Discovery of Branebrutinib (BMS-986195): A Strategy for Identifying a Highly Potent and Selective Covalent Inhibitor Providing Rapid in Vivo Inactivation of Bruton's Tyrosine Kinase (BTK)

Article abstract of DOI:10.1021/acs.jmedchem.9b00167

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec family of kinases and is essential for B cell receptor (BCR) mediated signaling. BTK also plays a critical role in the downstream signaling pathways for the Fcγ receptor in monocytes, the Fc receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as rheumatoid arthritis and lupus. This article will outline the evolution of our strategy to identify a covalent, irreversible inhibitor of BTK that has the intrinsic potency, selectivity, and pharmacokinetic properties necessary to provide a rapid rate of inactivation systemically following a very low dose. With excellent in vivo efficacy and a very desirable tolerability profile, 5a (branebrutinib, BMS-986195) has advanced into clinical studies.

Full text of DOI:10.1021/acs.jmedchem.9b00167

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Drug Annotation
Discovery of Branebrutinib (BMS-986195): A Strategy for Identifying
a Highly Potent and Selective Covalent Inhibitor Providing
Rapid In Vivo Inactivation of Bruton's Tyrosine Kinase (BTK)
Scott H. Watterson, Qingjie Liu, Myra Beaudoin Bertrand, Douglas G. Batt, Ling Li, Mark A. Pattoli, Stacey
Skala, Lihong Chen, Mary T. Obermeier, Robin Moore, Zheng Yang, Rodney Vickery, Paul A. Elzinga,
Lorell N. Discenza, Celia D'Arienzo, Kathleen M. Gillooly, Tracy L. Taylor, Claudine Pulicicchio, Yifan
Zhang, Elizabeth Heimrich, Kim W. McIntyre, Qian Ruan, Richard A Westhouse, Ian M. Catlett, Naiyu
Zheng, Charu Chaudhry, Jun Dai, Michael A. Galella, Andrew Joseph Tebben, Matt Pokross, Jianqing
Li, Rulin Zhao, Daniel Smith, Richard A. Rampulla, Alban Allentoff, Michael A. Wallace, Arvind Mathur,
Luisa Salter-Cid, John E. Macor, Percy H Carter, Aberra Fura, James R. Burke, and Joseph A Tino
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00167 • Publication Date (Web): 20 Mar 2019
Downloaded from http://pubs.acs.org on March 20, 2019
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Macor, John; Bristol-Myers Squibb Research and Development
Carter, Percy; Bristol-Myers Squibb Research and Development
Fura, Aberra; Bristol-Myers Squibb Research and Development
Burke, James; Bristol-Myers Squibb Research and Development
Tino, Joseph; Bristol-Myers Squibb Research and Development
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Discovery of Branebrutinib (BMS-986195): A
Strategy for Identifying a Highly Potent and
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Selective Covalent Inhibitor Providing Rapid In Vivo
Inactivation of Bruton's Tyrosine Kinase (BTK)
Scott H. Watterson,* Qingjie Liu, Myra Beaudoin Bertrand, Douglas G. Batt, Ling Li, Mark A. Pattoli,
Stacey Skala, Lihong Cheng, Mary T. Obermeier, Robin Moore, Zheng Yang, Rodney Vickery, Paul A.
Elzinga, Lorell Discenza, Celia D’Arienzo, Kathleen M. Gillooly, Tracy L. Taylor, Claudine Pulicicchio,
Yifan Zhang, Elizabeth Heimrich, Kim W. McIntyre, Qian Ruan, Richard A. Westhouse, Ian M. Catlett,
Naiyu Zheng, Charu Chaudhry, Jun Dai, Michael A. Galella, Andrew J. Tebben, Matt Pokross, Jianqing
Li, Rulin Zhao, Daniel Smith, Richard Rampulla, Alban Allentoff, Michael A. Wallace, Arvind Mathur,
Luisa Salter-Cid, John E. Macor, Percy H. Carter, Aberra Fura, James R. Burke, and Joseph A. Tino
Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543
RECEIVED DATE
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ABSTRACT: Bruton’s tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec
family of kinases and is essential for B cell receptor (BCR)-mediated signaling. BTK also plays a critical
role in the downstream signaling pathways for the Fc-gamma receptor in monocytes, the Fc-epsilon
receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition
of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases
such as rheumatoid arthritis and lupus. This article will outline the evolution of our strategy to identify a
covalent, irreversible inhibitor of BTK that has the intrinsic potency, selectivity, and pharmacokinetic
properties necessary to provide a rapid rate of inactivation systemically following a very low dose. With
excellent in vivo efficacy and a very desirable tolerability profile, 5a (branebrutinib, BMS-986195) has
advanced into clinical studies.
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Introduction
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Bruton’s tyrosine kinase (BTK) is a member of the Tec family of non-receptor tyrosine kinases and
is expressed in all hematopoietic cells, with the exception of T cells and terminally-differentiated plasma
cells. The underlying pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA) and
lupus relies on many of the pathways regulated by BTK, including B cell and myeloid cellular functions.1-3
In B cells, BTK kinase activity is essential in B cell receptor (BCR)-mediated activation, resulting in cell
proliferation, antibody and cytokine production, and costimulatory molecule expression (e.g. CD80,
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CD86, and CD69). In myeloid cells, BTK is a critical component in the signaling pathway of low-affinity
activating-Fc receptors (e.g. FcRIIa and FcRIIIa) for the immunoglobulin G (IgG)-containing immune
complexes in monocytic cells and Fc receptor signaling in mast cells and basophils, triggering the
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expression of pro-inflammatory cytokines, chemokines, and cell adhesion molecules. Of particular
interest for the clinical treatment of RA, BTK-dependent signaling is required for RANK-L controlled
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osteoclastogenesis in monocytic precursors. Overall, with its impact on both BCR signaling and Fc and
Fc receptor signaling, inhibition of the kinase activity of BTK7,8 is expected to provide an effective
strategy for the clinical treatment of autoimmune diseases such as lupus and RA, without depleting B
cells.
As a result of strong preclinical validation, there has been an intense effort to identify orally active
small molecule inhibitors of BTK as clinical therapeutic agents for the treatment of both oncological and
autoimmune diseases. With an appropriately positioned non-catalytic cysteine residue (Cys481) in the
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kinase domain, there has been particular interest in identifying inhibitors that covalently bind to Cys481.
Ibrutinib (1a), a covalent, irreversible inhibitor of BTK, is approved for the clinical treatment of chronic
lymphocytic leukemia, mantle cell lymphoma, Waldenstrom’s macroglobulinemia, and chronic graft
versus host disease.10 In spite of significant clinical efficacy in otherwise difficult to treat diseases,
administration of 1a has led to undesirable clinical side effects including rash and diarrhea, attributed to
the drug’s potent off-target inhibition of the epidermal growth factor receptors (EGFR), as well as
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bleeding and atrial fibrillation. Additionally, as a result of high clearance and a slow rate of inactivation,
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a requires high doses for clinical efficacy (420 – 560 mg QD), which when combined with the side effect
profile would likely limit its use to oncological diseases. In an effort to improve upon the overall profile
of 1a, several additional covalent inhibitors,12,13 as well reversible inhibitors,14,15 have advanced into
clinical trials for the treatment of both oncological and autoimmune disorders. One such drug,
acalabrutinib (1b) was recently approved for the treatment of mantle cell lymphoma.13
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We have previously reported on our efforts to identify reversible inhibitors of BTK.15c-d This effort
led to the discovery of BMS-986142 (2, Figure 2), as a single, stable atropisomer, which is currently being
evaluated clinically for the treatment of rheumatoid arthritis.15a-b Dosing was designed to provide
continuous coverage of the IC50 throughout the dosing interval, as supported by preclinical animal
studies.15 Recognizing that coverage as high as IC might be required for clinical efficacy, we initiated
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a covalent, irreversible discovery effort. It has been reported in the literature that covalent, irreversible
inhibition can provide advantages over traditional reversible inhibition such as increased biochemical
efficiency for target disruption and a prolonged pharmacodynamic (PD) effect that outlasts the
pharmacokinetics (PK) of the compound, both improving the potential for lower concentrations and
subsequent improved therapeutic margins. However, covalent, irreversible inhibition also enhances the
risk of off-target reactivity to biomolecules, potentially leading to hepatotoxicity, immunotoxicity, and
mutagenicity.17 As a result of their covalent nature, off-target interaction would be expected to be more
significant with the potential for prolonged recovery. Of particular concern with covalent inhibition is
the potential for idiosyncratic adverse drug reactions (IADRs), which are characterized as
immunogenicity of a protein adduct (haptenization) leading to an allergic response or drug
hypersensitivity reaction.17
In considering this strategy to develop a selective covalent, irreversible inhibitor for the treatment of
autoimmune diseases, a low human dose of ≤ 10 mg was desired to significantly reduce the potential for
off-target reactivity. Drugs with a dose ≤ 10 mg are unlikely to cause IADRs, including those that form
reactive metabolites.17a We reasoned that a low human dose would require that a covalent, irreversible
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inhibitor have both a rapid rate of enzyme inactivation and PK properties amenable to rapid target
engagement in vivo. As shown in Figure 3, covalent bonding requires a two-step process. The first step
involves non-covalent, reversible binding to the active site of BTK to establish [E, as measured by Ki.
The second step involves covalent, irreversible bonding of an electrophilic center on the inhibitor with
the nucleophilic Cys481 residue in the active site, resulting in complete enzyme inactivation [E, as
measured by kinact. The apparent second order rate of inactivation provided by a covalent, irreversible
inhibitor (k ) is represented by kinact/K . With this in mind, our strategy to attain a rapid rate of
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inactivation was to start with a high affinity, highly selective scaffold with reversible binding to the target
in the absence of covalent bonding, where the electrophilic acceptor enhances but does not drive potency
or selectivity. A high affinity compound (optimized K ) would be expected to provide high concentrations
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of [E, which would be essential for a rapid rate of inactivation and enhanced selectivity. Additionally,
a covalent, irreversible inhibitor with an appended acceptor with low intrinsic reactivity would be
desirable to mitigate any off-target interactions. This would require an optimal scaffold, linker, and
acceptor geometry to allow for precise positioning of the acceptor in close proximity to Cys481 to drive
rapid inactivation (kinact) while maintaining high affinity binding (K ) to the hinge region of the kinase
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domain.
To achieve a low human dose, a covalent, irreversible inhibitor would be expected to require PK
properties that provide a balance between clearance and the rate of target engagement and ultimate
enzymatic inactivation. In particular, the rate of target inactivation would need to occur at a rate faster
than that of drug elimination, but remaining compound would need to be cleared rapidly to mitigate any
off-target interactions (Figure 4). We expected that a compact, low molecular weight inhibitor with an
acceptor with low intrinsic reactivity would offer the best chance for success. Overall, this strategy was
anticipated to provide a rapid rate of inactivation of BTK, as well as minimal reactivity to small molecule
thiols, proteins, and DNA to mitigate any off-target interactions.
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As we began to focus on a covalent inhibition startegy, our deep understanding of non-covalent,
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reversible inhibition of BTK played a key role.15d,18-20 As the X-ray co-crystal structure of our highly
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potent and selective clinical asset 215d (Figures 2 and 5; BTK IC = 0.50 nM; hWB IC = 90 nM; 1.5 Å,
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PDB ID code: 5T18) bound to the kinase domain of BTK revealed, the tetrahydrocarbazole motif provides
strong hydrogen bonding interactions with the hinge region. Specifically, the tetrahydrocarbazole NH
and the carboxamide carbonyl engaged in hydrogen bonding interactions with Met477 in the hinge region
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of the active site, while the NH of the carboxamide formed hydrogen bonding interactions with Glu475
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and a conserved water, providing a bridging interaction with the gatekeeper Leu408 residue. In addition,
a weak hydrogen bonding interaction of the quinazolinedione to the Cys481 residue through a conserved
water was observed, highlighting the compound’s close proximity. As a result, our initial strategy, as
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recently reported, involved replacing the quinazolinedione with a simple acrylamide, as shown in Figure
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. Encouragingly, this provided compound 3a with highly potent, covalent inhibition. As recently
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disclosed, a truncated dimethyl indole scaffold, as represented by 3b and 3c, retained the activity seen
with 3a. Unfortunately, oral dosing of carbazole 3a in a mouse PK study resulted in low plasma
concentrations with an aniline metabolite, resulting from acrylamide hydrolysis, as the predominant drug-
related compound in circulation. Alternative acceptors did not resolve the PK issue while sustaining
desirable potency, so as a consequence, our revised strategy focused on replacing the aniline linker.
Although we explored covalent, irreversibility across the full range of our reversible scaffolds, this article
will focus on the SAR development of the dimethylindole carboxamide series since it provided a desirable
balance between potency/selectivity and PK properties, as well as overall favorable pharmaceutical
properties.21 This effort ultimately led to our covalent, irreversible clinical compound, branebrutinib (5a,
BMS-986195).
Biological Evaluation
The covalent, irreversible compounds presented in this article were evaluated in a human recombinant
BTK enzyme assay to determine their intrinsic activity against BTK. It should be noted that the potency
of a compound with a BTK IC significantly below 1 nM may be underestimated since this approaches
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the lower limit of potency that can be measured in the enzyme assay (limited by enzyme concentration).
Given the intrinsic potency of the parent scaffold, significant potency resulting from the addition of an
electrophilic acceptor suggests but does not prove covalent, irreversible inhibition. As a result, a dialysis
assay was used to determine whether or not a compound was irreversibly bound. In this assay, the
compound was incubated with BTK for 90 min. at concentrations approximately 25-fold higher than the
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IC , to ensure saturation of the active site, followed by overnight dialysis against assay buffer with two
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buffer exchanges. If BTK activity did not return, the compound was regarded as a likely covalent inhibitor
(later confirmed by active site occupancy, mass spectrometry, and crystallographic analysis of select
compounds). In this assay, a reversible BTK inhibitor showed almost complete recovery of activity. A
BCR stimulated calcium flux assay in Ramos B cells was used to establish activity in a cellular
environment. In light of the fact that IC determinations are dependent on both pre-incubation time and
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the rate of covalent bonding, the intrinsic potency of a covalent, irreversible inhibitor would need to be
evaluated in a time-dependent manner. As a result, the rate of BTK engagement and inactivation of
selected analogues was determined in an active site probe-based assay using a biotinylated covalent
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inactivator to quantify unmodified active sites following incubation with the compound in human whole
blood, as well as the kinetics and dose relationships of in vivo inactivation of BTK in mice and
cynomolgus monkeys after oral administration. In parallel with the in vitro potency assays, in vitro
liability profiling data was assessed to prioritize compounds. Selected compounds were evaluated in
multi-species PK studies and ultimately rodent efficacy models of human lupus and RA.
Results and Discussion
As seen in Table 1, replacing the tolyl or phenyl linker in 3b and 3c, respectively, with a 3-amino
piperidine ring appended with an acrylamide afforded 4a (racemic) which resulted in a 20-fold loss in
activity. Replacing the acrylamide acceptor with a more reactive vinyl sulfonamide (4b) resulted in a ~3-
fold increase in activity relative to 4a. Enantiomerically pure derivatives 4c and 4d were subsequently
prepared and evaluated, with 4d providing similar intrinsic potency as both 3b and 3c in both the
biochemical BTK assay and the Ramos cellular assay. As shown in Table 1, 4d had substantially
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improved stability in human liver microsomes when compared to 1a (90% remaining; T1/2 of 50 min vs.
% remaining; T1/2 of 1.5 min., respectively). However, in spite of the increased metabolic stability
relative to 1a, plasma concentrations for 4d in a mouse coarse PK study were low (Cmax: 127 nM; AUC:
.35 M h). Unfortunately, metabolite identification studies in human liver microsomes (10 uM
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incubation, 30 min.) revealed that the primary route of metabolism of 4d was through direct glutathione
addition not mediated through glutathione-S-transferase (GST), which would be undesirable for a
covalent, irreversible inhibitor. This suggested that the vinyl sulfonamide acceptor is too reactive and is
likely driving the observed potency. Interestingly, in spite of having a highly reactive acceptor, 4d
provided a slower rate of inactivation relative to 1a in a time-dependent, concentration-dependent BTK
inactivation assay in human whole blood, as shown in Figure 7. This clearly suggested that compound
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d has a less than optimal linker/acceptor geometry to provide both high affinity binding to the hinge
region of the kinase domain active site (K ) and optimal positioning of the acceptor for rapid inactivation
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(kinact).
As a result, our strategy evolved into the optimization of the linker and acceptor trajectory. Saturated
linkers with varying sizes, points of attachment, and chirality were paired with less reactive acceptors to
fine-tune the geometry (Figure 8), with a primary focus on optimizing kinact while maintaining the intrinsic
high affinity (K ) for the target. The SAR representing our effort to improve both potency and
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pharmacokinetic properties are outlined in Table 2. This effort resulted in two interesting leads,
compounds 4k and 4f, both providing less than 10 nM cellular potency. Further evaluation showed that
4
k inactivated BTK in the human whole blood assay at a rate similar to 1a (apparent second order rate =
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-4
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.0 x 10 nM min vs. 1.2 x 10 nM min , respectively). On a more intriguing note, compound 4f,
* *
with a propiolamide acceptor, provided 1.8 nM activity in the cellular assay and inactivated BTK in the
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human whole blood assay at a rate >10-fold faster than 1a (>10 x 10 nM min vs. 1.2 x 10 nM min
*
*
1)
. Unfortunately, low plasma concentrations were observed in a mouse PK study. In both human and
mouse liver microsomes, the primary metabolic pathway identified corresponded to direct glutathione
addition, likely contributing to the poor in vivo exposure, once again suggesting that the acceptor is too
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reactive. In an effort to mitigate the reactivity, the corresponding but-2-ynamide acceptor 4g was
investigated. Intriguingly, 4g was no longer a substrate for direct GSH addition and provided acceptable
plasma concentrations in vivo; however, 4g was ~20 fold less potent than 4f in the cellular assay, with an
rate of inactivation similar to that of 1a.
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Encouraged by the balance between potency and reactivity as well as PK and stability attained with
the but-2-ynamide acceptor paired with the (S)-3-aminopiperadine linker, we directed our attention toward
improving both the intrinsic potency and PK through core modifications. Consistent with our
observations in the reversible effort,15d addition of a fluoro substituent at C5 of the indole core, to give
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a, provided significant enhancements in potency, with 0.1 nM inhibition of BTK and 7 nM inhibition in
the cellular assay (Table 3). Additionally, 5a provided significantly higher plasma concentrations in a
mouse PK study relative to 4g (Cmax: 42 uM; AUC: 52. uM h vs. Cmax: 0.49 uM; AUC: 1.1 uM h,
*
*
respectively). As shown in Figure 9, 5a inactivated BTK in the human whole blood assay at a rate 3-fold
faster than 1a. Adding further support, biochemical kinetic evaluation of 5a demonstrated a rate of BTK
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inactivation 4-fold faster than 1a (3.5 x 10 nM min vs. 1.2 x 10 nM min , Table 4). This clearly
*
*
illustrated that we had optimized the geometry of the compound to allow for both high affinity binding
K ) and rapid inactivation through covalent bonding with Cys 481 (kinact). As expected, the des-methyl
(
i
derivative 5b was shown, when biochemically evaluated, to inactivate BTK at a rate 7-fold faster than 5a
and consequently would be expected to be too reactive in vivo.
An expanded view of the in vitro activity profile of 5a is outlined in Table 5. In B cells stimulated
through the B cell receptor (BCR), 5a potently inhibited signaling and functional endpoints, including
calcium flux (IC50 = 7 nM), production of cytokines, proliferation, and surface expression of the
costimulatory molecule CD86 (IC values < 1 nM). As expected, CD69 expression in peripheral B cells,
5
0
when stimulated through CD40, was not impacted by 5a since this pathway is not dependent on the kinase
activity of BTK to mediate downstream signaling, thus demonstrating the compound’s functional
selectivity. When evaluated against IgG-containing immune complex low affinity activating Fc (FcRIIa
and FcRIII) receptor endpoints in peripheral blood mononuclear cells (PBMC), 5a was highly effective
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at inhibiting TNF production with equivalent potency to those measured for the BCR-dependent
endpoints in B cells. In human whole blood assays, 5a potently inhibited BCR-stimulated expression of
CD69 on B cells with an IC of 11 nM. Measurements of BTK inactivation in these assays showed a
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similar IC of 5 nM. Since the concentration of BTK in human whole blood is approximately 10 nM,
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the compound effectively titrated the amount of BTK present in these assays. As previously mentioned,
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5a rapidly inactivated BTK in a time- and concentration-dependent manner when added to human whole
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blood, providing a second order rate of inactivation of 3.5 x 10 nM min .
*
In order to gain an understanding of the overall kinase selectivity, 5a was evaluated against a panel of
245 kinases (Figure 10). A partial list of kinase selectivity data is summarized in Table 6. Dimethylindole
5
a was highly selective, providing >5,000-fold selectivity for BTK over 240 kinases with only the 4
related Tec family kinases demonstrating less than 5000-fold selectivity (9 – 1,000-fold), as shown in
Figure 10. Interestingly, 10 kinases have a comparable cysteine residue in the active site, the Tec family,
the EGFR family, and JAK3. Out of these 10 kinases, 5a demonstrated activity of less than 5,000-fold
relative to BTK for only the 4 other members of the Tec family. Overall, when compared to reversible
inhibitor 2,15d covalent inhibition with 5a resulted in significant improvement in the overall magnitude of
selectivity, with the exception of Tec family kinases TEC, BMX, and TXK. On the other hand, the
selectivity of 5a against Tec family kinase ITK was significantly enhanced relative to 2 (1,000x vs. 30x,
respectively). The improved selectivity against ITK observed with 5a is likely attributed to both the
intrinsic structural selectivity of the compound and the impact of the unique cysteine micro-environment
of ITK on covalent bonding, compared to the other four Tec family kinases, BTK, TEC, BMX and TXK,
which all share a similar micro-environment.13c Of particular note, 5a was >10,000-fold selective for
BTK over members of the EGFR family (HER1 and HER4). Since a kinase assay does not fully capture
the time-dependent nature of a covalent inhibitor, compound 5a was evaluated against an EGFR-
dependent AU565 breast cancer cell line to assess functional EGFR kinase activity over a three day period.
Compound 5a failed to show inhibition at concentrations up to 25,000 nM in this assay. Compound 1a,
on the other hand, inhibited this assay with an IC of 30 nM.
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The X-ray co-crystal structure of 5a bound to the kinase domain of BTK (Figure 11, PDB ID code:
08I) clearly highlighted that the compact nature of 5a binds tightly in the active site to facilitate the rapid
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rate of BTK inactivation that is observed. Consistent with our reversible clinical inhibitor 2,15d the co-
crystal structure revealed that the tetrahydrocarbazole NH and the carboxamide carbonyl are engaged in
a hydrogen bonding interaction with Met477 in the hinge region of the active site. The NH of the
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carboxamide is not only forming a hydrogen bonding interaction with Glu475, but also with a conserved
water, providing a bridging interaction with the gatekeeper Leu408 residue. Most importantly, the
structure confirmed the covalent nature of the inhibitor through a clearly defined, albeit stereochemically
non-selective, covalent bond with the sulfur of Cys481. As shown in Figure 12, the overlay of the single
crystal X-ray structure with the co-crystal structure of 5a bound with BTK clearly illustrates that we have
designed a covalent, irreversible inhibitor that maintains both the high affinity binding of our reversible
compounds while orienting the acceptor in close proximity to the cysteine nucleophile for rapid
inactivation.
With a highly desirable activity and selectivity profile, a rapid rate of BTK inactivation, a favorable
liability profile (Table 7) particularly for a covalent inhibitor, and desirable compound plasma
concentrations in mice, 5a was further evaluated in vivo.23 In multi-species pharmacokinetic studies
(
Table 8), the absolute oral bioavailability of 5a was 100% in mice, 74% in rats, 46% in cynomolgus
monkeys, and 81% in dogs. The total body plasma clearance of 5a was low in all species. The steady-
state volume of distribution observed was greater than the plasma volume but less than the total body
water, indicative of extravascular distribution, in spite of the high protein binding (free fraction: 0.2 –
1
.2%, Table 7). However, brain penetration was very low in mice, rats, and dogs (<5% of plasma
concentration). Of particular interest, both the plasma T (i.v.) and T (p.o.) values were short across
1
/2
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species (0.46 – 4.3 h and 0.58 – 1.0 h, respectively, Table 8). We viewed this as important for a covalent
inhibitor since the compound would need to have adequate intrinsic stability to facilitate rapid enzymatic
inactivation in vivo, but remaining inhibitor would need to be cleared quickly to mitigate any off-target
interaction.
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When administered orally to BALB/c mice, 5a dose-dependently inactivated BTK in peripheral blood
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with a single dose of 0.5 mg/kg, providing 90% inactivation 4 hours after dosing (Figure 13A). Consistent
with a slow rate of BTK resynthesis, inactivation was sustained at this dose with 86% inactivation
remaining at 24 h post-dose. Evaluation of 5a in BALB/C mice (0.5 – 2.5 mg/kg p.o.) for 48 h indicated
that approximately 25% of BTK active sites were resynthesized over 24 h. In cynomolgus monkeys,
when administered orally with a single 0.5 mg/kg dose, 100% inactivation was achieved at the 4 hour
time point with 74% inactivation remaining at 72 hours (Figure 13B). The drop in inactivation over 72
hours is reflective of the slow resynthesis rate of BTK. Interestingly, the compound was largely cleared
within 4 - 6 hours after dosing in both studies, clearly highlighting one of the key advantages of covalent
inhibition. When compared with 1a, 5a was ~30 fold more potent at inactivating BTK in mice after a
single dose (Figure 13C).
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Dimethylindole 5a was evaluated in vivo in a mouse collagen-induced arthritis (CIA) model of human
RA, which is both B cell- and FcR-dependent. Once daily oral dosing of 5a, started at the time of the
initial collagen immunization and continued throughout the duration of the study (38 days), resulted in
dose-dependent inhibition of clinically evident disease with 21%, 83%, and 92% inhibition observed at
the end of the study at doses of 0.1, 0.5, and 2.5 mg/kg p.o., q.d., respectively (Figure 14). Of particular
note, the 0.5 mg/kg and 2.5 mg/kg dose levels provided robust protection from clinically evident disease
(Figure 14A and 14B). Measurements of BTK inactivation in blood samples collected 24 hours after the
last dose showed high levels of inactivation (0.1 mg/kg – 64%; 0.5 mg/kg – 97%, and 2.5 mg/kg – 99%,
Figure 14B). Histological evaluation of the right hind paws, evaluating bone resorption and inflammation,
was consistent with the clinical scores (data not shown). Micro-computed tomography of the hind limbs
(Figure 14C) illustrated that 5a provided a dose-dependent protection against pitting, loss of bone mass,
woven porous bone, and fusion of the small bones evident in the mice receiving only vehicle. The animals
receiving 0.5 mg/kg showed essentially complete protection, as illustrated by the presence of a smooth
bone surface and easily recognizable small individual bones of the foot and ankle. The compound was
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also effective at 0.5 mg/kg p.o, q.d. in blocking disease progression when dosed in a pseudo-established
dosing mode (data not shown).23
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Additionally, 5a was evaluated in vivo in a NZB/W lupus-prone mouse model, in which the mice
develop a disease closely resembling human systemic lupus erythematosus and lupus nephritis (Figure
1
5). Mice were dosed by once daily oral gavage for 14 weeks with either 5a (0.2, 0.5, and 1.5 mg/kg),
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prednisolone (10 mg/kg), or vehicle (80:20 PEG400:water). In this study, treatment with 5a provided
complete protection from disease-related death over the course of treatment (Figure 15A). Additionally,
compound 5a was shown to dose-dependently inhibit the increase in severe proteinuria (Figure 15B), a
measure of the underlying nephritis. Serum anti-double stranded deoxyribonucleic acid (anti-dsDNA)
titers, which progressively increased over the course of the study in vehicle control mice, were also
inhibited in a dose-dependent manner (Figure 15C) with 40%, 51%, and 61% reductions at the end of the
study at the 0.2, 0.5, and 1.5 mg/kg doses, respectively. Histological evaluation of the kidneys confirmed
that treatment with 5a at all doses provided robust protection against tubulo-interstitial and glomerular
nephritis, as well as inflammatory infiltration, at levels equivalent to those seen with prednisolone (Figure
1
5D). IgG immune complex deposition, critical in driving disease pathobiology in both this murine model
and human lupus nephritis, was prominent in the capillaries of the glomeruli of vehicle control mice. As
seen in Figure 15E, histological evaluation revealed that 5a reduced the level of immune complex deposits
as effectively as prednisolone. Measurements of BTK inactivation in blood samples collected 24 hours
after the last dose showed high levels of inactivation at all three doses (0.2 mg/kg – 92%; 0.5 mg/kg –
99%, and 1.5 mg/kg – 100%, Figure 15F).
In evaluating the in vivo efficacy results, achieving > 90% inactivation was associated with a very
robust response, with near complete protection from disease. On the basis of these results, we felt that a
projected human dose targeting BTK inactivation of ≥ 95% was desirable. Considering the rapid rate of
inactivation in human whole blood, the hepatocyte metabolism in human and other species, and the in
vivo PK results in multiple species, the human dose to achieve ≥ 95% inactivation was projected to be <
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With highly desirable pharmacology favoring a low projected human dose, we concentrated our
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efforts on assessing the off-target reactivity and overall toxicity. As previously mentioned, the concern
with covalent, irreversible inhibition is the potential for off-target reactivity to biomolecules. To assess
the risk for 5a, several studies were undertaken to evaluate intrinsic reactivity to thiols, proteins, and DNA
and to evaluate non-specific protein covalent binding both in vitro and in vivo. Evaluation of covalent
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binding in vitro in human hepatocytes incubated for 2 h at a concentration of 10 M using C-labeled 5a,
incorporated in the carbonyl of the acetylene acceptor (see experimental section), revealed very low levels
of protein covalent binding (1.0 %, 131±2 pmol/mg of protein). In addition, no covalent binding was
observed with off-target cysteine residues in protein mixtures containing BTK (10 cysteine residues),
human hemoglobin, or human serum albumin, up to 10 μM concentrations of 5a. In fact, only <10%
protein binding in human hemoglobin was observed at a concentration of 100 M. When incubated with
GSH in enzyme free phosphate buffer, no GSH addition was observed, indicating a low intrinsic chemical
reactivity to thiol containing nucleophiles. This is consistent with the previously reported GSH reactivity
data for acalabrutinib, which benefits from the same but-2-ynamide acceptor.13c
In a rat quantitative whole body autoradiography study (10 mg/kg, p.o.) evaluating tissue distribution
and retention, the majority of the compound was cleared within 24 h with only minor radioactivity
remaining in the liver, kidneys, and skin after 48 to 96 h, indicating low tissue binding. Consistent with
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this finding, a mass balance study with C-labeled 5a (10 mg/kg, PO) in intact rats (n = 3) showed
complete recovery of the total radioactivity in urine and feces over 72 h with only 0.7% (±0.2) of the
radioactivity remaining in the carcass. Furthermore, very low covalent binding was observed in the organs
studied, including liver (≤ 0.03% of dose), spleen (≤ 0.007% of dose), and pancreas (≤ 0.002% of dose)
in rats over a 7-24 h time point with a single, oral dose of 5a (1 and 10 mg/kg). Additionally, no significant
increase in covalent binding was observed in these tissues after 5 days of daily, oral dosing (1 and 10
mg/kg) in rats.
A recent report outlined a strategy to predict the in vivo daily covalent binding burden (DCBB) for
covalent, irreversible inhibitors derived from an analysis of several marketed covalent drugs that have
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established safe clinical profiles, as well as compounds that did not.24 The authors concluded that if a
compound has an estimated DCBB of ≤ 10 mg/day, it is more likely to be clinically safe. When applying
this strategy to 5a considering the low projected human dose of < 10 mg q.d., p.o. and the assessment of
both in vitro protein covalent binding (< 1%) in human hepatocytes and in vivo rat studies, 5a was
determined to have a DCBB of < 0.10 mg/day. This value is significantly lower than the literature reported
upper limit of 10 mg/day for clinically safe drugs. While this does not guarantee that 5a will be safe in
humans, it does instill a sense of confidence as the compound proceeds clinically. In evaluating
genotoxicity, 5a was negative in both Ames and in vitro micronucleus (CHO cells) assessments (Table
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). With an extremely low DCBB and a very desirable tolerability in toxicity studies in both rat and dog,
compound 5a was advanced into clinical development.25
Chemistry
The synthetic pathways utilized in the preparation of the compounds presented in this article are
provided in Schemes 1 – 2.26 Analogues 4 were prepared as outlined in Scheme 1. The desired
commercially available primary and secondary amines were coupled with 6 under Buchwald-Hartwig
2
7
amination conditions to provide intermediates 7a and 7b. Attempts to perform the amination step on
carboxamide intermediate 13 failed to provide any appreciable product. Heating a mixture of either 7a
o
or 7b in sulfuric acid at 60 C for 1 h resulted in concurrent hydrolysis of the carbonitrile to the
carboxamide and deprotection of the protecting group. The final covalent, irreversible analogues 4a – 4q
were prepared by an acylation step using chloromethanesulfonyl chloride, acryloyl chloride, propiolic
acid, or but-2-ynoic acid, as indicated in Scheme 1. C5 Fluoro substituted derivatives 5a and 5b were
prepared in a similar fashion as analogues 4 starting from compound 9, as outlined in Scheme 2. The
absolute structure of 5a was determined by single crystal X-ray structure analysis (CCDC # 1901788)
(see experimental for more detail). As previously reported, 21 dimethylindoles 3b and 3c were prepared
as depicted in Scheme 3.
The dimethylindole core intermediates 6 and 9 in Schemes 1 and 2, respectively, were prepared as
outlined in Scheme 4. Both the C5 hydrogen and the C5 fluoro substituted cores, utilized in the
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preparation of analogues 4 and 5, were prepared through a Fischer indole cyclization between hydrazines
215d,19-21 and 2-butanone in the presence of acetic acid to afford dimethylindoles 13 in good yield. The
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acids (13) were then activated with EDCI and HOBt and treated with ammonium hydroxide to afford
dimethylindole amides 14. The carbonitrile intermediates 6 (R = H) and 9 (R = F) utilized in Schemes 1
and 2 were prepared in high yield when the corresponding amines were stirred with POCl in
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tetrahydrofuran at room temperature overnight.
Conclusions
In summary, we have outlined a strategy to identify a covalent, irreversible inhibitor of BTK which
provides both a rapid in vivo enzymatic inactivation and a low projected human dose. The strategy
involved starting with a scaffold with high intrinsic affinity (K ) for both binding and selectivity, in the
i
absence of covalent inhibition, where the acceptor only enhances but does not drive potency and
selectivity. Electrophilic acceptors were explored to bond with Cys481 with low intrinsic reactivity to
mitigate off-target interactions. This required optimizing the linker and acceptor geometry to enhance
kinact while maintaining the overall binding affinity (K ). In addition, this strategy focused on identifying
i
a compound with PK/ADME properties amenable for rapid target engagement but would allow for excess
compound to be rapidly cleared. This effort resulted in the identification of branebrutinib, 5a, (S)-4-(3-
(but-2-ynamido)piperidin-1-yl)-5-fluoro-2,3-dimethyl-1H-indole-7-carboxamide, as a highly selective
covalent, irreversible inhibitor of BTK with rapid target inactivation in vivo, supporting a low projected
oral human dose of < 10 mg p.o., q.d. With a very low off-target/non-specific daily covalent binding
burden of < 0.10 mg/day and a desirable safety and tolerability profile, 5a was advanced for clinical
evaluation. In Phase I randomized, placebo-controlled multiple-ascending dose studies in healthy
participants, 5a was well tolerated providing >99% BTK occupancy (inactivation) at doses of ≥ 3 mg
2
5
dosed orally, once daily. This data is consistent with our preclinical dose projection and highly supports
the continued clinical investigation of this novel agent.
Experimental Section
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1
Proton magnetic resonance ( H) spectra were recorded on either a Bruker Avance 400 (400 MHz), a
Bruker Avance III 500 (500 MHz), or a JEOL Eclipse 500 (500 MHz) spectrometer and are reported in
ppm relative to the reference solvent of the sample in which they were run. LCMS analyses were
conducted using a Shimadzu SCL-10A liquid chromatograph and a SPD UV-vis detector at 220 or 254
nm with the MS detection performed on a Waters Micromass ZQ spectrometer or a Waters Acquity UPLC
with a MS detection performed on a Waters SQ spectrometer. Preparative, reverse-phase HPLC
purifications were performed using one of the following conditions: (1) Analogues 4a-d,g-j,m-q were
purified via preparative LC/MS with the following conditions: Column: Waters XBridge C18, 19 x 250
mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10 mM ammonium acetate; Mobile
Phase B: 95:5 acetonitrile: water with 10 mM ammonium acetate; Gradient: 5-100% or 15-55% B over
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5 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractions containing the desired product
were combined and dried via centrifugal evaporation; (2) Analogue 4e was purified via preparative
LC/MS with the following conditions: Column: Waters XBridge C18, 19 x 250 mm, 5-μm particles;
Mobile Phase A: 5:95 acetonitrile:water with 0.05% TFA; Mobile Phase B: 95:5 acetonitrile:water with
0
.05% TFA; Gradient: 0-100% B over 25 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min.
Fractions containing the desired product were combined and dried via centrifugal evaporation; or (3)
Analogue 4l was purified via preparative HPLC with the following conditions: Columns: Phenomenex
Axia C column with a binary solvent system where solvent A = 5% acetonitrile, 95% water, 0.05%
1
8
trifluoroacetic acid and solvent B = 95% acetonitrile, 5% water, and 0.05% trifluoroacetic acid and eluting
with a gradient of increasing concentration of acetonitrile suitable to the column size and separation to be
achieved. Fractions containing the product were neutralized with an aqueous solution of sodium
bicarbonate and concentrated under reduced pressure. The residue was dissolved in an organic solvent,
washed with water, dried over anhydrous sodium sulfate or magnesium sulfate, and concentrated under
reduced pressure. All flash column chromatography was performed on EM Science silica gel 60 (particle
size of 40 – 60 m) or with an ISCO column. All reagents were purchased from commercial sources and
1
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Journal of Medicinal Chemistry
used without further purification unless otherwise noted. All reactions were performed under an inert
atmosphere.
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Research involving animals was performed in accordance with institutional guidelines as defined in
the U.S. by the Institutional Animal Care and Use Committee.
HPLC analyses were performed using the following conditions. All final compounds had an HPLC
purity of ≥ 95%.
0
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Method A: A linear gradient using 5% acetonitrile, 95% water, and 0.05% trifluoroacetic acid (Solvent
A) and 95% acetonitrile, 5% water, and 0.05% trifluoroacetic acid (Solvent B); t = 0 min., 10% B, t = 12
min., 100% B (15 min.) was employed on a SunFire C18 3.5u, 3.0 x 150 mm column. Flow rate was 1.0
mL/min and UV detection was set to 220/254 nm. The LC column was maintained at ambient
temperature.
Method B: A linear gradient using 5% acetonitrile, 95% water, and 0.05% trifluoroacetic (Solvent A)
and 95% acetonitrile, 5% water, and 0.05% trifluoroacetic (Solvent B); t = 0 min., 10% B, t = 12 min.,
1
00% B (15 min.) was employed on a XBridge Ph 3.5u, 3.5 x 150 mm column. Flow rate was 1.0 mL/min
and UV detection was set to 220/254 nm. The LC column was maintained at ambient temperature.
Method C. Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7-μm particles; Mobile Phase A:
5
:95 acetonitrile:water with 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10
mM ammonium acetate; Temperature: 50 °C; Gradient: 0-100% B over 3 minutes, then a 0.75-minute
hold at 100% B; Flow: 1.11 mL/min; Detection: UV at 220 nm.
Method D. Column: Waters Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7-μm particles; Mobile Phase A:
5
:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%
trifluoroacetic acid; Temperature: 50 °C; Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at
100% B; Flow: 1.11 mL/min; Detection: UV at 220 nm.
Method E. A linear gradient using 10% methanol, 90% water, and 0.2% phosphoric acid (Solvent A) and
9
0% methanol, 10% water, and 0.2% phosphoric acid (Solvent B); t = 0 min., 0% B, t = 9 min., 100% B
o
(
8 min.) was employed on a YMC ProC18 S5 ODS 4.6 x 50 mm column (oven temperature: 40 C). Flow
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rate was 2.5 mL/min and UV detection was set to 220 nm. The LC column was maintained at ambient
temperature.
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7
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9
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1
1
1
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1
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o
Method F. Waters UPLC; Column: Waters Acquity BEH C18 1.7um 2.1 x 150 mm at 40 C; Flow rate:
0
.4 mL/min.; Mobile Phase: A: 0.05% trifuoroacetic acid in water and B: acetonitrile; Gradient: 0 min.
10% B), 15 min. (95% B), 17 min. (95% B), and 17.1 min. (10% B); Detection: UV 220 – 400 nm.
-(3-Acrylamido-2-methylphenyl)-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide
3a). A mixture of 3-bromo-2-methylaniline (6.20 g, 33.3 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-
(
0
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2
3
4
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7
8
9
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7
8
9
0
4
(
bi(1,3,2-dioxaborolane) (9.31 g, 36.7 mmol) and potassium acetate (6.54 g, 66.6 mmol) in 1,4-dioxane
83 mL) was purged with nitrogen for 10 min. The PdCl (dppf)-CH Cl adduct catalyst (0.653 g, 0.800
(
2
2
2
o
mmol) was added, and the mixture was purged for another 5 min. and then heated at reflux (110 C) for
.75 h. The reaction mixture was cooled and filtered through Celite®, and the pad was washed with ethyl
2
acetate. The filtrate was washed with water, washed with brine, and dried over anhydrous sodium sulfate.
Concentration under reduced pressure followed by purification by silica gel chromatography (ISCO 120
g column, 10-20% ethyl acetate in hexane) afforded 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2
-yl)aniline (7.09 g, 30.4 mmol, 91% yield) as a light yellow solid. LCMS (ESI) m/z Calcd for
+
1
C H BNO [M + H] 234.2. Found: 234.1. H-NMR (400 MHz, CDCl )  7.23 (dd, J=7.3, 1.1 Hz, 1H),
1
3
20
2
3
7
.04 (t, J=7.6 Hz, 1H), 6.77 (dd, J=7.9, 1.1 Hz, 1H), 3.61 (br. s., 2H), 2.39 (s, 3H), and 1.35 (s, 12H).
To a solution of 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.525 g, 2.25
mmol) and Hunig’s base (0.433 mL, 2.48 mmol) in dichloromethane (11.3 mL) was added acryloyl
chloride (0.192 mL, 2.37 mmol). The reaction mixture was stirred at room temperature for 2h, partitioned
between brine and dichloromethane, and the organic phase was collected and dried over sodium sulfate.
Concentration under reduced pressure followed by purification by silica gel chromatography (ISCO 40 g
column; 5-20% ethyl acetate in hexane) gave N-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
+
yl)phenyl)acrylamide as a pale yellow solid. LCMS (ESI) m/z Calcd for C H BNO [M + H] 288.2.
1
6
22
3
1
Found: 288.3. H-NMR (400 MHz, CDCl )  8.01 (br. s., 1H), 7.64 (d, J=5.9 Hz, 1H), 7.23 (t, J=7.7 Hz,
3
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Journal of Medicinal Chemistry
1
H), 7.07 (br. s., 1H), 6.48 - 6.40 (m, 1H), 6.32 (br. s., 1H), 5.78 (d, J=9.5 Hz, 1H), 2.49 (s, 3H), and 1.36
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
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3
4
4
4
4
4
4
4
4
4
4
5
5
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5
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5
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5
5
5
6
(s, 12H).
A mixture of 4-bromo-7-(2-hydroxypropan-2-yl)-9H-carbazole-1-carboxamide15d,19b (40 mg, 0.115
mmol), N-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acrylamide (36.4 mg, 0.127
mmol), and tetrakis(triphenylphosphine)palladium (6.66 mg, 5.76 µmol) in toluene (2.2 mL) and ethanol
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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7
8
9
0
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3
4
5
6
7
8
9
0
(
720 mL) was degassed with argon for 2-5 min. To the mixture was added 2 M aqueous sodium carbonate
146 µL, 0.291 mmol), followed by purging with argon. The reaction mixture was heated at 90 °C
(
overnight, concentrated under reduced pressure, and purified by reverse-phase, preparative HPLC to give
a (12 mg, 0.028 mmol, 24%) as a pale yellow solid. HPLC purity: 99% (t = 1.29 min. – Method C; t =
3
r
r
+
1
1.28 min. - Method D); LCMS (ESI) m/z Calcd for C H N O [M + H] 428.2. Found: 428.2. H-NMR
2
6
25
3
3
(500 MHz, DMSO-d )  11.42 (s, 1H), 9.69 (s, 1H), 8.16 (br. s., 1H), 7.95 (s, 1H), 7.86 (d, J=1.0 Hz, 1H),
6
7
.64 (d, J=7.9 Hz, 1H), 7.47 (br. s., 1H), 7.36 (t, J=7.7 Hz, 1H), 7.12 (d, J=7.4 Hz, 1H), 6.99 (dd, J=8.4,
1.5 Hz, 1H), 6.94 (d, J=7.4 Hz, 1H), 6.73 (d, J=8.4 Hz, 1H), 6.56 (dd, J=17.1, 10.2 Hz, 1H), 6.27 (dd,
J=16.8, 2.0 Hz, 1H), 5.78 - 5.74 (m, 1H), 4.98 (s, 1H), 1.88 (s, 3H), and 1.44 (s, 6H).
4
-(3-Acrylamido-2-methylphenyl)-2,3-dimethyl-1H-indole-7-carboxamide (3b).26 Following the
procedure used to prepare 3a, 2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (see
procedure for 3a) was converted into N-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
1
yl)phenyl)acrylamide in 80% yield. H-NMR (400 MHz, CDCl ) δ 8.01 (br. s., 1H), 7.64 (d, J=5.9 Hz,
3
1H), 7.23 (t, J=7.7 Hz, 1H), 7.07 (br. s., 1H), 6.48-6.40 (m, 1H), 6.32 (br. s., 1H), 5.78 (d, J=9.5 Hz, 1H),
2
.49 (s, 3H), and 1.36 (s, 12H).
A mixture of 4-bromo-2,3-dimethyl-1H-indole-7-carboxamide (50 mg, 0.112 mmol), N-(2-methyl-3-
(
4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acrylamide (35.5 mg, 0.124 mmol), and
tetrakis(triphenylphosphine)palladium (6.49 mg, 5.62 µmol) in a mixture of toluene (2.1 mL) and ethanol
0.70 mL) was degassed under argon for 2-5 min. To the mixture was added 2 M aqueous sodium
(
carbonate (140 µL, 0.281 mmol), followed by purging with argon. The reaction mixture was heated at 90
°C overnight, concentrated under reduced pressure, and purified by reverse-phase, preparative HPLC to
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provide 3b (10.8 mg, 0.031 mmol, 28%). HPLC purity: 97%; t = 1.41 min. (Method C); LCMS (ESI)
r
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
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3
3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
m/z Calcd for C H N O [M + H] 348.2. Found: 348.2. H-NMR (500 MHz, DMSO-d )  10.74 (s,
21 21
3
2
6
1H), 9.60 (s, 1H), 8.00 (br. s., 1H), 7.58 (d, J=7.9 Hz, 1H), 7.48 (d, J=7.9 Hz, 1H), 7.32 (br. s., 1H), 7.23
(
t, J=7.7 Hz, 1H), 7.01 (d, J=7.4 Hz, 1H), 6.69 (d, J=7.4 Hz, 1H), 6.53 (dd, J=17.1, 10.2 Hz, 1H), 6.24
dd, J=17.3, 2.0 Hz, 1H), 5.74 (dd, J=10.4, 2.0 Hz, 1H), 2.29 (s, 3H), 1.83 (s, 3H), and 1.50 (s, 3H).
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
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9
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9
0
4-(3-Acrylamidophenyl)-2,3-dimethyl-1H-indole-7-carboxamide (3c).26
bromoaniline (1.00 g, 5.81 mmol), 4,4,4ʹ,4ʹ,5,5,5ʹ,5ʹ-octamethyl-2,2ʹ-bi(1,3,2-dioxaborolane) (1.55 g,
.10 mmol), and potassium acetate (1.14 g, 11.6 mmol) in 1,4-dioxane (14.5 mL) was purged with
A mixture of 3-
6
nitrogen for 10 min. The mixture was treated with PdCl (dppf) dichloromethane adduct (0.114 g, 0.140
2
mmol) and bubbled with nitrogen for an additional 5 min. The mixture was heated at reflux for 2.75 h
and then cooled to room temperature and filtered through Celite®. The pad was washed with ethyl acetate
and tetrahydrofuran, and the filtrate was concentrated under reduced pressure and purified by silica gel
chromatography (ISCO 40 g, 10-25% ethyl acetate in hexanes) to provide 3-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)aniline as an off-white solid (1.27 g, >99%). LCMS (ESI) m/z Calcd for C H BNO
1
2
18
2
+
1
[
M + H] 220.2. Found: 220. H-NMR (400 MHz, CDCl ) δ 7.24-7.13 (m, 3H), 6.82-6.77 (m, 1H), 3.64
3
(br. s., 2H), and 1.35 (s, 12H).
A solution of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (0.300 g, 1.37 mmol) and
Hunig’s base (0.311 mL, 1.78 mmol) in dichloromethane (9.1 mL) was cooled in an ice-bath and treated
with acryloyl chloride (0.117 mL, 1.44 mmol). The mixture was stirred at room temperature for 40 min,
concentrated under reduced pressure, and purified by silica gel chromatography (ISCO 24 g, 15-40% ethyl
acetate in hexanes) to provide N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) acrylamide as a
+
white solid (0.292 g, 78% yield). LCMS (ESI) m/z Calcd for C H BNO [M + H] 274.2. Found: 274.0.
15 20
3
A mixture of 4-bromo-2,3-dimethyl-1H-indole-7-carboxamide (30 mg, 0.112 mmol), N-(3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acrylamide (32.2 mg, 0.118 mmol), and
tetrakis(triphenylphosphine)palladium (6.49 mg, 5.62 µmol) in toluene (2.1 mL) and ethanol (0.7 mL)
was degassed for 2-5 min. The mixture was treated with a 2 M aqueous sodium carbonate (142 µL, 0.284
2
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Journal of Medicinal Chemistry
mmol), degassed again, and the reaction mixture was heated at 90 °C overnight, concentrated under
reduced pressure, and purified by reverse-phase, preparative HPLC to give 3c (10.2 mg, 0.030 mmol,
27% yield). HPLC purity: 98% (t = 1.41 min. – Method C; t = 1.40 min. - Method D); LCMS (ESI) m/z
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
r
r
+
1
Calcd for C H N O [M + H] 334.2. Found: 334.1. H-NMR (500 MHz, DMSO-d )  10.82 (br. s.,
2
0
19
3
2
6
1
H), 10.23 (br. s., 1H), 8.02 (br. s., 1H), 7.73 (br. s., 1H), 7.68 (d, J=7.9 Hz, 1H), 7.59 (dd, J=7.4, 4.0 Hz,
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
1H), 7.42 - 7.31 (m, 2H), 7.11 - 7.05 (m, 1H), 6.80 (dd, J=7.7, 4.2 Hz, 1H), 6.49 - 6.39 (m, 1H), 6.25 (d,
J=17.3 Hz, 1H), 5.76 (d, J=9.9 Hz, 1H), 2.33 (d, J=4.0 Hz, 3H), and 1.71 (d, J=3.5 Hz, 3H).
(
RS)-4-(3-Acrylamidopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (4a).26 Following
the procedures used for the preparation of 4d but substituting (RS)-benzyl piperidin-3-ylcarbamate for
S)-benzyl piperidin-3-ylcarbamate, 4-bromo-2,3-dimethyl-1H-indole-7-carbonitrile (6) was converted
into (RS)-4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide. LCMS (ESI) m/z Calcd for
(
+
+
C H N O [M + H] 287.2. Found: 287 .
16 22
4
To a solution of 4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (35 mg, 0.092
mmol) and Hunig’s base (21 µL, 0.119 mmol) in a 1:1 mixture of dichloromethane and tetrahydrofuran
(1.8 mL), cooled in an ice-bath, was added acryloyl chloride (8.19 µL, 0.101 mmol). The resulting
reaction mixture was stirred at room temperature for 1h. Concentrated under reduce pressure followed
by purification by reverse-phase, preparative HPLC gave 4a (10 mg, 0.029 mmol, 32% yield). HPLC
purity: 99% (t = 1.29 min. – Method C; t = 1.21 min. - Method D); LCMS (ESI) m/z Calcd for
r
r
+
1
C H N O [M + H] 341.2. Found: 341.1. H-NMR (500 MHz, DMSO-d )  10.60 (br. s., 1H), 8.13 -
1
9
24
4
2
6
8
.01 (m, 1H), 7.82 (br. s., 1H), 7.46 (dd, J=7.9, 4.0 Hz, 1H), 7.12 (br. s., 1H), 6.53 (dd, J=8.2, 3.7 Hz,
H), 6.28 - 6.16 (m, 1H), 6.13 - 6.02 (m, 1H), 5.58 (dd, J=10.2, 2.2 Hz, 1H), 4.02 (br. s., 1H), 3.39 (br.
1
s., 1H), 3.17 (br. s., 1H), 2.63 (br. s., 1H), 2.43 - 2.26 (m, 7H), 1.99 - 1.67 (m, 3H), and 1.36 (d, J=11.4
Hz, 1H).
(
RS)-2,3-Dimethyl-4-(3-(vinylsulfonamido)piperidin-1-yl)-1H-indole-7-carboxamide (4b).26 To
a solution of 4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (30 mg, 0.105 mmol)
see procedure for 4a) and Hunig’s base (46 µL, 0.262 mmol) in dichloromethane (1.3 mL), cooled at
(
2
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0oC, was added 2-chloroethanesulfonyl chloride (22 µL, 0.210 mmol) dropwise. The resulting reaction
mixture was stirred at room temperature for 3h. Concentration under reduce pressure followed by
purification by reverse-phase, preparative HPLC provided 4b (7.2 mg, 0.105 mmol, 18% yield). HPLC
purity: 97% (t = 1.43 min. – Method C; t = 1.43 min. - Method D); LCMS (ESI) m/z Calcd for
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
r
r
+
1
C H N O S[M + H] 377.2. Found: 377.1. H-NMR (500 MHz, Methanol-d )  7.40 (d, J=7.9 Hz, 1H),
1
8
24
4
3
4
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
6.63 - 6.54 (m, 2H), 6.20 (d, J=16.8 Hz, 1H), 5.90 (d, J=9.9 Hz, 1H), 3.52 (br. s., 2H), 3.21 (br. s., 1H),
2
1
.75 - 2.50 (m, 2H), 2.41 (s, 3H), 2.34 (s, 3H), 2.09 - 2.00 (m, 1H), 1.90 - 1.77 (m, 2H), and 1.43 (br. s.,
H).
(R)-2,3-Dimethyl-4-(3-(vinylsulfonamido)piperidin-1-yl)-1H-indole-7-carboxamide
(4c).26
Following the procedures used to prepare 4d but substituting (R)-benzyl piperidin-3-ylcarbamate for (S)-
benzyl piperidin-3-ylcarbamate, 4-bromo-2,3-dimethyl-1H-indole-7-carbonitrile (6) was converted into
(R)-4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide. LCMS (ESI) m/z Calcd for
+
+
C H N O S [M + H] 287.2. Found: 287 .
1
8
24
4
3
To a solution of (R)-4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (50 mg, 0.105
mmol) and Hunig’s base (37 µL, 0.210 mmol) in a 1:1 mixture of dichloromethane and tetrahydrofuran
(1.9 mL), cooled in an ice-acetone-dry ice bath, was added a solution of 2-chloroethanesulfonyl chloride
(
20 µL, 0.189 mmol) in dichloromethane (273 µL). The resulting reaction mixture was stirred at 0°C for
h. Concentration under reduced pressure followed by purification by reverse-phase, preparative HPLC
afforded 4c (5.8 mg, 0.015 mmol, 15% yield). HPLC purity: 99% (t = 1.32 min. – Method C; t = 1.29
1
r
r
+
1
min. - Method D); LCMS (ESI) m/z Calcd for C H N O S [M + H] 377.2. Found: 377.2. H-NMR
1
8
24
4
3
(
500 MHz, DMSO-d )  10.61 (s, 1H), 7.82 (br. s., 1H), 7.47 (d, J=7.9 Hz, 2H), 7.13 (br. s., 1H), 6.76
6
(dd, J=16.5, 9.8 Hz, 1H), 6.52 (d, J=7.9 Hz, 1H), 6.05 (d, J=16.5 Hz, 1H), 5.94 (d, J=9.8 Hz, 1H), 3.39
br. s., 2H), 3.13 (br. s., 1H), 2.56 (br. s., 1H), 2.42 (d, J=11.0 Hz, 1H), 2.32 (d, J=10.4 Hz, 6H), 1.97 (d,
(
J=11.0 Hz, 1H), 1.78 (br. s., 1H), 1.68 (d, J=13.4 Hz, 1H), and 1.32 (d, J=13.4 Hz, 1H).
S)-2,3-Dimethyl-4-(3-(vinylsulfonamido)piperidin-1-yl)-1H-indole-7-carboxamide (4d).26
(
A
mixture of 4-bromo-2,3-dimethyl-1H-indole-7-carbonitrile (6) (2.50 g, 10.0 mmol), (S)-benzyl piperidin-
2
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Journal of Medicinal Chemistry
3
-ylcarbamate (2.47 g, 10.5 mmol), 2,2ʹ-bis(diphenylphosphino)-1,1ʹ-binaphthalene (0.312 g, 0.502
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
mmol), tris(dibenzylideneacetone)dipalladium (0.460 g, 0.502 mmol) and cesium carbonate (4.58 g, 14.1
mmol) in 1,4-dioxane (143 mL) was purged with nitrogen and then heated at 100 ºC for 16 h. The reaction
mixture was cooled to room temperature, diluted with tetrahydrofuran, filtered through Celite®, and the
solids were washed with tetrahydrofuran. The filtrate was concentrated under reduced pressure and
purified by silica gel chromatography (ISCO 80 g column, 15-30% ethyl acetate in hexanes) to provide
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
(S)-benzyl (1-(7-cyano-2,3-dimethyl-1H-indol-4-yl)piperidin-3-yl)carbamate as a light yellow solid (2.13
+
+
1
g, 53% yield). LCMS (ESI) m/z Calcd for C H N O [M + H] 403.2. Found: 403 . H-NMR (400
2
4
26
4
2
MHz, DMSO-d ) δ 11.43 (s, 1H), 7.40-7.26 (m, 7H), 6.62 (d, J=8.1 Hz, 1H), 5.08-4.94 (m, 2H), 3.79-
6
3.65 (m, 1H), 3.41 (d, J=10.1 Hz, 1H), 3.20 (d, J=11.0 Hz, 1H), 2.60 (t, J=10.7 Hz, 1H), 2.43-2.16 (m,
7
H), 1.92 (d, J=9.5 Hz, 1H), 1.86-1.78 (m, 1H), 1.71 (d, J=11.2 Hz, 1H), and 1.40-1.26 (m, 1H).
A suspension of (S)-benzyl (1-(7-cyano-2,3-dimethyl-1H-indol-4-yl)piperidin-3-yl)carbamate (1.69
g, 3.44 mmol) in 80% aqueous sulfuric acid (11 mL, 172 mmol) was heated at 60 ºC for 2.5 h. The
mixture was cooled to room temperature and then poured over ice. The pH of the mixture was adjusted
to about 9-10 with concentrated aqueous potassium hydroxide, and the resulting mixture was extracted
with a 3:1 mixture of chloroform and isopropanol. The organic phase was dried over anhydrous sodium
sulfate and concentrated to provide (S)-4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-
carboxamide as a brown solid (1.66 g, 99% yield), which was used without further purification. LCMS
+
+
(ESI) m/z Calcd for C H N O [M + H] 287.2. Found: 287 .
16 22 4
To a solution of (S)-4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (40 mg, 0.095
o
mmol) and Hunig’s base (58 µL, 0.332 mmol) in dichloromethane (1.9 mL), cooled at 0 C, was slowly
added 2-chloroethanesulfonyl chloride (30 µL, 0.285 mmol) dropwise. The resulting reaction mixture was
stirred at room temperature for 3 h, concentrated under reduced pressure, and purified by reverse-phase,
preparative HPLC to give 4d (13 mg, 0.033 mmol, 35%). HPLC purity: 99% (t = 1.36 min. - Method C;
r
+
1
t = 1.31 min. - Method D); LCMS (ESI) m/z Calcd for C H N4O S [M + H] 377.2. Found: 376.9. H-
r
18 24
3
NMR (500 MHz, DMSO-d )  10.62 (s, 1H), 7.83 (br. s., 1H), 7.52 - 7.40 (m, 2H), 7.14 (br. s., 1H), 6.76
6
2
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(
dd, J=16.3, 9.9 Hz, 1H), 6.58 - 6.46 (m, 1H), 6.06 (d, J=16.8 Hz, 1H), 5.94 (d, J=9.9 Hz, 1H), 3.42 - 3.36
m, 2H), 3.14 (d, J=9.9 Hz, 1H), 2.61 - 2.53 (m, 1H), 2.42 (d, J=10.9 Hz, 1H), 2.32 (d, J=10.9 Hz, 6H),
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
(
1.97 (d, J=10.9 Hz, 1H), 1.83 - 1.76 (m, 1H), 1.74 - 1.60 (m, 1H), and 1.40 - 1.26 (m, 1H).
Alternative Procedure: To a solution of (S)-4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-
carboxamide (2.2 g, 4.23 mmol) and Hunig’s base (1.26 mL, 7.18 mmol) in a 2:1 mixture of
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
o
dichloromethane and tetrahydrofuran (63 mL), cooled in ice-acetone-dry ice at -20 C, was slowly added
a solution of 2-chloroethaneulfonyl chloride (0.71 ml, 6.76 mmol) in dichloromethane (9.0 mL) The
resulting brown suspension was stirred at 0 °C for 1h. The reaction was filtered under reduce pressure,
and the brown solid was washed with tetrahydrofuran and ethyl acetate. The filtrates were concentrated
under reduced pressure and purified by silica gel chromatography (ISCO 80g column, 50-70%/15min,
7
0%/13min, 70-80%/12min, 80%/8 min, 80-90/5min, 90%/15min - ethyl acetate in hexane) to afford 4d
(0.519, 1.34 mmol, 32% yield) as a pale yellow solid. HPLC purity: 97.1%; t = 7.73 min. (Method A);
r
+
96.1%; t = 7.36 min. (Method B); LCMS (ESI) m/z Calcd for C H N4O S [M + H] 377.2. Found:
r
18 24
3
1
3
77.2. H-NMR (400 MHz, DMSO-d )  10.60 (s, 1H), 7.80 (br. s., 1H), 7.51 - 7.39 (m, 2H), 7.11 (br.
6
s., 1H), 6.76 (dd, J=16.5, 9.9 Hz, 1H), 6.53 (d, J=8.1 Hz, 1H), 6.06 (d, J=16.5 Hz, 1H), 5.94 (d, J=9.9 Hz,
1H), 3.39 (d, J=9.9 Hz, 2H), 3.14 (d, J=11.0 Hz, 1H), 2.62 - 2.55 (m, 1H), 2.46 - 2.38 (m, 1H), 2.32 (d,
J=8.6 Hz, 6H), 1.97 (d, J=12.1 Hz, 1H), 1.85 - 1.76 (m, 1H), 1.75 - 1.63 (m, 1H), and 1.39 - 1.27 (m, 1H).
(
R)-2,3-Dimethyl-4-(3-propiolamidopiperidin-1-yl)-1H-indole-7-carboxamide (4e).26 A solution
of (R)-4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (50 mg, 0.105 mmol) (see
procedure for 4c), HATU (80 mg, 0.210 mmol), Hunig’s base (55 µL, 0.314 mmol), and propiolic acid
(8.07 mg, 0.115 mmol) in N,N-dimethylformamide (1.7 mL) was stirred at room temperature for 2.5 h.
The reaction mixture was filtered, and the filtrate was concentrated and purified by reverse-phase,
preparative HPLC to provide 4e (21 mg, 0.062 mmol, 59% yield). HPLC purity: 96% (t = 1.34 min. –
r
+
Method C; t = 1.30 min. - Method D); LCMS (ESI) m/z Calcd for C H N O [M + H] 339.2. Found:
r
19 22
4
2
1
3
39.2. H-NMR (500 MHz, DMSO-d ) δ 10.62 (s, 1H), 8.83 - 8.72 (m, 1H), 7.48 (d, J=8.1 Hz, 1H), 6.54
6
(d, J=8.4 Hz, 1H), 3.99 (br. s., 1H), 2.65 - 2.55 (m, 1H), 2.36 (s, 3H), 2.30 (s, 3H), 1.93 - 1.65 (m, 3H),
2
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Journal of Medicinal Chemistry
and 1.37 (br. s., 1H) (Some alkyl and exchangable carboxamide protons were not observed due to water
suppression).
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
(S)-2,3-Dimethyl-4-(3-propiolamidopiperidin-1-yl)-1H-indole-7-carboxamide (4f).26 A mixture
of (S)-4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (0.077 g, 0.269 mmol) (see
procedure for 4d), propiolic acid (0.020 mL, 0.323 mmol), HATU (0.143 g, 0.376 mmol), and Hunig's
base (0.164 mL, 0.941 mmol) in N,N-dimethylformamide (2.0 mL) was stirred at room temperature for
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
4
0 min. The mixture was diluted with ethyl acetate, washed with 10% aqueous lithium chloride (2x), and
washed with brine. The organic layer was collected, and the aqueous layers were sequentially extracted
with ethyl acetate (2x). The combined organic layers were dried over anhydrous sodium sulfate and
concentrated. Purification by silica gel chromatography (ISCO 12 g column, 0% - 5% methanol in
dichloromethane) afforded (S)-2,3-dimethyl-4-(3-propiolamidopiperidin-1-yl)-1H-indole-7-carboxamide
(
0.061 g, 0.177 mmol, 66 % yield) as a pale yellow solid. HPLC purity: 98.1%; t = 7.82 min. (Method
r
+
A); 98.4%; t = 7.43 min. (Method B); LCMS (ESI) m/z Calcd for C H N O [M + H] 339.2. Found:
r
19 22
4
2
1
3
39.2. H-NMR (500 MHz, DMSO-d ) δ 10.61 (s, 1H), 8.74 (d, J=7.8 Hz, 1H), 7.91 - 7.73 (m, 1H), 7.48
6
(d, J=8.1 Hz, 1H), 7.19 - 7.04 (m, 1H), 6.54 (d, J=8.1 Hz, 1H), 4.05 - 3.93 (m, 1H), 3.36 - 3.33 (m, 1H),
3
2
.30 - 3.28 (m, 1H), 3.25 - 3.14 (m, 1H), 2.67 - 2.55 (m, 1H), 2.44 - 2.39 (m, 1H), 2.38 - 2.36 (m, 3H),
.34 - 2.30 (m, 3H), 1.95 - 1.67 (m, 3H), and 1.45 - 1.31 (m, 1H).
(
S)-4-(3-(But-2-ynamido)piperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (4g).26 To a
mixture of (S)-4-(3-aminopiperidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (60 mg, 0.210 mmol)
(see procedure for 4d), HATU (159 mg, 0.419 mmol), and Hunig’s base (110 µL, 0.629 mmol) in N,N-
dimethylformamide (3.1 mL) at room temperature was added but-2-ynoic acid (18.5 mg, 0.220 mmol).
The reaction mixture was stirred at room temperature for 2 h. Concentration under reduce pressure
followed by purification by silica gel flash chromatography (ISCO 12g column, 30-65% ethyl acetate in
hexane) afforded 4g (37 mg, 0.102 mmol, 49% yield) as a pale yellow solid. HPLC purity: 97.0%; t =
r
+
1
8
.74 min. (Method A); LCMS (ESI) m/z Calcd for C H N O [M + H] 353.2. Found: 353.2. H-NMR
20 24 4 2
(400 MHz, DMSO-d )  10.58 (s, 1H), 8.46 (d, J=7.9 Hz, 1H), 7.79 (br. s., 1H), 7.46 (d, J=7.9 Hz, 1H),
6
2
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7
.09 (br. s., 1H), 6.51 (d, J=8.1 Hz, 1H), 3.97 (br. s., 1H), 3.60 (t, J=6.4 Hz, 2H), 3.17 (d, J=11.9 Hz, 1H),
.57 (d, J=10.6 Hz, 1H), 2.37 - 2.29 (m, 6H), 1.94 (s, 3H), 1.91 - 1.77 (m, 3H), and 1.34 - 1.28 (m, 1H).
(RS)-4-(3-Acrylamidopyrrolidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (4h).26 Following
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
2
the procedures used to prepare 4d but substituting (RS)-benzyl pyrrolidin-3-ylcarbamate for (S)-benzyl
piperidin-3-ylcarbamate, 4-bromo-2,3-dimethyl-1H-indole-7-carbonitrile (6) was converted into (RS)-4-
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
(3-aminopyrrolidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide.
LCMS (ESI) m/z Calcd for
+
+
1
C H N O [M + H] 273.2. Found: 273 . H-NMR (400 MHz, DMSO-d ) δ 10.60 (s, 1H), 7.73 (dd,
1
5
20
4
6
J=8.7, 5.6 Hz, 1H), 7.44 (d, J=8.1 Hz, 1H), 6.96 (br. s., 3H), 6.44 (d, J=8.1 Hz, 1H), 3.56-3.46 (m, 1H),
.26-3.08 (m, 3H), 2.82 (dd, J=9.5, 5.3 Hz, 1H), 2.33 (s, 3H), 2.30 (s, 3H), 2.19-2.11 (m, 1H), 1.61-1.50
(m, 1H).
To a solution of 4-(3-aminopyrrolidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (35 mg, 0.129
3
mmol) and Hunig’s base (29 µL, 0.167 mmol) in a 1:1 mixture of dichloromethane and tetrahydrofuran
(2.6 mL), cooled in an ice-bath, was added acryloyl chloride (12 µL, 0.141 mmol). The reaction mixture
was stirred at room temperature for 3h, concentrated under reduced pressure, and purified by reverse-
phase, preparative HPLC to give 4h (12 mg, 0.037 mmol, 29% yield). HPLC purity: 99% (t = 1.15 min.
r
+
1
–
Method C); LCMS (ESI) m/z Calcd for C H N O [M + H] 327.2. Found: 327.1. H-NMR (500
18 22 4 2
MHz, DMSO-d6)  10.63 (s, 1H), 8.39 (d, J=6.4 Hz, 1H), 7.78 (br. s., 1H), 7.45 (d, J=7.9 Hz, 1H), 7.07
br. s., 1H), 6.48 (d, J=8.4 Hz, 1H), 6.33 - 6.20 (m, 1H), 6.14 - 6.04 (m, 1H), 5.59 (dd, J=9.9, 2.0 Hz, 1H),
4.45 - 4.30 (m, 1H), 3.30 - 3.23 (m, 2H), 3.14 - 2.98 (m, 2H), 2.38 - 2.22 (m, 7H), and 1.85 - 1.77 (m,
(
1
H).
(
RS)-2,3-Dimethyl-4-(3-(vinylsulfonamido)pyrrolidin-1-yl)-1H-indole-7-carboxamide (4i).26 To
a solution of 4-(3-aminopyrrolidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (30 mg, 0.088 mmol)
(see procedure for 4h) and Hunig’s base (39 µL, 0.220 mmol) in dichloromethane (1.1 mL), cooled at 0
oC, was added 2-chloroethanesulfonyl chloride (18 µL, 0.176 mmol) dropwise. The reaction mixture was
stirred at room temperature for 3 h, concentrated under reduce pressure, and purified by reverse-phase,
preparative HPLC to give 4i (9.8 mg, 0.029 mmol, 36% yield). HPLC purity: 99% (t = 1.30 min. –
r
2
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Journal of Medicinal Chemistry
+
Method C; t = 1.22 min. – Method D); LCMS (ESI) m/z Calcd for C H N O S [M + H] 363.2. Found:
r
17 22
4
3
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
63.1. H-NMR (500 MHz, DMSO-d )  10.64 (s, 1H), 7.77 (d, J=14.9 Hz, 1H), 7.69 (br. s., 1H), 7.45
6
(d, J=7.9 Hz, 1H), 7.08 (br. s., 1H), 6.78 (dd, J=16.3, 9.9 Hz, 1H), 6.46 (d, J=7.9 Hz, 1H), 6.08 (d, J=16.3
Hz, 1H), 5.99 (d, J=9.9 Hz, 1H), 3.83 (br. s., 1H), 3.42 - 3.38 (m, 1H), 3.21 - 3.09 (m, 2H), 3.00 (dd,
J=9.7, 6.2 Hz, 1H), 2.35 - 2.21 (m, 7H), and 1.88 - 1.78 (m, 1H).
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
6
(R)-2,3-Dimethyl-4-(3-propiolamidopyrrolidin-1-yl)-1H-indole-7-carboxamide (4j). Following
the procedures used to prepare 4d but substituting (R)-benzyl pyrrolidin-3-ylcarbamate for (S)-benzyl
piperidin-3-ylcarbamate, 4-bromo-2,3-dimethyl-1H-indole-7-carbonitrile (6) was converted into (R)-4-
(3-aminopyrrolidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide.
LCMS (ESI) m/z Calcd for
+
+
C H N O [M + H] 373.2. Found: 273 .
1
5
20
4
A solution of (R)-4-(3-aminopyrrolidin-1-yl)-2,3-dimethyl-1H-indole-7-carboxamide (30 mg, 0.094
mmol), HATU (71 mg, 0.187 mmol), Hunig’s base (49 µL, 0.281 mmol) and propiolic acid (7.2 mg, 0.103
mmol) in N,N-dimethylformamide (1.3 mL) was stirred at room temperature for 4 h, concentrated under
reduced pressure, and purified by reverse-phase, preparative HPLC to provide 4j (4.0 mg, 0.024 mmol,
1
3% yield). HPLC purity: 96% (t = 1.20 min. – Method C; t = 1.08 min. - Method D); LCMS (ESI) m/z
r r
+
1
Calcd for C H N O [M + H] 325.2. Found: 325.2. H-NMR (500 MHz, DMSO-d )  10.63 (s, 1H),
1
8
20
4
2
6
9
4
.08 (d, J=6.1 Hz, 1H), 7.79 (br. s., 1H), 7.45 (d, J=7.9 Hz, 1H), 7.07 (br. s., 1H), 6.47 (d, J=7.9 Hz, 1H),
.32 (br. s., 1H), 4.14 (s, 1H), 3.26 - 3.13 (m, 2H), 3.12 - 2.98 (m, 2H), 2.31 (d, J=13.4 Hz, 6H), 2.24 (dd,
J=13.7, 5.8 Hz, 1H), and 1.85 (dd, J=12.8, 6.1 Hz, 1H).
S)-4-((1-Acryloylpyrrolidin-3-yl)amino)-2,3-dimethyl-1H-indole-7-carboxamide (4k).26
(
A
mixture of 4-bromo-2,3-dimethyl-1H-indole-7-carbonitrile (6) (0.400 g, 1.61 mmol) and (S)-tert-butyl 3-
aminopyrrolidine-1-carboxylate (0.336 g, 1.80 mmol) in 1,4-dioxane (15 mL) was purged with nitrogen
for 5 min. To the mixture was added 2,2ʹ-bis(diphenylphosphino)-1,1ʹ-binaphthalene (0.050 g, 0.080
mmol), tris(dibenzylideneacetone)dipalladium (0.074 g, 0.080 mmol), and cesium carbonate (0.732 g,
2
.25 mmol), and the reaction mixture was sealed under nitrogen and heated at 100 ºC for 19 h. After
cooling to room temperature, the mixture was diluted with ethyl acetate (50 mL), washed with water (50
2
8
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