Illuminating a Dark Kinase: Structure-Guided Design, Synthesis, and Evaluation of a Potent Nek1 Inhibitor and Its Effects on the Embryonic Zebrafish Pronephros

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Illuminating a Dark Kinase: Structure-Guided Design, Synthesis, and

Evaluation of a Potent Nek1 Inhibitor and Its Eﬀects on the

Embryonic Zebraﬁsh Pronephros

Georg Baumann,* Tobias Meckel, Kevin B ö hm, Yung-Hsin Shih, Mirco Dickhaut, Torben Reichardt,

Johannes Pilakowski, Ulrich Pehl, and Boris Schmidt *

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ABSTRACT: NIMA-related kinase 1 (Nek1) has lately garnered

attention for its widespread function in ciliogenesis, apoptosis, and

the DNA-damage response. Despite its involvement in various

diseases and its potential as a cancer drug target, no directed

medicinal chemistry eﬀorts toward inhibitors against this dark

kinase are published. Here, we report the structure-guided design

of a potent small-molecule Nek1 inhibitor, starting from a scaﬀold

identiﬁed by kinase cross-screening analysis. Seven lead com-

pounds were identiﬁed in silico and evaluated for their inhibitory

activity. The top compound, 10f, was further proﬁled for eﬃcacy,

toxicity, and bioavailability in a zebraﬁsh polycystic kidney disease

model. Administration of 10f caused the expansion of

ﬂuorescence-labeled proximal convoluted tubules, supporting our hypothesis that Nek1-inhibition causes cystic kidneys in zebraﬁsh

embryos. Compound 10f displayed insigniﬁcant inhibition in 48 of 50 kinases in a selectivity test panel. The ﬁndings provide a

powerful tool to further elucidate the function and pharmacology of this neglected kinase.

Interestingly, NEK1 mutations have been linked not only to

defects of ciliogenesis, like PKD and short-rib polydactyly

syndrome type Majewski but also to other inheritable diseases

such as Mohr−Laussen syndrome or amyotrophic lateral

sclerosis (ALS), a neurodegenerative disorder that causes the

selective death of motor neurons and is associated with an

altered DNA damage response (DDR).¹⁰⁻¹³Like most of the

11 known members of the human Nek family, Nek1 plays a

crucial role in cell cycle control and in particular the DDR.¹⁴

Nek1 expression is upregulated upon chemical or radiative

induction of DNA double-strand breaks (DSBs) and acts as a

regulator of DNA repair by homologous recombination

(HR).^15,16Cell cycle phase-speciﬁc phosphorylation of

Rad54 by Nek1 either permits HR, by way of Rad51 removal

from chromatin in G2-phase, or ensures replication fork

stability in S-phase.¹⁷Moreover, functional Nek1 is indis-

pensable for proper cell cycle checkpoint activation through

phosphorylation of checkpoint kinases 1 and 2 (Chk1/Chk2)

INTRODUCTION

■

Nek1 is a dual serine/threonine protein kinase that also

possesses tyrosine kinase activity, albeit to a lesser degree.¹

Since its discovery in a murine cDNA expression library screen,

this mammalian homologue of the fungal NIMA cell cycle

regulator has attracted widespread attention due to its

pleiotropic function and its association with a number of

diseases in humans.²⁻⁴The ever-growing interest in this

remarkable kinase was ﬁrst sparked by ﬁndings that linked

Nek1 to two independent mutant alleles (kat and kat^2J), which

spontaneously arose from two inbred mouse strains, causing

various eﬀects such as male sterility, dwarﬁng, facial dys-

morphism, anemia, and a progressive polycystic kidney disease

(PKD).^5,6PKD is the most common genetic cause for renal

failure in humans and is characterized by the relentless growth

of large ﬂuid-ﬁlled cysts and the accompanying destruction of

the adjacent renal parenchyma.⁷The evidence for the causative

relation between Nek1 depletion and PKD was further

strengthened by an interactomic study, which indicated strong

interactions between the PKD-associated proteins KIF3A,

tuberin, and α-catulin and the central coiled-coil region of

Nek1.⁸Moreover, Nek1 expression levels are prominently

elevated in embryonic proximal tubule and podocyte precursor

cells and decrease with kidney maturation, supporting the

observation that Nek1 is of functional importance for the

etiology of PKD.⁹

Special Issue: New Horizons in Drug Discovery -

Understanding and Advancing Kinase Inhibitors

Received: December 8, 2020

American Chemical Society

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in response to radiative stress.¹⁸Eventually, Nek1-deﬁcient

cells accumulate unrepaired DSBs leading to genomic

instability, chromosomal damage, and ﬁnally cell death.¹⁹

A

remarkable feature of Nek1 is its ability to also limit apoptotic

cell death even after severe damage has occurred already.

Direct phosphorylation of voltage-dependent anion channel 1

(VDAC1) by Nek1 stabilizes the mitochondrial membrane

potential thus limiting the caspase-mediated killing of cells.^20,21

More recent evidence suggests that an activating phosphor-

ylation of Nek1 by tousled-like kinase 1 (Tlk1) contributes to

the phosphorylation of VDAC1 and that inhibition of the

Tlk1/Nek1/VDAC1 axis in combination with DNA damaging

agents can sensitize prostate cancer cells to apoptotic

killing.²²⁻²⁵Elevated levels of Nek1 expression have also

been identiﬁed in other cancer types like thyroid cancer,

human gliomas, or renal cell carcinoma (RCC).²⁶⁻²⁸In the

latter case, a decreased sensitivity to genotoxic treatment and

ionizing radiation was associated with high levels of Nek1,

emphasizing the importance of Nek1 inhibition as a

therapeutic strategy for drug development in the treatment

of RCC.^28,29More recently, Freund et al. have shown that high

Nek1 expression levels associate with impaired clinical

outcome in cervical cancer patients following chemo-radio-

therapy or brachytherapy and that a Nek1 knockdown impacts

the 3D clonogenic survival of HeLa cervical and HCT-15

colorectal carcinoma cells. Furthermore, the knockdown

sensitized those cells to ionizing radiation, underlining the

potential of Nek1 inhibitors to increase the radiation or

chemotherapy response in anticancer treatment.³⁰

Despite the crucial role of Nek1 in a broad variety of

diseases and its outstanding potential as a target for anticancer

therapeutics, no directed medicinal chemistry eﬀorts toward

potent and selective Nek1 inhibitors have been published in

the past.^4,31In a ﬁrst attempt to pinpoint structural elements

that inﬂuence Nek family inhibitor speciﬁcity, Moraes et al.

identiﬁed a c-Jun N-terminal kinase (JNK) inhibitor with

limited inhibitory activity against a truncated Nek1 activation

loop mutant in a thermal shift assay.³²Shortly thereafter, the

same group reported the crystal structure analysis of the

human Nek1 kinase domain both in its apo form and in

complex with an ATP-competitive CDK inhibitor.³³Until

today, only three members of the human Nek family, namely,

Nek1, -2, and -7, have been structurally solved. More recently,

a pyridin-2(5H)-one based inhibitor with a low micromolar

IC₅₀value against Nek6 was also shown to decrease Nek1

activity to a comparable degree.³⁵In the framework of

developing a comprehensive kinase chemogenomic set, Wells

et al. compiled chemical starting points for Nek-family

inhibitors by performing an intricate analysis of kinome

cross-screening data.³⁴In the case of Nek1, two series of

compounds with promising activities were identiﬁed from the

proﬁling data of a large inhibitor set (Published Kinase

Inhibitor Set 2, PKIS2).³⁶The most potent candidate

(UNC5078) of a series of 4-thiophene-7-azaindoles exhibited

91.9% Nek1 inhibition at 1 μM.^34,37Strikingly, the 4-aryl-7-

azaindole derivative UNC5452 (3a), which was originally

intended as an IκB kinase β (IKKβ) inhibitor, exhibited even

higher Nek1 activity (99.8% inhibition at 1 μM) while only

inhibiting 18 oﬀ-target kinases ≥90% at 1 μM (Figure 1).^34,38

Nevertheless, ﬁnding suitable inhibitors for Nek1 is not trivial,

as indicated by the low hit rates of previously reported

screenings.^33,34

Figure 1. Chemical starting points for the development of Nek1

inhibitors, as identiﬁed by Wells et al. in an analysis of kinome cross-

screening data.³⁴

In this work, we report the structure-guided design and

synthesis of several novel Nek1 inhibitors. The 4-phenyl-7-

azaindole moiety of UNC5452 (3a) served as a starting point

and was expanded upon by in silico analysis, molecular docking

simulations, and a virtual chemical library screen in P-loop

reconstructed Nek1 crystal structures (PDB codes 4APC and

4B9D) as well as a Nek1/Nek2 homology model. The most

potent compound 10f was further proﬁled in a kinase

selectivity panel, a Caco-2 permeability assay, and a zebraﬁsh

developmental toxicity assay. In vivo eﬃcacy of 10f was

evaluated by ﬂuorescence microscopy imaging of zebraﬁsh

pronephroi after inhibitor treatment, which is an established

model for cystic kidney diseases.³⁹

CHEMISTRY

■

The synthesis of UNC5452 (3a) and its derivatives

commenced with the amide formation between sulfonyl

chloride 1 and the respective primary or secondary amines

to aﬀord sulfonamides 2a−h and 2j−l. Acetyloxyester 2i was

prepared from alcohol 2f by acetylation with acetic anhydride.

Miyaura borylation of aryl halides 2a−l gave the corresponding

pinacol boronic acid esters, which were fused with 4-bromo-7-

azaindole by palladium catalyzed Suzuki cross-coupling

forthwith, yielding 4-phenyl-7-azaindoles 3a−l (Scheme 1).⁴⁰

In an attempt to synthesize the carboxylic amide analogue 6 of

sulfonamide 2f under identical conditions, 4-bromobenzoyl

chloride (4) was transformed to the undesired ester 5.

However, alkaline hydrolysis of 5 provided benzamide 6,

which was coupled to 4-bromo-7-azaindole, giving azaindole 7

in the ﬁnal Suzuki cross-coupling step. (Scheme 2). The amino

alcohols 8a−f were converted to ﬁnal compounds 10a−f

through a similar sequence described in Scheme 1.

Compounds 8a−c were obtained commercially, while 8d was

prepared by reduction of ^D-tyrosine methyl ester with sodium

borohydride and 8e and 8f were prepared by reduction of ^L-

tryptophan and ^D-tryptophan, following a procedure by Meyers

et al.^41,42Amide formation between 4-bromosulfonyl chloride

(1) and amino alcohols 8a−f provided aryl halides 9a−f, which

again underwent Miyaura borylation and subsequent Suzuki

cross-coupling to aﬀord azaindoles 10a−f (Scheme 3).

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a

Scheme 1

a

Reagents and conditions: (a) for 2a−h, DCM, amine, NEt₃, 0 °C−rt, 74−95%; (b) DMF, bis(pinacolato)diboron, KOAc, PdCl₂(dppf), 100 °C,

not isolated; (c) DMF, 4-bromo-7-azaindole, NaHCO₃(aq), Pd(PPh₃)₄, 100 °C, 11−69%; (d) for 2j, DCM, amine, NEt₃, 0 °C−rt, 64%; for 2k,

THF, 1-methylpiperazine, rt, 75%; for 2l, H₂O/acetone, L-phenylalanine, NaHCO₃, rt, 49%. *Compound 2i was prepared from 2f by acetylation

with acetic anhydride and NEt₃in DCM, 82%; see Experimental Section for more details.

a

Scheme 2

a

Reagents and conditions: (a) DCM, monoethanolamine, NEt₃, 0 °C−rt, 48%; (b) THF, 50% w/v KOH (aq), 60 °C, 92%; (c) DMF,

bis(pinacolato)diboron, KOAc, PdCl₂(dppf), 100 °C, not isolated; (d) DMF, 4-bromo-7-azaindole, NaHCO₃(aq), Pd(PPh₃)₄, 100 °C, 43%; see

Experimental Section for more details.

the Nek1 structures both crystallized as disulﬁde bridged

dimers.

RESULTS AND DISCUSSION

■

Molecular Modeling. As an initial step of the structure-

In an attempt to identify areas of conformational mobility in

the active site, we isolated and superimposed the monomeric

kinase domains using the Molecular Operating Environment

(MOE) software platform (Figure S1). Interestingly, the DFG-

motif aspartate (D146) and the gatekeeper (M80) formed

hydrogen bonds with an internal water molecule in the ATP

binding site in all but one of the isolated chains (PDB code

4APC.A). In this particular case, the side chains of D146 and

M80 extend outward in absence of the water molecule, thus

opening a hydrophobic back-pocket toward the DFG-motif.

This conformational mobility might be exploited by locking

Nek1 in an inactive conformation with a small molecule

inhibitor. In order to assess the ligand−protein interactions

and the amenability of the back-pocket, we docked UNC5452

guided design, the available Nek1 kinase domain crystal

structures were analyzed in detail. Both the apo (PDB code

4APC) and the CDK-inhibitor bound 3D structures (PDB

code 4B9D) were obtained from the same inactivating T162A

activation loop mutant and expectedly constitute an inactive

“DFG-out/C-helix-out” conformation. The activation loop

forms an ordered α helix, which is speculated to unfold

upon autophosphorylation, thus adopting a disordered

conformation more commonly found in active kinases.³³

This activation mechanism might to some degree be conserved

in the Nek-family, as a similar α-helical activation loop was

observed in Nek2 structures but not in Nek7, which is

activated through a diﬀerent mechanism that involves binding

of the noncatalytic C-terminal domain of Nek9.^43,44Moreover,

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a

Scheme 3

a

Reagents and conditions: (a) for 9a,b, DCM, amine, NEt₃, 0 °C−rt, 98−99%; for 9c,d, H₂O/acetone, L-phenylalanine, NaHCO₃, rt, 60−70%; for

9e,f, DCM/acetonitrile, N,N-diisopropylethylamine, 0 °C−rt, 30−43%; (b) DMF, bis(pinacolato)diboron, KOAc, PdCl₂(dppf), 100 °C, not

isolated; (c) DMF, 4-bromo-7-azaindole, NaHCO₃(aq), Pd(PPh₃)₄, 100 °C, 26−62%. *Compounds 8a−c were obtained commercially;

Compound 8d was prepared from ^D-tyrosine methyl ester hydrochloride by freeing the amine with KOAc in acetonitrile and subsequent reduction

with NaBH₄in methanol, 61%; compounds 8e,f were prepared by reduction of the corresponding tryptophan isomer using the method described

by Meyers et al.,⁴¹72−84%; see Experimental Section for more details.

Figure 2. In silico chemical library screening to identify hit compounds. See Experimental Section for more details.

(3a) into the Nek1 ATP-binding site (PDB code 4APC.A,

Figures S2 and S3).

region of Nek1 through a narrow channel formed by the side

chains of F135, A18, I10, and the catalytic lysine (K33).

Unfortunately, the terminal nitrogen atom of K33 is not

structurally resolved, but it appears that its side-chain ﬂexibility

and the close proximity to the sulfonamide oxygen allow for

hydrogen bonding. The proposed binding mode is supported

by the 9-fold reduction of activity of carboxylic acid 7 against

Nek1, when compared with sulfonamide 3f. It appears that the

tetrahedral geometry of the sulfonamide group is crucial for

proper orientation of the ligand. Consequently, the sulfona-

mide nitrogen is positioned downward, toward the catalytic

loop. As proposed by the docking experiment, the sulfonamide

NH is not acting as a hydrogen bond donor, as the methyl

The docking experiments suggest that the 7-azaindole

moiety positions itself between V31 and F135 side chains

and forms two hydrogen bonds with the backbone

heteroatoms of the hinge region C83. The pyrrole ring is

packed tightly against the side chain of Y82 and directed

toward the solvent exposed area of the ATP-binding site, thus

adopting the so-called “ﬂipped” binding mode. The “normal”

binding mode, where the pyrrole ring points toward the

gatekeeper M80, is also conceivable but was not favored in our

docking experiments.⁴⁵From the hinge-binding motif, the

phenyl ring of UNC5452 protrudes toward the ribose-binding

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substitution of the secondary sulfonamide hydrogen from 3f to

3j slightly increased potency.

reconstructed X-ray structure of Nek1 (Figure 3). In similar

fashion to 3a, the 7-azaindole moiety is tightly packed between

As expected, the highly ﬂexible dimethylaminoethyl frag-

ment was able to adopt multiple conformations. Unfortunately,

our experiments led to the conclusion that an entrance into the

hydrophobic back pocket from the 4-phenyl-7-azaindole

scaﬀold is not trivial. Due to the intrusion of the F135 and

Y82 side chains into the hinge-binding region, the 7-azaindole

moiety is angled in a way that hypothetically prevents access to

the back-pocket from the 5-position in the “ﬂipped” binding

mode. In the disfavored “normal” binding mode, the same

problem arises for the 3-position and an entry from the 2-

position is presumably blocked by the gatekeeper (M80).

Moreover, the docking shows that an approach from the ortho

position of the scaﬀold’s phenyl ring is likely also blocked by

M80 (Figure S4). It remains unclear if scaﬀold hopping to a

ﬁve membered aromatic ring as in UNC5078 would

suﬃciently widen the C−C−C bond angle to allow bypassing

of the gatekeeper. Laufer and colleagues have successfully

applied this strategy in the development of inhibitors for EGFR

Met gatekeeper mutants in the past.⁴⁶

As a consequence of these ﬁndings, we shifted our attention

to the derivatization of the dimethylaminoethyl fragment with

means to optimize existing and to establish new interactions in

the ribose-binding region. Depending on the protonation state

of the tertiary amine, interactions with the side chains of the

catalytic loop residues D128 or Q132 or the unresolved side

chains of the glycine-rich loop (P-Loop) residues E12 and S14

are conceivable. In order to establish a structure−activity

relationship (SAR), we synthesized and evaluated compounds

3b−k, with a range of steric demands as well as varying basicity

and hydrogen bond capabilities. The most potent compound

of the series, alcohol 3f, served as the starting point for an in

silico virtual library docking experiment to identify virtual hit

compounds (Figure 2). We reasoned that the structural

relation of the sulfonamide linked side chain to N-substituted

α-amino acids and their corresponding alcohols allows us to

make use of nature’s own toolkit for protein binding.

Moreover, this strategy ensured the convenient access to

stereochemically pure starting materials and a straightforward

synthetic approach.

The virtual library included a total of 80 compounds that

correspond to both isomers of the 20 canonical amino acids

and their respective amino alcohols. Molecular docking of the

virtual library into the Nek1 kinase domain required

reconstruction of the P-loop, which was performed using two

independent methods. Starting from the ligand-bound B-chain

(PDB code 4B9D), which was chosen because of its shorter

unresolved gap, we ﬁrst reconstructed the loop using the

SuperLooper2 web application.⁴⁷Second, a homology model

of Nek1 was created using the X-ray structure of Nek2 bound

to an aminopurine inhibitor (Figure S7).⁴⁸Seven virtual hit

compounds were identiﬁed by analysis of the docking results.

The selection was based on docking score (S-Score),

topological polar surface area (TPSA), ligand eﬃciency, and

visual inspection of the docked conformations. Both methods

provided similar results, favoring aromatic amino alcohol side

chains (10a−f) and an aromatic amino acid side chain (3l).

Interestingly, the structurally related Nek1 kinase cross

screening hit UNC5078 also possesses an equivalent benzyl

group, although the stereoinformation is missing.³⁴The

putative binding mode of the series most potent compound

10f was determined from molecular docking into the

Figure 3. Molecular docking of compound 10f into X-ray structure of

Nek1 (PDB code 4B9D.B). P-loop reconstruction was performed

using the SuperLooper2 web application.⁴⁷See Experimental Section

and Supporting Information for more details (Figures S5 and S6).

V31 and F135 in the “ﬂipped” binding mode and forms two

hydrogen bonds with the hinge region C83. Furthermore, the

docking experiment indicates C−H−π interactions of the

heterocyclic rings with G86 and V31 side chains as well as a

contingent parallel-displaced π−π stacking interaction with

F135. The phenyl ring of 10f once again protrudes toward the

ribose binding region and positions the sulfonamide moiety in

close proximity to the catalytic lysine (K33), forming a

hydrogen bond. Resulting from this interaction, the hydroxyl

group of 10f acts as a hydrogen donor toward the backbone

carbonyl oxygen of I10, while the indole moiety ﬁts tightly

between K17, Q132, and R161. From here, the indole NH

forms a hydrogen bond with the side chain of D128, while the

aromatic system establishes a C−H−π interaction with the

T166 backbone NH. Judging by these results, 10f can be

classiﬁed as a type IIB inhibitor that binds to an inactive DFG-

out conformation without occupying the back cleft.⁴⁹

Biological Assays and Structure−Activity Relation-

ship (SAR) Studies. As a ﬁrst step, we synthesized UNC5452

(3a) in order to benchmark a commercial in vitro Nek1 activity

assay by Euroﬁns Discovery (KinaseProﬁler Item 15-020

KP10). Compound 3a inhibited Nek1 activity by only 63% at

1 μM in this ATP-dependent radiometric assay, compared to

the 99.8% inhibitory activity reported by Wells et al.³⁴We

hypothesize that this diﬀerence results from the fact that

screening of the parent PKIS2 data set was performed using an

ATP-independent competition-binding assay (DiscoverX

KINOME-scan) that instead measures the ability of a small

molecule to compete for the active site with an immobilized

inhibitor.³⁶

The molecular modeling suggested interactions of 3a with

the Nek1 hinge C83, catalytic K33, and the catalytic loop

residues D128 and Q132 to be essential for potent inhibition.

Since our primary aim was the optimization of the

dimethylaminoethyl fragment, we ﬁrst opted for the improve-

ment of the terminal dimethylamino group. In the light of our

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docking experiments and the signiﬁcant drop in activity upon

removal of the two methyl groups, from 3a to primary amine

UNC5023 in the PKIS2 data set,³⁴we investigated the

inﬂuence of the amine pK_aby synthesis and evaluation of

compounds 3b−e (Table 1). Overall, the radiometric Nek1

loop residues D128 and Q132, as suggested by the docking

experiment, is elusive and that the introduction of an

alternative hydrogen bond donor might improve inhibitory

activity. Synthesis and evaluation of alcohol 3f revealed a

signiﬁcant decrease in residual Nek1 activity to 12% as well as

a more than 2-fold decreased IC₅₀of 3.2 μM, compared to 3a.

In order to evaluate the hydrogen bond characteristics of the

hydroxy group in 3f, methyl ether 3g and acetyl ester 3i

analogues were prepared and tested (Table 2). Both analogues

Table 1. Inhibitory Activity of Compounds 3a−f and 3k

against Nek1 and Nek2

Table 2. Inhibitory Activity of Compounds 3g−j and 7

against Nek1 and Nek2

a

b

Euroﬁns Discovery KinaseProﬁler service at 10 μM ATP. LANCE

a

b

Euroﬁns Discovery KinaseProﬁler service at 10 μM ATP. LANCE

#

Ultra TR-FRET kinase assay at K_m,ATP. A relative IC₅₀value was

provided because the determined IC₅₀value exceeded the employed

maximum test concentration (30 μM). See Experimental Section for

more details. See Supporting Information for raw data. CI, conﬁdence

interval; nd, not determined; ne, no eﬀect at maximum concentration

(30 μM).

#

Ultra TR-FRET kinase assay at K_m,ATP. A relative IC₅₀value was

provided because the determined IC₅₀value exceeded the employed

maximum test concentration (10 μM). See Experimental Section for

more details. See Supporting Information for raw data. CI, conﬁdence

interval; nd, not determined; ne, no eﬀect at maximum concentration

(30 μM). Euroﬁns Discovery IC50Proﬁler service at K_m,ATP

c

.

assay revealed no clear trend between the amine basicity and

kinase binding. Compounds 3b, 3d, and 3e displayed

decreased activity in comparison to 3a, while 3c showed

comparable activity. The results were conﬁrmed by the

determination of IC₅₀values for 3a−e in a TR-FRET based

showed decreased inhibitory activity compared to 3f, with an

IC₅₀larger than 30 μM for ether 3g and 8.8 μM for ester 3i.

The results clearly indicate that the hydroxy group in 3f does

indeed act as a hydrogen bond donor and not as an acceptor,

as the reduced acceptor capability in 3i did not further reduce

inhibitory activity. It remains unclear if the carbonyl oxygen in

3i acts as an alternative hydrogen bond acceptor but it is

unlikely to interact with the same region, due to its diﬀerent

position relative to the rest of the molecule. Moreover, the

introduction of a bulky tertiary alcohol in 3j led to a

signiﬁcantly decreased IC₅₀of 5.8 μM. With the improved

inhibitory activity of 3f in hand, we switched our focus to the

sulfonamide moiety (Table 2). As previously suggested by

docking of 3a into the Nek1 ATP binding site, the methyl

replacement of the secondary sulfonamide hydrogen from 3f to

3h did not inﬂuence Nek1 inhibition signiﬁcantly. However,

substitution of the tetrahedral sulfonamide group with a planar

carboxamide in 7 led to a 9-fold increased IC₅₀compared to 3f,

Nek1 activity assay (LANCE Ultra TR-FRET, K_m,ATP

,

PerkinElmer). Strikingly, no eﬀect on Nek1 activity was herein

observed at the maximum concentration (30 μM) for 3e,

which reduced Nek1 activity to 49% in the radiometric assay.

Furthermore, compound 3d displayed a signiﬁcant decrease in

inhibitory activity compared to 3b, in contrast to the previous

ﬁndings. Apart from these discrepancies, the results conﬁrm

that a variation of the terminal amine’s basicity did not

improve inhibitory activity against Nek1. Rigidiﬁcation of the

ﬂexible dimethylamino-ethyl fragment in compound 3k also

signiﬁcantly decreased inhibitory activity against Nek1 to over

10 μM from 7.1 μM in 3a. As a result, we reasoned that the

attempted optimization of interactions of 3a with the catalytic

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providing evidence for the proposed importance of the

sulfonamide geometry in hydrogen bonding to K33. In

addition, compounds 3a−k and 7 were proﬁled for their

Nek2 inhibitory activity using the same TR-FRET based assay

platform. In concordance with the Nek-family selectivity data

for 3a reported by Wells et al.,³⁴none of the tested compounds

exerted an observable eﬀect on the kinase activity of Nek2.

As described in the molecular modeling section, seven

virtual hit compounds (3l and 10a−f) were identiﬁed in an in

silico screening from a library of canonical N-substituted α-

amino acid derivatives, and their corresponding alcohols,

structurally based on 3f. Accordingly, phenylalanine derivative

3l and phenylalaninol, tyrosinol, and tryptophanol derivatives

10a−f were synthesized and evaluated (Table 3). The ^L-

phenylalanine derivative 3l did not notably decrease Nek1

activity, while the corresponding phenylalaninol derived

enantiomers 10a and 10b displayed decreased Nek1 IC₅₀

values of 1.9 μM and 1.3 μM, respectively. The tyrosinol

derivatives 10c and 10d performed slightly worse, while the ^L-

tryptophanol derivative 10e exhibited a decreased IC₅₀

compared to 10a (0.99 μM). Strikingly, the D-tryptophanol

derivative 10f showed 6-fold decreased IC₅₀of 0.33 μM against

Nek1 compared to 3f. Our docking experiments suggest that

this decrease results from the generally favored orientation of

the (R)-enantiomers, which in this particular case gives rise to

an additional hydrogen bond interaction of the indole NH with

the D128 side chain as well as a C−H−π interaction of the

aromatic system with the T166 backbone NH (Figure 3).

Kinase selectivity proﬁling of 10f against 50 kinases was

carried out with ATP concentrations set within 15 μM of the

apparent K_mfor ATP for each kinase tested, so that the

resulting selectivity proﬁle directly reﬂects the intrinsic

aﬃnities of 10f.⁵⁰Of the 50 kinases in this focused panel,

including several members of the Nek family, 47 showed

activity higher than 75%, whereas Nek1 displayed a residual

activity of only 49.7%. The only kinase that was also

signiﬁcantly inhibited by 10f was IKKβ with a residual activity

of 36.1% (Figure 4). However, given that the selectivity proﬁle

is based on a limited number of kinases (approximately 10% of

the kinome) and does not extend to enzymes beyond the

kinome, caution must be exercised in deducing general

selectivity from the data.

Table 3. Inhibitory Activity of Compounds 3l and 10a−f

against Nek1

Zebraﬁsh Developmental Toxicity Assay and Fluo-

rescence Microscopy Imaging of Zebraﬁsh Pronephroi.

In order to evaluate whether its in vitro inhibitory activity

translates into predictable eﬀects in vivo, compound 10f was

further proﬁled for its activity, safety, and toxicity in wild-type

zebraﬁsh (Danio rerio). For this purpose, we established a PKD

model based on a previously mentioned report by Chen et al.,

which indicates that Nek1 deﬁciency results in renal cyst

formation as a consequence of disordered kidney maturation in

kat^2jmice.⁹Indeed, the zebraﬁsh has proven itself as an

established model system for the evaluation of mammalian

kidney morphology, because of its simple pronephric structure,

which consists of two nephroi that share major anatomical and

functional features with the mammalian metanephros. Its

usefulness as a model for cystic kidney diseases has recently

been reviewed.^39,51Furthermore, it has been shown that the

majority of human kinases display high degrees of identity with

their zebraﬁsh homologues. Human Nek1, for example, shares

89% identity to zebraﬁsh Nek1 for the catalytically active

kinase domain, according to a study by Wlodarchak et al.⁵²

Moreover, all residues of the active site appear unaltered in

a

b

Euroﬁns Discovery KinaseProﬁler service at 10 μM ATP. Euroﬁns

Discovery IC50Proﬁler service at K_m,ATP. See Experimental Section for

more details. See Supporting Information for raw data. CI, conﬁdence

interval; nd, not determined; ne, no eﬀect at maximum concentration

c

(10 μM). Euroﬁns Discovery IC50Proﬁler service at 10 μM ATP.

sequence analysis (Figures S8 and S9). We therefore

hypothesized that treatment with 10f, during early stages of

embryonic organ development, leads to the formation of renal

cysts, thus indicating eﬃcient absorption and in vivo eﬃcacy of

10f. It is important to note here that our model is based on the

necessary assumption that Nek1 inhibition results in the same

phenotype that is associated with its depletion, as the

correlation between small-molecule inhibition and loss-of-

function phenotypes is not always straightforward.⁵³However,

our approach is substantiated by the reported link of aberrant

cystic growth to the absence of Nek1, as well as to its

overexpression.⁵⁴Hence, the authors conclude that healthy

ciliogenesis requires a delicate spatiotemporal activity of Nek1,

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Figure 4. Screening of Compound 10f against a panel of human protein kinases. Each bar represents the activity of one individual protein kinase.

Compound 10f was tested at a concentration of 1 μM. The ATP concentration was set to the approximate K_m,ATPfor each kinase tested. See

Supporting Information for more details.

which is likely to be substantially disturbed by inhibition and

depletion alike.

S12). After 48 h, all embryos were treated with 100 nM PT-

Yellow for 20 min, thoroughly washed with E3-medium, and

returned to the respective compound solution for a total

incubation time of 96 h. Finally, the embryos were

anesthetized and transferred to agarose molds, which were

prepared in 96-well plates according to a published procedure,

for ﬂuorescent imaging in a wideﬁeld microscope (Figure

S13).⁵⁵Administration of 10f caused a substantial increase in

ﬂuorescence intensity (Figure 5D) and expansion (Figure 5E)

of the proximal convoluted tubules, thus indicating the growth

of ﬂuid-ﬁlled cysts, alongside an impaired renal clearance. In

addition, our ﬁndings provide further evidence for the

adequate absorption and bioavailability of 10f. As hypothe-

sized, our experiments thus demonstrate that Nek1 inhibition

by 10f during early organ development causes cystic kidney

growth in zebraﬁsh embryos, despite our ﬁndings that 10f is

unselective against IKKβ. Park et al. have shown that levels of

phosphorylated-IKKα/β and its downstream factor NF-κB are

upregulated in the renal tissue of PKD2 transgenic mice

compared to wild-type controls.⁵⁸

Moreover, Booij et al. demonstrated that cystic growth was

in fact reduced by IKKα/β-inhibitors in a murine 3D cell

culture based screening platform.⁵⁹It therefore appears that

Nek1 inhibition overturns any presumed protective eﬀects of

IKKβ inhibition on cystogenesis in our experiments. It should

be noted here that, unlike in murine models, zebraﬁsh IKKα is

suspected to negatively regulate NF-κB activity. IKKα^−/−

zebraﬁsh embryos displayed severe head and tail malforma-

tions, as well as an upregulated NF-κB response and increased

NF-κB-dependent apoptosis, in a study by Correa et al.⁶⁰In

accordance with the low IKKα-activity of 10f (see Table S1),

this phenotype was not observed in our experiments. Overall,

this leads to the conclusion that the aberrant cystic growth in

10f-treated zebraﬁsh embryos is unlikely to be mediated by

IKK cross-reactivity. However, a causative relation to hitherto

In order to visualize the inﬂuence of 10f on larval zebraﬁsh

nephrogenesis, we implemented a ﬂuorescence-microscopy

based imaging pipeline developed by Westhoﬀ et al. (Figure

5).⁵⁵As a means of avoiding dependence on the transgenic

Tg(wt1b:EGFP) zebraﬁsh line, we instead opted for the use of

PT-Yellow (BDNCA3-D2), a ﬂuorescent probe that selectively

labels the renal proximal tubules in wild-type zebraﬁsh.⁵⁶This

approach allowed for zebraﬁsh maintenance and husbandry in

a standard laboratory, and it was furthermore reasoned that the

ingestion of an external probe is better suited to indicate the

impaired renal clearance associated with ﬂuid-ﬁlled cysts.⁵⁷As

a ﬁrst step, a bioavailability proﬁle of compound 10f was

evaluated. The aqueous solubility of 10f in E3-medium (13.9

0.1 μg/mL, Figure S10) was determined in a shake-ﬂask

solubility assay, and apical to basolateral transport was

determined in a Caco-2 permeability assay (3.4 × 10⁻⁶cm/

s, 45% recovery, Table S3). We then determined the minimal

lethal concentrations for compound 10f in a zebraﬁsh

developmental toxicity assay over a duration of 120 h.

Compound 10f was added to 6 h postfertilization (hpf)

embryos in concentrations ranging from 0.05 μM to 30 μM

(Figure S11). Full lethality was only observed close to the

solubility limit for a concentration of 30 μM after 72 h.

Treatment with 10f did not induce obvious phenotypes visible

to the eye. The treated embryos exhibited normal eye

development and body curvature and an ordinary heartbeat.

Based on these results, zebraﬁsh embryos were incubated

with (i) the negative control (E3-medium), (ii) 0.2% DMSO,

(iii) 10 μM 10f, and (iv) 20 μM 10f in E3-medium containing

75 μM 1-phenyl-2-thiourea (PTU) as a melanogenesis

inhibitor at 6 hpf. To ensure the stability of 10f against PTU

and PT-Yellow over the duration of the experiment, a stability-

indicating HPLC assay was performed in E3-medium (Figure

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Figure 5. Microscopic imaging of ﬂuorescence-labeled (PT-Yellow) zebraﬁsh pronephroi after treatment with Nek1-inhibitor 10f. (A) Schematic

representation of the workﬂow, entailing embryo selection, compound treatment, and image acquisition. (B) Schematic representation of the

(C) Exemplary maximum intensity projections of deconvolved Z-stacks (15× magniﬁcation; 33 Z-slices; dZ = 15 μm). The dotted line indicates

the cropped region of interest. (C, i) negative control E3-medium, (C, ii) negative control 0.2% DMSO, (C, iii) 10 μM 10f, and (C, iv) 20 μM 10f.

(D) Quantiﬁcation of ﬂuorescence intensity based on summed image projections of deconvolved Z-stacks using ImageJ. n = 3 for negative control,

0.2% DMSO, and 10 μM 10f; n = 5 for 20 μM 10f (^#P = 0.0516; *P < 0.05). (E) Quantiﬁcation of pronephroi size by summed pixel area

determination based on summed image projections of deconvolved Z-stacks using ImageJ. n = 3 for negative control, 0.2% DMSO, and 10 μM 10f;

n = 5 for 20 μM 10f (*P < 0.05; **P < 0.01). Abbreviations: au, arbitrary unit; C, Cloaca; CS, corpuscles of Stannius; DE, distal early tubule; DL,

distal late tubule; hpf, hours post fertilization; N, neck region; P, podocytes of the glomerulus; PCT, proximal convoluted tubule; PD, pronephric

duct; PST, Proximal straight tubule.

unidentiﬁed cross-reactivity cannot be ruled out entirely, due

to the aforementioned limitations of the selectivity test panel

and the fact that the complex PKD-related molecular pathways

and regulatory networks are not yet fully understood.⁶¹⁻⁶³

aryl-7-azaindole scaﬀold. The most potent compound of this

series, 3f, served as the structural basis of a virtual structure

library that encompassed 80 N-substituted amino acids and

their corresponding alcohols. Seven virtual hits were thereupon

identiﬁed in an in silico virtual library screen in two

independent P-loop reconstructed Nek1 3D-structures. This

natural product based approach allowed for convenient access

to enantiomerically pure starting materials and a straightfor-

ward synthetic route and provided the opportunity to exploit

nature’s own toolkit for protein binding. Our eﬀorts

culminated in the ATP-competitive Nek1 inhibitor 10f that

displayed IC₅₀values in the mid to low nanomolar range.

CONCLUSION

■

Based on a published chemical starting point, we generated

hypotheses for improved interactions with the Nek1 ATP-

binding site by molecular docking into the available crystal

structures. These hypotheses were challenged by a series of

structurally related inhibitors that were based on the same 4-

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(774 mg, 9.78 mmol) at 0 °C under cooling in an ice bath. The

cooling bath was removed, and the solution was stirred for 1 h at

ambient temperature. The organic layer was washed with water (3 × 5

mL) and brine (5 mL) and dried over MgSO₄. The solvent was

removed under reduced pressure to give 2a (1.14 g, 95%) as a light

tan solid. H NMR (CDCl₃, 300 MHz): δ = 7.78−7.71 (m, 2H),

7.69−7.62 (m, 2H), 3.01−2.95 (m, 2H), 2.40−2.33 (m, 2H), 2.11 (s,

6H).

Moreover, 48 out of 50 kinases were not substantially inhibited

by 10f in a kinase selectivity panel (Figure 4). Ample

bioavailability of 10f was conﬁrmed in a Caco-2 permeability

assay, and its toxicity was investigated in wild-type zebraﬁsh

embryos. Treatment with 10f was well tolerated, as full

lethality was only observed close to the solubility limit for a

concentration of 30 μM. In order to evaluate the in vivo

eﬃcacy of 10f, we modiﬁed a published procedure for

ﬂuorescence microscopy imaging of larval zebraﬁsh proneph-

roi. As hypothesized, our experiments demonstrated that Nek1

inhibition by 10f during early organ development causes cystic

kidney growth in zebraﬁsh embryos. Our study thus provides

the ﬁrst directed tool compound for a mostly neglected kinase

that has been shown to be a key player in numerous forms of

cancer and other illnesses, thereby helping to establish Nek1 as

a therapeutic target and providing a powerful tool to further

elucidate its biological function.

1

4-Bromo-N-(2-(diethylamino)ethyl)benzenesulfonamide

(2b). Synthesis of 2b was performed by following general procedure A

1

using N,N-dimethylethylenediamine. Yield 74%, yellow oil. H NMR

(CDCl₃, 300 MHz,): δ = 7.77−7.60 (m, 4H), 4.74 (s, 1H), 2.99−2.88

(m, 2H), 2.53−2.45 (m, 2H), 2.39 (q, J = 7.1 Hz, 4H), 0.92 (t, J = 7.1

Hz, 6H). ¹³C NMR (CDCl₃, 75 MHz,): δ = 139.0, 132.4, 128.8,

127.6, 51.1, 46.5, 40.3, 11.7. m/z (EI): 335 ([M]⁺).

4-Bromo-N-(2-(piperidin-1-yl)ethyl)benzenesulfonamide

(2c). Synthesis of 2c was performed by following general procedure A

1

using 1-(2-aminoethyl)piperidine. Yield 91%, orange solid. H NMR

(CDCl₃, 300 MHz): δ = 7.78−7.67 (m, 2H), 7.69−7.58 (m, 2H),

5.31 (s, 1H), 2.95 (dd, J = 6.6, 5.1 Hz, 2H), 2.34 (dd, J = 6.6, 5.1 Hz,

2H), 2.19 (t, J = 5.1 Hz, 4H), 1.43 (dp, J = 22.4, 5.7 Hz, 7H). ¹³C

NMR (CDCl_3,75 MHz,) δ = 138.9, 132.4, 128.8, 127.5, 56.2, 54.0,

39.4, 25.9, 24.3. m/z (APCI): 347.05 (M + H)⁺.

4-Bromo-N-(2-morpholinoethyl)benzenesulfonamide (2d).

Synthesis of 2d was performed by following general procedure A

using 4-(2-aminoethyl)morpholine. Yield 87%, colorless solid. ¹H

NMR (CDCl₃, 300 MHz): δ = 7.78−7.70 (m, 2H), 7.70−7.62 (m,

2H), 5.29 (s, 1H), 3.67−3.60 (m, 4H), 3.02 (dd, J = 6.7, 4.9 Hz, 2H),

2.46−2.40 (m, 2H), 2.33−2.27 (m, 4H). ¹³C NMR (CDCl₃, 75

MHz): δ = 138.8, 132.4, 128.6, 127.6, 66.7, 56.3, 53.0, 38.9. m/z

(APCI): 349.03 (M + H)⁺.

EXPERIMENTAL SECTION

■

General Information. Unless otherwise noted, starting materials

and reagents were purchased from commercial sources and used

without further puriﬁcation. All reactions employing anhydrous

conditions were performed in dried glassware with dry solvents

under argon atmosphere unless otherwise noted. All cross-coupling

reactions were performed with degassed solvents. NMR spectra were

1

recorded on a Bruker Avance II (300 MHz for H and 75 MHz for

¹³C NMR), a Bruker Avance III spectrometer (300 MHz for H and

1

75 MHz for ¹³C NMR), or a Bruker DRX 500 (500 MHz for ¹H and

126 MHz for ¹³C NMR) spectrometer. Chemical shifts are reported

as ppm by frequency downﬁeld of TMS. Coupling constants are

reported in Hertz (Hz). Mass spectrometry was performed on a

Bruker Daltonik Impact II quadrupol-TOF spectrometer for

atmospheric pressure chemical ionization (APCI) and ESI experi-

ments. EI experiments were performed on either a Finnigan MAT95

sector ﬁeld or a Fisons MD 800 quadrupol spectrometer. HPLC was

carried out on an Agilent 1100 system using a Phenomenex Synergi

Polar-RP reversed-phase column (4 μm particle size, 150 mm × 3.0

mm, pore size 80 Å) connected to a variable wavelength detector

(VWD) or a Phenomenex Synergi Hydro-RP reversed-phase column

(4 μm particle size, 150 mm × 3.0 mm, pore size 80 Å) connected to

a diode array detector (DAD). Solvent gradient = 30% A for 1 min,

linear gradient to 10% A for 10 min, 10% A for 1 min; solvent A =

0.1% TFA in water; solvent B = acetonitrile; ﬂow rate 1.0 mL/min.

TLC was performed on precoated 0.2 mm silica gel 60 F254

aluminum sheets (Merck) with detection by UV light (254 and 365

nm) or potassium permanganate staining. Flash chromatography was

carried out using the speciﬁed solvent systems on 40−63 μm

NORMASIL 60 silica gel (VWR). All compounds used in biological

or biochemical assays had >95% purity as determined in the HPLC

method described above.

General Procedure A: Synthesis of Phenylsulfonamides.

Procedure A is exempliﬁed by the triethylamine-mediated sulfona-

mide formation of compound 2a from 4-bromobenzenesulfonyl

chloride 1 and N,N-dimethylethylenediamine. Compounds 2b−h, 2j,

9a, and 9b were prepared in accordance with procedure A starting

from 4-bromobenzenesulfonyl chloride 1 and the corresponding

amine.

General Procedure B: Suzuki−Miyaura Coupling of Ar-

omatic Rings.⁴⁰Procedure B is exempliﬁed by the Suzuki−Miyaura

coupling of 4-bromo-7-azaindole and 4-bromo-N-(2-(dimethylami-

no)-ethyl)benzenesulfonamide (2a) to give target compound 3a.

Compounds 3b−k, 10a−f, and 7 were prepared in accordance with

procedure B starting from 4-bromo-7-azaindole and phenylsulfona-

mides 2b−k and 9a−f and benzamide 6, respectively.

tert-Butyl (2-(4-Bromophenyl)sulfonamido)ethyl)-

carbamate (2e). Synthesis of 2e was performed by following

general procedure A using N-Boc-ethylenediamine. Yield 78%,

1

colorless solid. H NMR (CDCl₃, 300 MHz): δ = 7.76−7.59 (m,

4H), 5.05 (s, 1H), 3.24−3.18 (m, 2H), 3.04 (dd, J = 6.3, 4.8 Hz, 2H),

1.41 (s, 9H). ¹³C NMR (CDCl₃, 75 MHz,): δ = 156.8, 139.2, 132.5,

128.7, 127.6, 52.6, 46.3, 43.9, 42.0, 40.4, 28.54, 28.47, 10.7. m/z (EI):

380 ([M⁺]).

4-Bromo-N-(2-hydroxyethyl)benzenesulfonamide (2f). Syn-

thesis of 2f was performed by following general procedure A using 2-

1

aminoethanol. Yield 81%, colorless solid. H NMR (DMSO-d6, 300

MHz): δ = 7.84−7.70 (m, 4H), 4.67 (t, J = 5.5 Hz, 1H), 3.40−3.32

(m, 2H), 2.80 (q, J = 6.1 Hz, 2H). m/z (APCI): 279.96 ([M + H]⁺).

4-Bromo-N-(2-methoxyethyl)benzenesulfonamide (2g).

Synthesis of 2g was performed by following general procedure A

using 2-methoxyethylamine. Yield 78%, yellow oil. ¹H NMR (DMSO-

d₆, 300 MHz,): δ = 7.82−7.78 (m, 2H), 7.75−7.69 (m, 2H), 3.32 (s,

3H), 3.29 (t, J = 5.6 Hz, 2H), 2.92 (q, J = 5.8 Hz, 2H).

4-Bromo-N-(2-hydroxyethyl)-N-methylbenzenesulfona-

mide (2h). Synthesis of 2h was performed by following general

procedure A using N-methylethanolamine. Yield 90%, colorless oil.

¹H NMR (CDCl₃, 300 MHz): δ = 7.68 (s, 4H), 3.78 (dd, J = 5.6, 4.9

Hz, 2H), 3.18 (t, J = 5.3 Hz, 2H), 2.85 (s, 3H). m/z (APCI): 293.98

([M + H]⁺).

2-((4-Bromophenyl)sulfonamido)ethyl acetate (2i). To a

stirred solution of 2f (0.77 g, 2.73 mmol) and triethylamine (0.33

g, 3.28 mmol) in 5 mL of DCM, acetanhydride (0.34 g, 3.28 mmol)

was added dropwise at 0 °C under cooling in an ice bath. The cooling

bath was removed, and the solution was stirred for 72 h at ambient

temperature. The reaction mixture was diluted with 20 mL of DCM

and quenched by bringing the pH to 9 through addition of saturated

NaHCO₃(aq). The organic layer was washed with saturated

NaHCO₃(aq, 3 × 5 mL) and brine (5 mL) and dried over

MgSO₄. The solvent was removed under reduced pressure to give 2i

(0.73 g, 82%) as a yellow oil that was used without further

4-Bromo-N-(2-(dimethylamino)ethyl)benzenesulfonamide

(2a). To a stirred solution of 4-bromobenzene-sulfonyl chloride (1,

1.00 g, 3.91 mmol) in 10 mL DCM were added N,N-

dimethylethylenediamine (414 mg, 4.70 mmol) and triethylamine

1

puriﬁcation. H NMR (DMSO-d₆, 300 MHz): δ = 7.85−7.80 (m,

2H), 7.75−7.69 (m, 2H), 3.95 (t, J = 5.6 Hz, 2H), 3.03 (q, J = 5.6 Hz,

2H), 1.93 (s, 3H). m/z (ESI): 343.96 ([M + Na]⁺).

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4 - B r o m o - N - ( 2 - h y d r o x y - 2 - m e t h y l p r o p y l ) -

benzenesulfonamide (2j). Synthesis of 2j was performed by

following general procedure A using 1-amino-2-methyl-2-propanol.

Yield 64%, yellow solid. ¹H NMR (DMSO-d₆, 500 MHz): δ = 7.83−

7.78 (m, 2H), 7.75−7.71 (m, 2H), 7.59 (t, J = 6.5 Hz, 1H), 4.40 (s,

1H), 2.62 (d, J = 6.5 Hz, 2H), 1.04 (s, 6H). ¹³C NMR (DMSO-d₆,

126 MHz): δ = 140.1, 132.1, 128.5, 125.9, 68.7, 53.6, 27.0. ¹³C NMR

(DMSO-d₆, 126 MHz, DEPT): δ = 132.1, 128.6, 53.6, 45.5, 27.1, 8.5.

m/z (APCI): 307.98 ([M + H]⁺).

1H), 8.04−7.90 (m, 4H), 7.60 (d, J = 3.5 Hz, 1H), 7.25 (d, J = 4.9

Hz, 1H), 6.63 (d, J = 3.5 Hz, 1H), 2.88 (dd, J = 8.1, 6.3 Hz, 2H), 2.43

(t, J = 7.2 Hz, 2H), 2.39 (q, J = 7.1 Hz, 4H), 0.86 (t, J = 7.1 Hz, 6H).

¹³C NMR (DMSO-d₆, 126 MHz): δ = 149.2, 142.9, 142.2, 140.3,

138.5, 128.9, 127.2, 127.1, 117.1, 114.3, 98.7, 51.8, 46.5, 41.0, 11.6.

¹³C NMR (126 MHz, DMSO-d₆, DEPT) δ = 142.9, 128.9, 127.1,

114.36, 98.7, 51.8, 46.5, 41.0, 11.6. m/z (ESI): 373.18 ([M + H]⁺),

187.09 ([M + 2H]²⁺). Mp: 143 °C.

N-(2-(Piperidin-1-yl)ethyl)-4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-

benzenesulfonamide (3c). Starting from 2c, the synthesis of 3c

was performed by following general procedure B. Puriﬁcation was

performed by column chromatography using a 15:1:0.1 mixture of

DCM/MeOH/NH₃in MeOH (7 N). Yield 58%, beige solid. ¹H

NMR (DMSO-d₆, 300 MHz): δ = 11.88 (s, 1H), 8.32 (d, J = 4.8 Hz,

1H), 7.97 (s, 4H), 7.60 (d, J = 3.4 Hz, 1H), 7.25 (d, J = 4.8 Hz, 1H),

6.63 (d, J = 3.4 Hz, 1H), 2.92 (t, J = 6.9 Hz, 2H), 2.29 (t, J = 6.9 Hz,

2H), 2.23 (t, J = 5.0 Hz, 4H), 1.45−1.21 (m, 6H). ¹³C NMR

(DMSO-d₆, 75 MHz,): δ = 149.2, 142.9, 142.1, 140.3, 138.5, 128.9,

127.2, 127.1, 117.1, 114.4, 98.7, 57.6, 53.9, 40.3, 25.4, 23.9. ¹³C NMR

(DMSO-d₆, 75 MHz, DEPT): δ = 142.9, 128.9, 127.2, 127.1, 114.4,

98.7, 57.6, 53.9, 40.2, 25.34, 23.9. m/z (APCI): 385.18 ([M + H]⁺).

Mp: 204 °C.

N-(2-Morpholinoethyl)-4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-

benzenesulfonamide (3d). Starting from 2d, the synthesis of 3d

was performed by following general procedure B. Puriﬁcation was

performed by column chromatography using a 50:1:0.5 mixture of

DCM/MeOH/NH₃in MeOH (7 N). Yield 69%, beige solid. ¹H

NMR (DMSO-d₆, 500 MHz,): δ = 11.88 (s, 1H), 8.33 (d, J = 4.9 Hz,

1H), 7.98 (s, 4H), 7.65 (t, J = 5.9 Hz, 1H), 7.60 (dd, J = 3.5, 2.5 Hz,

1H), 7.25 (d, J = 4.9 Hz, 1H), 6.64 (dd, J = 3.5, 1.8 Hz, 1H), 3.52−

3.46 (m, 4H), 2.95 (q, J = 6.3 Hz, 2H), 2.33 (t, J = 6.8 Hz, 2H), 2.27

(t, J = 4.7 Hz, 4H). ¹³C NMR (DMSO-d₆, 126 MHz) δ = 149.2,

142.9, 142.1, 140.3, 138.46, 128.9, 127.2, 127.1, 117.1, 114.4, 98.7,

66.0, 57.2, 53.1, 39.9. ¹³C NMR (DMSO-d₆, 126 MHz, DEPT) δ =

142.9, 128.9, 127.2, 127.1, 114.4, 98.7, 66.0, 57.2, 53.1, 39.8. m/z

(APCI): 387.16 ([M + H]⁺). Mp: 201 °C.

1-((4-Bromophenyl)sulfonyl)-4-methylpiperazine (2k). To a

solution of 4-bromobenzene-sulfonyl chloride (0.40 g, 1.47 mmol) in

9 mL of dry THF, 1-methyl-piperazine (1.41 g, 14.09 mmol) was

added, and the mixture was stirred for 16 h at ambient temperature.

The solvent was removed under reduced pressure, and the resulting

residue was dissolved in 10 mL of DCM. The organic layer was

washed with saturated NaHCO₃(aq, 3 × 2.5 mL) and dried over

MgSO₄. The solvent was removed under reduced pressure to give 2k

1

(0.37 g, 75%) as a yellow solid. H NMR (CDCl₃, 500 MHz): δ =

7.69−7.63 (m, 2H), 7.62−7.58 (m, 2H), 3.03 (t, J = 4.9 Hz, 4H),

2.47 (t, J = 5.0 Hz, 4H), 2.26 (s, 3H). ¹³C NMR (CDCl_3,126 MHz): δ

= 134.7, 132.4, 129.4, 128.0, 54.1, 46.0, 45.8. m/z (EI): 380 ([M]⁺).

((4-Bromophenyl)sulfonyl)-^L-phenylalanine (2l). To a stirred

solution of ^L-phenylalanine (194 mg, 1.17 mmol) in 4 mL of water

was added sodium bicarbonate (296 mg, 3.52 mmol). The mixture

was stirred at ambient temperature for 15 min, and a solution of 4-

bromobenzenesulfonyl chloride (1, 300 mg, 1.17 mmol) in 4 mL of

acetone was added at 0 °C under cooling in an ice bath. The cooling

bath was removed, and the solution was stirred at ambient

temperature for 20 h. After evaporation of the volatile solvent

under reduced pressure, the product was precipitated by bringing the

pH to 7 using hydrochloric acid (1 N). Filtration and drying under

1

reduced pressure gave 2l (220 mg, 49%) as a yellow solid. H NMR

(DMSO-d₆, 500 MHz): δ = 7.72−7.69 (m, 2H), 7.65−7.61 (m, 2H),

7.18−7.11 (m, 5H), 3.35 (t, J = 5.1 Hz, 1H), 2.98 (dd, J = 13.4, 5.0

Hz, 1H), 2.90 (dd, J = 13.4, 5.2 Hz, 1H). ¹³C NMR (DMSO-d₆, 126

MHz): δ = 170.8, 139.7, 138.7, 132.0, 129.9, 128.7, 127.5, 125.9,

125.6, 58.3, 38.5. m/z (ESI): 788.95 ([2M + Na]⁺), 405.97 ([M +

Na]⁺), 383.99 ([M + H]⁺).

tert-Butyl(2-((4-(1H-pyrrolo[2,3-b]pyridin-4-yl)phenyl)-

sulfonamido)ethyl)carbamate (3e). Starting from 2e, the syn-

thesis of 3e was performed by following general procedure B.

Puriﬁcation was performed by column chromatography using a

20:1:0.1 mixture of DCM/MeOH/NH₃in MeOH (7 N). Yield 60%,

N-(2-(Dimethylamino)ethyl)-4-(1H-pyrrolo[2,3-b]pyridin-4-

yl)benzenesulfonamide (3a). To a solution of sulfonamide 2a

(100 mg, 0.33 mmol) in 1.5 mL of DMF were added potassium

acetate (97 mg, 0.99 mmol), bis(pinacolato)diboron (100 mg, 0.40

mmol), and PdCl₂(dppf) (7 mg, 10 μmol), under argon atmosphere.

The reaction mixture was stirred at 100 °C for 3 h. After cooling to

ambient temperature, 15 mL of ethyl acetate was added, the solution

was ﬁltered through Celite, and the organic layer was washed with

brine (5 × 15 mL) before drying over NaSO₄. The solvent was

removed under reduced pressure to give the corresponding boronic

acid pinacolester as a crude solid that was dissolved in 1.5 mL of DMF

and treated with 4-bromo-7-azaindole (65 mg, 0.33 mmol), 0.5 mL of

saturated NaHCO₃(aq) and Pd(PPh₃)₄(38 mg, 36 μmol) under

argon atmosphere. The mixture was stirred at 100 °C for 18 h, cooled

to ambient temperature, and diluted with 20 mL of EtOAc. After

ﬁltration through Celite, the organic layer was washed with brine (5 ×

20 mL) and dried over MgSO₄, and the solvent was removed under

reduced pressure. Puriﬁcation was performed by column chromatog-

raphy using a 10:1:0.1 mixture of DCM/MeOH/NH₃in MeOH (7

N) to give 3a (47 mg, 42%) as a colorless solid. ¹H NMR (DMSO-d₆,

300 MHz): δ = 11.89 (s, 1H), 8.33 (d, J = 4.9 Hz, 1H), 7.98 (s, 4H),

7.60 (d, J = 3.5 Hz, 1H), 7.25 (d, J = 4.9 Hz, 1H), 6.64 (d, J = 3.5 Hz,

1H), 2.91 (t, J = 6.8 Hz, 2H), 2.28 (t, J = 6.8 Hz, 2H), 2.07 (s, 6H).

¹³C NMR (DMSO-d₆, 75 MHz,): δ = 149.2, 142.9, 142.2, 140.3,

1

colorless solid. H NMR (DMSO-d₆,500 MHz,): δ = 11.88 (s, 1H),

8.33 (d, J = 4.9 Hz, 1H), 8.01−7.93 (m, 4H), 7.76 (t, J = 6.0 Hz, 1H),

7.60 (dd, J = 3.5, 2.6 Hz, 1H), 7.26 (d, J = 4.9 Hz, 1H), 6.78 (t, J =

5.9 Hz, 1H), 6.66 (dd, J = 3.5, 1.8 Hz, 1H), 3.01 (q, J = 6.6 Hz, 2H),

2.84 (q, J = 6.4 Hz, 2H), 1.34 (s, 9H). ¹³C NMR (DMSO-d₆, 126

MHz): δ = 155.5, 149.2, 142.9, 142.2, 140.0, 138.4, 129.0, 127.2,

127.1, 117.1, 114.4, 98.8, 77.8, 42.3, 28.1. ¹³C NMR (DMSO-d₆, 126

MHz, DEPT): δ = 142.9, 129.0, 127.2, 127.1, 114.4, 98.8, 42.3, 39.8,

28.2. m/z (APCI): 417.16 ([M + H]⁺).

N-(2-Hydroxyethyl)-4-(1H-pyrrolo[2,3-b]pyridin-4-yl)ben-

zenesulfonamide (3f). Starting from 2f, the synthesis of 3f was

performed by following general procedure B. Puriﬁcation was

performed by column chromatography using a 10:1:0.1 mixture of

1

DCM/MeOH/NH₃in MeOH (7 N). Yield 66%, colorless solid. H

NMR (DMSO-d₆, 500 MHz): δ = 11.88 (s, 1H), 8.33 (d, J = 4.9 Hz,

1H), 8.01−7.94 (m, 4H), 7.71 (t, J = 5.9 Hz, 1H), 7.60 (t, J = 3.0 Hz,

1H), 7.26 (d, J = 5.0 Hz, 1H), 6.65 (dd, J = 3.5, 1.8 Hz, 1H), 4.70 (t, J

= 5.6 Hz, 1H), 3.42 (q, J = 6.0 Hz, 2H), 2.88 (q, J = 6.2 Hz, 2H). ¹³C

NMR (DMSO-d₆, 126 MHz): δ = 149.2, 142.9, 142.1, 140.2, 138.5,

128.9, 127.2, 127.12, 117.1, 114.4, 98.8, 59.9, 45.1. ¹³C NMR

(DMSO-d₆, 126 MHz, DEPT): δ = 142.9, 128.9, 127.2, 127.1, 114.4,

98.8, 60.0, 45.1. m/z (ESI): 318.09 ([M + H]⁺).

138.5, 128.9, 127.2, 127.2, 117.1, 114.4, 98.8, 58.1, 45.0, 40.7. m/z

(APCI): 345.13 ([M + H]⁺).

N-(2-Methoxyethyl)-4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-

benzenesulfonamide (3g). Starting from 2g, the synthesis of 3g

was performed by following general procedure B. Puriﬁcation was

performed by column chromatography using a 20:1:0.1 mixture of

N-(2-(Diethylamino)ethyl)-4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-

benzenesulfonamide (3b). Starting from 2b, the synthesis of 3b

was performed by following general procedure B. Puriﬁcation was

performed by column chromatography using a 10:1:0.1 mixture of

DCM/MeOH/NH₃in MeOH (7 N). Yield 41%, beige semisolid. ¹H

NMR (DMSO-d₆, 500 MHz): δ = 11.88 (s, 1H), 8.33 (d, J = 4.9 Hz,

1

DCM/MeOH/NH₃in MeOH (7 N). Yield 65%, colorless solid. H

NMR (DMSO-d₆, 500 MHz): δ = 11.88 (s, 1H), 8.33 (d, J = 4.9 Hz,

1H), 8.02−7.91 (m, 4H), 7.83 (t, J = 6.0 Hz, 1H), 7.60 (dd, J = 3.5,

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2.5 Hz, 1H), 7.25 (d, J = 4.9 Hz, 1H), 6.65 (dd, J = 3.6, 1.8 Hz, 1H),

3.34 (t, J = 5.7 Hz, 2H), 2.99 (q, J = 5.8 Hz, 2H). ¹³C NMR (DMSO-

d₆, 126 MHz): δ = 149.17, 142.92, 142.15, 140.35, 138.49, 128.87,

127.19, 127.10, 117.13, 114.38, 98.76, 70.56, 57.83, 42.24. ¹³C NMR

(DMSO-d₆, 126 MHz, DEPT): δ = 142.92, 128.87, 127.19, 127.10,

114.38, 98.76, 70.56, 57.83, 42.24. m/z (APCI): 332.10 ([M + H]⁺).

Mp: 215 °C.

treated with 4-bromo-7-azaindole (103 mg, 0.52 mmol), 1 mL of

saturated NaHCO₃(aq), and Pd(PPh₃)₄(60 mg, 52 μmol) under

argon atmosphere. The mixture was stirred at 100 °C for 17 h, cooled

to ambient temperature, and diluted with 20 mL of EtOAc. After

ﬁltration through Celite, the organic layer was washed with brine (5 ×

20 mL), and the product was precipitated by setting the pH to 4 using

hydrochloric acid (0.1 N). Filtration and drying under reduced

pressure gave 3l (55 mg, 25%) as a colorless solid. ¹H NMR (DMSO-

d₆, 500 MHz): δ = 11.89 (s, 1H), 8.41 (d, J = 9.0 Hz, 1H), 8.33 (d, J

= 4.9 Hz, 1H), 7.80 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.3 Hz, 2H), 7.61

(t, J = 3.0 Hz, 1H), 7.22 (d, J = 4.9 Hz, 1H), 7.20−7.10 (m, 5H), 6.62

(dd, J = 3.5, 1.7 Hz, 1H), 3.95 (dt, J = 8.8, 4.0 Hz, 1H), 2.99 (dd, J =

13.8, 5.5 Hz, 1H), 2.79−2.72 (m, 1H). ¹³C NMR (DMSO-d₆, 126

MHz): δ = 172.4, 149.2, 142.9, 141.9, 140.6, 138.6, 136.8, 129.1,

128.5, 128.1, 127.2, 126.8, 126.4, 117.1, 114.3, 98.8, 57.5, 57.5, 37.8.

m/z (APCI): 422.12 ([M + H]⁺).

2-(4-Bromobenzamido)ethyl 4-Bromobenzoate (5). A sol-

ution of 4-bromobenzoyl chloride (4, 2.00 g, 9.11 mmol) in 10 mL of

DCM was dropwise added to a solution of monoethanolamine (0.67

g, 10.94 mmol) and triethylamine (1.08 g, 6.78 mmol) in 10 mL of

DCM over the course of 20 min. The mixture was stirred at ambient

temperature for 24 h, diluted with 10 mL DCM, and washed with

saturated NaHCO₃(aq, 3 × 5 mL). The organic layer was dried over

MgSO₄, and the solvent was removed to give a colorless residue that

was recrystallized from EtOH to give 5 (0.60 g, 48%) as a colorless

solid. ¹H NMR (DMSO-d₆, 500 MHz): δ = 8.76 (t, J = 5.6 Hz, 1H),

7.92−7.87 (m, 2H), 7.81−7.76 (m, 2H), 7.75−7.70 (m, 2H), 7.70−

7.64 (m, 2H), 4.40 (t, J = 5.5 Hz, 2H), 3.64 (q, J = 5.6 Hz, 2H). ¹³C

NMR (DMSO-d₆, 126 MHz): δ = 165.7, 165.1, 133.4, 131.8, 131.3,

131.2, 129.3, 128.9, 127.3, 124.9, 63.6, 38.4. ¹³C NMR (DMSO-d₆,

126 MHz, DEPT): δ = 131.8, 131.3, 131.2, 129.3, 63.6, 38.4. m/z

(ESI): 425.93 ([M + H]⁺).

N-(2-Hydroxyethyl)-N-methyl-4-(1H-pyrrolo[2,3-b]pyridin-

4-yl)benzenesulfonamide (3h). Starting from 2h, the synthesis of

3h was performed by following general procedure B. Puriﬁcation was

performed by column chromatography using a 20:1:0.1 mixture of

1

DCM/MeOH/NH₃in MeOH (7 N). Yield 42%, colorless solid. H

NMR (DMSO-d₆, 500 MHz): δ = 11.89 (s, 1H), 8.33 (d, J = 4.9 Hz,

1H), 8.01 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 7.60 (t, J = 3.0

Hz, 1H), 7.27 (d, J = 5.0 Hz, 1H), 6.66 (dd, J = 3.6, 1.8 Hz, 1H), 4.81

(td, J = 5.5, 1.3 Hz, 1H), 3.56 (q, J = 5.8 Hz, 2H), 3.10 (t, J = 6.1 Hz,

2H), 2.80 (s, 3H). ¹³C NMR (DMSO-d₆, 126 MHz): δ = 149.2,

142.9, 142.6, 138.25, 136.82, 129.03, 127.71, 127.24, 117.07, 114.36,

98.7, 59.1, 51.94 35.6. ¹³C NMR (DMSO-d₆, 126 MHz, DEPT): δ =

142.9, 142.7, 129.0, 127.7, 127.2, 114.4, 98.7, 59.1, 51.94, 35.6. m/z

(APCI): 332.11 ([M + H]⁺).

2-((4-(1H-Pyrrolo[2,3-b]pyridin-4-yl)phenyl)sulfonamido)-

ethyl Acetate (3i). Starting from 2i, the synthesis of 3i was

performed by following general procedure B. Puriﬁcation was

performed by column chromatography using a 15:1:0.1 mixture of

1

DCM/MeOH/NH₃in MeOH (7 N). Yield 23%, colorless solid. H

NMR (DMSO-d₆, 500 MHz): δ = 11.90 (s, 1H), 8.35 (d, J = 5.0 Hz,

1H), 8.03−7.96 (m, 5H), 7.62 (dd, J = 3.5, 2.5 Hz, 1H), 7.28 (d, J =

5.0 Hz, 1H), 6.67 (dd, J = 3.5, 1.9 Hz, 1H), 4.02 (t, J = 5.6 Hz, 2H),

3.12 (q, J = 5.7 Hz, 2H), 1.96 (s, 3H). ¹³C NMR (DMSO-d₆, 126

MHz): δ = 170.2, 149.18, 142.94, 142.28, 140.22, 138.45, 128.98,

127.23, 127.1, 117.1, 114.4, 98.8, 62.5, 41.4, 20.6. m/z (APCI):

360.10 ([M + H]⁺).

4-Bromo-N-(2-hydroxyethyl)benzamide (6). To a solution of

ester 5 (0.50 g, 1.17 mmol) in 20 mL of THF was added 2 mL of 50%

w/v aq. KOH solution, and the mixture was stirred at 60 °C for 16 h.

The solvent was removed under reduced pressure, and the resulting

white residue was taken up in 20 mL of EtOAc and washed with water

(3 × 5 mL) and brine (5 mL). Puriﬁcation was performed by column

chromatography using a 15:1 mixture of DCM/MeOH to give 6 (262

N-(2-Hydroxy-2-methylpropyl)-4-(1H-pyrrolo[2,3-b]pyridin-

4-yl)benzenesulfonamide (3j). Starting from 2j, the synthesis of 3j

was performed by following general procedure B. Puriﬁcation was

performed by column chromatography using a 13:1:0.1 mixture of

1

DCM/MeOH/NH₃in MeOH (7 N). Yield 11%, colorless solid. H

NMR (DMSO-d₆, 500 MHz): δ = 11.87 (s, 1H), 8.33 (d, J = 4.9 Hz,

1H), 8.01−7.94 (m, 4H), 7.62−7.54 (m, 2H), 7.26 (d, J = 4.9 Hz,

1H), 6.65 (dd, J = 3.5, 1.9 Hz, 1H), 4.42 (s, 1H), 2.70 (d, J = 6.6 Hz,

2H), 1.08 (s, 6H). ¹³C NMR (DMSO-d₆, 126 MHz): δ = 149.2,

142.9, 142.1, 140.4, 138.5, 128.9, 127.18, 127.16, 117.1, 114.4, 98.8,

68.8, 53.7, 27.1. ¹³C NMR (DMSO-d₆, 126 MHz, DEPT): δ = 142.9,

128.9, 127.18, 127.16, 114.4, 98.8, 53.7, 27.1. m/z (EI): 345 ([M]⁺).

4-(4-((4-Methylpiperazin-1-yl)sulfonyl)phenyl)-1H-pyrrolo-

[2,3-b]pyridine (3k). Starting from 2k, the synthesis of 3k was

performed by following general procedure B. Puriﬁcation was

performed by column chromatography using a 20:1:0.1 mixture of

1

mg, 92%) as a colorless solid. H NMR (DMSO-d₆, 500 MHz): δ =

8.49 (t, J = 5.7 Hz, 1H), 7.82−7.77 (m, 2H), 7.68−7.65 (m, 2H),

4.70 (t, J = 5.6 Hz, 1H), 3.51 (q, J = 5.9 Hz, 2H), 3.32 (q, J = 6.1 Hz,

2H). ¹³C NMR (DMSO-d₆, 126 MHz): δ = 165.4, 133.7, 131.2,

129.3, 124.73, 59.6, 42.2. m/z (EI): 243 ([M]⁺).

N-(2-Hydroxyethyl)-4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-

benzamide (7). Starting from 6, the synthesis of 7 was performed by

following general procedure B. Puriﬁcation was performed by column

chromatography using a stepwise gradient from a 15:1:0.1 to a

10:1:0.1 mixture of DCM/MeOH/10% aq. NH₄OH (25%) in

MeOH. Yield 43%, colorless solid. ¹H NMR (DMSO-d₆, 500

MHz): δ = 11.83 (s, 1H), 8.53 (t, J = 5.6 Hz, 1H), 8.31 (d, J =

4.9 Hz, 1H), 8.07−8.00 (m, 2H), 7.89−7.82 (m, 2H), 7.60−7.55 (m,

1H), 7.23 (d, J = 4.9 Hz, 1H), 6.63 (dd, J = 3.6, 1.8 Hz, 1H), 4.75 (t, J

= 5.6 Hz, 1H), 3.56 (q, J = 6.0 Hz, 2H), 3.39 (q, J = 6.1 Hz, 2H). ¹³C

NMR (DMSO-d₆, 126 MHz): δ = 165.9, 149.2, 142.9, 141.0, 139.3,

134.2, 128.0, 127.8, 126.9, 117.2, 114.3, 99.0, 59.8, 42.2. ¹³C NMR

(DMSO-d₆, 126 MHz, DEPT): δ = 142.9, 128.0, 127.9, 126.9, 114.3,

98.9, 59.8, 42.3. m/z (ESI): 282.12 ([M + H]⁺).

(R)-4-(2-Amino-3-hydroxypropyl)phenol (8d). To a stirred

solution of ^D-tyrosine methyl ester hydrochloride (2.00 g, 8.63 mmol)

in 20 mL of acetonitrile was added potassium carbonate (2.98 g, 21.6

mmol), and the solution was heated to 50 °C for 16 h. Filtration of

the formed salt and evaporation of the solvent under reduced pressure

gave an orange oil that was taken up in 20 mL of methanol. Under

cooling to 0 °C in an ice bath, NaBH₄(491 mg, 12.99 mmol) was

added in small portions over 30 min. The cooling bath was removed,

and the solution was stirred at ambient temperature for 5 h. The

solvent was removed under reduced pressure, the resulting residue

was taken up in 50 mL of water, and the aqueous layer was extracted

with ethyl acetate (5 × 50 mL). The combined organic layers were

1

DCM/MeOH/NH₃in MeOH (7 N). Yield 58%, colorless solid. H

NMR (D₂O, as a TFA salt, 500 MHz): δ = 8.39 (d, J = 6.0 Hz, 1H),

8.00−7.87 (m, 4H), 7.72 (d, J = 3.6 Hz, 1H), 7.54 (d, J = 6.0 Hz,

1H), 6.82 (d, J = 3.6 Hz, 1H), 4.02 (d, J = 13.5 Hz, 2H), 3.68 (d, J =

12.7 Hz, 2H), 3.33 (td, J = 12.5, 3.3 Hz, 2H), 2.98 (s, 3H), 2.95−2.86

(m, 2H). ¹³C NMR (D₂O, as a TFA salt, 126 MHz,): δ = 146.8,

141.2, 139.6, 135.1, 133.6, 130.2, 130.0, 128.1, 122.9, 114.9, 101.7,

52.7, 43.2, 42.8. ¹³C NMR (D₂O, as a TFA salt, 126 MHz, DEPT): δ

= 133.5, 130.2, 130.0, 128.1, 114.9, 101.7, 52.7, 43.3, 42.8. m/z

(APCI): 357.15 ([M + H]⁺).

((4-(1H-Pyrrolo[2,3-b]pyridin-4-yl)phenyl)sulfonyl)-^L-phe-

nylalanine (3l). To a solution of sulfonamide 2l (200 mg, 0.52

mmol) in 3 mL of DMF were added potassium acetate (153 mg, 1.56

mmol), bis(pinacolato)diboron (159 mg, 0.62 mmol), and

PdCl₂(dppf) (13 mg, 15 μmol) under argon atmosphere. The

reaction mixture was stirred at 100 °C for 3 h. After cooling to

ambient temperature 20 mL of EtOAc was added, the solution was

ﬁltered through Celite, and the organic layer was washed with brine

(5 × 20 mL) before drying over NaSO₄. The solvent was removed

under reduced pressure to give the corresponding boronic acid

pinacolester as a crude solid that was dissolved in 3 mL of DMF and

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dried over MgSO₄, ﬁltered, and concentrated in vacuo to give 8d (878

solid. ¹H NMR (CDCl₃, 300 MHz): δ = 7.48 (s, 4H), 7.21−7.13 (m,

3H), 7.00−6.93 (m, 2H), 3.69 (dd, J = 11.1, 4.1 Hz, 1H), 3.59 (dd, J

= 11.1, 4.6 Hz, 1H), 3.53−3.39 (m, 1H), 2.83 (dd, J = 13.9, 6.3 Hz,

1H), 2.68 (dd, J = 14.0, 8.2 Hz, 1H). m/z (ESI): 370.01 ([M + H]⁺).

(S)-4-Bromo-N-(1-hydroxy-3-(4-hydroxyphenyl)propan-2-

yl)benzenesulfonamide (9c). To a solution of ^L-tyrosinol (469 mg,

2.15 mmol) in 10 mL of a 1:1 mixture of water/acetone was added

sodium bicarbonate (658 mg, 7.83 mmol), and the mixture was stirred

at ambient temperature for 15 min. 4-Bromobenzene-sulfonyl

chloride (1, 500 mg, 1.96 mmol) was added, and stirring was

continued for 17 h. After evaporation of the volatile solvent under

reduced pressure, 10 mL of ethyl acetate was added, and the layers

were separated. The aqueous layer was extracted with ethyl acetate (3

× 5 mL), and the combined organic layers were washed with

saturated NaHCO₃(aq, 3 × 5 mL) and dried over MgSO₄. The

solvent was removed under reduced pressure to give crude crystals

that were recrystallized from methanol to give 9c (455 mg, 60%) as

colorless crystals. ¹H NMR (DMSO-d₆, 500 MHz): δ = 9.09 (s, 1H),

7.67 (d, J = 7.3 Hz, 2H), 7.67−7.60 (m, 3H), 7.55−7.49 (m, 2H),

6.87−6.80 (m, 2H), 6.59−6.52 (m, 2H), 4.69 (t, J = 5.1 Hz, 1H),

3.30−3.25 (m, 2H), 3.22−3.14 (m, 2H), 2.69 (dd, J = 13.8, 5.4 Hz,

1H), 2.35 (dd, J = 13.8, 6.9 Hz, 1H). m/z (ESI): 386.01 ([M + H]⁺).

(R)-4-Bromo-N-(1-hydroxy-3-(4-hydroxyphenyl)propan-2-

yl)benzenesulfonamide (9d). To a solution of ^D-tyrosinol (8d, 229

mg, 1.37 mmol) in 7 mL of a 1:1 mixture of water/acetone was added

sodium bicarbonate (460 mg, 5.48 mmol), and the mixture was stirred

at ambient temperature for 15 min. 4-Bromobenzenesulfonyl chloride

(1, 350 mg, 1.37 mmol) was added, and the solution was heated to 40

°C for 4 h. After evaporation of the volatile solvent under reduced

pressure, 7 mL of ethyl acetate was added, and the layers were

separated. The aqueous layer was extracted with ethyl acetate (3 × 5

mL), and the combined organic layers were washed with saturated

NaHCO₃(aq, 3 × 5 mL) and dried over MgSO₄. The solvent was

removed under reduced pressure to give 9d (371 mg, 70%) as a crude

yellow solid that was used in the next step without further puriﬁcation.

¹H NMR (DMSO-d₆, 300 MHz): δ = 7.68−7.58 (m, 2H), 7.52 (d, J =

mg, 61%) as a crude yellow solid that was used for the next step

1

without further puriﬁcation. H NMR (DMSO-d₆, 500 MHz): δ =

6.68−6.65 (m, 2H), 6.37−6.34 (m, 2H), 2.97 (dd, J = 10.3, 4.5 Hz,

1H), 2.87−2.83 (m, 1H), 2.24 (dd, J = 13.4, 6.0 Hz, 1H), 2.20−2.09

(m, 1H), 2.02 (dd, J = 13.4, 7.5 Hz, 1H), 1.44 (s, 1H). ¹³C NMR

(DMSO-d₆, 500 MHz): δ = 155.5, 130.0, 129.5, 115.0, 65.5, 54.6,

39.0. m/z (EI): 167 ([M]⁺).

(S)-2-Amino-3-(1H-indol-3-yl)propan-1-ol (8e). An oven-dried

three-neck ﬂask was charged with ^L-tryptophan (2.04 g, 10.0 mmol),

sodium borohydride (757 mg, 20.0 mmol), and 20 mL of dry THF

and equipped with a dropping funnel and a reﬂux condenser under

argon atmosphere. A solution of iodine (2.54 g, 10.0 mmol) in 10 mL

dry THF was added dropwise over the course of 30 min at 0 °C under

cooling in an ice bath. Once the evolution of gas had stopped, the ice

bath was removed, and the mixture was heated to reﬂux for 20 h. After

cooling to ambient temperature, methanol was slowly added under

vigorous stirring until the solution became clear. After stirring for

another 15 min, removal of the solvents under reduced pressure gave

a white residue that was dissolved in 25 mL of aq. KOH solution

(20% w/v) and stirred for 16 h at ambient temperature. Adjustment

to pH 11 by addition of hydrochloric acid (2 M) was followed by

extraction of the aqueous layer with DCM (3 × 25 mL) and a 1:4

mixture of ethanol/chloroform (5 × 25 mL). The combined organic

layers were washed with water (3 × 25 mL) and brine (25 mL) and

dried over MgSO₄. The solvent was removed under reduced pressure

to give 8e (1.36 g, 72%) as a crude yellow oil that was used without

further puriﬁcation. ¹³C NMR (75 MHz, CDCl₃) δ = 136.5, 127.7,

122.9, 122.1, 119.4, 118.9, 112.4, 111.4, 66.6, 53.1, 30.1. m/z (ESI):

191.12 ([M + H]⁺).

(R)-2-Amino-3-(1H-indol-3-yl)propan-1-ol (8f). An oven-dried

three-neck ﬂask was charged with ^D-tryptophan (1.57 g, 7.69 mmol),

sodium borohydride (640 mg, 16.91 mmol), and 15 mL of dry THF

and equipped with a dropping funnel and a reﬂux condenser under

argon atmosphere. A solution of iodine (1.95 g, 7.69 mmol) in 8 mL

of dry THF was added dropwise over the course of 30 min at 0 °C

under cooling in an ice bath. Once the evolution of gas had stopped,

the ice bath was removed, and the mixture was heated to reﬂux for 19

h. After the mixture was cooled to ambient temperature, methanol

was slowly added under vigorous stirring until the solution became

clear. After stirring for another 15 min, removal of the solvents under

reduced pressure gave a white residue that was dissolved in 20 mL of

aq. KOH solution (20% w/v) and stirred for 15 h at ambient

temperature. Adjustment to pH 11 by addition of hydrochloric acid

(2 M) was followed by extraction of the aqueous layer with DCM (3

× 25 mL) and a 1:4 mixture of ethanol/chloroform (5 × 25 mL). The

combined organic layers were washed with water (3 × 25 mL) and

brine (25 mL) and dried over MgSO₄. The solvent was removed

under reduced pressure to give 8f (1.22 g, 84%) as a crude yellow oil

that was used for the next step without further puriﬁcation. ¹H NMR

(DMSO-d₆, 500 MHz): δ = 10.83 (s, 1H), 7.54 (d, J = 7.9 Hz, 1H),

7.34 (d, J = 8.0 Hz, 1H), 7.15 (d, J = 1.8 Hz, 1H), 7.06 (ddt, J = 8.2,

5.0, 1.4 Hz, 1H), 6.99−6.94 (m, 1H), 3.41−3.35 (m, 1H), 3.24 (m,

1H), 3.01 (q, J = 6.1 Hz, 1H), 2.81 (dd, J = 14.2, 6.1 Hz, 1H), 2.60

(dd, J = 14.2, 7.1 Hz, 1H). ¹³C NMR (DMSO-d₆, 126 MHz): δ =

136.3, 127.58, 123.3, 120.8, 118.4, 118.1, 111.6, 111.3, 65.7, 53.6,

29.4. m/z (ESI): 191.12 ([M + H]⁺).

8.5 Hz, 2H), 6.88−6.78 (m, 2H), 6.60−6.51 (m, 2H), 4.71 (s, 1H),

3.25−3.12 (m, 4H), 2.69 (dd, J = 13.7, 5.5 Hz, 1H), 2.35 (dd, J =

13.9, 6.9 Hz, 1H). ¹³C NMR (DMSO-d₆, 75 MHz) δ = 155.6, 141.0,

132.9, 131.8, 130.1, 130.0, 128.2, 114.8, 62.8, 57.4, 36.2. m/z (ESI):

386.01 ([M + H]⁺).

(S)-4-Bromo-N-(1-hydroxy-3-(1H-indol-3-yl)propan-2-yl)-

benzenesulfonamide (9e). To a stirred solution of ^L-tryptophanol

(8e, 400 mg, 2.10 mmol) in 40 mL of a 1:2 mixture of DCM/

acetonitrile was added N,N′-diisopropylethylamine (326 mg, 2.52

mmol). The mixture was stirred at ambient temperature for 1 h, and

4-bromobenzene-sulfonyl chloride (1, 537 mg, 2.10 mmol) was added

in small portions over 30 min at 0 °C under cooling in an ice bath.

The cooling bath was removed, and the solution was stirred at

ambient temperature for 24 h. Removal of the solvent under reduced

pressure gave a brown residue that was taken up in 30 mL of DCM,

washed with hydrochloric acid (1 N, 3 × 5 mL), water (3 × 5 mL),

and brine (5 mL) and dried over MgSO₄. The solvent was removed

under reduced pressure to give a crude brown oil that was puriﬁed by

column chromatography using a 1:1 mixture of ethyl acetate/

1

cyclohexane yielding 9e (257 mg, 30%) as a yellow oil. H NMR

(DMSO-d₆, 500 MHz): δ = 10.70 (s, 1H), 7.73 (d, J = 6.9 Hz, 1H),

7.50−7.43 (m, 4H), 7.28 (dt, J = 7.8, 1.0 Hz, 2H), 7.07−7.01 (m,

2H), 6.91 (tt, J = 6.9, 0.9 Hz, 1H), 4.79 (t, J = 5.3 Hz, 1H), 3.42−3.39

(m, 1H), 3.33−3.25 (m, 2H), 2.95 (dd, J = 14.3, 6.0 Hz, 1H), 2.61

(dd, J = 14.3, 7.0 Hz, 1H). ¹³C NMR (DMSO-d₆, 126 MHz): δ =

140.6, 136.1, 131.4, 127.9, 127.0, 125.4, 123.7, 120.6, 118.1, 117.9,

111.4, 110.3, 63.2, 56.1, 27.0. m/z (ESI): 409.02 ([M + H]⁺).

(R)-4-Bromo-N-(1-hydroxy-3-(1H-indol-3-yl)propan-2-yl)-

benzenesulfonamide (9f). To a stirred solution of ^D-tryptophanol

(8f, 625 mg, 3.29 mmol) in 60 mL of a 1:2 mixture of DCM/

acetonitrile was added N,N′-diisopropylethylamine (510 mg, 3.94

mmol). The mixture was stirred at ambient temperature for 1 h, and

4-bromobenzene-sulfonyl chloride (1, 839 mg, 3.29 mmol) was added

in small portions over 30 min at 0 °C under cooling in an ice bath.

(S)-4-Bromo-N-(1-hydroxy-3-phenylpropan-2-yl)benzene-

sulfonamide (9a). Synthesis of 9a was performed by following

general procedure A using ^L-phenylalaninol (8a). Yield 98%, colorless

solid. ¹H NMR (DMSO-d₆, 500 MHz) δ = 7.60−7.56 (m, 2H), 7.50−

7.46 (m, 2H), 7.12 (dd, J = 5.2, 1.9 Hz, 3H), 7.06−7.00 (m, 2H), 4.80

(s, 1H), 3.33 (dd, J = 10.1, 4.2 Hz, 1H), 3.30−3.24 (m, 1H), 3.21

(dd, J = 9.9, 6.6 Hz, 1H), 2.83 (dd, J = 13.7, 5.3 Hz, 1H), 2.46 (dd, J

= 13.7, 8.0 Hz, 1H). ¹³C NMR (DMSO-d₆, 126 MHz) δ = 140.9,

138.3, 131.8, 129.1, 128.0, 128.0, 125.7, 125.5, 63.2, 57.3, 37.0. m/z

(APCI): 370.01 ([M + H]⁺).

(R)-4-Bromo-N-(1-hydroxy-3-phenylpropan-2-yl)benzene-

sulfonamide (9b). Synthesis of 9b was performed by following

general procedure A using ^D-phenylalaninol (8b). Yield 99%, colorless

M

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The cooling bath was removed, and the solution was stirred at

ambient temperature for 24 h. Removal of the solvent under reduced

pressure gave a brown residue that was taken up in 40 mL of DCM,

washed with hydrochloric acid (1 N, 3 × 7 mL), water (3 × 7 mL),

and brine (7 mL) and dried over MgSO₄. The solvent was removed

under reduced pressure to give a crude brown oil that was puriﬁed by

column chromatography using a 1:1 mixture of ethyl acetate/

7.86−7.80 (m, 2H), 7.79−7.73 (m, 2H), 7.67 (d, J = 7.0 Hz, 1H),

7.59 (t, J = 2.9 Hz, 1H), 7.26 (dd, J = 5.0, 1.4 Hz, 1H), 6.87 (d, J =

7.1 Hz, 2H), 6.63 (q, J = 3.5, 1.6 Hz, 1H), 6.57 (d, J = 8.4 Hz, 2H),

4.75 (t, J = 5.2 Hz, 1H), 3.31−3.15 (m, 3H), 2.74 (dd, J = 13.7, 5.4

Hz, 1H), 2.40 (dd, J = 13.8, 6.8 Hz, 1H). ¹³C NMR (DMSO-d₆, 126

MHz): δ = 155.6, 149.1, 142.9, 141.7, 141.3, 138.6, 130.1, 128.6,

128.4, 127.1, 126.9, 117.2, 114.9, 114.4, 98.8, 62.8, 57.4, 36.3. ¹³C

NMR (DMSO-d₆, 126 MHz, DEPT): δ = 142.9, 130.1, 128.6, 127.1,

126.9, 114.9, 114.4, 98.8, 62.8, 36.3. m/z (ESI): 424.13 ([M + H]⁺).

m/z (ESI-HRMS): 424.1328 ([M + H]⁺).

(S)-N-(1-Hydroxy-3-(1H-indol-3-yl)propan-2-yl)-4-(1H-

pyrrolo[2,3-b]pyridin-4-yl)benzenesulfonamide (10e). Starting

from 9e, the synthesis of 10e was performed by following general

procedure B. Puriﬁcation was performed by column chromatography

using a 20:1:0.1 mixture of DCM/MeOH/10% aq. NH₄OH (25%) in

MeOH. Yield 60%, beige solid. ¹H NMR (DMSO-d₆, 500 MHz,): δ =

11.85 (s, 1H), 10.69 (s, 1H), 8.32 (d, J = 4.9 Hz, 1H), 7.79−7.73 (m,

4H), 7.70 (d, J = 6.5 Hz, 1H), 7.57 (t, J = 2.9 Hz, 1H), 7.31 (d, J = 7.9

Hz, 1H), 7.20 (dd, J = 7.0, 5.3 Hz, 2H), 7.07 (d, J = 1.8 Hz, 1H), 6.93

(td, J = 8.1, 1.3 Hz, 1H), 6.86 (t, J = 7.4 Hz, 1H), 6.59 (dd, J = 3.4,

1.7 Hz, 1H), 4.74 (s, 1H), 3.42−3.38 (m, 2H), 3.37−3.26 (m, 2H),

2.97 (dd, J = 14.3, 6.4 Hz, 1H), 2.67 (dd, J = 14.3, 6.0 Hz, 1H). ¹³C

NMR (DMSO-d₆, 126 MHz): δ = 149.1, 142.8, 141.6, 141.2, 138.6,

136.1, 128.4, 127.2, 127.1, 126.8, 123.8, 120.7, 118.1, 118.0, 117.1,

114.40, 111.3, 110.4, 98.9, 62.9, 56.2, 27.1. ¹³C NMR (DMSO-d₆, 126

MHz, DEPT): δ = 142.9, 128.4, 127.1, 126.8, 123.8, 120.7, 118.1,

118.0, 114.4, 111.3, 98.9, 62.9, 56.2, 27.1. m/z (ESI): 447.15 ([M +

H]⁺). m/z (ESI-HRMS): 447.1484 ([M + H]⁺).

(R)-N-(1-Hydroxy-3-(1H-indol-3-yl)propan-2-yl)-4-(1H-

pyrrolo[2,3-b]pyridin-4-yl)benzenesulfonamide (10f). Starting

from 9f, the synthesis of 10f was performed by following general

procedure B. Puriﬁcation was performed by column chromatography

using a 20:1:0.1 mixture of DCM/MeOH/10% aq. NH₄OH (25%) in

MeOH. Yield 62%, beige solid. ¹H NMR (DMSO-d₆, 500 MHz): δ =

11.86 (s, 1H), 10.71 (s, 1H), 8.33 (d, J = 4.9 Hz, 1H), 7.79−7.74 (m,

4H), 7.71 (d, J = 7.0 Hz, 1H), 7.58 (dd, J = 3.5, 2.6 Hz, 1H), 7.32 (d,

J = 7.0 Hz, 1H), 7.22 (d, J = 4.9 Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H),

7.08 (d, J = 2.3 Hz, 1H), 6.94 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 6.87

(ddd, J = 7.9, 7.0, 1.0 Hz, 1H), 6.60 (dd, J = 3.5, 1.9 Hz, 1H), 4.76 (t,

J = 5.4 Hz, 1H), 3.41−3.37 (m, 2H), 3.32−3.29 (m, 1H), 2.98 (dd, J

= 14.3, 6.5 Hz, 1H), 2.67 (dd, J = 14.3, 6.2 Hz, 1H). ¹³C NMR

(DMSO-d₆, 126 MHz): δ = 149.2, 142.9, 141.6, 141.2, 138.6, 136.1,

128.4, 127.2, 127.1, 126.8, 123.8, 120.7, 118.1, 118.0, 117.1, 114.4,

111.3, 110.4, 98.9, 62.9, 56.2, 27.0. m/z (ESI): 893.29 ([2M + H]⁺),

447.15 ([M + H]⁺). m/z (ESI-HRMS): 447.1485 ([M + H]⁺).

Molecular Modeling. Molecular modeling was performed using

the Molecular Operating Environment (Molecular Operating

Environment MOE, 2016.0802; Chemical Computing Group ULC,

1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A

2R7, 2021) software package, employing an “Amber10” force ﬁeld.

Isolation of the monomeric kinase domains was achieved by

manual deletion of the bridged chain using the “sequence editor”.

Superposition of the isolated chains was performed with the “align/

superpose” tool of the “sequence editor”.

1

cyclohexane yielding 9f (579 mg, 43%) as a yellow oil. H NMR

(DMSO-d₆, 500 MHz): δ = 10.66 (s, 1H), 7.69 (d, J = 7.0 Hz, 1H),

7.50−7.41 (m, 4H), 7.30−7.23 (m, 2H), 7.05−6.98 (m, 2H), 6.93−

6.85 (m, 1H), 4.75 (t, J = 5.4 Hz, 1H), 3.40−3.36 (m, 1H), 3.33−

3.24 (m, 2H), 2.94 (dd, J = 14.4, 6.0 Hz, 1H), 2.60 (dd, J = 14.3, 7.0

Hz, 1H). ¹³C NMR (DMSO-d₆, 126 MHz): δ = 140.6, 136.1, 131.4,

127.9, 127.0, 125.4, 123.7, 120.6, 118.1, 117.9, 111.4, 110.3, 63.2,

56.1, 27.0. m/z (ESI): 409.02 ([M + H]⁺).

(S)-N-(1-Hydroxy-3-phenylpropan-2-yl)-4-(1H-pyrrolo[2,3-

b]pyridin-4-yl)benzenesulfonamide (10a). Starting from 9a, the

synthesis of 10a was performed by following general procedure B.

Puriﬁcation was performed by column chromatography using a

20:1:0.1 mixture of DCM/MeOH/10% aq. NH₄OH (25%) in

1

MeOH. Yield 42%, beige solid. H NMR (DMSO-d₆, 500 MHz): δ

= 11.90 (s, 1H), 8.35 (d, J = 14.7, 5.0 Hz, 1H), 7.86−7.67 (m, 4H),

7.59 (t, J = 22.3, 3.1 Hz, 1H), 7.28 (d, J = 50.0, 4.9 Hz, 1H), 7.16−

6.99 (m, 4H), 6.58 (t, 1H), 4.87 (t, J = 5.5 Hz, 1H), 3.46−3.40 (m,

1H), 3.35−3.31 (m, 1H), 3.31−3.20 (m, 1H), 2.87 (dd, J = 13.6, 5.5

Hz, 1H), 2.47 (d, J = 8.4 Hz, 1H). ¹³C NMR (DMSO-d₆, 126 MHz):

δ = 149.2, 142.9, 141.6, 141.2, 138.6, 138.4, 129.1, 128.6, 128.0,

127.2, 126.8, 125.9, 117.1, 114.31, 98.9, 63.3, 57.3, 37.0. ¹³C NMR

(DMSO-d₆, 126 MHz, DEPT): δ = 142.9, 129.1, 128.6, 128.0, 127.1,

126.8, 125.8, 114.3, 98.8, 63.3, 57.3, 37.1. m/z (ESI): 408.14 ([M +

H]⁺). m/z (ESI-HRMS): 408.1378 ([M + H]⁺).

(R)-N-(1-Hydroxy-3-phenylpropan-2-yl)-4-(1H-pyrrolo[2,3-

b]pyridin-4-yl)benzenesulfonamide (10b). Starting from 9b, the

synthesis of 10b was performed by following general procedure B.

Puriﬁcation was performed by column chromatography using a

20:1:0.1 mixture of DCM/MeOH/10% aq. NH₄OH (25%) in

1

MeOH. Yield 56%, beige solid. H NMR (DMSO-d₆, 500 MHz): δ

= 11.88 (s, 1H), 8.34 (d, J = 4.9 Hz, 1H), 7.80−7.72 (m, 4H), 7.60 (t,

J = 2.9 Hz, 1H), 7.22 (dd, J = 4.9, 1.1 Hz, 1H), 7.15−6.98 (m, 5H),

6.72−6.54 (m, 1H), 4.86 (t, J = 5.4 Hz, 1H), 3.44−3.40 (m, 1H),

3.37−3.32 (m, 1H), 3.28 (dt, J = 11.1, 6.1 Hz, 1H), 2.88 (dd, J = 13.7,

5.4 Hz, 1H), 2.50 (dd, 1H). ¹³C NMR (DMSO-d₆, 126 MHz): δ =

149.15, 142.95, 141.64, 141.22, 138.63, 138.37, 129.14, 128.59,

127.97, 127.14, 126.76, 125.85, 117.13, 114.32, 98.80, 63.27, 57.28,

37.07. ¹³C NMR (DMSO-d₆, 126 MHz, DEPT): δ = 143.0, 129.2,

128.6, 128.0, 127.2, 126.8, 125.9, 114.4, 98.8, 63.3, 57.3, 37.1. m/z

(ESI): 815.09 ([2M + H]⁺), 408.14 ([M + H]⁺). m/z (ESI-HRMS):

408.1374 ([M + H]⁺).

(S)-N-(1-Hydroxy-3-(4-hydroxyphenyl)propan-2-yl)-4-(1H-

pyrrolo[2,3-b]pyridin-4-yl)benzenesulfonamide (10c). Starting

from 9c, the synthesis of 10c was performed by following general

procedure B. Puriﬁcation was performed by column chromatography

using a 10:1:0.1 mixture of DCM/MeOH/10% aq. NH₄OH (25%) in

1

MeOH. Yield 51%, colorless solid. H NMR (DMSO-d₆, 500 MHz):

Reconstruction of the glycine-rich loop (P-Loop) was performed

using the “SuperLooper2” web server.⁴⁷Starting from the crystal

structure of Nek1 (PDB code 4B9D.B), the missing loop sequence

EGSFG (between G11 and K17) was reconstructed by using the

AGPAG loop (between P311 and R317) of the Mycobacterium

tuberculosis probable periplasmic sugar-binding lipoprotein UspC

(PDB code 5K2Y) crystal structure as a template.

The Nek1 kinase domain homology model was created using the

“homology model” function and the crystal structure of Nek2 bound

to an aminopurine inhibitor (PDB code 5M53) as the template.⁴⁸

For all docking experiments, residual water molecules were deleted,

and the receptors were prepared using the “QuickPrep” function

without “automated structure preparation”. Using the respective

functions of the “MOE Database Viewer”, all ligands were “washed”

before partial charges were determined and the structures underwent

energy minimization. The binding sites were designated from the

δ = 11.88 (s, 1H), 9.14 (s, 1H), 8.33 (d, J = 4.9 Hz, 1H), 7.86−7.74

(m, 4H), 7.71 (d, J = 7.3 Hz, 1H), 7.59 (t, J = 3.0 Hz, 1H), 7.26 (d, J

= 4.9 Hz, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.63 (dd, J = 3.6, 1.8 Hz,

1H), 6.58 (d, J = 8.4 Hz, 2H), 4.79 (t, J = 5.3 Hz, 1H), 3.38−3.33 (m,

1H), 3.25 (p, J = 6.1 Hz, 2H), 2.75 (dd, J = 13.7, 5.5 Hz, 1H), 2.40

(dd, J = 13.7, 6.9 Hz, 1H). ¹³C NMR (DMSO-d₆, 126 MHz): δ =

155.7, 149.2, 143.0, 141.8, 141.4, 138.7, 130.1, 128.6, 128.4, 127.2,

126.9, 117.2, 114.9, 114.48, 98.9, 62.9, 57.5, 36.3. m/z (ESI): 424.13

([M + H]⁺). m/z (ESI-HRMS): 424.1326 ([M + H]⁺).

(R)-N-(1-Hydroxy-3-(4-hydroxyphenyl)propan-2-yl)-4-(1H-

pyrrolo[2,3-b]pyridin-4-yl)benzenesulfonamide (10d). Starting

from 9d, the synthesis of 10d was performed by following general

procedure B. Puriﬁcation was performed by column chromatography

using a 10:1:0.1 mixture of DCM/MeOH/10% aq. NH₄OH (25%) in

1

MeOH. Yield 26%, colorless solid. H NMR (DMSO-d₆, 500 MHz):

δ = 11.87 (s, 1H), 9.10 (d, J = 1.3 Hz, 1H), 8.33 (d, J = 4.9 Hz, 1H),

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cocrystallized ligand (PDB code 4B9D), which was placed in the

binding site by superposition in the case of the apo structure (PDB

code 4APC) and the homology model. Docking poses were placed

using the “Triangle Matcher” method and scored by a “London dG”

function. Reﬁnements of the poses were performed by using the

“Rigid Receptor” method and were ﬁnally scored using a “GBVI/WSA

dG” function. For the docking of 3a into the apo structure, a hinge

binding pharmacophore was deﬁned, based on the cocrystallized

ligand.

The sequence homology analysis between the human and zebraﬁsh

Nek1 kinase domains was performed using the “MOE protein

similarity monitor”. Identity and similarity values are equal to the

number of matches divided by the number of amino acids in the chain

corresponding to the cell column. Residues are considered similar if

their BLOSUM62 scores are greater than zero.

Software, San Diego, California USA, www.graphpad.com). For

compounds 3j and 3k, the maximum concentration data points were

dropped, as a consequence of solubility issues at 30 μM.

Caco-2 Cell Permeability Assay. A−B permeability (Caco-2, pH

6.5/7.4) and percent recovery were determined by HPLC-MS/MS

detection of the peak area response in a commercial assay by Euroﬁns

Discovery Services (Item 3318). Propranolol, labetalol, colchicine,

and ranitidine were tested concurrently as reference compounds.

Fish Maintenance and Husbandry Protocols. Fish main-

tenance and husbandry protocols were documented and approved by

the Darmstadt administrative authority. All animals were treated

humanely in accordance with the German animal protection standards

and the EU Directive 2010/63/EU of the European parliament and of

the council. Adult wild-type zebraﬁsh (Danio rerio) were maintained

in 60 L ﬁsh tanks in a laboratory with no daylight and a constant

temperature of 28 °C. Room light was programmed to a 12 h dark/12

h light cycle. Adult zebraﬁsh were bred in mating containers (2

animals/1 L) or mating tanks (6−8 animals/20 L) that were equipped

with plastic grass and a removable sieve at the bottom of the tank.

Embryos were collected 2−3 h after a light cycle started, rinsed with

E3-medium, and isolated in Petri dishes containing E3-medium. E3-

medium was prepared according to the Cold Spring Harbor Protocols

recipe for E3 medium for zebraﬁsh embryos (doi: 10.1101/

pdb.rec066449).

Zebraﬁsh Developmental Toxicity Assay. The aqueous

solubility of 10f was determined in a shake-ﬂask HPLC solubility

assay using the previously described HPLC system connected to a

diode array detector (DAD, see General Information). Absorption

was measured at 280 nm, and all measurements were performed in

triplicate. A saturated solution of 10f in E3-medium was prepared by

shaking for 24 h at 25 °C. A 10 mM stock solution of 10f in DMSO

was diluted to 1, 5, 10, 50, and 100 μM solutions in acetonitrile, and

the aqueous solubility of 10f was determined by interpolation using

Prism 8 by GraphPad Software, LLC.

In accordance with the author guidelines, all virtual screening hits

were ﬁltered for PAINS using the PAINS-remover online

application.⁶⁴All compounds passed the ﬁlter.

In Vitro Kinase Activity Assays. Residual kinase inhibition was

determined with the commercial Euroﬁns Discovery KinaseProﬁler

service at 1 μM compound concentration and either 10 μM ATP or

an ATP concentration within 15 μM of the apparent K_mfor ATP (see

Supporting Information for K_mvalues). In the case of Nek1

(KinaseProﬁler ITEM 15-020KP10 and ITEM 15-020KP), recombi-

nant human Nek1 (1−505) was incubated with 8 mM MOPS (pH

7.0), 0.2 mM EDTA, 250 μM RLGRDKYKTLRQIRQ, 10 mM

magnesium acetate, and [γ-³³P-ATP]. The reaction was initiated by

the addition of the Mg/ATP mix. After incubation for 40 min at room

temperature, the reaction was stopped by the addition of phosphoric

acid to a concentration of 0.5%. Ten microliters of the reaction was

then spotted onto a P30 ﬁltermat and washed four times for 4 min in

0.425% phosphoric acid and once in methanol prior to drying and

scintillation counting. Values are the mean from two independent

experiments.

IC₅₀values for Nek1 and Nek2 were determined in a TR-FRET

based activity assays (LANCE Ultra TR-FRET, PerkinElmer) at an

ATP concentration within 15 μM of the apparent K_mfor ATP in the

respective assay format (60 μM for both Nek1 and Nek2). The IC₅₀

curves consist of 10 test compound concentrations tested at half-log

dilutions from 30 μM to 1 nM with vehicle and inhibitory control

wells. For compounds 3j and 3k, the maximum concentration was set

to 10 μM as a consequence of solubility issues at 30 μM, and the

respective IC₅₀curves consist of 9 test compound concentrations

tested at half-log dilutions from 10 μM to 1 nM. Values are the mean

from two independent experiments. Recombinant human Nek1 (1−

505; 0.02 nM) or Nek2 (full length; 0.2 nM) (both from Thermo

Fisher Scientiﬁc) was incubated with 50 mM HEPES (pH 7.5), 1 mM

EGTA, 10 mM MgCl₂, 0.2 mM DTT, 0.01% Tween20, and 50 nM

ULight-p70 S6K (Thr389) peptide (sequence LGFTYVAP; Perki-

nElmer). The reaction was initiated by the addition of the p70 S6K

peptide/ATP mix. After incubation for 30 min at 22 °C, the reaction

was stopped by the addition of 10 mM EDTA and 0.3 nM europium-

antiphospho-p70 S6K (Thr389) antibody in CR97 detection buﬀer

(both PerkinElmer). After incubation for 1 h at 22 °C for signal

development, the plates were analyzed in a plate reader (PHERAstar

FSX, BMG LABTECH). For compounds 3g and 3k, relative IC₅₀

values were provided instead of absolute IC₅₀, as the determined IC₅₀

values exceeded the employed maximum test concentrations (30 μM

for 3g, 10 μM for 3k).

For compounds 3a, 3f, 10a−f, IC₅₀values were also determined

with the Euroﬁns Discovery IC50Proﬁler service at either 10 μM

(ITEM 15-020KP10) or an ATP concentration within 15 μM of the

apparent K_mfor ATP (90 μM, ITEM 15-020KP). The IC₅₀curves

consist of 9 test compound concentrations tested at half-log dilutions

from 10 μM to 1 nM with vehicle control wells. Values are the mean

from two independent experiments.

IC₅₀values and 95% conﬁdence intervals were determined from the

raw data provided in the Supporting Information by nonlinear

regression (log_inhibitor vs response_variable slope_four parame-

ters) using GraphPad Prism, version 8.4.3 (471) for OS X, (GraphPad

The zebraﬁsh developmental toxicity screen was performed in

accordance to guidelines published by M. Haldi et al. in Zebrafish:

Methods for Assessing Drug Saftey and Toxicity.⁶⁵

At 4 hpf (hours postfertilization), 15 embryos per group (0.05, 0.1,

0.5, 1.5, 2.5, 5, 10, 15, 20, 30 μM) that exhibited intact chorion

membranes were selected and reexamined after 2 h. A stock solution

(10 mM in DMSO) of the test compound was diluted to 10× ﬁnal

concentrations, and the embryos were sorted in 180 μL of E3

medium. At 6 hpf, 20 μL of the 10× solution was added to give the

ﬁnal concentrations. Survival was monitored every 24 h by

observation of the heartbeat. All survival rates were determined at

least in triplicate with the controls and the survival rate at 30 μM

being determined in 5 replicates.

Stability-Indicating HPLC Assay. The stability-indicating HPLC

assay was performed using the previously described HPLC system

connected to a diode array detector (DAD, see General Information).

A 15 μM solution of 10f in E3-medium containing 75 μM 2-

phenylthiourea (PTU) and a 15 μM solution of 10f containing 75 μM

PTU and 2.5 μM PT-Yellow (BDNCA3-D2) were prepared for

HPLC injection. Absorptions were measured every 24 h at 280 nm for

compound 10f and PTU and 548 nm for PT-Yellow over 48 or 120 h,

respectively. All measurements were performed in triplicate.

Fluorescence Microscopy Imaging of Zebraﬁsh Pronephroi.

The 96-well plate molding tool was manufactured by selective laser

melting on an EOS M290 (EOS GmbH Electro Optical Systems,

Krailing, Germany) metal 3D-printing system using EOS Stainless-

Steel 316L as the material with a layer height of 20 μM. The tool

consists of a base plate with 96 perpendicular pins that match the

positions of the wells of a microtiter plate. The agarose molds were

prepared using 1% NEEO ultraquality agarose (Carl Roth, Karlsruhe,

Germany, Art. No. 2267.1) in black-walled 96-well Nunc polymer

optical bottom plates (Thermo Fisher Scientiﬁc, Waltham,

Massachusetts, USA). The speciﬁcations of the molding tool and

the preparation of the agarose molds in 96-well microtiter plates

followed the procedure published by Westhoﬀ et al.⁵⁵

O

https://dx.doi.org/10.1021/acs.jmedchem.0c02118

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Image Acquisition. Imaging of ﬂuorescently labeled (PT-Yellow)

zebraﬁsh embryos was performed on an Olympus IX-81 wideﬁeld

microscope (Olympus, Shinjuku, Tokyo, Japan), using an Olympus

CPlanFL N, 10×, NA 0.3 objective in combination with a 1.5× tube

lens magniﬁcation, resulting in a total magniﬁcation of 15×.

Fluorescence was excited using a CoolLED pE300 Ultra (CoolLED

Ltd., Andover, NY, USA) and detected on a pco.edge 5.5 (PCO AG,

Kelheim, Germany) using appropriate ﬁlter sets. The whole setup was

controlled using Micromanager 1.4.22.⁶⁶to record 33 Z-slices with a

Z-distance of 15 μm per embryo after manual orientation in the

previously described agarose molds.

Torben Reichardt − Clemens Schöpf−Institute of Organic

Chemistry and Biochemistry, Technische Universität

Darmstadt, 64287 Darmstadt, Germany

Johannes Pilakowski − Clemens Schöpf−Institute of Organic

Chemistry and Biochemistry, Technische Universität

Darmstadt, 64287 Darmstadt, Germany

Ulrich Pehl − Merck Healthcare KGaA, Biopharma R&D,

Discovery and Development Technologies, 64293 Darmstadt,

Germany

Complete contact information is available at:

Image Processing and Deconvolution. Image processing was

performed using ImageJ, version 2.0.0.-rc-69/1.52p. Deconvolution

was performed using the DeconvolutionLab2 plugin and a point

spread function that was simulated in the PSF Generator plugin. The

image stacks are presented as colored maximum Z-projections using

the lookup table blue orange icb (Figure S14). As it was not possible to

remove all out of focus blur with the deconvolution procedure, the

sharpest 5 consecutive Z-slices of labeled pronephrons were manually

selected in each stack (Figures S15 and S16). Subsequently, the

following image analysis steps were applied using ImageJ: (1) subtract

background using a 150 pixel rolling ball, (2) sum all 5 images into

one, (3) threshold images above 100, (4) convert background to 8-bit

mask for the intensity quantiﬁcation or convert background to NaN

for the quantiﬁcation of the pronephron areas. The pronephron area

in the summed stacks was used as the quantity to estimate the size of

the pronephroi.

https://pubs.acs.org/10.1021/acs.jmedchem.0c02118

Author Contributions

G.B., B.S., and T.M. conceived and designed the experiments.

G.B., T.M., and B.S. wrote the manuscript. G.B., K.B., Y.S.,

M.D., U.D., and T.R. performed the experiments. G.B., B.S.,

U.P., and T.M. analyzed the data. G.B., B.S., T.M., and J.P.

gave scientiﬁc advice. All authors contributed to the article and

approved the submitted version.

Funding

The authors declare that this study received funding from the

German Research Foundation (DFG, GRK1657, Project

number: 161030019). The funders had no role in study

design, data collection and analysis, decision to publish, or

preparation of the manuscript.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02118.

■

Notes

sı

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Holger Hermann Merschroth for the design and

production of the 96-well plate molding tool and Nadine

Herget and Friedrich Baumgärtner for performing the TR-

FRET in vitro kinase activity assays. We furthermore thank

Prof. Markus Löbrich, Julian Spies, and Michael Ensminger for

discussions on the biological role of Nek1.

Molecular modeling, homology model coordinates, in

vitro pharmacology, NMR data, lead compound HPLC

traces, bioavailability proﬁle of compound 10f (PDF)

Molecular formula strings (CSV)

Nek1 homology model (PDB)

Superlooper2 reconstruction model (PDB)

ABBREVIATIONS

■

ALS, amyotrophic lateral sclerosis; CDK, cyclin-dependent

kinase; Chk1, checkpoint kinase 1; Chk2, checkpoint kinase 2;

cDNA, complementary DNA; DDR, DNA damage response;

HR, homologous recombination; IKK, IκB kinase; JNK, c-Jun

N-terminal kinase; DSB, double strand break; HR, homolo-

gous recombination; Nek1, NIMA-related kinase 1; NF-κB,

nuclear factor kappa-light-chain-enhancer of activated B-cells;

MOE, Molecular Operating Environment; NIMA, never-in-

mitosis gene A; PKD, polycystic kidney disease; PSF, point

square function; RCC, renal cell carcinoma; SAR, structure−

activity relationship; TLC, thin layer chromatography; TPSA,

topological polar surface area; Tlk1, tousled-like kinase 1

AUTHOR INFORMATION

Corresponding Authors

■

Boris Schmidt − Clemens Schöpf−Institute of Organic

Chemistry and Biochemistry, Technische Universität

Darmstadt, 64287 Darmstadt, Germany; orcid.org/

0000-0003-1662-2392; Phone: +496151-1623745;

Email: schmidt_boris@t-online.de; Fax: +496151-163278

Georg Baumann − Clemens Schöpf−Institute of Organic

Chemistry and Biochemistry, Technische Universität

Darmstadt, 64287 Darmstadt, Germany;

Email: georg.baumann@stud.tu-darmstadt.de

Authors

REFERENCES

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Yung-Hsin Shih − Clemens Schöpf−Institute of Organic

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Darmstadt, 64287 Darmstadt, Germany

Mirco Dickhaut − Clemens Schöpf−Institute of Organic

Chemistry and Biochemistry, Technische Universität

Darmstadt, 64287 Darmstadt, Germany

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https://dx.doi.org/10.1021/acs.jmedchem.0c02118

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Illuminating a Dark Kinase: Structure-Guided Design, Synthesis, and Evaluation of a Potent Nek1 Inhibitor and Its Effects on the Embryonic Zebrafish Pronephros

DOI: 10.1021/acs.jmedchem.0c02118

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Full text of DOI:10.1021/acs.jmedchem.0c02118

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