Small Molecule Reversible Inhibitors of Bruton's Tyrosine Kinase (BTK): Structure-Activity Relationships Leading to the Identification of 7-(2-Hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-dihydroquinazolin-3-yl)phenyl]-9H-carbazole-1-carboxamide (BMS-935177)

Article abstract of DOI:10.1021/acs.jmedchem.6b00722

Bruton's tyrosine kinase (BTK) belongs to the TEC family of nonreceptor tyrosine kinases and plays a critical role in multiple cell types responsible for numerous autoimmune diseases. This article will detail the structure-activity relationships (SARs) leading to a novel second generation series of potent and selective reversible carbazole inhibitors of BTK. With an excellent pharmacokinetic profile as well as demonstrated in vivo activity and an acceptable safety profile, 7-(2-hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-dihydroquinazolin-3-yl)phenyl]-9H-carbazole-1-carboxamide 6 (BMS-935177) was selected to advance into clinical development.

Full text of DOI:10.1021/acs.jmedchem.6b00722

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Article
Small Molecule Reversible Inhibitors of Bruton’s Tyrosine
Kinase (BTK): Structure-Activity Relationships Leading to the
Identification of 7-(2-hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-
dihydroquinazolin-3-yl)phenyl]-9H-carbazole-1-carboxamide (BMS-935177)
George Vincent De Lucca, Qing Shi, Qingjie Liu, Douglas G. Batt, Myra Beaudoin Bertrand, Richard Rampulla,
Arvind Mathur, Lorell N. Discenza, Celia J. D´Arienzo, Jun Dai, Mary T. Obermeier, Rodney Vickery, Yingru
Zhang, Zheng Yang, Punit Marathe, Andrew Joseph Tebben, Jodi K. Muckelbauer, ChiehYing J Chang,
Huiping Zhang, Kathleen M. Gillooly, Tracy L. Taylor, Mark A. Pattoli, Stacey Skala, Daniel W Kukral, Kim W.
McIntyre, Luisa Salter-Cid, Aberra Fura, James R. Burke, Joel C. Barrish, Percy H Carter, and Joseph A Tino
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00722 • Publication Date (Web): 17 Aug 2016
Downloaded from http://pubs.acs.org on August 17, 2016
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Development, Metabolism and Pharmacokinetic Department,
Pharmaceutical Candidate Optimization
Tebben, Andrew; Bristol-Myers Squibb Pharmaceutical Research and
Development, Molecular Structure and Design, Molecular Discovery
Technologies
Muckelbauer, Jodi; Bristol-Myers Squibb Pharmaceutical Research and
Development, Molecular Structure and Design, Molecular Discovery
Technologies
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Chang, ChiehYing; Bristol-Myers Squibb Pharmaceutical Research and
Development, Molecular Structure and Design, Molecular Discovery
Technologies
Zhang, Huiping; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Chemistry
Gillooly, Kathleen; Bristol-Myers Squibb, Immunology biology
Taylor, Tracy; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Biology
Pattoli, Mark; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Biology
Skala, Stacey; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Biology
Kukral, Daniel; Bristol-Myers Squibb Pharmaceutical Research and
Development, ECTR/CTTO Imaging Department
McIntyre, Kim; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Biology
Salter-Cid, Luisa; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Biology
Fura, Aberra; Bristol-Myers Squibb Pharmaceutical Research and
Development, Metabolism and Pharmacokinetic Department,
Pharmaceutical Candidate Optimization
Burke, James; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Biology
Barrish, Joel; Bristol-Myers Squibb Pharmaceutical Research and
Development , Discovery Chemistry
Carter, Percy; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Chemistry
Tino, Joseph; Bristol-Myers Squibb Pharmaceutical Research and
Development, Discovery Chemistry
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Small Molecule Reversible Inhibitors of Bruton’s
Tyrosine Kinase (BTK): StructureꢀActivity
Relationships Leading to the Identification of 7ꢀ(2ꢀ
hydroxypropanꢀ2ꢀyl)ꢀ4ꢀ[2ꢀmethylꢀ3ꢀ(4ꢀoxoꢀ3,4ꢀ
dihydroquinazolinꢀ3ꢀyl)phenyl]ꢀ9Hꢀcarbazoleꢀ1ꢀ
carboxamide (BMSꢀ935177)
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George V. De Lucca,* Qing Shi, Qingjie Liu, Douglas G. Batt, Myra Beaudoin Bertrand, Rick
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Rampulla, Arvind Mathur, Lorell Discenza, Celia D’Arienzo, Jun Dai, Mary Obermeier, Rodney
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Vickery, Yingru Zhang, Zheng Yang, Punit Marathe, Andrew J. Tebben, Jodi K. Muckelbauer,
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ChiehYing J. Chang, Huiping Zhang, Kathleen Gillooly, Tracy Taylor, Mark A. Pattoli, Stacey
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Skala, Daniel W. Kukral, Kim W. McIntyre, Luisa Salter-Cid, Aberra Fura, James R. Burke, Joel
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C. Barrish, Percy H. Carter, and Joseph A. Tino*
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Immunosciences Discovery Chemistry, Immunoscience Discovery Biology, Molecular Structure and
d
Design, Molecular Discovery Technologies, Metabolism and Pharmacokinetic Department,
Pharmaceutical Candidate Optimization, and ECTR/CTTO Imaging Department, BristolꢀMyers Squibb
Research and Development, P.O. Box 4000, Princeton, New Jersey 08543
e
RECEIVED DATE
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ABSTRACT: Bruton’s Tyrosine Kinase (BTK) belongs to the TEC family of nonꢀreceptor tyrosine
kinases, and plays a critical role in multiple cell types responsible for numerous autoimmune diseases.
This article will detail the structureꢀactivity relationships (SAR) leading to a novel second generation
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series of potent and selective reversible carbazole inhibitors of BTK.
With an excellent
pharmacokinetic profile as well as demonstrated in vivo activity and an acceptable safety profile, 7ꢀ(2ꢀ
hydroxypropanꢀ2ꢀyl)ꢀ4ꢀ[2ꢀmethylꢀ3ꢀ(4ꢀoxoꢀ3,4ꢀdihydroquinazolinꢀ3ꢀyl)phenyl]ꢀ9Hꢀcarbazoleꢀ1ꢀ
carboxamide 6 (BMSꢀ935177) was selected to advance into clinical development.
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INTRODUCTION
Kinases continue to be a very active area for drug discovery, targeting treatments in a wide range of
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therapeutic areas. Bruton’s Tyrosine Kinase (BTK) is a nonꢀreceptor tyrosine kinase expressed in all
hematopoietic cells except plasma and T cells. Although only one of the five mammalian TEC family
members, BTK is the most important from a target perspective since it is critical for B cell signaling
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through the B cell receptor (BCR). In addition to B cells, BTK is vital for signaling in low affinity
activating Fcγ receptors (e.g., FcγRIII and FcγRIIa) in monocytic cells. BTK also regulates the
expression of proinflammatory cytokines, chemokines and cell adhesion molecules in response to
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receptor activation through FcεRI signaling in mast cells and basophils. RANK signaling, which
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controls osteoclastogenesis from monocytic precursors, is also BTKꢀdependent.
B cells are essential to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA)
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as demonstrated by the clinical results for the B cellꢀdepleting antiꢀCD20 antibody rituximab. In
addition to producing autoantibodies, B cells can control autoimmunity as sources of proinflammatory
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chemokines and cytokines, such as ILꢀ6 and TNFα, and through their role as antigenꢀpresenting cells.
Because BTK kinase activity is necessary for BCRꢀdependent proliferation of B cells as well as
production of proinflammatory cytokines and costimulatory molecules (e.g., CD86), pharmacological
inhibition of BTK is expected to affect several pathways by which B cells may mediate autoimmunity,
but without depleting B cells. Pharmacological and genetic studies in animal models strengthen the case
that BTK inhibitors would be efficacious against autoimmune indications such as RA or systemic lupus
erythematosus (SLE). BTKꢀdeficient mice are less sensitive to both the (NZB X NZW)F1 model of
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lupus and the collagenꢀinduced arthritis (CIA) model. BTK inhibitors reduce disease progression in the
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collagen antibodyꢀinduced arthritis (CAIA) and CIA models, in addition to murine lupus models. The
vital role of BTK in B cells is also observed in human xꢀlinked agammaglobulinemia (XLA)
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immunodeficiency. XLA patients have inactivating mutations in the BTK gene leading to a loss of
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mature B cells and circulating antibodies.
A key structural feature of the BTK kinase domain is the nonꢀcatalytic cysteine (Cys481) residue in
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the extended hinge region. Numerous companies have targeted this residue to prepare irreversible
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BTK inhibitors, with the most successful being ibrutinib (1; Pharmacyclics/J&J), which is approved for
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various nonꢀHodgkin’s lymphomas (Figure 1).
Several additional BTK inhibitors have entered
clinical trials for the treatment of autoimmune indications, including covalent inhibitors from Celgene
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(
2), Hanmi (3) and Principia (4, structure unknown, 2014 press release), with the most advanced
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appearing to be 2, currently in Phase II trials in RA (Figure 1). Efficacy results for these and other
compounds entering clinical trials are eagerly awaited, since small molecule inhibitors of this
mechanism have the potential to compete with biologics for the treatment of RA and several other
autoimmune diseases. In addition to these covalent inhibitors, recent reports from Roche and Merck
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show continued interest in reversible BTK inhibitors.
We have previously disclosed the discovery of a novel carbazole series of BTK inhibitors, as well as
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the initial structureꢀactivity relationships (SAR) leading to 5 (Figure 2). Compound 5 inhibited BTK
with modest kinase selectivity. Having identified a novel carbazole series of BTK inhibitors, we began
an effort to address the liabilities of 5 and similar analogs. In the current paper, we disclose carbazoles
with improved oral exposure, kinase selectivity and BTK potency, leading to the discovery of the
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clinical development candidate 6 (BMSꢀ935177) .
RESULTS AND DISCUSSION
As we have reported previously, Xꢀray structural data for a member of the carbazole carboxamide
series showed that the carbazole NH and both the carbonyl and NHs of the primary amide formed
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critical hydrogen bonds to the key hinge region of BTK. It was also noted that a small orthoꢀ
substituent on the Cꢀ4 phenyl ring improved both BTK potency and kinase selectivity when compared
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to the parent unsubstituted analog. In addition, Xꢀray structures showed the Cꢀ7 substituent bound in the
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solvent exposed extended hinge region of BTK. Several initial issues for the carbazole series required
optimization, including kinase potency and selectivity, as well as oral exposure in mouse. Our efforts to
address these concerns involved the use of a combination of focused library and individual analog
synthesis as outlined in Figure 3. The 4ꢀfluorophenyl amide of 5 was replaced with a variety of alkyl,
aryl, and heteroaryl amides as well as ureas and carbamates using the aniline 7a. To explore the SAR at
the Cꢀ7 position, the piperazine of amide of 5 was replaced with a variety of alkyl, aryl, and heteroaryl
amides starting from the acid 8a. The acid substituent of a 8a precursor was converted to aniline 9a (see
experimental section) which was used as the starting material to further explore the SAR at Cꢀ7 to give
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b, incorporating a variety of alkyl anilines as well as a range of alkyl, aryl, and heteroaryl amides,
ureas and carbamates. From this thorough exercise two analogs were noteworthy, 10 and 11 (Table 1).
The polarity of the piperazine amide Cꢀ7 substituent coupled with the large number of hydrogen
bond donors and acceptors in 5 led to low permeability as measured in PAMPA, contributing to low oral
exposure in mouse (Table 1). The ortho–pyridyl analog 10, designed to form an intraꢀmolecular Hꢀbond
with the amide NH to improve permeability, maintained good BTK potency with improved PAMPA
and oral exposure as shown in Table 1, although selectivity over the SRC family of kinases (LCK as an
example found on Tꢀcells) was still unacceptable. The goal of >50ꢀfold selectivity over the SRC family
was set to minimize the chances of being too immunosuppressive in vivo by targeting both Bꢀ and Tꢀ
cells. Recognizing that the Cꢀ7 substituents could potentially bind in the solvent exposed extended hinge
region of BTK, modification of the side chain could be used to “tune” the properties of inhibitors.
Analog exploration at Cꢀ7 (Figure 3) often showed a dramatic effect on kinase selectivity and potency.
However, many of these analogs were of high molecular weight with low oral plasma concentrations in
mouse PK studies (results not shown). The amino acid at the Cꢀterminal end of the hinge in BTK,
Ala478, is often an acidic residue in SRC kinase family members such as LCK (Glu 320). We predicted
that a less basic Cꢀ7 group could improve selectivity over the piperazine amide. In order to keep the
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molecular weight low, compound 11 was prepared and found to be a potent BTK inhibitor with
improved oral plasma exposure and LCK kinase selectivity (Table 1). However, analogs with a pendant
metaꢀaryl or heteroaryl amide substituent on the Cꢀ4 phenyl, as in 10 and 11, showed hydrolysis to an
aniline metabolite as the major metabolic pathway in human and rat liver microsomes. In addition, in
the case of 11, concomitant Nꢀdealkylation of the isoꢀpropyl group led to the corresponding bisꢀaniline
metabolite, observed in both liver microsomes and as a circulating metabolite after oral dosing in mice.
Upon multiple day dosing in mice, compound 11 showed toxicity in vivo that could be attributed to the
bisꢀaniline metabolite (data not shown). Predicting the extent of amide hydrolysis in humans based on
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preꢀclinical data is potentially challenging, supporting the decision to stop further progression of these
analogs.
To avoid any possible aniline release, numerous medicinal chemistry approaches to stabilize amide
hydrolysis were investigated. The most direct approach, synthesis of the cyclic amides, was also the
most effective. The fiveꢀmembered ring lactam 12 maintained BTK potency with acceptable
permeability (PAMPA Pc 494 nm/s, Table 2). As expected, 12 was stable to hydrolysis in vivo, however
the plasma concentration in mouse was similar to 5, which also contained the piperazine amide. It
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should be noted that the mouse liver microsome metabolic stability of the three compound 5, 10, and
12 containing the piperazine amide were comparable. It was surprising that the exposure for lactam 12
was significantly worse than that of ortho–pyridyl analog 10, designed to form an intraꢀmolecular Hꢀ
bond with the amide NH, which points to the difficulty of predicting exposure based solely on one
parameter, such as numbers of hydrogen bonds.
The SAR results from our previous Cꢀ7 analogs prepared for compound 5 (outlined in Figure 3),
suggested that small polar nonꢀbasic or weakly basic groups would be best for potency, selectivity, and
PAMPA. Combining the Cꢀ4 lactam with a tertiary carbinol as a Cꢀ7 substituent led to analog 13 which
maintained BTK potency and improved potency in the Ramos B cell calcium flux assay (Table 2). In
order to further optimize 13, a second survey of alternate Cꢀ7 substituents was undertaken focusing on
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small polar groups. Ultimately, no other substituent was found to give better overall properties than the
carbinol. An investigation of substituents on the phenyl ring of the lactam identified the fluoroꢀ
substituted analog 14, which had improved BTK cell potency and mouse liver microsome metabolic
stability compared to 13. Compound 14 showed significantly improved oral plasma concentrations in
mouse compared to 12 (Table 2), suggesting that it was suitable for further in vivo studies.
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BTK regulates antigen receptor (BCR) signaling in B cells, and one of the hallmarks of xid (xꢀlinked
immune defect) mice deficient in BTK is a defective neoantigenꢀinduced antibody response. As a result,
it was anticipated that activity against a keyhole limpet hemocyanin (KLH) induced antibody response
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in mice would provide an important pharmacodynamics (PD) readout of BTK inhibition. In this
model, antiꢀKLH antibodies of the IgM isotype appear in the first week after challenge, followed by
isotype switching to IgG antiꢀKLH antibodies evident by day 14. However, 14 showed only a nonꢀ
statistically significant decrease in IgG when dosed orally at 30 mg/kg BID in the chronic KLH mouse
model. The weak suppression of the antiꢀKLH response, coupled with a nonꢀoptimal PK profile,
prohibited further advancement of compound 14.
An extensive exploration of heterocyclic analogs of 14 was conducted to find compounds with
improved potency and oral plasma concentrations (Table 2). BTK potency was maintained in several 6,
6 bicyclic ring systems, such as in analogs 15 and 16, but they were either less LCK (or other kinase)
selective or less potent in cells than 14. The unsubstituted quinazolinone 6 showed improved Ramos B
cell potency with similar LCK selectivity to 14. Quinazolinones were substituted with an array of
electron donating and withdrawing groups, as well as a range of both polar and lipophilic moieties.
However, in none of these cases was the overall profile improved over the unsubstituted parent analog
6
. For example, a fluoro scan on the quinazolinone ring provided 17ꢀ20. Although all of the fluoro
analogs were potent BTK inhibitors, none showed significant improvements over 6. All five analogs
were investigated in mouse PK studies at 10 mpk, showing improved oral plasma concentrations
compared to 14 (Table 2). Quinazolinone analogs 21 (Table 2) and 22 (Table 3) were also prepared to
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investigate the requirements of the tertiary carbinol and the orthoꢀmethyl substituent in the Cꢀ4 aryl
linker, respectively. Compound 21 maintained BTK enzyme potency, but showed lower oral plasma
concentrations in mouse compared to 6. Analog 22 had a significant drop in BTK enzyme potency,
highlighting the importance of the Cꢀ4 aryl orthoꢀmethyl substituent.
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Attempts to obtain an Xꢀray coꢀcrystal structure of 6 bound to BTK were unsuccessful. However,
close analog 23 (Table 3) provided a 1.9 Å Xꢀray crystal structure bound to the active site of BTK (PDB
ID 5JRS. Authors will release the atomic coordinates and experimental data upon article publication.),
17a
as shown in Figure 4. The carbazole NH and primary amide were bound to the hinge as expected,
forming crucial hydrogen bonds with the backbone carbonyl of Metꢀ477 and both the backbone NH of
Metꢀ477 and the carbonyl of Gluꢀ475, respectively. In addition, the primary amide formed a third
hydrogen bond to a water in the gate keeper region, which was linked to an extensive water network that
filled the pocket. The fluorophenyl ring was nearly orthogonal to the carbazole, with the ortho fluoro
filling a small hydrophobic pocket formed by Leuꢀ528 at the base of the ATP pocket and the backbone
of Cysꢀ481. The quinazolinone ring was in turn orthogonal to the Cꢀ4 phenyl linker, with the carbonyl
interacting with a conserved water in the extended hinge region. Finally, the Cꢀ7 tertiary carbinol was
waterꢀexposed in the extended hinge region as predicted, stabilizing a water network that also included
the quinazolinone carbonyl.
Although several analogs displayed in Table 2 showed promising plasma concentrations in mouse,
none displayed a superior profile for progression compared to the parent quinazolinone 6. With the low
aqueous solubility (<1 µg/mL) of 6, further lipophilic fluoro substituents in 17ꢀ20 could be problematic
in achieving dose linear oral exposures in further animal studies. Therefore, compound 6 was selected
for detailed in vitro and in vivo characterization.
A partial list of kinase selectivity data for compound 6 is summarized in Table 4. Compound 6 is a
potent, reversible inhibitor of BTK (IC 2.8 nM) and demonstrated good kinase selectivity when tested
5
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against a screening panel of 384 kinases at DiscoveRx (Fremont, CA; formally Ambit Biosciences),
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with only 16 kinases showing less than 15% control binding at 1000 nM. Consistent with this measure
of selectivity, biochemical kinase assays against 60 kinases showed 6 to be more potent against BTK
than any other kinase, including the other Tec family kinases (TEC, BMX, ITK and TXK) over which
the compound is between 5ꢀ and 67ꢀfold selective. With the exception of 7ꢀfold selectivity over BLK,
expressed specifically in B cells, the compound showed greater than 50ꢀfold selectivity over the SRC
family of kinases, including 1,100ꢀfold selectivity over SRC itself. Other kinases inhibited with a
potency less than 150 nM (50ꢀfold selectivity) included TRKA, HER4, TRKB, and RET.
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Cell potency data for compound 6 is summarized in Table 5. In B cells stimulated through the BCR,
6
selectively inhibited several different readouts. Compound 6 inhibited calcium flux in human Ramos
B cells (IC 27 nM) and inhibited CD69 surface expression in peripheral B cells stimulated with antiꢀ
5
0
IgM and antiꢀIgG. However, 6 had no effect on CD69 surface expression in B cells stimulated through
the CD40 receptor with CD40 ligand. Against IgGꢀcontaining immune complexꢀdriven low affinity
activating Fcγ receptor (FcγRIIa and FcγRIII) endpoints in peripheral blood mononuclear cells
(PBMCs), 6 effectively inhibited TNFα production with an IC50 value of 14 nM. To determine the
whole blood potency, the ability of the compound to inhibit BCRꢀstimulated expression of CD69 on the
surface of B cells in whole blood was measured in a FACSꢀbased assay which allowed for gating on the
B cells. Against this BTKꢀdependent endpoint, 6 showed mean IC values of 550 ± 100 (n=11) and
5
0
2
060 ± 240 nM (n=3) in human and mouse whole blood, respectively. The somewhat more potent
inhibition in human compared to mouse whole blood may reflect both an intrinsic human potency
advantage, as well as protein binding differences between species (Table 6).
Some of the detailed in vitro profiling data for compound 6 is summarized in Table 6. Plasma
protein binding for 6 was high for all species, with less than 1% free for human. The high protein
binding could mitigate the effects of the hERG patch clamp IC of ~10 µM. Although the crystalline
5
0
aqueous solubility was <1 µg/mL for 6, simulated gastric fluid showed improved solubility, both in fed
and fasted state solutions. The high PAMPA permeability and low aqueous solubility placed 6 in the
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BCS Class 2 category. The Cacoꢀ2 values showed that 6 is a PgP substrate, although this is mitigated by
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the high passive permeability.
In the KLH assay, 6 showed superior in vivo potency compared to 14. As shown in Figure 5a, when
dosed orally once daily at 5, 20 and 45 mg/kg to mice the compound inhibited antiꢀKLH antibodies of
the IgG isotype at day 14, with statistically significant reductions at all doses. In satellite mice from this
study dosed with 6 at 5 mg/kg, the plasma concentration was maintained above the mouse whole blood
BCRꢀstimulated CD69 IC value of 2 µM for only approximately five hours (see Figure 5b). However,
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this transient daily coverage at the low dose yielded a significant inhibition of IgG antiꢀKLH titers,
indicating that significant pharmacological activity can be observed even with incomplete coverage of
the whole blood IC potency.
5
0
Figure 6 shows the results of fully preventative dosing with 6 in a mouse CIA model. At once daily
oral doses of 10, 20 and 30 mg/kg beginning on the day of primary immunization, 6 provided a clear
doseꢀdependent reduction in both the severity and incidence of clinically evident disease in this rodent
model of RA. At 10 mg/kg of 6, disease severity was reduced about 40% compared to vehicle treatment,
and the percentage of animals showing any signs of disease was reduced by a third. At the 10 mg/kg
dose the plasma level of 6 was maintained above the mouse WB IC of 2.06 ꢀM (Table 5) for 12 hours,
50
with a 24 hour trough level of 277 nM. At the two higher doses of 20 and 30 mg/kg, disease severity
was reduced by 85ꢀ90% compared to vehicle treatment, and disease incidence was reduced by twoꢀ
thirds. At the 20 mg/kg dose the plasma level of 6 was maintained above the mouse WB IC for 20
5
0
hours, with a 24 hour trough plasma level of 1171 nM. At the end of the study, the tibiotarsal joints
were evaluated histologically and graded semiꢀquantitatively for severity of inflammation, synovial
hyperplasia, bone resorption, and cartilage erosion. Consistent with the clinically observed effects on
disease incidence and severity, 6 doseꢀdependently inhibited both inflammation and bone resorption
endpoints (Figure 7). Microꢀcomputed tomography of the hind limbs also showed that 6 provided a
doseꢀdependent protection against the pitting, loss of bone mass, woven porous bone, and fusion of the
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Page 12 of 65
small bones evident in the mice receiving only vehicle, with the animals receiving 20 mg/kg showing
essentially complete protection as evidenced by the presence of a smooth bone surface and easily
recognizable small individual bones of the foot and ankle (representative images in Figure 8). The
compound was also effective at 30 mg/kg PO QD in blocking disease progression when administration
of 6 is initiated after the booster immunization (pseudoꢀestablished dosing mode; data not shown).
In the B cell independent mouse CAIA disease model, 6 was also efficacious, reflecting the
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21
involvement of activating Fcγ receptor pathways which were blocked by BTK inhibition. Figure 9
shows that QD oral doses of 10 and 30 mg/kg provided a significant, doseꢀdependent reduction in paw
clinical scores. Indeed, mice receiving 6 at 30 mg/kg were virtually diseaseꢀfree throughout the period
of the study, and this regimen was more effective in this model than dexamethasone. These results
indicate that inhibition of BTK by 6 in signal transduction pathways and cells (e.g., activating Fcγ
receptors in monocytic cells) other than BCR signaling is likely contributing to the profound efficacy
seen in the CAIA and CIA models of arthritis.
One of the strongest attributes of compound 6 is excellent oral bioavailability in all preꢀclinical
species, both from suspension and solution dosing, despite its low aqueous solubility. The oral
bioavailability for 6 with solution dosing ranges from 84 to 100% in rat, mouse, dog and cynomolgus
monkey, with low clearance in single intravenous (IV) infusion studies (summarized in Table 7). When
dosed as a crystalline microꢀsuspension in rats (5 mpk in citrate buffer at pH 4, with 0.02% DOSS and
0
.5% methocel) 6 maintained excellent oral bioavailability of ~100%. Projections based on the good
pharmacokinetics across species as well as efficacy in mouse models suggested that compound 6 could
provide significant clinical benefit with once a day dosing.
Chemistry
The general synthetic route utilized in the preparation of the bicyclic amide replacement carbazole
analogs in Tables 2 and 3 is outlined in Scheme 1. The key reaction was a Suzuki coupling between the
1
appropriate partners 24 and 25. Bromo carbazoles 24 (Y =Br) were reacted with boronic esters 25
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Journal of Medicinal Chemistry
1
22,23
(
Z =boronic ester) under Suzuki coupling conditions to give final compounds 6 and 12ꢀ23.
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1
Alternatively, the partners could be reversed with the carbazole boronic esters 24 (Y = boronic ester)
1
reacting with the bromide 25 (Z =Br) using the same coupling condition to give the final products in
generally good yields. The Cꢀ7 functionalized carbazole intermediates were prepared as shown in
Schemes 2ꢀ3. The Fischer indole cyclization using ketone 26 and hydrazine 27 was accomplished by
heating in acetic acid to give the tetrayhdrocarbazole 28 in 58% yield. Acid intermediate 28 was
activated with EDC and HOBt, then treated with ammonium hydroxide to give amide 29 in 76% yield.
Aromatization of 29 with DDQ gave ester carbazole 30 in good yield. Reduction of 30 with lithium
aluminum hydride gave the benzyl alcohol intermediate 32 (64% yield). The tertiary carbinol
intermediate 31 was prepared directly from ester 30 using methyl lithium in 84% yield. Ester 30 was
hydrolyzed with LiOH to acid 34 which was then coupled with Nꢀmethyl piperazine in the presence of
EDC and HOBt to provide bromo amide 35 in excellent yield (Scheme 3). Alternatively, acid 34 was
subjected to a Curtius rearrangement to give the Cbzꢀprotected intermediate 36, allowing access to Cꢀ7
amino analogs 9a and 9b shown in Figure 3.
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The bromo amide intermediate 35 underwent a Suzuki reaction with 2ꢀmethylꢀ3ꢀ(4,4,5,5ꢀ
tetramethylꢀ1,3,2ꢀdioxaboralanꢀ2ꢀyl)aniline to give intermediate 7a in 77% yield as shown in Scheme 4.
Coupling of 7a with the appropriate acid then provided the final amides 5 or 10 in good yields.
Alternatively, the bromo intermediate 35 was subjected to a Suzuki reaction with boronic ester 40a to
give the lactam 12 in 60% yield (Scheme 4). The synthesis of 40a is summarized in Scheme 5. The
bromo aniline 37 was acylated with 2ꢀ(chloromethyl)benzoyl chloride 38 and the resulting amide
intermediate was cyclized in the presence of a base to give the bromo lactam 39a. Treatment with
bis(pinacolato)diboron under standard conditions gave high yields of the boronic ester 40a.
The other key Suzuki partners required for the preparation of final compounds 6 and 13ꢀ23 were
synthesized as shown in Schemes 6ꢀ7. The preparations of isoquinolinone 44 and 3,4ꢀ
dihydroisoquinolinone 47 are summarized in Scheme 6. The condensation of bromo aniline 41 with
cyclic anhydride 42 provided the corresponding cyclic imide which was subsequently reduced with
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NaBH and dehydrated under acidic conditions to give bromoꢀisoquinolinone 43. The bromo
4
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isoquinolinone was then converted to the boronic ester 44 in good yield. The reaction of 1,3ꢀdibromoꢀ2ꢀ
methylbenzene 45 with 3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀone 46 in the presence of CuI gave the bromo
dihydroisoquinolinꢀ1(2H)ꢀone 47 used to synthesize final compound 15. The quinazolinones were
obtained by the reaction of bromo anilines 48 with anthranilic acids 49 in the presence of triethyl
orthoformate to give the bromo quinazolinones 50 in moderate to good yields (Scheme 7). The bromo
quinazolinones were then converted to the required boronic esters 51 in good yields. With the
appropriate Suzuki partners prepared, the assembly of the final compounds (6 and 12ꢀ23, Tables 2ꢀ3)
was carried out under standard crossꢀcoupling conditions in good yields as summarized in Scheme 1.
During the analysis of the NMR data of final analogs such as 6, complex splitting patterns were
observed. Hindered rotation of the carbazole Cꢀ4 bond as well as the bond to the quinazolinone caused
by the orthoꢀmethyl substituent on the phenyl linker (Figure 10) created atropisomers. Chiral SFC
chromatography was able to separate the four possible atropisomers, although they could not be isolated
without racemization back to the original mixture due to the low barrier to rotation at room temperature.
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Conclusion
In summary, a novel second generation series of carbazole BTK inhibitors has been identified. The
potential problem of aniline formation from benzamides such as 5, 10 and 11 was solved by conversion
to the corresponding lactams (such as 12). Oral exposure was improved by replacing the Cꢀ7 substituent
with the lessꢀpolar tertiary carbinol. Extensive exploration of other heteroaryl modifications of the
lactam moiety provided 7ꢀ(2ꢀhydroxypropanꢀ2ꢀyl)ꢀ4ꢀ[2ꢀmethylꢀ3ꢀ(4ꢀoxoꢀ3,4ꢀdihydroquinazolinꢀ3ꢀ
yl)phenyl]ꢀ9Hꢀcarbazoleꢀ1ꢀcarboxamide 6, a potent BTK inhibitor with improved kinase selectivity and
superior oral exposure in multiple species. These features, coupled with robust efficacy in mouse
arthritis models dependent on both B cell and nonꢀB cell mechanisms, predict that 6 should provide
useful clinical efficacy in autoimmune diseases. Based on in vitro potency, in vivo activity and
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pharmacokinetic profile, compound 6 was selected for further studies in support of clinical
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development.
Experimental Section
General Procedures. All reagents were purchased from commercial sources and used without further
purification unless otherwise noted. All reactions were performed under an inert atmosphere of nitrogen
or argon. Chromatographic purification was conducted on preꢀpacked silica gel cartridges using a flash
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®
chromatography system such as the CombiFlash Rf+System (Teledyne ISCO). HPLC and LCMS
analyses were conducted using a Shimadzu SCLꢀ10A liquid chromatograph and a SPD UVꢀVis detector
at 220 or 254 nm with the MS detection performed with either a Waters Micromass ZQ spectrometer or
a Micromass Platform LC spectrometer. Unless otherwise reported, preparative reverseꢀphase HPLC
purifications were performed using the following conditions: YMC S5 ODS 20 x 100 mm column with
a binary solvent system where solvent A = 10% methanol, 90% water, 0.1% trifluoroacetic acid and
solvent B = 90% methanol, 10% water, and 0.1% trifluoroacetic acid, flow rate = 20 mL/min, linear
gradient time = 10 min, start %B = 20, final %B = 100. Fractions containing the product were
concentrated in vacuo to remove solvent. The resulting product could be further neutralized with
aqueous sodium bicarbonate, extracted into organic solvent, dried and concentrated under reduced
1
pressure. Proton nuclear magnetic resonance ( H NMR) spectra were recorded on either a JEOL Eclipse
500 or a Bruker Avance 400 spectrometer and are reported in ppm relative to either residual protioꢀ
solvent of the sample in which they were run or to TMS as internal standard. Coupling constants are
provided in Hz with standard abbreviations for the spectral pattern designations.
HPLC analyses for purity were performed using the following two conditions for all final compounds
and were determined to have an HPLC purity of ≥ 95% unless otherwise noted.
Method A: A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (Solvent A) and 95%
acetonitrile, 5% water, and 0.05% TFA (Solvent B); t = 0 min., 10% B, t = 12 min., 100% B (15 min.)
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was employed on a Waters SunFire C 3.5µ 4.6 x 150 mm column. Flow rate was 1.0 ml/min and UV
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detection was set to 220/254 nm. The column was maintained at ambient temperature.
Method B: A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (Solvent A) and 95%
acetonitrile, 5% water, and 0.05% TFA (Solvent B); t = 0 min., 10% B, t = 12 min., 100% B (15 min.)
was employed on a Waters XBridge Ph 3.5µ 4.6 x 150 mm column. Flow rate was 1.0 ml/min and UV
detection was set to 220/254 nm. The column was maintained at ambient temperature.
0
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4ꢀ(3ꢀ(4ꢀFluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ7ꢀ(4ꢀmethylpiperazineꢀ1ꢀcarbonyl)ꢀ9Hꢀcarbazoleꢀ1ꢀ
carboxamide, TFA (5).
Step A. Ethyl 3ꢀhydroxycyclohexanecarboxylate. To an EtOH (500 mL) solution of ethyl 3ꢀ
hydroxybenzoate (50 g, 0.3 mol) was added 5% rhodium on alumina (7.5 g) and the mixture was stirred
in an autoclave under 12 kg hydrogen pressure at RT overnight. The reaction mixture was filtered
through Celite and the volatiles removed to give ethyl 3ꢀhydroxycyclohexanecarboxylate (50 g, 96%) as
a liquid and a mixture of diastereomers. The material was used without further purification. Mass
+
1
spectrum m/z 173 (M+H) . H NMR (CDCl , 400 MHz) δ 4.03ꢀ4.21 (m, 2.3H), 3.54ꢀ3.71 (m, 0.7H),
3
2.67ꢀ2.83 (m, 0.3H), 2.36 (m, 0.7H), 2.20 (m, 1H), 1.76ꢀ2.01 (m, 3H), 1.16ꢀ1.76 (m, 8H).
Step B. Ethyl 3ꢀoxocyclohexanecarboxylate (26). To a DCM (1.1 L, dry) solution of ethyl 3ꢀ
o
hydroxycyclohexanecarboxylate (51 g, 0.3 mol) at 0 C was added carefully portionꢀwise DessꢀMartin
periodinane (251 g, 0.59 mol). After the addition was complete, the reaction mixture was allowed to
slowly come to RT overnight. The reaction was basified by the addition of Na CO solution, the
2
3
resulting solids removed by filtration through Celite, and the filtrate concentrated. The resulting residue
was diluted with EtOAc, the organic layer washed with Na CO solution, water, brine, dried over
2
3
Na SO and concentrated to give 26 (48 g, 95%) as a lightly yellow colored oil. The ester 26 had a very
2
4
+
1
strong odor and should be handled in a fume hood. Mass spectrum m/z 171 (M+H) . H NMR (400
MHz, CDCl ) δ 4.16 (q, J=7.1 Hz, 2H), 2.78 (s, 1H), 2.55 (d, J=8.3 Hz, 2H), 2.28ꢀ2.43 (m, 2H), 2.03ꢀ
3
2
.16 (m, 2H), 1.68ꢀ1.90 (m, 2H), 1.27 (t, J=7.0 Hz, 3H).
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Step C. 4ꢀBromoꢀ2ꢀhydrazinylbenzoic acid (27). A solution of sodium nitrite (2.4 g, 35.5 mmol) in
water (12 mL) was added dropꢀwise to a suspension of 2ꢀaminoꢀ4ꢀbromobenzoic acid (7.3 g, 33.8
mmol) in concentrated aqueous hydrochloric acid (34 mL) at ꢀ5 °C, at such rate that the temperature did
not exceed 0 °C. The resulting suspension was stirred at ꢀ5 °C for 10 min and was then added dropꢀwise
to a rapidly stirred solution of tin (II) chloride (13.5 g, 71.0 mmol) in concentrated aqueous hydrochloric
acid (10 mL) at ꢀ5 °C at such a rate that the temperature did not exceed 0 °C. The resulting suspension
was warmed to RT and stirred for 1h. The precipitate was collected by filtration, washed with water, and
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
airꢀdried to afford 27 (7.8 g, 86%) as a lightꢀcolored solid. Mass spectrum m/z 231.0, 233.0 (M+H) .
Step D. 5ꢀBromoꢀ2ꢀ(ethoxycarbonyl)ꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀcarbazoleꢀ8ꢀcarboxylic acid (28). A
stirred suspension of 27 (16.4 g, 58.1 mmol) in acetic acid (171 mL) was treated with 26 (9.9 g, 58.1
mmol) at RT. The mixture was stirred at reflux for 2.5 h. The mixture was cooled to RT and
concentrated to afford a brown solid. The solid was suspended in ethyl acetate (20 mL) and the
precipitate was collected by filtration, washed with ethyl acetate and air dried to provide 28 (11.5 g) as a
colorless solid. The filtrate was concentrated, the residue resuspended in ethyl acetate, and the
precipitate collected by filtration to provide additional 28 (0.8 g, total 12.3 g, 58%). Mass spectrum m/z
+
1
366.0, 368.0 (M+H) . H NMR (400 MHz, DMSOꢀd ) δ 13.10 (br s, 1H), 11.06 (s, 1H), 7.50 (d, J=8.13
6
Hz, 1H), 7.19 (d, J=8.13 Hz, 1H), 4.07ꢀ4.15 (m, 2H), 3.08ꢀ3.19 (m, 1H), 2.99ꢀ3.08 (m, 1H), 2.89ꢀ2.99
(m, 2H), 2.79ꢀ2.89 (m, 1H), 2.09ꢀ2.22 (m, 1H), 1.75ꢀ1.89 (m, 1H), 1.20 (t, J=7.14 Hz, 3H).
Step E. Ethyl 5ꢀbromoꢀ8ꢀcarbamoylꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀcarbazoleꢀ2ꢀcarboxylate (29). A
suspension of acid 28 (12.3 g, 33.5 mmol), EDC (7.7 g, 40.2 mmol), and 1ꢀhydroxybenzotriazole
hydrate (6.2 g, 40.2 mmol) in THFꢀDCM (4:1, 335 mL) was treated with aqueous ammonium hydroxide
(7.83 mL, 201 mmol), and the resulting suspension was stirred at RT overnight. The mixture was
concentrated and the residue was suspended in water. The precipitate was collected by filtration, washed
with water and ethyl acetate and air dried to give 29 (8.9 g). The filtrate was concentrated and the
residue was suspended in methanol. A solid was collected by filtration, washed with methanol and air
+
1
dried to afford additional 29 (0.4 g, total 9.3 g, 76%). Mass spectrum m/z 365.1, 367.1 (M+H) . H
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NMR (400 MHz, DMSOꢀd ) δ 11.08 (s, 1H) 8.02 (br s, 1H), 7.43 (d, J=8.13 Hz, 1H), 7.39 (br s, 1H),
6
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7.14 (d, J=8.13 Hz, 1H), 4.02ꢀ4.17 (m, 2H), 3.07ꢀ3.18 (m, 1H), 2.97ꢀ3.06 (m, 1H), 2.86ꢀ2.98 (m, 2H),
2
.77ꢀ2.86 (m, 1H), 2.09ꢀ2.19 (m, 1H), 1.72ꢀ1.86 (m, 1H), 1.20 (t, J=7.14 Hz, 3H).
Step F. Ethyl 5ꢀbromoꢀ8ꢀcarbamoylꢀ9Hꢀcarbazoleꢀ2ꢀcarboxylate (30). To a suspension of 29 (60 g,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
164 mmol) in THF (400 mL) was added 4,5ꢀdichloroꢀ3,6ꢀdioxocyclohexaꢀ1,4ꢀdieneꢀ1,2ꢀdicarbonitrile
(78 g, 345 mmol) portion wise at RT. After 1 h, the reaction mixture was added to an aqueous NaOH
solution (1N NaOH (500 mL) and water (725 mL)), the resulting suspension filtered and washed with
water. The resulting material was dried in vacuo at 50 °C to obtain 30 (51 g, 86%) as a yellow solid.
+
1
LCMS m/z 360.9, 362.9 (M+H) . H NMR (400 MHz, DMSOꢀd ) δ 12.00 (s, 1H), 8.69 (d, J = 8.4 Hz,
6
1H), 8.50 (d, J = 0.9 Hz, 1H), 8.23 (br s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.86 (dd, J = 8.4, 1.5 Hz, 1H),
7
.59 (br s, 1H), 7.50 (d, J = 8.1 Hz, 1H), 4.36 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.0 Hz, 3H).
Step G. 5ꢀBromoꢀ8ꢀcarbamoylꢀ9Hꢀcarbazoleꢀ2ꢀcarboxylic acid (34). A solution of 30 (7.2 g, 19.1
mmol) and LiOH monohydrate (1.4 g, 57.2 mmol) in THF/EtOH/H O (3:1:1, 201 mL) was heated at
2
reflux for 6 h. The reaction mixture was cooled to RT and concentrated. The resulting residue was
suspended in water and the pH was adjusted to ~2ꢀ3 with 1N aqueous HCl. The resulting precipitate was
collected by filtration, washed with water and dried to provide 34 (7 g, 99%) as an offꢀwhite solid.
+
+
LCMS m/z 333, 335 (M+H) , 316, 318 (M+HꢀNH ) .
3
Step H. 4ꢀBromoꢀ7ꢀ(4ꢀmethylpiperazineꢀ1ꢀcarbonyl)ꢀ9Hꢀcarbazoleꢀ1ꢀcarboxamide (35).
A
suspension of 34 (7.0 g, 18.9 mmol), EDC (5.1 g, 26.5 mmol), and 1ꢀhydroxybenzotriazole hydrate (4.1
g, 26.5 mmol) in THF/DCM/DMF (378 mL, 4:1:1) was treated with 1ꢀmethylpiperazine (6.3 mL, 56.7
mmol). The mixture was stirred at RT overnight, then was concentrated under vacuum. The resulting
residue was partitioned between DCM:MeOH (8:1) and saturated aqueous NaHCO . The aqueous phase
3
was extracted twice again with DCM:MeOH (8:1), and the combined organic phases were washed with
brine, dried over Na SO and concentrated under vacuum. The resulting residue was suspended in ethyl
2
4
acetate and allowed to stand overnight, forming a precipitate which was collected by filtration, washed
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Journal of Medicinal Chemistry
with ethyl acetate and dried. The filtrates were concentrated under vacuum and the residue was
triturated with ethyl acetateꢀmethanol to provide additional precipitate, which was collected by filtration
and dried. The process was repeated to yield additional precipitate. The dried precipitates were
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
combined to provide 35 (7.5 g, 96%) as a peachꢀcolored solid. LCMS m/z 415, 417 (M+H) . H NMR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(400 MHz, DMSOꢀd ) δ 11.83 (s, 1H), 8.57 (d, J=8.1 Hz, 1H), 8.20 (br s, 1H), 7.84 (d, J=8.3 Hz, 1H),
6
7
.79 (s, 1H), 7.56 (br s, 1H), 7.42 (d, J=8.1 Hz, 1H), 7.22 (d, J=8.1 Hz, 1H), 3.70ꢀ3.30 (m, 4H), 2.39ꢀ
2
.20 (m, 4H), 2.16 (s, 3H).
Step I. 2ꢀMethylꢀ3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)aniline: A mixture of 3ꢀbromoꢀ2ꢀ
methylaniline (4.0 g, 21.5 mmol), bis(pinacolato)diboron (6.5 g, 25.8 mmol) and potassium acetate (4.2
g, 43.0 mmol) in 1,4ꢀdioxane (44.8 mL) and DMSO (9 mL) was bubbled with nitrogen for 10 min.
PdCl (dppf)ꢀCH Cl adduct (0.53 g, 0.64 mmol) was added and the mixture was degassed for another 5
2
2
2
min, then was heated to reflux. After 2 h, the mixture was cooled to RT, filtered through Celite, and the
solids were washed with ethyl acetate. The combined filtrates were washed sequentially with water and
brine. The organic phase was dried over Na SO and concentrated under vacuum. The resulting residue
2
4
was purified by chromatography (eluting with ethyl acetateꢀhexanes, 5:95, then 15:85), to provide 2ꢀ
methylꢀ3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)aniline as an offꢀwhite solid (4.4 g, 88%).
Step
J.
4ꢀ(3ꢀAminoꢀ2ꢀmethylphenyl)ꢀ7ꢀ(4ꢀmethylpiperazineꢀ1ꢀcarbonyl)ꢀ9Hꢀcarbazoleꢀ1ꢀ
carboxamide (7a). A mixture of 35 (3.0 g, 7.2 mmol), Pd(Ph P) (0.4 g, 0.36 mmol), 2 M aqueous
3
4
Na CO (9 mL, 18.1 mmol), and 2ꢀmethylꢀ3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)aniline (2 g,
2
3
8
.7 mmol) in tolueneꢀethanol (4:1, 181 mL) under nitrogen was heated at 90 °C for 11 h. The mixture
was cooled to RT and treated with water, saturated aqueous NaHCO and ethyl acetate. An insoluble
3
gummy solid was formed. The supernatant phases were decanted, and the organic phase was separated
and washed with brine. The gummy solid was dissolved in MeOH, combined with the ethyl acetate
phase, and the combined solution was dried over Na SO and concentrated under vacuum. The residue
2
4
was suspended in ethyl acetate, forming a precipitate which was collected by filtration, washed with
ethyl acetate and dried. Additional solid was isolated from the filtrates, and the combined solids were
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+
1
triturated with ethyl acetate to provide 7a (2.45 g, 77%) as a white solid. LCMS m/z 442.2 (M+H) . H
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NMR (400 MHz, DMSOꢀd ) δ 11.65 (s, 1H), 8.19 (br s, 1H), 8.00 (d, J=7.9 Hz, 1H), 7.75 (s, 1H), 7.51
6
(
br s, 1H), 7.09ꢀ7.01 (m, 1H), 6.98ꢀ6.85 (m, 3H), 6.82ꢀ6.76 (m, 1H), 6.47 (d, J=6.2 Hz, 1H), 5.02 (s,
H), 3.74ꢀ3.22 (m, 4H), 2.29 (s, 4H), 2.18 (s, 3H), 1.71 (s, 3H).
Step K. 4ꢀ(3ꢀ(4ꢀFluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ7ꢀ(4ꢀmethylpiperazineꢀ1ꢀcarbonyl)ꢀ9Hꢀ
carbazoleꢀ1ꢀcarboxamide, TFA (5). To a solution of 7a (26 mg, 0.059 mmol) and DIEA (0.031 mL,
.177 mmol) in THF (2 mL) was added 4ꢀfluorobenzoyl chloride (0.014 mL, 0.118 mmol). The reaction
mixture was stirred at RT overnight and concentrated. The resulting crude residue was purified by
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
+
1
preparative HPLC to give a TFA salt of 5 (23 mg, 59%) as a white solid. LCMS m/z 564.3 (M+H) . H
NMR (400 MHz, DMSOꢀd ) δ 11.77 (s, 1H), 10.15 (s, 1H), 9.81 (br s, 1H), 8.26 (br s, 1H), 8.14ꢀ8.03
6
(m, 3H), 7.85 (s, 1H), 7.63ꢀ7.48 (m, 2H), 7.46ꢀ7.32 (m, 3H), 7.22 (d, J=7.3 Hz, 1H), 7.08ꢀ6.99 (m, 3H),
3
.32ꢀ2.99 (m, 7H), 2.82 (s, 3H), 1.91 (s, 3H).
7
ꢀ(2ꢀHydroxypropanꢀ2yl)ꢀ4ꢀ(2ꢀmethylꢀ3ꢀ(4ꢀoxoquinazolinꢀ3(4H)ꢀyl))phenylꢀ9Hꢀcarbazoleꢀ1ꢀ
carboxamide (6).
Step A. 4ꢀBromoꢀ7ꢀ(2ꢀhydroxypropanꢀ2ꢀyl)ꢀ9Hꢀcarbazoleꢀ1ꢀcarboxamide (31). To a solution of 30
in THF (600 mL) at ꢀ75 °C was added drop wise 1.6 N methyl lithium solution in hexane (529 mL, 847
mmol). The addition temperature was maintained below ꢀ60 °C. There was a heavy yellow suspension
observed, requiring the addition of more THF (200 mL) to maintain stirring. The reaction mixture was
stirred for 30 min. at ꢀ75 °C, then quenched by the slow addition of MeOH (132 mL), followed by 6N
HCl (190 mL) to adjust the pH to 2ꢀ3. The reaction mixture was allowed to warm to RT and the layers
were separated. The acidic aqueous layer was back extracted with EtOAc (300 mL) and both organic
layers were combined, washed with saturated NaHCO solution, dried over Na SO , and treated with
3
2
4
activated charcoal and stirred for 30 min at RT. The suspension was filtered through Celite, the filtrate
concentrated and the resulting residue was triturated with anhydrous ether (250 mL) and stirred for 1 h
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Journal of Medicinal Chemistry
+
at RT. The resulting solid was filtered and dried to give 31 (40 g, 84%). LCMS m/z 331 (M+HꢀH O) .
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H NMR (400 MHz, DMSOꢀd ) δ 11.56 (s, 1H), 8.48 (d, J=8.4 Hz, 1H), 8.16 (br s, 1H), 7.93 (d, J=1.1
6
Hz, 1H), 7.80 (d, J=8.1 Hz, 1H), 7.57ꢀ7.44 (m, 1H), 7.42ꢀ7.32 (m, 2H), 5.07 (s, 1H), 1.50 (s, 6H).
Step B. 3ꢀ(3ꢀBromoꢀ2ꢀmethylphenyl)quinazolinꢀ4(3H)ꢀone (50). A mixture of anthranilic acid (80 g,
583 mmol), 3ꢀbromoꢀ2ꢀmethylaniline (109 g, 583 mmol), and triethyl orthoformate (86 g, 583 mmol) in
toluene (800 mL) was heated to 85 °C to distill off ethanol. After the distillation was complete, the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reaction mixture was refluxed at 110 °C for 16 hours with a DeanꢀStark apparatus to collect water. After
cooling to RT, the reaction mixture was concentrated and the resulting residue was dissolved in EtOAc
(
600 mL). The organic layer was washed with saturated NaHCO solution, water, dried over Na SO
3 2 4
and concentrated. The resulting solid was triturated with anhydrous ether for 30 min at RT and collected
+
1
by filtration to give 50 (115 g, 63%) as an off white solid. LCMS m/z 314.0, 316.0 (M+H) . H NMR
(400 MHz, CDCl ) δ 8.38 (dd, J=7.9, 0.7 Hz, 1H), 7.97 (s, 1H), 7.94 (dd, J=7.2, 1.7 Hz, 1H), 7.85ꢀ7.77
3
(m, 2H), 7.56 (ddd, J=8.0, 6.4, 1.9 Hz, 1H), 7.38ꢀ7.33 (m, 1H), 7.33ꢀ7.29 (m, 1H), 2.37 (s, 3H).
Step C. 3ꢀ(2ꢀMethylꢀ3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)quinazolinꢀ4(3H)ꢀone
(51). A mixture of 50 (60 g, 190 mmol), PdCl (dppf)ꢀCH Cl adduct (3.9 g, 4.8 mmol),
2
2
2
4
,4,4',4',5,5,5',5'ꢀoctamethylꢀ2,2'ꢀbi(1,3,2ꢀdioxaborolane) (53.2 g, 209 mmol), and potassium acetate
(56.1 g, 571 mmol) in dioxane (500 mL) was heated to reflux for 1 h. The reaction mixture was cooled
to RT and concentrated. The resulting dark residue was dissolved in EtOAc (400 mL), washed twice
with water, the organic layer filtered through Celite and dried over Na SO with activated charcoal for
2
4
30 min at RT. The mixture was filtered through Celite, concentrated and purified in three portions by
chromatography (eluting with EtOAc/hexanes, 0ꢀ35% gradient) to obtain 51 (45 g, 65%). LCMS m/z
+
1
3
63.1 (M+H) . H NMR (400 MHz, CDCl ) δ 8.38 (dq, J=8.0, 0.6 Hz, 1H), 7.96 (s, 1H), 7.94 (dd,
3
J=7.2, 1.7 Hz, 1H), 7.86ꢀ7.74 (m, 2H), 7.56 (ddd, J=8.0, 6.4, 1.9 Hz, 1H), 7.39ꢀ7.32 (m, 1H), 7.32ꢀ7.29
m, 1H), 2.37 (s, 3H), 1.35 (s, 12H).
(
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Step D. 7ꢀ(2ꢀHydroxypropanꢀ2yl)ꢀ4ꢀ(2ꢀmethylꢀ3ꢀ(4ꢀoxoquinazolinꢀ3(4H)ꢀyl))phenylꢀ9Hꢀcarbazoleꢀ
ꢀcarboxamide (6). To a mixture of 31 (24 g, 69.1 mmol) and 51 (27.5 g, 76 mmol) in toluene (360
mL) and ethanol (120 mL) was added 2 M aqueous K PO (104 mL, 208 mmol) and Pd(PPh ) (3.99 g,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
4
3 4
3
.46 mmol). The reaction mixture was then heated to 75ꢀ80 °C for 16 h. After cooling to RT, the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
precipitated gray solid was collected by filtration, was suspended in THF (120 mL) and heated at 65 °C
for 2 h. After cooling to RT, the dark solution was filtered through Celite to remove Pd material. The
filtrate was divided into 3 equal portions and purified by chromatography (eluting with MeOHꢀDCM, 2ꢀ
7
% gradient) to give 6 (18 g, 52%) as a colorless solid, and as a mixture of four atropisomers. CHN
analysis, Calcd for C H N O 0.79H O: C, 72.04, H, 5.38, N, 10.84, H O, 2.76; Found: C, 72.27, H,
3
1
26
4
3
2
2
+
1
5.25, N, 10.88, H O, 2.76; Pd 27 ppm. LCMS m/z 503.0 (M+H) . H NMR (500 MHz, DMSO-d6) δ
2
1
1.43 (s, 0.6H), 11.42 (s, 0.4H), 8.48 (s, 0.4H), 8.41 (s, 0.6H), 8.26 (dd, J=8.04, 1.22 Hz, 0.4H), 8.23
dd, J=8.04, 1.22 Hz, 0.6H), 8.15 (br s, 2H), 8.00 (d, J=7.79 Hz, 0.4H), 7.99 (d, J=7.79 Hz, 0.6H), 7.88ꢀ
7.92 (m, 1H), 7.86 (dd, J=7.91, 1.10 Hz, 1H), 7.79 (d, J=5.11 Hz, 0.6H), 7.77 (d, J=5.36 Hz, 0.4H),
.62ꢀ7.67 (m, 1H), 7.60 ꢀ 7.62 (m, 1H), 7.58 (t, J=7.30 Hz, 1H), 7.48 (dd, J=7.55, 1.22 Hz, 0.4H), 7.46
dd, J=7.43, 1.10 Hz, 0.6H), 7.12ꢀ7.14 (m, 0.4H), 7.11 (dd, J=5.84, 1.70 Hz, 0.6H), 7.06 (d, J=8.28 Hz,
(
7
(
0
.6H), 7.03 (d, J=7.79 Hz, 0.4H), 7.00 (d, J=7.79 Hz, 0.6H), 6.85 (d, J=8.52 Hz, 0.4H), 5.00 (s, 0.6H),
13
4
.97 (s, 0.4H), 1.79 (s, 1.5H), 1.76 (s, 1.5H), 1.47 (d, J=4.38 Hz, 3H), 1.46 (d, J=1.22 Hz, 3H). C
NMR (126 MHz, DMSO-d6) δ 169.00, 168.97, 159.80, 159.78, 148.89 (s, 2C), 147.94 (s, 2C), 147.34,
1
1
47.29, 141.51 (s, 2C), 140.36 (s, 2C), 139.01, 138.96, 137.81, 137.70, 137.42, 137.24, 134.75, 134.70,
34.00, 133.56, 130.21, 130.13, 128.19, 128.08, 127.52, 127.50, 127.43, 127.39, 127.18, 127.08,
126.48, 126.40, 124.13, 124.03, 121.88, 121.84, 121.80, 121.62, 120.42, 119.94, 119.07, 119.02,
1
3
18.48, 118.37, 116.88, 116.60, 114.97, 114.93, 108.01, 107.87, 70.94, 70.92, 32.39, 32.27, 32.12,
2.05, 14.55, 14.48. Numerous carbons were split due to the diastereomeric nature of the mixture of
atropisomers.
ꢀ(3ꢀ(5ꢀFluoropicolinamido)ꢀ2ꢀmethylphenyl)ꢀ7ꢀ(4ꢀmethylpiperazineꢀ1ꢀcarbonyl)ꢀ9Hꢀcarbazoleꢀ1ꢀ
4
carboxamide (10). A mixture of 7a (100 mg, 0.2 mmol), 5ꢀfluoropicolinic acid (43.1 mg, 0.31 mmol),
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Journal of Medicinal Chemistry
and 1ꢀhydroxyꢀ7ꢀazabenzotriazole (41.6 mg, 0.31 mmol) in acetonitrile (2 mL) was treated with
diisopropylethylamine (0.053 mL, 0.31 mmol) and EDC (78 mg, 0.41 mmol). After 18 h at RT, the
mixture was diluted with methanol and purified by preparative HPLC (Phenomenex AXIA C 21.2 x
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
8
1
00 mm, 5ꢀ95% methanolꢀwater containing 0.1% TFA, 10 min, 20 mL/min, 220 nm). The aqueous
residue from partial concentration of the appropriate effluent fractions was made basic with saturated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aqueous NaHCO and extracted with ethyl acetate (3x). The combined organic phases were dried over
3
Na SO and concentrated under vacuum to provide 10 as a light gray powder (111.5 mg, 92%). LCMS
2
4
+
1
m/z 565.2 (M+H) . H NMR (400 MHz, CD OD) δ 8.58 (d, J=3.1 Hz, 1H) 8.31 (dd, J=8.8, 4.8 Hz, 1H)
3
7
.96 ꢀ 8.03 (m, 2H) 7.82 (td, J=8.6, 2.6 Hz, 1H) 7.68 (d, J=0.9 Hz, 1H) 7.45 (t, J=7.9 Hz, 1H) 7.23 (dd,
J=7.5, 0.9 Hz, 1H) 7.09 (d, J=7.9 Hz, 1H) 7.03ꢀ7.08 (m, 1H) 6.95ꢀ7.00 (m, 1H) 3.78 (br s, 2H) 3.49 (br
s, 2H) 2.52 (br s, 2H) 2.40 (br s, 2H) 2.31 (s, 3H) 2.04 (s, 3H). Contains 0.33 equiv. (5% by weight)
residual ethyl acetate.
4
ꢀ(3ꢀ(4ꢀFluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ7ꢀ(isopropylamino)ꢀ9Hꢀcarbazoleꢀ1ꢀcarboxamide (11).
Step A. Benzyl 5ꢀbromoꢀ8ꢀcarbamoylꢀ9Hꢀcarbazolꢀ2ꢀylꢀcarbamate (36). To a suspension of 34 (4.6
g, 12.6 mmol) and 4Å molecular sieves (4.6 g) in 1,4ꢀdioxane (126 mL) at 50 °C was added Et N (4.3
3
o
mL, 31 mmol) and diphenylphosphoryl azide (6.7 mL, 31 mmol). The mixture was stirred at 50 C for
1
.5 h at which point phenylmethanol (13 mL, 126 mmol) was added, and the reaction temperature was
increased to 85 °C. After 18 h, the reaction mixture was cooled to RT, filtered through Celite, the solid
washed with MeOH, EtOAc, and acetone. The filtrates were concentrated to afford crude 36 as a light
brown syrup. The syrup was triturated with MeOH, the resulting solid collected by filtration, washed
with MeOH, and dried. The resulting filtrates were concentrated, triturated with MeOH again, and the
solid collected by filtration. The pale solids were combined to give 36 (5.1 g, 91%). LCMS m/z 437.9,
+
1
4
39.9 (M+H) . H NMR (400 MHz, DMSOꢀd ) δ 11.63 (s, 1H), 9.98 (s, 1H), 8.43 (d, J=8.6 Hz, 1H),
6
8.16 (br s, 1H), 8.06 (s, 1H), 7.76 (d, J=8.3 Hz, 1H), 7.60ꢀ7.23 (m, 8H), 5.19 (s, 2H).
Step B. Benzyl 5ꢀ(3ꢀaminoꢀ2ꢀmethylphenyl)ꢀ8ꢀcarbamoylꢀ9Hꢀcarbazolꢀ2ꢀylcarbamate.
A
suspension of 36 (1 g, 2.3 mmol), (Ph P) Pd (0.13 g, 0.11 mmol), 2 M aqueous Na CO (2.8 mL, 5.7
3
4
2
3
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mmol), and 2ꢀmethylꢀ3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)aniline (0.64 g, 2.7 mmol) in
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
o
toluene/EtOH (4/1) (57 mL) under nitrogen was heated at 90 C for 10 h. After cooling to RT, the
reaction mixture was filtered through Celite, which was washed with EtOAc. The filtrates were
partitioned between EtOAc and water, the organic phase washed with brine, dried over Na SO , and
2
4
concentrated to afford a yellow colored solid. The crude product was triturated with MeOH to give
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
benzyl 5ꢀ(3ꢀaminoꢀ2ꢀmethylphenyl)ꢀ8ꢀcarbamoylꢀ9Hꢀcarbazolꢀ2ꢀylcarbamate as an offꢀwhite solid
+
1
(
0.86 g in 82%). LCMS m/z 465.1 (M+H) . H NMR (400 MHz, DMSOꢀd ) δ 11.47 (s, 1H), 9.82 (s,
6
1H), 8.14 (br s, 1H), 7.97 (d, J=1.3 Hz, 1H), 7.91 (d, J=7.5 Hz, 1H), 7.47 (br s, 1H), 7.45ꢀ7.31 (m, 5H),
.30ꢀ7.24 (m, 1H), 7.17 (d, J=7.5 Hz, 1H), 6.97ꢀ6.86 (m, 3H), 6.67 (d, J=8.8 Hz, 1H), 5.15 (s, 2H), 1.84
s, 3H) (some exchangeable protons diffuse and not observed).
Step C. Benzyl 8ꢀcarbamoylꢀ5ꢀ(3ꢀ(4ꢀfluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ9Hꢀcarbazolꢀ2ꢀ
7
(
ylcarbamate. To a mixture of benzyl 5ꢀ(3ꢀaminoꢀ2ꢀmethylphenyl)ꢀ8ꢀcarbamoylꢀ9Hꢀcarbazolꢀ2ꢀ
ylcarbamate (700 mg, 1.5 mmol) and Hunig's Base (1 mL, 6 mmol) in THF (80 mL) at RT was added 4ꢀ
fluorobenzoyl chloride (0.36 mL, 3 mmol). After 1 hr the reaction mixture was concentrated, then
partitioned between DCM, water, and sat. NaHCO . The organic phases were separated, washed with
3
brine, dried over Na SO , and concentrated. The resulting residue was purified by chromatography
2
4
(eluting with hexane/EtOAc, 70/30 ꢀ 50/50 ꢀ 30/70) to give benzyl 8ꢀcarbamoylꢀ5ꢀ(3ꢀ(4ꢀ
fluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ9Hꢀcarbazolꢀ2ꢀylcarbamate (721 mg, 82%) as an offꢀwhite foamy
+
1
solid. LCMS m/z 587.3 (M+H) . H NMR (400 MHz, DMSOꢀd ) δ 11.47 (s, 1H), 10.13 (s, 1H), 9.82 (s,
6
1
H), 8.08 (dd, J=9.0, 5.5 Hz, 2H), 7.98 (d, J=1.3 Hz, 1H), 7.93 (d, J=7.7 Hz, 1H), 7.50ꢀ7.33 (m, 11H),
7.20 (dd, J=7.5, 1.1 Hz, 1H), 6.95 (d, J=7.7 Hz, 2H), 6.79 (d, J=8.8 Hz, 1H), 5.16 (s, 2H), 1.89 (s, 3H).
Step D. 7ꢀAminoꢀ4ꢀ(3ꢀ(4ꢀfluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ9Hꢀcarbazoleꢀ1ꢀcarboxamide (9a). A
suspension
of
benzyl
8ꢀcarbamoylꢀ5ꢀ(3ꢀ(4ꢀfluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ9Hꢀcarbazolꢀ2ꢀ
ylcarbamate (720 mg, 1.2 mmol) in hydrogen bromide in acetic acid (2.45 mL, 12.3 mmol) was stirred
at RT for 1 h. To the above suspension was added EtOAc, water, and 1 M NaOH to give a basic
aqueous layer (pH ~9). The organic phases were washed with brine, dried over Na SO , filtered, and
2
4
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Journal of Medicinal Chemistry
concentrated. The crude product was then triturated with EtOAc to give 7ꢀaminoꢀ4ꢀ(3ꢀ(4ꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
fluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ9Hꢀcarbazoleꢀ1ꢀcarboxamide (9a) (0.33 g, 59%) as a greenishꢀ
+
1
yellow solid. LCMS m/z 453.0 (M+H) . H NMR (400 MHz, DMSOꢀd ) δ 11.02 (s, 1H), 10.11 (s, 1H),
6
8
.09 (dd, J=8.8, 5.3 Hz, 3H), 7.78 (d, J=7.9 Hz, 1H), 7.48ꢀ7.43 (m, 1H), 7.42ꢀ7.32 (m, 4H), 7.18 (dd,
J=7.5, 0.9 Hz, 1H), 6.89ꢀ6.75 (m, 2H), 6.60 (d, J=8.8 Hz, 1H), 6.21 (dd, J=8.6, 2.0 Hz, 1H), 5.18 (s,
H), 1.90 (s, 3H).
Step E.
carboxamide (11).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4ꢀ(3ꢀ(4ꢀFluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ7ꢀ(isopropylamino)ꢀ9Hꢀcarbazoleꢀ1ꢀ
A suspension of 7ꢀaminoꢀ4ꢀ(3ꢀ(4ꢀfluorobenzamido)ꢀ2ꢀmethylphenyl)ꢀ9Hꢀ
carbazoleꢀ1ꢀcarboxamide (30 mg, 0.066 mmol), acetone (0.019 mL, 0.265 mmol), and sodium
triacetoxyborohydride (35.1 mg, 0.166 mmol) in DCM/THF (3 mL, 2/1) was stirred at RT overnight.
After 18 h, the reaction mixture was concentrated, dissolved in DMF, and purified by prep HPLC to
+
1
give 11 (11 mg, 24%) as a pale solid. LCMS m/z 495.0 (M+H) . H NMR (500 MHz, CD OD) δ 8.12ꢀ
3
8.01 (m, 3H), 7.66 (d, J=1.4 Hz, 1H), 7.55ꢀ7.50 (m, 1H), 7.46 (t, J=7.8 Hz, 1H), 7.33ꢀ7.20 (m, 4H), 7.15
(
d, J=7.8 Hz, 1H), 6.97 (dd, J=8.6, 1.9 Hz, 1H), 3.80 (dt, J=13.0, 6.5 Hz, 1H), 1.99 (s, 3H), 1.35 (dd,
J=6.2, 5.1 Hz, 6H).
ꢀ(2ꢀMethylꢀ3ꢀ(1ꢀoxoisoindolinꢀ2ꢀyl)phenyl)ꢀ7ꢀ(4ꢀmethylpiperazineꢀ1ꢀcarbonyl)ꢀ9Hꢀcarbazoleꢀ1ꢀ
4
carboxamide (12).
Step A. 2ꢀ(3ꢀBromoꢀ2ꢀmethylphenyl)isoindolinꢀ1ꢀone (39a). To a solution of 3ꢀbromoꢀ2ꢀ
methylaniline (2 g, 10.8 mmol) in DCM (25 mL) and TEA (3 mL, 21.5 mmol) at RT was slowly added
a DCM (10 mL) solution of 2ꢀ(chloromethyl)benzoyl chloride (2 g, 10.8 mmol) over 1.5 h. The
reaction mixture was then washed with water and brine, and concentrated to give Nꢀ(3ꢀbromoꢀ2ꢀ
methylphenyl)ꢀ2ꢀ(chloromethyl)benzamide as a tan solid. A 60% oil dispersion of NaH (0.86 g, 21.5
mmol) was washed twice with hexanes and suspended in tetrahydrofuran (25 mL). To this suspension
was slowly added a THF (50 mL) solution of Nꢀ(3ꢀbromoꢀ2ꢀmethylphenyl)ꢀ2ꢀ(chloromethyl)benzamide.
After the addition was complete the mixture was stirred at RT for 1 h. The reaction mixture was then
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diluted with DCM and quenched carefully with methanol, washed with water, brine and concentrated to
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
give 39a (1.9 g, 60%) as a tan solid. LCMS m/z (M+H) 301.9, 303.9.
Step B. 2ꢀ(2ꢀMethylꢀ3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)isoindolinꢀ1ꢀone (40a).
Nitrogen was bubbled into a mixture of 39a (1.5 g, 5 mmol), 4,4,4',4',5,5,5',5'ꢀoctamethylꢀ2,2'ꢀbi(1,3,2ꢀ
dioxaborolane) (1.9 g, 7.4 mmol), and potassium acetate (1.5 g, 14.9 mmol) in dioxane (40 mL) for 10
min, followed by the addition of PdCl2(dppf)ꢀCH Cl adduct (0.12 g, 0.15 mmol). The resulting mixture
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
was heated to reflux for 4 h and diluted with ethyl acetate. The resulting mixture was filtered, the filtrate
washed with water and again filtered. The resulting solution was concentrated and the residue purified
by chromatography (eluting with EtOAc/hexane, 10ꢀ50% gradient) to give 40a (1.2 g, 69%) as a light
+
yellow sticky foam. LCMS m/z (M+H) 350.1.
Step
carbazoleꢀ1ꢀcarboxamide (12). To a mixture of 35 (52 mg, 0.12 mmol) and 40a (52 mg, 0.15 mmol) in
toluene (3 mL) and ethanol (1 mL) was added 2 M aqueous K PO (0.19 mL, 0.38 mmol) and then
C.
4ꢀ(2ꢀMethylꢀ3ꢀ(1ꢀoxoisoindolinꢀ2ꢀyl)phenyl)ꢀ7ꢀ(4ꢀmethylpiperazineꢀ1ꢀcarbonyl)ꢀ9Hꢀ
3
4
Pd(Ph P) (7.2 mg, 6.3 µmol). The reaction mixture was heated at 100 °C for 8 h and then diluted with
3
4
ethyl acetate. The mixture was washed with water and the organic layer concentrated. The resulting
residue was purified by chromatography (eluting with 90:9:1 DCM:MeOH:NH OH/DCM, 50ꢀ100%
4
gradient) to give 12 (44 mg), which was further purified by prep HPLC (Phenomenex Luna Axia, 5µ,
2
1x100 mm, solvent A: 10% MeOHꢀ 90% H Oꢀ 0.1% TFA; solvent B: 90% MeOHꢀ 10% H Oꢀ 0.1%
2 2
TFA, gradient: 0ꢀ100% B, gradient time: 10 min, flow rate: 30 ml/min, wavelength 220 nm) to give 12
+
1
(
30 mg, 44%) as a white solid. LCMS m/z (M+H) 558.2; H NMR (400 MHz, CDCl ) δ 10.68 (s, 1 H),
3
7
.97 (d, J=8.35 Hz, 1H), 7.70 (d, J=7.47 Hz, 1H), 7.63 (t, J=6.81 Hz, 1H), 7.51ꢀ7.58 (m, 3H), 7.46 (d,
J=3.95 Hz, 2H), 7.33ꢀ7.39 (m, 1H), 7.08ꢀ7.16 (m, 3H), 4.83 (s, 2H), 3.30ꢀ4.04 (m, 4H), 2.34ꢀ2.61 (m,
H), 2.32 (s, 3H), 1.93 (s, 3H) (some exchangeable protons diffuse and not observed).
4
7ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)ꢀ4ꢀ(2ꢀmethylꢀ3ꢀ(1ꢀoxoisoindolinꢀ2ꢀyl)phenyl)ꢀ9Hꢀcarbazoleꢀ1ꢀ
carboxamide (13). A mixture of 31 (40 mg, 0.12 mmol), 40a (40 mg, 0.12 mmol), Pd(Ph P) (6.7 mg,
3
4
5
.8 umol) and 2 M aqueous K PO (0.14 mL, 0.28 mmol) in toluene (2 mL) and EtOH (0.5 mL) was
3 4
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Journal of Medicinal Chemistry
heated at 90 °C for 16 h. After cooling to RT, the reaction mixture was diluted with DMF (1 mL),
filtered and purified by preparative HPLC. The product containing fractions were collected, basified
with 1N NaOH and extracted twice with DCM. The combined organic phases were washed with water
and concentrated. The resulting residue was purified further by chromatography (eluting with a gradient
from 100% DCM to 0.8:7.2:92 ammoniaꢀMeOHꢀDCM) to give 13 as a white solid (25 mg, 42%). Mass
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
spectrum m/z 472.2 (MꢀOH) . H NMR (400 MHz, CD OD) δ 7.95 (d, J=7.92 Hz, 1H), 7.89 (d, J=7.48
3
Hz, 1H), 7.76 (d, J=1.10 Hz, 1H), 7.65ꢀ7.73 (m, 2H), 7.48 ꢀ 7.61 (m, 3H), 7.41 (dd, J=7.04, 1.76 Hz,
1H), 7.17 (dd, J=8.36, 1.54 Hz, 1H), 7.04ꢀ7.10 (m, 2H), 4.96 (s, 2H), 1.92 (s, 3H), 1.61 (d, J=3.74 Hz,
6
H).
4
ꢀ(3ꢀ(6ꢀFluoroꢀ1ꢀoxoisoindolinꢀ2ꢀyl)ꢀ2ꢀmethylphenyl)ꢀ7ꢀ(2ꢀhydroxypropanꢀ2ꢀyl)ꢀ9Hꢀcarbazoleꢀ1ꢀ
carboxamide (14).
Step A. Nꢀ(3ꢀBromoꢀ2ꢀmethylphenyl)ꢀ2ꢀ(bromomethyl)ꢀ5ꢀfluorobenzamide. To a solution of 2ꢀ
(bromomethyl)ꢀ5ꢀfluorobenzoic acid (3.1 g, 13.1 mmol) in DCM (50 mL) at RT was added 6 drops of
DMF and oxalyl dichloride (1.7 g, 13.1 mmol). After 1 h, the reaction was concentrated, redissolved in
DCM and concentrated again. The resulting residue was redissolved in DCM (50 mL) and 3ꢀbromoꢀ2ꢀ
methylaniline (1.7 g, 9.1 mmol) was added. After stirring for 1 h, Et N (2.9 mL, 15.7 mmol) was added
3
portionꢀwise. The reaction mixture was diluted with DCM (100 mL) after 2 h, the organic layer washed
with satd. NaHCO , water, and concentrated. The resulting solid residue was triturated with DCM to
3
give Nꢀ(3ꢀbromoꢀ2ꢀmethylphenyl)ꢀ2ꢀ(bromomethyl)ꢀ5ꢀfluorobenzamide (1.4 g). The mother liquor was
purified by chromatography (eluting with ethyl acetate/hexane, 0ꢀ70% gradient) to give additional
material (2.5 g total, 48%) suitable for the subsequent reaction.
Step B. 2ꢀ(3ꢀBromoꢀ2ꢀmethylphenyl)ꢀ6ꢀfluoroisoindolinꢀ1ꢀone (39b). A mixture of Nꢀ(3ꢀbromoꢀ2ꢀ
methylphenyl)ꢀ2ꢀ(bromomethyl)ꢀ5ꢀfluorobenzamide (2.5 g, 6.3 mmol) and sodium tertꢀbutoxide (0.91
g, 9.5 mmol) in THF (80 mL) was stirred at RT for 30 min. The reaction mixture was quenched with
water, extracted with DCM twice, the combined organic phases washed with water and concentrated.
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The resulting residue was purified by chromatography (eluting with hexane/ethyl acetate) to give 39b
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
(
1.18 g, 59%) as a white solid. LCMS m/z (M+H) 319.9, 321.9.
Step C. 6ꢀFluoroꢀ2ꢀ(2ꢀmethylꢀ3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)isoindolinꢀ1ꢀ
one (40b). A solution of 39b (1.3 g, 4.1 mmol), 4,4,4',4',5,5,5',5'ꢀoctamethylꢀ2,2'ꢀbi(1,3,2ꢀ
dioxaborolane) (1.6 g, 6.1 mmol), PdCl (dppf)ꢀCH Cl adduct (0.17 g, 0.2 mmol), and potassium acetate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
(1.2 g, 12.2 mmol) in dioxane (20 mL) was heated at 90 °C overnight. The reaction mixture was diluted
with DCM, washed with water, the organic layer separated, and the aqueous layer was extracted twice
more with DCM. The combined organic extracts were concentrated and the crude material was purified
by chromatography (eluting with EtOAc/hexane, 0ꢀ50% gradient) to give 40b (1.35 g, 86%) as a white
+
1
solid. LCMS m/z (M+H) 368.0. H NMR (400 MHz, CDCl ) δ 7.82 (dd, J=6.93, 1.87 Hz, 1H), 7.63
3
(
dd, J=7.59, 2.31 Hz, 1H), 7.47 (dd, J=8.25, 4.29 Hz, 1H), 7.27ꢀ7.34 (m, 3H), 4.65 (s, 2H), 2.41 (s, 3H),
.32ꢀ1.37 (m, 12H).
Step D. 4ꢀ(3ꢀ(6ꢀFluoroꢀ1ꢀoxoisoindolinꢀ2ꢀyl)ꢀ2ꢀmethylphenyl)ꢀ7ꢀ(2ꢀhydroxypropanꢀ2ꢀyl)ꢀ9Hꢀ
1
carbazoleꢀ1ꢀcarboxamide (14). A mixture of 31 (30 mg, 0.09 mmol), 40b (41 mg, 0.11 mmol),
Pd(Ph P) (5 mg, 4.3 µmol), and 2 M aqueous K PO (0.13 mL, 0.26 mmol) in toluene (3 mL) and
3
4
3
4
ethanol (1 mL) was heated at 100 °C for 9 h. The reaction mixture was cooled to RT, partitioned
between water and ethyl acetate, and the organics concentrated to give yellow solid. The crude material
was purified by chromatography (eluting with 90:9:1 DCM:MeOH:NH OH/DCM, 10ꢀ30% gradient) to
4
+
1
give 14 (28 mg, 59%) as a white solid. LCMS m/z (M+Hꢀ H 0) = 490.3. H NMR (400 MHz, CDCl ) δ
2
3
1
0.60 (s, 1H), 7.70 (d, J=1.10 Hz, 1H), 7.63ꢀ7.68 (m, 2H), 7.52 (dd, J=8.36, 4.40 Hz, 1H), 7.44ꢀ7.48 (m,
2H), 7.31ꢀ7.41 (m, 2H), 7.21 (dd, J=8.36, 1.76 Hz, 1H), 7.06ꢀ7.11 (m, 2H), 4.82 (s, 2H), 2.01 (s, 1H),
1
.97 (s, 3H), 1.66 (s, 6H) (some exchangeable protons diffuse and not observed).
7
ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)ꢀ4ꢀ(2ꢀmethylꢀ3ꢀ(1ꢀoxoꢀ3,4ꢀdihydroisoquinolinꢀ2(1H)ꢀyl)phenyl)ꢀ9Hꢀ
carbazoleꢀ1ꢀcarboxamide (15).
Step A. 7ꢀ(2ꢀHydroxypropanꢀ2ꢀyl)ꢀ4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ9Hꢀcarbazoleꢀ
1
ꢀcarboxamide (33). A mixture of 31 (5 g, 14.4 mmol), 4,4,4',4',5,5,5',5'ꢀoctamethylꢀ2,2'ꢀbi(1,3,2ꢀ
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Journal of Medicinal Chemistry
dioxaborolane) (4.4 g, 17.3 mmol), potassium acetate (4.2 g, 43.2 mmol) and PdCl (dppf)ꢀCH Cl
2
2
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
adduct (0.59 g, 0.72 mmol) was equally divided into two large pressure reaction vials, suspended in
dioxane (30 mL), sealed and heated at 110 °C for 4.5 h. The two reaction mixtures were then combined,
diluted with ethyl acetate, washed with water, filtered through Celite and concentrated. The resulting
black residue was purified by chromatography (eluting with EtOAc/hexane, 50ꢀ100% gradient) to give
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
1
3
3 (3.9 g, 67%). LCMS m/z 376.9 (M+HꢀOH) . H NMR (400 MHz, CDCl ) δ 10.55 (br s, 1H), 8.92
3
(d, J=8.6 Hz, 1H), 7.71 (d, J=7.7 Hz, 1H), 7.59ꢀ7.52 (m, 2H), 7.43 (dd, J=8.5, 1.7 Hz, 1H), 1.49 (s,
12H) (some exchangeable protons diffuse and not observed).
Step B. 3,4ꢀDihydroisoquinolinꢀl (2H)ꢀone (46). A solution of 2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀone (1 g, 7.6
mmol) in DCM (10 mL) was treated with methane sulfonic acid (10 mL) and cooled to 0 °C. Sodium
azide (0. 98 g, 15.1 mmol) was added and the mixture was stirred at 0 °C for 2 h, then at RT overnight.
The mixture was made basic with 20% aqueous sodium hydroxide and extracted with DCM. The
organic phase was washed with water, dried and concentrated. The resulting residue was purified by
chromatography (eluting with hexaneꢀEtOAc) to provide 3,4ꢀdihydroisoquinolinꢀl (2H)ꢀone 46 as a
+
colorless oil (162 mg, 15%). Mass spectrum m/z 148.1 (M+H) . 1H NMR (400 MHz, CD3OD) δ 7.93
(dd, J=7.8, 1.0 Hz, 1H), 7.44ꢀ7.52 (m, 1H), 7.35 (td, J=7.6, 1.2 Hz, 1H), 7.29 (d, J=7.7 Hz, 1H), 3.50 (t,
J=6.6 Hz, 2H), 2.98 (t, J=6.7 Hz, 2H).
Step C. 2ꢀ(3ꢀBromoꢀ2ꢀmethylphenyl)ꢀ3,4ꢀdihydroisoquinolinꢀ1(2H)ꢀone (47). A mixture of 1,3ꢀ
dibromoꢀ2ꢀmethylbenzene (340 mg, 1.36 mmol), 46 (100 mg, 0.68 mmol) and potassium carbonate (94
mg, 0.68 mmol) in DMSO (2 mL) was purged with nitrogen, treated with copper (I) iodide (26 mg, 0.14
mmol) and heated at 150 °C for 3.5 h. The mixture was combined with that from a second identical
reaction using 1, 3ꢀdibromoꢀ2ꢀmethylbenzene (2.6 g, 10.4 mmol) and 3, 4ꢀdihydroisoquinolinꢀl (2H)ꢀ
one (508 mg, 3.4 mmol), diluted with DCM and filtered through Celite. The filtrate was washed with
5
% aqueous ammonium hydroxide, dried and concentrated. The residue was purified by
chromatography (eluting with hexaneꢀEtOAc) to provide 47 as a yellow solid (142 mg, 11%). Mass
+
spectrum m/z 315.9, 317.9 (M+H) . 1H NMR (400 MHz, CDCl ) δ 8. 15 (dd, J=7.70, 1.10 Hz, 1H), 7.
3
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