Comprehensive structure-activity-relationship of azaindoles as highly potent FLT3 inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.01.006

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.

Full text of DOI:10.1016/j.bmc.2019.01.006

Accepted Manuscript
Comprehensive structure-activity-relationship of azaindoles as highly potent
FLT3 inhibitors
Sebastian H. Grimm, Berend Gagestein, Jordi F. Keijzer, Nora Liu, Ruud H.
Wijdeven, Eelke B. Lenselink, Adriaan W. Tuin, Adrianus M.C.H. van den
Nieuwendijk, Gerard J.P. van Westen, Constant A.A. van Boeckel, Herman S.
Overkleeft, Jacques Neefjes, Mario van der Stelt
PII:
S0968-0896(18)31968-0
https://doi.org/10.1016/j.bmc.2019.01.006
BMC 14698
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
25 November 2018
8 January 2019
10 January 2019
Please cite this article as: Grimm, S.H., Gagestein, B., Keijzer, J.F., Liu, N., Wijdeven, R.H., Lenselink, E.B., Tuin,
A.W., van den Nieuwendijk, A.M.C., van Westen, G.J.P., van Boeckel, C.A.A., Overkleeft, H.S., Neefjes, J., van
der Stelt, M., Comprehensive structure-activity-relationship of azaindoles as highly potent FLT3 inhibitors,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Comprehensive structure-activity-relationship
of azaindoles as highly potent FLT3 inhibitors
a
a
a
a
Sebastian H. Grimm , Berend Gagestein , Jordi F. Keijzer , Nora Liu , Ruud H.
b
c
d
Wijdeven , Eelke B. Lenselink , Adriaan W. Tuin , Adrianus M. C. H. van den
Nieuwendijk , Gerard J. P. van Westen , Constant A. A. van Boeckel , Herman S.
Overkleeft , Jacques Neefjes , Mario van der Stelt .
d
c
e
d
b
a,*
a
Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands
Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands
Computational Drug Discovery, Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden
b
c
University, Leiden, the Netherlands
d
Department of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands
Pivot Park Screening Center, Oss, the Netherlands
e
*
Correspondence: m.van.der.stelt@chem.leidenuniv.nl
KEYWORDS
H-89 analogs, Fms-like tyrosine kinase 3 (FLT3), acute myeloid leukemia (AML), inhibitors
Abstract
Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in
which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver
mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown
emergence of drug resistant mutants, emphasizing the ongoing need for new chemical
matter to enable the treatment of this disease. Here, we present the discovery and
topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide
H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was
not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair
analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel
FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an
alternative, flipped binding orientation of the new inhibitors.
1. Introduction
Acute myeloid leukemia (AML) is a cancer of the blood and bone marrow that is
characterized by a failure in differentiation of stem cells during hematopoiesis, resulting in
flooding of the bloodstream with immature myeloid blood cells. These blast cells fatally
disrupt normal hematopoietic function and their abundance in blood obstruct the normal

1
,2
flow in capillaries resulting in a high mortality. While in younger patients cure rates can
reach up to 35-40%, elderly patients, who are often unable to cope with the intensive
3
chemotherapy regimen, do not experience this benefit. AML is a genetically diverse disease,
but in 20-30% of patients an internal tandem duplication (ITD) in the juxtamembrane
domain of the Fms-like tyrosine kinase 3 (FLT3) receptor has been identified as a driver
4,5
mutation. The validation of FLT3 as a drug target led to clinical development of several
small molecule inhibitors, culminating in the recent FDA approval of midostaurin for
6
–9
treatment of FLT3-dependent AML in conjunction with standard treatment. Although the
initial response to treatment with FLT3 inhibitors shows therapeutic promise, many AML
1
0–12
patients relapse due to the emergence of drug-resistant cancer cells.
Resistance-
inducing mutations have thus far been observed in treatments with several FLT3 inhibitors,
1
2–14
among which the highly potent experimental drug quizartinib.
The discovery of new
chemical entities to target FLT3 represents, therefore, a medical need.
15
Figure 1: FLT3 screening hits (1-4) from an H-89 library. Data represent residual in vitro FLT3 activity
at 2 µM.
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) is a prototypical and
intensely-studied kinase inhibitor (Figure 1A). It was one of the first non-natural, synthetic
inhibitors that competitively inhibited the binding of ATP to the structurally conserved
1
6,17
binding domain of cAMP-dependent protein kinase (PKA).
The binding mode of H-89 to
1
8
PKA has been studied in great detail at the atomic level using crystallization studies. This
contributed to the understanding of kinase function and provided general principles to
develop drug-like kinase inhibitors. The isoquinoline sulfonamide mimics the binding mode
of adenosine. The nitrogen of the isoquinoline ring forms a crucial H-bond bridge to the
1
8
backbone of Val-123, located in the hinge region of PKA. This binding mode of H-89 is not
specific to PKA, but has also been observed with Haspin, as shown in structural data (PDB:
3
FMD). Furthermore, H-89 activity has been shown for several other kinases, including S6K1,
1
9,20
MSK1 and ROCK-II.
Consequently, H-89 is used as a starting point in several drug
discovery programs. For example, this lab has previously described the use of H-89 and its
analogs as RAC-alpha serine/threonine-protein kinase (AKT1) inhibitors to combat bacterial
1
5,21
infections, such as Salmonella typhimurium and Mycobacterium tuberculosis.
During the

hit optimization program of H-89 analogs as AKT1 inhibitors, four compounds (1-4) were
1
5
identified that demonstrated substantial activity against FLT3 (Figure 1B). In this study the
optimization and structure-activity relationships of H-89-derived compounds as new FLT3
inhibitors is presented.
2. Results and Discussion
To confirm the structure and activity of compound 1, the synthesis was commenced with the
commercially available building blocks as outlined in Scheme 1. After methylation and
reduction, the resulting alcohol was exchanged for a chlorine and a trityl protected
ethylenediamine linker was introduced via nucleophilic substitution. Subsequent Boc-
protection, Suzuki-coupling with 3-pyridinylboronic acid and trityl-deprotection yielded the
primary amine, which could be coupled with isoquinoline sulfonyl chloride to provide the
desired product 1. The activity of compound 1 was confirmed in a biochemical assay using
purified, recombinantly expressed human FLT3 with a time-resolved fluorescence resonance
energy transfer (FRET) method. Compound 1 showed potent inhibition with a half maximum
inhibitory concentration (IC ) in the low nanomolar range (pIC = 8.02 ± 0.05), which was
5
0
50
comparable to the inhibitory activity of the reference inhibitor quizartinib (pIC = 8.30 ±
5
0
0
.07). Compound 1 demonstrated favorable physico-chemical properties with a molecular
2
2
weight (MW) of 445 and a logD (pH 7.4) of 1.5. This resulted in a lipophilic efficiency (LipE =
pIC - logD) of 6.5. In summary, compound 1 was defined as an excellent starting point to
2
3
5
0
develop new FLT3 inhibitors.
a
Scheme 1: Synthetic route towards the derivatives 1, 5-16.
a
Reagents and conditions: (a) K CO , dimethyl sulfate, ACN, 80°C, overnight; (b) DIBAL-H, toluene, -80
2
3
–
0°C; (c) SOCl , DCM, RT; (d) 60, K CO , ACN, 70°C, 2 h; (e) TrtCl, K CO , RT, 40 min; (f) NaHCO ,
2
2
3
2
3
3
Boc O, THF, RT, overnight; (g) 3-pyridinylboronic acid, Pd(PPh ) , K CO , DCM/DMF, 85°C, 6 h; (h) TFA,
2
3 4
2
3
TES, DCM 0°C – RT, 5 h; (i) heteroaryl-bromide, K S O , HCOONa, Pd(OAc) , PPh , 1,10-
2
2
5
2
3
phenanthroline, DMSO then DiPEA, 63, NBS, THF, 0°C – RT, 1 h; (j) TFA, CHCl , 1 h; (k) aryl-
3
sulfonylchloride, Et N, DCM/DMF, 0°C – RT.
3
A topological exploration of the structure-activity relationship of isoquinolinesulfonamides
1
8
was employed guided by the observed binding mode of H-89 in other kinases. First, the

isoquinoline substituent was replaced by various other hinge binding moieties inspired by
2
4–27
kinase drugs, including indolones (sunitinib and nintedanib),
aminoisoquinolines
2
4,28,29
24,30
24,31,32
(crizotinib and palbociclib),
indazoles (axitinib)
and picolinamides (sorafenib).
The analogs (5-16) were synthesized in a similar manner as compound 1 using a palladium-
catalyzed sulfination of heteroaryl halides and subsequent coupling with the primary amine
3
3
as shown in Scheme 1. Interestingly, compounds 5-12 displayed similar or slightly weaker
activity compared to compound 1 with a range of pIC s between 7.6 and 8.0 (Table 1).
5
0
Indazole 6 was the most potent compound of the series with a pIC of 8.01 ± 0.02.
5
0
Table 1: In vitro FLT3 activity and lipophilic efficiency (LipE) of compounds 1 – 18.
R =
Entry
1
pIC ± SEM LipE
Entry
11
pIC ± SEM LipE
5
0
50
8.02 ± 0.05 6.5
7.70 ± 0.11 7.0
7.71 ± 0.10 6.4
7.62 ± 0.16 6.3
5
6
7
8
9
12
13
14
15
16
8.01 ± 0.08 6.6
7.77 ± 0.09 6.6
7.74 ± 0.11 7.0
7.32 ± 0.12 6.6
7.21 ± 0.14 4.6
6.19 ± 0.15 6.2
8.07 ± 0.07 6.6
7.57 ± 0.18 6.6
1
0
7.86 ± 0.10 7.6
Entry
pIC ± SEM LipE
5
0
1
7
< 5
n.a.

1
8
< 5
n.a.
Moreover, substantially more polar groups such as picolinamide were well tolerated (as
observed in compound 10), resulting in a high lipE of 7.6. Surprisingly, the nitrogen atom,
which plays an important role in the hinge binding to other kinases, was not required for
activity. Compounds 13 and 14 retained activity with a pIC of 7.21 ± 0.34 and 6.19 ± 0.15,
5
0
respectively. The same was true for the nitro and amino phenyl derivatives 15 and 16. All
together, these results suggested that the binding orientation of the
isoquinolinesulfonamides might be different than the one of H-89 in PKA. It was envisioned
that the nitrogen atom of the pyridyl ring could act as a potential H-bond acceptor to
interact with the hinge region, which may potentially explain the activity of compounds 13-
1
6. To test this hypothesis compounds (17-18), in which the pyridine ring was substituted for
a carbacycle, were synthesized (SI Scheme 1). The pIC of these novel derivatives dropped
5
0
to < 5 (Table 1). This suggested that the nitrogen in the pyridine is indeed important for the
interaction with FLT3 and the isoquinolinesulfonamide may have a flipped binding
orientation in the ATP-pocket of FLT3 compared to PKA.
Table 2: FLT3 activity and LipE of compounds 19 – 31.
1
2
R =
R =
Entry
pIC ± SEM LipE
5
0
1
2
2
2
2
2
2
2
2
9
0
1
2
3
4
5
6
7
6
6
6
6
6
8
7
6
7
.74 ± 0.26 5.0
.87 ± 0.20 4.6
.90 ± 0.19 4.3
.57 ± 0.21 5.4
.80 ± 0.17 5.6
.08 ± 0.09 5.3
.49 ± 0.14 5.3
.77 ± 0.17 2.2
.32 ± 0.15 5.5

2
2
3
8
9
0
7
8
.41 ± 0.13 4.3
.05 ± 0.07 6.4
6
<
.25 ± 0.21 3.7
3
1
5
n.a.
To further understand the SAR of our chemical series, the importance of the linker between
the isoquinoline and the pyridyl moieties was investigated (19-31). The results from this
study are summarized in Table 2. The synthetic schemes for these compounds (19-31) are
shown in the SI (SI Scheme 1-4). Several analogs were made to investigate possible hydrogen
bond donor capability of the sulfonamide and secondary amine group. To this end, the
nitrogens of sulfonamide (19), amine (20) or both (21) were substituted with a methyl group.
This led to a > 10-fold drop in potency for all compounds, which indicated that these NH
donors could be important for the interaction with FLT3. Next, the linker length between the
secondary amine and the phenyl was investigated. Compounds with reduced length of one
(
22) and two (23) methylene groups showed decreased activity. The importance of the
basicity of the linker moiety was tested by replacing the amine with an ether (24), amide
25), or a methylene (26) containing linker. 24 and 25 were equally active as the
(
corresponding amine derivative, while 26 was > 10-fold less active (Table 2). These results
suggested that the basic center of the linker is not required. Of note, reduction of the double
bond (27 - 29) in the linker resulted in an almost identical inhibitory activity as the parent
compound, whereas increasing the conformational restriction in compound 30 reduced its
activity. This indicated that the reduced conformational flexibility by the double bond in
compound 1 is not beneficial for its activity as has recently been noted for other kinase
3
4
inhibitors. Finally, the substitution of the sulfonamide for an amide did result in an inactive
compound (31) (pIC < 5), which could possibly be due to a difference in the spatial
5
0
orientation of the (sulfon)amide substituents. These data indicate that a flexible linker of 6
atoms with or without a basic amine is optimal between the sulfonamide and phenyl-pyridyl
rings.

a
Scheme 2: Synthetic route towards the derivatives 32 - 36 and 38 - 54.
a
Reagents and conditions: (a) SOCl , DMF, reflux, 4 h; (b) ethylenediamine, DCM, 0°C – RT; (c) B Pin ,
2
2
2
KOAc, Pd(dppf)Cl , 1,4-dioxane, 100°C, overnight; (d) 105, EDC, HOBt, DiPEA, DCM, 4 h; (e)
2
heteroaryl-bromide, Pd(PPh ) , K CO , DMF, 85°C, overnight; (f) 60, EDC, HOBt, DiPEA, DCM, 4 h; (g)
3
4
2
3
1
12, Pd(PPh ) , K CO , DMF, 90°C; (h) TFA, TES, DCM, 0°C – RT, 16 h; (i) aryl-sulfonylchloride, Et N,
3
4
2
3
3
DCM/DMF, 0°C – RT, 16 h; (j) NaBH , BF , THF, 0° - RT, 16 h; (k) SOCl , DMF, 0°C – RT, 19 h; (l) 60,
4
3
2
K CO , ACN, 70°C, 72 h; (m) NaHCO , Boc O, THF, RT, 36 h; (n) 112, Pd(PPh ) , K CO , DMF, 90°C; (o)
2
3
3
2
3 4
2
3
TFA, TES, DCM, 0°C – RT, 20 h; (p) aryl-sulfonylchloride, Et N, DCM/DMF, 0°C – 30°C, 16 h (q) TFA,
3
DCM, 0°C – RT, 16 h.
Having established the optimal linker features, an additional array of compounds (32-37)
was synthesized in which the pyridyl ring was replaced with other (substituted) heteroaryls
to optimize the hinge-binding interaction (Scheme 2). In contrast to the isoquinoline
replacements, a wide range of activities was observed (pIC : 5 – 8.9) (Table 3). While the
5
0
picolinamide variations (34-35) were inactive (pIC < 5), the azaindoles 36 and 37
5
0
demonstrated a significantly increased pIC of 8.87 ± 0.06. and 8.78 ± 0.05, respectively. Of
5
0
note, 37 demonstrated a LipE of 6.7. All together, the optimization of the potential hinge-
binding pyridyl moiety resulted in the discovery of the azaindoles as a potent FLT3 inhibitor
scaffold.

Table 3: FLT3 activity and LipE of compounds 32 - 37.
R =
Entry
X
pIC ± SEM
LipE
5
0
3
3
2
3
CO
6.82 ± 0.14
7.63 ± 0.11
4.8
CO
CO
5.6
3
3
4
5
< 5
< 5
n.a.
CO
n.a.
3
3
6
7
CO
8.87 ± 0.06
8.78 ± 0.05
6.2
6.7
CH2
Next, a matched-molecular pair analysis was performed using the azaindole scaffold with
3
5
amide (38-49) and amine linker (50-54) series. The goal was to study the influence of the
3
6
substitution pattern of the phenyl ring. Compounds (38-54) were prepared as shown in
Scheme 2. Compounds with electron-withdrawing groups, such as Cl (39), p-NO (43), p-F
2
(45), or electron donating groups (p-Me (41) and p-OMe (42)) both displayed high potency
(
pIC > 8.0). No correlation could be found between the Hammett constants of the
5
0
substituents and the activity of the compounds (SI Figure 1). In fact, non-substituted
compound 38 was the most potent compound identified in this study with a pIC of 9.49 ±
5
0
0
.08. The matched-molecular pair analysis of LipE values of the amine and amide series
showed good correlation, which supports the hypothesis that both series bind in a similar
fashion to FLT3 (Figure 2).

Table 4: FLT3 activity and LipE of compounds 38 - 54
R =
Entry
X
pIC ± SEM
LipE
5
0
3
3
8
9
CO
9.49 ± 0.08
8.62 ± 0.05
6.5
CO
CO
CO
CO
5.0
4.1
5.2
5.8
4
4
4
0
1
2
8.39 ± 0.05
8.67 ± 0.06
8.74 ± 0.08
4
4
3
4
CO
CO
8.80 ± 0.08
8.72 ± 0.06
5.9
5.1
4
5
CO
9.39 ± 0.18
6.2
4
4
6
7
CO
CO
9.32 ± 0.09
8.16 ± 0.08
5.7
3.9
4
8
CO
7.97 ± 0.09
5.0
4
5
9
0
CO
8.37 ± 0.09
8.88 ± 0.06
4.5
6.5
CH2
5
1
CH2
8.36 ± 0.08
6.4

5
5
5
2
3
4
CH2
CH2
CH2
8.13 ± 0.09
8.69 ± 0.07
8.62 ± 0.10
4.5
5.9
6.3
A m in e v s A m id e L ip E
8
7
6
5
4
3
2
R
= 0.8191
Ph e n y l
Is o q u in o lin e
- O CH 3
is o q u in o lin e - Py r id in e
4
4
- Ch lo r o
4 - CH 3
3
,4 - Dic h lo r o
3
4
5
6
7
8
L ip E A m ine series
Figure 2: Matched molecular pair analysis of amine and amide containing compounds. Data shows a
2
high correlation (R = 0.82), indicating a similar binding mode for both linker series.
Finally, to explain our structure-activity relationships a structure based study was performed
with compound 1 and compound 38 using a published DFG-out crystal structure (4RT7), and
a DFG-in model (SI figure 2). Induced fit docking was performed in combination with a
4
5
previously established binding pose metadynamics protocol , in order to determine a
feasible binding mode. On the basis of these results and overlap in binding mode with
quizartinib (SI Figure 2) it was established that compound 1 and compound 38 bind DFG-out
(
Figure 3A). The pyridine moiety of 1 is engaged in a hydrogen bond interaction with the
backbone of C694 (hinge) and the adjacent phenyl engages in a π-interaction with F691
Figure 3A). Moreover, in the induced fit experiments no poses were observed in which the
(
isoquinoline interacted with the hinge of FLT3. As shown in Figure 3B the resulting docking
pose of 38 is similar to the binding mode of 1 with an additional hydrogen-bond-interaction
to C694, which may explain the increased potency. To conclude, the observed binding mode
is in agreement with the obtained structure-activity relations.

Figure 3: Proposed “flipped” binding mode of 1 and 38 in FLT3. (A-left) 1 and (B-left) 38 docked in
FLT3 crystal structure (PDB: 4RT7). On the right a 2D-interaction diagram is shown depicting the
interactions between the ligand and FLT3.
3. Conclusion
In this study, azaindole 38 was identified as a new, highly potent inhibitor of FLT3-ITD with
favorable physico-chemical properties. Our structure-activity relationships and modeling
studies suggest that 38 has an alternative flipped binding mode compared to other kinase
inhibitors derived from the prototypical kinase inhibitor H-89. 38 forms an excellent starting
point for further lead optimization studies to obtain clinical candidates to modulate FLT3-ITD
in AML patients.
4. Experimental
4
.1. Chemistry
All of the compounds were synthesized as shown in schemes 1-2 and supplementary
schemes 1-5. Detailed procedures and characterization data are described in the
Supplementary information.
4
.2 Biochemical Evaluation of FLT3 inhibitors
In a 384-wells plate (PerkinElmer 384 Flat White), 5 µL kinase/peptide mix (0.06 ng/µL FLT3
Life Technologies; PV3182; Lot: 1614759F), 200 nM peptide (PerkinElmer; Lance® Ultra
(
ULightTM TK-peptide; TRFO127-M; Lot: 2178856)) in assay buffer (50 mM HEPES pH 7.5, 1
mM EGTA, 10 mM MgCl , 0.01% Tween-20, 2 mM DTT) was dispensed. Separately inhibitor
2

solutions (10 µM – 0.1 pM) were prepared in assay buffer containing 400 µM ATP and 1%
DMSO. 5 µL of these solutions were dispensed and the plate was incubated in the dark at
room temperature. After 90 minutes the reaction was quenched by the addition of 10 µL of
2
0 mM EDTA containing 4 nM antibody (PerkinElmer; Lance® Eu-W1024-anti-
phosphotyrosine(PT66); AD0068; Lot: 2342358). After mixing, samples were incubated for 60
minutes in the dark. The FRET fluorescence was measured on a Tecan Infinite M1000 Pro
plate reader (excitation 320 nm, emission donor 615 nm, emission acceptor 665 nm). Data
was processed using Microsoft Excel 2016, pIC values were fitted using GraphPad Prism
5
0
7
.0. Final assay concentrations during reaction: 200 µM ATP, 0.03 ng/µL FLT3, 100 nM Lance
TK-peptide, 0.5% DMSO. Compounds were tested in n=2 and N=2.
4
.3 Structure based modeling on FLT3
All structure based modeling was performed in the Schrödinger suite (Schrödinger Release
017-4: Maestro, Schrödinger, LLC, New York, NY, 2017). Crystal structures were prepared
2
3
7
38
using the protein preparation wizard, ligands were prepared using LigPrep. Both the DFG-
3
9
out structure co-crystalized with quizartinib (4RT7) and a DFG-in model were used in order
to dock our initial compound 1. The DFG-in model was constructed on the basis of 4RT7 and
4
0
3
LCD, in a similar fashion as has been done before, using the knowledge based potential in
4
3
1, 42
prime.
Docking was done using induced fit docking and using H-bond constraints on
C694. In order to determine to correct binding pose, induced fit docking was followed by
4
44
the conformer cluster script, using the Kelley criterion to determine the optimal number of
clusters. The highest scoring poses of every cluster were used in a previously published
workflow to determine binding poses , which is based on metadynamics. The highest
scoring pose was selected by adding the Metadynamics CompScore to the docking score.
Based on this workflow the highest scoring pose was visualized and rendered using PyMol.
4
5
4
6
Acknowledgements
Contributions
Cancer Drug Discovery Initiative (CDDI) is acknowledged for funding. SG, BG, JK, NL and RW
performed the organic synthesis and biochemical evaluation. EL and GW performed the
docking studies and provided LogD calculations. SG, RW, CB, HO, JN and MS designed the
experiments. SG and MS wrote the manuscript.
Competing interests
Declarations of interest: none.
Corresponding Author
*Phone: +31 (0)71 527 4768. E-mail: m.van.der.stelt@chem.leidenuniv.nl.

Appendix A. Supplementary data
Supplementary data to this article can be found online.
References
(1)
(2)
(3)
Shipley, J. L.; Butera, J. N. Acute Myelogenous Leukemia. Exp. Hematol. 2009, 37 (6),
49–658.
6
Döhner, H.; Weisdorf, D. J.; Bloomfield, C. D. Acute Myeloid Leukemia. N. Engl. J. Med.
015, 373 (12), 1136–1152.
2
Döhner, H.; Estey, E. H.; Amadori, S.; Appelbaum, F. R.; Büchner, T.; Burnett, A. K.;
Dombret, H.; Fenaux, P.; Grimwade, D.; Larson, R. A.; et al. Diagnosis and
Management of Acute Myeloid Leukemia in Adults: Recommendations from an
International Expert Panel, on Behalf of the European LeukemiaNet. Blood 2010, 115
(3), 453–474.
(
(
(
(
(
(
(
4)
5)
6)
7)
8)
9)
Fathi, A. T.; Chen, Y.-B. The Role of FLT3 Inhibitors in the Treatment of FLT3-Mutated
Acute Myeloid Leukemia. Eur. J. Haematol. 2017, 98 (4), 330–336.
Stirewalt, D. L.; Radich, J. P. The Role of FLT3 in Haematopoietic Malignancies. Nat.
Rev. Cancer 2003, 3 (9), 650–665.
Leung, a Y. H.; Man, C.-H.; Kwong, Y.-L. FLT3 Inhibition: A Moving and Evolving Target
in Acute Myeloid Leukaemia. Leukemia 2012, 27 (2), 260–268.
Pemmaraju, N.; Kantarjian, H.; Ravandi, F.; Cortes, J. FLT3 Inhibitors in the Treatment
of Acute Myeloid Leukemia: The Start of an Era? Cancer 2011, 117 (15), 3293–3304.
Kayser, S.; Levis, M. J. Advances in Targeted Therapy for Acute Myeloid Leukaemia. Br.
J. Haematol. 2018, 180 (4), 484–500.
Levis, M. Midostaurin Approved for FLT3-Mutated AML. Blood 2017, 129 (26), 3403–
3
406.
10) Zimmerman, E. I.; Turner, D. C.; Buaboonnam, J.; Hu, S.; Orwick, S.; Roberts, M. S.;
Janke, L. J.; Ramachandran, A.; Stewart, C. F.; Inaba, H.; et al. Crenolanib Is Active
against Models of Drug-Resistant FLT3-ITD-Positive Acute Myeloid Leukemia. Blood
2
013, 122 (22), 3607–3615.
(
11) Baker, S. D.; Zimmerman, E. I.; Wang, Y.-D.; Orwick, S.; Zatechka, D. S.; Buaboonnam,
J.; Neale, G. A.; Olsen, S. R.; Enemark, E. J.; Shurtleff, S.; et al. Emergence of Polyclonal
FLT3 Tyrosine Kinase Domain Mutations during Sequential Therapy with Sorafenib and
Sunitinib in FLT3-ITD-Positive Acute Myeloid Leukemia. Clin. Cancer Res. 2013, 19 (20),
5
758–5768.
(
12) Smith, C. C.; Wang, Q.; Chin, C.-S.; Salerno, S.; Damon, L. E.; Levis, M. J.; Perl, A. E.;
Travers, K. J.; Wang, S.; Hunt, J. P.; et al. Validation of ITD Mutations in FLT3 as a
Therapeutic Target in Human Acute Myeloid Leukaemia. Nature 2012, 485 (7397),

2
60–263.
(
13) Man, C. H.; Fung, T. K.; Ho, C.; Han, H. H. C.; Chow, H. C. H.; Ma, A. C. H.; Choi, W. W.
L.; Lok, S.; Cheung, A. M. S.; Eaves, C.; et al. Sorafenib Treatment of FLT3-ITD(+) Acute
Myeloid Leukemia: Favorable Initial Outcome and Mechanisms of Subsequent
Nonresponsiveness Associated with the Emergence of a D835 Mutation. Blood 2012,
1
19 (22), 5133–5143.
(
14) Smith, C. C.; Lasater, E. a.; Zhu, X.; Lin, K. C.; Stewart, W. K.; Damon, L. E.; Salerno, S.;
Shah, N. P. Activity of Ponatinib against Clinically-Relevant AC220-Resistant Kinase
Domain Mutants of FLT3-ITD. Blood 2013, 121 (16), 3165–3171.
(
15) Liu, N. Development of Kinase Inhibitors and Activity-Based Probes, Leiden University,
2
016.
(
16) Fedorov, O.; Marsden, B.; Pogacic, V.; Rellos, P.; Müller, S.; Bullock, A. N.; Schwaller, J.;
Sundström, M.; Knapp, S. A Systematic Interaction Map of Validated Kinase Inhibitors
with Ser/Thr Kinases. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (51), 20523–20528.
(
17) Chijiwa, T.; Mishima, A.; Hagiwara, M.; Sano, M.; Hayashi, K.; Inoue, T.; Naito, K.;
Toshioka, T.; Hidaka, H. Inhibition of Forskolin-Induced Neurite Outgrowth and Protein
Phosphorylation by a Newly Synthesized Selective Inhibitor of Cyclic AMP-Dependent
Protein Kinase, N-[2-(P-Bromocinnamylamino)ethyl]-5-Isoquinolinesulfonamide (H-
8
9), of PC12D Pheochromocytoma Cells. J. Biol. Chem. 1990, 265 (9), 5267–5272.
(
18) Pflug, A.; Johnson, K. A.; Engh, R. A. Anomalous Dispersion Analysis of Inhibitor
Flexibility: A Case Study of the Kinase Inhibitor H-89. Acta Crystallogr. Sect. F Struct.
Biol. Cryst. Commun. 2012, 68 (8), 873–877.
(
19) Gao, Y.; Davies, S. P.; Augustin, M.; Woodward, A.; Patel, U. A.; Kovelman, R.; Harvey,
K. J. A Broad Activity Screen in Support of a Chemogenomic Map for Kinase Signalling
Research and Drug Discovery. Biochem. J. 2013, 451 (2), 313–328.
(
20) Lochner, A.; Moolman, J. A. The Many Faces of H89: A Review. Cardiovasc. Drug Rev.
2
006, 24 (3–4), 261–274.
(
21) Kuijl, C.; Savage, N. D. L.; Marsman, M.; Tuin, A. W.; Janssen, L.; Egan, D. A.; Ketema,
M.; van den Nieuwendijk, R.; van den Eeden, S. J. F.; Geluk, A.; et al. Intracellular
Bacterial Growth Is Controlled by a Kinase Network around PKB/AKT1. Nature 2007,
4
50 (7170), 725–730.
(
(
(
(
22) Csizmadia, F.; Tsantili-Kakoulidou, A.; Panderi, I.; Darvas, F. Prediction of Distribution
Coefficient from Structure. 1. Estimation Method. J. Pharm. Sci. 1997, 86 (7), 865–871.
23) Johnson, T. W.; Gallego, R. A.; Edwards, M. P. Lipophilic Efficiency as an Important
Metric in Drug Design. J. Med. Chem. 2018, acs.jmedchem.8b00077.
24) Wu, P.; Nielsen, T. E.; Clausen, M. H. FDA-Approved Small-Molecule Kinase Inhibitors.
Trends Pharmacol. Sci. 2015, 36 (7), 422–439.
25) Martin, M. P.; Alam, R.; Betzi, S.; Ingles, D. J.; Zhu, J.-Y.; Schönbrunn, E. A Novel
Approach to the Discovery of Small-Molecule Ligands of CDK2. ChemBioChem 2012,
1
3 (14), 2128–2136.

(
26) Gajiwala, K. S.; Wu, J. C.; Christensen, J.; Deshmukh, G. D.; Diehl, W.; DiNitto, J. P.;
English, J. M.; Greig, M. J.; He, Y.-A.; Jacques, S. L.; et al. KIT Kinase Mutants Show
Unique Mechanisms of Drug Resistance to Imatinib and Sunitinib in Gastrointestinal
Stromal Tumor Patients. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (5), 1542–1547.
(
27) Hilberg, F.; Roth, G. J.; Krssak, M.; Kautschitsch, S.; Sommergruber, W.; Tontsch-Grunt,
U.; Garin-Chesa, P.; Bader, G.; Zoephel, A.; Quant, J.; et al. BIBF 1120: Triple
Angiokinase Inhibitor with Sustained Receptor Blockade and Good Antitumor Efficacy.
Cancer Res. 2008, 68 (12), 4774–4782.
(
(
(
(
28) Awad, M. M.; Katayama, R.; McTigue, M.; Liu, W.; Deng, Y.-L.; Brooun, A.; Friboulet, L.;
Huang, D.; Falk, M. D.; Timofeevski, S.; et al. Acquired Resistance to Crizotinib from a
Mutation in CD74 – ROS1. N. Engl. J. Med. 2013, 368 (25), 2395–2401.
29) Chen, P.; Lee, N. V; Hu, W.; Xu, M.; Ferre, R. A.; Lam, H.; Bergqvist, S.; Solowiej, J.;
Diehl, W.; He, Y.-A.; et al. Spectrum and Degree of CDK Drug Interactions Predicts
Clinical Performance. Mol. Cancer Ther. 2016, 15 (10), 2273–2281.
30) Pemovska, T.; Johnson, E.; Kontro, M.; Repasky, G. A.; Chen, J.; Wells, P.; Cronin, C. N.;
McTigue, M.; Kallioniemi, O.; Porkka, K.; et al. Axitinib Effectively Inhibits BCR-
ABL1(T315I) with a Distinct Binding Conformation. Nature 2015, 519 (7541), 102–105.
31) McTigue, M.; Murray, B. W.; Chen, J. H.; Deng, Y.-L.; Solowiej, J.; Kania, R. S. Molecular
Conformations, Interactions, and Properties Associated with Drug Efficiency and
Clinical Performance among VEGFR TK Inhibitors. Proc. Natl. Acad. Sci. U. S. A. 2012,
1
09 (45), 18281–18289.
(
32) Xing, L.; Rai, B.; Lunney, E. A. Scaffold Mining of Kinase Hinge Binders in Crystal
Structure Database. J. Comput. Aided. Mol. Des. 2014, 28 (1), 13–23.
(
33) Shavnya, A.; Coffey, S. B.; Smith, A. C.; Mascitti, V. Palladium-Catalyzed Sulfination of
Aryl and Heteroaryl Halides: Direct Access to Sulfones and Sulfonamides. Org. Lett.
2
013, 15 (24), 6226–6229.
(
34) Wienen-Schmidt, B.; Jonker, H. R. A.; Wulsdorf, T.; Gerber, H.-D.; Saxena, K.; Kudlinzki,
D.; Sreeramulu, S.; Parigi, G.; Luchinat, C.; Heine, A.; et al. Paradoxically, Most Flexible
Ligand Binds Most Entropy-Favored: Intriguing Impact of Ligand Flexibility and
Solvation on Drug–Kinase Binding. J. Med. Chem. 2018, acs.jmedchem.8b00105.
(
(
(
35) Dossetter, A. G.; Griffen, E. J.; Leach, A. G. Matched Molecular Pair Analysis in Drug
Discovery. Drug Discov. Today 2013, 18 (15–16), 724–731.
36) Topliss, J. G. Utilization of Operational Schemes for Analog Synthesis in Drug Design. J.
Med. Chem. 1972, 15 (10), 1006–1011.
37) Schrödinger Release 2017-4: Schrödinger Suite 2017-4 Protein Preparation Wizard;
Epik, Schrödinger, LLC, New York, NY, 2017; Impact, Schrödinger, LLC, New York, NY,
2
017; Prime, Schrödinger, LLC, New York, NY, 2017.
(38) Schrödinger Release 2017-4: LigPrep, Schrödinger, LLC, New York, NY, 2018.
(39) Smith, C. C.; Zhang, C.; Lin, K. C.; Lasater, E. A.; Zhang, Y.; Massi, E.; Damon, L. E.;
Pendleton, M.; Bashir, A.; Sebra, R., Characterizing and overriding the structural

mechanism of the quizartinib-resistant FLT3 “gatekeeper” F691L mutation with
PLX3397. Cancer discovery 2015, 5, 668-679.
(
40) Ke, Y.-Y.; Singh, V. K.; Coumar, M. S.; Hsu, Y. C.; Wang, W.-C.; Song, J.-S.; Chen, C.-H.;
Lin, W.-H.; Wu, S.-H.; Hsu, J. T., Homology modeling of DFG-in FMS-like tyrosine kinase
3
(FLT3) and structure-based virtual screening for inhibitor identification. Scientific
reports 2015, 5, 11702.
(
(
(
(
(
(
41) Jacobson, M. P.; Pincus, D. L.; Rapp, C. S.; Day, T. J.; Honig, B.; Shaw, D. E.; Friesner, R.
A., A hierarchical approach to all‐atom protein loop prediction. Proteins: Structure,
Function, and Bioinformatics 2004, 55, 351-367.
42) Jacobson, M. P.; Friesner, R. A.; Xiang, Z.; Honig, B., On the role of the crystal
environment in determining protein side-chain conformations. Journal of molecular
biology 2002, 320, 597-608.
43) Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R., Novel procedure for
modeling ligand/receptor induced fit effects. Journal of medicinal chemistry 2006, 49,
5
34-553.
44) Kelley, L. A.; Gardner, S. P.; Sutcliffe, M. J., An automated approach for clustering an
ensemble of NMR-derived protein structures into conformationally related
subfamilies. Protein Engineering, Design and Selection 1996, 9, 1063-1065.
45) A.J. Clark; P. Tiwary; al., e., Prediction of protein–ligand binding poses via a
combination of induced fit docking and metadynamics simulations. J. Chem. Theory
Comput. 2016, 12, 2990-2998.
46) The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.

Supporting information
Comprehensive structure-activity-relationship of
azaindoles as highly potent FLT3 inhibitors
†
†
†
†
‡
Sebastian H. Grimm , Berend Gagestein , Jordi F. Keijzer , Nora Liu , Ruud H. Wijdeven , Eelke
§
§
⊥
B. Lenselink , Adriaan W. Tuin , Adrianus M. C. H. van den Nieuwendijk , Gerard J. P. van
§
‡
⊥
∥
Westen , Constant A. A. van Boeckel , Herman S. Overkleeft , Jacques Neefjes , Mario van
†
,*
der Stelt .
†
Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University,
‡
Leiden, the Netherlands Oncode Institute and Department of Cell and Chemical Biology,
⊥
Leiden University Medical Center, Leiden, the Netherlands Computational Drug Discovery,
Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden
§
University, Leiden, the Netherlands Department of Bio-Organic Synthesis, Leiden Institute
∥
of Chemistry, Leiden University, Leiden, the Netherlands Pivot Park Screening Center, Oss,
the Netherlands
Table of Contents
I. Supplementary figures and schemes................................................................................... S2
II. General methods for synthesis and characterization of compounds ............................... S5
III. Synthetic procedures and characterization data of compounds...................................... S6

I. Supplementary figures and schemes
SI Figure 1: Plot of pIC -values of the amide series versus the corresponding substituent σ-values and
5
0
36
the used σ-values for each substituent.
SI Figure 2: Proposed binding mode of 1 (purple) overlaid with the crystal structure of FLT3 co-
crystalized with quizartinib (yellow) (PDB: 4RT7).

a
SI Scheme 1: Synthetic route towards the derivatives 16 - 18, 31.
a
Reagents and conditions: (a) Fe, AcOH, EtOH/H O; (b) diethyl cyanomethylphosphonate, NaH, DMF,
2
0
°C – RT; (c) DiBAL-H, Et O, -80°C – 0°C, then 105, NaBH , MeOH, -100°C – 0°C, then Boc O; (d)
2
4
2
arylboronic acid, Pd(PPh ) , K CO , DMF/DCM/H O, 90°C; (e) TFA, DCM, 0°C – RT; (f) isoquinoline-5-
3
4
2
3
2
carboxylic acid, SOCl , then DiPEA, DMAP, substrate, DCM, 0°C – RT.
2
a
SI Scheme 2: Synthetic route towards the derivatives 19 – 21, 27.
a
Reagents and conditions: (a) MeI, Cs CO , DMF, RT; (b) arylboronic acid, Pd(PPh ) , K CO ,
2
3
3 4
2
3
DMF/DCM/H O, 80°C; (c) TFA, CHCl , 0°C – RT; (d) formaldehyde, NaHB(OAc) , THF/MeOH, RT; (e)
2
3
3
Pd/C, H , MeOH.
2

a
SI Scheme 3: Synthetic route towards the derivatives 22, 23, 29 and 30.
a
Reagents and conditions: (a) arylboronic acid, Pd(PPh ) , K CO , DMF/DCM/H O, 90°C; (b) DMP,
3
4
2
3
2
DCM, 0°C – RT; (c) 105, NaHB(OAc) , THF or DCM, RT; (d) Boc O, NaHCO , THF, RT; (e) TFA, DCM, 0°C
3
2
3
–
RT; (f) NaBH , BF , THF, 0° - RT; (g) LiAlH , THF, 0° - RT.
4
3
4
a
SI Scheme 4: Synthetic route towards the derivatives 24, 26, 28.
a
Reagents and conditions: (a) 86, NaH, ACN, 70°C; (b) arylboronic acid, Pd(PPh ) , K CO ,
3
4
2
3
DMF/DCM/H O, 80°C; (c) TFA, TES, DCM, 0°C – RT; (d) 104, Et N, DCM, 0°C – RT; (e) TrtCl, K CO ,
2
3
2
3
DCM, RT; (f) p-toluenesulfonyl hydrazide, NaOAc, THF, 66°C; (g) benzaldehyde, NaBH(OAc) , THF, RT;
3
(
0
h) oxalyl chloride, DMSO, Et N, DCM, -80°C – RT; (i) diethyl (4-bromobenzyl)phosphonate, NaH, THF,
3
°C – RT; (j) arylboronic acid, Pd(PPh ) , K CO , DMF/DCM/H O, 80°C; (k) Pd(OH) , t-BuOH/1,4-
3
4
2
3
2
2
dioxane/H O, H , RT; (l) 104, Et N, DCM, 0°C – RT.
2
2
3

a
SI Scheme 5: Synthetic route towards the derivatives 25 and 37.
a
Reagents and conditions: (a) 105, EDC, HOBt, DiPEA, DCM, RT; (b) arylboronic acid, Pd(PPh ) , K CO ,
3
4
2
3
DMF/DCM/H O, 85°C; (c) NaBH , BF , THF, 0° - RT; (d) B Pin , KOAc, Pd(dppf)Cl , 1,4-dioxane, 100°C,
2
4
3
2
2
2
overnight; (e) arylbromide, Pd(PPh ) , K CO , DMF/DCM/H O, 85°C; (f) DMP, DCM, 0°C – RT; (g) 105,
3
4
2
3
2
NaHB(OAc) , DCM, RT.
3
II. General methods for synthesis and characterization of
compounds
Solvents were purchased from Biosolve, Sigma Aldrich or Fluka and, if necessary dried over
3
Å or 4Å molecular sieves. Reagents purchased from chemical suppliers were used without
further purification, unless stated otherwise. Oxygen or H O sensitive reactions were
2
performed under argon or nitrogen atmosphere and/or under exclusion of H O. Reactions
2
were followed by thin layer chromatography which was performed using TLC silica gel 60 F245
on aluminium sheets, supplied by Merck. Compounds were visualized by UV absorption
(
254 nm) or spray reagent (permanganate (5 g/L KMnO , 25 g/L K CO )). TLCMS was
4
2
3
measured with a thin layer chromatography-mass spectrometer (Advion, Expression LCMS;
1
13
Advion, Plate Express). H- and C-NMR spectra were performed on one of the following
Bruker spectrometers: DPX 300 NMR spectrometer (300 MHz), equipped with 5mm-BBO-z-
gradient-probe; AV-400 NMR spectrometer (400 MHz), equipped with 5mm-BBO-z-gradient-
probe; AV-500 NMR spectrometer (500 MHz), equipped with BBFO-z-gradient-probe; AV-600
NMR spectrometer (600 MHz), equipped with 5mm-Cryo-z-gradient probe. NMR spectra
were measured in deuterated methanol, chloroform or DMSO and were referenced to the
1
residual protonated solvent signals as internal standards (chloroform-d = 7.260 ( H), 77.160
1
3
1
13
1
13
(
C); methanol-d = 3.310 ( H), 49.000 ( C); DMSO-d = 2.500 ( H), 39.520 ( C)). Signals
4
6
multiplicities are written as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), p
pentet) or m (multiplet). Coupling constants (J) are given in Hz. Preparative HPLC (Waters,
15 HPLC pump M; Waters, 515 HPLC pump L; Waters, 2767 sample manager; Waters SFO
(
5
System Fluidics Organizer; Waters Acquity Ultra Performance LC, SQ Detector; Waters Binary
Gradient Module) was performed on a Phenomenex Gemini column (5 μM C18, 150 x 4.6
mm) or a Waters XBridgeTM column (5 μM C18, 150 x 19 mm). Diode detection was done
between 210 and 600 nm. Gradient: ACN in (H O + 0.2% TFA). HRMS (Thermo, Finnigan LTQ
2
Orbitrap; Thermo, Finnigan LTQ Pump; Thermo, Finnigan Surveyor MS Pump PLUS Thermo,
Finnigan Surveyor Autosampler; NESLAB, Merlin M25). Data acquired through direct
injection of 1 mM of the sample in ACN/H O/t-BuOH (1:1:1), with mass spectrometer
2
equipped with an electrospray ion source in positive mode (source voltage 3.5 kV, sheath

gas low 10, capillary temperature 275°C) with resolution R = 60.000 at m/z = 400 (mass
range = 150-2000) and dioctylphtalate (m/z = 391.28428) as lock mass. All tested compounds
were checked for purity by HPLC, either on a Thermo (Thermo Finnigan LCQ Advantage Max;
Thermo Finnigan Surveyor LC-pump Plus; Thermo Finnigan Surveyor Autosampler Plus;
Thermo Finnigan Surveyor PDA Plus Detector; Phenomenex Gemini column (5 μm C18, 50 x
4
.6 mm)) or a Waters (Waters 515 HPLC pump M; Waters 515 HPLC pump L; Waters 2767
sample manager; Waters SFO System Fluidics Organizer; Waters Acquity Ultra Performance
LC, SQ Detector; Waters binary gradient module; Phenomenex Gemini column (5 μm C18,
1
50 x 4.6 mm)) system and were determined to be >95% pure by integrating UV intensity
recorded.
III. Synthetic procedures and characterization data of compounds
General procedure A: Sulfonamide coupling
Step 1: A glass vial was charged with corresponding bromo-heteroaryl compound
(
0.20 mmol, 1 eq), potassium metabisulfite (88 mg, 0.40 mmol, 2 eq), tetrabutylammonium
bromide (70 mg, 0.22 mmol, 1.1 eq), sodium formate (15 mg, 0.22 mmol, 1.1 eq),
palladium(II) acetate (5 mg, 0.02 mmol, 0.1 eq), triphenylphosphine (16 mg, 0.06 mmol,
0
.3 eq), 1,10-phenanthroline (11 mg, 0.06 mmol, 0.3 eq). After sealing, the vial was flushed
with argon for 30 min and the reagents were suspended in dry, degassed DMSO (1 mL) and
the reaction mixture was stirred for 4 h at 70°C. After cooling to RT N,N-
Diisopropylethylamine (70 µL, 0.40 mmol, 2 eq) and a solution of tert-butyl (E)-(2-
aminoethyl)(3-(4-(pyridin-3-yl)phenyl)allyl)carbamate (63) (106 mg, 0.30 mmol, 1.5 eq) in dry
THF (1 mL) were added and the reaction mixture was cooled to 0°C. Subsequently a solution
of N-bromosuccinimide (62 mg, 0.40 mmol, 2 eq) in dry THF (1 mL) was added and the
reaction mixture was allowed to come to RT. After stirring for 1 h the reaction was quenched
by adding H O (1 mL) and brine (2 mL). The resulting mixture was extracted with EtOAc. The
2
combined organic layers were dried over Na SO , filtered and the solvent removed under
2
4
reduced pressure. The residue was purified via flash-column-chromatography (SiO , 0% to
2
5
% MeOH in DCM) to yield the desired Boc-protected product, which was used directly in
step 2.
Step 2: The Boc-protected product was dissolved in chloroform (1.6 mL) and cooled to 0°C.
After drop-wise addition of TFA (0.4 mL), the reaction mixture was allowed to come to RT
and stirred for 1 h. Chloroform (10 mL) was added to the reaction mixture and subsequently
concentrated in vacuum. After co-evaporating with chloroform (1x10 mL), the residue was
purified by reverse phase HPLC.

General procedure B: Suzuki Coupling
A glass vial was charged with the corresponding bromo-heteroaryl compound (0.15 mmol,
1
.5 eq), N-(2-(isoquinoline-5-sulfonamido)ethyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)propanamide (107) (51 mg, 0.10 mmol, 1 eq) and Pd(PPh ) (6 mg,
3
4
0
.005 mmol, 0.05 eq). The vial was put under an argon atmosphere and degassed DMF
(0.35 mL) and 2 M degassed aqueous K CO (0.125 mL, 0.25 mmol, 2.5 eq) were added. The
2
3
reaction mixture was stirred at 85°C overnight, diluted with DCM (10 mL) and half-saturated
aq. NaHCO solution (10 mL), extracted with DCM (3x10 mL), dried over MgSO , filtered and
3
4
concentrated under reduced pressure. The residue was purified by reverse phase HPLC.
General procedure C: Sulfonamide formation
3
0
-(4-(1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)-N-(2-aminoethyl)propanamide (113) (50 mg,
.16 mmol, 1.0 eq) and Et N (45 μL,0.32 mmol, 2.0 eq) were dissolved in DMF (1.6 mL). The
3
reaction mixture was cooled to 0°C and corresponding sulfonylchloride (194.6 μmol, 1.2 eq)
dissolved in DCM (1.6 mL) or DMF (1.6 mL) was added. After 15 min the mixture was
warmed up to RT and stirred for 5-16 h. The mixture was quenched with saturated aqueous
NaHCO (50 mL), the phases were separated and the aqueous layer was extracted with DCM
3
or with a mixture of 10% MeOH in CHCl (3x40 mL). The combined organic layers were
3
washed with brine (1x100 mL), dried over Na SO , filtered and concentrated under reduced
2
4
pressure. The residue was purified via flash column chromatography and preparative HPLC.

General Procedure D: Sulfonamide formation and debocylation
Step 1: tert-Butyl (3-(4-(1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)propyl)(2-aminoethyl)
carbamate (117) (90 mg, 228.3 μmol, 1.0 eq) and Et N (63 μL, 456.3 μmol, 2.0 eq) were
3
dissolved in DCM (1 mL). The mixture was cooled to 0°C, corresponding sulfonylchloride
(
0.27 mmol, 1.2 eq) dissolved in DCM (1 mL) was added and the mixture was allowed to
warm up and stirred at 30°C until full conversion was confirmed by TLC (4 – 40 h). The
mixture was quenched with saturated aqueous NaHCO (50 mL), the phases were separated
3
and the aqueous layer was extracted with DCM (3x70 mL). The combined organic layers
were washed with brine (1x120 mL), dried over Na SO , filtered and concentrated under
2
4
reduced pressure. The resulting residue was purified by flash-column-chromatography (SiO2,
dry-loading, 5% to 7% (10% of sat. aqueous NH in MeOH) in DCM) and used in step 2.
3
Step 2: The product from step 1 was dissolved in DCM (1 mL) and subsequently cooled to
0
°C. TFA (250 μL) was added dropwise to the solution and warmed to RT and stirred for 19 h.
The mixture was diluted with 15 mL CHCl and concentrated under reduced pressure. The
3
resulting crude was purified by flash-column-chromatography and preparative HPLC to yield
the desired compound after lyophilisation.
(E)-N-(2-((3-(4-(Pyridin-3-yl)phenyl)allyl)amino)ethyl)isoquinoline-5-sulfonamide (1)
A round-bottom-flask was charged with tert-butyl
(
(
E)-(2-(isoquinoline-5-sulfonamido)ethyl)(3-(4-
pyridin-3-yl)phenyl) allyl)carbamate (64) (610 mg,
1
.12 mmol, 1 eq) dissolved in CHCl (50 mL). After
3
cooling the solution to 0°C and dropwise addition of
TFA (12.5 mL), it was allowed to warm to RT and
stirred for 30 min. The reaction was quenched by slow addition of sat. aqueous Na CO3
2
solution (70 mL) until a pH of ~12 was reached and the mixture was extracted with DCM
(
3x50 mL). The combined organic layers were dried over Na SO , filtered and concentrated
2
4
under reduced pressure. The residue was purified via flash-column-chromatography (SiO2,
0
6
8
1
% to 15% (10% of sat. aqueous NH in MeOH) in DCM) to yield the desired product (329 mg,
3
1
6%). H NMR (400 MHz, methanol-d ) δ 9.32 (d, J = 0.7 Hz, 1H), 8.80 (dd, J = 2.3, 0.7 Hz, 1H),
4
.61 (d, J = 6.2 Hz, 1H), 8.55 (d, J = 6.2 Hz, 1H), 8.50 (dd, J = 4.9, 1.5 Hz, 1H), 8.47 (dd, J = 7.4,
.2 Hz, 1H), 8.33 (d, J = 8.3 Hz, 1H), 8.11 – 8.06 (m, 1H), 7.81 – 7.74 (m, 1H), 7.62 (d, J = 8.3
Hz, 2H), 7.53 – 7.48 (m, 1H), 7.46 (d, J = 8.3 Hz, 2H), 6.44 (d, J = 15.9 Hz, 1H), 6.17 (dt, J =
1
5.9, 6.5 Hz, 1H), 3.21 (dd, J = 6.5, 1.1 Hz, 2H), 3.03 (t, J = 6.4 Hz, 2H), 2.60 (t, J = 6.4 Hz, 2H).
1
3
C NMR (101 MHz, methanol-d ) δ 154.33, 148.68, 148.12, 144.87, 138.41, 138.10, 137.49,
4
1
1
36.36, 136.24, 134.88, 134.75, 132.65, 132.60, 130.62, 128.72, 128.25, 128.21, 127.72,
+
25.49, 119.15, 68.12, 51.73, 43.06. HRMS calculated for C H N O S 445.16927 [M+H] ,
2
5 25 4 2
found 445.16891. LCMS (ESI, Waters, C , linear gradient, 5% to 50% ACN in H O 0.2% TFA,
1
8
2
+
1
0 min): t = 5.17 min; m/z : 445 [M+H] .
R

(E)-1-oxo-N-(2-((3-(4-(Pyridin-3-yl)phenyl)allyl)amino)ethyl)isoindoline-4-sulfonamide (5)
The title compound was synthesized from 4-
bromoisoindolin-1-one following general procedure A on
a 0.2 mmol scale and purified by preparative HPLC
(
XBridge, C , 0% to 20% ACN in H O 0.2% TFA, 10 min
1
8
2
gradient) to yield the compound as a TFA salt after
lyophilisation (27 mg, 24%). H NMR (600 MHz, methanol-
1
d4) δ 8.98 (s, 1H), 8.66 (d, J = 4.8 Hz, 1H), 8.45 (dt, J = 8.1,
.6 Hz, 1H), 8.07 (dd, J = 17.2, 7.6 Hz, 2H), 7.82 – 7.72 (m, 4H), 7.66 (d, J = 8.3 Hz, 2H), 6.95
1
(
d, J = 15.9 Hz, 1H), 6.41 (dt, J = 15.7, 7.2 Hz, 1H), 4.76 (s, 2H), 3.90 (d, J = 7.1 Hz, 2H), 3.23 (s,
1
3
4
1
1
H). C NMR (151 MHz, methanol-d ) δ 170.09, 144.52, 143.92, 141.94, 138.51, 137.84,
4
37.75, 136.29, 136.01, 134.98, 134.39, 130.70, 129.02, 127.63, 127.55, 127.28, 125.41,
+
19.05, 49.03, 46.21, 45.73, 38.79. HRMS calculated for C H N O S 449.16419 [M+H] ,
2
4 25 4 3
found 449.16397. LCMS (ESI, Waters, C , linear gradient, 5% to 50% ACN in H O 0.2% TFA,
1
8
2
+
1
0 min): t = 5.59 min; m/z : 449 [M+H] .
R
(E)-N-(2-((3-(4-(Pyridin-3-yl)phenyl)allyl)amino)ethyl)-1H-indazole-5-sulfonamide (6)
The title compound was synthesized from 5-bromo-1H-
indazole following general procedure A on a 0.2 mmol scale
and purified by preparative HPLC (XBridge, C , 0% to 20%
1
8
ACN in H O 0.2% TFA, 10 min gradient) to yield the
compound as a TFA salt after lyophilisation (11 mg, 10%). H
2
1
NMR (600 MHz, methanol-d ) δ 9.03 (s, 1H), 8.69 (d, J = 4.7
4
Hz, 1H), 8.54 (d, J = 8.1 Hz, 1H), 8.43 – 8.42 (m, 1H), 8.25 (s,
H), 7.88 – 7.84 (m, 2H), 7.79 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 8.9 Hz, 1H), 7.68 (d, J = 8.3 Hz,
H), 6.96 (d, J = 15.9 Hz, 1H), 6.42 (dt, J = 15.8, 7.2 Hz, 1H), 3.90 (d, J = 7.1 Hz, 2H), 3.22 (t, J =
1
2
5
1
1
1
3
.5 Hz, 2H), 3.17 (t, J = 5.5 Hz, 2H). C NMR (151 MHz, methanol-d ) δ 145.10, 144.55,
4
42.82, 140.89, 139.64, 139.03, 137.93, 137.01, 136.53, 132.92, 129.00, 128.74, 127.18,
25.40, 123.58, 123.43, 120.67, 112.49, 50.36, 47.65, 40.33. HRMS calculated for
+
C H N O S 434.16452 [M+H] , found 434.16414. LCMS (ESI, Waters, C , linear gradient, 5%
2
3
24
5
2
18
+
to 50% ACN in H O 0.2% TFA, 10 min): t = 5.80 min; m/z : 434 [M+H] .
2
R
(E)-N-(2-((3-(4-(Pyridin-3-yl)phenyl)allyl)amino)ethyl)-1H-indazole-6-sulfonamide (7)
The title compound was synthesized from 6-bromo-1H-
indazole following general procedure A on a 0.2 mmol scale
and purified by preparative HPLC (XBridge C , 10% to 35%
1
8
ACN in H O 0.2% TFA, 10 min gradient) to yield the compound
2
1
as a TFA salt after lyophilisation (27 mg, 25%). H NMR (600
MHz, methanol-d ) δ 9.02 (d, J = 1.9 Hz, 1H), 8.71 – 8.68 (m,
4
1
H), 8.54 (dt, J = 8.1, 1.6 Hz, 1H), 8.20 (d, J = 0.9 Hz, 1H), 8.15
(
s, 1H), 8.03 – 7.96 (m, 1H), 7.86 (dd, J = 8.1, 5.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.68 (d, J =
8
1
.3 Hz, 2H), 7.62 (dd, J = 8.5, 1.5 Hz, 1H), 6.96 (d, J = 15.9 Hz, 1H), 6.42 (dt, J = 15.8, 7.2 Hz,
H), 3.90 (d, J = 7.2 Hz, 2H), 3.22 (d, J = 4.9 Hz, 2H), 3.19 (d, J = 4.7 Hz, 2H). C NMR (151
1
3
MHz, methanol-d ) δ 143.67, 143.12, 139.51, 139.06, 138.23, 137.65, 137.34, 136.51,
4
1
4
35.59, 133.75, 127.59, 127.33, 125.77, 125.07, 122.03, 119.22, 117.78, 110.40, 48.97,
+
6.24, 38.96. HRMS calculated for C H N O S 434.16452 [M+H] , found 434.16410. LCMS
23 24 5 2
(
ESI, Waters, C , linear gradient, 5% to 50% ACN in H O 0.2% TFA, 10 min): t = 6.02 min;
m/z : 434 [M+H] .
18
2
R
+

(E)-1-oxo-N-(2-((3-(4-(Pyridin-3-yl)phenyl)allyl)amino)ethyl)isoindoline-5-sulfonamide (8)
The title compound was synthesized from 5-bromoisoindolin-
1
-one following general procedure A on a 0.2 mmol scale and
purified by preparative HPLC (Gemini C , 0% to 20% ACN in
1
8
H O 0.2% TFA, 10 min gradient) to yield the compound as a
2
1
TFA salt after lyophilisation (9 mg, 8%). H NMR (600 MHz,
methanol-d ) δ 8.99 (s, 1H), 8.67 (d, J = 5.0 Hz, 1H), 8.48 (d, J =
4
7
.7 Hz, 1H), 8.13 (s, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.98 (d, J = 8.0
Hz, 1H), 7.84 – 7.80 (m, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 6.97 (d, J = 15.9
1
3
Hz, 1H), 6.45 – 6.37 (m, 1H), 4.56 (s, 2H), 3.91 (d, J = 7.1 Hz, 2H), 3.22 (s, 4H). C NMR (126
MHz, methanol-d ) δ 170.33, 145.49, 145.25, 144.82, 142.88, 137.93, 137.60, 137.53,
4
1
4
36.56, 136.15, 136.06, 127.59, 127.33, 126.64, 125.14, 124.05, 122.52, 118.94, 49.10,
+
6.30, 45.60, 39.02. HRMS calculated for C H N O S 449.16419 [M+H] , found 449.16390.
2
4 25 4 3
LCMS (ESI, Waters, C , linear gradient, 5% to 50% ACN in H O 0.2% TFA, 10 min): t =
5
1
8
2
R
+
.35 min; m/z : 449 [M+H] .
(E)-3-oxo-N-(2-((3-(4-(Pyridin-3-yl)phenyl)allyl)amino)ethyl)isoindoline-5-sulfonamide (9)
The title compound was synthesized from 6-
bromoisoindolin-1-one following general procedure A on a
0
.2 mmol scale and purified by preparative HPLC (XBridge,
C , 0% to 20% ACN in H O 0.2% TFA, 10 min gradient) to
1
8
2
yield the compound as a TFA salt after lyophilisation
1
(
16 mg, 14%). H NMR (600 MHz, methanol-d ) δ 9.04 (s,
4
1
H), 8.70 (d, J = 5.0 Hz, 1H), 8.57 (d, J = 8.2 Hz, 1H), 8.27 (s,
1
H), 8.13 (dd, J = 8.0, 1.6 Hz, 1H), 7.88 (dd, J = 8.1, 5.4 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.79
(
d, J = 8.3 Hz, 2H), 7.69 (d, J = 8.3 Hz, 2H), 6.97 (d, J = 15.9 Hz, 1H), 6.47 – 6.37 (m, 1H), 4.57
1
3
(s, 2H), 3.91 (d, J = 7.2 Hz, 2H), 3.26 – 3.22 (m, 2H), 3.22 – 3.17 (m, 2H). C NMR (151 MHz,
methanol-d ) δ 171.74, 150.24, 144.77, 144.25, 141.27, 141.26, 139.78, 139.04, 138.01,
4
1
4
36.84, 134.58, 131.36, 129.02, 128.76, 127.30, 126.15, 123.29, 120.70, 50.40, 47.67, 47.05,
+
0.34. HRMS calculated for C H N O S 449.16419 [M+H] , found 449.16386. LCMS (ESI,
2
4 25 4 3
Waters, C , linear gradient, 5% to 50% ACN in H O 0.2% TFA, 10 min): t = 5.39 min; m/z :
4
1
8
+
2
R
49 [M+H] .
(E)-N-Methyl-5-(N-(2-((3-(4-(pyridin-3-yl)phenyl)allyl)amino)ethyl)sulfamoyl) picolinamide
(
10)
The title compound was synthesized from 5-bromo-N-
methylpicolinamide following general procedure A on a
0
.2 mmol scale and purified by preparative HPLC (XBridge C ,
1
8
0
% to 20% ACN in H O 0.2% TFA, 10 min gradient) to yield the
2
1
compound as a TFA salt after lyophilisation (17 mg, 15%). H
NMR (600 MHz, methanol-d ) δ 9.07 (d, J = 2.0 Hz, 1H), 9.03
4
(
(
s, 1H), 8.70 (d, J = 5.2 Hz, 1H), 8.57 (d, J = 8.2 Hz, 1H), 8.40
dd, J = 8.2, 2.2 Hz, 1H), 8.27 (d, J = 8.2 Hz, 1H), 7.88 (dd, J =
8
.0, 5.4 Hz, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H), 6.97 (d, J = 15.9 Hz, 1H), 6.42
1
3
(dt, J = 15.7, 7.2 Hz, 1H), 3.91 (d, J = 7.1 Hz, 2H), 3.29 – 3.23 (m, 4H), 2.98 (s, 3H). C NMR
(
1
2
151 MHz, methanol-d ) δ 165.72, 154.28, 148.04, 144.92, 144.39, 141.12, 139.78, 139.70,
4
39.10, 137.95, 137.76, 136.94, 129.01, 128.76, 127.25, 123.41, 120.64, 50.43, 47.67, 40.30,
+
6.53. HRMS calculated for C H N O S 452.17509 [M+H] , found 452.17469. LCMS (ESI,
2
3 26 5 3

Waters, C , linear gradient, 5% to 50% ACN in H O 0.2% TFA, 10 min): t = 5.62 min; m/z :
1
8
+
2
R
4
52 [M+H] .
(E)-3-Amino-N-(2-((3-(4-(pyridin-3-yl)phenyl)allyl)amino)ethyl)isoquinoline-5-sulfonamide
(
11)
The title compound was synthesized from 5-
bromoisoquinolin-3-amine following general
procedure A on a 0.2 mmol scale and purified by
preparative HPLC (XBridge C , 10% to 20% ACN in
1
8
H2O 0.2% TFA, 10 min gradient) to yield the
compound as a TFA salt after lyophilisation (19 mg,
1
1
7%). H NMR (400 MHz, methanol-d ) δ 9.06 (s, 1H),
4
8
=
7
7
1
1
.98 (s, 1H), 8.72 (d, J = 5.2 Hz, 1H), 8.61 (d, J = 8.2 Hz, 1H), 8.29 (d, J = 7.3 Hz, 1H), 8.13 (d, J
8.2 Hz, 1H), 7.92 (dd, J = 8.0, 5.5 Hz, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H),
.60 (s, 1H), 7.36 (t, J = 7.8 Hz, 1H), 6.94 (d, J = 15.9 Hz, 1H), 6.46 – 6.36 (m, 1H), 3.89 (d, J =
1
3
.2 Hz, 2H), 3.19 (m, J = 8.6, 4.4 Hz, 4H). C NMR (101 MHz, methanol-d ) δ 156.42, 150.93,
4
44.36, 143.86, 141.72, 139.95, 138.95, 138.10, 136.93, 136.62, 136.47, 136.13, 132.35,
29.04, 128.77, 127.47, 124.30, 122.66, 120.79, 99.56, 50.39, 47.72, 40.16. HRMS calculated
+
for C H N O S 460.18017 [M+H] , found 460.17998. LCMS (ESI, Waters, C , linear gradient,
5
2
5
26
5
2
18
+
% to 50% ACN in H O 0.2% TFA, 10 min): t = 5.51 min; m/z : 460 [M+H] .
2
R
(E)-1-Amino-N-(2-((3-(4-(pyridin-3-yl)phenyl)allyl)amino)ethyl)isoquinoline-5-sulfonamide
(
12)
The title compound was synthesized from 5-
bromoisoquinolin-1-amine following general procedure A
on a 0.2 mmol scale and purified by preparative HPLC
(
XBridge C , 0% to 20% ACN in H O 0.2% TFA, 10 min
1
8
2
gradient) to yield the compound as a TFA salt after
lyophilisation (32 mg, 28%). H NMR (600 MHz, methanol-
1
d4) δ 8.96 (s, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.64 (d, J = 4.3
Hz, 1H), 8.60 (d, J = 8.7 Hz, 1H), 8.41 (dt, J = 8.1, 1.8 Hz, 1H), 7.94 – 7.88 (m, 2H), 7.79 – 7.73
(
m, 4H), 7.66 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 15.9 Hz, 1H), 6.41 (dt, J = 15.8, 7.2 Hz, 1H), 3.90
1
3
(d, J = 7.1 Hz, 2H), 3.22 (s, 4H). C NMR (151 MHz, methanol-d ) δ 156.25, 146.32, 145.70,
4
1
1
39.44, 139.12, 139.05, 137.61, 137.58, 137.46, 137.05, 135.36, 131.46, 130.51, 128.98,
28.93, 128.65, 126.65, 121.02, 120.43, 109.12, 50.47, 47.69, 40.15. HRMS calculated for
+
C H N O S 460.18017 [M+H] , found 460.18005. LCMS (ESI, Waters, C , linear gradient, 5%
to 50% ACN in H O 0.2% TFA, 10 min): t = 5.45 min; m/z : 460 [M+H] .
2
5
26
5
2
18
+
2
R
(E)-N-(2-((3-(4-(Pyridin-3-yl)phenyl)allyl)amino)ethyl)naphthalene-1-sulfonamide (13)
To a solution of tert-butyl (E)-(2-(naphthalene-1-
sulfonamido)ethyl)(3-(4-(pyridin-3-yl)phenyl) allyl)
carbamate (65) (0.270 g, 0.50 mmol, 1 eq) in DCM
(
5 mL) at 0 °C was added TFA (1 mL). The reaction
was allowed to warm to RT and stirred for 1 h before
it was concentrated under reduced pressure and re-
dissolved in DCM (20 mL) and sat. aqueous Na CO solution (20 mL). The organic layer was
2
3
collected and the aqueous layer extracted with DCM (4x20 mL). The combined organic layers
were dried over MgSO , filtered and concentrated under reduced pressure. The residue was
4

purified via flash-column-chromatography (SiO (neutralized with 1% Et N in DCM), 1.25% to
2
3
1
1
8
1
.5% MeOH in DCM) to yield the product (0.18 g, 81%). H NMR (400 MHz, chloroform-d) δ
.85 (d, J = 1.9 Hz, 1H), 8.70 (d, J = 8.6 Hz, 1H), 8.58 (dd, J = 4.8, 1.6 Hz, 1H), 8.29 (dd, J = 7.3,
.1 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.89 – 7.84 (m, 1H), 7.67 – 7.61
(m, 1H), 7.59 – 7.54 (m, 1H), 7.53 – 7.49 (m, 3H), 7.39 – 7.33 (m, 3H), 6.35 (d, J = 15.9 Hz,
1
2
1
1
H), 6.06 (dt, J = 15.9, 6.3 Hz, 1H), 3.17 (bs, 2H), 3.09 (dd, J = 6.3, 1.1 Hz, 2H), 3.03 – 2.98 (m,
H), 2.66 – 2.61 (m, 2H). C NMR (101 MHz, chloroform-d) δ 148.45, 148.07, 136.81, 136.69,
36.20, 134.52, 134.30, 134.29, 134.22, 130.69, 129.82, 129.20, 128.50, 128.42, 128.18,
1
3
27.26, 127.01, 126.95, 124.45, 124.26, 123.71, 50.87, 47.34, 42.51. HRMS calculated for
+
C H N O S 444.17402 [M+H] , found 444.17370. LCMS (ESI, Waters, C , linear gradient, 5%
to 90% ACN in H O 0.2% TFA, 10 min): t = 5.32 min; m/z : 444 [M+H] .
2
6
26
3
2
18
+
2
R
(E)-N-(2-((3-(4-(Pyridin-3-yl)phenyl)allyl)amino)ethyl)methanesulfonamide (14)
A round-bottom-flask was charged with tert-butyl (E)-(2-
(
isoquinoline-5-sulfonamido)ethyl)(3-(4-(pyridin-3-
yl)phenyl) allyl)carbamate (66) (107 mg, 0.25 mmol,
eq) dissolved in CHCl (8 mL). After cooling the
1
3
solution to 0°C and dropwise addition of TFA (2 mL), it
was allowed to warm to RT and stirred for 60 min. The
reaction was quenched by slow addition of sat. aqueous Na CO solution (12 mL) until a pH
2
3
of ~12 was reached and the mixture was extracted with DCM (3x10 mL). The combined
organic layers were dried over Na SO , filtered and concentrated under reduced pressure.
2
4
The residue was purified via flash-column-chromatography (SiO , 0% to 15% (10% of sat.
2
1
aqueous NH in MeOH) in DCM) to yield the product (52 mg, 63%). H NMR (600 MHz,
3
methanol-d ) δ 8.80 (d, J = 2.3 Hz, 1H), 8.50 (dd, J = 4.9, 1.4 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H),
4
7
.63 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.51 (dd, J = 8.0, 4.9 Hz, 1H), 6.65 (d, J = 15.9
Hz, 1H), 6.40 (dt, J = 15.9, 6.5 Hz, 1H), 3.46 – 3.42 (m, 2H), 3.23 (t, J = 6.3 Hz, 2H), 2.96 (s, 3H),
1
3
2
1
.80 (t, J = 6.3 Hz, 2H). C NMR (151 MHz, methanol-d ) δ 148.66, 148.11, 138.58, 138.15,
4
37.49, 136.26, 132.78, 129.03, 128.26, 128.23, 125.49, 51.91, 49.46, 43.26, 39.68. HRMS
+
calculated for C H N O S 332.14272 [M+H] , found 332.14267. LCMS (ESI, Waters, C ,
1
7
22
3
2
18
+
linear gradient, 5% to 50% ACN in H O 0.2% TFA, 10 min): t = 4.49 min; m/z : 332 [M+H] .
2
R
(E)-2-Nitro-N-(2-((3-(4-(pyridin-3-yl)phenyl)allyl)amino)ethyl)benzenesulfonamide (15)
To a solution of tert-butyl (E)-(2-((2-
nitrophenyl)sulfonamido)ethyl)(3-(4-(pyridin-3-
yl)phenyl)allyl)carbamate (67) (0.347 g, 0.64 mmol,
1
eq) dissolved in CHCl (4.8 mL) at 0 °C was added
3
dropwise TFA (1.2 mL). The reaction was allowed to
warm to RT and stirred for 2 h before it was
concentrated under reduced pressure. It was re-dissolved in DCM (20 mL) and sat. aqueous
Na CO solution (20 mL). The organic layer was collected and the aqueous layer extracted
2
3
with DCM (3x20 mL). The combined organic layers were dried over MgSO , filtered,
4
concentrated under reduced pressure and purified by preparative HPLC (Gemini, C , 10% to
1
8
3
5% ACN in H O 0.2% TFA, 10 min gradient) to yield the compound as a TFA salt after
2
1
lyophilisation (11 mg, 3%). H NMR (600 MHz, DMSO-d ) δ 8.99 (s, 1H), 8.86 (bs, 2H), 8.63
6
(
dd, J = 3.5, 1.3 Hz, 1H), 8.36 (d, J = 5.4 Hz, 1H), 8.23 (d, J = 6.9 Hz, 1H), 8.03 (dt, J = 6.1, 3.2
Hz, 2H), 7.91 (dd, J = 5.9, 3.3 Hz, 2H), 7.81 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 8.3 Hz, 3H), 6.87 (d, J
15.9 Hz, 1H), 6.40 – 6.30 (m, 1H), 3.81 (d, J = 5.1 Hz, 2H), 3.23 (d, J = 6.1 Hz, 2H), 3.10 (s,
=

1
3
2
1
4
H). C NMR (101 MHz, chloroform-d) δ 148.40, 147.98, 136.77, 136.61, 136.06, 134.11,
33.57, 133.39, 132.69, 131.07, 130.73, 128.68, 127.20, 126.98, 125.25, 123.65, 51.03,
7.46, 43.16. HRMS calculated for C H N O S 439.14345 [M+H] , found 439.14302. LCMS
+
22 23 4 4
(
ESI, Waters, C , linear gradient, 5% to 50% ACN in H O 0.2% TFA, 10 min): t = 6.71min;
18
2
R
+
m/z : 439 [M+H] .
E)-2-Amino-N-(2-((3-(4-(pyridin-3-yl)phenyl)allyl)amino)ethyl) benzenesulfonamide (16)
E)-2-Nitro-N-(2-((3-(4-(pyridin-3-yl)phenyl) allyl)
(
(
amino) ethyl)benzenesulfonamide (15) (84 mg,
.19 mmol, 1 eq) was dissolved in EtOH (0.32 mL),
0
AcOH (0.32 mL) and H O (0.16 mL) after which iron
2
powder (30 mg) was added and the vial was
sonicated for 2.5 h. The mixture was basified with
aqueous NaOH (1 M, 5.5 mL) solution, concentrated under reduced pressure, re-suspended
in DCM (5 mL) and sat. aqueous Na CO (5 mL) and filtered over filter paper. The filter was
2
3
rinsed with sat. aqueous Na CO (50 mL) and the filtrate was extracted with DCM (5x40 mL).
2
3
The combined organic layers were dried over MgSO , concentrated under reduced pressure
4
and purified by preparative HPLC (XBridge C , 10% to 35% ACN in H O 0.2% TFA, 10 min
1
8
2
1
gradient) to yield the compound as a TFA salt after lyophilisation (48 mg, 48%). H NMR (500
MHz, DMSO-d ) δ 9.00 (d, J = 1.8 Hz, 1H), 8.78 (bs, 2H), 8.65 (dd, J = 4.9, 1.5 Hz, 1H), 8.26 (d,
6
J = 8.1 Hz, 1H), 7.86 – 7.79 (m, 3H), 7.65 – 7.59 (m, 3H), 7.50 (dd, J = 8.0, 1.5 Hz, 1H), 7.31 –
7
.27 (m, 1H), 6.88 – 6.82 (m, 2H), 6.64 (t, J = 7.0 Hz, 1H), 6.34 (dt, J = 15.8, 6.9 Hz, 1H), 4.37
1
3
(bs, 2H), 3.83 – 3.72 (m, 2H), 3.08 – 2.96 (m, 4H). C NMR (126 MHz, DMSO-d ) δ 147.26,
6
1
1
46.41, 146.19, 136.36, 136.18, 135.59, 135.56, 135.41, 133.90, 129.13, 127.40, 127.35,
24.52, 120.42, 118.72, 117.12, 115.31, 48.38, 45.42, 38.47. HRMS calculated for
+
C H N O S 409.16927 [M+H] , found 409.16884. LCMS (ESI, Waters, C , linear gradient, 5%
to 90% ACN in H O 0.2% TFA, 10 min): t = 4.62 min; m/z : 409 [M+H] .
2
2
25
4
2
18
+
2
R
(E)-N-(2-((3-([1,1'-Biphenyl]-4-yl)allyl)amino)ethyl)isoquinoline-5-sulfonamide (17)
To a solution of tert-butyl (E)-(3-([1,1'-biphenyl]-4-
yl)allyl)(2-(isoquinoline-5-sulfonamido) ethyl)
carbamate (71) (0.387 g, 0.70 mmol, 1 eq) in DCM
(3.1 mL) at 0 °C was added TFA (3.1 mL) after which
the mixture was allowed to warm to RT. After
stirring for 30 min it was concentrated under
reduced pressure, re-dissolved in sat. aqueous NaHCO (30 mL) and DCM (30 mL), the
3
organic layer was collected and the aqueous layer extracted with DCM (3x30 mL). The
combined organic layers were washed with brine (1x50 mL), dried over MgSO , filtered and
4
concentrated under reduced pressure. The crude was purified via flash-column-
chromatography (SiO , 3% to 4% (10% of sat. aqueous NH in MeOH) in DCM) to yield the
2
3
1
desired product (0.150 g, 48%). H NMR (400 MHz, chloroform-d) δ 9.35 (d, J = 0.8 Hz, 1H),
8
–
1
.71 (d, J = 6.1 Hz, 1H), 8.48 – 8.42 (m, 2H), 8.18 (d, J = 8.2 Hz, 1H), 7.72 – 7.64 (m, 1H), 7.63
7.58 (m, 2H), 7.55 (d, J = 8.3 Hz, 2H), 7.44 (t, J = 7.6 Hz, 2H), 7.40 – 7.32 (m, 3H), 6.40 (d, J =
5.9 Hz, 1H), 6.08 (dt, J = 15.9, 6.4 Hz, 1H), 3.31 (bs, 2H), 3.17 (dd, J = 6.4, 1.3 Hz, 2H), 3.01
1
3
(dd, J = 6.4, 4.8 Hz, 2H), 2.69 (dd, J = 6.4, 4.9 Hz, 2H). C NMR (101 MHz, chloroform-d) δ
1
1
53.49, 145.39, 140.69, 140.44, 135.78, 134.27, 133.70, 133.49, 131.49, 131.36, 129.13,
28.92, 127.46, 127.40, 127.39, 127.02, 126.81, 126.02, 117.30, 51.01, 47.18, 42.41. HRMS