the present invention.
##STR00154##
Methods of Treatment
[0352] Compounds of the invention may be used for the treatment of a disease or condition (e.g., a cancer overexpressing CCNE1 or having an inactivating mutation in the FBXW7 gene) which depend on the activity of Myt1 (Gene name PKMYT1).
[0353] The disease or condition may have the symptom of cell hyperproliferation. For example, the disease or condition may be a cancer (e.g., a cancer overexpressing CCNE1 or having an inactivating mutation in the FBXW7 gene).
[0354] Cancers which have a high incidence of CCNE1 overexpression include e.g., uterine cancer, ovarian cancer, breast cancer, stomach cancer, esophageal cancer, lung cancer, and endometrial cancer.
[0355] Cancers which have a deficiency in FBXW7 include, e.g., uterine cancer, colorectal cancer, breast cancer, lung cancer, and esophageal cancer.
[0356] A compound of the invention may be administered by a route selected from the group consisting of oral, sublingual, buccal, transdermal, intradermal, intramuscular, parenteral, intravenous, intra-arterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, intranasal, inhalation, intratumoral, and topical administration.
Pharmaceutical Compositions
[0357] The compounds used in the methods described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Pharmaceutical compositions typically include a compound as described herein and a pharmaceutically acceptable excipient. Certain pharmaceutical compositions may include one or more additional pharmaceutically active agents described herein.
[0358] The compounds described herein can also be used in the form of the free base, in the form of salts, zwitterions, solvates, or as prodrugs, or pharmaceutical compositions thereof. All forms are within the scope of the invention. The compounds, salts, zwitterions, solvates, prodrugs, or pharmaceutical compositions thereof, may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds used in the methods described herein may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration, and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
[0359] For human use, a compound of the invention can be administered alone or in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present invention thus can be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries that facilitate processing of a compound of the invention into preparations which can be used pharmaceutically.
[0360] This invention also includes pharmaceutical compositions which can contain one or more pharmaceutically acceptable carriers. In making the pharmaceutical compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, and soft and hard gelatin capsules. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, e.g., preservatives.
[0361] The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005), a well-known reference text in this field, and in the USP/NF (United States Pharmacopeia and the National Formulary). Examples of suitable excipients are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents, e.g., talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents, e.g., methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. Other exemplary excipients are described in Handbook of Pharmaceutical Excipients, 6th Edition, Rowe et al., Eds., Pharmaceutical Press (2009).
[0362] These pharmaceutical compositions can be manufactured in a conventional manner, e.g., by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Methods well known in the art for making formulations are found, for example, in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005), and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York. Proper formulation is dependent upon the route of administration chosen. The formulation and preparation of such compositions is well-known to those skilled in the art of pharmaceutical formulation. In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.
Dosages
[0363] The dosage of the compound used in the methods described herein, or pharmaceutically acceptable salts or prodrugs thereof, or pharmaceutical compositions thereof, can vary depending on many factors, e.g., the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compounds used in the methods described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In general, a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
[0364] A compound of the invention may be administered to the patient in a single dose or in multiple doses. When multiple doses are administered, the doses may be separated from one another by, for example, 1-24 hours, 1-7 days, 1-4 weeks, or 1-12 months. The compound may be administered according to a schedule or the compound may be administered without a predetermined schedule. An active compound may be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per day, every 2nd, 3rd, 4th, 5th, or 6th day, 1, 2, 3, 4, 5, 6, or 7 times per week, 1, 2, 3, 4, 5, or 6 times per month, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per year. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted overtime according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
[0365] While the attending physician ultimately will decide the appropriate amount and dosage regimen, an effective amount of a compound of the invention may be, for example, a total daily dosage of, e.g., between 0.05 mg and 3000 mg of any of the compounds described herein. Alternatively, the dosage amount can be calculated using the body weight of the patient. Such dose ranges may include, for example, between 10-1000 mg (e.g., 50-800 mg). In some embodiments, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg of the compound is administered.
[0366] In the methods of the invention, the time period during which multiple doses of a compound of the invention are administered to a patient can vary. For example, in some embodiments, doses of the compounds of the invention are administered to a patient over a time period that is 1-7 days; 1-12 weeks; or 1-3 months. In some embodiments, the compounds are administered to the patient over a time period that is, for example, 4-11 months or 1-30 years. In some embodiments, the compounds are administered to a patient at the onset of symptoms. In any of these embodiments, the amount of compound that is administered may vary during the time period of administration. When a compound is administered daily, administration may occur, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per day.
[0367] Formulations
[0368] A compound identified as capable of treating any of the conditions described herein, using any of the methods described herein, may be administered to patients or animals with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. The chemical compounds for use in such therapies may be produced and isolated by any standard technique known to those in the field of medicinal chemistry. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the identified compound to patients suffering from a disease or condition. Administration may begin before the patient is symptomatic.
[0369] Exemplary routes of administration of the compounds (e.g., a compound of the invention), or pharmaceutical compositions thereof, used in the present invention include oral, sublingual, buccal, transdermal, intradermal, intramuscular, parenteral, intravenous, intra-arterial, intracranial, subcutaneous, intraorbital, intraventricular, intraspinal, intraperitoneal, intranasal, inhalation, and topical administration. The compounds desirably are administered with a pharmaceutically acceptable carrier. Pharmaceutical formulations of the compounds described herein formulated for treatment of the disorders described herein are also part of the present invention.
[0370] Formulations for Oral Administration
[0371] The pharmaceutical compositions contemplated by the invention include those formulated for oral administration (“oral dosage forms”). Oral dosage forms can be, for example, in the form of tablets, capsules, a liquid solution or suspension, a powder, or liquid or solid crystals, which contain the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.
[0372] Formulations for oral administration may also be presented as chewable tablets, as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules where the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.
[0373] Controlled release compositions for oral use may be constructed to release the active drug by controlling the dissolution and/or the diffusion of the active drug substance. Any of a number of strategies can be pursued in order to obtain controlled release and the targeted plasma concentration versus time profile. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes. In some embodiments, compositions include biodegradable, pH, and/or temperature-sensitive polymer coatings.
[0374] Dissolution or diffusion-controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.
[0375] The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils, e.g., cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
[0376] Formulations for Parenteral Administration
[0377] The compounds described herein for use in the methods of the invention can be administered in a pharmaceutically acceptable parenteral (e.g., intravenous or intramuscular) formulation as described herein. The pharmaceutical formulation may also be administered parenterally (intravenous, intramuscular, subcutaneous or the like) in dosage forms or formulations containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. In particular, formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. For example, to prepare such a composition, the compounds of the invention may be dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. The aqueous formulation may also contain one or more preservatives, for example, methyl, ethyl, or n-propyl p-hydroxybenzoate. Additional information regarding parenteral formulations can be found, for example, in the United States Pharmacopeia-National Formulary (USP-NF), herein incorporated by reference.
[0378] The parenteral formulation can be any of the five general types of preparations identified by the USP-NF as suitable for parenteral administration:
[0379] (1) Drug Injection: a liquid preparation that is a drug substance (e.g., a compound of the invention), or a solution thereof;
[0380] (2) Drug for Injection: the drug substance (e.g., a compound of the invention) as a dry solid that will be combined with the appropriate sterile vehicle for parenteral administration as a drug injection;
[0381] (3) Drug Injectable Emulsion: a liquid preparation of the drug substance (e.g., a compound of the invention) that is dissolved or dispersed in a suitable emulsion medium;
[0382] (4) Drug Injectable Suspension: a liquid preparation of the drug substance (e.g., a compound of the invention) suspended in a suitable liquid medium; and
[0383] (5) Drug for Injectable Suspension: the drug substance (e.g., a compound of the invention) as a dry solid that will be combined with the appropriate sterile vehicle for parenteral administration as a drug injectable suspension.
[0384] Formulations for parenteral administration include solutions of the compound prepared in water suitably mixed with a surfactant, e.g., hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippincott Williams & Wilkins (2005) and in The United States Pharmacopeia: The National Formulary (USP 36 NF31), published in 2013.
[0385] Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols, e.g., polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
[0386] The parenteral formulation can be formulated for prompt release or for sustained/extended release of the compound. Exemplary formulations for parenteral release of the compound include: aqueous solutions, powders for reconstitution, cosolvent solutions, oil/water emulsions, suspensions, oil-based solutions, liposomes, microspheres, and polymeric gels.
Combinations
[0387] Compounds of the present invention may be administered to a subject in combination with one or more additional agents, e.g.: [0388] (a) a cytotoxic agent; [0389] (b) an antimetabolite; [0390] (c) an alkylating agent; [0391] (d) an anthracycline; [0392] (e) an antibiotic; [0393] (f) an anti-mitotic agent; [0394] (g) a hormone therapy; [0395] (h) a signal transduction inhibitor; [0396] (i) a gene expression modulator; [0397] (j) an apoptosis inducer; [0398] (k) an angiogenesis inhibitor; [0399] (l) an immunotherapy agent; [0400] (m) a DNA damage repair inhibitor; [0401] or [0402] a combination thereof.
[0403] The cytotoxic agent may be, e.g., actinomycin-D, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, amphotericin, amsacrine, arsenic trioxide, asparaginase, azacitidine, azathioprine, Bacille Calmette-Guerin (BCG), bendamustine, bexarotene, bevacuzimab, bleomycin, bortezomib, busulphan, capecitabine, carboplatin, carfilzomib, carmustine, cetuximab, cisplatin, chlorambucil, cladribine, clofarabine, colchicine, crisantaspase, cyclophosphamide, cyclosporine, cytarabine, cytochalasin B, dacarbazine, dactinomycin, darbepoetin alfa, dasatinib, daunorubicin, 1-dehydrotestosterone, denileukin, dexamethasone, dexrazoxane, dihydroxy anthracin dione, disulfiram, docetaxel, doxorubicin, emetine, epirubicin, erlotinib, epigallocatechin gallate, epoetin alfa, estramustine, ethidium bromide, etoposide, everolimus, filgrastim, finasunate, floxuridine, fludarabine, flurouracil (5-FU), fulvestrant, ganciclovir, geldanamycin, gemcitabine, glucocorticoids, gramicidin D, histrelin acetate, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib, irinotecan, interferons, interferon alfa-2a, interferon alfa-2b, ixabepilone, lactate dehydrogenase A (LDH-A), lenalidomide, letrozole, leucovorin, levamisole, lidocaine, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, methoxsalen, metoprine, metronidazole, mithramycin, mitomycin-C, mitoxantrone, nandrolone, nelarabine, nilotinib, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, pemetrexed, pentostatin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, procaine, procarbazine, propranolol, puromycin, quinacrine, radicicol, radioactive isotopes, raltitrexed, rapamycin, rasburicase, salinosporamide A, sargramostim, sunitinib, temozolomide, teniposide, tetracaine, 6-thioguanine, thiotepa, topotecan, toremifene, trastuzumab, treosulfan, tretinoin, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, zoledronate, or a combination thereof.
[0404] The antimetabolites may be, e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine, cladribine, pemetrexed, gemcitabine, capecitabine, hydroxyurea, mercaptopurine, fludarabine, pralatrexate, clofarabine, cytarabine, decitabine, floxuridine, nelarabine, trimetrexate, thioguanine, pentostatin, or a combination thereof.
[0405] The alkylating agent may be, e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, altretamine, cyclophosphamide, ifosfamide, hexamethylmelamine, altretamine, procarbazine, dacarbazine, temozolomide, streptozocin, carboplatin, cisplatin, oxaliplatin, uramustine, bendamustine, trabectedin, semustine, or a combination thereof.
[0406] The anthracycline may be, e.g., daunorubicin, doxorubicin, aclarubicin, aldoxorubicin, amrubicin, annamycin, carubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, or a combination thereof.
[0407] The antibiotic may be, e.g., dactinomycin, bleomycin, mithramycin, anthramycin (AMC), ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, piperacillin, pivampicillin, pivmecillinam, ticarcillin, aztreonam, imipenem, doripenem, ertapenem, meropenem, cephalosporins, clarithromycin, dirithromycin, roxithromycin, telithromycin, lincomycin, pristinamycin, quinupristin, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, tobramycin, streptomycin, sulfamethizole, sulfamethoxazole, sulfisoxazole, demeclocycline, minocycline, oxytetracycline, tetracycline, penicillin, amoxicillin, cephalexin, erythromycin, clarithromycin, azithromycin, ciprofloxacin, levofloxacin, ofloxacin, doxycycline, clindamycin, metronidazole, tigecycline, chloramphenicol, metronidazole, tinidazole, nitrofurantoin, vancomycin, teicoplanin, telavancin, linezolid, cycloserine, rifamycins, polymyxin B, bacitracin, viomycin, capreomycin, quinolones, daunorubicin, doxorubicin, 4′-deoxydoxorubicin, epirubicin, idarubicin, plicamycin, mitomycin-c, mitoxantrone, or a combination thereof.
[0408] The anti-mitotic agent may be, e.g., vincristine, vinblastine, vinorelbine, docetaxel, estramustine, ixabepilone, paclitaxel, maytansinoid, a dolastatin, a cryptophycin, or a combination thereof.
[0409] The signal transduction inhibitor may be, e.g., imatinib, trastuzumab, erlotinib, sorafenib, sunitinib, temsirolimus, vemurafenib, lapatinib, bortezomib, cetuximab panitumumab, matuzumab, gefitinib, STI 571, rapamycin, flavopiridol, imatinib mesylate, vatalanib, semaxinib, motesanib, axitinib, afatinib, bosutinib, crizotinib, cabozantinib, dasatinib, entrectinib, pazopanib, lapatinib, vandetanib, or a combination thereof.
[0410] The gene expression modulator may be, e.g., a siRNA, a shRNA, an antisense oligonucleotide, an HDAC inhibitor, or a combination thereof. An HDAC inhibitor may be, e.g., trichostatin A, trapoxin B, valproic acid, vorinostat, belinostat, LAQ824, panobinostat, entinostat, tacedinaline, mocetionstat, givinostat, resminostat, abexinostat, quisinostat, rocilinostat, practinostat, CHR-3996, butyric acid, phenylbutyric acid, 4SC202, romidepsin, sirtinol, cambinol, EX-527, nicotinamide, or a combination thereof. An antisense oligonucleotide may be, e.g., custirsen, apatorsen, AZD9150, trabadersen, EZN-2968, LErafAON-ETU, or a combination thereof. An siRNA may be, e.g., ALN-VSP, CALAA-01, Atu-027, SPC2996, or a combination thereof.
[0411] The hormone therapy may be, e.g., a luteinizing hormone-releasing hormone (LHRH) antagonist. The hormone therapy may be, e.g., firmagon, leuproline, goserelin, buserelin, flutamide, bicalutadmide, ketoconazole, aminoglutethimide, prednisone, hydroxyl-progesterone caproate, medroxy-progesterone acetate, megestrol acetate, diethylstil-bestrol, ethinyl estradiol, tamoxifen, testosterone propionate, fluoxymesterone, flutamide, raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, toremifine citrate, megestrol acetate, exemestane, fadrozole, vorozole, letrozole, anastrozole, nilutamide, tripterelin, histerelin, arbiraterone, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, tretinoin, fenretinide, troxacitabine, or a combination thereof.
[0412] The apoptosis inducers may be, e.g., a recombinant human TNF-related apoptosis-inducing ligand (TRAIL), camptothecin, bortezomib, etoposide, tamoxifen, or a combination thereof.
[0413] The angiogenesis inhibitors may be, e.g., sorafenib, sunitinib, pazopanib, everolimus or a combination thereof.
[0414] The immunotherapy agent may be, e.g., a monoclonal antibody, cancer vaccine (e.g., a dendritic cell (DC) vaccine), oncolytic virus, cytokine, adoptive T cell therapy, Bacille Calmette-Guerin (BCG), GM-CSF, thalidomide, lenalidomide, pomalidomide, imiquimod, or a combination thereof. The monoclonal antibody may be, e.g., anti-CTLA4, anti-PD1, anti-PD-L1, anti-LAG3, anti-KIR, or a combination thereof.
[0415] The monoclonal antibody may be, e.g., alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, trastuzumab, ado-trastuzumab emtansine, blinatumomab, bevacizumab, cetuximab, pertuzumab, panitumumab, ramucirumab, obinutuzumab, ofatumumab, rituximab, pertuzumab, tositumomab, gemtuzumab ozogamicin, tositumomab, or a combination thereof. The cancer vaccine may be, e.g., Sipuleucel-T, BioVaxlD, NeuVax, DCVax, SuVaxM, CIMAvax®, Provenge,®, hsp110 chaperone complex vaccine, CDX-1401, MIS416, CDX-110, GVAX Pancreas, HyperAcute™ Pancreas, GTOP-99 (MyVax®), or Imprime PGG®. The oncolytic virus may be, e.g., talimogene laherparepvec. The cytokine may be, e.g., IL-2, IFNα, or a combination thereof. The adoptive T cell therapy may be, e.g., tisagenlecleucel, axicabtagene ciloleucel, or a combination thereof.
[0416] The DNA damage repair inhibitor may be, e.g., a PARP inhibitor, a cell checkpoint kinase inhibitor, or a combination thereof. The PARP inhibitor may be, e.g., olaparib, rucaparib, veliparib (ABT-888), niraparib (ZL-2306), iniparib (BSI-201), talazoparib (BMN 673), 2X-121, CEP-9722, KU-0059436 (AZD2281), PF-01367338 or a combination thereof. The cell checkpoint kinase inhibitor may be, e.g., MK-1775 or AZD1775, AZD7762, LY2606368, PF-0477736, AZD0156, GDC-0575, ARRY-575, CCT245737, PNT-737 or a combination thereof.
EXAMPLES
[0417] The following examples were meant to illustrate the invention. They were not meant to limit the invention in any way.
[0418] Reactions were typically performed at room temperature (rt or RT) under a nitrogen atmosphere using dry solvents (Sure/Seal™) if not described otherwise in the Examples below. Reactions were monitored by TLC or by injection of a small aliquot on a Waters Acquity-H UPLC® Class system using an Acquity® UPLC HSS C18 2.1×30 mm column eluting with a gradient (1.86 min) of acetonitrile (15% to 98%) in water (both containing 0.1% formic acid). Purifications by preparative HPLC were performed on a Teledyne Isco Combi Flash® EZ Prep system using either Phenomenex Gemini@ 5 μm NX-C18 110 Å 150×21.2 mm column at a flow of 40 mL/min over 12 min (<100 mg or multiple injections of <100 mg) or HP C18 RediSep® Rfgold column (>100 mg) eluting with an appropriate gradient of acetonitrile in water (both containing 0.1% formic acid) unless otherwise specified. The gradient was selected based on the retention time observed by reaction monitoring on the Waters Acquity-H UPLC® Class system (see above). Fractions containing the desired compounds were combined and finally lyophilized. Purifications by silica gel chromatography were performed on a Teledyne Isco Combi Flash® Rf system using RediSep® Rf silica gel columns of appropriate sizes. Purity of final Compounds was assessed by injection of a small aliquot on a Waters Acquity-H UPLC® Class system using an Acquity® UPLC BEH C18 2.1×50 mm column eluting with a gradient (7 min) of acetonitrile (2% to 98%) in water (both containing 0.1% formic acid).
Example 1. Preparation of Compounds
General Procedure 1 for Nitrile Hydrolysis
[0419] The nitrile intermediate was stirred in concentrated H.sub.2SO.sub.4 for an appropriate time to complete nitrile hydrolysis and concomitant removal of acid labile protecting groups such as benzyl, THP and PMB in some cases. The reaction mixture was then quenched with ice water and/or crushed ice and neutralized or made slightly alkaline with aqueous NH.sub.4OH (7 or 14M). The solid formed was collected by filtration and washed with portions of H.sub.2O. It was then air-dried then dried in vacuo. Alternatively, the product was extracted from the aqueous solution using DCM or EtOAc. When a subsequent deprotection step was needed, the crude product was often used as such. In other cases, the crude product was purified by prep HPLC, reverse phase flash chromatography on C18 cartridge or flash chromatography on silica gel as appropriate.
General Procedure 2 for Methoxy Deprotection
[0420] To a solution of the appropriate methoxy phenyl intermediate in DCM was added excess tribromoborane (1M solution in DCM)—typically 3-6 eq. After stirring for the appropriate time at rt (sometime heating was required to complete deprotection), the reaction mixture was concentrated to dryness and coevaporated with DCM/MeOH and or MeOH a few times, then the residue was taken in MeOH, made basic with Et.sub.3N and concentrated again. The residue was then purified by prep HPLC, reverse phase flash chromatography on C18 or flash chromatography on silica gel as appropriate.
##STR00155##
Compound 1 (2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide), Compound 2 ((R)-2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide) and Compound 3 ((S)-2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide)
[0421] Step 1. Malononitrile (6.83 g, 104 mmol) was carefully added by portions with vigorous stirring to a suspension of sodium hydride (60% dispersion in mineral oil, 4.07 g, 106 mmol) in DME (200 mL). After the addition, the stirring was continued for 30 min. at RT and then 2,3-dichloroquinoxaline (10.16 g, 51.1 mmol) was added. The reaction mixture was stirred at RT for 30 min and then refluxed for 1 h. The DME was evaporated and cold aqueous 1M HCl was added to the resulting residue to give a precipitate that was filtered, washed with cold water and a minimum of cold ethanol to afford 2-(3-chloroquinoxalin-2-yl)propanedinitrile (6.70 g, 57% yield) as a yellow solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 7.74-7.67 (m, 2H), 7.63-7.57 (m, 1H), 7.43-7.37 (m, 1H). MS: [M−1]: 227.0.
[0422] Step 2. A solution of 2-(3-chloroquinoxalin-2-yl)propanedinitrile (998 mg, 4.36 mmol) and 3-methoxy-2,6-dimethyl-aniline (arylamine AA1, 2.07 g, 13.7 mmol) in NMP (10 mL) was heated to 150° C. for 1 h. The reaction mixture was cooled down to RT, added dropwise to saturated NaHCO.sub.3, stirred and extracted with EtOAc (3×). The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was adsorbed on silica using DCM and purified by flash chromatography on silica gel (0-100% EtOAc in hexanes). The relevant fractions were combined, concentrated and dried in vacuo to provide 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carbonitrile (758 mg, 51% yield) as a dark orange solid. MS: [M+1]: 344.3.
[0423] Step 3. Following general procedure 1, treatment of 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carbonitrile (130 mg, 0.379 mmol) with H.sub.2SO.sub.4 provided crude 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (135 mg, 99% yield) as an orange/brown solid which was used as such for the next step. MS: [M+1]: 362.2.
[0424] Step 4. Following general procedure 2, treatment of 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (784 mg, 2.17 mmol) with BBr.sub.3 provided 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (Compound 1, 619 mg, 82% yield) as an orange solid, after trituration of the residue in saturated NaHCO.sub.3 and purification of the solid by flash chromatography on silica gel (1-20% MeOH in DCM). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 7.99 (br s, 3H), 7.97-7.90 (m, 1H), 7.81-7.69 (m, 2H), 7.58 (ddd, J=8.4, 6.9, 1.5 Hz, 1H), 7.46 (ddd, J=8.3, 6.9, 1.5 Hz, 1H), 7.37 (br s, 1H), 7.12 (dt, J=8.3, 0.8 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 1.83 (s, 3H), 1.75 (s, 3H). MS: [M+1]: 348.3.
[0425] Chiral SFC separation of 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (619 mg), (Column: Chiral technologies IC, 10×250 mm, 5 μm; Conditions: Isocratic at 30% MeOH with 70% CO.sub.2; Flow Rate: 10 mL/min; outlet pressure 100 bar) provided Compound 2 and Compound 3.
##STR00156##
[0426] Compound 2 from chiral SFC separation of 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide. Peak 1 (7.67 min (chiral SFC), 99.9%). The fractions were concentrated then lyophilized from MeCN/H.sub.2O, providing (R)-2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (222 mg). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 7.99 (br s, 3H), 7.97-7.90 (m, 1H), 7.81-7.69 (m, 2H), 7.58 (ddd, J=8.4, 6.9, 1.5 Hz, 1H), 7.46 (ddd, J=8.3, 6.9, 1.5 Hz, 1H), 7.37 (br s, 1H), 7.12 (dt, J=8.3, 0.8 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 1.83 (s, 3H), 1.75 (s, 3H). MS: [M+1]: 348.3.
##STR00157##
[0427] Compound 3 from chiral SFC separation of 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide. Peak 2 (11.20 min (chiral SFC), 99.7%). The fractions were concentrated then lyophilized from MeCN/H.sub.2O, providing (S)-2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (227 mg). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 7.99 (br s, 3H), 7.97-7.90 (m, 1H), 7.81-7.69 (m, 2H), 7.58 (ddd, J=8.4, 6.9, 1.5 Hz, 1H), 7.46 (ddd, J=8.3, 6.9, 1.5 Hz, 1H), 7.37 (br s, 1H), 7.12 (dt, J=8.3, 0.8 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 1.83 (s, 3H), 1.75 (s, 3H). MS: [M+1]: 348.3.
##STR00158##
Compound 15 (2-amino-1-(2,6-dichloro-3-hydroxy-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide)
[0428] Step 1. To a cold (0° C.) mixture 2,3-dichloroquinoxaline (98 mg, 0.492 mmol) and 2,6-dichloro-3-[(4-methoxyphenyl)methoxy]aniline (arylamine AA7, 292 mg, 0.979 mmol) in THE (4 mL) was added slowly a solution of potassium tert-butoxide in THE (1 M, 1.50 mL). After stirring for 1 h at 0° C., the reaction mixture was quenched with saturated NH.sub.4Cl and diluted with EtOAc and H.sub.2O. The layers were separated and the aqueous layer was extracted with EtOAc (2×). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified twice by flash chromatography on silica gel (0-50% EtOAc in hexanes). The desired fractions were combined, concentrated and dried in vacuo, affording 3-chloro-N-[2,6-dichloro-3-[(4-methoxyphenyl)methoxy]phenyl]quinoxalin-2-amine (109 mg, 48% yield) as a sticky off-white foam. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.88 (ddd, J=8.3, 1.5, 0.6 Hz, 1H), 7.67 (ddd, J=8.4, 1.5, 0.7 Hz, 1H), 7.58 (ddd, J=8.4, 6.9, 1.5 Hz, 1H), 7.49 (ddd, J=8.4, 7.0, 1.5 Hz, 1H), 7.43-7.38 (m, 2H), 7.36 (d, J=9.0 Hz, 1H), 7.12 (s, 1H), 6.99-6.90 (m, 3H), 5.14 (s, 2H), 3.82 (s, 3H). MS: [M+1]: 462.0.
[0429] Step 2. To a MW vial containing sodium hydride (60% dispersion in mineral oil, 15 mg, 0.391 mmol) in dioxane (2 mL), was added a solution of malononitrile (17 mg, 0.26 mmol) in dioxane (0.5 mL). After 20 min, 3-chloro-N-[2,6-dichloro-3-[(4-methoxyphenyl)methoxy]phenyl]quinoxalin-2-amine (59 mg, 0.128 mmol) in dioxane (1 mL) and Pd(PPh.sub.3).sub.4 (15 mg, 0.013 mmol) were added, the reaction mixture was flushed with N.sub.2, the vial was capped and heated to 100° C. for 1 h. The reaction mixture was cooled to RT, quenched with saturated NH.sub.4Cl, extracted with EtOAc (2×). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by flash chromatography on silica gel (0-50% EtOAc in hexanes). The desired fractions were combined, concentrated and dried in vacuo, affording 2-amino-1-[2,6-dichloro-3-[(4-methoxyphenyl)methoxy]phenyl]pyrrolo[3,2-b]quinoxaline-3-carbonitrile (40 mg, 63% yield) as a yellow solid. MS: [M+1]: 490.1.
[0430] Step 3. Following general procedure 1, treatment of 2-amino-1-[2,6-dichloro-3-[(4-methoxyphenyl)methoxy]phenyl]pyrrolo[3,2-b]quinoxaline-3-carbonitrile (71 mg, 0.145 mmol) with H.sub.2SO.sub.4 provided 2-amino-1-(2,6-dichloro-3-hydroxy-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (Compound 15, 29 mg, 52% yield) as a fluffy yellow solid, after precipitation from the reaction mixture and purification by prep HPLC (35-65% MeCN in H.sub.2O, 0.1% formic acid modifier. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.33 (br s, 2H), 7.95 (dd, J=8.3, 1.4 Hz, 1H), 7.79 (dd, J=8.3, 1.4 Hz, 1H), 7.74 (br s, J=3.1 Hz, 1H), 7.60 (ddd, J=8.4, 6.9, 1.5 Hz, 1H), 7.56 (d, J=9.0 Hz, 1H), 7.48 (ddd, J=8.4, 6.9, 1.5 Hz, 1H), 7.41 (br d, J=3.1 Hz, 1H), 7.26 (d, J=9.0 Hz, 1H). MS: [M+1]: 388.1.
##STR00159##
Compound 16 (2,6-diamino-5-(5-hydroxy-2-methylphenyl)-5H-pyrrolo[2,3-b]thiazolo[4,5-e]pyrazine-7-carboxamide)
[0431] Step 1. To a solution of 3,5-dibromo-6-chloro-pyrazin-2-amine (2.87 g, 9.99 mmol) in acetone (50 mL) was added 1-(isothiocyanatomethyl)-4-methoxy-benzene (2.30 g, 12.8 mmol, 2 mL), followed by sodium hydroxide (1.4 g, 35.0 mmol). The mixture was stirred at rt for 1 h, then it was neutralized with acetic acid and diluted with water. The mixture was stirred at rt for 20 min and filtered to collect the solid which was washed with water, dried in vacuo and purified by flash chromatography on silica gel (0-20% EtOAc in DCM) to provide 5-bromo-6-chloro-N-[(4-methoxyphenyl)methyl]thiazolo[4,5-b]pyrazin-2-amine (1.05 g, 27% yield) as an off-white solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 7.27 (d, J=8.7 Hz, 2H), 6.98-6.76 (m, 2H), 4.56 (s, 2H), 3.68 (s, 3H). MS: [M+1]: 385.0.
[0432] Step 2. To a RBF under N.sub.2 containing sodium hydride (60% dispersion in mineral oil, 220 mg, 5.74 mmol) and THE (15 mL) in an ice bath was added malononitrile (172 mg, 2.60 mmol) and the cold bath was removed. After stirring for 30 min, 5-bromo-6-chloro-N-[(4-methoxyphenyl)methyl]thiazolo[4,5-b]pyrazin-2-amine (500 mg, 1.30 mmol) and Pd(PPh.sub.3).sub.4 (150 mg, 0.13 mmol) were added. The mixture was heated to 60° C. for 18 h. 4 g of silica was added, the mixture was evaporated and the residue was purified by flash chromatography on silica gel (0-15% MeOH in DCM) to provide 2-[6-chloro-2-[(4-methoxyphenyl)methylamino]thiazolo[4,5-b]pyrazin-5-yl]propanedinitrile (325 mg, 68% yield) as a yellow solid. MS: [M+1]: 371.1.
[0433] Step 3. Potassium tert-butoxide (182 mg, 1.62 mmol) was added to a solution of 2-[6-chloro-2-[(4-methoxyphenyl)methylamino]thiazolo[4,5-b]pyrazin-5-yl]propanedinitrile (500 mg, 1.35 mmol), 5-methoxy-2-methyl-aniline (225 mg, 1.64 mmol), Pd.sub.2dba.sub.3 (105 mg, 0.115 mmol), and Xantphos (133 mg, 0.230 mmol) in toluene (10 mL). The mixture was heated to reflux for 1 h. The solvent was evaporated and the brown residue was purified by flash chromatography on silica gel (0-20% EtOAc in hexanes) to provide 6-amino-5-(5-methoxy-2-methylphenyl)-2-((4-methoxybenzyl)amino)-5H-pyrrolo[2,3-b]thiazolo[4,5-e]pyrazine-7-carbonitrile (430 mg, 68% yield). MS: [M+1]: 472.1.
[0434] Step 4. Following general procedure 1, 6-amino-5-(5-methoxy-2-methylphenyl)-2-((4-methoxybenzyl)amino)-5H-pyrrolo[2,3-b]thiazolo[4,5-e]pyrazine-7-carbonitrile (550 mg, 1.17 mmol) was treated with H.sub.2SO.sub.4 to provide crude 2,6-diamino-5-(5-methoxy-2-methylphenyl)-5H-pyrrolo[2,3-b]thiazolo[4,5-e]pyrazine-7-carboxamide (405 mg, 94% yield) as a fluffy yellow solid after precipitation from the reaction mixture. MS: [M+1]: 370.2.
[0435] Step 5. Following general procedure 2, 2,6-diamino-5-(5-methoxy-2-methylphenyl)-5H-pyrrolo[2,3-b]thiazolo[4,5-e]pyrazine-7-carboxamide (405 mg, 1.10 mmol) was treated with BBr.sub.3, to provide 2,6-diamino-5-(5-hydroxy-2-methylphenyl)-5H-pyrrolo[2,3-b]thiazolo[4,5-e]pyrazine-7-carboxamide (Compound 16, 26 mg, 7% yield) as a light yellow solid, after purification by prep HPLC (20-60% MeCN in H.sub.2O, 0.1% formic acid modifier). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 7.72 (s, 2H), 7.24 (dd, J=8.3, 0.8 Hz, 1H), 7.11 (s, 4H), 6.87 (dd, J=8.3, 2.6 Hz, 1H), 6.68 (d, J=2.5 Hz, 1H), 1.80 (s, 3H). MS: [M+1]: 356.1.
##STR00160##
Compound 17 (2-amino-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[3,2-b]quinoline-3-carbonitrile)
[0436] Step 1. In a MW vial, 3-bromo-2-chloroquinoline (0.5 g, 2.066 mmol) and 5-(methoxymethoxy)-2-methylaniline (arylamine AA8, 0.24 g 1.445 mmol) was dissolved in toluene (10 mL) at room temperature followed by the addition of sodium tert-butoxide (0.23 g, 2.48 mmol). The reaction mixture was purged with N.sub.2 gas, Pd.sub.2(dba).sub.3 (0.15 g, 0.21 mmol) and Xantphos (0.12 g, 0.21 mmol) were added and again reaction mixture was purged with N.sub.2 gas again, then submitted to microwave irradiation for 20 min at 110° C. The reaction mixture was quenched in water and extracted with EtOAc (3×). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum to get crude product which was purified by flash chromatography on silica gel (5% EtOAc in hexanes). The pure product fractions were collected and evaporated to provide 2-chloro-N-(5-(methoxymethoxy)-2-methylphenyl)quinolin-3-amine (0.18 g, 27% yield).
[0437] Step 2. In a MW vial, malononitrile (54 mg, 0.82 mmol) was dissolved in DME (10 mL) at 0° C. followed by the addition of potassium tert-butoxide (0.36 g, 3.3 mmol). The reaction mixture was stirred at same temperature for 30 min, after which 2-chloro-N-(5-(methoxymethoxy)-2-methylphenyl)quinolin-3-amine (0.18 g, 0.55 mmol) was added and reaction mixture was submitted to microwave irradiation for 3 h at 150° C. The reaction mixture was quenched in water and extracted with EtOAc (3×). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum to get crude product which was purified by flash chromatography on silica gel (70% EtOAc in hexanes). The pure product fractions were collected and evaporated to get of 2-amino-1-(5-(methoxymethoxy)-2-methylphenyl)-1H-pyrrolo[3,2-b]quinoline-3-carbonitrile (50 mg, 25% yield).
[0438] Step 3. 2-amino-1-(5-(methoxymethoxy)-2-methylphenyl)-1H-pyrrolo[3,2-b]quinoline-3-carbonitrile (50 mg, 0.14 mmol) was stirred in 4M HCl in dioxane (2 mL) for 3 h at room temperature. The reaction mixture was concentrated under vacuum to get the crude product which was triturated with n-pentane to obtain 2-amino-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[3,2-b]quinoline-3-carbonitrile (HCl salt, 50 mg), which was used in next step without further purification.
[0439] Step 4. Following general procedure 1, treatment of 2-amino-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[3,2-b]quinoline-3-carbonitrile (50 mg, 0.16 mmol) in H.sub.2SO.sub.4 at 0° C. provided 2-amino-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[3,2-b]quinoline-3-carbonitrile (Compound 17, 3 mg, 6% yield) after aqueous work-up and purification by prep HPLC (SUNFIRE C18 250×19 mm, 5 μm, 0-100% MeCN in H.sub.2O, 0.1% TFA modifier). .sup.1H NMR (500 MHz, DMSO-d6) δ 9.80 (s, 1H), 8.29 (d, J=3.7 Hz, 1H), 7.90 (d, J=8.5 Hz, 1H), 7.85 (dd, J=8.1, 1.4 Hz, 1H), 7.52 (br s+ddd, J=8.4, 6.8, 1.5 Hz, 3H), 7.36-7.31 (m, 2H), 7.29 (ddd, J=8.1, 6.8, 1.3 Hz, 1H), 7.18 (d, J=3.5 Hz, 1H), 6.96 (dd, J=8.4, 2.6 Hz, 1H), 6.79 (d, J=2.5 Hz, 1H), 1.87 (s, 3H). MS: [M+1]: 333.3.
##STR00161##
Compound 18 (2-amino-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoline-3-carboxamide)
[0440] Step 1. In a MW vial, 3-bromo-2-chloroquinoline (0.2 g, 0.82 mmol) and 5-(methoxymethoxy)-2-methylaniline (arylamine AA8, 0.166 g, 0.99 mmol) was dissolved in toluene (2 mL) at room temperature followed by the addition of sodium tert-butoxide (94 mg, 0.98 mmol). The reaction mixture was purged with N.sub.2 gas, Pd(OAc).sub.2 (28 mg, 0.04 mmol) and Xantphos (48 mg, 0.099 mmol) were added, purged with N.sub.2 gas again, then submitted to microwave irradiation for 3 h at 85° C. The reaction mixture was filtered and the filtrate was concentrated under vacuum to get crude product, which was purified by flash chromatography on silica gel (0-2% EtOAc in hexanes). The pure product fractions were collected and evaporated to provide 3-bromo-N-(5-(methoxymethoxy)-2-methylphenyl)quinolin-2-amine (0.49 g, 16% yield).
[0441] Step 2. In a MW vial, malononitrile (14 mg 0.21 mmol) was dissolved in THE (3 mL) at 0° C. followed by the addition of sodium hydride (60% wt dispersion in mineral oil, 17 mg, 0.42 mmol). The reaction mixture was stirred at same temperature for 30 min, after which 3-bromo-N-(5-(methoxymethoxy)-2-methylphenyl)quinolin-2-amine (0.04 g, 0.10 mmol) and Pd(PPh.sub.3).sub.4 (12 mg, 0.010 mmol) were added and the reaction mixture was submitted to microwave irradiation for 4 h at 80° C. The reaction mixture was diluted with water and extracted with EtOAc (3×). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum to get crude product which was purified by flash chromatography on silica gel (20-50% EtOAc in hexanes). The pure product fractions were collected and evaporated to get 2-amino-1-(5-(methoxymethoxy)-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoline-3-carbonitrile (39 mg, 25% yield).
[0442] Step 3. 2-amino-1-(5-(methoxymethoxy)-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoline-3-carbonitrile (0.038 g, 0.10 mmol) was stirred in 3M HCl in MeOH (2 mL) for 3 h at rt. The reaction mixture was concentrated under vacuum to get the crude product which was triturated with n-pentane to obtain 2-amino-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoline-3-carbonitrile (HCl salt, 32 mg, 90% yield) which was used for next step without further purification.
[0443] Step 4. Following general procedure 1, treatment of 2-amino-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoline-3-carbonitrile (0.032 g, 0.10 mmol) in H.sub.2SO.sub.4 at 0° C. provided 2-amino-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoline-3-carboxamide (Compound 18, 6 mg, 17% yield) after aqueous work-up, purification by flash chromatography on silica gel (20-50% EtOAc in hexanes) and purification by prep HPLC (X-select Phenyl Hexyl (250×19) 5 μm, 0-100% MeCN in H.sub.2O, 0.1% TFA modifier). .sup.1H NMR (500 MHz, DMSO-d6) δ 9.65 (s, 1H), 8.44 (s, 1H), 7.85 (dd, J=8.1, 1.5 Hz, 1H), 7.70 (d, J=8.3 Hz, 1H), 7.45 (br s, 2H), 7.45-7.41 (m, 1H), 7.38 (ddd, J=8.1, 6.7, 1.4 Hz, 1H), 7.29 (d, J=8.4 Hz, 1H), 6.92 (dd, J=8.4, 2.5 Hz, 1H), 6.86 (br s, 2H), 6.73 (d, J=2.6 Hz, 1H), 1.84 (s, 3H). MS: [M+1]: 333.2.
##STR00162##
Compound 20 (2-amino-N-cyclopropyl-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carboxamide)
[0444] Step 1. In a sealed tube, to a solution of 2,3-dichloroquinoxaline (3 g, 15 mmol) and 5-(methoxymethoxy)-2-methylaniline (arylamine AA8, 1.76 g, 10.55 mmol) in toluene (25 mL) was added sodium tert-butoxide (1.73 g, 18.1 mmol). The reaction mixture was purged with N.sub.2 gas for 15 min, then Pd.sub.2(dba).sub.3 (0.41 g, 0.45 mmol) and Xantphos (0.8 g, 1.5 mmol) were added. Then the reaction mixture was stirred at 110° C. for 16 h. After cooling down to rt, he reaction mixture was poured in water (90 mL) and extracted with EtOAc (3×). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum to get the crude product which was purified by flash chromatography on silica gel (15% EtOAc in hexanes) The pure product fractions were collected and concentrated to get 3-chloro-N-(5-(methoxymethoxy)-2-methylphenyl) quinoxalin-2-amine (0.23 g, 4.63% yield). MS: [M+1]: 330.24.
[0445] Step 2. A MW vial containing 3-chloro-N-(5-(methoxymethoxy)-2-methylphenyl)quinoxalin-2-amine (0.15 g, 0.45 mmol), 2-cyano-N-cyclopropylacetamide (0.084 g 0.68 mmol) and Cs.sub.2CO.sub.3 (0.741 g, 2.27 mmol) in DMF (10 mL) was submitted to microwave irradiation for 1 h at 110° C. The reaction mixture was diluted with water and extracted with EtOAc (3×). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum to get crude product which was purified by flash chromatography on silica gel (20% EtOAc in hexanes). The pure product fractions were collected and evaporated to get 2-amino-N-cyclopropyl-1-(5-(methoxymethoxy)-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carboxamide (0.1 g, 52% yield). MS: [M+1]: 418.34.
[0446] Step 3. To a cold (0° C.) solution of 2-amino-N-cyclopropyl-1-(5-(methoxymethoxy)-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carboxamide (0.1 g 0.2395 mmol in 1,4-dioxane (1 mL) at 0° C. was HCl in 1,4-dioxane (4M, 2 mL). The reaction mixture was stirred at rt for 30 min and the reaction mixture was concentrated under vacuum to get the crude product which was purified trituration with diethyl ether to get 2-amino-N-cyclopropyl-1-(5-hydroxy-2-methylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carboxamide (Compound 20, 55 mg, 68% yield). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.75 (bs, 1H), 8.35 (s, 1H), 8.18 (bs, 1H), 8.00 (d, J=7.6 Hz, 1H), 7.80 (d, J=7.6 Hz, 1H), 7.60 (t, J=7.2 Hz, 1H), 7.49 (t, J=7.2 Hz, 1H), 7.31 (d, J=8.4 Hz, 1H), 6.96 (d, J=8.0 Hz, 1H), 6.83 (s, 1H), 2.88 (s, 1H), 1.89 (s, 3H), 0.82 (d, J=6.0 Hz, 2H), 0.67 (bs, 2H). MS: [M+1]: 374.32.
##STR00163##
Compound 24 (1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide)
[0447] Step 1. To a solution of 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carbonitrile from Compound 1 step 2 (400 mg, 1.16 mmol) in THE (10 mL) was added tert-butyl nitrite (599 mg, 5.81 mmol). The reaction mixture was stirred at RT for 30 min then refluxed for 4.75 h, cooled down to room temperature and concentrated to dryness. The residue was purified by flash chromatography on silica (0-100% EtOAc in hexanes), providing 1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carbonitrile (367 mg, 96% yield) as a light orange solid. MS: [M+1]: 329.3.
[0448] Step 2. Following general procedure 1, treatment of 1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carbonitrile (367 mg, 1.12 mmol) with H.sub.2SO.sub.4 provided crude 1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (356 mg, 92% yield) as a beige solid which was used as such for the next step. MS: [M+1]: 347.3.
[0449] Step 3. Following general procedure 2, deprotection of 1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (356 mg, 1.03 mmol) afforded 1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (Compound 24, 189 mg, 55% yield) as a beige fluffy solid, after purification by reverse phase flash chromatography on C18 cartridge (10-100% MeCN in H.sub.2O, 0.1% formic acid modifier). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.67 (s, 1H), 8.82 (s, 1H), 8.37-8.24 (m, 1H), 8.11-8.04 (m, 2H), 7.91-7.73 (m, 3H), 7.11 (dt, J=8.3, 0.7 Hz, 1H), 6.97 (d, J=8.3 Hz, 1H), 1.81 (s, 3H), 1.71 (s, 3H). MS: [M+1]: 333.4.
##STR00164##
Compound 29 (2-amino-5-bromo-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide) and Compound 59 (2-amino-8-bromo-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide)
[0450] Step 1. Malononitrile (16.8 g, 254 mmol) was added portionwise to a vigorously stirred suspension of sodium hydride (60% dispersion in mineral oil, 10.3 g, 269 mmol) in DME (600 mL). After the addition, the stirring was continued for 30 min and then 5-bromo-2,3-dichloro-quinoxaline (intermediate R, 35.4 g, 127 mmol) was added (a small exotherm was observed). The reaction mixture was stirred at RT for 15 min and then heated under reflux for 4 h. The DME was evaporated and the resulting residue was poured by portion in cold aqueous 1M HCl to give a yellow precipitate that was filtered and washed with water to afford a mixture of 2-(5-bromo-3-chloro-quinoxalin-2-yl)propanedinitrile and 2-(8-bromo-3-chloro-quinoxalin-2-yl)propanedinitrile (36 g, 92% yield) (in about 1:1 ratio estimated by UPLCMS) as a yellow solid. MS: [M−1]: 306.9.
[0451] Step 2. 3-methoxy-2,6-dimethyl-aniline (arylamine AA1, 7.4 g, 48.9 mmol) was added to a mixture of 2-(5-bromo-3-chloro-quinoxalin-2-yl)propanedinitrile and 2-(8-bromo-3-chloro-quinoxalin-2-yl)propanedinitrile (5.00 g, 16.3 mmol) in NMP (50 mL). The reaction mixture was heated to 130° C. for 6 h, cooled, and the mixture was poured into vigorously stirring aqueous NaHCO.sub.3 sat. The precipitate was collected by filtration, washed with water and dried by codistillation with toluene twice. The brown residue was taken in 250 mL of 15% MeOH in CH.sub.2Cl.sub.2 and 25 g of silica gel was added. The mixture was evaporated and the brown residue was purified by silica gel chromatography (dry load) using a gradient of 20 to 60% EtOAc in hexanes to provide a mixture of 2-amino-8-bromo-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carbonitrile and 2-amino-5-bromo-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carbonitrile (5.0 g, 73% yield) as an orange solid. MS: [M+1]: 424.1.
[0452] Step 3. Following general procedure 1, treatment of 2-amino-8-bromo-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carbonitrile and 2-amino-5-bromo-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carbonitrile (5.0 g, 11.8 mmol) with H.sub.2SO.sub.4 provided a crude mixture of 2-amino-8-bromo-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide and 2-amino-5-bromo-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide (5.2 g, quantitative yield) as a yellow solid, after precipitation from the reaction mixture. MS: [M+1]: 442.0.
[0453] Step 4. Following general procedure 2, deprotection of a mixture of 2-amino-8-bromo-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide and 2-amino-5-bromo-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide (2.4 g, 5.45 mmol) led to a mixture of 2-amino-8-bromo-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (1.8 g, 56% yield) as a red solid after purification by flash chromatography on silica gel (0-10% MeOH in DCM). The regiomers in the mixture were separated by SFC (Column: ZymorSPHER HA-Dipyridyl, 30×150 mm, 5 μm; Conditions: Isocratic at 50% MeOH+0.1% Formic Acid with 50% CO.sub.2; Flow Rate: 70 mL/min; outlet pressure 100 bar) providing 2-amino-5-bromo-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide (P1, Compound 29, RT 4.33 min, 550 mg, 46% yield).sup.1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.12 (m, 2H), 7.89 (dd, J=7.6, 1.3 Hz, 1H), 7.82 (d, J=3.3 Hz, 1H), 7.77 (dd, J=8.3, 1.3 Hz, 1H), 7.48 (d, J=3.3 Hz, 1H), 7.33 (dd, J=8.3, 7.6 Hz, 1H), 7.08 (dt, J=8.3, 0.8 Hz, 1H), 6.94 (d, J=8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). MS: [M+1]: 428.0; and 2-amino-8-bromo-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (P2, Compound 59, RT 4.99 min, 250 mg, 22% yield). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 8.12 (s, 2H), 7.96 (dd, J=8.3, 1.3 Hz, 1H), 7.80 (dd, J=7.6, 1.3 Hz, 1H), 7.70 (d, J=3.1 Hz, 1H), 7.49 (dd, J=8.3, 7.6 Hz, 1H), 7.44 (d, J=3.2 Hz, 1H), 7.13 (dt, J=8.3, 0.8 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 1.86 (d, J=0.7 Hz, 3H), 1.78 (s, 3H). MS: [M+1]: 428.0. The regiochemistry was confirmed by Xray structure of Compound 29.
##STR00165##
Compound 32 (2-amino-5-cyano-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide)
[0454] Copper (I) cyanide (32 mg, 0.36 mmol) was added to a solution of 2-amino-5-bromo-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide (Compound 29, 50 mg, 0.117 mmol) in DMF (1 mL) and the suspension was stirred at 80° C. for 8 h. The mixture was filtered through a 0.45 micron PTFE filter and purified by preparative HPLC (30-80% MeCN in H.sub.2O, 0.1% formic acid modifier). The recovered tubes were combined and lyophilized to provide 2-amino-5-cyano-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide (Compound 32, 12 mg, 26% yield, 95% purity) as a pale yellow solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 8.31 (s, 2H), 8.18-8.02 (m, 2H), 7.69 (d, J=3.1 Hz, 1H), 7.55 (d, J=6.7 Hz, 1H), 7.51 (dd, J=8.4, 7.3 Hz, 1H), 7.08 (d, J=8.3 Hz, 1H), 6.95 (d, J=8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). MS: [M+1]: 373.2.
##STR00166##
Compound 45 (2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-(3-hydroxyprop-1-ynyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide)
[0455] Diisopropylethylamine (163 mg, 1.26 mmol, 220 μL) was added to a mixture of 2-amino-6-bromo-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide (Compound 38, 100 mg, 0.235 mmol), prop-2-yn-1-ol (31 mg, 0.55 mmol, 32 μL), Copper (I) iodide (12 mg, 0.063 mmol) and Pd(PPh.sub.3).sub.4 (56 mg, 0.048 mmol) in acetonitrile (1.5 mL). The mixture was heated to 80° C. for 30 min. After cooling down to RT, the mixture was filtered on a 0.45 micron PTFE filter and the filtrate was purified by preparative HPLC (30-60% MeCN in H.sub.2O, 0.1% formic acid modifier) The recovered tubes were combined and lyophilized to provide 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-(3-hydroxyprop-1-ynyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide (Compound 45, 16 mg, 17% yield) as a pale yellow solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.06 (s, 2H), 7.93 (d, J=1.8 Hz, 1H), 7.70 (d, J=8.5 Hz, 1H), 7.66 (d, J=3.2 Hz, 1H), 7.44-7.33 (m, 2H), 7.08 (d, J=8.3 Hz, 1H), 6.94 (d, J=8.3 Hz, 1H), 4.31 (s, 2H), 1.79 (s, 3H), 1.71 (s, 3H). MS: [M+1]: 402.4.
##STR00167##
Compound 46 (2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-(3-hydroxypropyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide)
[0456] A mixture of 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-(3-hydroxyprop-1-ynyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide (Compound 45, 5 mg, 0.012 mmol) and palladium on carbon (10% w/w, 5 mg) in EtOH (2 mL) was stirred under H.sub.2 atmosphere (balloon) for 2 h, the mixture was filtered on a 0.45 micron PTFE filter and the filtrate was evaporated to provide 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-(3-hydroxypropyl)pyrrolo[2,3-b]quinoxaline-3-carboxamide (Compound 46, 2 mg, 34% yield) as a pale yellow solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.22-7.69 (m, 5H), 7.64 (dd, J=8.4, 1.5 Hz, 1H), 7.40-7.23 (m, 2H), 7.07 (d, J=8.3 Hz, 1H), 6.94 (dd, J=8.3, 1.4 Hz, 1H), 3.41 (t, J=6.4 Hz, 4H), 2.76 (t, J=7.6 Hz, 3H), 1.78 (d, J=6.1 Hz, 5H), 1.71 (d, J=1.5 Hz, 3H). MS: [M+1]: 406.2.
##STR00168##
Compound 49 (2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-7-(3-pyridyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide)
[0457] Pd(dppf)Cl.sub.2.DCM (18 mg, 0.023 mmol) was added to a mixture of 2-amino-7-bromo-1-(3-hydroxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (Compound 38, 100 mg, 0.235 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (96 mg, 0.468 mmol), and aqueous K.sub.2CO.sub.3 (2 M, 350 μL) in DMF (0.8 mL). The mixture was heated to 80° C. for 2 h. After cooling to rt, the mixture was filtered on a 0.45 micron PTFE filter and purified by prep HPLC (20-60% MeCN in H.sub.2O, 0.1% formic acid modifier). The recovered tubes were combined and lyophilized to provide 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-7-(3-pyridyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (Compound 49, 25 mg, 25% yield) as a pale yellow solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 9.03 (dd, J=2.4, 0.8 Hz, 1H), 8.56 (dd, J=4.7, 1.6 Hz, 1H), 8.27 (dd, J=2.0, 0.6 Hz, 1H), 8.22 (m, 1H), 8.12-7.91 (m, 2H), 7.89-7.78 (m, 2H), 7.76 (d, J=3.0 Hz, 1H), 7.48 m, 1H), 7.45-7.35 (m, 1H), 7.09 (dt, J=8.3, 0.7 Hz, 1H), 6.96 (d, J=8.3 Hz, 1H), 1.81 (d, J=0.7 Hz, 3H), 1.73 (s, 3H). MS: [M+1]: 425.3.
##STR00169##
Compound 66 (2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-7-(morpholine-4-carbonyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide or 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-(morpholine-4-carbonyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide)
[0458] ##STR00170## ##STR00171##
[0459] Steps 1, 2, 3. Applying the steps outlined in Method A and using Intermediate Y and arylamine AA1 and following general procedure 1 for hydrolysis of the nitrile, a single regioisomer of unknown regiochemistry which can be either 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-7-carboxylate or 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-6-carboxylate was obtained as a yellow solid (Ester Intermediate 1, 889 mg). .sup.1H NMR (400 MHz, DMSO-d6) δ 8.26 (dd, J=1.9, 0.6 Hz, 1H), 8.02 (dd, J=8.7, 1.9 Hz, 1H), 7.96 (dd, J=8.7, 0.5 Hz, 1H), 7.70 (br s, 1H), 7.45 (br s, 1H), 7.27 (d, J=7.7 Hz, 1H), 7.14 (d, J=8.5 Hz, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 1.84 (s, 3H), 1.76 (s, 3H). MS: [M+1]: 420.2.
[0460] Step 4. To a suspension of 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-7-carboxylate or 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-6-carboxylate (Ester Intermediate 1, 443 mg, 1.06 mmol) in MeOH (6 mL) and THE (9 mL) was added aqueous LiOH (1 M, 3.2 mL) and the mixture was stirred overnight, more aqueous LiOH (1 M, 1 mL) was added and the reaction was allowed to continue for another 5 days. The reaction mixture was concentrated to remove volatiles, acidified to pH 4 with 1N HCl and the solid formed was collected by filtration, washed with H.sub.2O, air-dried then dried in vacuo, affording crude product as an orange-brown solid (413 mg, 96% yield). The product consists of a single regioisomer that is 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-7-carboxylic acid or 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-6-carboxylic acid (Acid Intermediate 1). MS: [M+1]: 406.4.
[0461] Step 5. To a vial charged with 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-7-carboxylic acid or 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-6-carboxylic acid (Acid Intermediate 1, 45 mg, 0.11 mmol) and HATU (46 mg, 0.12 mmol) was added DMF (0.5 mL), morpholine (13 mg, 0.15 mmol, 13 μL) then DIPEA (43 mg, 0.33 mmol, 58 μL). The mixture was stirred for 45 min, diluted with H.sub.2O (3 mL) and the resulting solid was collected by filtration and washed with H.sub.2O, air-dried then dried in vacuo, affording a pale yellow solid (37 mg, 70% yield). The product consists of a single regioisomer that is 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-7-(morpholine-4-carbonyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide or 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-6-(morpholine-4-carbonyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide. MS: [M+1]: 475.4.
[0462] Step 6. Following general procedure 2, treatment of 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-7-(morpholine-4-carbonyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide or 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-6-(morpholine-4-carbonyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (37 mg, 0.078 mmol) with BBr.sub.3 led to a pale yellow fluffy solid (17 mg, 47% yield) after purification by prep HPLC (25-55% MeCN in H.sub.2O, 0.1% formic acid modifier). The product consists of a single regioisomer that is 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-7-(morpholine-4-carbonyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide or 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-(morpholine-4-carbonyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (Compound 66). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 8.12 (brs, 2H), 7.98 (d, J=8.5 Hz, 1H), 7.79 (d, J=1.8 Hz, 1H), 7.74 (br d, J=3.3 Hz, 1H), 7.59 (dd, J=8.5, 1.9 Hz, 1H), 7.43 (br s, 1H), 7.13 (dt, J=8.3, 0.8 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 3.55 (br m, 8H), 1.83 (s, 3H), 1.76 (s, 3H). MS: [M+1]: 461.4.
##STR00172##
Compound 67 (2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-7-(1-hydroxy-1-methyl-ethyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide or 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-(1-hydroxy-1-methyl-ethyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide) and Compound 68 (2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-7-isopropenyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide or either 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-isopropenyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide)
[0463] Step 1. To a solution of 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-7-carboxylate or 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-6-carboxylate (Ester Intermediate 1, 82 mg, 0.20 mmol) in THE (2.5 mL) at −40° C. was added methylmagnesium chloride in THE (3 M, 655 μL) dropwise. The reaction mixture was stirred under N.sub.2, allowing to warm up to rt and stirred overnight. It was then quenched with saturated aqueous NH.sub.4Cl and diluted with EtOAc. The layers separated and the aqueous layer was extracted with EtOAc (2×). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude product was adsorbed on silica and purified by flash chromatography on silica gel (50-100% EtOAc in hexanes). The appropriate fractions were combined, concentrated and dried in vacuo to provide a yellow solid (54 mg, 66% yield) which was not pure but was carried through the next step without further purification. The product consists of a single regioisomer that is 2-amino-7-(1-hydroxy-1-methyl-ethyl)-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide or 2-amino-6-(1-hydroxy-1-methyl-ethyl)-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide. MS: [M+1]: 420.3.
[0464] Step 2. Following general procedure 2, treatment of 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-7-carboxylate or 2-amino-3-carbamoyl-1-(3-methoxy-2,6-dimethyl-phenyl)pyrrolo[3,2-b]quinoxaline-6-carboxylate (54 mg, 0.13 mmol) with BBr.sub.3 led to the final product (5 mg, 10% yield) as a pale yellow fluffy solid after purification by prep HPLC (30-60% MeCN in H.sub.2O, 0.1% formic acid modifier). The product consists of a single regioisomer that is 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-7-(1-hydroxy-1-methyl-ethyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide or 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-(1-hydroxy-1-methyl-ethyl)pyrrolo[3,2-b]quinoxaline-3-carboxamide (Compound 67). .sup.1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.03-7.90 (m, 2H), 7.88 (dd, J=8.6, 0.5 Hz, 1H), 7.77 (dd, J=2.2, 0.5 Hz, 1H), 7.76-7.66 (m, 2H), 7.34 (br s, 1H), 7.12 (d, J=8.3 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 5.09 (s, 1H), 1.83 (s, 3H), 1.75 (s, 3H), 1.48 (s, 6H). MS: [M+1]: 406.5. Upon dissolving the crude product in a mixture of MeCN/H.sub.2O/dmso for the purification by prep HPLC, the formation of a dehydration side product was observed. This side-product was also isolated and repurified by prep HPLC 50-80% MeCN in H.sub.2O, 0.1% formic acid modifier) to afford a single regioisomer that is 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-7-isopropenyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide or 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-6-isopropenyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide (Compound 68, 13 mg, 26% yield) as a pale yellow fluffy solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.01 (br s, 2H), 7.89 (dd, J=7.8, 1.8 Hz, 1H), 7.85-7.78 (m, 2H), 7.73 (d, J=3.2 Hz, 1H), 7.38 (d, J=3.2 Hz, 1H), 7.12 (dt, J=8.3, 0.8 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 5.60 (s, 1H), 5.16 (t, J=1.5 Hz, 1H), 2.18 (s, 3H), 1.83 (s, 3H), 1.75 (s, 3H). MS: [M+1]: 388.4.
Preparation of Ester Intermediate 2 and Acid Intermediate 2
[0465] ##STR00173##
[0466] Steps 1, 2, 3. Applying the steps outlined in Method A and using Intermediate X and 5-methoxy-2-methyl-aniline and following general procedure 1 for hydrolysis of the nitrile, a major regioisomer of unknown regiochemistry which can be either methyl 2-amino-3-carbamoyl-1-(5-methoxy-2-methyl-phenyl)pyrrolo[2,3-b]quinoxaline-5-carboxylate or methyl 2-amino-3-carbamoyl-1-(5-methoxy-2-methyl-phenyl)pyrrolo[2,3-b]quinoxaline-8-carboxylate was obtained as a dark yellow solid, (Ester Intermediate 2, 1.78 g). .sup.1H NMR (400 MHz, DMSO-d6) δ 7.95 (dd, J=8.2, 1.5 Hz, 1H), 7.93-7.87 (m, 2H), 7.50 (dd, J=8.3, 7.3 Hz, 1H), 7.43 (dt, J=8.2, 0.7 Hz, 1H), 7.37 (br d, J=3.3 Hz, 1H), 7.16-7.09 (m, 2H), 3.92 (s, 3H), 3.78 (s, 3H), 1.94 (s, 3H). MS: [M+1]: 406.2.
[0467] Step 4. To a solution of methyl 2-amino-3-carbamoyl-1-(5-methoxy-2-methyl-phenyl)pyrrolo[2,3-b]quinoxaline-5-carboxylate or methyl 2-amino-3-carbamoyl-1-(5-methoxy-2-methyl-phenyl)pyrrolo[2,3-b]quinoxaline-8-carboxylate (300 mg, 0.740 mmol) in THE (4.5 mL) and MeOH (3 mL) was added aqueous lithium hydroxide (1 M, 1.5 mL) and the resulting mixture was stirred for 24 at rt, more aqueous lithium hydroxide (1 M, 1.5 mL) was added and the mixture was stirred for another 6 h. The reaction mixture was concentrated to remove the volatiles, acidified to pH 4-5 with aqueous 1N HCl and the resulting solid collected by filtration and washed with H.sub.2O, air-dried then dried in vacuo to afford crude product as a yellow solid (Acid Intermediate 2, 259 mg, 89% yield), consisting of a single regioisomer that is 2-amino-3-carbamoyl-1-(5-methoxy-2-methyl-phenyl)pyrrolo[2,3-b]quinoxaline-5-carboxylic acid or 2-amino-3-carbamoyl-1-(5-methoxy-2-methyl-phenyl)pyrrolo[2,3-b]quinoxaline-8.-carboxylic acid. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.31 (br s, 2H), 8.03 (dd, J=7.3, 1.5 Hz, 1H), 7.97 (dd, J=8.3, 1.5 Hz, 1H), 7.53 (dd, J=8.3, 7.3 Hz, 1H), 7.47 (br s, 3H), 7.43 (dt, J=8.1, 0.8 Hz, 1H), 7.19-7.07 (m, 2H), 3.78 (s, 3H), 1.95 (s, 3H). MS: [M+1]: 392.2.
##STR00174##
Compound 77 and Compound 78 (2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-5-methyl-pyrrolo[2,3-b]quinoxaline-3-carboxamide and 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-8-methyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide)
[0468] ##STR00175##
[0469] Step 1. Malononitrile (4.1 g, 62.1 mmol) was added portionwise to a vigorously stirred suspension of sodium hydride (60% dispersion in mineral oil, 2.47 g, 64.4 mmol) in DME (100 mL). After the addition, the stirring was continued for 30 min and then 2,3-dichloro-5-methyl-quinoxaline (intermediate T, 6.5 g, 30.5 mmol) was added. The reaction mixture was stirred at rt for 15 min and then heated under reflux for 4 h. The DME was evaporated and cold aqueous 1M HCl was added to give a yellow precipitate that was filtered and washed with water to afford a mixture of 2-(3-chloro-5-methyl-quinoxalin-2-yl)propanedinitrile and 2-(3-chloro-8-methyl-quinoxalin-2-yl)propanedinitrile (5.8 g, 78% yield) (in about 1:1 ratio estimated by UPLCMS) as a yellow solid. MS: [M−1]: 241.1.
[0470] Step 2. 3-methoxy-2,6-dimethyl-aniline (arylamine AA1, 1.9 g, 12.5 mmol) was added to a mixture of 2-(3-chloro-5-methyl-quinoxalin-2-yl)propanedinitrile and 2-(3-chloro-8-methyl-quinoxalin-2-yl)propanedinitrile (1.00 g, 4.12 mmol) in NMP (10 mL). The reaction mixture was heated to 130° C. for 6 h, cooled, and the mixture was poured into aqueous NaHCO.sub.3 sat. The precipitate was collected by filtration, washed with water and dried by codistillation with toluene twice. The brown residue was adsorbed on silica using 15% MeOH in DCM and purified by flash chromatography on silica gel (20-60% EtOAc in hexanes to provide a mixture of 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-8-methyl-pyrrolo[3,2-b]quinoxaline-3-carbonitrile and 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-5-methyl-pyrrolo[2,3-b]quinoxaline-3-carbonitrile (1.04 g, 71% yield) as a brown solid. MS: [M+1]: 358.2.
[0471] Step 3. Following general procedure 1, treatment of the mixture of 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-8-methyl-pyrrolo[3,2-b]quinoxaline-3-carbonitrile and 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-5-methyl-pyrrolo[2,3-b]quinoxaline-3-carbonitrile (1.04 g, 2.90 mmol) with H.sub.2SO.sub.4 led to a mixture of 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-8-methyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide and 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-5-methyl-pyrrolo[2,3-b]quinoxaline-3-carboxamide (960 mg, 89% yield) as a yellow solid. MS: [M+1]: 376.2.
[0472] Step 4. Deprotection of the mixture of 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-8-methyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide and 2-amino-1-(3-methoxy-2,6-dimethyl-phenyl)-5-methyl-pyrrolo[2,3-b]quinoxaline-3-carboxamide (600 mg, 1.60 mmol) with BBr.sub.3 following general procedure 2 provided crude product which was purified by flash chromatography on silica gel (dry load, 0-10% MeOH in DCM) to obtain a mixture of the desired products as an orange solid. Separation of the regioisomers was achieved by preparative HPLC (30-80% MeCN in H.sub.2O, 0.1% formic acid modifier). The recovered tubes were combined and lyophilized, providing 2 regioisomers. The first to elute, (P1, Compound 77, 20 mg, 7% yield) as a pale yellow solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.86-9.36 (m, 1H), 7.93 (s, 2H), 7.76 (d, J=3.4 Hz, 1H), 7.57 (ddd, J=8.2, 1.5, 0.7 Hz, 1H), 7.42 (ddd, J=7.1, 1.6, 0.9 Hz, 1H), 7.35 (d, J=3.4 Hz, 1H), 7.31 (dd, J=8.3, 7.0 Hz, 1H), 7.08 (dt, J=8.3, 0.8 Hz, 1H), 6.94 (d, J=8.3 Hz, 1H), 2.69 (s, 3H), 1.84-1.75 (m, 3H), 1.71 (s, 3H). MS: [M+1]: 362.2. The last to elute (P2, Compound 78, 9 mg, 3% yield) as a yellow solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.12 (s, 1H), 7.89 (s, 2H), 7.77-7.66 (m, 2H), 7.42 (dd, J=8.3, 7.0 Hz, 1H), 7.36-7.26 (m, 2H), 7.09 (dt, J=8.3, 0.7 Hz, 1H), 6.93 (d, J=8.3 Hz, 1H), 2.42 (d, J=0.8 Hz, 3H), 1.81 (d, J=0.7 Hz, 3H), 1.74 (s, 3H). MS: [M+1]: 362.2. Compound 77 is one of the regioisomers 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-5-methyl-pyrrolo[2,3-b]quinoxaline-3-carboxamide and 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-8-methyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide, and compound 78 is the other of the regioisomers 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-5-methyl-pyrrolo[2,3-b]quinoxaline-3-carboxamide and 2-amino-1-(3-hydroxy-2,6-dimethyl-phenyl)-8-methyl-pyrrolo[3,2-b]quinoxaline-3-carboxamide.
##STR00176##
Compound 79 (2-amino-7-bromo-1-(3-hydroxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[2,3-e]pyrazine-3-carboxamide or 2-amino-6-bromo-1-(3-hydroxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[3,2-e]pyrazine-3-carboxamide)
[0473] Step 1. Malononitrile (4.75 g, 71.9 mmol) was added to a stirred suspension of sodium hydride (60% dispersion in mineral oil, 2.85 g, 74.4) in DME (300 mL). After the addition, the stirring was continued for 30 min, then 7-bromo-2,3-dichloro-pyrido[2,3-b]pyrazine (intermediate W, 10 g, 35.9 mmol) was added. The reaction mixture was stirred at room temperature for 10 min and then refluxed for 3 h. The DME was evaporated and the resulting residue was treated with cold aqueous hydrochloric acid to give a brown solid which was collected by filtration, adsorbed on silica using 10% MeOH in DCM and purified by flash chromatography on silica gel (0-30% MeOH in DCM), affording a brown solid (6.8 g, 61% yield). The product consists of a single regioisomer that is 2-(7-bromo-2-chloro-pyrido[2,3-b]pyrazin-3-yl)propanedinitrile or 2-(7-bromo-3-chloro-pyrido[2,3-b]pyrazin-2-yl)propanedinitrile. MS: [M−1]: 307.9.
[0474] Step 2. A mixture of 3-methoxy-2,6-dimethyl-aniline (arylamine AA1, 735 mg, 4.86 mmol) and 2-(7-bromo-2-chloro-pyrido[2,3-b]pyrazin-3-yl)propanedinitrile or 2-(7-bromo-3-chloro-pyrido[2,3-b]pyrazin-2-yl)propanedinitrile (500 mg, 1.62 mmol) in NMP (5 mL) was heated to 130° C. for 6 h, cooled, and the mixture was poured into saturated aqueous NaHCO.sub.3. The precipitate was collected by filtration, washed with water and dried by codistillation with toluene twice. The brown residue was adsorbed on silica using 15% MeOH in DCM and purified by flash chromatography on silica gel (20-100% EtOAc in hexanes), affording a yellow solid (330 mg, 48% yield). The product consists of a single regioisomer that is 2-amino-7-bromo-1-(3-methoxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[2,3-e]pyrazine-3-carbonitrile or 2-amino-6-bromo-1-(3-methoxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[3,2-e]pyrazine-3-carbonitrile. MS: [M+1]: 425.1.
[0475] Step 3. Following general procedure 1, treatment of 2-amino-7-bromo-1-(3-methoxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[2,3-e]pyrazine-3-carbonitrile or 2-amino-6-bromo-1-(3-methoxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[3,2-e]pyrazine-3-carbonitrile (330 mg, 0.780 mmol) with H.sub.2SO.sub.4 led to a crude product (330 mg, 95% yield) as a yellow solid. The product consists of a single regioisomer that is 2-amino-7-bromo-1-(3-methoxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[2,3-e]pyrazine-3-carboxamide or 2-amino-6-bromo-1-(3-methoxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[3,2-e]pyrazine-3-carboxamide. MS: [M+1]: 441.1.
[0476] Step 4. Deprotection of 2-amino-7-bromo-1-(3-methoxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[2,3-e]pyrazine-3-carboxamide or 2-amino-6-bromo-1-(3-methoxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[3,2-e]pyrazine-3-carboxamide (330 mg, 0.748 mmol) with BBr.sub.3 following general procedure 2 provided a crude product which was purified by flash chromatography on silica gel (dry load, 0-10% MeOH in DCM) then by preparative HPLC (25-70% MeCN in H.sub.2O, 0.1% formic acid modifier). The recovered tubes were combined and lyophilized to provide a product (Compound 79, 24 mg, 7% yield). The product is a single regioisomer that is either 2-amino-7-bromo-1-(3-hydroxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[2,3-e]pyrazine-3-carboxamide or 2-amino-6-bromo-1-(3-hydroxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[3,2-e]pyrazine-3-carboxamide. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.78 (d, J=2.4 Hz, 1H), 8.45 (d, J=2.3 Hz, 1H), 8.30 (s, 2H), 7.75-7.57 (m, 1H), 7.44 (s, 1H), 7.05 (d, J=8.3 Hz, 1H), 6.92 (d, J=8.3 Hz, 1H), 1.77 (s, 3H), 1.69 (s, 3H). MS: [M+1]: 429.0.
##STR00177##
Compound 82 (2-amino-1-(3-hydroxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[2,3-e]pyrazine-3-carboxamide or 2-amino-1-(3-hydroxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[3,2-e]pyrazine-3-carboxamide)
[0477] A mixture of Compound 79 (100 mg, 0.234 mmol) and palladium on carbon (10% w/w, 25 mg) in EtOH (5 mL) was stirred under H.sub.2 atmosphere (balloon) for 2 h. The reaction mixture was filtrated on a 0.45 micron PTFE filter and the filtrate was purified by prep HPLC (25-70% MeCN in H.sub.2O, 0.1% formic acid modifier). The recovered tubes were combined and lyophilized to provide a pale yellow solid (Compound 82, 41 mg, 50% yield). The product consists of a single regioisomer that is 2-amino-1-(3-hydroxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[2,3-e]pyrazine-3-carboxamide or 2-amino-1-(3-hydroxy-2,6-dimethylphenyl)-1H-pyrido[2,3-b]pyrrolo[3,2-e]pyrazine-3-carboxamide. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 9.09 (s, 1H), 8.96-8.84 (m, 2H), 8.82 (dd, J=8.2, 1.5 Hz, 1H), 7.78 (dd, J=8.2, 5.7 Hz, 2H), 7.48 (s, 1H), 7.07 (d, J=8.3 Hz, 1H), 6.96 (d, J=8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). MS: [M+1]: 349.2.
##STR00178##
Compound 88 (3-(3-hydroxy-2,6-dimethylphenyl)-3H-pyrrolo[2,3-c]isoquinoline-1-carboxamide)
[0478] Step 1. To a solution of intermediate P (410 mg, 1.81 mmol) and arylamine AA1 (302 mg, 2.00 mmol) in THE (6.5 mL) was added dropwise a solution of LiHMDS in THE (1M, 3.81 mL, 3.81 mmol) at RT under nitrogen. The reaction mixture was stirred at 65° C. for 1 h. The reaction was cooled to RT and partitioned between EtOAc and sat aq NH.sub.4Cl. The organic phase was washed with brine, dried over Na.sub.2SO.sub.4 and evaporated. The residue was purified by silica gel chromatography using EtOAc in heptanes (0 to 20%), and then by reverse phase flash chromatography (C18) using MeOH in H.sub.2O (25 to 100%) to get 4-bromo-N-(3-methoxy-2,6-dimethylphenyl)isoquinolin-3-amine (418.0 mg, 64% yield) as a beige foam. MS: [M+1]: 357.0, 359.0.
[0479] Step 2. In a sealed tube, to a solution of malononitrile (155 mg, 2.35 mmol) in 1,2-dimethoxyethane (5 mL) was added sodium tert-butoxide (215 mg, 2.24 mmol) and the resulting solution was stirred for 20 min then, 4-bromo-N-(3-methoxy-2,6-dimethylphenyl)isoquinolin-3-amine (400 mg, 1.12 mmol) and Pd(dppf)Cl.sub.2.DCM (69 mg, 0.084 mmol) were added. The reaction mixture was stirred at 105° C. for 22 hours. The resulting suspension was cooled to RT and filtered through a plug of silica gel, eluted with EtOAc. The filtrate was washed with water/brine and brine, dried over Na.sub.2SO.sub.4 and evaporated. The residue was purified by silica gel chromatography using EtOAc in heptanes (5 to 50%) to get 2-amino-3-(3-methoxy-2,6-dimethylphenyl)-3H-pyrrolo[2,3-c]isoquinoline-1-carbonitrile (297 mg, 77% yield) as a yellow solid. MS: [M+1]: 343.2.
[0480] Step 3. Following the procedure described for compound 24 step 1 using 2-amino-3-(3-methoxy-2,6-dimethylphenyl)-3H-pyrrolo[2,3-c]isoquinoline-1-carbonitrile (200 mg, 0.584 mmol) as starting material, 3-(3-methoxy-2,6-dimethylphenyl)-3H-pyrrolo[2,3-c]isoquinoline-1-carbonitrile (154 mg, 80% yield) was obtained as a beige solid after purification by silica gel chromatography using EtOAc in heptanes (0 to 50%), and then by reverse phase flash chromatography (C18) using MeOH in H.sub.2O (15 to 100%). MS: [M+1]: 328.2.
[0481] Step 4. To a solution of 3-(3-methoxy-2,6-dimethylphenyl)-3H-pyrrolo[2,3-c]isoquinoline-1-carbonitrile (150 mg, 0.458 mmol) in ethanol (3 mL) and water (250 μL) was added Ghaffar-Parkins catalyst (7.0 mg, 0.016 mmol). The reaction mixture was heated to 80° C. to get a solution. After 23 hours of stirring, the reaction mixture was poured into EtOAc and washed with water and brine, dried over Na.sub.2SO.sub.4 and evaporated to get 3-(3-methoxy-2,6-dimethylphenyl)-3H-pyrrolo[2,3-c]isoquinoline-1-carboxamide (164 mg, quantitative) as a yellow solid. MS: [M+1]: 346.2.
[0482] Step 5. Following general procedure 2, deprotection of 3-(3-methoxy-2,6-dimethylphenyl)-3H-pyrrolo[2,3-c]isoquinoline-1-carboxamide (156 mg, 0.452 mmol) afforded 3-(3-hydroxy-2,6-dimethylphenyl)-3H-pyrrolo[2,3-c]isoquinoline-1-carboxamide (compound 88, 100 mg, 66% yield) as a light brown fluffy solid after purification by reverse phase flash chromatography on C18 cartridge (15-100% MeOH in H.sub.2O). .sup.1H NMR (400 MHz, DMSO-d6) δ ppm 1.67 (s, 3H), 1.78 (s, 3H), 6.94 (d, J=8.3 Hz, 1H), 7.08 (d, J=8.3 Hz, 1H), 7.12-7.27 (m, 1H), 7.59 (ddd, J=8.0, 6.9, 1.0 Hz, 1H), 7.73 (br. s, 1H), 7.81 (ddd, J=8.5, 7.0, 1.3 Hz, 1H), 8.09 (s, 1H), 8.16 (d, J=8.1 Hz, 1H), 8.92 (s, 1H), 9.56 (s, 1H), 9.65 (d, J=8.6 Hz, 1H). MS: [M+1]: 332.1.
##STR00179##
Compound 89 (5-(3-hydroxy-2,6-dimethylphenyl)-1-methyl-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine-7-carboxamide)
[0483] Step 1. A vial was charged with arylamine AA1 (240 mg, 1.59 mmol), intermediate Q (420 mg, 1.44 mmol), Xantphos (63 mg, 0.11 mmol), cesium carbonate (1.00 g, 3.07 mmol) and 1,2-dimethoxyethane (5 mL). The suspension was sparged with nitrogen over the sonic bath for 5 min and Pd.sub.2(dba).sub.3 (100 mg, 0.109 mmol) was added. The reaction mixture was heated to 82° C. for 16 h. The suspension was cooled to RT and filtered through a small pad of silica gel, rinsed with EtOAc. The filtrate was evaporated, and the crude product was purified by silica gel chromatography using EtOAc in heptanes (0 to 40%) to provide 6-bromo-N-(3-methoxy-2,6-dimethylphenyl)-1-methyl-1H-pyrazolo[4,3-b]pyridin-5-amine (280 mg, 53% yield) as an off-white solid. MS: [M+1]: 361.0, 363.0
[0484] Step 2. Following the procedure described for compound 88 step 2 using 6-bromo-N-(3-methoxy-2,6-dimethylphenyl)-1-methyl-1H-pyrazolo[4,3-b]pyridin-5-amine (240 mg, 0.664 mmol) as starting material, 6-amino-5-(3-methoxy-2,6-dimethylphenyl)-1-methyl-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine-7-carbonitrile (141 mg, 61% yield) was obtained as an orange solid after purification by silica gel chromatography using EtOAc in heptanes (25 to 100%). MS: [M+1]: 347.2.
[0485] Step 3. Following the procedure described for compound 24 step 1 using 6-amino-5-(3-methoxy-2,6-dimethylphenyl)-1-methyl-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine-7-carbonitrile (141 mg, 0.407 mmol) as starting material, 5-(3-methoxy-2,6-dimethylphenyl)-1-methyl-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine-7-carbonitrile (68 mg, 50% yield) was obtained as a yellow foam after purification by silica gel chromatography using EtOAc in heptanes (20 to 90%). MS: [M+1]: 332.2.
[0486] Step 4. Following the procedure described for compound 88 step 4 using 5-(3-methoxy-2,6-dimethylphenyl)-1-methyl-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine-7-carbonitrile (67 mg, 0.20 mmol) as starting material, 5-(3-methoxy-2,6-dimethylphenyl)-1-methyl-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine-7-carboxamide (70 mg, 99% yield) was obtained as a red oil. MS: [M+1]: 350.2.
[0487] Step 5. Following general procedure 2, deprotection of 5-(3-methoxy-2,6-dimethylphenyl)-1-methyl-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine-7-carboxamide (70 mg, 0.20 mmol) afforded 5-(3-hydroxy-2,6-dimethylphenyl)-1-methyl-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine-7-carboxamide (compound 89, 31 mg, 46% yield) as white fluffy solid after purification by reverse phase flash chromatography on 018 cartridge (10-100% MeOH in H.sub.2O). .sup.1H NMR (400 MHz, DMSO-d6) b ppm 1.67 (s, 3H), 1.77 (s, 3H), 4.15 (s, 3H), 6.93 (d, J=8.3 Hz, 1H), 7.07 (d, J=8.3 Hz, 1H), 7.21 (br. s, 1H), 7.55 (br. s, 1H), 8.13 (d, J=1.0 Hz, 1H), 8.34 (s, 1H), 8.77 (d, J=0.7 Hz, 1H), 9.56 (s, 1H). MS: [M+1]: 336.3.
Examples of Arylamines Preparation
[0488] A variety of arylamines were used to prepare compounds of the present invention. Some of these arylamines were commercially available and some were prepared. Table 2 lists some examples of such arylamines for which the preparation is described herein.
TABLE-US-00002 TABLE 2
Preparation of Arylamine AA1
[0489] Compounds of the present invention can be prepared from arylamine AA1 which can be prepared as shown in Scheme AA1 and described herein. Commercially available 1,3-dimethyl-2-nitrobenzene can be brominated under suitable bromination conditions. The resulting bromo can be converted to a methoxy upon treatment with sodium methoxide and copper(I) bromide. The nitro can be reduced to generate arylamine AA1.
##STR00191##
[0490] Step 1. A 3 necked 3 L round-bottom flask was equipped with a mechanical stirrer, reflux condenser and addition funnel and loaded with 1,3-dimethyl-2-nitro-benzene (300 g, 1.98 mol), DCM (900 mL), iron powder (28.0 g, 501 mmol) and iron(III) bromide (11.9 g, 40.3 mmol). Bromine (112 mL, 2.19 mol) was added dropwise via an addition funnel over 45-60 min. Internal monitoring of the temperature showed an exotherm to 30° C. 90 min after the addition of bromine was complete, more bromine (5 mL, 98 mmol) was added and the reaction mixture was stirred for another 45 min to complete conversion. The reaction mixture was diluted with ice water (1.5 L) and Et.sub.2O (1.5 L). The layers were separated. The aqueous layer was back extracted with Et.sub.2O (0.5 L). The combined organic layers were washed with aqueous 20% Na.sub.2S.sub.2O.sub.3 (1 L), brine (500 mL), dried over Na.sub.2SO.sub.4, filtered over a silica gel pad (300 cc), concentrated then dried in vacuo to provide 1-bromo-2,4-dimethyl-3-nitro-benzene (451.5 g, 99% yield) as an off-white solid.
[0491] Step 2. A 5 L 4 neck round bottom flask equipped with a mechanical stirrer and a reflux condenser was charged with 1-bromo-2,4-dimethyl-3-nitro-benzene (451.5 g, 1.96 mol) in DMF (1.6 L). CuBr (28.0 g, 195 mmol) was added followed by sodium methoxide (1.31 L, 5.89 mol, 25% in MeOH). The reaction mixture was slowly heated to 95° C., achieving a mild reflux. After 6 h, the reaction mixture was left to cool to rt overnight. The reaction mixture was diluted with Et.sub.2O and saturated aqueous NH.sub.4Cl (1.5 L each). The layers were separated, and the aqueous layer was back extracted with Et.sub.2O (750 mL). The combined organic extracts were washed with brine (750 mL), dried over Na.sub.2SO.sub.4 and filtered over a silica pad, rinsed with Et.sub.2O and concentrated, dried in vacuo to provide 1-methoxy-2,4-dimethyl-3-nitro-benzene (352 g, 99% yield) as an ochre solid.
[0492] Step 3. To a solution of 1-methoxy-2,4-dimethyl-3-nitro-benzene (115 g, 635 mmol) in EtOH (1.5 L) in a 3 neck 3 L flask equipped with a mechanical stirrer was added iron powder (213 g, 3.81 mol), then a solution of ammonium chloride (204 g, 3.81 mol) in water (500 mL) was added portion wise. The mixture was heated to 85° C. for 8 h. The mixture was cooled down to rt and filtered on Celite. The volume of the filtrate was reduced (most of the EtOH was evaporated) and the resulting mixture was diluted with Et.sub.2O (800 mL) and water (150 mL). The layers were separated, and the aqueous layer was back extracted with Et.sub.2O (500 mL). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4, filtered, concentrated and dried in vacuo to provide 3-methoxy-2,6-dimethyl-aniline (89.1 g, 93% yield) as a brown oil. .sup.1H NMR (400 MHz, Chloroform-d) δ 6.88 (dq, J=8.3, 0.7 Hz, 1H), 6.31 (d, J=8.2 Hz, 1H), 3.79 (s, 3H), 3.61 (brs, 2H), 2.14 (d, J=0.7 Hz, 3H), 2.07 (s, 3H). MS: [M+1]: 152.3.
Preparation of Arylamine AA5
[0493] Compounds of the present invention can be prepared from arylamine AA5 which can be prepared as shown in Scheme AA5 and described herein. Commercially available 2-chloro-3-methoxy-benzoic acid can be brominated with a suitable bromination reagent and the carboxylic acid can be converted to a NHBoc under Curtius conditions. The bromo can be converted to a methyl and the NHBoc can be cleaved under acidic conditions to generate arylamine AA5.
##STR00192##
[0494] Step 1. To a solution of 2-chloro-3-methoxy-benzoic acid (50 g, 268 mmol) in AcOH (250 mL) and water (250 mL) was added bromine (27.5 mL, 537 mmol) dropwise. The mixture was stirred at 60° C. for 18 h, cooled to rt, brine was added, and the mixture was extracted twice with DCM. The combined organic extracts were dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to provide 6-bromo-2-chloro-3-methoxy-benzoic acid (71 g, quantitative yield) as a brown oil which solidified upon standing under vacuum over the weekend.
[0495] Step 2. To a solution of 6-bromo-2-chloro-3-methoxy-benzoic acid (23.6 g, 88.9 mmol). Et.sub.3N (38 mL, 271 mmol) and tert-butanol (42.5 mL, 450 mmol) in toluene (500 mL) was added [azido(phenoxy)phosphoryl]oxybenzene (29.5 mL, 136 mmol). The mixture was heated at 100° C. for 16 h, cooled down to rt then the volatiles were removed in vacuo. The residue was diluted with EtOAc (100 mL), and the organic layer was washed with 5% citric acid, water, saturated aqueous NaHCO.sub.3, brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness. The residue was purified by silica gel chromatography eluting with a gradient of 0 to 20% EtOAc in hexanes to provide tert-butyl N-(6-bromo-2-chloro-3-methoxy-phenyl)carbamate (16.2 g, 54% yield) as a yellowish solid.
[0496] Step 3. To a solution of tert-butyl N-(6-bromo-2-chloro-3-methoxy-phenyl)carbamate (25 g, 74.3 mmol) in dioxane (500 mL) was added trimethylboroxine (50% w/w in THF, 20.51 g, 81.7 mmol). PdCl.sub.2(dppf.CH.sub.2Cl.sub.2 (5.22 g, 7.43 mmol) and aqueous Na.sub.2CO.sub.3 (2 M, 111 mL, 223 mmol). The mixture was heated at 100° C. for 16 h, cooled down to rt then volatiles were removed in vacuo. EtOAc and water were added. The organic layer was separated and washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to dryness. The residue was purified by silica gel chromatography eluting with a gradient of 0 to 30% EtOAc in heptane to provide tert-butyl N-(2-chloro-3-methoxy-6-methyl-phenyl)carbamate (13.8 g, 68% yield) as a yellowish solid.
[0497] Step 4. HCl in dioxane (4 M, 100 mL) was added to a solution of tert-butyl N-(2-chloro-3-methoxy-6-methyl-phenyl)carbamate (13.8 g, 50.8 mmol) in MeOH (100 mL). After 3 h, the volatiles were evaporated to dryness under vacuum to provide a white solid to which was added under vigorous stirring 250 mL of EtOAc and 250 mL of aqueous saturated NaHCO.sub.3. The organic layer was separated. The aqueous layer was back extracted with EtOAc. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with a gradient of 0 to 30% EtOAc in heptane to provide 2-chloro-3-methoxy-6-methyl-aniline (7.9 g, 91% yield) as a clear oil which solidified upon standing. .sup.1H NMR (400 MHz, Chloroform-d) δ 6.95-6.79 (m, 1H), 6.27 (dd, J=8.3, 1.5 Hz, 1H), 4.06 (br s, 2H), 3.83 (d, J=1.6 Hz, 3H), 2.12 (d, J=0.8 Hz, 3H). MS: [M+1]: 172.2.
Preparation of Arylamine AA6
[0498] Compounds of the present invention can be prepared from arylamine AA6 which can be prepared as shown in Scheme AA6 and described herein. Commercially available 3-methoxy-2-methyl-aniline can be chlorinated with a chlorination reagent to generate arylamine AA6.
##STR00193##
[0499] Step 1. NCS (98 g, 734 mmol) was added in 4 portions (15 minutes between each addition) to a solution of 3-methoxy-2-methyl-aniline (100 g, 729 mmol) in DCM (500 mL) at 0° C. 30 min after the last addition, 100 g of silica gel was added, the mixture was evaporated under vacuum and the black residue was purified by silica gel chromatography (dry load) in eluting with a gradient of 0 to 10% EtOAc in hexanes to provide 6-chloro-3-methoxy-2-methyl-aniline (55.6 g, 44% yield) as an orange solid. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.08 (d, J=8.8 Hz, 1H), 6.28 (d, J=8.8 Hz, 1H), 4.02 (br s, 2H), 3.78 (s, 3H), 2.07 (s, 3H). MS: [M+1]: 172.3.
Preparation of Arylamine AA7
[0500] Compounds of the present invention can be prepared from arylamine AA7 which can be prepared as shown in Scheme AA7 and described herein. Commercially available 3-amino-2,4-dichloro-phenol can be O-protected with a suitable protecting group such as O-PMB to generate arylamine AA7.
##STR00194##
[0501] To a suspension of 3-amino-2,4-dichloro-phenol.HCl salt (20 g, 93.3 mmol) in DMF (150 mL) was added 1-(chloromethyl)-4-methoxy-benzene (14.0 mL, 103 mmol), tetrabutylammonium iodide (1 g, 3.00 mmol) and Cs.sub.2CO.sub.3 (64.0 g, 196 mmol). The mixture was stirred at 40° C. overnight, then it was diluted with water, stirred for 20 min, and filtered. The precipitate was washed with water and dried in vacuo. The resulting crude product was purified by silica gel chromatography eluting with a gradient of 0 to 100% DCM in hexanes to provide 2,6-dichloro-3-[(4-methoxyphenyl)methoxy]aniline (20 g, 72% yield) as an off-white solid. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.38-7.30 (m, 2H), 7.05 (d, J=8.9 Hz, 1H), 6.92-6.77 (m, 2H), 6.32 (s, 1H), 5.01 (s, 2H), 4.46 (s, 2H), 3.79 (s, 3H). MS: [M+1]: 298.0.
Preparation of Arylamine AA8
[0502] Compounds of the present invention can be prepared from arylamine AA8 which can be prepared as shown in Scheme AA8 and described herein. The commercially available 4-methyl-3-nitro-phenol can be O-protected with a suitable protecting group such as O-MOM. The nitro can be reduced to generate arylamine AA8.
##STR00195##
[0503] Step 1. To a suspension of 4-methyl-3-nitro-phenol (25 g, 163 mmol) in DCM (250 mL) was added DIPEA (34 mL, 195 mmol) followed by chloro(methoxy)methane (26.0 g, 323 mmol, 24.5 mL) added dropwise. After stirring 18 h, the reaction mixture was washed with water. The layers were separated. The organic layer was washed with 0.2N HCl (2×), brine, dried over MgSO.sub.4, filtered and concentrated, then dried in vacuo affording 4-(methoxymethoxy)-1-methyl-2-nitro-benzene (31.4 g, 98% yield) as a dark red oil.
[0504] Step 2. To a suspension of 4-(methoxymethoxy)-1-methyl-2-nitro-benzene (31.4 g, 159 mmol) in EtOH (200 mL) and water (75 mL) was added ammonium chloride (43.3 g, 809 mmol) then iron powder (44.5 g, 796 mmol). The reaction mixture was heated to 80° C. for 3.5 h, then temperature was increased to 90° C., stirring for 4 days. The reaction mixture was cooled to rt, filtered and rinsed with EtOAc. The filtrate was concentrated and diluted with EtOAc and saturated aqueous NaHCO.sub.3. The layers were separated, and the aqueous layer was back extracted with EtOAc (2×). The combined organic extracts were washed with brine, dried over MgSO.sub.4, filtered and concentrated, affording 26.3 g crude product as a dark brown oil which was purified on a silica gel pad, eluting with 20-30% EtOAc in hexanes. The pure fractions were combined, concentrated, then dried in vacuo, affording 5-(methoxymethoxy)-2-methyl-aniline (25.4 g, 95% yield) as a purple oil. .sup.1H NMR (400 MHz, Chloroform-d) δ 6.94 (dd, J=8.0, 1.7 Hz, 1H), 6.45-6.30 (m, 2H), 5.12 (s, 2H), 3.47 (s, 3H), 2.10 (s, 3H). MS: [M+1]: 168.3.
Preparation of Arylamine AA9
[0505] Compounds of the present invention can be prepared from arylamine AA9 which can be prepared as shown in Scheme AA9 and described herein. The commercially available 4-nitro-1H-indazole can be fluorinated with a suitable fluorinating agent. The indazole can be protected with a SEM protecting group and the nitro can be reduced to generate arylamine AA10.
##STR00196##
[0506] Step 1. To a solution of 4-nitro-1H-indazole (5.0 g, 30.7 mmol) in MeCN (75 mL) was added Selectfluor (21.72 g, 61.30 mmol). The mixture was stirred at 100° C. for 10 h, cooled down to rt and diluted with water and 2M Na.sub.2CO.sub.3 and stirred at rt for 20 min. The solid was collected by filtration, washed with water and dried in vacuo to provide 3-fluoro-4-nitro-1H-indazole (510 mg, 9% yield) as an off-white solid which contained about 10% starting material and was used as such for the next step. .sup.1H NMR (400 MHz, DMSO-d6) δ 13.43 (s, 1H), 8.07 (d, J=7.7 Hz, 1H), 7.94 (d, J=8.5 Hz, 1H), 7.59 (dd, J=8.5, 7.7 Hz, 1H). MS: [M−1]: 180.0.
[0507] Step 2. To a solution of 3-fluoro-4-nitro-1H-indazole (3.46 g, 19.1 mmol) in DMF (15 mL) were added cesium carbonate (7 g, 21 mmol), 2-(2-chloroethoxy)ethyl-trimethyl-silane (4.14 g, 24.8 mmol, 4.4 mL) and tetrabutylammoniumiodide (450 mg, 1.35 mmol). The mixture was stirred at rt for 1 h, then it was diluted with EtOAc, washed with water (2×), followed by brine, dried over sodium sulfate, filtered and the filtrate was concentrated to dryness. The residue was purified by flash chromatography on silica gel (0-35% EtOAc in hexanes) to provide 2-[(3-fluoro-4-nitro-indazol-1-yl)methoxy]ethyl-trimethyl-silane (3.1 g, 52% yield) and in about 10:1 ratio as a brown solid (10:1 ratio with minor regioisomer 2-[(3-fluoro-4-nitro-indazol-2-yl)methoxy]ethyl-trimethyl-silane). .sup.1H NMR (400 MHz, Chloroform-d) δ 8.10 (m 1H), 7.84 (m, 1H), 7.55 (m, 1H), 5.64 (s, 2H), 3.61-3.47 (m, 2H), 0.93-0.75 (m, 2H), −0.07 (s, 9H).
[0508] Step 3. To a solution of 2-[(3-fluoro-4-nitro-indazol-1-yl)methoxy]ethyl-trimethyl-silane (3 g, 9.6 mmol) in MeOH (20 mL) was added palladium on carbon (10% w/w, 600 mg, 0.564 mmol). The mixture was stirred at rt under a hydrogen atmosphere (balloon) for 5 h. Then the mixture was filtered over a pad of celite. The filtrate was concentrated and dried in vacuo to provide 3-fluoro-1-(2-trimethylsilylethoxymethyl)indazol-4-amine (2.5 g, 92% yield) as a brown thick oil. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.18 (dd, J=8.4, 7.6 Hz, 1H), 6.76 (m, 1H), 6.32-6.24 (m, 1H), 5.46 (s, 2H), 4.31 (s, 2H), 3.61-3.48 (m, 2H), 0.99-0.63 (m, 2H), −0.07 (s, 9H).
Preparation of Arylamine AA10
[0509] Compounds of the present invention can be prepared from arylamine AA10 which can be prepared as shown in Scheme AA10 and described herein. The commercially available 4-nitro-1H-indazole can be chlorinated with a suitable chlorinating agent. The nitro can be reduced to generate arylamine AA10.
##STR00197##
[0510] Step 1. To a solution of sodium hydroxide (3.04 g, 76.0 mmol) in H.sub.2O (100 mL) was added 4-nitro-1H-indazole (3.0 g, 18 mmol). The resulting mixture was heated to 50° C. for 35 min, then cooled in an ice bath. Aqueous sodium hypochlorite (0.806 M, 35 mL) was added and the cold bath was removed. After 6 h, more aqueous sodium hypochlorite (0.806 M, 15 mL) was added and the mixture was stirred overnight. It was then cooled in an ice bath, acidified to pH 2 with 3M HCl (about 30 mL). The resulting precipitate was stirred in the ice bath then the solids were collected by filtration and washed with H.sub.2O and air-dried, affording 3-chloro-4-nitro-1H-indazole (3.39 g, 93% yield) as a light tan solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.02 (dd, J=8.5, 0.8 Hz, 1H), 7.99 (dd, J=7.6, 0.8 Hz, 1H), 7.64 (dd, J=8.5, 7.6 Hz, 1H). MS: [M−1]: 196.0.
[0511] Step 2. To a RBF equipped with a condenser and charged with 3-chloro-4-nitro-1H-indazole (1.0 g, 5.06 mmol), ammonium chloride (1.35 g, 25.3 mmol), H.sub.2O (5 mL) and EtOH (10 mL) was added iron powder (1.40 g, 25.1 mmol). The reaction mixture was stirred at to 80° C. for 45 min and brought back to RT. The reaction mixture was filtered, the solids were washed with EtOAc. The filtrate was diluted with H.sub.2O, the layers were separated and the aqueous layer extracted with EtOAc (2×). The combined organic extracts were washed with H.sub.2O then brine, dried over Na.sub.2SO.sub.4 and filtered through a silica pad (ca 40 cc), eluting with EtOAc, concentrated and dried in vacuo, providing 3-chloro-1H-indazol-4-amine (757 mg, 89% yield) as a dark olive solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 12.82 (br s, 1H), 7.05 (dd, J=8.3, 7.5 Hz, 1H), 6.61 (dd, J=8.3, 0.7 Hz, 1H), 6.21 (dd, J=7.6, 0.7 Hz, 1H), 5.58 (br s, 2H). MS: [M+1]: 168.2.
Examples of 2,3-dichloropyrazine preparation and fused 2,3-dihalopyridines
[0512] A variety of 2,3-dichloropyrazine intermediates and fused 2,3-dihalopyridines intermediates were used to prepare compounds of the present invention. Table 3 lists some examples of such 2,3-dichloropyrazine intermediates and fused 2,3-dihalopyridines intermediates for which the preparation is described herein.
TABLE-US-00003 TABLE 3
Preparation of Intermediate P
[0513] Bromination of isoquinolin-3-amine followed by diazotization of the amine and treatment with HF-pyridine gives access to the required 4-bromo-3-fluoroisoquinoline as illustrated in Scheme 5.
##STR00209##
##STR00210##
[0514] Step 1. To a solution isoquinolin-3-amine (2.20 g, 15.3 mmol) in dichloromethane (50 mL) and methanol (50 mL) was added portion wise NBS (3.26 g, 18.3 mmol) below 20° C. After 1 h of stirring, the reaction mixture was diluted in 250 mL of DCM and was washed with 5% sodium thiosulfate (aq), water and brine, dried over Na.sub.2SO.sub.4 and evaporated. The crude product was purified by silica gel chromatography using EtOAc in heptanes (0 to 50%) to get 4-bromoisoquinolin-3-amine (1.80 g, 52% yield) as brown solid.
[0515] Step 2. To a solution of 4-bromoisoquinolin-3-amine (500 mg, 2.24 mmol) in 70% hydrogen fluoride-pyridine (5 g, 50.5 mmol) was added portion wise sodium nitrite (201 mg, 2.91 mmol) at 0-5° C. After 20 minutes of stirring, the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was added dropwise to a mixture of EtOAc and K.sub.2CO.sub.3 (30 g) in water (200 mL). The two layers were separated, and the aqueous phase was extracted with EtOAc. The combined organic layers were washed with sat aq NaHCO.sub.3 and brine, dried over Na.sub.2SO.sub.4 and evaporated. The crude product was purified by silica gel chromatography using EtOAc in heptanes (0 to 20%), affording 4-bromo-3-fluoroisoquinoline (439 mg, 86% yield) as white solid. .sup.1H NMR (400 MHz, CDCl3) δ ppm 7.64 (ddd, J=8.1, 7.0, 1.0 Hz, 1H), 7.77-7.93 (m, 1H), 8.02 (d, J=8.3 Hz, 1H), 8.20 (dd, J=8.7, 0.9 Hz, 1H), 8.90 (s, 1H). .sup.19F NMR (377 MHz, CDCl3) δ ppm −72.12 (br. s, 1 F). MS: [M+1]: 226.0, 228.0.
Preparation of Intermediate Q
[0516] Bromination of 5-bromo-2-methylpyridin-3-amine followed by formation of the fused pyrazole ring and N-methylation gives access to the required dibromo fused pyridine as illustrated in Scheme 6.
##STR00211##
##STR00212##
[0517] Step 1. To a solution of 5-bromo-2-methylpyridin-3-amine (5.00 g, 26.7 mmol) in acetonitrile (50 mL) was added portion wise NBS (5.00 g, 28.1 mmol) at 18-22° C. Once the addition was complete, the suspension was stirred at RT 1 h. The product was filtered (first crop) and the filtrate was evaporated to one third the initial volume. The solution was partitioned between DCM and water and the organic phase was washed with brine, dried over sodium sulfate, filtered, and adsorbed on silica. The crude product was purified by silica gel chromatography using EtOAc in heptanes (10 to 30%), providing a second crop of material which was merged with the first crop, affording 5,6-dibromo-2-methylpyridin-3-amine (5.46 g, 76% yield) as a solid.
[0518] Step 2. To a solution of 5,6-dibromo-2-methylpyridin-3-amine (3.00 g, 11.3 mmol) in CHCl.sub.3 (70 mL) were added potassium acetate (1.33 g, 13.5 mmol) and acetic anhydride (4.27 mL, 45.1 mmol). The reaction mixture was heated to reflux for 3 h to get a suspension then cooled to RT and 18-Crown-6 (298 mg, 1.13 mmol) and isoamylnitrite (1.52 mL, 11.3 mmol) were added. The suspension was heated to reflux for 12 h. Methanol (20 mL) and a solution of K.sub.2CO.sub.3 (8.0 g) in 20 mL of water were added. The biphasic solution was stirred at RT for 3 h. The precipitate was collected by filtration and the solid was dissolved into warm EtOAc (˜200 mL) and filtered through a pad of silica gel. The filter cake was rinsed with EtOAc. The filtrate was evaporated to get 5,6-dibromo-1H-pyrazolo[4,3-b]pyridine (1.11 g, 35% yield) as a beige solid.
[0519] Step 3. To a solution of 5,6-dibromo-1H-pyrazolo[4,3-b]pyridine (813 mg, 2.94 mmol) in N,N-dimethylformamide (16 mL, 207 mmol) was added dropwise a solution of NaHMDS in THE (1M, 3.8 mL, 3.8 mmol) at 0-5° C. under nitrogen. After 5 minutes of stirring, iodomethane (240 μL, 3.86 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was partitioned between EtOAc and water/brine (3:1, 140 mL). The aqueous layer was extracted with EtOAc once and the combined organic phases were washed with water and brine, dried over Na.sub.2SO.sub.4 and evaporated. The crude product was purified by silica gel chromatography using EtOAc in heptanes (5 to 100%). The major, less polar regioisomer was collected to get 5,6-dibromo-1-methyl-1H-pyrazolo[4,3-b]pyridine (432 mg, 50% yield). .sup.1H NMR (400 MHz, CDCl3) δ ppm 4.08 (s, 3H), 8.06 (s, 1H), 8.14 (s, 1H). MS: [M+1]: 292.0.
Preparation of Intermediates R-Z
[0520] Condensation of a 2,3-diaminoaryl compound with diethyloxalate followed by chlorination of the resulting dione intermediate gives access to the required substituted fused 2,3-dichloropyrazine as illustrated in the Scheme 4.
##STR00213##
##STR00214##
[0521] Step 1. A solution of 3-bromobenzene-1,2-diamine (37.50 g, 200.5 mmol) in diethyl oxalate (205 g, 1.40 mol, 190 mL) was refluxed for 4 h. The reaction mixture was allowed to cool to room temperature, after which EtOAc (500 mL) was added. The precipitate was filtered, washed with EtOAc three times and dried under vacuum to give 5-bromo-1,4-dihydroquinoxaline-2,3-dione (42 g, 87% yield) as a brown powder.
[0522] Step 2. To a mixture of 5-bromo-1,4-dihydroquinoxaline-2,3-dione (42 g, 174 mmol) in thionyl chloride (623 g, 5.24 mol, 380 mL) was added DMF (2.36 g, 32.3 mmol, 2.5 mL). The reaction mixture was heated under reflux for 4 h, cooled to room temperature, and poured very slowly into an ice/water bath with vigorous stirring. The precipitate was filtered, dissolved in EtOAc (750 mL), and dried with Na.sub.2SO.sub.4. 40 g of silica gel was added and the mixture was evaporated to afford a brown residue that was purified by chromatography on silica gel (0-20% EtOAc in hexanes) to provide 5-bromo-2,3-dichloro-quinoxaline (35.4 g, 73% yield) as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.24-8.19 (m, 1H), 8.05-8.00 (m, 1H), 7.81-7.74 (m, 1H).
##STR00215##
[0523] 6-bromo-2,3-dichloro-quinoxaline (15.5 g) as a light orange-brown solid was obtained in two steps from 4-bromobenzene-1,2-diamine. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.29 (dd, J=2.1, 0.5 Hz, 1H), 8.00 (d, J=2.1 Hz, 1H), 7.97 (d, J=0.5 Hz, 1H).
##STR00216##
[0524] 2,3-dichloro-5-methyl-quinoxaline (6.5 g) was obtained as a white solid in two steps from 3-methylbenzene-1,2-diamine. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.81 (dd, J=8.4, 1.4, 1H), 7.65 (dd, J=8.3, 7.1 Hz, 1H), 7.59 (m, 1H), 2.72 (s, 3H). MS: [M+1]: 213.1.
##STR00217##
[0525] 2,3-dichloro-5,8-difluoro-quinoxaline (1.1 g) was obtained as a white solid in two steps from 3,6-difluorobenzene-1,2-diamine. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.55-7.35 (m, 2H).
##STR00218##
[0526] 7-bromo-2,3-dichloro-8-methyl-pyrido[2,3-b]pyrazine (1.6 g) was obtained as a white solid in two steps from 5-bromo-4-methyl-pyridine-2,3-diamine. .sup.1H NMR (400 MHz, Chloroform-d) δ 9.19 (s, 1H), 2.82 (s, 3H). MS: [M+1]: 293.9.
##STR00219##
[0527] 7-bromo-2,3-dichloro-pyrido[2,3-b]pyrazine (14.2 g) was obtained as a white solid in two steps from 5-bromo-pyridine-2,3-diamine. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.20 (d, J=2.4 Hz, 1H), 8.89 (d, J=2.4 Hz, 1H). MS: [M+1]: 280.0.
##STR00220##
[0528] Methyl 2,3-dichloroquinoxaline-5-carboxylate (3.02 g) was obtained as a light reddish brown solid in two steps from methyl 2,3-diaminobenzoate. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.27 (dd, J=8.4, 1.4 Hz, 1H), 8.22 (dd, J=7.3, 1.4 Hz, 1H), 8.00 (dd, J=8.4, 7.3 Hz, 1H), 3.95 (s, 3H). MS: [M+1]: 257.1.
##STR00221##
[0529] Methyl 2,3-dichloroquinoxaline-6-carboxylate (10.9 g) was obtained as a white solid in two steps from methyl 3,4-diaminobenzoate. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.52 (dd, J=1.9, 0.6 Hz, 1H), 8.34 (dd, J=8.7, 1.9 Hz, 1H), 8.18 (dd, J=8.8, 0.6 Hz, 1H), 3.96 (s, 3H).
##STR00222##
[0530] 2,3,5-trichloroquinoxaline (5.5 g) was obtained as a white solid in two steps from 3-chlorobenzene-1,2-diamine. .sup.1H NMR (400 MHz, Chloroform-d) δ 7.92 (dd, J=8.4, 1.3 Hz, 1H), 7.85 (dd, J=7.7, 1.3 Hz, 1H), 7.70 (m, 1H).
Chiral Separation of Selected Compounds
[0531] Racemic mixtures of atropisomers were separated using chiral SFC methods on a Mettler Toledo Minigram SFC (MTM), a Waters Prep 15 SFC-MS (WP15), a Waters Prep 100 SFC-MS (WP100) or a Pic Solution Hybrid 10-150 (PSH) (Table 4). An appropriate column was selected to achieve a satisfactory resolution of the peaks. The appropriate fractions for each peak were combined, concentrated and usually taken in a mixture of water and a suitable water miscible organic solvent such as EtOH, IPA, CH.sub.3CN or a mixture thereof and freeze-dried. The separated products were reanalyzed by chiral SFC to assess chiral purity.
[0532] C1A is Phenomenex Lux Cellulose-2, 10×250 mm, 5 μm; C1B is Phenomenex Lux Cellulose-2, 30×250 mm, 5 μm; C2 is Chiral Technologies IA, 10×250 mm, 5 μm; C3 is Chiral Technologies IC, 10×250 mm, 5 μm; C4 is Chiral Technologies ID, 10×250 mm, 5 μm; C5 is Chiral Technologies IG, 10×250 mm, 5 μm; C6 is Chiral Technologies AS, 10×250 mm, 5 μm; C7 is Phenomenex Lux Cellulose-4, 10×250 mm, 5 μm; C8 is Phenomenex Lux Cellulose-1, 21.2×250 mm, 5 μm.
[0533] Structural assignments of the separated atropisomers were confirmed by biological activity where the biologically active enantiomer was assigned to have the (S) configuration, which was confirmed by X-ray crystallography of key compounds.
TABLE-US-00004 TABLE 4 Peak 1 Peak 2 Mixture Cmpd # Cmpd # Eluent (%, Flow Rate Cmpd # (RT) (RT) Instrument Column (mL/min)) 1 2 3 MTM C3 MeOH (30%, 10) (7.67 min) (11.20 min) 5 6 7 MTM C1A MeOH + 10 mM Ammonium (7.03 min) (13.10 min) Formate (55%, 10) 24 25 26 MTM C2 IPA + 10 mM Ammonium (3.06 min) (5.38 min) Formate (40%, 10) 29 30 31 MTM C3 MeOH + 10 mM Ammonium (6.00 min) (8.31 min) Formate (35%, 10) 40 41 42 MTM C6 MeOH + 10 mM Ammonium (5.88 min) (8.13 min) Formate (35%, 10) 55 56 57 MTM C1A 1:1 ACN/EtOH (55%, 10) (6.81 min) (10.03 min)
Example 2. Enzymatic Assay
[0534] Detection of Myt1 kinase activity utilized a recombinant human Myt1 kinase assay measuring the hydrolysis of ATP using a commercially available ADP-Glo Assay (ADP-Glo™ Kinase Assay from Promega, 10 000 assays, #V9102). Briefly, 5 μL recombinant human Myt1 (full length PKMYT1 recombinant human protein expressed in insect cells from Thermo Fisher #A33387; ˜80% purity) was prepared in reaction buffer (70 mM HEPES, 3 mM MgCl.sub.2, 3 mM MnCl.sub.2, 50 μg/ml PEG 20000, 3 μM Na-orthovanadate, 1.2 mM DTT) and added to 384 well white polystyrene, flat bottom well, non-treated, microplate (Corning #3572). After this, 5 μL of compounds (diluted in reaction buffer to 0.5% DMSO) was added to the microplate and the plate was spun briefly and incubated at 22° C. for 15 minutes. Ultra-Pure Adenosine Triphosphate (ATP) solution (ADP-Glo kit from Promega) was diluted in reaction buffer and 5 μL was added to the microplate, spun down briefly and incubated for 60 minutes at 30° C. The final Myt1 enzyme concentration was 18 nM and the final ATP concentration was 10 μM. After the 60-minute incubation, 15 μL of ADP-Glo reagent was added and the plate was spun briefly and sealed and incubated in the dark for 40 minutes at 22° C. Following this, 30 μL of kinase detection reagent was added per well and the plate was spun briefly, sealed and incubated for 45-60 minutes at 22° C. in the dark. Luminescence was read using the Envision (250 ms integration). The IC.sub.50 and the % max inhibition were calculated for each inhibitor compound tested.
[0535] Exemplary prepared compounds and their activities were shown in Table 5 below.
TABLE-US-00005 TABLE 5 Myt1 MS IC.sub.50 (+ESI) Cpd. Method (nM) [M + 1] NMR 1 A <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (brs, 1H), 348.2 8.01 (brs, 2H), 7.95 (ddd, J = 8.3, 1.5, 0.5 Hz, 1H), 7.81-7.71 (m, 2H), 7.57 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.46 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H), 7.38 (br s, 1H), 7.12 (dt, J = 8.3, 0.7 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 1.83 (s, 3H), 1.76 (s, 3H). 2 A 5000 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 7.99 348.3 (brs, 3H), 7.97-7.90 (m, 1H), 7.81-7.69 (m, 2H), 7.58 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.46 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H), 7.37 (brs, 1H), 7.12 (dt, J = 8.3, 0.8 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 1.83 (s, 3H), 1.75 (s, 3H). 3 A <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 7.99 348.3 (brs, 3H), 7.97-7.90 (m, 1H), 7.81-7.69 (m, 2H), 7.58 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.46 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H), 7.37 (brs, 1H), 7.12 (dt, J = 8.3, 0.8 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 1.83 (s, 3H), 1.75 (s, 3H). 4 A 130 m/z .sup.1H NMR (500 MHz, DMSO-d6) δ 8.16 (brs, 2H), 354.1 7.93 (dd, J = 8.4, 1.4 Hz, 1H), 7.78 (dd, J = 8.3, 1.4 Hz, 1H), 7.73 (br s, 1H), 7.57 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H), 7.46 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H), 7.36 (brs, 1H), 7.10-7.00 (m, 2H). 5 A from arylamine <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 10.56 (brs, 1H), AA5 368.3 8.17 (brs, 2H), 7.95 (ddd, J = 8.3, 1.5, 0.5 Hz, 1H), 7.78 (ddd, J = 8.2, 1.5, 0.5 Hz, 1H), 7.75 (brd, J = 3.1 Hz, 1H), 7.58 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.47 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.39 (brs, 1H), 7.28 (dd, J = 8.5, 0.8 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 1.94 (s, 3H). 6 A from arylamine 8250 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 10.56 (brs, 1H), AA5 368.3 8.17 (brs, 2H), 7.95 (ddd, J = 8.3, 1.5, 0.5 Hz, 1H), 7.78 (ddd, J = 8.2, 1.5, 0.5 Hz, 1H), 7.75 (brd, J = 3.1 Hz, 1H), 7.58 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.47 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.39 (brs, 1H), 7.28 (dd, J = 8.5, 0.8 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 1.94 (s, 3H). 7 A from arylamine <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 10.56 (brs, 1H), AA5 368.3 8.17 (brs, 2H), 7.95 (ddd, J = 8.3, 1.5, 0.5 Hz, 1H), 7.78 (ddd, J = 8.2, 1.5, 0.5 Hz, 1H), 7.75 (brd, J = 3.1 Hz, 1H), 7.58 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.47 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.39 (brs, 1H), 7.28 (dd, J = 8.5, 0.8 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 1.94 (s, 3H). 8 A from arylamine 289 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 13.73 (brs, 1H), AA10 378.2 8.05 (brs, 2H), 7.95 (ddd, J = 8.3, 1.5, 0.5 Hz, 1H), 7.86 (dd, J = 8.6, 0.7 Hz, 1H), 7.79 (brd, J = 3.0 Hz, 1H), 7.71-7.63 (m, 2H), 7.57 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.47-7.40 (m, 2H), 7.38 (br s, 1H). 9 A from arylamine 281 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 13.03 (brs, 1H), AA9 362.2 8.19 (br s, 2H), 7.96 (d, J = 8.3 Hz, 1H), 7.84-7.63 (m, 4H), 7.62 - 7.52 (m, 1H), 7.50-7.33 (m, 3H). 10 A 182 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 13.45 (s, 1H), 8.07 344.1 (brs, 2H), 7.96 (ddd, J = 8.3, 1.5, 0.5 Hz, 1H), 7.89- 7.75 (m, 3H), 7.69 (ddd, J = 8.2, 1.5, 0.5 Hz, 1H), 7.63-7.51 (m, 2H), 7.44 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H), 7.42 - 7.38 (m, 1H), 7.35 (dd, J = 7.3, 0.7 Hz, 1H). 11 A from arylamine 2170 m/z AA13 374.4 12 A from arylamine 98 m/z AA11 362.3 13 A from arylamine 427 m/z AA12 362.3 14 B from arylamine 10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 10.23 (brs, 1H), AA6 368.3 8.18 (br s, 2H), 7.95 (dd, J = 8.5, 1.4 Hz, 1H), 7.82- 7.76 (m, 1H), 7.75 (brd, J = 2.7 Hz, 1H), 7.58 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.47 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H), 7.42 - 7.34 (m, 2H), 7.09 (d, J = 8.8 Hz, 1H), 1.86 (s, 3H). 15 B from arylamine <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) 6 8.33 (brs, 2H), AA7 388.1 7.95 (dd, J = 8.3, 1.4 Hz, 1H), 7.79 (dd, J = 8.3, 1.4 Hz, 1H), 7.74 (brs, J = 3.1 Hz, 1H), 7.60 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.48 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.41 (brd, J = 3.1 Hz, 1H), 7.26 (d, J = 9.0 Hz, 1H). 16 C 94 m/z .sup.1H NMR (400 MHz, DMSO-d6) 6 9.64 (s, 1H), 7.72 356.1 (s, 2H), 7.24 (dd, J = 8.3, 0.8 Hz, 1H), 7.11 (s, 4H), 6.87 (dd, J = 8.3, 2.6 Hz, 1H), 6.68 (d, J = 2.5 Hz, 1H), 1.80 (s, 3H). 17 D 29 m/z .sup.1H NMR (500 MHz, DMSO-d6) δ 9.80 (s, 1H), 8.29 333.3 (d, J = 3.7 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.85 (dd, J = 8.1, 1.4 Hz, 1H), 7.52 (br s + ddd, J = 8.4, 6.8, 1.5 Hz, 3H), 7.36-7.31 (m, 2H), 7.29 (ddd, J = 8.1,6.8, 1.3 Hz, 1H), 7.18 (d, J = 3.5 Hz, 1H), 6.96 (dd, J = 8.4, 2.6 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H), 1.87 (s, 3H). 18 E 12 m/z .sup.1H NMR (500 MHz, DMSO-de) δ 9.65 (s, 1H), 8.44 333.2 (s, 1H), 7.85 (dd, J= 8.1, 1.5 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.45 (br s, 2H), 7.45 - 7.41 (m, 1H), 7.38 (ddd, J = 8.1,6.7, 1.4 Hz, 1H), 7.29 (d, J= 8.4 Hz, 1H), 6.92 (dd, J= 8.4, 2.5 Hz, 1H), 6.86 (brs, 2H), 6.73 (d, J = 2.6 Hz, 1H), 1.84 (s, 3H). 19 F 4060 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.72 (s, 1H), 8.20 362.32 (d, J = 5.6 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.58 (t, J = 7.2 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.94 (dd, J = 8.4, 2.4 Hz, 1H), 6.81 (d, J = 2.4 Hz, 1H), 3.48 - 3.40 (m, 2H), 1.88 (s, 3H), 1.23 (t, J = 7.2 Hz, 3H). 20 F 7580 m/z .sup.1H NMR (400 MHz, DMSO-de) δ 9.75 (bs, 1H), 8.35 374.32 (s, 1H), 8.18 (bs, 1H), 8.00 (d, J= 7.6 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 7.49 (t, J = 7.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.83 (s, 1H), 2.88 (s, 1H), 1.89 (s, 3H), 0.82 (d, J = 6.0 Hz, 2H), 0.67 (bs, 2H). 21 F 1160 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.40 378.26 (d, J = 5.2 Hz, 1H), 8.04-7.92 (m, 3H), 7.79 (d, J = 8.0 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.83 (s, 1H), 4.91 (t, J = 4.4 Hz, 1H), 3.62 (t, J = 6.0 Hz, 2H), 3.52 (t, J = 5.2 Hz, 2H), 1.89 (s, 3H). 22 F 690 m/z .sup.1HNMR(400 MHz, DMSO-d6) δ 9.75 (s, 1H), 8.11- 348.46 7.96 (m, 3H), 7.79 (d, J = 8.4 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.50-7.56 (m, 1H), 7.32 (d, J = 8.0 Hz, 1H), 6.96 (dd, J = 8.4, 2.4 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 2.97 (d, J = 4.4 Hz, 3H), 1.89 (s, 3H). 23 A then G 323 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 8.91 (brs, 1H), 319.1 8.03 (s, 1H), 7.53 - 7.40 (m, 1H), 7.32-7.20 (m, 2H), 7.09 - 6.90 (m, 3H), 6.46 (d, J = 8.2 Hz, 1H), 6.17-6.00 (m, 2H), 1.14 (s, 3H). 24 A then G 11 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.67 (s, 1H), 8.82 333.4 (s, 1H), 8.37-8.24 (m, 1H), 8.11-8.04 (m, 2H), 7.91-7.73 (m, 3H), 7.11 (dt, J = 8.3, 0.7 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 1.81 (s, 3H), 1.71 (s, 3H). 25 A then G 5000 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.67 (s, 1H), 8.82 333.4 (s, 1H), 8.37-8.24 (m, 1H), 8.11-8.04 (m, 2H), 7.91 - 7.73 (m, 3H), 7.11 (dt, J = 8.3, 0.7 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 1.81 (s, 3H), 1.71 (s, 3H). 26 A then G <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.67 (s, 1H), 8.82 333.4 (s, 1H), 8.37-8.24 (m, 1H), 8.11-8.04 (m, 2H), 7.91-7.73 (m, 3H), 7.11 (dt, J = 8.3, 0.7 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 1.81 (s, 3H), 1.71 (s, 3H). 28 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.22 intermediate U 384.2 (s, 2H), 7.63 (d, J = 3.1 Hz, 1H), 7.46 (d, J = 3.1 Hz, 1H), 7.36 (ddd, J = 10.3, 8.7, 4.3 Hz, 1H), 7.23 (ddd, J = 10.2, 8.7, 4.2 Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 1.80 (s, 3H), 1.72 (s, 3H). 29 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.12 intermediate R 428.0 (m, 2H), 7.89 (dd, J = 7.6, 1.3 Hz, 1H), 7.82 (d, J = 3.3 Hz, 1H), 7.77 (dd, J = 8.3, 1.3 Hz, 1H), 7.48 (d, J = 3.3 Hz, 1H), 7.33 (dd, J = 8.3, 7.6 Hz, 1H), 7.08 (dt, J = 8.3, 0.8 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). 30 A from 1180 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.12 intermediate R 428.0 (m, 2H), 7.89 (dd, J = 7.6, 1.3 Hz, 1H), 7.82 (d, J = 3.3 Hz, 1H), 7.77 (dd, J = 8.3, 1.3 Hz, 1H), 7.48 (d, J = 3.3 Hz, 1H), 7.33 (dd, J = 8.3, 7.6 Hz, 1H), 7.08 (dt, J = 8.3, 0.8 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). 31 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.12 intermediate R 428.0 (m, 2H), 7.89 (dd, J = 7.6, 1.3 Hz, 1H), 7.82 (d, J = 3.3 Hz, 1H), 7.77 (dd, J = 8.3, 1.3 Hz, 1H), 7.48 (d, J = 3.3 Hz, 1H), 7.33 (dd, J = 8.3, 7.6 Hz, 1H), 7.08 (dt, J = 8.3, 0.8 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). 32 H on compound 29 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 8.31 373.2 (s, 2H), 8.18-8.02 (m, 2H), 7.69 (d, J = 3.1 Hz, 1H), 7.55 (d, J = 6.7 Hz, 1H), 7.51 (dd, J = 8.4, 7.3 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). 33 I on compound 29 16 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 8.02 412.3 (s, 2H), 7.88 (d, J = 3.4 Hz, 1H), 7.68 (dd, J = 8.3, 1.4 Hz, 1H), 7.59 (dd, J = 7.3, 1.4 Hz, 1H), 7.54- 7.47 (m, 1H), 7.33 (dd, J = 8.3, 7.3 Hz, 1H), 7.08 (dt, J = 8.3, 0.7 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 1.82- 1.75 (m, 3H), 1.71 (s, 3H), 1.61 (tt, J = 8.2, 5.0 Hz, 1H), 0.98-0.88 (m, 2H), 0.81 - 0.69 (m, 2H). 34 J on compound 29 12 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 7.95 430.3 (s, 2H), 7.74-7.60 (m, 2H), 7.44-7.33 (m, 3H), 7.08 (dt, J = 8.3, 0.8 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 5.91 (m, 1H), 4.26 (m, 2H), 3.85 (t, J = 5.3 Hz, 2H), 2.66 (m, 2H), 1.79 (d, J = 0.7 Hz, 3H), 1.71 (s, 3H). 35 J on compound 29 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 7.95 428.3 (s, 2H), 7.76 (dd, J = 8.2, 1.6 Hz, 1H), 7.73-7.67 (m, 2H), 7.64-7.31 (m, 6H), 7.19 (d, J = 3.4 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 1.81 (s, 3H), 1.73 (s, 3H). 36 J on compound 29 17 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 7.95 424.3 (s, 2H), 7.76 (dd, J = 8.2, 1.6 Hz, 1H), 7.73 - 7.67 (m, 2H), 7.64 - 7.31 (m, 6H), 7.19 (d, J = 3.4 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 1.81 (s, 3H), 1.73 (s, 3H). 37 J on compound 29 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 7.95 414.3 (s, 2H), 7.73 - 7.57 (m, 2H), 7.47 (dd, J = 7.3, 1.6 Hz, 1H), 7.43 - 7.32 (m, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.48 (m, 1H), 2.91 (m, 2H), 2.54 (m, 2H), 1.98 (m, 2H), 1.79 (s, 3H), 1.72 (s, 3H). 38 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 8.09 intermediate S 426.1 (d, J = 2.3 Hz, 3H), 7.66 (d, J = 8.8 Hz, 1H), 7.63 (dq, J = 4.9, 2.3 Hz, 1H), 7.50 (dd, J = 8.8, 2.3 Hz, 1H), 7.43 - 7.32 (m, 1H), 7.06 (dt, J = 8.2, 0.8 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 1.81 - 1.73 (m, 3H), 1.69 (s, 3H). 39 H on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.42 373.2 (d, J = 1.8 Hz, 1H), 8.39-8.12 (m, 2H), 7.87 (d, J = 8.5 Hz, 1H), 7.71 (dd, J = 8.5, 1.9 Hz, 1H), 7.56 (s, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 1.79 (s, 3H), 1.71 (s, 3H). 40 1 on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.03 412.3 (s, 2H), 7.86 (dd, J = 1.9, 0.5 Hz, 1H), 7.68 - 7.62 (m, 2H), 7.39-7.29 (m, 2H), 7.08 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 1.82-1.74 (m, 3H), 1.70 (s, 3H), 1.55 (m, 1H), 0.92 - 0.82 (m, 2H), 0.78-0.67 (m, 2H). 41 I on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.03 412.3 (s, 2H), 7.86 (dd, J = 1.9, 0.5 Hz, 1H), 7.68-7.62 (m, 2H), 7.39-7.29 (m, 2H), 7.08 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 1.82-1.74 (m, 3H), 1.70 (s, 3H), 1.55 (m, 1H), 0.92-0.82 (m, 2H), 0.78-0.67 (m, 2H). 42 I on compound 38 1330 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.03 412.3 (s, 2H), 7.86 (dd, J = 1.9, 0.5 Hz, 1H), 7.68-7.62 (m, 2H), 7.39-7.29 (m, 2H), 7.08 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 1.82-1.74 (m, 3H), 1.70 (s, 3H), 1.55 (m, 1H), 0.92-0.82 (m, 2H), 0.78-0.67 (m, 2H). 43 I on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.76 - 9.54 (m, 450.2 1H), 8.91 (d, J = 1.5 Hz, 1H), 8.68 (dd, J = 2.6, 1.5 Hz, 1H), 8.67-8.58 (m, 1H), 8.34-7.99 (m, 3H), 7.80 (d, J = 8.5 Hz, 1H), 7.70 (d, J = 13.4 Hz, 1H), 7.63-7.48 (m, 1H), 7.41 (s, 1H), 7.13-7.05 (m, 1H), 6.95 (d, J = 8.3 Hz, 1H), 1.85-1.76 (m, 3H), 1.73 (s, 3H). 44 I on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 8.06 471.3 (s, 2H), 7.96 (d, J = 1.9 Hz, 1H), 7.73-7.61 (m, 2H), 7.39 (td, J = 8.9, 2.4 Hz, 2H), 7.08 (dt, J = 8.3, 0.8 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 3.59 (t, J = 4.7 Hz, 4H), 3.52 (s, 2H), 2.52 (dd, J = 5.6, 3.6 Hz, 4H), 1.84-1.75 (m, 3H), 1.71 (s, 3H). 45 I on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.06 402.4 (s, 2H), 7.93 (d, J = 1.8 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 3.2 Hz, 1H), 7.44-7.33 (m, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 4.31 (s, 2H), 1.79 (s, 3H), 1.71 (s, 3H). 46 Reduction of <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 8.22-7.69 (m, compound 45 406.2 5H), 7.64 (dd, J = 8.4, 1.5 Hz, 1H), 7.40-7.23 (m, 2H), 7.07 (d, J = 8.3 Hz, 1H), 6.94 (dd, J = 8.3, 1.4 Hz, 1H), 3.41 (t, J = 6.4 Hz, 4H), 2.76 (t, J = 7.6 Hz, 3H), 1.78 (d, J = 6.1 Hz, 5H), 1.71 (d, J = 1.5 Hz, 3H). 47 J on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 8.19 424.3 (d, J = 2.0 Hz, 1H), 8.13-7.91 (m, 2H), 7.87-7.67 (m, 5H), 7.47 (m, 2H), 7.43-7.31 (m, 2H), 7.09 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 1.81 (s, 3H), 1.73 (s, 3H). 48 J on compound 38 11 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 8.31 449.3 (s, 2H), 8.17 (m, 1H), 8.03 (m, 2H), 7.92-7.61 (m, 4H), 7.47 (m, 2H), 7.09 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H), 1.81 (s, 3H), 1.73 (s, 3H) 49 J on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 9.03 425.3 (dd, J = 2.4, 0.8 Hz, 1H), 8.56 (dd, J = 4.7, 1.6 Hz, 1H), 8.27 (dd, J = 2.0, 0.6 Hz, 1H), 8.22 (m, 1H), 8.12-7.91 (m, 2H), 7.89-7.78 (m, 2H), 7.76 (d, J = 3.0 Hz, 1H), 7.48 m, 1H), 7.45-7.35 (m, 1H), 7.09 (dt, J = 8.3, 0.7 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 1.81 (d, J = 0.7 Hz, 3H), 1.73 (s, 3H). 50 J on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.63 (s, 1H), 8.18 - 428.3 7.93 (m, 3H), 7.83 (dd, J = 8.5, 0.5 Hz, 1H), 7.72 (d, J = 4.9 Hz, 1H), 7.57 (dd, J = 8.6, 2.1 Hz, 1H), 7.47 (d, J = 1.9 Hz, 1H), 7.39 (d, J = 3.0 Hz, 1H), 7.09 (dt, J = 8.3, 0.7 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.50 (d, J = 1.9 Hz, 1H), 3.92 (s, 3H), 1.81 (d, J = 0.7 Hz, 3H), 1.73 (s, 3H). 51 J on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 8.13- 430.3 7.87 (m, 3H), 7.76-7.71 (m, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.61 (m, 1H), 7.35 (d, J = 3.2 Hz, 1H), 7.08 (m, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.43 (m, 1H), 4.23 (m, 2H), 3.83 (t, J = 5.5 Hz, 2H), 2.56 (m, 2H), 1.79 (s, 3H), 1.71 (s, 3H). 52 J on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) 6 9.61 (s, 1H), 8.10 552.3 (dd, J = 2.0, 0.6 Hz, 1H), 7.93 (d, J = 16.6 Hz, 2H), 7.76 (m, 1H), 7.74 (d, J = 0.5 Hz, 1H), 7.72 (d, J = 2.0 Hz, 1H), 7.69 (m, 1H), 7.67 (d, J = 1.9 Hz, 1H), 7.40 - 7.32 (m, 1H), 7.09 (dt, J = 8.3, 0.8 Hz, 1H), 7.04 - 6.98 (m, 2H), 6.95 (d, J = 8.3 Hz, 1H), 4.42 (s, 1H), 3.51 (d, J = 12.4 Hz, 2H), 3.17 (m, 4H), 2.55 (m, 4H), 2.42 (t, J = 6.2 Hz, 2H), 1.85-1.76 (m, 3H), 1.73 (s, 3H). 53 J on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.11- 414.3 7.84 (m, 2H), 7.81 (m, 1H), 7.75-7.68 (m, 1H), 7.66 (m, 2H), 7.33 (d, J = 3.3 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.43 (m, 1H), 2.82-2.72 (m, 2H), 2.56-2.48 (m, 2H), 1.97 (p, J = 7.6 Hz, 2H), 1.79 (s, 3H), 1.71 (s, 3H). 54 J on compound 38 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 9.68 414.2 (s, 1H), 8.35 - 8.03 (m, 3H), 7.91 (s, 2H), 7.74 (d, J = 3.3 Hz, 1H), 7.73 - 7.66 (m, 2H), 7.35 (d, J = 3.3 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 1.79 (s, 3H), 1.71 (s, 3H). 55 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 8.13 intermediate S 426.1 (d, J = 23.0 Hz, 2H), 7.97 (d, J = 2.3 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.74 - 7.61 (m, 2H), 7.41 (d, J = 3.3 Hz, 1H), 7.11 (dt, J = 8.3, 0.8 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 1.85 - 1.78 (m, 3H), 1.73 (s, 3H). 56 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 7.94 intermediate S 426.1 (m, 3H), 7.84 (d, J = 8.8 Hz, 1H), 7.73-7.55 (m, 2H), 7.38 (d, J = 3.1 Hz, 1H), 7.08 (dt, J = 8.2, 0.7 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 1.83-1.74 (s, 3H), 1.71 (s, 3H). 57 A from 1140 m/z .sup.1H NMR (400 MHz, DMSO-d6) 5 9.62 (s, 1H), 8.42- intermediate S 426.1 7.91 (m, 3H), 7.84 (d, J = 8.9 Hz, 1H), 7.65 (m, 2H), 7.38 (s, 1H), 7.08 (dt, J = 8.3, 0.8 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 1.84-1.74 (s, 3H), 1.71 (s, 3H). 58 I on compound 55 11 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.78 (s, 1H), 7.99 402.4 (d, J = 67.1 Hz, 2H), 7.86 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 1.9 Hz, 1H), 7.67 (s, 1H), 7.52 (dd, J = 8.6, 1.9 Hz, 1H), 7.39 (s, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 4.28 (s, 2H), 1.79 (s, 3H), 1.71 (s, 3H). 59 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 8.12 intermediate R 428.0 (s, 2H), 7.96 (dd, J = 8.3, 1.3 Hz, 1H), 7.80 (dd, J = 7.6, 1.3 Hz, 1H), 7.70 (d, J = 3.1 Hz, 1H), 7.49 (dd, J = 8.3, 7.6 Hz, 1H), 7.44 (d, J = 3.2 Hz, 1H), 7.13 (dt, J = 8.3, 0.8 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 1.86 (d, J = 0.7 Hz, 3H), 1.78 (s, 3H). 60 H on compound 59 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 8.44- 373.2 8.03 (m, 3H), 7.96 (dd, J = 7.3, 1.3 Hz, 1H), 7.79- 7.32 (m, 3H), 7.10 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 1.82 (s, 3H), 1.74 (s, 3H). 61 J on compound 59 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.02 428.3 (dd, J = 8.3, 1.5 Hz, 3H), 7.71 (d, J = 3.1 Hz, 1H), 7.61 (dd, J = 8.3, 7.2 Hz, 1H), 7.44 (dd, J = 7.2, 1.5 Hz, 1H), 7.41-7.35 (m, 1H), 7.32 (d, J = 1.8 Hz, 1H), 7.06-6.99 (m, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.23 (d, J = 1.8 Hz, 1H), 3.40 (s, 3H), 1.75 (d, J = 0.7 Hz, 3H), 1.68 (s, 3H). 62 J on compound 59 <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 7.98 414.3 (s, 2H), 7.79 (dd, J = 8.3, 1.4 Hz, 1H), 7.71-7.59 (m, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.38 (dd, J = 7.4, 1.4 Hz, 1H), 7.35-7.27 (m, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.47 (t, J = 2.2 Hz, 1H), 2.71 (m, 2H), 2.36 - 2.23 (m, 2H), 1.82 (s, 3H), 1.75 (m, 5H). 63 K on acid 31 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 8.22 (s, 1H), 8.11 intermediate 1 Arl+.A: (br s, 2H), 7.97 (dd, J = 8.5, 2.1 Hz, 1H), 7.78-7.66 (m, 2H), 7.56 (dd, J = 8.5, 2.0 Hz, 1H), 7.49-7.32 (m, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 3.39 (brs, 4H), 2.31 (brs, 4H), 2.17 (s, 3H), 1.83 (s, 3H), 1.76 (s, 3H). 64 K on acid <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.69 (brs, 1H), intermediate 1 391.4 8.41 (d, J = 2.0 Hz, 1H), 8.16 (brs, 2H), 8.04 (dd, J = 8.7, 2.0 Hz, 1H), 8.01 (brs, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.74 (brs, J = 3.1 Hz, 1H), 7.43 (brs, 1H), 7.35 (brs, 1H), 7.12 (dt, J = 8.3, 0.8 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 1.84 (s, 3H), 1.76 (s, 3H). 65 K on acid 11 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.57 - intermediate 1 449.3 8.43 (m, 1H), 8.37 (d, J = 1.9 Hz, 1H), 8.17 (brs, 2H), 8.01 (dd, J = 8.7, 2.0 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.73 (brs, 1H), 7.43 (brs, 1H), 7.12 (dt, J = 8.3, 0.7 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 4.45 (t, J = 5.2 Hz, 1H), 3.45 (q, J = 6.1 Hz, 2H), 3.39-3.26 (m, 2H), 1.84 (s, 3H), 1.76 (s, 3H), 1.67 (d, J = 6.8 Hz, 2H). 66 K on acid 13 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 8.12 intermediate 1 461.4 (brs, 2H), 7.98 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.74 (brd, J = 3.3 Hz, 1H), 7.59 (dd, J = 8.5, 1.9 Hz, 1H), 7.43 (brs, 1H), 7.13 (dt, J = 8.3, 0.8 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 3.55 (br m, 8H), 1.83 (s, 3H), 1.76 (s, 3H). 67 L on ester 10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.03- intermediate 1 406.5 7.90 (m, 2H), 7.88 (dd, J = 8.6, 0.5 Hz, 1H), 7.77 (dd, J = 2.2, 0.5 Hz, 1H), 7.76-7.66 (m, 2H), 7.34 (brs, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 5.09 (s, 1H), 1.83 (s, 3H), 1.75 (s, 3H), 1.48 (s, 6H). 68 L on ester 13 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.01 intermediate 1 388.4 (br s, 2H), 7.89 (dd, J = 7.8, 1.8 Hz, 1H), 7.85 - 7.78 (m, 2H), 7.73 (d, J = 3.2 Hz, 1H), 7.38 (d, J = 3.2 Hz, 1H), 7.12 (dt, J = 8.3, 0.8 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 5.60 (s, 1H), 5.16 (t, J = 1.5 Hz, 1H), 2.18 (s, 3H), 1.83 (s, 3H), 1.75 (s, 3H). 69 A, from 1010 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.29 intermediate X 378.2 (brs, 2H), 8.03 (dd, J = 7.3, 1.5 Hz, 1H), 7.97 (dd, J = 8.3, 1.5 Hz, 1H), 7.53 (dd, J = 8.3, 7.3 Hz, 1H), 7.46 (brs, 2H), 7.31 (dd, J = 8.3, 0.8 Hz, 1H), 6.95 (dd, J = 8.3, 2.6 Hz, 1H), 6.84 (d, J = 2.6 Hz, 1H), 1.90 (s, 3H). 70 A, from 2300 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.79 (brs, 1H), intermediate X 378.2 8.35 (brs, 2H), 8.21 (dd, J = 8.3, 1.5 Hz, 1H), 8.11 (d, J = 7.3 Hz, 1H), 7.71 (dd, J = 8.3, 7.4 Hz, 1H), 7.66 (s, 1H), 7.49 (s, 1H), 7.34 (d, J = 8.5 Hz, 1H), 6.97 (dd, J = 8.3, 2.6 Hz, 1H), 6.89 (d, J = 2.5 Hz, 1H), 1.94 (s, 3H). 71 A from 377 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.72 (s, 1H), 8.14 intermediate X 392.2 (br s, 2H), 7.95 (dd, J = 8.3, 1.5 Hz, 1H), 7.92-7.84 (m, 2H), 7.50 (dd, J = 8.3, 7.3 Hz, 1H), 7.41-7.33 (m, 1H), 7.30 (dd, J = 8.4, 0.8 Hz, 1H), 6.95 (dd, J = 8.3, 2.6 Hz, 1H), 6.83 (d, J = 2.6 Hz, 1H), 3.92 (s, 3H), 1.89 (s, 3H). 72 K on acid 62 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.76 (brs, 1H), intermediate 2 377.3 8.46 (s, 1H), 8.17 (brs, 2H), 7.92 (dd, J = 7.3, 1.6 Hz, 1H), 7.87 (dd, J = 8.2, 1.6 Hz, 1H), 7.79 (s, 1H), 7.49 (brs and dd, J = 8.2, 7.3 Hz, 2H), 7.31 (dd, J = 8.4, 0.8 Hz, 1H), 6.95 (dd, J = 8.3, 2.6 Hz, 1H), 6.82 (d, J = 2.5 Hz, 1H), 1.89(S, 3H). 73 K on acid 339 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.98 intermediate 2 405.4 (t, J = 5.5 Hz, 1H), 8.17 (br s, 2H), 7.93-7.77 (m, 2H), 7.63-7.38 (m, 3H), 7.31 (dd, J = 8.3, 0.8 Hz, 1H), 6.95 (dd, J = 8.3, 2.5 Hz, 1H), 6.82 (d, J = 2.6 Hz, 1H), 3.47-3.36 (m, 2H), 1.88 (s, 3H), 1.21 (t, J = 7.2 Hz, 3H). 74 K on acid 2270 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.74 (brs, 1H), intermediate 2 405.2 8.08 (brs, 2H), 7.81 (dd, J = 7.1,2.6 Hz, 1H), 7.60 (d, J = 3.3 Hz, 1H), 7.51-7.42 (m, 2H), 7.35 (d, J = 3.2 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 6.95 (dd, J = 8.3, 2.6 Hz, 1H), 6.89-6.69 (m, 1H), 3.11 (s, 3H), 2.73 (d, J = 2.3 Hz, 3H), 1.92 and 1.88 (2 s, 3H). Splitting coalesced at 60° C. 75 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 8.12 intermediate Z 382.2 (s, 3H), 7.80 (d, J = 3.3 Hz, 1H), 7.76-7.69 (m, 2H), 7.45 (d, J = 3.3 Hz, 1H), 7.39 (dd, J = 8.3, 7.7 Hz, 1H), 7.08 (dt, J = 8.3, 0.7 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 1.79 (d, J = 0.7 Hz, 3H), 1.72 (s, 3H). 76 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.13 intermediate Z 382.2 (m, 3H), 7.88 (dd, J = 8.3, 1.4 Hz, 1H), 7.66 (d, J = 3.2 Hz, 1H), 7.58 (dd, J = 7.6, 1.5 Hz, 1H), 7.53- 7.47 (m, 1H), 7.45-7.39 (m, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 1.81 (s, 3H), 1.73 (s, 3H). 77 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 7.95 intermediate T 362.2 (s, 2H), 7.74-7.60 (m, 2H), 7.44-7.33 (m, 3H), 7.08 (dt, J = 8.3, 0.8 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 5.91 (m, 1H), 4.26 (m, 2H), 3.85 (t, J = 5.3 Hz, 2H), 2.66 (m, 2H), 1.79 (d, J = 0.7 Hz, 3H), 1.71 (s, 3H). 78 A from <10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.12 intermediate T 362.2 (s, 1H), 7.89 (s, 2H), 7.77-7.66 (m, 2H), 7.42 (dd, J = 8.3, 7.0 Hz, 1H), 7.36-7.26 (m, 2H), 7.09 (dt, J = 8.3, 0.7 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 2.42 (d, J = 0.8 Hz, 3H), 1.81 (d, J = 0.7 Hz, 3H), 1.74 (s, 3H). 79 A from 30 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.78 intermediate W 429.0 (d, J = 2.4 Hz, 1H), 8.45 (d, J = 2.3 Hz, 1H), 8.30 (s, 2H), 7.75-7.57 (m, 1H), 7.44 (s, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 1.77 (s, 3H), 1.69 (s, 3H). 80 A from 12 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 8.83 intermediate W 429.0 (s, 1H), 8.50 (d, J = 2.2 Hz, 1H), 8.35 (s, 2H), 7.78-7.34 (m, 2H), 7.08 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). 81 A from 5000 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.62 (s, 1H), 8.83 intermediate W 429.0 (s, 1H), 8.51 (d, J = 2.2 Hz, 1H), 8.36 (s, 2H), 7.57 (d, J = 63.4 Hz, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). 82 Reduction of 21 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.65 (s, 1H), 9.09 compound 79 349.2 (s, 1H), 8.96-8.84 (m, 2H), 8.82 (dd, J = 8.2, 1.5 Hz, 1H), 7.78 (dd, J = 8.2, 5.7 Hz, 2H), 7.48 (s, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 1.79 (s, 3H), 1.72 (s, 3H). 83 A from 12 m/z intermediate V, 363.3 then reduction of Br 84 A 11 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 7.38 352.4 (s, 1H), 7.14 (s, 2H), 7.08 (s, 1H), 7.05-6.99 (m, 1H), 6.89 (d, J = 8.3 Hz, 1H), 2.84 (t, J = 6.0 Hz, 2H), 2.66 (d, J = 6.1 Hz, 3H), 1.76 (d, J = 6.0 Hz, 3H), 1.73 (s, 3H), 1.65 (s, 3H). 85 A from arylamine 48900 m/z AA14 374.3 86 A 53 m/z .sup.1H NMR (500 MHz, DMSO-d6) δ 13.33 (s, 1H), 8.03 358.2 (brs, 2H), 7.96 (dd, J = 8.4, 1.4 Hz, 1H), 7.81 (brd, J = 3.3 Hz, 1H), 7.76-7.71 (m, 2H), 7.68 (dd, J = 8.3, 1.4 Hz, 1H), 7.57 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.43 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H), 7.39 (brs, 1H), 2.12 (s, 3H). 87 A 10 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 7.90 334.1 (dd, J = 8.3, 1.5 Hz, 2H), 7.78-7.67 (m, 2H), 7.53 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H), 7.42 (ddd, J = 8.3, 7.0, 1.5 Hz, 1H), 7.33 (s, 1H), 7.21 (t, J = 7.9 Hz, 1H), 7.03 (dd, J = 8.2, 1.2 Hz, 1H), 6.85 (dd, J = 7.8, 1.1 Hz, 1H), 1.76 (s, 3H). 88 E then G, from 32 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ ppm 1.67 (s, 3 H), arylamine AA1 and 332.1 1.78 (s, 3 H), 6.94 (d, J = 8.3 Hz, 1 H), 7.08 (d, J = intermediate P 8.3 Hz, 1 H), 7.12 - 7.27 (m, 1 H), 7.59 (ddd, J = 8.0, 6.9, 1.0 Hz, 1 H), 7.73 (br. s, 1 H), 7.81 (ddd, J = 8.5, 7.0, 1.3 Hz, 1 H), 8.09 (s, 1 H), 8.16 (d, J = 8.1 Hz, 1 H), 8.92 (s, 1 H), 9.56 (s, 1 H), 9.65 (d, J = 8.6 Hz, 1 H). 89 E then G, from 31 m/z .sup.1H NMR (400 MHz, DMSO-d6) δ ppm 1.67 (s, 3 H), arylamine AA1 and 336.3 1.77 (s, 3 H), 4.15 (s, 3 H), 6.93 (d, J = 8.3Hz, 1 H), intermediate Q 7.07 (d, J = 8.3 Hz, 1 H), 7.21 (br. s, 1H), 7.55 (br. s, 1 H), 8.13 (d, J = 1.0 Hz, 1 H), 8.34 (s, 1 H), 8.77 (d, J = 0.7 Hz, 1 H), 9.56 (s, 1 H).
In Table 5, the Method column indicates a preparatory method described above used in the preparation of the compounds.
Example 3. Genetic Validation
[0536] Two sgRNAs for PKMYT1 and one sgRNA for LacZ (control) were transduced into the RPE1-hTERT Cas9 TP53−/− parental (WT) and CCNE1-overexpressing clones. Infected cells were plated at low density to measure their ability to form colonies of <50 cells. After 10 days of growth, the colonies were stained, imaged, and quantified. Using clonogenic survival assays, we observed a profound cellular fitness defect in CCNE1-overexpressing cells compared to parental cells transduced with PKMYT1 sgRNAs (
[0537] To determine if the kinase activity of PKMYT1 was responsible for maintaining the viability of CCNE1-overexpressing RPE1-hTERT Cas9 TP53−/− cells, the PKMYT1 open reading frame (ORF) was cloned into an inducible mammalian expression vector. sgRNA-resistant silent mutations in the PKMYT1 ORF sequence were then created by PCR mutagenesis. A single point mutation was generated that resulted in an asparagine (N) to alanine (A) amino acid change at residue 238. The N.sub.238A amino acid change in the kinase domain resulted in a catalytically inactive PKMYT1 mutant. Stable cell lines in the RPE1-hTERT Cas9 TP53−/− parental and CCNE1-overexpressing clones were generated that either expressed the wild type PKMYT1 ORF or the kinase-dead N238A mutant (
Example 4. Pharmacological Validation
[0538] ##STR00223##
[0539] RPE1-hTERT Cas9 TP53−/− parental (WT) and CCNE1-overexpressing clones were treated with compound A in a dose titration and cell viability was determined. The CCNE1-overexpressing cells were found to be more sensitive to compound A than the corresponding WT cells (
[0540] A panel of 16 cancer cell lines with either normal (n=8) or elevated levels of CCNE1 (n=8) was evaluated for their sensitivity to compound B in a cell proliferation assay (
[0541] A similar experiment was conducted in a panel of 8 cancer cell lines with either wild-type FBXW7 (n=5) or FBXW7-mutations (n=3) in which these cells were evaluated for their sensitivity to compound C in a cell proliferation assay (
Other Embodiments
[0542] Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.
[0543] Other embodiments are in the claims.