Discovery of a novel class of covalent dual inhibitors targeting the protein kinases bmx and btk

Article abstract of DOI:10.3390/ijms21239269

The nonreceptor tyrosine TEC kinases are key regulators of the immune system and play a crucial role in the pathogenesis of diverse hematological malignancies. In contrast to the substantial efforts in inhibitor development for Bruton’s tyrosine kinase (BTK), specific inhibitors of the other TEC kinases, including the bone marrow tyrosine kinase on chromosome X (BMX), remain sparse. Here we present a novel class of dual BMX/BTK inhibitors, which were designed from irreversible inhibitors of Janus kinase (JAK) 3 targeting a cysteine located within the solvent-exposed front region of the ATP binding pocket. Structure-guided design exploiting the differences in the gatekeeper residues enabled the achievement of high selectivity over JAK3 and certain other kinases harboring a sterically demanding residue at this position. The most active compounds inhibited BMX and BTK with apparent IC50 values in the single digit nanomolar range or below showing moderate selectivity within the TEC family and potent cellular target engagement. These compounds represent an important first step towards selective chemical probes for the protein kinase BMX.

Full text of DOI:10.3390/ijms21239269

International Journal of
Molecular Sciences
Article
Discovery of a Novel Class of Covalent Dual
Inhibitors Targeting the Protein Kinases BMX
and BTK
Michael Forster 1,2,†, Xiaojun Julia Liang 1,2,†, Martin Schröder 3,4, Stefan Gerstenecker 1,
Apirat Chaikuad 3,4 , Stefan Knapp 3,4,5 , Stefan Laufer 1,2,6 and Matthias Gehringer 1,2,
*
1
Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmaceutical Sciences,
Faculty of Sciences, University of Tübingen, 72076 Tübingen, Germany;
michael.forster@uni-tuebingen.de (M.F.); xiaojun-julia.liang@uni-tuebingen.de (X.J.L.);
stefan.gerstenecker@uni-tuebingen.de (S.G.); stefan.laufer@uni-tuebingen.de (S.L.)
Cluster of Excellence iFIT (EXC 2180) ‘Image-Guided & Functionally Instructed Tumor Therapies’,
2
University of Tübingen, 72076 Tübingen, Germany
Structural Genomics Consortium, Goethe University Frankfurt, Buchmann Institute for Molecular
3
Life Sciences, Max-von-Laue-Straße 15, 60438 Frankfurt am Main, Germany;
m.schroeder@pharmchem.uni-frankfurt.de (M.S.); chaikuad@pharmchem.uni-frankfurt.de (A.C.);
knapp@pharmchem.uni-frankfurt.de (S.K.)
4
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Buchmann Institute for Molecular Life
Sciences, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany
Frankfurt Cancer Institute (FCI) and German Translational Cancer Network (DKTK) Site Frankfurt/Mainz,
60438 Frankfurt am Main, Germany
Tübingen Center for Academic Drug Discovery (TüCAD2), 72076 Tübingen, Germany
Correspondence: matthias.gehringer@uni-tuebingen.de; Tel.: +49-7071-2974582
These authors contributed equally to this work.
5
6
*
†
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 22 October 2020; Accepted: 1 December 2020; Published: 4 December 2020
Abstract: The nonreceptor tyrosine TEC kinases are key regulators of the immune system and play a
crucial role in the pathogenesis of diverse hematological malignancies. In contrast to the substantial
efforts in inhibitor development for Bruton’s tyrosine kinase (BTK), specific inhibitors of the other
TEC kinases, including the bone marrow tyrosine kinase on chromosome X (BMX), remain sparse.
Here we present a novel class of dual BMX/BTK inhibitors, which were designed from irreversible
inhibitors of Janus kinase (JAK) 3 targeting a cysteine located within the solvent-exposed front region
of the ATP binding pocket. Structure-guided design exploiting the differences in the gatekeeper
residues enabled the achievement of high selectivity over JAK3 and certain other kinases harboring
a sterically demanding residue at this position. The most active compounds inhibited BMX and
BTK with apparent IC50 values in the single digit nanomolar range or below showing moderate
selectivity within the TEC family and potent cellular target engagement. These compounds represent
an important first step towards selective chemical probes for the protein kinase BMX.
Keywords: tyrosine kinases; bone marrow tyrosine kinase on chromosome X; Bruton’s tyrosine
kinase; Janus kinase 3; covalent inhibitors; chemical probes
1. Introduction
The TEC kinase family constitutes the second largest family of nonreceptor tyrosine kinases
including the five members Bruton’s tyrosine kinase (BTK), interleukin (IL)-2-inducible T-cell kinase
(ITK or TSK/EMT), tyrosine kinase expressed in hepatocellular carcinoma (TEC), bone marrow tyrosine
kinase on chromosome X (BMX or ETK) and tyrosine-protein kinase (TXK or RLK) [1]. All TEC family
Int. J. Mol. Sci. 2020, 21, 9269; doi:10.3390/ijms21239269
www.mdpi.com/journal/ijms

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members except TXK commonly harbor an N-terminal pleckstrin homology (PH) domain required for
membrane association via binding to phosphatidylinositols. The PH domain is followed by a TEC
homology (TH) domain which includes a BTK-homology motif and two proline-rich regions in BTK,
ITK and TEC, one of which is absent in BMX. In contrast to the other TEC family members, TXK is
devoid of the PH and TH domains and membrane targeting is promoted by a palmitoylated cysteine
string motif (CC) instead. All Tec family kinases also contain two SRC homology domains, SH3 and
SH2, preceding the highly conserved C-terminal kinase domain [2]. This makes TEC kinases closely
related to the SRC kinase family, which represents the largest family of nonreceptor tyrosine kinases.
TEC kinases differ in their expression pattern. While TEC and BMX are widely expressed in
hematopoietic and endothelial cells, expression of BTK appears to be more restricted, mainly to
myeloid and B-cells. On the contrary, ITK and TXK are predominantly expressed in T-lymphocytes [1].
Among the TEC kinases, BTK stands out as the best-studied family member due to its key function
in B-cell receptor signaling required for B-cell development. Mutations in the btk gene are causative
to human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency, which are
characterized by a lack of mature B-lymphocytes and defective humoral immunity [3]. BTK is essential
for the proliferation and survival of leukemic B-cells making this kinase not only a promising target
for treatment of inflammatory and autoimmune diseases but also for B-cell malignancies. Currently,
the three BTK inhibitors ibrutinib (
for the treatment of various B-cell lymphoma and chronic graft-versus-host disease (only ibrutinib)
and many more are in preclinical or clinical development [ ]. Despite different selectivity profiles,
these current drugs share a common mode of inhibition by covalently targeting BTK C481 located
within the front pocket of the ATP binding site in the proximity of the D helix. By comparison,
1, Figure 1), acalabrutinib (2) and zanubrutinib (3) are approved
4–6
α
a cysteine in this position exists also in other members of the TEC family and few additional kinases
(vide infra).
Figure 1. Examples of covalent Bruton’s tyrosine kinase (BTK), Janus kinase 3 (JAK3) and bone marrow
tyrosine kinase on chromosome X (BMX) inhibitors including the FDA-approved BTK-targeted drugs
ibrutinib, acalabrutinib and zanubrutinib. The electrophilic warhead group is highlighted in red.

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Contrary to BTK, the effort on inhibitor development for other TEC kinases has remained rather
limited despite several lines of evidence suggesting important roles in hematopoiesis as well as
their potential as therapeutic targets. For instance, the function of ITK is crucial in T-cell receptor
signaling. As such, it is required for T-cell development and response, controlling the production
of proinflammatory cytokines [
inflammatory and autoimmune diseases; however, so far, no selective ITK inhibitors have entered
clinical development [ ]. The role of other TEC kinases, including BTK’s closest relative BMX, in health
7]. ITK has primarily been considered as a target in the treatment of
4
and disease remains less clear. This highlights the need for specific chemical probes [
8] to explore the
biology of these understudied TEC kinases.
In recent years, targeting protein kinases with covalent inhibitors has become a mature strategy
not only for the development of selective chemical probes, but also drugs [ ]. Although protein
4,5
kinases do not feature a catalytic nucleophile, a substantial fraction of human protein kinases are
known to possess one or several accessible cysteine moieties inside or in close proximity to the ATP
binding site [4,9–11]. These cysteines are located at diverse positions with a low degree of conservation.
The mechanism of covalent binding typically follows a two-step process with an initial reversible
binding event that positions the inhibitor’s “warhead” in the spatial proximity of a reactive amino
acid residue [12]. High affinity noncovalent interaction along with suitable preorientation between the
target kinase and the covalent inhibitor in the first step are essential for efficiency of the subsequent
covalent bond formation, and hence on-target specificity [13]. Thus, covalent targeting of a poorly
conserved amino acid residue may be employed as an additional selectivity filter complementing
reversible recognition. Such a strategy has been very valuable in the context of protein kinase inhibitor
design, where achieving selectivity is intrinsically challenging due to the high degree of conservation
within this target class, especially in the ATP binding site [4,5]. Besides the possible advantages in
terms of selectivity, covalent inhibitors possess other potential benefits. For example, persistent target
occupancy enables an increased, time-dependent potency, lowering a hurdle on ATP competition.
Such property may also decouple pharmacodynamics from pharmacokinetics, which can be particularly
useful in a drug discovery setting [14]. Moreover, initial concerns about the safety of targeted covalent
inhibitor drugs have not materialized so far [15]. Beyond drug discovery, the attachment of reporter
tags (e.g., fluorescent dyes, affinity labels or click handles) to covalent ligands yields very useful tool
compounds to explore various facets of target biology [13].
The presence of a nonconserved cysteine within the solvent-exposed front region at the N-terminus
of the
α
D helix, often referred to as the
α
D-1 position [9], in all five TEC kinases (C496 in BMX; C481 in
BTK) opens an opportunity for irreversible targeting. However, cysteines at the
α
D-1 position exist
also in six other kinases, namely most members of the HER family (EGFR, HER2 and HER4), the Janus
kinase JAK3, the SRC kinase BLK and the mitogen activated protein kinase kinase MKK7 (MAP2K7).
Currently, the
inhibitors afatinib, dacomitinib and osimertinib, the HER2 inhibitor neratinib and the aforementioned
BTK inhibitors ibrutinib ( ), acalabrutinib ( ) and zanubrutinib ( ) [ ]. Covalent BTK inhibitors
αD-1 cysteine has been targeted by seven approved drugs, including the EGFR/pan-HER
1
2
3
4,5
often feature a certain cross-reactivity with other TEC family members. For example, ibrutinib shows
strong off-target (re)activity in the entire TEC kinase family, but also for other kinases sharing the
α
D-1 cysteine [16] while acalabrutinib is more specific [17]. Covalent JAK3 inhibitors have attracted
considerable interest [18 19] since the D-1 cysteine (C909) is the key feature distinguishing JAK3
from the other JAK family members, JAK1, JAK2 and TYK2. For example, the acrylamide-based JAK3
inhibitor PF-06651600 (Ritlecitinib, , Figure 1) [20] is currently being investigated in several phase II
and III clinical trials. In our own JAK3 program, we generated highly selective covalent-reversible
inhibitors FM-381 (5a) and FM-409 (5b) [21 22]. The equivalent cysteine in MKK7 (C218) is modified
by hypothemicin analogs [4] but also by recently discovered acrylamide-based inhibitors [16,23,24].
,
α
4
,

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As exemplified by BTK, irreversible inhibition represents an excellent strategy for targeting
TEC kinases. Nevertheless, the development of inhibitors for other members of this family remains
challenging due to the subtly different nature of their binding sites as well as the lack of understanding
on these pockets. Design of specific ITK inhibitors, for example, is hindered by lower cysteine
reactivity [25] and the larger gatekeeper residue. Only few reports describing the development of
selective inhibitors of the less prominent TEC kinases, namely TXK, TEC and BMX, have been published
so far. While no inhibitors addressing TXK or TEC as the primary target are known, two structural
series of covalent BMX inhibitors targeting C496 have been disclosed. In 2013, Gray and colleagues
identified the benzo[h] [1,6]naphthyridin-2(1H)-one derivative BMX-IN-1 (6, Figure 1), a dual covalent
BMX/BTK inhibitor [26]. This compound has recently been complemented by a series of analogs from
Bernardes and co-workers [27] (preprint on Chemrxiv), which feature increased potency but limited
selectivity against JAK3, BLK and within the TEC family. JS25 (7a), the most potent BMX inhibitor from
this set (IC50 = 3.5 nM, kinact/KI = 19
µM
−1s−1) showed increased cellular target engagement compared
to
6
in a NanoBRETTM assay [28] while covalent binding has been demonstrated for the close analog
JS24 (7b) by mass spectrometry and crystal structure. A structurally distinct series of covalent BMX
inhibitors has been published in 2017 by Liu and colleagues [29]. These compounds exemplified by
CHMFL-BMX-078 (8) showed high affinity for the inactive (nonphosphorylated) kinase while affinity
for the active (phosphorylated) kinase was weak (apparent Kd > 10
µM). It is assumed that the latter
compound binds BMX and related kinases in a type II binding mode addressing the hydrophobic
back-pocket sometimes referred to as “deep pocket” [30], which is only accessible in the DFG-out
conformation. However, there is still a need for expanding the scope of novel BMX/TEC family kinase
inhibitors with high selectivity or complementary selectivity patterns to study biology. Here we present
a novel and structurally unrelated series of covalent BMX/TEC-family kinase inhibitors featuring a
distinct selectivity profile among the kinases with a αD-1 cysteine.
2. Results
2.1. Discovery of a New Class of Covalent JAK3/TEC Family Kinase Inhibitors
Our previous study demonstrated the successful use of the tricyclic imidazo
[5,4-d]pyrrolo[2,3-b]pyridine scaffold for the design of selective covalent-reversible JAK3 probes FM-381
(5a, see Figure 1) and FM-409 (5b) targeting JAK3 C909 via an α-cyano acrylamide warhead [21,22].
Based on this, we aimed to expand the inhibitor series and explore their potentials as irreversible
inhibitors for JAK3 and other kinases that harbor a cysteine at the equivalent position. With no
success in generating acrylamide-derived irreversible inhibitors based on the aforementioned tricyclic
scaffold, we tested alternative hinge binding motifs and synthesized 1H-pyrrolo[2,3-b]pyridine-derived
compound 9a (Figure 2, left). This derivative retained excellent JAK inhibitory potency (IC50 = 0.2 nM)
and high selectivity against the other JAK family members and EGFR, demonstrating the hinge binder
replacement as a potential strategy for optimizing the parent inhibitor towards other kinases (Table 1).
Figure 2. Design of covalent JAK3 inhibitor 10a from analog 9a by introduction of a carboxamide group
at the 5-postion of the 7-azaindole core.

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Table 1. Selectivity of 9a and 10a in the JAK family and against EGFR.
Kinase
IC50 [nM] 1
9a
10a
JAK1
JAK2
JAK3
TYK2
EGFR
2970
3580
0.2
5540
5250
460
1120
<0.1
1900
>10,000
1
IC50 values were determined in a radiometric assay (Reaction Biology HotSpot
™ [31]). Data were obtained as
five-dose singlicate with 10-fold serial dilution starting at 10 µM. [ATP] = 10 µM.
We hypothesized that the 1H-pyrrolo[2,3-b]pyridine (7-azaindole) core in 9a might adopt an
inverted binding orientation when compared to 5a or common 7H-pyrrolo[2,3-d]pyrimidine derived
JAK inhibitors like with two hydrogen bonds between the 7-azaindole and the backbone of L905
,
b
4
(Figure 3). On this basis, we introduced a carboxamide group in the 5-position of the 7-azaindole
scaffold to establish a third hydrogen bond towards the hinge region [32]. As expected, the resulting
compound 10a (Figure 2, right) exhibited an increased potency for JAK3 (IC50 < 0.1 nM) while
maintaining a similar degree of selectivity against other JAKs. Interestingly, we observed that this
compound demonstrated increased selectivity against EGFR compared to 9a. In line with covalent
binding, nonreactive analogs 9b,c and 10b,c showed a strongly decreased potency with JAK3 IC50’s
in the micromolar range (not shown). The determined crystal structures of 9a and 10a (Figure 4) in
complex with JAK3 confirmed the predicted hinge interaction pattern and covalent modification of
C909. As expected, the 7-azaindole core in both compounds was anchored to the hinge region via two
hydrogen bonds with the L905 backbone NH and carbonyl oxygen atom, respectively. Compound
10a established an additional hydrogen bond between a carboxamide NH and the E903 backbone
carbonyl oxygen atom. The second amide proton of the 5-carboxamide was involved in a presumably
weak interaction with the methionine sulfur atom. The warhead amide was present in two flipped
conformations (see Figure 4b) and involved in water-mediated hydrogen bonds, most notably with the
backbone of C909. The latter interactions may assist in pre-orienting the Michael acceptor to facilitate
cysteine addition.
Figure 3. Differences in the hinge binding of different scaffolds exemplified by 5a and 9a. Usually,
the protein backbone in the hinge region of a kinase provides three potential hydrogen bonding partners
(two carbonyl-acceptors (A) and one NH-donor (D)). Depending on the orientation of the hinge binding
heterocycle different hydrogen-bonding patterns are possible.

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(a)
(b)
Figure 4. X-ray crystal structures of compounds 9a (PDB-code 7APG) and 10a (PDB-code 7APF) in
complex with JAK3. ( ) Compound 9a covalently bound to JAK3-C909. An ethylene glycol molecule
a
(depicted in gray) fills the space between the ligand and the DFG motif forming a hydrogen bond to
D967 but no direct hydrogen bonds with the ligand. Certain water-mediated hydrogen bonds as well
as an alternative pose with a flipped amide conformation were omitted for clarity; (b) Compound 10a
covalently bound to JAK3-C909. Two poses with a flipped warhead amide conformation were observed
(depicted in salmon and blue). The carbonyl group of the 5-carboxamide moiety forms a hydrogen
bond to an ethylene glycol molecule in the back pocket, which is further anchored by hydrogen bonding
to E871 in the αC-helix and to the DFG motif. Certain water-mediated hydrogen bonds were omitted
for clarity.
To examine whether our replacement strategy of the hinge binding motif for expanding the
profile of the JAK3 covalent inhibitors to other kinases succeeded, we characterized the activity of
inhibitors 9a and 10a against all kinases featuring the αD-1 cysteine. This screening revealed that the
compounds strongly inhibited BMX and TXK with no or less significant binding to other kinases at
200 nM (see Table 2). Notably, the observed selectivity pattern did not seem to rely on differences in
the gatekeeper moiety which is methionine in JAK3 and MKK7 and a threonine in the HER and TEC
family kinases (except ITK).
Table 2. Selectivity of 9a and 10a in among the protein kinases with an equivalent cysteine.
Kinase
Gatekeeper Residue
Residual Activity @ 200 nM 1
9a
10a
BMX
TXK
BTK
TEC
ITK
JAK3
MKK7
EGFR
HER2
HER4
BLK
T
T
T
T
F
M
M
T
T
T
2.7%
1.8%
18.2%
37.1%
88.1%
0.8%
37.3%
>100%
95.0%
66.1%
95.8%
1.6%
3.2%
18.9%
42.1%
90.8%
0.5%
28.3%
95.5%
97.0%
66.6%
82.4%
T
1
Residual activities were determined in a radiometric assay (Reaction Biology HotSpot
™ [31]). Data were obtained
from duplicate measurements and reported as the arithmetic mean of the individual values. [ATP] = 10 µM.

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2.2. Evaluation of N-Substitution in the 5-Carboxamide Series
To exploit the steric nature of the gatekeeper moiety as an additional selectivity filter, we introduced
N-substituents at the 5-carboxamide’s nitrogen atom to preserve the triple hydrogen bonding interaction
with the hinge region while forcing a steric clash with the bulky methionine gatekeeper in JAK3
or MKK7 and the phenylalanine in ITK. The first analog from this series with a relatively bulky
N-cyclopentyl substituent (11a, Figure 5) did not show substantial activity at 200 nM on any of the
kinases harboring an
αD-1 cysteine. Nevertheless, we determined the IC50 values for JAK3, BMX
and TXK to compare acrylamide 11a with nonreactive propionamide 11b (Table 3). While 11a still
showed residual activity on BMX, no JAK3 inhibitory activity was observed (IC50 > 10
analog 11b did not inhibit any of the latter kinases up to 10 µM.
µM). In contrast,
Figure 5. N-alkylation of the 5-carboxamide moiety.
Table 3. Inhibitory activity of acrylamides 10a and 11a and unreactive analog 11b.
Kinase
IC50 [nM] 1
10a
11a
11b
BMX
TXK
JAK3
29
28
<0.1
539
>10,000
>10,000
n.d.
>1000 2
>10,000 3
1
IC50 values were determined in a radiometric assay (Reaction Biology HotSpot
five-dose singlicate with 10-fold serial dilution starting at 1 M (for 10a 11a) or 10
72% residual activity at 1 µM. 3 Determined in a JAK3 ELISA assay [33].
™
[
31]). Data were obtained as
µ
/
µ
M (11b). [ATP] = 10 M.
µ
2
To test whether smaller N-substituents could prevent the loss in BMX potency, we prepared the
corresponding N-methyl and N-ethyl analogs 11c and 11d (Table 4). In addition, we synthesized
compounds 11e
moieties of variable size to the hydrophobic pocket behind the threonine gatekeeper often termed
“hydrophobic region I” [34]. The binding of these derivatives was assessed using a thermal shift ( Tm)
assay, which unfortunately revealed a dramatic decrease of Tm for all compounds in relation to the
–g to evaluate whether a methylene linker would be suitable to direct apolar cyclic
∆
∆
starting point 10a (Table 4). These results suggested that the applied modification strategy was not
suitable to generate potent BMX inhibitors with high selectivity against JAK3.

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Table 4. Thermal stabilization of JAK3 and BMX by compound 10a and N-alkylated analogs 11a
and 11c–g.
Compound
10a
R
JAK3 ∆Tm [K] 1
21.07 ± 0.03
BMX ∆Tm [K] 1
10.49 ± 0.09
11a
9.12 ± 0.02
5.31 ± 0.18
11c
11d
11e
10.27 ± 0.37
10.35 ± 0.26
9.64 ± 0.28
7.84 ± 0.04
7.35 ± 0.19
6.94 ± 0.07
11f
5.54 ± 0.82
8.29 ± 1.26
3.68 ± 0.13
6.84 ± 0.23
11g
1
Mean of at least three independent measurements ± standard deviation.
2.3. Design and Structure–Activity Exploration of the 5-Acylamino Series
In search for alternative approaches exploiting the gatekeeper residue as a selectivity filter,
molecular modeling studies were performed. Docking simulations suggested that inverting the
5-carboxamide substituent would enable a favorable NH···O hydrogen bond to the hydroxy group
of the threonine gatekeeper in BMX. While the third hydrogen bond to the hinge region would not
be compatible with this arrangement, the interaction with the gatekeeper was predicted to direct
moieties attached at the carbonyl group towards the hydrophobic region I behind the gatekeeper
moiety (see Figure 6).
We thus prepared a set of analogs with an N-linked amide moiety (compounds 12a–i, see Figure 6
and Table 5) to validate this hypothesis. Satisfactorily, the compounds from this series showed increased
BMX thermal shifts while JAK3 thermal shifts remained in a moderate range suggesting low inhibitory
potency on the latter. To confirm these results, the IC50 values of a subset of compounds were determined
for BMX and JAK3. In agreement, all tested inhibitors exhibited high inhibitory activities for BMX
with IC50 values in a low nanomolar range. Moreover, all compounds displayed excellent selectivity
against JAK3, which was most pronounced for the most active analog 12c (>9000-fold selectivity).

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(a)
(b)
(c)
(d)
Figure 6. Amide inversion as a strategy to rescue BMX inhibitory activity. (
inversion and representative analogs 12a and 12b; ( ) Schematic depiction of the expected binding
mode of analog 12a; ( ) Representative noncovalent docking pose of compound 12a modelled into the
a) General strategy of amide
b
c
BMX active site using the BMX structure with the PDB-code 3SXR (BMX in complex with dasatinib)
as a template. Similar poses were obtained using an alternative crystal structure (PDB-code 3SXS) in
which the P-loop region is completely resolved. (d) Overlay of the docking pose of 12a with the crystal
structure of JAK3 (7APF) highlighting the clash with the JAK3 gatekeeper moiety. Only the gatekeeper
methionine (transparent spheres) of the JAK3 structure is shown for clarity.
We selected potent cyclopentanoic amide 12a and compound 12c with a thiophene 2-carboxylic
acid amide moiety for further profiling against the kinases that have an equivalent cysteine at the αD-1
position. At a concentration of 200 nM, 12a strongly inhibited BMX, BTK and TXK while TEC, BLK,
HER4 and EGFR were less affected (Table 6). As expected, no inhibition of JAK3, MKK7 and ITK was
observed, which may be attributed to their bulkier gatekeeper incapable of forming the predicted
hydrogen bond to the inverted amide moiety. In comparison, we observed that compound 12c had
a slightly better potency than 12a, yet both shared a similar selectivity profile. Moreover, saturated
analog 12b expectedly showed a much weaker BMX inhibition with an IC50 value of 582 nM while
being devoid any significant activity on other kinases in this set except BTK and TXK (70% and 89%
residual activity at 200 nM, respectively). For a better selectivity ranking, we determined IC50 values
of 12a and 12c against all kinases that were significantly inhibited at 200 nM (Table 6). The results
confirmed that both compounds were highly potent inhibitors of BMX demonstrated by IC50 values of
2 nM and 1 nM, respectively. However, these inhibitors also inhibited BTK with similar potencies,
suggesting that the moderate selectivity against BTK observed earlier for the parent compounds 9a
and 10a was unfortunately compromised. Nevertheless, moderate selectivity of 12a and 12c over the
other kinases in this test set, which included TXK, TEC, EGFR, HER2, HER4 and BLK, was evident and
both had a slightly different selectivity profile. In comparison to 12c, which exhibited 8 30-fold lower
−
potencies for the tested kinases (>100-fold only for HER2), compound 12a showed a better selectivity
against HER family kinases (72-, 280- and 48-fold against EGFR, HER2 and HER4, respectively),
TEC (14-fold) and BLK (24-fold), while selectivity against TXK (6-fold) remained moderate.

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Table 5. Thermal stabilization of compounds 12a and 12c–i and inhibitory potency of selected inhibitors
on JAK3 and BMX.
Compound
R
JAK3 ∆Tm [K] 1
13.80 ± 0.13
BMX ∆Tm [K] 1
11.92 ± 0.03
IC50 JAK3 [nM] 2
IC50 BMX [nM] 2
12a
1340
2
1
12c
12d
12e
7.36 ± 0.44
9.93 ± 0.12
10.88 ± 0.95
11.52 ± 0.08
10.75 ± 0.09
11.37 ± 0.03
>10,000
1080
4
n.d.
n.d.
12f
13.44 ± 0.43
11.89 ± 0.23
10.57 ± 0.12
11.88 ± 0.07
n.d.
n.d.
6
12g
>10,000
12h
12.1
10.1
11.9
11.5
n.d.
n.d.
16
12i
n.d.
1
2
Mean of at least three independent measurements
±
standard deviation. IC50 values were determined in a
radiometric assay (Reaction Biology HotSpot
dilution starting at 10 M (JAK3) or 1 M (12d
0.5 µM was used. [ATP] to 10 µM.
™
[
g
31]). Data were obtained as five-dose singlicate with 10-fold serial
vs. BMX). For 12a vs. BMX, 5-fold serial dilution starting from
µ
µ
,
,
i
,c
Table 6. Inhibitory activity of compounds 12a–c on all protein kinases with an αD-1 cysteine.
Kinase
Gatekeeper Residue
Residual Activity @ 200 nM 1
IC50 [nM] 2
12b
12a
12b
12c
12a
12c
BMX
TXK
BTK
TEC
T
T
T
T
F
M
M
T
T
T
4.8%
5.9%
2.2%
n.d.
88.8%
69.7%
99.2%
n.d.
n.d.
n.d.
>100%
>100%
95.4%
95.1%
0.2%
0.3%
0.8%
17.2%
94.0%
>100%
>100%
14.9%
41.1%
4.7%
2.2
13
2.2
582
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.1
10
<1
13
25.4%
98.5%
94.5%
100%
51.3%
81.9%
15.9%
14.7%
31
ITK
n.d.
1340
n.d.
158
612
106
52
n.d
JAK3
MKK7
EGFR
HER2
HER4
BLK
1
>10,000
n.d.
12
118
8.5
T
8.6%
30
Residual activities were determined in a radiometric assay (Reaction Biology HotSpot
™
[
31]). Data were obtained
M. 2 IC50
values were determined in the above radiometric assay. Data were obtained as five-dose singlicate with 5-fold serial
dilution starting at 0.5 M (12a vs. BMX, BTK, TEC, TXK and HER4), 1 M (12a vs. EGFR and HER2) or 10
(12b vs. BMX). A 10-fold serial dilution starting at 10 µM was employed for 12a,b vs. JAK3. [ATP] = 10 µM.
from duplicate measurements and reported as the arithmetic mean of the individual values. [ATP] = 10
µ
µ
,
c
µ
,c
µM
Cellular target engagement was evaluated by a bioluminescence resonance energy transfer
(NanoBRET ) assay [28] using HEK293T cells (see Figure 7). Lead compounds 12a and 12c showed
™
favorable low nanomolar IC50 values against both, BMX and BTK with a slight bias towards BMX.
High potency was also observed for compounds 12g,i and early hit compound 9a while the close

Int. J. Mol. Sci. 2020, 21, 9269
11 of 37
analog 10a showed decreased potency, which might be a result of lower cell penetration due to the
primary amide functionality. As expected, and in line with the data from enzymatic assays, N-alkylated
analog 11a and non-reactive control compounds 9b, 10b, 11b and 12b showed weak or no affinity for
both kinases in this cellular model.
Figure 7. (a) Exemplified dose–response curve of 12a titrated versus BTK (blue squares) or BMX
(magenta circles) in a NanoBRET™ assay using HEK293T cells. Each point represents the average of
triplicates with standard deviation. For dose response curves of the other inhibitors tested, see the
supporting information. (b) IC50 values determined in NanoBRET™ assays in HEK293T cells.
2.4. Validation of Covalent Modification and Binding Mode Prediction
Covalent adduction between BMX and the inhibitors was investigated by mass spectrometry
(
see Supplementary Table S1). When incubating highly potent acrylamide-derived inhibitors 9a
, 10a
and 12a
,c,g,i
at 1.5-fold molar excess with BMX at 4 ◦C, complete labeling was observed readily after
30 min indicating a high efficiency of covalent bond formation. In contrast, analog 11a, which was less
potent in the activity assay required prolonged exposure (240 min) to approach full target modification.
As expected, no modification was detectable for propionamides 9b, 10b, 11b and 12b, which were
employed as negative controls. To examine general reactivity towards thiols, compound 12c was
tested for its stability against glutathione (GSH). In the presence of 5 mM GSH at pH 7.4, 12c showed a
half-life of approx. 10 h which compares favorably to Afatinib (<1 h) tested under the same conditions
(see Supplementary Figure S2). It can therefore be concluded that covalent target modification is
facilitated by the preceding reversible binding event and not a result of nonspecific thiol addition.
To gain a detailed insight in binding interactions, we aimed to crystallize the complex of BMX and the
inhibitors, yet unsuccessfully. Thus, covalent docking analyses were instead performed. Suggested
covalent binding modes of the two representative compounds 12a and 12c are depicted in Figure 8.
As expected, the docking poses show the dual hinge interaction pattern along with an additional
hydrogen bond between the amide NH and the hydroxy group of T489 orienting the nonpolar
substituent at the 5-acylamino group towards the hydrophobic region I.

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(a)
Figure 8. Covalent docking models of compounds 12a (panel (
(b)
a
)) and 12c (panel (b)). Poses were
generated using the BMX crystal structure with the PDB-code 3SXR as the template.
Although we did not experimentally prove selective modification of C496 by tryptic digestion/MS
experiments, crystal structure or mutation of the target cysteine, the efficiency and stoichiometry of
covalent modification in conjunction with modeling data and the binding modes of analogs 9a and 10a
in JAK3 strongly support the specific labeling of this residue, which is the only accessible cysteine in or
proximal to the BMX ATP binding site. In addition, much lower activity of non-reactive analog 12b
compared to acrylamide 12a on all enzymes tested complies with a covalent mode of action on other
kinases with an equivalent cysteine placement.
2.5. Compound Synthesis
The key transformation in the synthetic route to the inhibitors disclosed herein was the late
stage introduction of the entire N-aryl acrylamide warhead/linker fragment via a Suzuki coupling.
This reaction could be performed under very mild conditions preventing possible side reactions
involving the electrophilic Michael acceptor system. The synthesis of the key boronic acid ester
14 was realized as a high yielding two step sequence starting from 3-bromoaniline. The latter was
smoothly converted to the pinacol boronate 13 under Miyaura conditions [35] followed by the acylation
with acryloyl chloride to deliver the bench stable building block 14 in a good overall yield of 71%
(Scheme 1, top).
For the facile derivatization in position 5 of the 7-azaindole scaffold, the corresponding 5-amino
and 5-carboxy functionalized key intermediates 18 and 22 were prepared. The synthesis of 18 started
with the SEM-protection of commercially available 5-bromo-7-azaindole (15) to deliver compound 16
.
The carboxy group was then introduced via lithium–halogen-exchange followed by quenching the
intermediate aryllithium species with gaseous carbon dioxide at low temperatures. The treatment with
elemental bromine in methylene chloride converted acid 17 cleanly to the desired intermediate 18 in
excellent yields (Scheme 1, middle).

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Scheme 1. Synthesis of key building blocks: (a) B2pin2, XPhos Pd G4, KOAc, dioxane, 90 ◦C (84%);
(b) acryloyl chloride, Et3N, DCM,
10◦C (85%); (c) NaH, SEM-Cl, DMF, 0◦C to rt (56–78%) (d) n-BuLi,
78 ◦C (51%); (e) Br2, DCM, 0◦C (89%); (f) NBS, DMF, rt (70%); (g) Zn0, ammonium formate,
−
CO2, THF.
−
EtOH, 50 ◦C (51%).
The corresponding 5-amino derivative 22 was prepared from 5-nitro-7-azaindole 19, which is
accessible via a previously reported scalable three-step process [36]. Starting from intermediate 19
the 3-bromo substituent was introduced via electrophilic bromination with NBS (20) followed by the
SEM-protection of the indole nitrogen atom (21). Finally, the nitro group was reduced under mild
conditions with zinc and ammonium formate to obtain the key building block 22 (Scheme 1, bottom).
The early hit compounds 9a and 10a and their corresponding unsubstituted phenyl analogs 9c
and 10c were synthesized following slightly modified routes (Schemes 2 and 3). Starting from plain
7-azaindole, the bromination of position 3 was performed with NBS to give compound 23 in almost
quantitative yield. In the following step, the indole nitrogen atom was protected with a tosyl group
,
via deprotonation with sodium hydride and reaction with tosyl chloride to obtain intermediate 24
.
The latter was coupled with phenylboronic acid or building block 14 under Suzuki conditions to yield
the compounds 25 and 26, respectively. Tosyl deprotection under basic conditions in methanol or
tert-butanol, respectively, delivered the final compounds 9c and 9a.
Scheme 2. Synthesis of compounds 9c and 9a. (a) NBS, DMF, rt (96%); (b) NaH, TsCl, THF, 0 ◦C to rt
(85%); (c) phenylboronic acid, Pd(OAc)2, XPhos, Na2CO3, dioxane/H2O, 70 ◦C (82%); (d) 14, XPhos Pd
G3, K2CO3, dioxane/H2O, 50 ◦C (quant.); (e) for 9c: KOH, MeOH, 60◦C (85%); (f) for 9a: KOH, tBuOH,
50 ◦C (81%).

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Scheme 3. Synthesis of compounds 10c and 10a. (a) CDI, NH3(aq), DMF, rt (72%); (b) Br2, DCM, 0 ◦C
(68%); (c) phenylboronic acid, tBu3P Pd G3, K3PO4, dioxane/H2O, rt (quant.); (d) 14, tBu3P Pd G3,
K2CO3, dioxane/H2O, rt (93%); (e) TFA, DCM, rt (71–92%).
The corresponding derivatives with an unsubstituted carboxamide in position 5 (10c and 10a) were
accessible from the SEM-protected acid 17. CDI-mediated coupling of the latter with ammonia led to
carboxamide 27. Bromination in position 3 with elemental bromine afforded intermediate 28, which was
then Suzuki-coupled with phenylboronic acid or building block 14, respectively. The precursors 29
and 30 were finally deprotected under acidic conditions with TFA in DCM to obtain compounds 10c
and 10a (Scheme 3).
The series of N-substituted azaindole 5-carboxamides (compounds 11a and 11c
–g) was accessible
via a three-step procedure starting from key intermediate 18 (Scheme 4). First, amides 31a
–
g
were
prepared via CDI-mediated acid activation and reaction with the corresponding amines. The latter
intermediates were then coupled with 14 under mild Suzuki conditions at ambient temperature to
give 32a–g in favorable yields. The final compounds 11a and 11c–g were obtained after acid-promoted
SEM-cleavage using TFA in DCM at ambient temperature (Scheme 4).
Scheme 4. Synthesis of N-alkylated 7-azaindole-5-carboxamides. (a) CDI, corresponding amine, DMF,
rt (46–80%); (b) 14, tBu3P Pd G3, K3PO4 or K2CO3, dioxane/H2O, rt (53–79%); (c) TFA, DCM, rt (23–97%).
The final inhibitor series 12a and 12c
via a similar route as described for 11a and 11c
corresponding acid chlorides to deliver the amide derivatives 33a
The Suzuki coupling with boronate ester 14 was performed under the mild conditions established
before to afford precursors 34a . The latter were converted to the final compounds 12a and 12c
under the same acidic conditions as described above (Scheme 5).
–i
featuring an inverted amide functionality was prepared
(Scheme 5). Building block 22 was reacted with the
in moderate to excellent yields.
–
g
–
i
–
i
–i

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Scheme 5. Synthesis of 5-acylamino-7-azaindoles. (a) corresponding acid chloride, Et3N, DCM,
rt (47–90%); (b) 14, tBu3P Pd G3, K3PO4 or K2CO3, dioxane/H2O, rt (45–73%); (c) TFA, DCM,
rt (29–77%).
To access the propionamide analogs of selected inhibitors as negative control compounds,
the corresponding acrylamides were hydrogenated in the presence of a Pd/C catalyst. Due to the
absence of sensitive functional groups, these conversions usually proceeded smoothly and gave the
desired propionamides 9b, 10b, 11b and 12b in good to excellent yields (Scheme 6).
Scheme 6. Synthesis of propionamides 9b
THF/MeOH, (60–95%).
, 10b, 11b and 12b as negative controls. (a) H2 (1 bar), Pd/C,
3. Discussion
Here we describe the discovery of a novel class of covalent inhibitors of the TEC family kinases,
most notably BMX and BTK. Development started from compounds 9a and 10a belonging to an
unprecedented class of covalent JAK3 inhibitors, which possessed potent off-target activity on the
TEC kinases BMX and TXK while showing moderate to high selectivity against the other eight protein
kinases with an equivalent cysteine placement. Guided by the crystal structures of 9a and 10a in
complex with JAK3, modification of the hinge binding motif as well as back pocket binding moieties was
successfully exploited as a strategy to fine tune the binding profiles of these irreversible inhibitors that
target a cysteine located at the αD-1 position. This approach enabled the development of irreversible
inhibitors for BMX and BTK, which showed a general preference for kinases with a less bulky threonine
gatekeeper residue, enabling high selectivity over JAK3 and MKK7 that harbor a methionine, and ITK
that possess a phenylalanine at this position.
The key step in the synthetic access to these inhibitors was the late stage introduction of the
N-phenyl acrylamide warhead via Suzuki coupling under exceptionally mild conditions. This facilitated
broad SAR evaluation and fast library synthesis. Starting from unsubstituted carboxamide 10a
we introduced N-alkyl substituents to induce a clash with bulkier gatekeeper moieties, but this
modification led to a drop in potency on both JAK3 and BMX. In contrast, inversion of the carboxamide
in the 5-position of the azaindole scaffold retained BMX inhibitory potency, while activity on JAK3 was
,

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strongly decreased. Besides the expected steric clash with the methionine gatekeeper moiety in JAK3,
molecular modeling suggested inverted amides to form an additional hydrogen bond towards the
hydroxy group of threonine gatekeeper moieties, which drives the carbonyl’s substituent towards the
hydrophobic region I.
The most promising compounds 12a and 12c were further profiled against the TEC kinase family
and other kinases with an αD-1 cysteine. As expected, low activity was detected on kinases with a
non-threonine gatekeeper. However, the promising selectivity pattern observed for starting compounds
9a and 10a was partially lost. Most notably, optimization was accompanied by a strong increase in BTK
potency with compound 12a being equipotent on BMX and BTK, while 12c slightly favored BTK with
subnanomolar inhibitory activity in the enzymatic assay. Moderate selectivity was observed against
the other TEC kinases and BLK. However, compound 12c also showed significant off-target activity
on HER family kinases while 12a maintained good selectivity against the latter. Comparison with
unreactive analog 12b, which showed weak activity on all tested kinases underlined the importance
of the covalent mode of action. Highly efficient covalent modification of BMX was experimentally
shown for several analogs via mass spectrometry while potent cellular BMX and BTK engagement was
demonstrated by means of a NanoBRETTM assay.
Targeting BMX has been proposed as a strategy for treatment of various diseases including
cardiovascular disorders [37] and certain cancers [26
been developed to date: BMX-IN-1 ( , see Figure 1), the analog JS25 (7a) and the type II inhibitor
CHMFL-BMX-078 ( ). However, these inhibitors still exhibit pronounced affinities to several other
,38,39]. Nevertheless, only three inhibitors have
6
8
kinases with different selectivity profiles. Off-target activities could thus limit their use as a sole
tool for biological study. Moreover, their chemical structures suggest unfavorable physicochemical
properties due to size and the high portion of sp2 atoms. The dual covalent BMX/BTK inhibitors
presented here thus complement the repository of available BMX inhibitors. It should be mentioned,
however, that caution must be taken when comparing covalent inhibitors based on (apparent) IC50
data since the latter depend on incubation time. For example, it has recently been demonstrated in the
case of MKK7 and ibrutinib that a longer incubation could result in a misled nanomolar inhibitory
activity observed for an inhibitor that has only weak reversible binding affinity to the kinase [16].
Nonetheless, determination of meaningful kinetic data, i.e., kinact and KI, to disentangle contributions
of reversible binding and the subsequent bond-forming step remains highly elaborate, thus for BMX
such values have only been reported for JS25 and a few related compounds from the same study [27].
Moreover, and in contrast to the aforementioned studies, the compounds presented herein have
not been characterized in terms of wider kinome selectivity. Improvements also need to be made
concerning the intra-TEC family selectivity. Future efforts will primarily focus on increasing the
selectivity against BTK, for example by modifying the residues targeting the hydrophobic region I or
by extending the latter towards the DFG-out pocket to generate type II inhibitors [16].
Overall, the presented optimization approach offers a strategy that can be exploited to fine tune
existing covalent inhibitors towards unrelated kinases. Compared to previous compounds, the BMX
inhibitors discovered here are based on an alternative and readily optimizable chemotype, which may
be beneficial for further development of probes that exclusively target BMX. Currently, there is no
selective inhibitor or chemical probe available for BMX. However, combinatorial use of our dual
irreversible BMX/BTK inhibitors along with other inhibitors with different off-target profiles as a set of
chemogenomic compounds would nevertheless allow dissecting biological functions of BMX as well
as its role in disease development, which will enable further validation of this kinase as a potential
therapeutic target.

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4. Materials and Methods
4.1. Chemical Synthesis
4.1.1. General Information
Chemical synthesis was carried out using commonly applied techniques and general procedures.
All starting materials and reagents were of commercial quality and were used without further
purification. Thin layer chromatography (TLC) was carried out on Merck 60 F254 silica plates
(Merck KGaA, Darmstadt, Germany) and were visualized under UV light (254 nm and 366 nm) or
developed with an appropriate staining reagent. Preparative column chromatography was carried
out with an Interchim PuriFlash 430 or PuriFlash XS420 (Interchim S.A., Montlucon, Allier, France)
automated flash chromatography system on normal phase silica gel (Grace Davison Davisil LC60A
20–45 micron (W.R. Grace and Company, Columbia, MD, USA) or Merck Geduran Si60 63–200-micron
silica (Merck KGaA, Darmstadt, Germany).
Nuclear magnetic resonance (NMR) spectral analysis was performed on Bruker Avance 200 or
Bruker Avance 400 instruments (Bruker Corporation, Billerica, MA, USA). The samples were dissolved
in deuterated solvents and chemical shifts are given in relation to tetramethylsilane (TMS). Spectra were
calibrated using the residual peaks of the used solvent.
Mass spectrometry (MS) was carried out with an Advion TLC-MS interface (Advion, Ithaca, NY,
USA) with electron spray ionization (ESI) in positive and/or negative mode. Instrument settings were
as follows: ESI voltage 3.50 kV, capillary voltage 187 V, source voltage 44 V, capillary temperature
250 ◦C, desolvation gas temperature 250 ◦C, gas flow 5 L/min nitrogen.
Purities of final compounds were determined via high performance liquid chromatography
(HPLC) using an Agilent 1100 Series LC system (Agilent Technologies, Santa Clara, CA, USA) with
Phenomenex Luna C8 columns (150
×
4.6 mm, 5 µm) (Phenomenex Inc. Torrance, CA, USA) and
detection was performed with a UV DAD at 254 nm and 230 nm wavelength. Elution was carried
out with the following gradient: 0.01 M KH2PO4, pH 2.30 (solvent A), MeOH (solvent B), 40% B
to 85% B in 8 min, 85% B for 5 min, 85% to 40% B in 1 min, 40% B for 2 min, stop time 16 min,
flow 1.5 mL/min. All final compounds showed a purity above 95% in the means of peak area at the
two different wavelengths.
4.1.2. General Synthetic Procedures
General Procedure A (Suzuki coupling): In a screw-top reaction vial were combined the corresponding
boronic acid or ester and the aryl bromide under argon atmosphere. Dioxane and the aqueous base were
added subsequently, and the mixture was degassed carefully via several vacuum/argon cycles. Finally,
the catalyst system was added to the reaction and the degassing procedure was repeated. The vial was
sealed and heated to the indicated temperature until reaction control (TLC or HPLC) showed complete
consumption of starting materials. The reaction was cooled to ambient temperature, diluted with
EtOAc and washed with brine. The organic phase was dried over Na2SO4 and evaporated to dryness.
The residue was usually purified via flash chromatography using an appropriate solvent system.
General Procedure B (SEM cleavage): The SEM-protected substrate was dissolved in dry DCM and
TFA was added subsequently. The reaction was stirred until TLC indicated complete consumption of
the starting material. The majority of TFA was removed under reduced pressure and the residue was
taken up in EtOH and was basified with aqueous NH3 solution. The mixture was stirred at ambient
temperature until complete consumption of the hydroxymethyl intermediate (usually overnight) was
observed. The volatiles were stripped of under reduced pressure and the residue purified via flash
chromatography as indicated.
General Procedure C (amide coupling with CDI): Carboxylic acid 18 was dissolved in dry DMF
(ca. 0.1 M) and CDI was added at ambient temperature. The mixture was stirred until gas evolution
ceased (usually about 1 h) before the corresponding amine was added to the reaction. Stirring was

Int. J. Mol. Sci. 2020, 21, 9269
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continued until reaction control indicated complete conversion and the reaction was quenched by
addition of water followed by dilution with EtOAc. The organic phase was washed with sat. NaHCO3
(two times) and brine, prior to solvent evaporation and purification by flash chromatography.
General Procedure D (amide coupling with acid chlorides): To a solution of 22 and Et3N in dry DCM
(ca. 0.1 M) was added the corresponding acid chloride under ice-cooling. After complete addition,
the cooling bath was removed and stirring was continued at ambient temperature until reaction control
(HPLC or TLC) indicated complete conversion. The reaction was quenched by addition of water
followed by dilution with EtOAc. The organic phase was washed with sat. NaHCO3, and brine,
prior to drying over Na2SO4 and evaporation. The residue was purified via flash chromatography
with an appropriate eluent system.
4.1.3. Synthesis and Characterization of Intermediates and Final Compounds
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (13): In a flame-dried Schlenk flask were suspended
1.47 g dry KOAc (15.0 mmol), 860 mg 3-bromoaniline (5.00 mmol) and 1.27 g bis(pinacolato)diboron
(5.00 mmol) in 20 mL dry dioxane. The mixture was carefully degassed via three vacuum/argon cycles
and 9 mg XPhos Pd G4 (10 µmol) was added subsequently. The degassing procedure was repeated
and then the reaction was heated to 90 ◦C oil-bath temperature overnight. After cooling to ambient
temperature, the mixture was diluted with EtOAc and filtered over a bed of celite. The filtrate was
washed with water (three times) and brine, prior to drying over Na2SO4 and evaporation. The residue
was triturated with heptane and the precipitate isolated by filtration to yield 914 mg (84%) of the
1
product as a slightly brownish solid. H NMR (200 MHz, DMSO)
δ
7.06–6.97 (m, 1H), 6.95 (d, J = 1.9 Hz
,
1H), 6.82 (dt, J = 7.2, 1.1 Hz, 1H), 6.66 (ddd, J = 7.9, 2.4, 1.1 Hz, 1H), 5.02 (br s, 2H), 1.26 (s, 12H)
13C NMR (50 MHz, DMSO)
[M + H]+ HPLC tret = 6.97 min.
δ 148.0, 128.3, 128.3, 121.9, 120.1, 116.8, 83.2, 24.7 TLC-MS (ESI) m/z: 219.9
N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acrylamide (14): To a solution of 891 mg 13
(4.07 mmol) and 715
chloride (4.48 mmol) at
µ
L Et3N (5.09 mmol) in 16 mL dry DCM were dropwise added 368
−
µL acryloyl
10 ◦C. After 1 h, the reaction was quenched by addition of sat. NH4Cl and
transferred to a separatory funnel. The phases were separated and the organic phase was washed with
sat NaHCO3 and brine prior to drying over Na2SO4 and evaporation. The crude product was purified
via flash chromatography (petrol ether/EtOAc + 5% MeOH (10–40%)) to obtain 780 mg (85%) of the
1
title compound as a white solid. H NMR (200 MHz, DMSO)
δ 10.14 (br s, 1H), 7.98 (d, J = 1.4 Hz, 1H),
7.85 (dt, J = 6.3, 2.4 Hz, 1H), 7.41–7.26 (m, 2H), 6.43 (dd, J = 17.0, 9.7 Hz, 1H), 6.25 (dd, J = 17.0, 2.4 Hz,
1H), 5.75 (dd, J = 9.7, 2.4 Hz, 1H), 1.29 (s, 12H) 13C NMR (50 MHz, CDCl3)
δ 163.1, 138.6, 131.8, 129.3,
128.4, 126.8, 125.3, 122.2, 83.7, 24.7 TLC-MS (ESI) m/z: 296.4 [M + H]+ HPLC tret = 7.45 min.
5-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine (16): To an ice-cooled solution of
1.96 g 5-bromo-1H-pyrrolo[2,3-b]pyridine (10 mmol) in 10 mL dry DMF were added 518 mg sodium
hydride (60%wt dispersion in min. oil, 12.9 mmol) and stirring was continued for about 30 min.
Subsequently were added 1.95 mL SEM-Cl (10.9 mmol) and stirring was continued until TLC indicated
complete conversion. The reaction was diluted with 120 mL EtOAc and transferred to a separatory
funnel. The organic phase was successively washed with water (four times) and brine, prior to drying
over Na2SO4 and evaporation. The residue was purified via flash chromatography (petrol ether/EtOAc
1
(0–15%)) to obtain 2.55 g (78%) as a colorless oil. H NMR (200 MHz, CDCl3)
δ 8.34 (d, J = 1.9 Hz, 1H),
8.02 (d, J = 1.9 Hz, 1H), 7.35 (d, J = 3.6 Hz, 1H), 6.46 (d, J = 3.6 Hz, 1H), 5.64 (s, 2H), 3.52 (t, J = 8.2 Hz,
2H), 0.89 (t, J = 8.2 Hz, 2H), δ 146.6, 143.8, 131.1, 129.5, 122.3,
0.07 (s, 9H) 13C NMR (50 MHz, CDCl3)
−
112.3, 100.7, 73.2, 66.5, 17.9, −1.3 TLC-MS (ESI) m/z: 349.3 [M + Na]+ HPLC tret = 10.84 min.
1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxylic acid (17): Under argon
atmosphere were dissolved 2.49 g 16 (7.6 mmol) in 80 mL dry THF in a flame-dried Schlenk tube.
At –78 ◦C, 3.3 mL of a 2.5 M n-BuLi solution in hexane (8.36 mmol) was added dropwise. After complete
addition, stirring was continued for 30 min before dry CO2 gas was bubbled through the solution.

Int. J. Mol. Sci. 2020, 21, 9269
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After another 30 min, the reaction was quenched with water and slowly warmed to ambient temperature.
After dilution with EtOAc, the organic phase was extracted with 0.5 M aqueous NaOH (three times).
The combined extracts were acidified with 2M HCl and back-extracted with EtOAc (three times).
The combined organic phases were washed with brine, dried over Na2SO4 and evaporated to dryness.
The crude product was purified via flash chromatography (petrol ether/EtOAc + 5% MeOH + 2%
1
AcOH (25–75%)) to yield 1.14 g (51%) of pure 17 as an off-white semisolid. H NMR (200 MHz, CDCl3)
δ
10.39 (br s, 1H), 9.10 (s, 1H), 8.65 (s, 1H), 7.43 (d, J = 3.5 Hz, 1H), 6.64 (d, J = 3.5 Hz, 1H), 5.73 (s, 2H),
3.55 (t, J = 8.2 Hz, 2H), 0.90 (t, J = 8.2 Hz, 2H), -0.09 (s, 9H) 13C NMR (50 MHz, CDCl3)
171.4, 150.0,
H]− HPLC tret
δ
145.8, 132.0, 129.8, 120.3, 118.7, 102.8, 73.3, 66.6, 17.8,
= 8.66 min.
−
1.4 TLC-MS (ESI) m/z: 291.4 [M
−
3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxylic acid (18): In a 100 mL
round-bottomed flask were dissolved 1.14 g 17 (3.89 mmol) in 50 mL dry DCM and the solution was
cooled in an ice/water bath. To this were added 684 mg bromine (4.28 mmol) as 0.5 M solution in DCM
in small portions until the persistence of yellow coloration was observed. The reaction was quenched
by addition of aqueous Na2S2O3 solution and extracted with DCM (three times). The combined
extracts were washed with sat. NH4Cl, water and brine, prior to drying over Na2SO4 and evaporation.
The residue was subjected to flash purification (petrol ether/EtOAc + 5% MeOH + 2% AcOH (25–75%))
1
to obtain 1.29 g (89%) of 18 as a brownish oil. H NMR (200 MHz, CDCl3)
δ
11.16 (br s, 1H), 9.10 (s,
0.07 (s, 9H)
1.4
1H), 8.59 (s, 1H), 7.48 (s, 1H), 5.70 (s, 2H), 3.56 (t, J = 8.2 Hz, 2H), 0.92 (t, J = 8.2 Hz, 2H),
−
13C NMR (50 MHz, CDCl3) δ δ 171.1, 149.1, 146.8, 130.9, 128.7, 119.8, 119.3, 91.8, 73.2, 66.9, 17.8,
TLC-MS (ESI) m/z: 369.2 [M − H]− HPLC tret = 9.82 min.
−
3-bromo-5-nitro-1H-pyrrolo[2,3-b]pyridine (20): To an ice-cooled suspension of 1.8 g 5-nitro-1H-
pyrrolo[2,3-b]pyridine (11.0 mmol) in 40 mL dry DMF were added 2.36 g N-bromosuccinimide
(13.2 mmol) as solid in small portions. After complete addition, the cooling bath was removed and the
reaction was stirred for 3 h at ambient temperature. The mixture was diluted with water and aqueous
Na2S2O3 and the precipitate was isolate by filtration. The filter cake was washed with water and dried
1
at 60 ◦C in a convection oven to yield 1.86 g (70%) of the title compound as a yellow solid. H NMR
(200 MHz, DMSO-d6)
δ 12.95 (s, 1H), 9.18 (d, J = 2.5 Hz, 1H), 8.64 (d, J = 2.5 Hz, 1H), 8.09 (d, J = 2.6 Hz,
1H). TLC-MS (ESI) m/z: 239.1 [M − H]− HPLC tret = 7.73 min.
3-bromo-5-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine (21): To an ice-cooled
solution of 2.14 g 20 (8.9 mmol) in 20 mL dry DMF were added 462 mg sodium hydride (60%wt
dispersion in min. oil, 11.5 mmol) and stirring was continued for about one h. Subsequently were
added 1.7 mL SEM-Cl (9.7 mmol) and stirring was continued until TLC indicated complete conversion.
The reaction was diluted with 120 mL EtOAc and transferred to a separatory funnel. The organic
phase was successively washed with water (four times) and brine, prior to drying over Na2SO4 and
evaporation. The residue was purified via flash chromatography (petrol ether/EtOAc (0–20%)) to
1
obtain 1.85 g (56%) 21 as a yellow oil. H NMR (200 MHz, CDCl3)
δ
9.24 (d, J = 2.4 Hz, 1H), 8.73 (d,
J = 2.4 Hz, 1H), 7.58 (s, 1H), 5.70 (s, 2H), 3.54 (m, 2H), 0.92 (m, 2H),
−
0.05 (s, 9H). HPLC tret = 11.56 min.
3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-amine (22): To a solution of 1.85 g
21 (5.0 mmol) in 40 mL abs. EtOH were added 1.63 g finely powdered zinc (24.9 mmol) and 1.57 g
ammonium formate (24.9 mmol). The mixture was stirred for 22 h at 50 ◦C and was then filtered over a
celite pad to remove unreacted zinc. The filtrate was concentrated under reduced pressure and taken
up in EtOAc. The organic phase was washed with sat NH4Cl and brine, prior to drying over Na2SO4
and evaporation. The residue was purified via flash chromatography (petrol ether/EtOAc 20–60%)
to obtain 0.86 g (51%) of the title compound as a brown oil. 1H NMR (200 MHz, CDCl3)
J = 2.6 Hz, 1H), 7.27 (s, 1H), 7.14 (d, J = 2.6 Hz, 1H), 5.55 (s, 2H), 4.60 (s, 2H), 3.51 (t, 2H), 0.88 (t, 2H),
0.08 (s, 9H). 13C NMR (50 MHz, CDCl3)
142.1, 137.5, 134.6, 127.2, 120.2, 112.5, 88.5, 72.9, 66.2, 17.7,
−1.6. HPLC tret = 8.65 min.
δ 7.93 (d,
−
δ

Int. J. Mol. Sci. 2020, 21, 9269
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3-bromo-1H-pyrrolo[2,3-b]pyridine (23): To an ice-cooled solution of 473 mg 1H-pyrrolo[2,3-b]pyridine
(4.0 mmol) in 8 mL DMF were added 726 mg N-bromosuccinimide (4.1 mmol) in several portions.
After complete addition, the cooling bath was removed and stirring was continued for 4 h. The reaction
mixture was poured on sat. NaHCO3/ice and the resulting suspension was stirred for ca. 10 min until
a homogenous precipitate was formed. The solids were collected by filtration, washed with water
1
and dried in vacuo to yield 753 mg (96%) of the title compound as a white solid. H NMR (200 MHz,
CDCl3)
δ 12.07 (br s, 1H), 8.29 (dd, J = 4.7, 1.5 Hz, 1H), 7.83 (dd, J = 7.9, 1.5 Hz, 1H), 7.70 (s, 1H),
7.16 (dd, J = 7.9, 4.7 Hz, 1H) 13C NMR (50 MHz, DMSO)
TLC-MS (ESI) m/z: 197.1 [M + H]+ HPLC tret = 6.56 min.
δ 147.2, 143.9, 126.4, 125.6, 118.7, 116.3, 87.1
3-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (24): To an ice-cooled solution of 197 mg 23 (1.0 mmol) in 5 mL
dry THF were added 50 mg sodium hydride (60%wt dispersion in min. oil, 1.25 mmol) and stirring
was continued for about 30 min. Subsequently were added 210 mg TsCl (1.1 mmol) and stirring was
continued until TLC indicated complete conversion. The reaction was diluted with 25 mL EtOAc and
transferred to a separatory funnel. The organic phase was successively washed with water and brine,
prior to drying over Na2SO4 and evaporation. The residue was triturated with chilled MeOH and
1
filtered to obtain 298 mg (85%) 24 as a white solid. H NMR (200 MHz, DMSO)
δ
8.49–8.40 (m, 1H),
8.20 (s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.96–7.86 (m, 1H), 7.46–7.33 (m, 3H), 2.31 (s, 3H) 13C NMR (50 MHz,
DMSO) 146.1, 146.0, 145.4, 134.2, 130.2, 128.7, 127.8, 125.7, 121.7, 120.2, 95.1, 21.1 HPLC tret = 8.86 min.
δ
3-phenyl-1-tosyl-1H-pyrrolo[2,3-b]pyridine (25): The preparation was performed following General
Procedure A from 100 mg 24 (0.25 mmol) and 38 mg phenylboronic acid (0.31 mmol), catalyzed by 1 mg
Pd(OAc)2 (5 µmol) and 5 mg XPhos (10 µmol) in 3 mL dioxane and 0.75 mL of an 1 M Na2CO3 solution.
The reaction was conducted at 70 ◦C and the crude product was purified via flash chromatography
(petrol ether/EtOAc + 5% MeOH (5–30%)) to obtain 72 mg (82%) of the title compound as a colorless
1
semisolid. H NMR (200 MHz, CDCl3)
δ
8.50 (dd, J = 4.7, 1.2 Hz, 1H), 8.25–8.04 (m, 3H), 7.92 (s, 1H),
147.6, 145.3, 145.1, 135.4,
7.68–7.56 (m, 2H), 7.55–7.17 (m, 6H), 2.36 (s, 3H) 13C NMR (50 MHz, CDCl3)
δ
132.6, 129.7, 129.1, 128.9, 128.1, 127.7, 127.4, 122.7, 121.6, 120.3, 119.1, 21.6 TLC-MS (ESI) m/z: 403.0
[M + Na + MeOH]+ HPLC tret = 9.02 min.
N-(3-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)acrylamide (26): The preparation was performed
following General Procedure A from 53 mg 24 (0.15 mmol) and 51 mg 14 (0.19 mmol), catalyzed
by 3 mg XPhos Pd G3 (5◦ µmol) in 3.6 mL dioxane and 0.9 mL of an 0.5 M K2CO3 solution. The reaction
was conducted at 50 C and the crude product was purified via flash chromatography (petrol
ether/EtOAc + 5% MeOH (40–70%)) to obtain 63 mg (quant.) of the title compound as a colorless waxy
1
solid. H NMR (200 MHz, CDCl3)
δ 8.47–8.35 (m, 5H), 8.23–7.95 (m, 5H), 7.85 (s, 1H), 7.57–7.45 (m,
1H), 7.40–7.11 (m, 5H), 6.52–6.24 (m, 2H), 5.74 (dd, J = 8.7, 2.5 Hz, 1H), 2.34 (s, 3H) 13C NMR (50 MHz,
CDCl3)
δ 164.3, 147.5, 145.5, 145.1, 138.8, 135.2, 133.2, 131.3, 129.8, 129.6, 129.2, 128.0, 127.9, 123.3, 122.8,
121.5, 120.1, 119.3, 119.2, 21.6 TLC-MS (ESI) m/z: 416.5 [M − H]− HPLC tret = 8.00 min.
3-phenyl-1H-pyrrolo[2,3-b]pyridine (9c): In a 25 mL round-bottomed flask were dissolved 72 mg 25
(0.3 mmol) in 6 mL of a 1 M solution of KOH in MeOH at 60 ◦C oil-bath temperature. The reaction was
stirred for 3 h and was then quenched by the addition of sat. NH4Cl solution. The aqueous phase was
extracted with EtOAc (4
×
15 mL) and the combined extracts were washed with brine. After drying over
Na2SO4 and evaporation, the residue was purified via flash chromatography (DCM/EtOAc (10–80%))
to yield 34 mg (85%) of the final compound as a white solid. H NMR (400 MHz, DMSO)
1
δ
11.92 (br
s, 1H), 8.35–8.20 (m, 2H), 7.87 (s, 1H), 7.77–7.64 (m, 2H), 7.49–7.36 (m, 2H), 7.25 (t, J = 7.9 Hz, 1H),
7.17–7.08 (m, 1H) 13C NMR (100 MHz, DMSO)
δ 149.1, 142.9, 135.0, 128.8, 127.4, 126.2, 125.6, 123.6,
117.3, 116.0, 114.3 TLC-MS (ESI) m/z: 193.0 [M − H]− HPLC tret = 7.60 min.
N-(3-(1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)acrylamide (9a): To a suspension of 63 mg 26 (0.15 mmol) in
10 mL tBuOH were added 561 mg finely powdered KOH (10 mmol) and the mixture was heated to
50 ◦C oil-bath temperature until TLC indicated complete conversion. The reaction was quenched by the

Int. J. Mol. Sci. 2020, 21, 9269
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addition of sat. NH4Cl solution, followed by extraction with EtOAc (3
were washed with brine and dried over Na2SO4. After evaporation of the volatiles, the residue was
×
20 mL). The combined extracts
purified via flash chromatography (DCM/MeOH (2–8%)) to yield 32 mg (81%) of the final compound
1
as a white solid. H NMR (200 MHz, DMSO)
δ 11.94 (s, 1H), 10.22 (s, 1H), 8.39–8.23 (m, 2H), 8.14 (br s,
1H), 7.86 (d, J = 2.5 Hz, 1H), 7.63–7.50 (m, 1H), 7.47–7.30 (m, 2H), 7.19 (dd, J = 7.9, 4.9 Hz, 1H), 6.48 (dd,
J = 16.8, 9.7 Hz, 1H), 6.29 (dd, J = 16.8, 2.1 Hz, 1H), 5.78 (dd, J = 9.7, 2.1 Hz, 1H) 13C NMR (50 MHz,
DMSO)
δ 163.3, 149.1, 143.0, 139.5, 135.5, 132.0, 129.3, 127.4, 126.9, 123.7, 121.4, 117.2, 117.1, 116.6, 116.1,
114.1 TLC-MS (ESI) m/z: 318.5 [M + Na + MeOH]+ HPLC tret = 5.42 min.
1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (27): To a solution of 235 mg
17 (0.8 mmol) were added 156 mg CDI (0.96 mmol) and stirring was continued at ambient temperature
for 30 min. Then 5 mL conc. NH3 solution were added and after another hour of stirring the reaction
was further diluted with water and transferred to a separatory funnel. The aqueous phase was extracted
with EtOAc (3
×
20 mL) and the combined organic phases were backwashed two times with brine.
After drying over Na2SO4 and evaporation, the residue was purified via flash chromatography (petrol
ether/EtOAc + 5% MeOH (40–100%)) to obtain 169 mg (72%) of the title compound as a white solid.
1H NMR (200 MHz, CDCl3)
δ
8.82 (s, 1H), 8.40 (s, 1H), 7.38 (d, J = 3.3 Hz, 1H), 6.60 (br s, 2H), 6.55 (d,
J = 3.3 Hz, 1H), 5.66 (s, 2H), 3.51 (t, J = 8.2 Hz, 2H), 0.87 (t, J = 8.2 Hz, 2 H),
0.11 (s, 9H) 13C NMR
(50 MHz, CDCl3) 169.4, 149.5, 142.9, 129.6, 129.1, 122.4, 120.1, 102.3, 73.2, 66.5, 17.8, -1.4 TLC-MS (ESI)
m/z: 314.4 [M + Na]+ HPLC tret = 7.58 min.
−
δ
3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (28): An ice-cooled
solution of 150 mg 27 (0.52 mmol) in 10 mL dry DCM was treated dropwise with a 1 M bromine
solution in DCM until a yellow coloration persisted. TLC indicated complete conversion at this point
and the reaction was quenched by addition of aqueous Na2SO3 solution. After dilution with 40 mL
EtOAc, the organic phase was washed with sat. NaHCO3 and brine, prior to drying over Na2SO4 and
evaporation. The residue was purified via flash chromatography (petrol ether/EtOAc + 5% MeOH
1
(20–100%)) to obtain 130 mg (68%) of the title compound as a white solid. H NMR (200 MHz, CDCl3)
δ
8.85 (s, 1H), 8.31 (s, 1H), 7.42 (s, 1H), 6.81–6.45 (m, 2H), 5.64 (s, 2H), 3.52 (t, J = 8.2 Hz, 2H), 0.88 (t,
J = 8.2 Hz, 2H), δ 168.9, 148.4, 144.2, 128.5, 127.8, 123.1, 119.5,
0.09 (s, 9H) 13C NMR (50 MHz, CDCl3)
−
91.3, 73.2, 66.8, 17.8, −1.4 TLC-MS (ESI) m/z: 424.3 [M + Na + MeOH]+ HPLC tret = 8.77 min.
3-phenyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (29): The preparation
was performed following General Procedure A from 62 mg 28 (0.17 mmol) and 24 mg phenylboronic
acid (0.20 mmol), catalyzed by 2 mg tBu3P Pd G3 (3 µmol) in 4 mL dioxane and 1 mL of an 0.5 M
K3PO4 solution. The reaction was conducted at ambient temperature and the crude product was of
sufficient purity to be used directly in the next step. Yield: 62 mg (quant.) as a colorless semisolid.
1H NMR (200 MHz, CDCl3/MeOD)
7.50–7.27 (m, 3 H), 5.71 (s, 2 H), 3.59 (t, J = 8.3 Hz, 2H), 0.93 (t, J = 8.3 Hz, 2H),
δ
8.85 (d, J = 1.6 Hz, 1H), 8.73 (d, J = 1.9 Hz, 1H), 7.67–7.56 (m, 3H),
−
0.06 (s, 9H). 13C NMR
(50 MHz, CDCl3/MeOD)
δ 169.3, 149.6, 143.0, 133.5, 128.9, 128.6, 127.1, 126.8, 126.2, 122.5, 118.3, 117.7,
73.1, 66.5, 17.6, −1.6. TLC-MS (ESI) m/z: 422.2 [M + Na + MeOH]+ HPLC tret = 9.44 min.
3-(3-acrylamidophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (30):
The preparation was performed following General Procedure A from 61 mg 28 (0.17 mmol) and 56 mg
14 (0.21 mmol), catalyzed by 3 mg tBu3P Pd G3 (5 µmol) in 4 mL dioxane and 1 mL of an 0.5 M K2CO3
solution. The reaction was conducted at ambient temperature and the crude product was purified via
flash chromatography (petrol ether/EtOAc + 5% MeOH (40–100%)). Yield: 67 mg (93%) as colorless a
1
semisolid. H NMR (200 MHz, DMSO)
δ 10.28 (br s, 1H), 8.87 (s, 1H), 8.78 (s, 1H), 8.18 (br s, 1H), 8.07
(s, 1H), 8.03 (s, 1H), 7.77–7.60 (m, 1 H), 7.45 (d, J = 4.7 Hz, 3H), 6.49 (dd, J = 17.0, 9.8 Hz, 1 H), 6.29 (dd,
J = 17.0, 1.7 Hz, 1H), 5.85–5.53 (m, 3H), 3.58 (t, J = 7.8 Hz, 2H), 0.85 (t, J = 7.8 Hz, 2H),
0.10 (s, 9H) 13
NMR (50 MHz, DMSO) 167.4, 163.3, 149.2, 143.5, 143.5, 139.7, 134.2, 131.9, 129.5, 127.9, 127.8, 127.0,
−
C
δ

Int. J. Mol. Sci. 2020, 21, 9269
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123.5, 122.0, 117.6, 117.0, 115.6, 72.7, 65.7, 17.2,
8.53 min.
−
1.4 TLC-MS (ESI) m/z: 459.5 [M + Na]+ HPLC tret
=
3-phenyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (10c): The preparation was carried out following
General Procedure B starting from 62 mg 29 (0.17 mmol) in 6 mL DCM and 2 mL TFA. Flash purification
(DCM/MeOH (8–16%)) afforded 37 mg (92%) of the final compound as a white solid. 1H NMR
(200 MHz, DMSO) δ 12.18 (br s, 1H), 8.82 (d, J = 1.4 Hz, 1H), 8.77 (d, J = 1.4 Hz, 1H), 8.18 (br s, 1H),
7.96 (d, J = 2.0 Hz, 1H), 7.79 (s, 1H), 7.76 (s, 1H), 7.54–7.21 (m, 4H). 13C NMR (50 MHz, DMSO)
δ 167.7,
150.2, 143.4, 134.5, 128.9, 127.2, 126.5, 126.0, 125.0, 122.4, 116.4, 115.4. TLC-MS (ESI) m/z: 238.0 [M + H]+
HPLC tret = 5.19 min.
3-(3-acrylamidophenyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (10a): The preparation was carried out
following General Procedure B starting from 90 mg 30 (0.21 mmol) in 8 mL DCM and 2 mL TFA.
Flash purification (DCM/MeOH (6–16%)) afforded 45 mg (71%) of the final compound as a white solid.
1H NMR (400 MHz, DMSO)
δ
12.17 (s, 1H), 10.26 (s, 1H), 8.81 (s, 1H), 8.76 (s, 1H), 8.12 (br s, 1H), 7.99
(s, 1H), 7.88 (s, 1H), 7.68 (d, J = 7.4 Hz, 1H), 7.50–7.31 (m, 3H), 6.55–6.42 (m, 1H), 6.35–6.22 (m, 1H),
5.83–5.72 (m, 1H) 13C NMR (100 MHz, DMSO)
δ 167.7, 163.2, 150.1, 143.2, 139.5, 134.9, 131.9, 129.3,
127.2, 126.7, 124.8, 122.6, 121.9, 117.5, 117.2, 116.4, 115.3 TLC-MS (ESI) m/z: 329.3 [M + Na]+ HPLC tret
= 3.94 min.
3-bromo-N-cyclopentyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (31a):
The amide coupling was performed following General Procedure C starting from 111 mg 18 (0.30 mmol)
and 58 mg CDI (0.36 mmol) followed by reaction with 59
product was purified via flash chromatography (hexane/EtOAc (10–40%)). Yield: 105 mg (80%) as
a colorless oil. 1H NMR (200 MHz, CDCl3)
8.73 (d, J = 1.9 Hz, 1H), 8.15 (d, J = 1.9 Hz, 1H), 7.36
(s, 1H), 6.60 (d, J = 7.2 Hz, 1H), 5.58 (s, 2H), 4.49–4.28 (m, 1H), 3.56–3.37 (m, 2H), 2.15–1.93 (m, 2H),
1.80–1.38 (m, 6H), 0.90–0.76 (m, 2H), 166.2, 148.1, 144.0,
0.11 (s, 9H). 13C NMR (50 MHz, CDCl3)
1.4. TLC-MS (ESI) m/z: 492.3
µL cyclopentylamine (0.60 mmol). The crude
δ
−
δ
128.1, 126.7, 124.5, 119.1, 90.9, 73.0, 66.6, 51.9, 33.2, 23.9, 23.9, 17.8,
−
[M + Na + MeOH]+ HPLC tret = 10.49 min.
3-bromo-N-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
The amide coupling was performed following General Procedure C starting from 100 mg 18 (0.27 mmol)
and 53 mg CDI (0.32 mmol) followed by addition with 270 L of a 2 M solution of methylamine in
(31c):
µ
THF (0.54 mmol). The crude product was purified via flash chromatography (hexane/EtOAc (10–50%)).
1
Yield: 60 mg (58%) as a white solid. H NMR (200 MHz, CDCl3)
δ 8.77 (d, J = 1.9 Hz, 1H), 8.21 (d,
J = 2.0 Hz, 1H), 7.38 (s, 1H), 6.90 (d, J = 5.1 Hz, 1H), 5.60 (s, 2H), 3.49 (t, J = 8.3 Hz, 2H), 3.01 (d, J = 4.7
Hz, 3H), 0.86 (t, J = 8.2 Hz, 2H), -0.11 (s, 9H). 13C NMR (50 MHz, CDCl3)
δ
167.4, 148.2, 143.9, 128.3,
126.9, 124.2, 119.2, 91.0, 73.1, 66.7, 27.1, 17.8,
9.47 min.
−
1.4 TLC-MS (ESI) m/z: 405.9 [M + Na]+ HPLC tret
=
3-bromo-N-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
(31d):
The amide coupling was performed following General Procedure C starting from 150 mg 18 (0.41 mmol)
and 86 mg CDI (0.53 mmol) followed by addition with 1 mL of a 2 M solution of ethylamine in MeOH
(2.0 mmol). The crude product was purified via flash chromatography (hexane/EtOAc (10–50%)).
1
Yield: 74 mg (46%) as a yellowish solid. H NMR (200 MHz, CDCl3)
δ 9.02 (d, J = 1.8 Hz, 1H), 8.53 (d,
J = 2.0 Hz, 1H), 7.45 (s, 1H), 7.38 (s, 1H), 5.67 (s, 2H), 3.70 (dd, J = 16.2, 10.0 Hz, 2H), 3.53 (t, J = 8.3 Hz,
2H), 1.01–0.77 (m, 5H), 166.3, 148.0, 143.6, 128.2, 126.8,
0.07 (s, 9H). 13C NMR (50 MHz, CDCl3)
124.2, 119.2, 90.9, 73.0, 66.6, 35.0, 17.7, 14.9, −1.5. HPLC tret = 9.85 min.
−
δ
3-bromo-N-(cyclopropylmethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
31e): The amide coupling was performed following General Procedure C starting from 100 mg 18
(0.27 mmol) and 53 mg CDI (0.32 mmol) followed by reaction with 94 L cyclopropylmethanamine
(
µ
(1.35 mmol). The crude product was purified via flash chromatography (hexane/EtOAc (20–80%)).
1
Yield: 83 mg (80%) as a white solid. H NMR (200 MHz, CDCl3 + MeOD)
δ
8.77 (d, J = 2.0 Hz, 1H),

Int. J. Mol. Sci. 2020, 21, 9269
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8.20 (d, J = 2.0 Hz, 1H), 7.44 (s, 1H), 6.42 (s, 1H), 5.65 (s, 2H), 3.70 (s, 2H), 3.60–3.42 (m, 2H), 3.04–2.85
(m, 1H), 0.99–0.80 (m, 4H), 0.76–0.58 (t, 2H), 168.0,
0.07 (s, 9H). 13C NMR (50 MHz, CDCl3 + MeOD)
1.3. TLC-MS (ESI) m/z: 446.0
−
δ
148.5, 144.0, 128.4, 126.9, 124.1, 119.3, 91.2, 73.1, 67.2, 66.8, 23.4, 17.9, 7.0,
−
[M + Na]+ HPLC tret = 9.88 min.
3-bromo-N-(cyclohexylmethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
31f): The amide coupling was performed following General Procedure C starting from 100 mg 18
(0.27 mmol) and 53 mg CDI (0.32 mmol) followed by reaction with 175 L cyclohexylmethanamine
(1.35 mmol). The crude product was purified via flash chromatography (hexane/EtOAc (0–40%)).
Yield: 72 mg (53%) as a brown oil. 1H NMR (200 MHz, CDCl3)
8.82 (d, J = 2.1 Hz, 1H), 8.26 (d,
J = 2.1 Hz, 1H), 7.46 (s, 1H), 6.27 (s, 1H), 5.67 (s, 2H), 3.53 (t, 2H), 3.37 (dd, J = 7.1, 5.4 Hz, 2H), 1.37
(
µ
δ
–1.06 (m, 4H), 0.99–0.82 (m, 4H), 0.65–0.53 (m, 2H), 0.33 (t, J = 5.2 Hz, 2H), 0.07 (s, 1H),
−
0.06 (s, 9H).
TLC-MS (ESI) m/z: 488.0 [M + Na]+ HPLC tret = 12.44 min.
N-benzyl-3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
The amide coupling was performed following General Procedure C starting from 300 mg 18 (0.81 mmol)
and 171 mg CDI (1.05 mmol) followed by reaction with 441 L benzylamine (1.35 mmol). The crude
(31g):
µ
product was purified via flash chromatography (hexane/EtOAc (10–50%)). Yield: 205 mg (55%) as a
1
brown viscous oil. H NMR (200 MHz, CDCl3)
δ 8.83 (d, J = 2.1 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 7.42
(s, 1H), 7.41–7.27 (m, 5H), 6.73 (t, J = 5.7 Hz, 1H), 5.64 (s, 2H), 4.67 (d, J = 5.6 Hz, 2H), 3.58–3.41 (m, 2H),
0.96–0.81 (m, 2H), −0.07 (s, 9H). TLC-MS (ESI) m/z: 481.9 [M + Na]+ HPLC tret = 10.60 min.
3-(3-acrylamidophenyl)-N-cyclopentyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-
carboxamide (32a): The preparation was performed following General Procedure A from 105 mg 31a
(0.24 mmol) and 71 mg 14 (0.26 mmol), catalyzed by 3 mg tBu3P Pd G3 (5 µmol) in 5.6 mL dioxane and
1.4 mL of a 0.5 M K3PO4 solution. The reaction was conducted at ambient temperature and the crude
product was purified via flash chromatography (hexane/EtOAc (30–70%)). Yield: 92 mg (78%) as a
1
white solid. H NMR (200 MHz, DMSO)
δ
10.28 (s, 1H), 8.82 (d, J = 1.9 Hz, 1H), 8.72 (d, J = 1.9 Hz, 1H),
8.44 (d, J = 7.3 Hz, 1H), 8.10–8.00 (m, 2H), 7.71–7.60 (m, 1H), 7.52–7.40 (m, 2H), 6.49 (dd, J = 17.0, 9.8
Hz, 1H), 6.29 (dd, J = 17.0, 2.2 Hz, 1H), 5.79 (dd, J = 9.8, 2.2 Hz, 1H), 5.71 (s, 2H), 4.38–4.18 (m, 1H), 3.58
(t, J = 7.9 Hz, 2H), 2.02–1.84 (m, 2H), 1.76–1.39 (m, 6H), 0.84 (t, J = 7.9 Hz, 2H),
−
0.10 (s, 9H). 13C NMR
(50 MHz, DMSO) 165.4, 163.3, 149.0, 143.2, 139.7, 134.3, 131.9, 129.5, 127.8, 127.4, 126.9, 124.2, 121.9,
δ
117.6, 117.5, 116.9, 115.6, 72.7, 65.7, 51.0, 32.2, 23.7, 17.2,
1.4. TLC-MS (ESI) m/z: 527.5 [M + Na]+
−
HPLC tret = 9.90 min.
3-(3-acrylamidophenyl)-N-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-
carboxamide (32c): The preparation was performed following General Procedure A from 60 mg 31c
(0.16 mmol) and 64 mg 14 (0.23 mmol), catalyzed by 3 mg tBu3P Pd G3 (5 µmol) in 4 mL dioxane and
0.9 mL of a 0.5 M K3PO4 solution. The reaction was conducted at ambient temperature and the crude
product was purified via flash chromatography (petrol ether/EtOAc + 5% MeOH (40–100%)). Yield:
48 mg (69%) as a white solid. The material was carried on directly to the next step. TLC-MS (ESI) m/z:
473.0 [M + Na]+ HPLC tret = 8.97 min.
3-(3-acrylamidophenyl)-N-ethyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
(32d): The preparation was performed following General Procedure A from 74 mg 31d (0.19 mmol)
and 64 mg 14 (0.23 mmol), catalyzed by 3 mg tBu3P Pd G3 (5 mol) in 4 mL dioxane and 1.1 mL of a
µ
0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the crude product was
purified via flash chromatography (petrol ether/EtOAc (40–100%)). Yield: 48 mg (69%) as white solid.
The material was carried on directly to the next step. TLC-MS (ESI) m/z: 487.2 [M + Na]+ HPLC tret
=
9.11 min.
3-(3-acrylamidophenyl)-N-(cyclopropylmethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-
5-carboxamide (32e): The preparation was performed following General Procedure A from 83 mg 31e
(0.20 mmol) and 83 mg 14 (0.30 mmol), catalyzed by 3 mg tBu3P Pd G3 (5 µmol) in 5 mL dioxane and

Int. J. Mol. Sci. 2020, 21, 9269
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1.2 mL of a 0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the crude
product was purified via flash chromatography (petrol ether/EtOAc + 5% MeOH (40–100%)). Yield:
1
78 mg (59%) as a white solid. H NMR (200 MHz, CDCl3)
δ 8.82 (d, J = 2.0 Hz, 1H), 8.68 (d, J = 2.0 Hz,
1H), 8.08 (s, 1H), 7.93 (s, 1H), 7.55 (s, 1H), 7.49 (s, 1H), 7.34 (q, J = 7.4 Hz, 2H), 6.73 (t, J = 5.5 Hz, 1H),
6.55–6.33 (m, 2H), 5.76 (dd, J = 8.3, 3.3 Hz, 1H), 5.69 (s, 2H), 3.56 (t, 2H), 3.37 (t, 2H), 1.13 (d, J = 9.8 Hz,
1H), 1.01–0.82 (m, 2H), 0.65–0.46 (m, 2H), 0.30 (d, J = 5.1 Hz, 2H),
−
0.07 (s, 9H). TLC-MS (ESI) m/z:
513.2 [M + Na]+ HPLC tret = 8.82 min.
3-(3-acrylamidophenyl)-N-(cyclohexylmethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-
5-carboxamide (32f): The preparation was performed following General Procedure A from 72 mg 31f
(0.16 mmol) and 64 mg 14 (0.23 mmol), catalyzed by 3 mg tBu3P Pd G3 (5 µmol) in 4 mL dioxane and
0.9 mL of a 0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the crude
product was purified via flash chromatography (petrol ether/EtOAc (20–60%)). Yield: 57 mg (69%) as
1
a yellowish solid. H NMR (200 MHz, CDCl3)
δ 8.81 (s, 1H), 8.70 (s, 1H), 8.10 (s, 1H), 7.99 (s, 1H),
7.78–7.30 (m, 4H), 6.74 (s, 1H), 6.42 (s, 1H), 6.37 (s, 1H), 5.77 (s, 1H), 5.68 (s, 2H), 3.55 (t, 2H), 3.34 (t, 2H),
1.73 (d, J = 16.2 Hz, 9H), 1.10 (s, 1H), 0.91 (t, J = 8.1 Hz, 2H), 0.07 (s, 1H),
m/z: 555.3 [M + Na]+ HPLC tret = 10.98 min.
−
0.07 (s, 9H). TLC-MS (ESI)
3-(3-acrylamidophenyl)-N-benzyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
32g): The preparation was performed following General Procedure A from 200 mg 31g (0.43 mmol)
and 149 mg 14 (0.54 mmol), catalyzed by 8 mg tBu3P Pd G3 (13 mol) in 12 mL dioxane and 2.6 mL of a
(
µ
0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the crude product was
purified via flash chromatography (petrol ether/EtOAc + 5% MeOH (40–100%)). Yield: 120 mg (53%)
1
as a brown solid. H NMR (200 MHz, CDCl3 + MeOD)
δ
8.61 (s, 1H), 8.51 (s, 1H), 8.36 (t, J = 5.4 Hz
,
1H), 7.78 (s, 1H), 7.36 (s, 1H), 7.30–7.19 (m, 1H), 7.17–6.95 (m, 8H), 6.18–6.10 (m, 2H), 5.51–5.44 (m,
1H), 5.41 (s, 2H), 4.08 (s, 2H), 3.33 (t, J = 8.2 Hz, 2H), 0.66 (t, J = 8.2 Hz, 2H),
0.31 (s, 9H). 13C NMR
(50 MHz, CDCl3 + MeOD) 167.4, 164.7, 149.2, 143.1, 138.6, 138.3, 134.1, 130.9, 129.3, 128.3, 127.9, 127.4,
−
δ
127.3, 127.0, 126.4, 123.3, 122.8, 118.7, 118.3, 118.1, 117.0, 73.0, 66.4, 43.6, 17.5, 1.9. TLC-MS (ESI) m/z:
−
548.9 [M+Na]+ HPLC tret = 10.08 min.
3-(3-acrylamidophenyl)-N-cyclopentyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11a): The preparation was
carried out following General Procedure B starting from 81 mg 32a (0.16 mmol) in 7.5 mL DCM and
2.5 mL TFA. Flash purification (DCM/MeOH (4–12%)) afforded 49 mg (82%) of the final compound as
1
a white solid. H NMR (400 MHz, DMSO)
δ 12.16 (br s, 1H), 10.26 (s, 1H), 8.77 (d, J = 1.6 Hz, 1H), 8.70
(d, J = 1.6 Hz, 1H), 8.39 (d, J = 7.1 Hz, 1H), 8.06–7.95 (m, 1H), 7.88 (d, J = 2.4 Hz, 1H), 7.72–7.61 (m,
1H), 7.49–7.34 (m, 2H), 6.48 (dd, J = 17.0, 10.1 Hz, 1H), 6.29 (dd, J = 17.0, 1.8 Hz, 1H), 5.78 (dd, J = 10.1,
1.8 Hz, 1H), 4.34–4.20 (m, 1H), 2.00–1.81 (m, 2H), 1.79–1.64 (m, 2H), 1.63–1.46 (m, 4H). 13C NMR (101
MHz, DMSO)
δ 165.6, 163.2, 149.9, 142.9, 139.5, 135.0, 131.9, 129.3, 126.9, 126.7, 124.8, 123.3, 121.8, 117.5,
117.1, 116.3, 115.3, 51.0, 32.1, 23.6. TLC-MS (ESI) m/z: 397.0 [M + Na]+ HPLC tret = 6.94 min.
3-(3-acrylamidophenyl)-N-methyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11c): The preparation was
carried out following General Procedure B starting from 48 mg 32c (0.11 mmol) in 7 mL DCM and 3 mL
TFA. Flash purification (DCM/MeOH (6–16%)) afforded 31 mg (91%) of the final compound as a white
1
solid. H NMR (400 MHz, DMSO-d6)
δ 12.18 (s, 1H), 10.27 (s, 1H), 8.76 (d, J = 2.0 Hz, 1H), 8.71 (d,
J = 2.0 Hz, 1H), 8.57 (d, J = 4.6 Hz, 1H), 7.98 (s, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H),
7.51–7.37 (m, 2H), 6.49 (dd, J = 16.9, 10.1 Hz, 1H), 6.29 (dd, J = 16.9, 2.0 Hz, 1H), 5.78 (dd, 1H), 2.83 (d,
J = 4.4 Hz, 3H) 13C NMR (101 MHz, DMSO-d6)
δ 166.9, 163.7, 150.5., 143.3, 140.0, 135.4, 132.5, 129.8,
127.3, 127.1, 125.4, 123.5, 122.4, 118.0, 117.7, 116.9, 115.8, 26.7. TLC-MS (ESI) m/z: 343.0 [M + Na]+.
HRMS ESI-TOF [M + H]+ m/z calcd. for C18H16N4O2: 321.1346, found: 321.1351. HPLC tret = 4.86 min.
3-(3-acrylamidophenyl)-N-ethyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11d): The preparation was
carried out following General Procedure B starting from 68 mg 32d (0.15 mmol) in 4.8 mL DCM
and 1.2 mL TFA. Flash purification (DCM/MeOH (6–10%)) afforded 34 mg (70%) of the final compound

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1
as a white solid. H NMR (400 MHz, DMSO-d6)
δ
12.16 (s, 1H), 10.25 (s, 1H), 8.77 (d, J = 2.0 Hz, 1H),
8.71 (d, J = 2.0 Hz, 1H), 8.58 (t, J = 5.5 Hz, 1H), 7.98 (d, J = 1.7 Hz, 1H), 7.88 (d, J = 2.6 Hz, 1H), 7.68
(dt, J = 7.1, 2.1 Hz, 1H), 7.49–7.38 (m, 2H), 6.48 (dd, J = 16.9, 10.1 Hz, 1H), 6.29 (dd, J = 17.0, 2.1 Hz,
1H), 5.78 (dd, J = 10.1, 2.1 Hz, 1H), 3.40–3.33 (m, 2H), 1.16 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 166.2, 163.7, 150.5, 143.3, 140.0, 135.5, 132.4, 129.8, 127.3, 127.2, 125.4, 123.6, 122.4, 118.0,
117.7, 116.9, 115.8, 34.5, 15.3. TLC-MS (ESI) m/z: 357.0 [M + Na]+. HRMS ESI-TOF [M + H]+ m/z calcd.
for C19H18N4O2: 335,1503, found: 335.1508. HPLC tret = 5.53 min.
3-(3-acrylamidophenyl)-N-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
(
11e):
The preparation was carried out following General Procedure B starting from 78 mg 32e
(0.16 mmol) in
4.2 mL DCM and 1.8 mL TFA. Flash purification (DCM/MeOH (6–16%)) afforded 13 mg (23%) of the
1
final compound as a yellowish solid. H NMR (400 MHz, DMSO-d6)
δ 12.18 (s, 1H), 10.30 (s, 1H), 8.80
(d, J = 2.0 Hz, 1H), 8.74 (d, J = 2.1 Hz, 1H), 8.71 (t, J = 5.6 Hz, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.90 (s, 1H),
7.70 (dt, J = 7.3, 1.9 Hz, 1H), 7.50–7.38 (m, 2H), 6.49 (dd, J = 17.0, 10.1 Hz, 1H), 6.29 (dd, J = 17.0, 2.1 Hz,
1H), 5.79 (dd, J = 10.0, 2.1 Hz, 1H), 3.20 (t, J = 6.2 Hz, 2H), 1.07 (ttd, J = 12.9, 6.8, 3.7 Hz, 1H), 0.51–0.37
(m, 2H), 0.36–0.22 (m, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 165.8, 163.2, 149.9, 142.8, 139.5, 134.9,
131.9, 129.2, 126.8, 126.7, 124.8, 123.0, 121.9, 117.5, 117.1, 116.4, 115.3, 43.5, 11.0, 3.3. TLC-MS (ESI) m/z:
383.1 [M + Na]+. HRMS ESI-TOF [M + H]+ m/z calcd. for C21H20N4O2: 361.1659, found: 361.1662.
HPLC tret = 6.54 min.
3-(3-acrylamidophenyl)-N-(cyclohexylmethyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide
(11f):
The preparation was carried out following General Procedure B starting from 30 mg 32f (0.06 mmol) in
7 mL DCM and 3 mL TFA. Flash purification (DCM/MeOH (5–10%)) afforded 15 mg (64%) of the final
1
compound as a yellowish solid. H NMR (400 MHz, DMSO-d6)
δ
12.16 (s, 1H), 10.27 (s, 1H), 8.78 (s,
1H), 8.72 (s, 1H), 8.54 (s, 1H), 8.02 (s, 1H), 7.88 (d, J = 3.0 Hz, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.44 (s, 2H),
6.49 (ddd, J = 17.2, 10.1, 3.3 Hz, 1H), 6.29 (d, J = 16.8 Hz, 1H), 5.78 (d, J = 10.2 Hz, 1H), 3.16 (q, J = 6.5,
5.0 Hz, 2H), 1.72 (dd, J = 24.7, 11.6 Hz, 4H), 1.61 (d, J = 10.0 Hz, 2H), 1.22 (s, 1H), 1.17 (d, J = 9.2 Hz,
2H), 0.95 (t, J = 12.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 165.9, 163.2, 149.9, 142.8, 139.5, 134.9,
131.9, 129.2, 126.7, 126.6, 124.8, 123.1, 121.8, 117.5, 117.1, 116.3, 115.2, 45.4, 37.4, 30.5, 26.0, 25.4. TLC-MS
(ESI) m/z: 425.3 [M + Na]+. HRMS ESI-TOF [M + H]+ m/z calcd. for C24H26N4O2: 403.2129, found:
403.2133. HPLC tret = 8.50 min.
3-(3-acrylamidophenyl)-N-benzyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11g): The preparation was
carried out following General Procedure B starting from 80 mg 32g (0.15 mmol) in 7 mL DCM and 3 mL
TFA. Flash purification (DCM/MeOH (6–8%)) afforded 58 mg (97%) of the final compound as a pale
1
red solid. H NMR (400 MHz, DMSO-d6)
δ
12.20 (d, J = 2.7 Hz, 1H), 10.26 (s, 1H), 9.18 (t, J = 6.0 Hz,
1H), 8.84 (d, J = 2.0 Hz, 1H), 8.79 (d, J = 2.0 Hz, 1H), 8.00 (t, J = 1.9 Hz, 1H), 7.90 (d, J = 2.6 Hz, 1H), 7.67
(dt, J = 7.7, 1.8 Hz, 1H), 7.52–7.18 (m, 7H), 6.48 (q, J = 16.9, 10.1 Hz, 1H), 6.29 (dd, J = 17.0, 2.1 Hz, 1H),
5.78 (dd, J = 10.1, 2.0 Hz, 1H), 4.54 (d, J = 5.9 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 166.0, 163.2,
150.1, 142.9, 139.7, 139.5, 134.9, 131.9, 129.3, 128.2, 127.2, 126.9, 126.8, 126.7, 124.9, 122.7, 121.9, 117.5,
117.2, 116.4, 115.4, 42.6. TLC-MS (ESI) m/z: 419.3 [M + Na]+. HRMS ESI-TOF [M + H]+ m/z calcd. for
C24H20N4O2: 397.1659, found: 397.1663. HPLC tret = 7.20 min.
N-(3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)cyclopentanecarboxamide (33a):
The preparation was carried out following General Procedure D with 90 mg 22 (0.26 mmol), 111 µL
Et3N (0.79 mmol) and 70 mg cyclopentanecarboxylic acid chloride (0.53 mmol). Flash purification
1
(hexane/EtOAc (10–50%)) afforded 90 mg (78%) of the product as a colorless oily residue. H NMR
(200 MHz, CDCl3)
δ
8.30–8.21 (m, 1H), 8.20–8.15 (m, 1H), 8.11 (br s, 1H), 7.29 (s, 1H), 5.54 (s, 2H), 3.48
0.10
(t, J = 8.2 Hz, 2H), 2.82–2.60 (m, 1H), 1.95–1.80 (m, 4H), 1.78–1.43 (m, 4H), 0.86 (t, J = 8.2 Hz, 2H),
−
(s, 9H). 13C NMR (50 MHz, CDCl3)
δ 175.6, 144.1, 138.3, 129.5, 127.6, 120.0, 119.7, 89.9, 73.0, 66.5, 46.4,
30.7, 26.1, 17.8, −1.4. TLC-MS (ESI) m/z: 492.1 [M + Na + MeOH]+ HPLC tret = 10.84 min.

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N-(3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)thiophene-2-carboxamide (33c):
The preparation was carried out following General Procedure D with 100 mg 22 (0.29 mmol), 123
Et3N (0.88 mmol) and 41 L thiophene-2-carbonyl chloride (0.38 mmol). Flash purification (petrol
ether/EtOAc + 10% THF (0–50%)) afforded 62 mg (47%) of the product as a white solid. 1H NMR
(200 MHz, CDCl3) 8.39 (d, J = 2.3 Hz, 1H), 8.24 (d, J = 2.3 Hz, 1H), 8.22 (s, 1H), 7.71 (dd, J = 3.8, 1.1
Hz, 1H), 7.54 (dd, J = 5.0, 1.1 Hz, 1H), 7.36 (s, 1H), 7.10 (dd, J = 5.0, 3.7 Hz, 1H), 5.59 (s, 2H), 3.52 (t, 2H),
0.89 (t, J = 11.1, 2.2, 0.9 Hz, 2H), 161.4, 145.1, 139.5, 139.3,
0.07 (s, 9H). 13C NMR (50 MHz, CDCl3)
0.7. TLC-MS (ESI) m/z: 474.3 [M + Na]+
µL
µ
δ
−
δ
131.8, 129.6, 129.5, 128.6, 121.5, 120.6, 90.8, 73.8, 67.3, 18.5,
HPLC tret = 10.07 min.
−
N-(3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)furan-2-carboxamide (33d):
The preparation was carried out following General Procedure D with 100 mg 22 (0.29 mmol), 123 L Et3N
(0.88 mmol) and 44 L furane-2-carbonyl chloride (0.38 mmol). Flash purification (petrol ether/EtOAc
(40–50%)) afforded 101 mg (80%) of the product as a yellow oil. H NMR (200 MHz, CDCl3)
J = 2.4 Hz, 1H), 8.33 (d, J = 2.4 Hz, 1H), 8.29 (s, 1H), 7.51 (dd, J = 1.7, 0.8 Hz, 1H), 7.37 (s, 1H), 7.25 (dd,
J = 3.3, 1.1 Hz, 1H), 6.56 (dd, J = 3.6, 1.8 Hz, 1H), 5.61 (s, 2H), 3.52 (t, 2H), 0.89 (t, 2H), 0.07 (s, 9H).
µ
µ
1
δ
8.44 (d,
−
13C NMR (50 MHz, CDCl3)
δ 156.4, 147.5, 144.4, 144.3, 138.0, 128.5, 127.8, 119.9, 119.7, 115.4, 112.6, 89.9,
72.9, 66.4, 17.7, −1.5. TLC-MS (ESI) m/z: 458.1 [M + Na]+ HPLC tret = 9.92 min.
N-(3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide
(33e):
The preparation was carried out following General Procedure D with 100 mg 22 (0.29 mmol),
123
µ
L Et3N (0.88 mmol) and 68
µ
L benzoyl chloride (0.58 mmol). Flash purification (petrol
1
ether/EtOAc (30–60%)) afforded 91 mg (70%) of the product as a yellow solid. H NMR (200 MHz,
CDCl3)
δ
8.40 (s, 1H), 8.38 (s, 1H), 8.28 (s, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.49 (q, J = 13.4, 11.4 Hz, 3H),
0.07 (s, 9H). 13C NMR (50 MHz,
7.35 (s, 1H), 5.59 (s, 2H), 3.52 (t, J = 8.3 Hz, 2H), 0.89 (t, J = 8.0 Hz, 2H),
−
CDCl3)
δ 166.2, 144.3, 138.5, 134.3, 131.9, 129.0, 128.7, 127.8, 127.1, 120.5, 119.7, 89.9, 72.9, 66.4, 17.7,
−1.5. TLC-MS (ESI) m/z: 468.2 [M + Na]+ HPLC tret = 10.20 min.
N-(3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)acetamide
(33f):
The preparation was carried out following General Procedure D with 130 mg 22 (0.38 mmol),
160
µ
L Et3N (1.14 mmol) and 54
µ
L acetyl chloride (0.76 mmol). Flash purification (petrol ether/EtOAc
1
(30–80%)) afforded 134 mg (83%) of the product as a yellow solid. H NMR (200 MHz, CDCl3)
δ
8.25
(d, J = 2.3 Hz, 1H), 8.19 (s, 1H), 8.12 (d, J = 2.3 Hz, 1H), 7.32 (s, 1H), 5.56 (s, 2H), 3.59–3.40 (t, 2H), 2.19
(s, 3H), 0.99–0.73 (t, 2H), δ 169.4, 144.3, 138.4, 129.2, 127.9,
0.08 (s, 9H). 13C NMR (50 MHz, CDCl3)
−
120.4, 119.8, 90.0, 73.1, 66.6, 24.3, 17.8, −1.4. TLC-MS (ESI) m/z: 406.1 [M + Na]+ HPLC tret = 9.44 min.
N-(3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-2-cyclopentylacetamide (33g):
The preparation was carried out following General Procedure D with 100 mg 22 (0.29 mmol), 123
(0.88 mmol) and 79 L 2-cyclopentylacetyl chloride (0.58 mmol). Flash purification (petrol ether/EtOAc
(0–40%)) afforded 120 mg (91%) of the product as a brown oil. H NMR (200 MHz, CDCl3)
µL Et3N
µ
1
δ
8.30 (d,
J = 2.2 Hz, 1H), 8.27 (d, J = 2.1 Hz, 1H), 7.68–7.52 (m, 1H), 7.36 (s, 1H), 5.59 (s, 2H), 3.65–3.36 (m, 2H),
2.36 (d, J = 6.5 Hz, 2H), 2.23 (p, J = 7.3 Hz, 1H), 2.01–1.75 (m, 4H), 1.72–1.45 (m, 4H), 0.91 (td, J = 8.3,
7.7, 4.8 Hz, 2H), −0.06 (d, J = 2.5 Hz, 9H). TLC-MS (ESI) m/z: 474.2 [M + Na]+ HPLC tret = 11.32 min.
N-(3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-2-phenylacetamide (33h):
The preparation was carried out following General Procedure D with 100 mg 22 (0.29 mmol), 123
(0.88 mmol) and 77 L 2-phenylacetyl chloride (0.58 mmol). Flash purification (petrol ether/EtOAc
(40–70%)) afforded 111 mg (82%) of the product as a yellow oil. H NMR (200 MHz, CDCl3)
(d, J = 2.4 Hz, 1H), 8.11 (d, J = 2.4 Hz, 1H), 7.45 (d, J = 2.5 Hz, 1H), 7.41 (d, J = 3.4 Hz, 2H), 7.37 (d,
J = 2.1 Hz, 2H), 7.34 (s, 2H), 5.57 (s, 2H), 3.77 (s, 2H), 3.49 (t, 2H), 0.88 (t, J = 8.2 Hz, 2H), 0.07 (s, 9H).
µL Et3N
µ
1
δ
8.18
−
13C NMR (50 MHz, CDCl3)
δ 169.6, 144.4, 138.2, 134.3, 129.5, 129.2, 128.7, 127.7, 127.6, 120.1, 119.5, 89.8,
72.9, 66.4, 44.4, 17.7, −1.5. TLC-MS (ESI) m/z: 482.1 [M + Na]+ HPLC tret = 10.42 min.

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N-(3-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-2-(thiophen-2-yl)acetamide
33i): The preparation was carried out following General Procedure D with 100 mg 22 (0.29 mmol),
(
123
µ
L Et3N (0.88 mmol) and 72
µ
L 2-(thiophen-2-yl)acetyl chloride (0.58 mmol). Flash purification
1
(petrol ether/EtOAc (20–50%)) afforded 123 mg (90%) of the product as a brown oil. H NMR (200 MHz,
CDCl3)
δ 8.22 (d, J = 2.3 Hz, 1H), 8.12 (d, J = 2.3 Hz, 1H), 7.73 (s, 1H), 7.34 (s, 1H), 7.30 (dd, 1H), 7.05 (s,
1H), 7.03 (d, J = 1.6 Hz, 1H), 5.57 (s, 2H), 3.97 (s, 2H), 3.49 (t, 2H), 0.88 (t, 2H), −0.08 (s, 9H). 13C NMR
(50 MHz, CDCl3)
δ 168.6, 144.5, 138.4, 135.6, 128.7, 128.0, 127.9, 127.7, 126.1, 120.4, 119.8, 90.0, 73.1, 66.6,
38.3, 17.9, −1.3. TLC-MS (ESI) m/z: 488.1 [M + Na]+ HPLC tret = 10.15 min.
N-(3-(3-acrylamidophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)cyclopentane
carboxamide (34a): The preparation was performed following General Procedure A from 90 mg 33a
(0.21 mmol) and 64 mg 14 (0.24 mmol), catalyzed by 2 mg tBu3P Pd G3 (4 µmol) in 3.3 mL dioxane and
0.4 mL of a 1.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the crude
product was purified via flash chromatography (hexane/EtOAc (30–80%)). Yield: 76 mg (73%) as a
1
white solid. H NMR (200 MHz, DMSO)
δ 10.25 (s, 1H), 10.04 (s, 1H), 8.57 (d, J = 2.1 Hz, 1H), 8.49 (d,
J = 2.1 Hz, 1H), 8.02–7.95 (m, 1H), 7.93 (s, 1H), 7.66–7.56 (m, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.38–7.28 (m,
1H), 6.48 (dd, J = 16.9, 9.9 Hz, 1H), 6.28 (dd, J = 16.9, 2.2 Hz, 1H), 5.78 (dd, J = 9.9, 2.2 Hz, 1H), 5.65 (s,
2H), 3.56 (t, J = 8.0 Hz, 2H), 2.91–2.70 (m, 1H), 1.97–1.46 (m, 8H), 0.84 (t, J = 8.0 Hz, 2H),
−
0.10 (s, 9H).
13C NMR (50 MHz, DMSO)
δ
174.6, 163.3, 144.8, 139.6, 136.7, 134.8, 131.9, 130.6, 129.4, 127.3, 127.0,
121.8, 121.8, 118.6, 117.4, 117.3, 114.5, 72.6, 65.6, 45.1, 30.1, 25.7, 17.2, 1.4. TLC-MS (ESI) m/z: 527.1
−
[M + Na]+ HPLC tret = 10.24 min.
N-(3-(3-acrylamidophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)thiophene-2-
carboxamide (34c): The preparation was performed following General Procedure A from 90 mg 33c
(0.20 mmol) and 81 mg 14 (0.30 mmol), catalyzed by 3 mg tBu3P Pd G3 (5 µmol) in 5.0 mL dioxane and
1.2 mL of a 0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the crude
product was purified via flash chromatography (petrol ether/EtOAc + 10% THF (20–50%)). Yield:
1
46 mg (45%) as a white solid. H NMR (200 MHz, DMSO-d6)
δ 10.44 (s, 1H), 10.24 (s, 1H), 8.61 (s, 1H),
8.60 (s, 1H), 8.14–7.97 (m, 2H), 7.99 (s, 1H), 7.87 (d, J = 5.0 Hz, 1H), 7.63 (d, J = 7.4 Hz, 1H), 7.42 (d,
J = 8.1 Hz, 2H), 7.25 (t, J = 4.4 Hz, 1H), 6.48 (dd, J = 16.9, 9.9 Hz, 1H), 6.27 (dd, J = 16.8, 1.7 Hz, 1H),
5.77 (d, J = 10.0 Hz, 1H), 5.68 (s, 2H), 3.59 (t, J = 7.9 Hz, 2H), 0.86 (t, J = 7.9 Hz, 2H),
−
0.08 (s, 9H).
13C NMR (50 MHz, -CDCl3 + MeOD)
δ
164.8, 161.5, 145.6, 139.2, 138.7, 137.7, 134.7, 131.2, 131.1, 129.5,
129.2, 128.9, 127.9, 127.4, 126.1, 122.4, 121.5, 118.5, 118.3, 118.0, 115.9, 73.2, 66.4, 17.7, 1.7 TLC-MS (ESI)
−
m/z: 541.5 [M + Na]+ HPLC tret = 9.48 min.
N-(3-(3-acrylamidophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)furan-2-
carboxamide (34d): The preparation was performed following General Procedure A from 101 mg 33d
(0.23 mmol) and 95 mg 14 (0.35 mmol), catalyzed by 4 mg tBu3P Pd G3 (7 µmol) in 6.0 mL dioxane
and 1.4 mL of a 0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the
crude product was purified via flash chromatography (petrol ether/EtOAc + 10% MeOH (40–60%)).
1
Yield: 58 mg (50%) as a white solid. H NMR (200 MHz, CDCl3 + MeOD)
δ
9.38 (s, 1H), 9.19 (s, 1H),
8.63 (d, J = 2.3 Hz, 1H), 8.46 (d, J = 2.3 Hz, 1H), 7.85 (s, 1H), 7.65 (d, J = 7.0 Hz, 1H), 7.59–7.46 (m,
2H), 7.40–7.23 (m, 2H), 7.22 (dd, J = 3.5, 0.8 Hz, 1H), 6.51 (dd, J = 3.5, 1.8 Hz, 1H), 6.38 (s, 1H), 6.35
(d, J = 3.3 Hz, 1H), 5.69 (dd, J = 7.5, 4.3 Hz, 1H), 5.60 (s, 2H), 3.62–3.43 (m, 2H), 0.96–0.78 (m, 2H),
−
0.12 (s, 9H). 13C NMR (50 MHz, CDCl3 + MeOD)
δ 222.3, 221.4, 164.6, 147.5, 145.9, 144.9, 138.9, 137.4,
134.8, 131.3, 129.6, 128.4, 127.5, 126.3, 122.4, 121.2, 118.5, 118.2, 116.0, 115.5, 112.5, 73.2, 66.5, 17.8, 1.5.
−
TLC-MS (ESI) m/z: 525.4 [M + Na]+ HPLC tret = 9.34 min.
N-(3-(3-acrylamidophenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide
34e): The preparation was performed following General Procedure A from 91 mg 33e (0.20 mmol) and
84 mg 14 (0.31 mmol), catalyzed by 4 mg tBu3P Pd G3 (7 mol) in 5.0 mL dioxane and 1.2 mL of a
0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the crude product was
(
µ

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purified via flash chromatography (petrol ether/EtOAc + 10% MeOH (20–60%)). Yield: 47 mg (45%) as
1
a white solid. H NMR (200 MHz, CDCl3 + MeOD)
δ 9.65 (s, 1H), 9.47 (s, 1H), 8.57 (d, J = 2.1 Hz, 1H),
8.49 (d, J = 2.1 Hz, 1H), 7.99–7.81 (m, 3H), 7.63–7.25 (m, 6H), 7.23 (s, 1H), 6.32 (s, 1H), 6.29 (d, J = 1.5
Hz, 1H), 5.63 (dd, J = 6.6, 5.1 Hz, 1H), 5.55 (s, 2H), 3.47 (t, 2H), 0.81 (t, J = 8.3 Hz, 2H), 0.17 (s, 9H).
13C NMR (50 MHz, CDCl3 + MeOD)
224.0, 167.3, 164.7, 145.7, 138.8, 137.7, 134.8, 134.5, 131.9, 131.2,
−
δ
129.5, 129.4, 128.6, 127.5, 126.1, 122.3, 121.5, 118.5, 118.3, 118.0, 116.0, 73.2, 66.5, 17.8, 1.6. TLC-MS
−
(ESI) m/z: 535.1 [M + Na]+ HPLC tret = 9.77 min.
N-(3-(5-acetamido-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)acrylamide (34f):
The preparation was performed following General Procedure A from 121 mg 33f (0.32 mmol) and 129 mg
14 (0.47 mmol), catalyzed by 6 mg tBu3P Pd G3 (9 µmol) in 6.0 mL dioxane and 1.9 mL of a 0.5 M K2CO3
solution. The reaction was conducted at ambient temperature and the crude product was purified via
1
flash chromatography (DCM/MeOH (0–5%)). Yield: 90 mg (63%) as a white solid. H NMR (200 MHz,
CDCl3 + MeOD)
J = 6.9, 3.8 Hz, 2H), 6.57 (dt, J = 28.2, 8.8 Hz, 1H), 6.14 (s, 1H), 6.11 (s, 1H), 5.45 (t, J = 5.8 Hz, 1H), 5.32
(s, 2H), 3.26 (t, J = 8.1 Hz, 2H), 1.90 (s, 3H), 0.60 (t, J = 8.2 Hz, 2H),
0.37 (s, 9H). 13C NMR (50 MHz,
CDCl3 + MeOD) 170.1, 164.7, 145.4, 138.6, 137.0, 134.7, 131.2, 129.5, 129.3, 127.2, 126.0, 122.2, 120.5,
δ 9.44 (s, 1H), 8.23 (s, 1H), 8.19 (s, 1H), 7.75 (s, 1H), 7.24 (d, J = 9.3 Hz, 2H), 7.04 (dd,
−
δ
118.4, 118.3, 117.8, 115.7, 73.1, 66.3, 23.4, 17.6,
9.18 min.
−
1.7. TLC-MS (ESI) m/z: 473.1 [M + Na]+ HPLC tret
=
N-(3-(5-(2-cyclopentylacetamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)
acrylamide (34g): The preparation was performed following General Procedure A from 120 mg 33g
(0.27 mmol) and 109 mg 14 (0.40 mmol), catalyzed by 5 mg tBu3P Pd G3 (8 µmol) in 6.0 mL dioxane
and 1.6 mL of a 0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the
crude product was purified via flash chromatography (DCM/MeOH (0–5%)). Yield: 85 mg (62%) as a
1
white solid. H NMR (200 MHz, DMSO-d6)
δ 10.26 (s, 1H), 10.04 (s, 1H), 8.54 (s, 1H), 8.47 (s, 1H), 8.01
(s, 1H), 7.94 (s, 1H), 7.58 (d, J = 6.7 Hz, 1H), 7.53–7.34 (m, 2H), 6.68–6.37 (m, 1H), 6.27 (d, J = 17.8 Hz,
1H), 5.78 (d, J = 12.2 Hz, 1H), 5.65 (s, 2H), 4.12 (t, 2H), 2.34 (s, 2H), 1.67 (d, J = 38.3 Hz, 5H), 1.23 (s, 4H),
0.84 (t, 2H), δ 172.8, 164.7, 145.3, 138.8, 136.7, 134.8,
0.10 (s, 9H). 13C NMR (50 MHz, CDCl3 + MeOD)
−
131.3, 129.7, 129.5, 127.4, 126.0, 122.2, 120.5, 118.6, 118.3, 117.9, 115.8, 73.2, 66.4, 43.2, 37.2, 32.5, 24.9,
17.8, −1.6. TLC-MS (ESI) m/z: 541.5 [M + Na]+ HPLC tret = 10.60 min.
N-(3-(5-(2-phenylacetamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)
acrylamide (34h): The preparation was performed following General Procedure A from 111 mg 33h
(0.24 mmol) and 98 mg 14 (0.36 mmol), catalyzed by 4 mg tBu3P Pd G3 (7 µmol) in 5.0 mL dioxane and
1.4 mL of a 0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the crude
product was purified via flash chromatography (petrol ether/EtOAc + 10% MeOH (20–60%)). Yield:
1
59 mg (47%) as a white solid. H NMR (200 MHz, CDCl3 + MeOD)
δ 9.79 (s, 1H), 9.72 (s, 1H), 8.53 (d,
J = 2.3 Hz, 1H), 8.37 (s, 1H), 7.85 (s, 1H), 7.50 (s, 1H), 7.47 (s, 1H), 7.31 (d, J = 5.3 Hz, 1H), 7.28–7.18 (m,
2H), 7.24–7.07 (m, 2H), 7.10–6.81 (m, 1H), 6.32 (s, 1H), 6.29 (s, 1H), 6.24 (d, J = 5.8 Hz, 1H), 5.62 (dt,
J = 11.8, 6.3 Hz, 1H), 5.54 (s, 2H), 4.22 (s, 2H), 3.44 (t, 2H), 0.78 (t, 2H),
−
0.19 (s, 9H). 13C NMR (50 MHz,
CDCl3 + MeOD) 170.8, 164.8, 144.9, 138.6, 136.2, 134.8, 134.5, 131.1, 129.6, 129.3, 129.0, 128.5, 127.1,
δ
126.9, 126.2, 122.3, 120.8, 118.7, 118.3, 118.0, 115.9, 73.2, 66.3, 43.6, 17.6, 1.9. TLC-MS (ESI) m/z: 549.1
−
[M + Na]+ HPLC tret = 10.02 min.
N-(3-(5-(2-(thiophen-2-yl)acetamido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)
phenyl)acrylamide (34i): The preparation was performed following General Procedure A from 123 mg
33i (0.26 mmol) and 108 mg 14 (0.39 mmol), catalyzed by 5 mg tBu3P Pd G3 (8 µmol) in 6.0 mL dioxane
and 1.6 mL of a 0.5 M K2CO3 solution. The reaction was conducted at ambient temperature and the
crude product was purified via flash chromatography (DCM/MeOH (0–5%)). Yield: 87 mg (62%) as a
1
white solid. H NMR (200 MHz, CDCl3)
δ 8.62 (d, J = 2.3 Hz, 1H), 8.49 (d, J = 2.3 Hz, 1H), 8.35 (s, 1H),
8.23 (s, 1H), 8.05 (s, 1H), 7.73 (s, 1H), 7.68 (s, 1H), 7.52 (d, J = 3.3 Hz, 4H), 7.28 (s, 1H), 6.69 (dd, J = 16.6,

Int. J. Mol. Sci. 2020, 21, 9269
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1.8 Hz, 1H), 6.55 (dd, J = 16.7, 9.4 Hz, 1H), 5.97 (dd, J = 9.5, 1.8 Hz, 1H), 5.84 (s, 2H), 4.21 (s, 2H), 3.79 (t,
J = 8.1 Hz, 2H), 1.16 (t, J = 8.2 Hz, 2H), 0.19 (s, 9H). 13C NMR (50 MHz, CDCl3)
168.8, 146.0, 142.7,
δ
141.4, 138.3, 137.7, 135.6, 134.9, 129.4, 128.2, 127.6, 127.4, 126.2, 125.8, 121.2, 118.2, 118.0, 115.9, 92.4, 84.8,
84.3, 73.0, 66.3, 38.0, 17.7, −1.5. TLC-MS (ESI) m/z: 555.1 [M + Na]+ HPLC tret = 9.71 min.
N-(3-(3-acrylamidophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)cyclopentanecarboxamide (12a): The preparation
was carried out following General Procedure B starting from 70 mg 34a (0.14 mmol) in 5 mL DCM and
2 mL TFA. Flash purification (DCM/MeOH (2–8%)) afforded 40 mg (77%) of the final compound as a
1
white solid. H NMR (200 MHz, DMSO)
δ 11.84 (br s, 1H), 10.24 (s, 1H), 9.97 (s, 1H), 8.62–8.47 (m, 1H),
8.47–8.32 (m, 1H), 7.93 (s, 1H), 7.75 (d, J = 2.2 Hz, 1H), 7.68–7.54 (m, 1H), 7.49–7.26 (m, 2H), 6.49 (dd,
J = 16.9, 9.9 Hz, 1H), 6.28 (dd, J = 16.9, 1.6 Hz, 1H), 5.78 (dd, J = 9.9, 1.6 Hz, 1H), 2.91–2.71 (m, 1H),
1.99–1.46 (m, 8H). 13C NMR (50 MHz, DMSO)
126.9, 124.4, 121.7, 118.3, 117.3, 117.0, 116.7, 114.3, 45.1, 30.2, 25.8. TLC-MS (ESI) m/z: 397.0 [M + Na
δ
174.5, 163.3, 145.7, 139.5, 136.8, 135.6, 132.0, 129.7, 129.3,
+
]
HPLC tret = 7.38 min.
N-(3-(3-acrylamidophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)thiophene-2-carboxamide (12c): The preparation
was carried out following General Procedure B starting from 46 mg 34c (0.09 mmol) in 4.2 mL DCM and
1.8 mL TFA. Flash purification (DCM/MeOH (4–10%)) afforded 15 mg (44%) of the final compound as a
1
white solid. H NMR (200 MHz, DMSO-d6)
δ 11.94 (s, 1H), 10.42 (s, 1H), 10.27 (s, 1H), 8.55 (s, 2H), 8.06
(d, J = 3.7 Hz, 1H), 7.97 (s, 1H), 7.87 (d, J = 5.1 Hz, 1H), 7.82 (s, 1H), 7.65 (dd, J = 5.8, 2.1 Hz, 1H), 7.41 (s,
1H), 7.39 (s, 1H), 7.25 (t, J = 4.4 Hz, 1H), 6.49 (dd, J = 16.9, 9.8 Hz, 1H), 6.27 (dd, J = 17.0, 1.8 Hz, 1H),
5.77 (dd, J = 9.8, 2.2 Hz, 1H). 13C NMR (50 MHz, DMSO-d6)
δ 163.2, 160.1, 146.2, 139.9, 139.5, 138.0,
135.4, 131.9, 131.7, 129.3, 129.0, 128.7, 128.1, 126.9, 124.6, 121.6, 120.2, 117.2, 116.9, 116.7, 114.3. TLC-MS
(ESI) m/z: 411.3 [M + Na]+. HRMS ESI-TOF [M + H]+ m/z calcd. for C21H16N4O2S: 389.1067, found:
389.1072. HPLC tret = 6.44 min.
N-(3-(3-acrylamidophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)furan-2-carboxamide (12d): The preparation was
carried out following General Procedure B starting from 58 mg 34d (0.12 mmol) in 4.2 mL DCM and
1.8 mL TFA. Flash purification (DCM/MeOH (4–10%)) afforded 23 mg (54%) of the final compound as a
1
white solid. H NMR (200 MHz, DMSO-d6)
δ 11.92 (s, 1H), 10.34 (s, 1H), 10.22 (s, 1H), 8.57 (s, 2H), 7.96
(s, 2H), 7.86–7.74 (m, 1H), 7.62 (d, J = 5.3 Hz, 1H), 7.50–7.33 (m, 2H), 7.33 (d, J = 3.5 Hz, 1H), 6.72 (dd,
J = 3.2, 1.4 Hz, 1H), 6.48 (dd, J = 17.0, 9.8 Hz, 1H), 6.29 (dd, 1H), 5.77 (dd, J = 9.6, 2.0 Hz, 1H). 13C NMR
(50 MHz, DMSO-d6)
δ 163.2, 156.5, 147.6, 146.2, 145.6, 139.5, 138.0, 135.4, 131.9, 129.3, 128.4, 126.9, 124.5,
121.6, 120.2, 117.2, 116.9, 116.7, 114.6, 114.2, 112.1. TLC-MS (ESI) m/z: 395.2 [M + Na]+. HRMS ESI-TOF
[M + H]+ m/z calcd. for C21H16N4O3: 373.1295, found: 373.1300. HPLC tret = 5.88 min
N-(3-(3-acrylamidophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (12e): The preparation was carried out
following General Procedure B starting from 47 mg 34e (0.09 mmol) in 4.2 mL DCM and 1.8 mL TFA.
Flash purification (DCM/MeOH (4–10%)) afforded 10 mg (29%) of the final compound as a white solid.
1H NMR (400 MHz, DMSO-d6)
δ 11.92 (d, 1H), 10.39 (s, 1H), 10.23 (s, 1H), 8.62 (d, J = 2.3 Hz, 1H), 8.59
(d, J = 2.2 Hz, 1H), 8.03 (s, 1H), 8.01 (d, J = 1.6 Hz, 1H), 7.96 (s, 1H), 7.81 (d, J = 2.6 Hz, 1H), 7.66–7.61
(m, 1H), 7.60 (d, J = 6.7 Hz, 1H), 7.55 (t, J = 7.2 Hz, 2H), 7.41 (d, J = 6.5 Hz, 2H), 6.47 (dd, J = 16.9, 10.1
Hz, 1H), 6.27 (dd, J = 17.0, 2.1 Hz, 1H), 5.76 (dd, J = 10.0, 2.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
δ
165.5, 163.2, 146.1, 139.5, 138.0, 135.4, 134.6, 131.9, 131.5, 129.2, 129.1, 128.3, 127.5, 126.7, 124.4, 121.6,
120.0, 117.2, 116.9, 116.6, 114.3. TLC-MS (ESI) m/z: 405.3 [M + Na]+. HRMS ESI-TOF [M + H]+ m/z
calcd. for C23H18N4O2: 383.1503, found: 383.1509. HPLC tret = 6.74 min.
N-(3-(5-acetamido-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)acrylamide (12f): The preparation was carried out
following General Procedure B starting from 90 mg 34f (0.20 mmol) in 4.2 mL DCM and 1.8 mL TFA.
Flash purification (DCM/MeOH (10–16%)) afforded 26 mg (41%) of the final compound as a white
1
solid. H NMR (200 MHz, DMSO-d6)
δ
11.85 (s, 1H), 10.22 (s, 1H), 10.03 (s, 1H), 8.46 (s, 1H), 8.39 (s,
1H), 7.95 (s, 1H), 7.76 (s, 1H), 7.60 (d, J = 7.3 Hz, 1H), 7.38 (q, J = 7.7 Hz, 2H), 6.49 (dd, J = 16.9, 9.8 Hz,
1H), 6.28 (d, J = 16.5 Hz, 1H), 5.78 (d, J = 9.7 Hz, 1H), 2.08 (s, 3H). 13C NMR (50 MHz, DMSO-d6)
δ

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168.3, 163.2, 145.8, 139.5, 136.8, 135.5, 131.9, 129.4, 129.3, 126.9, 124.4, 121.6, 118.4, 117.2, 116.9, 116.7,
114.2, 23.7. TLC-MS (ESI) m/z: 343.1 [M + Na]+. HRMS ESI-TOF [M + H]+ m/z calcd. for C18H16N4O2:
321.1346, found: 321.1352. HPLC tret = 4.74 min.
N-(3-(5-(2-cyclopentylacetamido)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)acrylamide (12g): The preparation
was carried out following General Procedure B starting from 85 mg 34g (0.16 mmol) in 7 mL DCM and
3 mL TFA. Flash purification (DCM/MeOH (5–6%)) afforded 27 mg (42%) of the final compound as a
1
white solid. H NMR (400 MHz, DMSO-d6)
δ
11.84 (s, 1H), 10.22 (s, 1H), 9.94 (s, 1H), 8.49 (d, J = 2.2 Hz,
1H), 8.42 (d, J = 2.2 Hz, 1H), 7.96 (s, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.41 (t, J =
7.8 Hz, 1H), 7.35 (d, J = 7.7 Hz, 1H), 6.48 (dd, J = 16.9, 10.1 Hz, 1H), 6.29 (dd, J = 17.0, 2.0 Hz, 1H),
5.78 (dd, J = 10.0, 2.0 Hz, 1H), 2.35 (s, 1H), 2.33 (s, 1H), 2.27 (dt, J = 15.1, 7.4 Hz, 1H), 1.78 (dq, J = 11.8,
6.2 Hz, 2H), 1.62 (pd, J = 9.0, 8.3, 5.0 Hz, 2H), 1.59–1.45 (m, 2H), 1.22 (dq, J = 11.5, 7.3 Hz, 2H). 13
C
NMR (101 MHz, DMSO-d6)
δ 171.0, 163.3, 145.9, 139.6, 137.0, 135.7, 132.1, 129.6, 129.3, 126.9, 124.4,
121.8, 118.6, 117.5, 117.1, 116.8, 114.4, 42.5, 36.8, 32.1, 24.7. TLC-MS (ESI) m/z: 411.4 [M + Na]+. HRMS
ESI-TOF [M + H]+ m/z calcd. for C23H24N4O2: 389.1972, found: 389.1977. HPLC tret = 7.85 min.
N-(3-(5-(2-phenylacetamido)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)acrylamide (12h): The preparation was
carried out following General Procedure B starting from 59 mg 34h (0.11 mmol) in 4.2 mL DCM and
1.8 mL TFA. Flash purification (DCM/MeOH (4–10%)) afforded 15 mg (34%) of the final compound
1
as a white solid. H NMR (400 MHz, DMSO-d6)
δ 11.86 (s, 1H), 10.28 (s, 1H), 10.22 (s, 1H), 8.51 (d,
J = 2.3 Hz, 1H), 8.43 (d, J = 2.3 Hz, 1H), 7.93 (t, J = 1.9 Hz, 1H), 7.76 (d, J = 2.3 Hz, 1H), 7.60 (dt, J = 8.1,
1.6 Hz, 1H), 7.36 (ddt, J = 14.8, 12.3, 7.7 Hz, 6H), 7.28–7.22 (m, 1H), 6.47 (dd, J = 17.0, 10.1 Hz, 1H), 6.28
(dd, J = 17.0, 2.1 Hz, 1H), 5.77 (dd, J = 10.1, 2.1 Hz, 1H), 3.68 (s, 2H). 13C NMR (101 MHz, DMSO-d6)
δ
169.0, 163.1, 145.8, 139.4, 136.6, 135.9, 135.4, 131.9, 129.4, 129.1, 129.1, 128.2, 126.7, 126.4, 124.3, 121.6,
118.3, 117.2, 116.9, 116.6, 114.2, 43.0. TLC-MS (ESI) m/z: 419.4 [M + Na]+. HRMS ESI-TOF [M + H]+
m/z calcd. for C24H20N4O2: 397.1659, found: 397.1662. HPLC tret = 6.98 min.
N-(3-(5-(2-(thiophen-2-yl)acetamido)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)acrylamide (12i): The preparation
was carried out following General Procedure B starting from 87 mg 34i (0.16 mmol) in 4.2 mL DCM and
1.8 mL TFA. Flash purification (DCM/MeOH (6–8%)) afforded 15 mg (34%) of the final compound as a
1
white solid. H NMR (200 MHz, DMSO-d6)
δ 11.88 (s, 1H), 10.33 (s, 1H), 10.23 (s, 1H), 8.49 (s, 1H), 8.43
(s, 1H), 7.94 (s, 1H), 7.77 (s, 1H), 7.61 (d, J = 6.8 Hz, 1H), 7.38 (d, J = 7.9 Hz, 3H), 7.01 (s, 2H), 6.48 (dd,
1H), 6.27 (d, J = 17.2 Hz, 1H), 5.78 (d, J = 9.6 Hz, 1H), 3.92 (s, 2H). 13C NMR (50 MHz, DMSO-d6)
δ
168.1, 163.2, 145.9, 139.5, 137.1, 136.6, 135.4, 131.9, 129.2, 129.2, 126.9, 126.6, 126.3, 125.0, 124.5, 121.6,
118.4, 117.2, 116.9, 116.7, 114.2, 37.2. TLC-MS (ESI) m/z: 425.2 [M + Na]+. HRMS ESI-TOF [M + H]+
m/z calcd. for C22H18N4O2S: 403.1223, found: 403.1227. HPLC tret = 6.71 min.
N-(3-(1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)propionamide (9b): To a solution of 17 mg 9a (0.07 mmol)
in THF/MeOH (6 mL each) were added 8 mg Pd/C and the reaction was purged with hydrogen.
The mixture was then stirred under hydrogen until HPLC indicated complete conversion. The catalyst
was filtered off, the filtrate evaporated and the residue purified via flash chromatography (DCM/MeOH
1
(4–8%)). Yield: 12 mg (69%) as a white solid. H NMR (200 MHz, DMSO)
δ
11.89 (br s, 1H), 9.90 (s, 1H),
8.37–8.19 (m, 2H), 8.10–7.98 (m, 1H), 7.87–7.75 (m, 1H), 7.56–7.41 (m, 1H), 7.41–7.28 (m, 2H), 7.25–7.06
(m, 1H), 2.35 (q, J = 7.4 Hz, 2H), 1.11 (t, J = 7.4 Hz, 3H) 13C NMR (50 MHz, DMSO)
δ
172.1, 149.1, 142.9,
139.8, 135.3, 129.1, 127.4, 123.6, 120.8, 117.2, 116.8, 116.3, 116.0, 114.2, 29.6, 9.7 TLC-MS (ESI) m/z: 266.4
[M + H]+ HPLC tret = 5.47 min.
3-(3-propionamidophenyl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (10b): To a solution of 25 mg 10a
(0.08 mmol) in THF/MeOH (5 mL each) were added 8 mg Pd/C and the reaction was purged with
hydrogen. The mixture was then stirred under hydrogen until HPLC indicated complete conversion.
The catalyst was filtered off, the filtrate evaporated and the residue purified via flash chromatography
1
(DCM/MeOH (6–16%)). Yield: 15 mg (60%) as a white solid. H NMR (400 MHz, DMSO)
δ
12.15 (s,
1H), 9.95 (s, 1H), 8.81 (s, J = 24.3 Hz, 1H), 8.75 (s, 1H), 8.10 (br s, 1H), 7.88 (d, J = 19.3 Hz, 2H), 7.61 (d,