Fragment-Derived Selective Inhibitors of Dual-Specificity Kinases DYRK1A and DYRK1B

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Fragment-Derived Selective Inhibitors of Dual-Speciﬁcity Kinases

DYRK1A and DYRK1B

David Lee Walmsley, James B. Murray, Pawel Dokurno, Andrew J. Massey, Karen Benwell,

Andrea Fiumana, Nicolas Foloppe, Stuart Ray, Julia Smith, Allan E. Surgenor, Thomas Edmonds,

Didier Demarles, Mike Burbridge, Francisco Cruzalegui, Andras Kotschy, and Roderick E. Hubbard*

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ABSTRACT: The serine/threonine kinase DYRK1A has been implicated in

regulation of a variety of cellular processes associated with cancer

progression, including cell cycle control, DNA damage repair, protection

from apoptosis, cell diﬀerentiation, and metastasis. In addition, elevated-level

DYRK1A activity has been associated with increased severity of symptoms in

Down’s syndrome. A selective inhibitor of DYRK1A could therefore be of

therapeutic beneﬁt. We have used fragment and structure-based discovery

methods to identify a highly selective, well-tolerated, brain-penetrant

DYRK1A inhibitor which showed in vivo activity in a tumor model. The

inhibitor provides a useful tool compound for further exploration of the eﬀect of DYRK1A inhibition in models of disease.

implicated to have multiple roles in cancer development

including a role in the G1/S and G2/M cell cycle transition,

epithelial−mesenchymal transition, cancer stemness, and DNA

damage response through p53 phosphorylation. In lower

eukaryotes, DYRK2 has been implicated in tissue development,

thereby implying a role for this kinase in cell diﬀerentiation

and homeostasis in mammals.^19,20These observations suggest

that targeting DYRK kinase activity could be of therapeutic

beneﬁt for patients presenting with a diverse range of cancers.

There has also been interest in DYRK1A in patients with

Down’s syndrome. The gene is located on chromosome 21,

and the subsequent elevated levels of DYRK1A in Down’s

syndrome patients has been correlated with an increase in

severity of symptoms and a poorer prognosis.²¹Recently, it has

been suggested that a natural product inhibitor of DYRK1A,

the polyphenol from green tea, epigallocatechin gallate

(EGCG), has eﬃcacy in a murine model of Down’s syndrome

through chromosome 16 trisomy.²²Although the wider

activity, instability, and poor compound properties of EGCG

complicate interpretation of these results, inhibiting DYRK

kinase activity could also be of therapeutic beneﬁt to Down’s

syndrome patients.

INTRODUCTION

■

DYRK1A is the best-studied member of the evolutionarily

conserved DYRK (dual-speciﬁcity tyrosine-regulated kinase)

branch of the cyclin-dependent, mitogen-activated, glycogen

synthase, and CDC-like (CMGC) family of serine/threonine

kinases that consists of two classes: DYRK1A and DYRK1B

(Class 1) and DYRK2, DYRK3, and DYRK4 (Class 2).

DYRK1A is activated by self-phosphorylation of S520, leading

to 14-3-3β binding which stimulates the catalytic activity.¹

DYRK1A and 1B play an important role in cell cycle control

and modulation of DYRK1A/B in sarcoma,^2,3and lung,⁴

pancreatic,⁵and ovarian cancer⁶can result in dysregulation of

cell cycle control. These kinases regulate the induction of

quiescence via control of the DREAM complex,⁷thereby

controlling the G0-G1 transition and exit from quiescence.⁸⁻¹⁰

DYRK1A and 1B can also phosphorylate Cyclin D1 (resulting

in its destabilization)^11,12and p27Kip1,^13,14thereby altering

the cell’s decision to enter the cell cycle. Additionally,

DYRK1A kinase activity stabilizes epidermal growth factor

(EGFR) in glioblastoma through regulation of Sprouty2, and

inhibition of DYRK1A reduces EGFR-dependent glioblastoma

growth.¹⁵DYRK1A and 1B control the activity of the

Hedgehog signaling eﬀector GLI through direct phosphor-

ylation, resulting in increased nuclear accumulation and

transcriptional activity.¹⁶Pharmacological inhibition of

DYRK1B repressed Hedgehog signaling and inhibited the

proliferation of GLI1-dependent pancreatic cancer cells.¹⁷

Recent work suggests that suppression of DYRK1A restrains

myeloid cell leukemia 1 (MCL-1) expression and sensitizes

nonsmall cell lung cancer cells to B-cell lymphoma 2 (Bcl-2)

inhibitors.¹⁸Of the Class 2 DYRKs, DYRK2 has been

In addition to Down’s syndrome, recent studies have

suggested a role for DYRK1A in other neurodegenerative

Received: January 7, 2021

Published: June 18, 2021

American Chemical Society

https://doi.org/10.1021/acs.jmedchem.1c00024

J. Med. Chem. 2021, 64, 8971−8991

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Figure 1. (a) Ribbon structure of Dyrk1a, highlighting hinge (red) and ADPNP bound in the stick (monomer B in the crystal structure, PDB:

7A4O) and the portion of the structure (light blue) removed in all subsequent ﬁgures (residues E160 to D178 which include the glycine loop). (b)

Closeup of the ATP binding site showing side by side ADPNP bound to Dyrk1a (main chain white) and PAK4 (main chain pink) (PDB: 4FIG).

(c) Standard orientation and convention (unless stated) used for ﬁgures of compounds (element coloring with purple carbons) binding to

Dyrk1aremove the glycine loop from E160 to D178 to view compound binding, peptide backbone as a ribbon structure for the remainder of the

protein except for Dyrk1a F238 to N244 (hinge region) and V306 to F308 with additional selected side chains and water molecules (none in Figure

1) colored on the element in stick (gray carbons) and protein backbone and side chain hydrogen bonds as blue and green dashed lines with

nonstandard interactions in orange dashed lines.

disorders such as Alzheimer’s disease (including early onset

AD in Down’s syndrome patients²³⁻²⁵) and Parkinson’s

disease.^26,27DYRK1A has also been demonstrated to regulate

the proliferation and cell size of beta cells (and therefore their

to that used to generate the crystal structure deposited with

PDB code 2vx3³¹(see the Experimental Section for details).

The kinase p21-associated kinase 4 (4PAK4) was used as a

counterscreen throughout the project, and selectivity against

other kinases was measured as the project progressed. The

inhibition of a selection of protein kinases was measured using

either an adenosine diphosphate (ADP) Hunter or time-

resolved ﬂuorescence energy transfer (TR-FRET) assay, and

the inhibition of GSK3-β was measured by a radiometric assay

(details in the Experimental Section).

We solved the crystal structure of Dyrk1a with 5′-adenylyl-

β,γ-imidodiphosphate (ADPNP) bound. Figure 1 shows a

comparison between this structure of Dyrk1a and the

published structure of PAK4 with ADPNP.³³

Dyrk1a has the conventional fold of a protein kinase with a

larger, predominantly α-helical subdomain connected to a

predominantly β-sheet subdomain, forming a cleft that binds

ATP. The subdomains are connected by a so-called “hinge”

region where the main chain atoms hydrogen-bond to the

donor−acceptor−donor motif of the adenosine moiety of

ATP, positioning the sugar and phosphate groups adjacent to

the catalytic machinery. The structure reveals an unusual

conformation for the main chain carbonyl of L241 in Dyrk1a

compared to that seen for the equivalent amino acid (L398) in

PAK4 (Figure 1b) and other kinases (data not shown).

Identifying and Characterizing the Initial Fragment

Hits. The Vernalis fragment library consisting of 1063

compounds at the time^34,35was screened using three ligand-

observed nuclear magnetic resonance (NMR) experiments³⁶

for binding to the ATP-binding site of Dyrk1a in competition

with the nonspeciﬁc kinase inhibitor, staurosporine. Com-

pounds that were conﬁrmed as binding competitively in all

three NMR experiments were assayed in an ADP Hunter

biochemical assay (hereafter the functional assay) to generate

166 fragment hits, a hit rate of 15%.

regenerative capacity) in the pancreas²⁸⁻³⁰

.

Our objective was to use fragment and structure-based lead

discovery methods to identify potent and selective adenosine

triphosphate (ATP) competitive inhibitors of DYRK1A and

DYRK1B to be used to probe the therapeutic beneﬁt of

inhibiting this class of kinase. Our work was initiated

concurrently to the release of the ﬁrst structure of the kinase

domain of DYRK1A (protein databank (PDB) code 2VX3³¹)

by the Structural Genomics Consortium in 2009. Here, we

describe the initial fragment-based hit discovery eﬀorts that

identiﬁed multiple series of hit compounds and the lead

optimization aided by molecular modeling that generated

pyrrolopyrimidine compounds that were proﬁled in several

cellular and in vivo models. An accompanying paper³²

describes a companion optimization project to generate

imidazopyridine compounds that were similarly proﬁled.

DYRK1A and DYRK1B vary by only one amino acid in the

ATP-binding pocket; the Class 2 DYRKs have more variation.

The lead molecule, 34, from this pyrrolopyrimidine hit series

inhibited DYRK1A and 1B with low nM potency and exhibited

excellent selectivity versus the Class 2 DYRKs. It potently

inhibited DYRK1A autophosphorylation in a cellular model

and had good oral bioavailability with a robust pharmacoki-

netic (PK)/pharmacodynamic (PD) relationship with in vivo

activity in a glioblastoma xenograft model. This molecule is a

potentially useful tool compound for further study of the eﬀect

of DYRK1A inhibition in models of human disease.

RESULTS AND DISCUSSION

Fragment screening and structural studies used a construct of

the DYRK1A kinase domain (hereafter termed Dyrk1a) similar

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Table 1. Fragment Hits Ranked by Ligand Eﬃciency Measured in ADP Hunter Assay

a

% inhibition in PAK4 assay with the 200 μM (400 μM) compound; binding pose in the crystal structure in the same orientation as Figure 1c. See

Tables S1 and S2 for structure determination and reﬁnement statistics; the accession codes for coordinates deposited to the PDB are listed at the

end of the manuscript. All 2D chemical structures are oriented to approximate the binding pose seen in crystal structures.

Table 1 lists the 8 hits with the highest ligand eﬃciency

[LE,³⁷deﬁned as free energy of binding (ΔG)/heavy atom

count (HAC)]. A high hit rate in an NMR fragment screen

indicates^38,39a highly eﬃcient site for binding small molecules

(often termed druggable⁴⁰), and the high LE of the hits

reinforces this for the ATP binding site in Dyrk1a. In addition,

these fragment hits show remarkable selectivity, given some

contain relatively standard kinase ATP binding motifs. This

can be explained by inspection of the Dyrk1a structure (Figure

1c) which shows a distinctive conformation for the carbonyl of

the hinge amino acid, L241.

We determined the crystal structures for the eight fragment

hits bound to Dyrk1a (Tables 1, S1 and S2). The fragments

exhibited both “classical” hinge binding with a donor−

acceptor−donor hydrogen bonding motif and “nonclassical”

where as few as one hydrogen bond anchors the fragment to

the hinge in the binding site. This diversity of binding modes

and motifs demonstrates one of the beneﬁts of fragment-based

discovery: even for the widely studied ATP binding site of a

kinase, a fragment screen can identify compounds that explore

features that can be exploited for selectivity, identify novel

binding modes that are not often discovered when screening

larger compounds, and provide a wide diversity of hit

compounds from which the medicinal chemist can choose

the most appropriate one for optimization.

with E203 through the NH₂of the pendant amino pyrimidine

(Figure 2b). Fragment 5 (Figure 2c) was selected for further

study as it makes a comprehensive set of interactions not only

with the hinge but also with the catalytic residues at the base of

the binding site through a well-deﬁned network of hydrogen

bonds to water.

Optimization from the Fragment to Lead Pyrrolopyr-

imidine Series. The ﬁrst step of optimization was to exploit

the pocket proximal to the carbonyl of L241. Inspection of the

crystal structures (Figure 2) suggested that a methyl group

could be incorporated into the 2-position of the pyrrolopyr-

imidine scaﬀold of 5. As can be seen in Table 2, this improved

the aﬃnity of compounds for DYRK1A and retained selectivity

over PAK4. We also investigated substitution of the 6-position

bromine with various aryls and heteroaryls to improve potency

further by building groups that replaced the solvent to interact

with K188 and E203.

The crystal structure (Figure 3) of the most ligand-eﬃcient

compound of the series (16) bound to Dyrk1a conﬁrmed the

success of the structure-based design and the structural basis

for retention of good selectivity against PAK4. The 2-methyl

occupies the pocket adjacent to carbonyl of L241, and the

pyridine nitrogen hydrogen-bonds to K188 with the other

polar atoms of the ligand making interactions with deﬁned

solvent positions.

As the fragment screen was completed, the crystal structure

(PDB: 3ANR) and properties of harmine (9) on Dyrk1a were

published⁴¹and the activity of meriolin derivatives was

reported.⁴²

It was noted in the structures of 10 (Figure 2b) and the

most ligand eﬃcient fragment (1) that the 4-amino group of

the pyrimidine ring of 16 is within a pyrrole ring, making a

“classical” hinge binding interaction with the carbonyl of E239.

A series of 2-methyl-5-aryl-7H-pyrrolo[2,3-d]pyrimidines were

therefore synthesized as listed in Table 3, keeping the methyl

group to target the L241 pocket and designed to retain the

main interactions with the remainder of the binding site. A 4-

position methoxy was also included for further elaboration.

The three most potent compounds (18, 19, and 20) were

proﬁled for their inhibition of a mini-panel of kinases (Table

4).

Figure 2 compares the binding of harmine (9) and meriolin-

3 (10) to Dyrk1a with some of the fragment hits. As shown in

Figure 2a, fragment 3 and harmine both have a “nonclassical”

binding mode, making just one hydrogen bond to the hinge

and with the methyl group of the methoxy in the pocket

formed by the unusual conformation of the main chain of L241

and one to E203. This fragment is very similar to fragment 4.

The optimization of fragment 3 with a similar binding mode to

harmine is described in a separate publication.³²The fragment

1 (Figure 2b) makes a “classical” set of hydrogen bonds with

the hinge, overlapping with the core of 10 which also interacts

The most potent compound (19) combines high target

aﬃnity (and LE of 0.57), almost equipotent inhibition of the

other proﬁled DYRK enzymes and with good selectivity against

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Figure 2. Detailed comparison of the binding mode of literature compounds and selected fragments to Dyrk1a showing the protein structure

(white ribbon), selected waters, and key interactions with the fragment (a) harmine (9green carbons, PDB: 3ANR) and fragment 3 (PDB:

7A4W); (b) meriolin-3 (10green carbons, PDB: 7A5B) and fragment 1 (PDB: 7A4R); and (c) fragment 5 (PDB: 7A51). See the legend to

Figure 1 for conventions used in the representation.

other kinases. The 4-position methoxy on the pyrimidine

presents a vector toward the amino acids in the sugar pocket of

the ATP site and the glycine loop regions. A selection of single-

ring aromatic moieties were synthesized at this 4-position

(Table 5).

A number of 5-membered heterocycles had similar potency

and selectivity (Table 6) and were found to be stable under a

range of aqueous conditions.

Lead Optimization to the Compound Suitable for in

Vivo Studies. The next stage in optimization was to

investigate further substituents at the 4-position of the

pyrimidine aromatic ring. A number of analogues were

synthesized, and a biaryl compound (37) was introduced by

direct coupling. These were all successful in retaining potency

but with an altered selectivity proﬁle, particularly against

DYRK2. The cellular activity and metabolic stability of this set

of compounds were also proﬁled (Table 7).

As can be seen in Figure 5, introduction of ortho

substituents on the pendant benzyl ring induces a twist of

the aromatic to be perpendicular to the plane of the

pyrrolopyrimidine core. This twist displaces a water molecule

in the structure. 34 was taken as the most promising lead

compound. Although the metabolic stability is moderate, it

shows good cellular activity with the IC₅₀for inhibition of the

autophosphorylation of S520 on DYRK1A in U2OS cells only

threefold less than the IC₅₀in the functional assay and with an

Simple aromatics such as thiophene (24) achieve single-digit

nanomolar potency in the DYRK1A functional assay, more

than 2 orders of magnitude greater than inhibition of Gsk3 β

and cyclin-dependent kinase 9 (CDK9). The crystal structure

of 24 (Figure 4) bound to Dyrk1a conﬁrmed the predicted

interactions that were made with additional Van Der Waals

interactions made in a lipophilic region under the glycine loop

(not shown in Figure 4) contributed to by G168 and the side

chains of I165, F170, V173, and K188. However, the 4-alkoxy

compounds were unstable in aqueous media at pH < 5 over a

relatively short period of a few hours due to hydrolysis of the

pendant methoxy (as analyzed by liquid chromatography−

mass spectrometry, LCMS), presumably via protonation of the

pyrimidine ring.

Similar benzylic motifs were introduced to make glycine

loop interactions by installing benzylamines at the 4-position.

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Table 2. Improving Interactions with the Carbonyl of L241 and the Catalytic Site; NDNot Determined

Figure 3. Structure of 16 bound to Dyrk1a (PDB: 7A5D). See the legend to Figure 1 for conventions used in the representation.

EC₅₀of less than 1 nM in a nano-BRET assay (Figure S1).

These results are consistent with the cell permeability

properties of the compound, measured in a Caco2 assay (see

Table 7). In addition, the very high plasma protein binding

(unbound fraction f_u<1% in mouse and human plasma)

should mitigate the measured eﬀect on human ether-a-go-go-

related gene (hERG), suggesting that the compound was

suitable for further in vitro and in vivo proﬁling.

Selectivity. The selectivity proﬁle for inhibition of other

protein kinases by 34 was characterized in an Ambit kinase

assay which measured the percentage activity remaining for

each of 442 kinases (Table S4). Only two kinases were

inhibited to less than 10% activity at 10 nM compound,

DYRK1A (0.4%) and DYRK1B (5%), and only eight other

kinases to less than 90% activity, most notably CLK1 (24%),

CLK2 (10%), CLK4 (23%), and HIPK2 (53%), showing

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compound was absorbed slowly, giving a Cmax a few hours

after dosing and showing moderate exposure.

Table 3. Changing to a Pyrrolo[2,3-d]pyrimidine Scaﬀold

In vivo Eﬃcacy. These properties of 34, namely, kinase

selectivity (particularly against DYRK2), moderate exposure,

and brain penetration, and the fact that the compound is

tolerated in vivo make 34 a suitable tool for probing the

potential of DYRK1A/1B inhibition in various disease models,

keeping in mind potential interference from inhibition of the

CLK family at high concentrations. In the study reported here,

the in vivo eﬃcacy was assessed in a human xenograft tumor

model.

Previous work has demonstrated that glioblastoma cells are

dependent on DYRK1A for growth, both in vitro and in vivo.¹⁵

Compound 34 inhibited the proliferation of U87MG

glioblastoma cells grown either as a 2D monolayer (Figure

S4A) or as multicellular tumor spheroids (Figure S4B).

Activity was dependent on serum concentration with increased

activity observed under lower serum conditions. 34 was

administered po at 25, 100, and 200 mg/kg to female nude

mice bearing established U87MG tumors and tumor

concentration measured at 6 and 16 h (Figure 3C) post

dose. Good plasma and tumor exposure were observed

following either a 100 or 200 mg/kg oral dose with compound

levels in the tumor above that needed to inhibit U87MG cell

growth in vitro for at least 16 h following the administration of

the highest dose. It was not possible to measure the DYRK1A

biomarker (pS520 phosphorylation) due to the low level of

expression in these cells. Further work is ongoing to identify

novel biomarkers of DYRK1A inhibition. As shown in Figure

6A, 34 signiﬁcantly inhibited the proliferation of established

U87MG xenografts with tumor growth delays (TGDs) of 93%

(P < 0.01) and 224% (P < 0.001) following daily oral

administration of 100 and 200 mg/kg, respectively. Little to no

toxicity was observed at these doses (Figure 6B).

Comparison of the Binding of 34 with Other Recently

Published DYRK1A Inhibitors. A number of recent studies

have generated crystal structures of other series of inhibitors of

DYRK1A, derived from natural products such as the

leucettines⁴⁴and harmines^41,45or from proﬁling⁴⁶or

elaboration⁴⁷of known kinase binding motifs and fragment

molecules.⁴⁸Figure 7 shows the chemical structures of

representatives of these chemical series and 34, oriented

approximately as they bind in Dyrk1a and highlighting various

features, in particular, the interactions with amino acids in the

kinase hinge and with K188. The binding pose as seen in the

crystal structures for these compounds is shown in Figure S5.

The most similar hinge-binding heterocycle to the

pyrrollopyrimidine of 34 is the pyrollopyridine of 6EIF⁴⁶

(38) and 6S1H (39) with two of the heterocyclic nitrogens of

the amino-pyrimidine in 6EIL (45) making the same hydrogen

bonds. Similarly, 6QU2 (40) has a pyrimidine as part of a

tricycle in an equivalent position as that in 34 with a

cyclopropyl group occupying the position of the methyl. A

number of methyl amino pyrimidines such as 6S1H (39)

position the methyl in the same position but with diﬀerent

excellent selectivity against DYRK2 (88%). At 1 μM

compound, only 15 kinases were inhibited to less than 50%

activity. Although IC₅₀values for compounds inhibiting a

particular kinase can be compared, it is more diﬃcult to

compare such values across enzymes where there will be

diﬀerent K_Ms for ATP, substrate, and assay conditions. We

therefore determined the K_dfor 34 binding to DYRK1A,

DYRK1B, CLK1, CLK2, and CLK4 in a DiscoveRx

Kinomescan binding assay which gave values of 0.24, 0.05,

2.6, 0.9, and 0.8 nM, respectively, conﬁrming the selectivity for

DYRK1A and the high aﬃnity for CLK kinases. An overlay

(Figure S2) of a published structure of CLK1 (PDB code

6Q8K⁴³) with that of 34 bound to Dyrk1a shows that L244 in

CLK1 (equivalent to L241 in Dyrk1a) adopts a similar

conformation, providing some explanation for the observed

selectivity, which is a feature seen for other DYRK1A

inhibitors.⁴³

Brain Permeability. Preliminary assessment of oral versus iv

PK (Figure S3) showed that the compound has moderate oral

bioavailability in the mouse (% F = 33). Three animals (female

SCID mice) were dosed with compound 34 at po 25 mg/kg,

and the mean plasma and brain concentrations (2 h after

dosing) of the compound were measured as 1849 and 133 ng/

mL, respectively (see Table S5). The calculated brain to

plasma partition was 0.1.

ADME-PK. Female nude BALB/c mice were dosed po at 25

and 100 mg/kg, and the plasma concentration of 34 was

measured as shown in Table 8 (details in Table S6). The

Table 4. Selectivity in a Small Assay Panel of Protein Kinases (IC₅₀μM)

compd no ADP DYRK1A TR FRET DYRK1A TR FRET DYRK1B ADP DYRK2 TR FRET CDK9 ADP PAK4 ADP Pim1 ADP Aurora A

18

19

20

0.05

0.02

0.04

>50

2.8

9.6

>50

0.02

0.03

0.02

0.04

0.06

0.30

4.4

>10

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Table 5. SAR and Selectivity of O-Linked Aryls at 4-Position of the Pyrimidine Ring (IC₅₀μM)

Figure 4. Structure of 24 bound to Dyrk1a (PDB: 7A5L). See the legend to Figure 1 for conventions used in the representation.

positions and orientations of the pyrimidine. There are a large

number of other structures where atoms or small groups ﬁll the

“methyl” holesome of which are derived from natural

compounds such as harmine as in 3ANR (9) and leucettine as

in 4AZE (42) and others where a methoxy benzene on

benzothiazole, as in 5A4L (43), or halogen in the methyl

position, such as Br in 4YLL (44).

The interaction made with K188 through compound

optimization (as seen in Figures 3 and 4) is a common feature

of many DYRK1A inhibitors such as the equivalent pyridine

ring in 6EIF (38) and 6QU2 (40), although just past the

conventional limit of hydrogen bonding distance (3.5 Å) for

34 and for 6EIL (45). There are, however, no published

structures where the substituents mimic the full extent of the

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Table 6. SAR and Selectivity of Aryls at 4-Amino Position (IC₅₀μM)

interactions made by the diﬂuorobenzene substituent in 34

In a similar manner, we used the appropriate primary amines

to install 4-aminoaryls such as 34 of which the preparation is

outlined in Scheme 3, using triethylamine as a tertiary base.

Compounds shown in Table 6 were prepared via a similar

route, starting with the SEM-protected intermediate 19a.

In order to investigate pyrimidines at the 5-position, we

needed to change the protection and deprotection strategy for

the pyrrole ring 7 N. To prepare 35, we ﬁrst installed the

benzene sulfonyl-protecting group and reversed the coupling

partners to install the 5-postion aminopyridine. This route

involved the preparation of the intermediate 5-boronate ester

(50) prior to the Suzuki coupling with amino-4-chloropyr-

imidine, as shown in Scheme 4.

To investigate 4-aryl substituents, we again modiﬁed the 7 N

protection strategy employing boc-protection after installation

of the 4 aryls via Suzuki coupling. We found that bromination

in the 5-position and protection of the 7 N with boc-anhydride

could be performed in a single step isolating the intermediate

53 by puriﬁcation, as shown in Scheme 5. A second Suzuki

coupling under conditions similar to previous examples

installed the 5-position aminopyridine moiety.

that sits within a hydrophobic region. The only compound for

which a substructure reaches the same pocket is the iodine

substituent of 4YLL (44); however, this does not fully occupy

the available space. One interesting comparison is between the

pose for 41 and that for 39the change from a pyrolo-

pyridine (41) to a pyrazolo-pyradazine (39) leads to a ﬂip of

the binding mode, presumably because of changed hydrogen

bonding potential.

CHEMISTRY

■

Compounds 11−17 shown in Table 2 introduced aryls and

heteroaryls into the 6-position, and these were prepared in an

analogous manner to 16 (Scheme 1).

16 was prepared starting from commercially available

1,1,2,2-ethenetetracarbonitrile and cyclization to form the

amino pyrrole 38 using HBr in acetic acid (Scheme 1). Ring

closure to form the pyrimidine ring was performed using

triethyl orthoacetate under reﬂuxing conditions followed by

methanolic ammonia in a sealed vessel to isolate key

intermediate 39. The 6-position bromo facilitated in

installation of aromatics and heteroaromatics such as the 3-

pyridinyl moiety via a Suzuki coupling to form compound 16.

To investigate diﬀerent substituents at the 4-position, we

prepared a series of 4-alkoxy compounds (Table 3). These

were prepared via a similar route to 19, as outlined in Scheme

2. Starting from commercially available 4-chloro-2-methyl-7h-

pyrrolo[2,3-d]pyrimidine, the pyrrole-7H was ﬁrst protected

with a SEM group and then brominated at the 5-position using

N-bromosuccinamide. The formation of the appropriate

alkoxide with sodium hydride and SNAr to substitute the 4-

chloro occurs. The 5-position pyridines were installed via

Suzuki coupling and ﬁnally deprotection of the SEM using

tetrabutyl ammonium ﬂuoride solution.

All tested compounds were of >95% purity (NMR spectra

for 34 and MS traces for 34−37 in the Supporting

Information).

CONCLUSIONS

■

We have used fragment and structure-based discovery methods

to identify 34, a potent, in vivo-tolerated, selective inhibitor of

DYRK1A kinase.

The methyl group on the pyryllopyrimidine of 34 is an

important determinant of selectivity for DYRK1A against other

kinases, as it occupies a small hydrophobic pocket adjacent to

Leu 244 for which the carbonyl of the backbone peptide bond

has a diﬀerent orientation to that seen in most protein kinases.

As shown in Figure 7, the introduction of other hydrophobic

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Table 7. Investigation of the SAR and Cellular Activity of Aryls at the 4-Position

Figure 5. Structure of 34 (PDB code: 7A5N) bound to Dyrk1a. (a) See the legend in Figure 1 for conventions used in the representation. (b).

Alternate view to show hydrophobic interactions with underside of the glycine loop.

groups in this region is a feature of other DYRK1A inhibitors

identiﬁed elsewhere. However, other features of 34 are

distinctive. The diﬂuoro-benzene moiety is in a portion of

the active site of Dyrk1a not occupied by compounds in other

published structures; similarly, the amino group on the

pyridine moiety makes a hydrogen bond not mirrored by

other compounds for which structures are available. Although

34 has similar potency to an inhibitor of DYRK1A and

DYRK1B enzymes, it is highly selective against most other

kinases; one of the exceptions is CLK1 for which a crystal

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Table 8. Mean Plasma Concentration SD in ng/mL of 34 Measured after po Administration (Three Animals/Time Point)

0.5 h

2h

6h

16h

25 mpk

850 246

1828 805

451 503

100 mpk

12176 7809

15177 2430

322 503

Figure 6. Compound 34 inhibits the growth of U87MG glioblastoma xenografts. Nude mice with established U87MG subcutaneous tumor

xenografts were dosed with either 100 or 200 mg/kg PO 34 daily for 21 days (qdx21). Tumor volume (A) was determined by twice weekly caliper

measurement, and body weight (B) was recorded.

Figure 7. 34 and representatives of diﬀerent classes of DYRK1A inhibitors drawn in approximately the relative orientation seen in the available

crystal structures for binding to Dyrk1a. Shading highlights equivalent features: greenequivalent to pyrollopyrimidine hinge binding; gray

equivalent to the methyl of 34, binding in the small hydrophobic pocket adjacent to carbonyl of L241; yellowequivalent pyrimidine; blue−

equivalent interactions to the hydrophobic pocket; and pinkinteraction with K188. See the text for references.

structure demonstrates a similar hydrophobic pocket in the

kinase hinge due to a similar peptide conformation in the

kinase hinge to that seen in Dyrk1a.

34 has many of the properties desired for a tool compound

to probe the biological eﬀects of DYRK1A inhibition. In vitro,

it demonstrates potent inhibition of DYRK1A with concom-

itant target engagement in cells. It is orally bioavailable and

tolerated at doses over 100 mg/kg and achieves suﬃcient

plasma and tumor exposure to show eﬃcacy in an in vivo

model of glioblastoma with a PD marker (phosphorylation of

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a

Scheme 1. Preparation of Compound 16

a

Reagents and conditions: (a) 33% HBr in acetic acid, acetone, −10 to −5 °C, 3 h; (b) triethyl orthoacetate, MeCN, reﬂux, yield 63%; (c) 7 N

NH₃in MeOH, sealed vessel, 105 °C, 6 h, yield 59%; (d) NaH, SEM-Cl, DMF, yield 57%, yield 48%; (e) pyridine-3-boronic acid, K₂CO₃, THF,

water [1,1_-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (10 mol %), 130 °C; and (e) ethylenediamine, TBAF (1 M in THF), 120 °C

microwave, 30 min, yield 38%.

a

Scheme 2. Synthesis of 19

a

Reagents and conditions: (a) SEM-Cl, NaH, DMF, 0 °C, 2 h, yield quant; (b) NBS, DMF, rt, yield 72%; (c) NaH, MeOH, 0 °C, yield quant; (d)

(2-aminopyridin-4-yl)boronic acid, THF, water, K₂CO₃, cat [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), 1 h microwave, 140 °C,

yield 32%; and (e) TBAF (1 M in THF), ethylenediamine, THF, yield 43%.

triplet, q = quartet, quin = quintet, and m = multiplet. All compounds

were determined as having purity >95% unless stated otherwise, and

all ﬁnal tested compounds were puriﬁed to >95% by reverse-phase

high-performance liquid chromatography (HPLC), or normal-phase

silica gel column chromatography. The purity was assessed by reverse-

phase ultraperformance liquid chromatography with a gradient of 5−

98% acetonitrile in water (with either the acid or base modiﬁer) and

monitored by UV absorption at 210−450 nm.

Unless otherwise described, the following conditions were used for

compound synthesis, puriﬁcation, and analysis. A CEM microwave

was used for the microwave reaction conditions. OH-type silica gel

column chromatography was performed using ISCO Combi-ﬂash RF

or RF Gold. The prep-HPLC puriﬁcations were performed using

S520), conﬁrming cellular target engagement. This compound

will be a useful tool to explore further the functional

consequence of DYRK1A inhibition which may lead to new

therapeutic strategies in oncology and Down’s syndrome.

EXPERIMENTAL SECTION

■

All chemicals were used directly as received from commercial

1

suppliers. H spectra were recorded on AVANCE III HD 400 (400

1

MHz for H, BRUKER). Chemical shifts for H are reported in parts

per million (ppm) referenced to the residual solvent peak (DMSO-d₆,

δ = 2.50 ppm). Abbreviations used to describe the peak signals in ¹H

NMR data are s = singlet, br = broad, d = doublet, dd = double

doublet, dt = double triplet, t = triplet, td = triple doublet, tt = triple

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a

Scheme 3. Synthesis of 34

a

Reagents and conditions: (a) 2,6-diﬂuorobenzylamine, TEA, EtOH, 80 °C, 16 h, yield 87%; (b) 2-aminopyridine-4-boronic acid pinacol ester,

K₂CO₃, Pd-118 (cat), THF, water, 60 °C, 16 h, yield: 76%; and (c) TBAF (1 M in THF) ethylenediamine, THF, 80 °C, 14 h, yield 59%.

a

Scheme 4. Synthesis of 35

a

Reagents and conditions: (a) benzene sulfonyl chloride, NaH, DMF, 0 °C, 16 h, yield 91%; (b) 2,6-diﬂuorobenzylamine, TEA, ethanol, 80 °C, 16

h, yield 93%; (c) bis(pinacolato)diboron, KOAc, PdCl₂(PPh₃)₂, THF, 140 °C, 1 h, yield 61%; (d) 2-amino-4-chloropyrimidine, K₂CO₃, Pd118,

THF, 90 °C, 2 h, yield 64%; and (e) K₂CO₃, methanol, RT, 16 h, yield 40%.

Waters prep LCMS [ionization method: electron spray ionization

(ESI)].

The following compounds 11−15 were prepared in an analogous

manner to compound 16.

4.10 (q, J = 7.14 Hz, 2H), 2.47 (s, 3H), 1.15 (t, J = 7.15 Hz, 3H). MS

(ESI⁺) m/z: 314[M + H]⁺. HRMS (ESI⁺/APCI): calcd for

[C₁₆H₁₃N₅F₂+H]⁺, 314.1139; found, 314.1217.

4-Amino-6-(2,3-diﬂuorophenyl)-7-methyl-2-methyl-7H-

pyrrolo[2,3-d]pyrimidine-5-carbonitrile (13). It was isolated as a

colorless solid, 18 mg (0.06 mmol) (yield 32%). ¹H NMR (400 MHz,

DMSO-d₆): δ 7.82−7.70 (m, 1H), 7.59−7.44 (m, 2H), 6.89 (s, 2H),

3.61 (d, J = 0.97 Hz, 3H), 2.46 (s, 3H). MS (ESI⁺) m/z: 300[M +

H]⁺. HRMS (ESI⁺/APCI): calcd for [C₁₅H₁₁N₅F2+H]⁺, 300.0983;

found, 300.1059.

4-Amino-6-(5-methoxypyridin-3-yl)-2,7-dimethyl-7H-

pyrrolo[2,3-d]pyrimidine-5-carbonitrile (14). It was isolated as a

colorless solid, 28 mg (0.10 mmol) (yield 13%). ¹H NMR (400 MHz,

DMSO-d₆): δ 8.52 (d, J = 2.9 Hz, 1H), 8.47 (d, J = 1.8 Hz, 1H), 7.76

(dd, J = 2.9, 1.8 Hz, 1H), 3.93 (s, 3H), 3.69 (s, 3H), 3.35 (s, 2H),

4-Amino-7-ethyl-2-methyl-6-(pyridine-3-yl)-7H-pyrrolo[2,3-

d]pyrimidine-5-carbonitrile (11). It was isolated as a colorless

1

solid, 32 mg (0.11 mmol) (yield 7%). H NMR (400 MHz, DMSO-

d₆): δ 8.89−8.78 (m, 2H), 8.14 (m, 1H), 7.73−7.58 (m, 2H), 6.84 (s,

2H), 4.17 (q, J = 7.16 Hz, 2H), 2.47 (s, 3H), 1.15 (t, J = 7.16 Hz,

3H). MS (ESI⁺) m/z: 279[M + H]⁺. HRMS (ESI⁺/APCI): calcd for

[C₁₅H₁₄N₆+H]⁺, 279.1284; found, 279.1356.

4-Amino-6-(2,3-diﬂuorophenyl)-7-ethyl-2-methyl-7H-

pyrrolo[2,3-d]pyrimidine-5-carbonitrile (12). It was isolated as a

colorless solid, 15 mg (0.05 mmol) (yield 3%). ¹H NMR (400 MHz,

DMSO-d₆): δ 7.83−7.71 (m, 1H), 7.58−7.45 (m, 2H), 6.88 (s, 2H),

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a

Scheme 5. Synthesis of 37

a

Reagents and conditions: (a) 3,4-(methylenedioxy)-phenyl boronic acid, Cs₂CO₃, palladium(dppf)Cl₂, THF, water, 130 °C, 45 min, yield quant;

(b) NBS, DMF 0 °C, 2 h, yield 54%; (c) boc-anhydride, DMAP, Et₃N; (d), boc-2-aminopyridine-4-boronic acid pinacol ester, K₂CO₃, Pd118,

THF, water, 65 °C, 16 h; and (e) HCl in MeOH, microwave 90 °C, 1 h, yield 27% (over two steps).

2.47 (s, 3H). MS (ESI⁺) m/z: 295[M + H]⁺. HRMS (ESI⁺/APCI):

calcd for [C₁₅H₁₄N₆O + H]⁺, 295.1229; found, 295.1307.

Puriﬁcation was performed by ﬂash column chromatography to

furnish the title compound as a mixture of regio-isomers (ratio ∼1:1),

172 mg (yield 57%). MS (ESI⁺) m/z: 384[M + H]⁺(2 overlapping

peaks, isomers seen by LCMS).

4-Amino-2,7-dimethyl-6-(pyridine-3-yl)-7H-pyrrolo[2,3-d]-

pyrimidine-5-carbonitrile (15). It was isolated as a colorless solid,

1

28 mg (0.11 mmol) (yield 28%). H NMR (400 MHz, DMSO): δ

4-Amino-2-methyl-6-(pyridine-3-yl)-7-{[2-(trimethylsilyl)ethoxy]-

methyl}-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (41). 40 (100

mg, 0.26 mmol) and pyridine-3-boronic acid (48 mg, 0.39 mmol, 1.5

equiv) were dissolved in a mixture of THF/water (5:1, 6 mL) under

nitrogen. The mixture was treated with potassium carbonate (311 mg,

0.52 mmol, 2 equiv), and [1,1-bis(diphenylphosphino)ferrocene]-

dichloropalladium(II) (19 mg, 10 mol %) was added, and the

resulting mixture was degassed with a stream of nitrogen for 5 min.

The reaction mixture was heated at 130 °C in a CEM microwave for

60 min. The whole mixture was then diluted with dichloromethane

(20 mL), and water was added (20 mL). The organic layer was

separated, washed with brine, dried over magnesium sulfate, and

concentrated under vacuum. The crude residue was puriﬁed using

ﬂash column chromatography to furnish the title compound as a

yellow solid, 48 mg (yield 48%). MS (ESI⁺) m/z: 381[M + H]⁺.

4-Amino-2-methyl-6-(pyridine-3-yl)-7H-pyrrolo[2,3-d]-

pyrimidine-5-carbonitrile (16). To a solution of 3001-AF-085 4-

amino-2-methyl-6-(pyridine-3-yl)-7-{[2-(trimethylsilyl)ethoxy]-

methyl}-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (48 mg, 0.13

mmol) in tetrahydrofuran (THF) (3 mL) was added ethylenediamine

(42 μL, 0.63 mmol, 5 equiv) followed by TBAF (1 M solution in

THF) (0.63 mL, 0.63 mmol, 5 equiv). The reaction was heated to 120

°C for 30 min in a microwave. The reaction mixture was poured into a

mixture of water and ethyl acetate (10 mL, 1:1) and stirred for ∼15

min. The aqueous layer was separated and concentrated under

vacuum. The resulting precipitate was triturated in acetonitrile, and

the solid was collected by ﬁltration. The solid was washed lightly with

diethyl ether and dried under vacuum to give the title compound as a

colorless solid, 12 mg (yield 38%). ¹H NMR (400 MHz, DMSO-d₆):

δ 13.02 (s, 1H), 9.11 (d, J = 2.3 Hz, 1H), 8.70 (dd, J = 4.8, 1.6 Hz,

1H), 8.29 (dt, J = 8.1, 2.0 Hz, 1H), 7.64 (dd, J = 8.0, 4.7 Hz, 1H),

6.79 (s, 2H), 2.43 (s, 3H). MS (ESI⁺) m/z: 251[M + H]⁺. HRMS

(ESI⁺/APCI): calcd for [C₁₃H₁₀N₆+H]⁺, 251.0967; found, 251.1042.

4-Amino-6-(2,3-diﬂuorophenyl)-2-methyl-7H-pyrrolo[2,3-d]-

pyrimidine-5-carbonitrile (17). Compound 17 was prepared in an

analogous manner to compound 16. The title compound was isolated

8.90 (dd, J = 2.3, 0.9 Hz, 1H), 8.80 (dd, J = 4.9, 1.6 Hz, 1H), 8.17

(dd, J = 2.4, 1.7 Hz, 1H), 7.68 (dd, J = 4.9, 0.9 Hz, 1H), 6.84 (s, 2H),

3.68 (s, 3H), 2.47 (s, 3H). MS (ESI⁺) m/z: 265[M + H]⁺. HRMS

(ESI⁺/APCI): calcd for [C₁₄H₁₂N₆+H]⁺, 265.1123; found, 265.1199.

4-Amino-2-methyl-6-(pyridine-3-yl)-7H-pyrrolo[2,3-d]-

pyrimidine-5-carbonitrile (16). Step 1. 2-Amino-5-bromo-1H-

pyrrole-3,4-dicarbonitrile (38). A 3 L ﬂask was ﬁtted with a

condenser, a drying tube, and an internal thermometer. The ﬂask

was cooled in a methanol/ice bath. Ethyl acetate (1 L), acetone (185

mL), and 1,1,2,2-ethenetetracarbonitrile, Sigma-Aldrich T8809 (50 g,

0.39 mol), were added. The solution was cooled to −10 °C

(methanol/ice bath), and HBr (287 mL, 33% in acetic acid solution, 3

equiv) was added dropwise at such a rate as to maintain −5 °C (over

∼1 h). A yellow solid began to precipitate during the addition. After

the addition was complete, the suspension was stirred for an

additional 3 h. The yellow precipitate was collected by ﬁltration

and washed with ethyl acetate (2 × 100 mL) and diethyl ether (2 ×

100 mL). The solid cake was then suspended in water (600 mL), and

the pH was adjusted to 11 with 50% NaOH, giving a dark solution.

Decolorizing charcoal (ca. 15 g) was then added to the solution and

ﬁltered through celite, and the ﬁltrate was acidiﬁed to pH 5 with

glacial acetic acid. The mixture was allowed to stand at rt overnight.

The product was then collected by ﬁltration and dried at 40 °C in a

vacuum oven for 5 days to give the title compound as a brown solid,

1

52 g (yield 63%). H NMR (400 MHz, DMSO-d₆): δ 12.33 (s, 1H),

6.49 (s, 2H).

4-Amino-6-bromo-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-car-

bonitrile (39). 38 (8.44 g, 40 mmol) was suspended in dry acetonitrile

(150 mL). Triethyl orthoacetate (11 mL, 60 mmol, 1.5 equiv) was

added, and the mixture was heated at rt for ∼1 h. The dark brown

solution was cooled to rt and ﬁltered through a ﬁlter paper.

Acetonitrile was removed under vacuum to aﬀord a brown solid which

was transferred to a steel reaction vessel. 7 N NH₃in MeOH (200

mL) was added, and the vessel was then sealed and heated to 105 °C

for 6 h. The vessel was cooled to rt overnight, and the resulting solid

was collected by ﬁltration. The crude solid was washed several times

with methanol followed by diethyl ether to furnish the title compound

1

as a colorless solid, 10 mg (0.03 mmol) (yield 31%). H NMR (400

1

MHz, DMSO-d₆): δ 13.04 (s, 1H), 7.66 (q, J = 8.7 Hz, 1H), 7.61−

7.52 (m, 1H), 7.45 (q, J = 5.4, 3.4 Hz, 1H), 6.81 (s, 2H), 2.43 (s,

3H). MS (ESI⁺) m/z: 286[M + H]⁺. HRMS (ESI⁺/APCI): calcd for

[C₁₄H₉N₅F₂+H]⁺, 286.0826; found, 286.0902.

as a gray solid, 6 g (yield 59%). H NMR (400 MHz, DMSO-d₆): δ

13.81 (s, 1H), 7.34 (s, 2H), 2.43 (s, 3H). MS (ESI⁺) m/z: 254[M +

H]⁺.

4-Amino-6-bromo-2-methyl-7-²³-7H-pyrrolo[2,3-d]pyrimidine-5-

carbonitrile (40). To a suspension of 39 (200 mg, 0.8 mmol) in dry

dimethylformamide (DMF) (10 mL) was added sodium hydride

(60% in mineral oil (32 mg, 0.8 mmol, 1 equiv) at rt). The suspension

became a dark clear solution after ca. 5 min. The mixture was stirred

for 30 min before adding SEM-Cl (140 μL, 0.8 mmol, 1 equiv) at rt

and stirred under nitrogen for 2 h. The reaction mixture was diluted

with ethyl acetate (50 mL), washed with brine (2 × 50 mL), dried

over anhydrous magnesium sulfate, and concentrated under vacuum.

4-{4-Methoxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl}pyridine

(18). 4-{4-methoxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl}-

pyridine (18) was as prepared in an analogous manner to compound

19. The title compound was isolated as a colorless solid, 15 mg (yield

1

21%). H NMR (400 MHz, DMSO-d₆): δ 12.33 (s, 1H), 8.58−8.50

(m, 2H), 7.85 (s, 1H), 7.78−7.72 (m, 2H), 4.05 (s, 3H), 2.57 (s, 3H).

MS (ESI⁺): m/z 241[M + H]⁺. HRMS (ESI⁺/APCI): calcd for

[C₁₃H₁₂N₄O + H]⁺, 241.1011; found, 241.1082.

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4-{4-Methoxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl}-

pyridine-2-amine (19). Step 1. 4-Chloro-2-methyl-7-{[2-

(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine (42).

To a solution of 4-chloro-2-methyl-7h-pyrrolo[2,3-d]pyrimidine

(Combi-Blocks ST-3008) (1.85 g, 11 mmol) in dry DMF (30 mL)

was added sodium hydride (60% in mineral oil) (290 mg, 12 mmol)

at 0 °C under nitrogen. The mixture was stirred for 30 min before

adding SEM-Cl (2.34 mL, 13 mmol 1.2 equiv) dropwise at 0 °C, and

after addition, the whole mixture was stirred under nitrogen for 2 h.

The reaction mixture was diluted with diethyl ether (100 mL),

washed with brine (2 × 50 mL), dried over anhydrous magnesium

sulfate, and concentrated under vacuum to give the title compound as

brown oil, 3.57 g (yield quant) (purity determined by LCMS 90%)

The crude product title compound was used in the subsequent step

without further puriﬁcation. MS (ESI⁺) m/z: 298 [M + H]⁺.

5-Bromo-4-chloro-2-methyl-7-{[2-(trimethylsilyl)ethoxy]methyl}-

7H-pyrrolo[2,3-d]pyrimidine (43). To a solution of crude 42 (3.3 g,

11 mmol) in DMF (30 mL) was added N-bromosuccinamide (2 g, 11

mmol, 1 equiv) in DMF (5 mL) at 0 °C under N₂. It was stirred for

30 min at 0 °C and then allowed to warm to rt over 30 min. The

reaction mixture was diluted with water (20 mL) and extracted with

diethyl ether (30 mL). The organic layer was washed several times

with water, dried over anhydrous magnesium sulfate, and concen-

trated in vacuum. The residue was puriﬁed by ﬂash column

chromatography to give the title compound as a colorless solid, 3 g

(yield 72% over two steps). ¹H NMR (400 MHz, DMSO-d₆): δ 8.13

(s, 1H), 5.65 (s, 2H), 3.66−3.57 (m, 2H), 2.74 (s, 3H), 0.98−0.88

(m, 2H). MS (ESI⁺) m/z: 378[M + H]⁺.

5-Bromo-4-methoxy-2-methyl-7-{[2-(trimethylsilyl)ethoxy]-

methyl}-7H-pyrrolo[2,3-d]pyrimidine (44). To a suspension of

sodium hydride (60% suspension in mineral oil) (530 mg, 13

mmol, 2 equiv) in THF (10 mL) was added methanol (346 μL, 8.6

mmol, 1.3 equiv) dropwise at 0 °C (ice bath cooling). The mixture

was stirred for approximately 10 min before adding 43 (2.5 g, 6.6

mmol) in THF (10 mL) under N₂at 0 °C. The reaction mixture was

stirred at 0 °C for 30 min and allowed to warm to rt over 1 h. The

reaction was quenched with saturated NH₄Cl_(aq)(50 mL) and

extracted with ethyl acetate (50 mL). The organic layer was washed

with brine, dried over anhydrous magnesium sulfate, and concentrated

under vacuum to give the title compound as light yellow oil (2.4 g,

yield quant). MS (ESI⁺) m/z: 372[M + H]⁺.

4-(4-Methoxy-2-methyl-7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-

pyrrolo[2,3-d]pyrimidin-5-yl)pyridine-2-amine (45). 44 (150 mg, 0.4

mmol) and (2-aminopyridin-4-yl)boronic acid (133 mg, 0.6 mmol,

1.5 equiv) were dissolved in a mixture of THF and water (6:1, 5 mL)

under nitrogen. Potassium carbonate (166 mg, 1.2 mmol, 3 equiv)

and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)

(15 mg, 5 mol %) were added, and the resulting mixture was

degassed with a stream of N₂for 5 min. The reaction mixture was

heated at 140 °C in a CEM microwave for 1 h. The product mixture

was diluted with water (10 mL) and extracted with ethyl acetate. The

organic layer was washed with brine, dried over anhydrous

magnesium sulfate, and concentrated under vacuum. The resulting

crude product oil was taken up in dichloromethane and dry-loaded

onto ISOLUTE before puriﬁcation by ﬂash column chromatography

to aﬀord the title compound as a colorless solid, 50 mg (yield 32%).

MS (ESI⁺) m/z: 386[M + H]⁺.

4-{4-Methoxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl}-

pyridine-2-amine (19). To a solution of 45 (50 mg, 0.13 mmol) in

THF (3 mL) was added ethylenediamine (43 μL 0.65 mmol, 5 equiv)

followed by TBAF (1 M in THF, 5 equiv). The reaction was stirred at

80 °C for 2 h in the microwave. The reaction mixture was poured into

water and ethyl acetate (20 mL, 1:1) and stirred for 15 min. The

organic layer was separated, washed with brine, dried over anhydrous

magnesium sulfate, and concentrated under vacuum. The residue was

then triturated with acetonitrile and collected by ﬁltration to furnish

the title compound as a colorless solid, 15 mg (yield 43%). ¹H NMR

(400 MHz, DMSO-d₆): δ 12.16 (s, 1H), 7.86 (dd, J = 5.4, 0.7 Hz,

1H), 7.63 (d, J = 2.5 Hz, 1H), 6.89−6.79 (m, 2H), 5.83 (s, 2H), 4.04

(s, 3H), 2.56 (s, 3H). MS (ESI⁺) m/z: 256[M + H]⁺. HRMS (ESI⁺/

APCI): calcd for [C₁₃H₁₃N₅O + H]⁺, 256.1123; found, 256.1196.

The following compounds 20−28 were prepared in an analogous

manner to compound 19.

4-{4-Methoxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl}-2-

methylpyridine (20). It was isolated as a colorless solid, 15 mg (yield

45%). ¹H NMR (400 MHz, DMSO-d₆): δ 12.29 (s, 1H), 8.39 (dd, J =

5.3, 0.7 Hz, 1H), 7.81 (s, 1H), 7.64−7.59 (m, 1H), 7.54 (dd, J = 5.4,

1.8 Hz, 1H), 4.04 (s, 3H), 2.57 (s, 3H), 2.50 (s, 2H). MS (ESI⁺) m/z:

255[M + H]⁺. HRMS (ESI⁺/APCI): calcd for [C₁₄H₁₄N₄O + H]⁺,

255.1168; found, 255.1238.

3-{4-Methoxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl}-

benzamide (21). It was isolated as a colorless solid, 16 mg (yield

26%). ¹H NMR (400 MHz, DMSO): δ 12.10 (s, 1H), 8.19 (t, J = 1.8

Hz, 1H), 7.99 (s, 1H), 7.83 (dd, J = 7.8, 1.4 Hz, 1H), 7.74 (dd, J =

7.8, 1.4 Hz, 1H), 7.58 (s, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.38 (s, 1H),

3.99 (s, 3H), 2.57 (s, 3H). MS (ESI⁺) m/z: 283[M + H]⁺. HRMS

(ESI⁺/APCI): calcd for [C₁₅H₁₄N₄O₂+H]⁺, 283.1117; found,

283.1193.

2-Fluoro-4-{4-methoxy-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-

yl}pyridine (22). It was isolated as a colorless solid, 20 mg (yield

40%). ¹H NMR (400 MHz, DMSO-d₆): δ 12.52 (s, 1H), 8.26 (d, J =

5.3 Hz, 1H), 8.08 (s, 1H), 7.83 (dd, J = 5.4, 2.2, 1.4 Hz, 1H), 7.64−

7.58 (m, 1H), 4.13 (s, 3H), 2.64 (s, 3H). MS (ESI⁺) m/z: 259[M +

H]⁺. HRMS (ESI⁺/APCI): calcd for [C₁₃H₁₁N₄OF + H]⁺, 259.0917;

found, 259.0996.

5-(4-ﬂuorophenyl)-4-methoxy-2-methyl-7H-pyrrolo[2,3-d]-

pyrimidine (23). It was isolated as a colorless solid, 40 mg (yield

1

55%). H NMR (400 MHz, DMSO-d₆): δ 12.05 (s, 1H), 7.74−7.64

(m, 2H), 7.49 (s, 1H), 7.27−7.16 (m, 2H), 3.99 (s, 3H), 2.56 (s, 3H).

MS (ESI⁺) m/z: 258[M + H]⁺. HRMS (ESI⁺/APCI): calcd for

[C₁₄H₁₂N₃OF + H]⁺, 258.0964; found, 258.1043.

4-{2-Methyl-4-[(thiophen-3-yl)methoxy]-7H-pyrrolo[2,3-d]-

pyrimidin-5-yl}pyridine-2-amine (24). It was isolated as a colorless

1

solid, 15 mg (yield 37%). H NMR (400 MHz, DMSO-d₆): δ 12.26

(s, 1H), 7.88 (dd, J = 5.3, 0.7 Hz, 1H), 7.69 (s, 2H), 7.74−7.60 (m,

2H), 7.37 (dd, J = 4.9, 1.3 Hz, 1H), 6.95−6.85 (m, 2H), 5.76 (s, 2H),

5.63 (s, 2H), 2.67 (s, 3H). MS (ESI⁺) m/z: 338[M + H]⁺. HRMS

(ESI⁺/APCI): calcd for [C₁₇H₁₅N₅OS + H]⁺, 338.0995; found,

338.1068.

4-{2-Methyl-4-[(5-methyl-1,2-oxazol-3-yl)methoxy]-7H-pyrrolo-

[2,3-d]pyrimidin-5-yl}pyridine (25). It was isolated as a colorless

solid, 8 mg (yield 38%). ¹H NMR (400 MHz, DMSO-d₆): δ 12.41 (s,

1H), 8.50−8.44 (m, 2H), 7.89 (s, 1H), 7.79−7.72 (m, 2H), 6.38 (d, J

= 1.1 Hz, 1H), 5.59 (s, 2H), 2.59 (s, 3H), 2.45 (s, 3H). MS (ESI⁺) m/

z: 322[M + H]⁺. HRMS (ESI⁺/APCI): calcd for [C₁₇H₁₅N₅O₂+H]⁺,

322.1226; found, 322.1299.

{2-Methyl-4-[(1,3-thiazol-4-yl)methoxy]-7H-pyrrolo[2,3-d]-

pyrimidin-5-yl}pyridine-2-amine (26). It was isolated as a colorless

1

solid, 3 mg (yield 6%). H NMR (400 MHz, DMSO-d₆): δ 12.28 (s,

1H), 9.24 (d, J = 2.0 Hz, 1H), 7.88−7.80 (m, 2H), 7.72 (s, 1H),

6.97−6.90 (m, 1H), 6.92 (s, 2H), 5.79−5.71 (m, 4H), 2.66 (s, 3H).

MS (ESI⁺) m/z: 339[M + H]⁺. HRMS (ESI⁺/APCI): calcd for

[C₁₆H₁₄N₆OS + H]⁺, 339.0948; found, 339.1019.

4-{2-Methyl-4-[(1,3-thiazol-5-yl)methoxy]-7H-pyrrolo[2,3-d]-

pyrimidin-5-yl}pyridine-2-amine (27). It was isolated as a colorless

1

solid, 10 mg (yield 40%). H NMR (399 MHz, DMSO): δ 12.24 (s,

1H), 9.10 (d, J = 0.8 Hz, 1H), 8.09 (d, J = 0.9 Hz, 1H), 7.85−7.79 (d,

1H), 7.64 (s, 1H), 6.81 (dd, 2H), 5.82 (s, 2H), 5.75 (s, 2H), 2.63 (s,

3H). MS (ESI⁺) m/z: 339[M + H]⁺. HRMS (ESI⁺/APCI): calcd for

[C₁₆H₁₄N₆OS + H]⁺, 339.0948; found, 339.1025.

4-[4-(Benzyloxy)-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-

pyridine (28). It was isolated as a colorless solid, 22 mg (yield 75%).

¹H NMR (400 MHz, DMSO-d₆): δ 12.35 (s, 1H), 8.41−8.35 (m,

2H), 7.86 (s, 1H), 7.77−7.70 (m, 2H), 7.57−7.49 (m, 2H), 7.47−

7.32 (m, 3H), 5.57 (s, 2H), 2.60 (s, 3H). MS (ESI⁺) m/z: 317[M +

H]⁺. HRMS (ESI⁺/APCI): calcd for [C₁₉H₁₆N₄O + H]⁺, 317.1324;

found, 317.1391.

The following compounds were prepared in parallel in an

analogous manner to 34. To a solution of the SEM-protected

precursor in THF (5 mL) was added ethylenediamine (5 equiv)

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followed by TBAF (1 M in THF, 5 equiv), and the mixture was

stirred at 80 °C for 3 h. The individual reaction mixtures were

concentrated under vacuum and evaporated onto ISOLUTE.

Puriﬁcation was performed by ﬂash column chromatography followed

by trituration with acetonitrile (3 × 2 mL), and the resultant solids

were collected via ﬁltration. The solids were washed lightly with

diethyl ether (2 mL) and then dried under vacuum. The puriﬁed

(m, 4H), 2.49 (s, 6H), 1.27 (t, J = 7.1 Hz, 1H), 1.00−0.85 (m, 4H).

MS (ESI⁺) m/z: 485[M + H]⁺.

5-Bromo-N-[(3,5-diﬂuorophenyl)methyl]-2-methyl-7-{[2-

(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-amine

(47). 46 (5.6 g, 11.5 mmol) and 2-aminopyridine-4-boronic acid

pinacol ester (Combi-Blocks PN-0151) (4.3 g, 13.8 mmol, 1.2 equiv)

were dissolved in a mixture of THF and water (4:1, 200 mL) under

N₂. The mixture was treated with potassium carbonate (4.9 g, 36

mmol, 3 equiv) and Pd-118 (375 mg, 5 mol %), and the mixture was

degassed with a stream of N₂for 5 min. The whole mixture was

heated up to 60 °C for 16 h, and the reaction was monitored by

LCMS. The reaction mixture was cooled to rt, ﬁltered through a plug

of celite, and diluted with ethyl acetate. The organic layer was washed

with brine, dried over anhydrous magnesium sulfate, concentrated

under vacuum, and dry-loaded by evaporation onto ISOLUTE.

Puriﬁcation was performed by ﬂash column chromatography (120 g

silica). Trituration with iso-hexane gave a suspended solid which was

collected by ﬁltration and dried under vacuum to furnish the title

compound as a colorless solid, 4.33 g (yield: 76%). MS (ESI⁺) m/z:

497[M + H]⁺.

1

compounds were characterized by LCMS and H NMR.

4-[4-(benzylamino)-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl]-

pyridine-2-amine (29). It was isolated as a colorless solid, 51 mg

(0.15 mmol) (yield 21%). ¹H NMR (400 MHz, DMSO-d₆): δ 11.78−

11.72 (m, 1H), 7.88 (dd, J = 0.71, 5.18 Hz, 1H), 7.42−7.28 (m, 4H),

7.33−7.19 (m, 2H), 6.60−6.49 (m, 2H), 5.99−5.88 (m, 3H), 4.70 (d,

J = 5.61 Hz, 2H), 2.42 (s, 3H). MS (ESI⁺) m/z: 331[M + H]⁺.

HRMS (ESI⁺/APCI): calcd for [C₁₉H₁₈N₆+H]⁺, 331.1593; found,

331.1589.

4-(2-Methyl-4-{[(thiophen-2-yl)methyl]amino}-7H-pyrrolo[2,3-

d]pyrimidin-5-yl)pyridine-2-amine (30). It was isolated as a colorless

solid, 31 mg (0.09 mmol) (yield 24%). ¹H NMR (400 MHz, DMSO-

d₆): δ 11.77 (s, 1H), 7.91 (dd, J = 0.73, 5.24 Hz, 1H), 7.36 (dd, J =

1.26, 5.14 Hz, 1H), 7.25 (d, J = 1.65 Hz, 1H), 7.08−7.01 (m, 1H),

6.94 (dd, J = 3.43, 5.11 Hz, 1H), 6.58−6.46 (m, 2H), 6.07 (t, J = 5.86

Hz, 1H), 5.94 (s, 2H), 4.86 (d, J = 5.60 Hz, 2H), 2.47 (s, 3H). MS

(ESI⁺) m/z: 337[M + H]⁺. HRMS (ESI⁺/APCI): calcd for

[C₁₇H₁₆N₆S + H]⁺, 337.1149; found, 337.1222.

4-(2-Methyl-4-{[(thiophen-3-yl)methyl]amino}-7H-pyrrolo[2,3-

d]pyrimidin-5-yl)pyridine-2-amine (31). It was isolated as a colorless

solid, 54 mg (0.16 mmol) (yield 45%). ¹H NMR (400 MHz, DMSO-

d₆): δ 11.75 (s, 1H), 7.90 (d, J = 5.20 Hz, 1H), 7.49 (dd, J = 2.93,

4.95 Hz, 1H), 7.38 (d, J = 3.33 Hz, 1H), 7.24 (s, 1H), 7.15 (dd, J =

1.35, 4.91 Hz, 1H), 6.57 (dd, J = 1.55, 5.17 Hz, 1H), 6.51 (s, 1H),

5.98 (s, 2H), 5.83 (t, J = 5.57 Hz, 1H), 4.68 (d, J = 5.42 Hz, 2H), 2.45

(s, 3H). MS (ESI⁺) m/z: 337[M + H]⁺. HRMS (ESI⁺/APCI): calcd

for [C₁₇H₁₆N₆S + H]⁺, 337.1157; found, 337.1231.

4-(4-{[(Furan-3-yl)methyl]amino}-2-methyl-7H-pyrrolo[2,3-d]-

pyrimidin-5-yl)pyridine-2-amine (32). It was isolated as a colorless

solid, 30 mg (0.09 mmol) (yield 24%). ¹H NMR (400 MHz, DMSO-

d₆): δ 11.78−11.72 (m, 1H), 7.91 (dd, J = 0.70, 5.18 Hz, 1H), 7.66−

7.57 (m, 2H), 7.24 (d, J = 2.03 Hz, 1H), 6.59−6.52 (m, 2H), 6.50

(dd, J = 0.77, 1.60 Hz, 1H), 5.99 (s, 2H), 5.68 (t, J = 5.45 Hz, 1H),

4.51 (d, J = 5.23 Hz, 2H), 2.46 (s, 3H). MS (ESI⁺) m/z: 321[M +

H]⁺. HRMS (ESI⁺/APCI): calcd for [C₁₇H₁₆N₆O + H]⁺, 320.1394;

found, 321.1466.

4-(4-{[(3,5-Diﬂuorophenyl)methyl]amino}-2-methyl-7H-pyrrolo-

[2,3-d]pyrimidin-5-yl)pyridine-2-amine (34).

To a solution of 47 (1 equiv) in THF (100 mL) was added

ethylenediamine (6 equiv) followed by TBAF (1 M solution in THF,

6 equiv), and the reaction mixture was heated to 80 °C for a total of

14 h. The reaction mixture was cooled, diluted with ethyl acetate (100

mL), washed with water (2 × 100 mL) and brine, dried over

anhydrous magnesium sulfate, and concentrated under vacuum. The

residue was triturated to a solid using ethyl acetate and acetonitrile,

followed by diethyl ether, and the solid was collected by ﬁltration. The

solid product was dried under vacuum to give the title compound as a

1

colorless solid, 1.89 g (yield 59%). H NMR (400 MHz, DMSO-d₆):

δ ppm 11.75 (s, 1H), 7.90 (d, J = 5.18 Hz, 1H), 7.37 (m, J = 6.61,

8.41 Hz, 1H), 7.24 (d, J = 2.29 Hz, 1H), 7.09 (t, J = 7.94 Hz, 2H),

6.54−6.45 (m, 2H), 5.93 (s, 2H), 5.85 (t, J = 5.79 Hz, 1H), 4.77 (d, J

= 5.65 Hz, 2H), 2.43 (s, 3H). MS (ESI⁺) m/z: 367[M + H]⁺. HRMS

(ESI⁺/APCI): calcd for [C₁₉H₁₆N₆F₂+H]⁺, 367.1405; found,

367.1471.

4-(4-{[(3,5-Diﬂuorophenyl)methyl]amino}-2-methyl-7H-

pyrrolo[2,3-d]pyrimidin-5-yl)pyrimidin-2-amine (35). Step 1.

7-(Benzenesulfonyl)-5-bromo-4-chloro-2-methyl-7H-pyrrolo[2,3-

d]pyrimidine (48). To a solution of 5-bromo-4-chloro-2-methyl-7H-

pyrrolo[2,3-d]pyrimidine (Combi-Blocks QV-2886) (1.86 g, 7.55

mmol) in DMF (40 mL) was added sodium hydride (60% oil

dispersion) (332 mg, 8.3 mmol, 1.1 equiv) at 0 °C under nitrogen.

The reaction mixture was stirred at 0 °C for 30 min before adding

benzene sulfonyl chloride (1.16 mL, 9 mmol, 1.2 equiv) at 0 °C. The

reaction mixture was allowed to warm to room temperature gradually,

and stirring was continued under nitrogen for 16 h. The reaction

mixture was poured into water (50 mL) and stirred at room

temperature for 20 min to break up the precipitated solid. The

resulting ﬁne precipitate was collected by ﬁltration and washed several

times with water and air-dried on a sintered funnel to furnish the title

compound as a white solid, 2.64 g (yield 91%). MS (ESI⁺) m/z:

387[M + H]⁺.

4-(2-Methyl-4-{[(1,3-thiazol-4-yl)methyl]amino}-7H-pyrrolo[2,3-

d]pyrimidin-5-yl)pyridine-2-amine (33). It was isolated as a colorless

1

solid, 6 mg (0.02 mmol) (yield 3%). H NMR (400 MHz, DMSO-

d₆): δ 11.78 (s, 1H), 9.07 (d, J = 1.96 Hz, 1H), 7.92 (d, J = 5.26 Hz,

1H), 7.53 (dd, J = 1.02, 2.03 Hz, 1H), 7.26 (d, J = 2.44 Hz, 1H), 6.60

(dd, J = 1.53, 5.19 Hz, 1H), 6.53 (d, J = 1.72 Hz, 1H), 6.09 (q, J =

4.64, 5.62 Hz, 1H), 5.96 (s, 2H), 4.88−4.79 (m, 2H), 3.22−3.12 (m,

3H), 2.43 (s, 3H), 1.57 (dt, J = 7.32, 15.32 Hz, 3H), 1.32 (h, J = 7.35

Hz, 3H), 0.94 (t, J = 7.35 Hz, 4H). MS (ESI⁺) m/z: 338[M + H]⁺.

HRMS (ESI⁺/APCI): calcd for [C₁₆H₁₅N₇S + H]⁺, 337.1114; found,

338.1189.

4-(4-{[(3,5-Diﬂuorophenyl)methyl]amino}-2-methyl-7H-

pyrrolo[2,3-d]pyrimidin-5-yl)pyridine-2-amine (34). Step 1. 5-

Bromo-N-[(3,5-diﬂuorophenyl)methyl]-2-methyl-7-{[2-

(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-amine

(46). To a solution of 43 (5 g, 13.3 mmol) in ethanol (50 mL) was

added 2,6-diﬂuorobenzylamine (Combi-Blocks SS-4198) (3.17 mL,

26.5 mmol, 2 equiv) and TEA (5.5 mL, 39.8 mmol, 5 equiv) and

heated at 80 °C for 16 h. The reaction mixture was concentrated

under vacuum, and the residue was retaken up in EtOAc (100 mL),

washed with water and brine, and dried over anhydrous magnesium

sulfate. The ﬁltered organics were evaporated and dry-loaded onto

ISOLUTE, and puriﬁcation was performed by ﬂash column

chromatography (120 g silica column) to furnish the title compound

7-(Benzenesulfonyl)-5-bromo-N-[(3,5-diﬂuorophenyl)methyl]-2-

methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (49). To a suspension

of 48 (1 g, 2.59 mmol) in ethanol (20 mL) was added 2,6-

diﬂuorobenzylamine (0.62 mL, 5.17 mmol, 2 equiv) and triethylamine

(1 mL, 7.8 mmol, 3 equiv), and the mixture was heated at 80 °C for

16 h. The clear reaction mixture was cooled to rt upon which a yellow

precipitate was formed. Water (30 mL) was added, and the

suspension was stirred for ∼20 min before the precipitate was

collected by ﬁltration. The solid ﬁlter cake was washed further with

water and air-dried on the ﬁlter thoroughly before washing with iso-

hexane. The solid was then dried under vacuum to furnish the title

1

compound as a colorless solid, 1.19 g (yield 93%). H NMR (400

1

as clear oil, 5.56 g (yield 87%). H NMR (400 MHz, DMSO-d₆): δ

MHz, DMSO-d₆): δ 8.17−8.09 (m, 2H), 7.81−7.71 (m, 2H), 7.65

(dd, J = 8.5, 7.2 Hz, 2H), 7.35 (m, J = 8.4, 6.6 Hz, 1H), 7.13−6.97

(m, 3H), 4.77 (d, J = 5.7 Hz, 2H), 3.31 (s, 1H), 2.41 (s, 3H).

7.55 (s, 2H), 7.46 (tt, J = 8.6, 6.6 Hz, 2H), 7.23−7.11 (m, 4H), 6.80

(t, J = 5.9 Hz, 2H), 5.51 (s, 4H), 4.92 (d, J = 5.8 Hz, 4H), 3.62−3.53

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7-(Benzenesulfonyl)-N-[(3,5-diﬂuorophenyl)methyl]-2-methyl-5-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7H-pyrrolo[2,3-d]-

pyrimidin-4-amine (50). To a microwave vial was added 49 (1 g 2

mmol), bis(pinacolato)diboron (617 mg, 2.43 mmol, 1.2 equiv), and

KOAc (600 mg 6 mmol 3 equiv), and the mixture was dissolved in

THF (15 mL) and sparged with a stream of N₂for 10 min.

PdCl₂(PPh₃)₂(70 mg, 5 mol %) was then added, and the mixture was

heated to 140 °C for 1 h. LCMS showed the target product and also

some des-bromo byproduct. The cooled reaction mixture was diluted

with ethyl acetate (50 mL) and washed with saturated brine solution,

and the organics were dried over anhydrous magnesium sulfate. The

ﬁltered organics were reduced in volume under vacuum and dry-

loaded onto ISOLUTE by evaporation. The product was puriﬁed via

automated chromatography, eluting gradient 0−20% ethyl acetate/

iso-hexane, and fractions were evaporated to furnish the title

compound as a colorless solid, 0.68 g (yield 61%). MS (ESI⁺) m/z:

541[M + H]⁺.

added portionwise over ∼15 min. The reaction mixture was allowed

to stir, slowly warming to room temperature over 2 h. The mixture

was then treated with boc-anhydride (800 mg 3.67 mmol 1.2 equiv),

DMAP (5 mg 0.01 equiv), and triethylamine, and stirring at room

temperature was continued for a further 16 h. Water was added, and

the mixture was extracted with ethyl acetate twice. The combined

organics were washed with saturated brine solution (aq) and dried

over anhydrous magnesium sulfate. The ﬁltered organics were

concentrated under vacuum and puriﬁed via automated column

chromatography on 24 g SiO₂cartridge eluting hexane55% ethyl

acetate/hexane mixture. Selected fractions were evaporated, and the

resultant solid was dried under vacuum to furnish the title compound

as oﬀ-white powder, 697 mg (yield 54%). ¹H NMR (400 MHz,

DMSO-d₆): δ 8.00 (s, 1H), 7.23−7.13 (m, 2H), 7.07 (d, J = 8.0 Hz,

1H), 6.14 (s, 2H), 2.72 (s, 3H), 1.63 (s, 9H). MS (ESI⁺) m/z:

431.8[M + H]⁺.

4-[4-(2H-1,3-benzodioxol-5-yl)-2-methyl-7H-pyrrolo[2,3-d]-

pyrimidin-5-yl]pyridine-2-amine (37). 53 (432 mg 1 mmol), K₂CO₃

(415 mg 3 mmol 3 equiv), and boc-2-aminopyridine-4-boronic acid

pinacol ester (Combi-Blocks PN-6264) (352 mg 1.1 mmol 1.1 equiv)

were combined and dissolved in a mixture of THF (15 mL) and water

(4 mL). The mixture was sparged with N₂for 10 min, and the catalyst

Pd118 (33 mg, 5 mol %) was then added. The whole mixture was

then heated to 65 °C for 16 h. The reaction mixture was diluted with

dichloromethane and washed with water, followed by saturated brine

solution. The organics were then dried over anhydrous magnesium

sulfate and concentrated under vacuum. The residue was puriﬁed via

automated column chromatography on the 24 g SiO₂cartridge eluting

neat hexane to 10% ethyl acetate/hexane. The puriﬁed intermediate

boc-protected compound was dissolved in methanol (2 mL) and

treated with 1.25 M HCl in MeOH (4 mL, 5 mmol, 5 equiv), and the

solution was heated in a CEM microwave at 90 °C for 1 h. Solvents

were then removed in vacuo, residues were taken up in 2:1

dichloromethane/isopropanol and washed with saturated sodium

bicarbonate solution (aq), and the organics were dried over

anhydrous magnesium sulfate and evaporated to dryness. The residue

solid was triturated in diethyl ether and collected by ﬁltration. The

solid ﬁlter cake was dried under vacuum to furnish the title compound

as a colorless solid, 95 mg (yield 27%).¹H NMR (400 MHz, DMSO-

d₆): δ ppm 12.28 (s, 1H), 7.68 (s, 1H), 7.60 (dd, J = 0.72, 5.30 Hz,

1H), 6.98 (d, J = 1.66 Hz, 1H), 6.85 (dd, J = 1.68, 8.04 Hz, 1H), 6.74

(d, J = 8.02 Hz, 1H), 6.16 (dd, J = 0.77, 1.57 Hz, 1H), 6.02 (s, 2H),

6.05−5.98 (m, 1H), 5.65 (s, 2H), 2.69 (s, 3H). MS (ESI⁺) m/z:

346[M + H]⁺. HRMS (ESI⁺/APCI): calcd for [C₁₉H₁₅N₅O₂+H]⁺,

346.1226; found, 346.1127.

4-[7-(Benzenesulfonyl)-4-{[(3,5-diﬂuorophenyl)methyl]amino}-2-

methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl]pyrimidin-2-amine (51). 50

(915 mg, 1.69 mmol), 2-amino-4-chloropyrimidine (329 mg, 2.54

mmol, 1.5 equiv), and K₂CO₃(700 mg 5 mmol, 3 equiv) were

combined in a microwave vial and dissolved in a mixture of THF (16

mL)/water (4 mL). The mixture was sparged with a stream of N₂for

10 min before treatment with Pd118 (55 mg 5 mol %), and the

reaction mixture was heated to 90 °C for 2 h in a CEM microwave.

The reaction mixture was diluted with dichloromethane (200 mL)

and washed with water (200 mL). The organics were separated, dried

over MgSO4, and concentrated under vacuum. The residue was

triturated with acetonitrile to furnish a light beige solid, 551 mg (yield

1

64%). H NMR (400 MHz, DMSO-d₆): δ 8.44 (s, 1H), 8.29 (d, J =

5.3 Hz, 1H), 8.20−8.13 (m, 2H), 7.80−7.71 (m, 1H), 7.65 (dd, J =

8.5, 7.1 Hz, 2H), 7.37 (d, J = 5.4 Hz, 1H), 7.30 (m, J = 14.8, 8.4, 6.5

Hz, 1H), 7.01 (t, J = 7.9 Hz, 2H), 6.70 (s, 2H), 4.90 (d, J = 5.9 Hz,

2H), 3.31 (s, 1H), 2.38 (s, 3H). MS (ESI⁺) m/z: 508[M + H]⁺.

4-(4-{[(3,5-Diﬂuorophenyl)methyl]amino}-2-methyl-7H-pyrrolo-

[2,3-d]pyrimidin-5-yl)pyrimidin-2-amine (35). To a suspension of 51

(550 mg, 1.08 mmol) in methanol (10 mL) was added K₂CO₃(∼1.4

g, 10 mmol, excess) and stirred at room temperature for 16 h. The

suspension was ﬁltered, and the solids were washed with further

methanol. The solid residue was then washed several times with water

to remove the remaining inorganics, and the remaining solid was air-

dried on the ﬁlter thoroughly before lightly washing with hexane. The

resultant solid was dried under a vacuum oven at 60 °C to give the

1

title compound as a colorless solid, 159 mg, (yield 40%). H NMR

(400 MHz, DMSO-d₆): δ ppm 11.96 (s, 1H), 8.14 (d, J = 5.41 Hz,

1H), 8.04 (s, 1H), 7.33 (m, J = 7.25 Hz, 1H), 7.12 (d, J = 5.44 Hz,

1H), 7.05 (t, J = 7.80 Hz, 2H), 6.34 (s, 2H), 4.91 (d, J = 5.84 Hz,

2H), 3.39 (s, 1H), 2.36 (s, 3H), 1.24 (s, 1H). MS (ESI⁺) m/z: 368[M

+ H]⁺. HRMS (ESI⁺/APCI): calcd for [C₁₈H₁₅N₇F₂+H]⁺, 368.1357;

found, 368.1413.

Protein Production. Three protein constructs of DYRK1A were

used for crystal structure determination.

His-TEV-DYRK1A(127-485) was used to generate crystals of

Dyrk1a with ADPNP, 3, 8, and 10 bound. This is the same construct

as that used for the published structure (2VX3³¹), removing the

DYRK homology (DH) domain and the C terminus. Electron density

was not observed for the initial N-terminal amino acids; a construct

was therefore designed with slightly truncated N and C terminal

sequences with a thrombin cleavage site (His-thrombin-DYRK1A-

(148-479-ala-ala)) which generated crystals of Dyrk1a with 1 and 6

bound. A further construct was designed (His-thrombin-

DYRK1A(148−485)) for all other crystal structures reported here.

A clone codon optimized for expression in Escherichia coli for His−

TEV−DYRK1A(127−485) in pET21a was generated by gene

synthesis (Celtek BioScience, USA). This clone was used as a

template for PCR to generate an N-terminal truncation ligated to

pET15b, His-thrombin-DYRK1A(148−485), and subsequent site-

directed mutagenesis (Agilent) yielded a C-terminal truncate, His-

Thrombin-DYRK1A(148−479-ala-ala). E. coli BL21(DE3) pLysS

were transformed with the DYRK1A encoding plasmid and cultured

at 37 °C in Luria−Bertani (LB) supplemented with 100 μg/mL

ampicillin. Once the culture had reached an O.D.600 nm of 0.4, the

incubator temperature was reduced to 22 °C. At O.D.600 nm of 0.6,

expression was induced by the addition of 1 mM IPTG (ﬁnal

4-[4-(2H-1,3-benzodioxol-5-yl)-2-methyl-7H-pyrrolo[2,3-d]-

pyrimidin-5-yl]pyridine-2-amine (37). Step 1. 4-(2H-1,3-benzo-

dioxol-5-yl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine (52). A mixture

of 4-chloro-2-methyl-7h-pyrrolo[2,3-d]pyrimidine (Combi-Blocks

ST-3008) (502 mg, 3 mmol), 3,4-(methylenedioxy)-phenyl boronic

acid (522 mg, 3.15 mmol), and (2 g, 6 mmol, 2 equiv) of cesium

carbonate was treated with a spatula tip of the palladium(dppf)Cl₂

catalyst, and the whole mixture was suspended in 10 mL of

tetrahydrofuran and 1 mL of water in a microwave vessel. The

whole mixture was degassed by a stream of nitrogen for 10 min. The

sealed vessel and contents were heated to 130 °C for 45 min, and the

reaction mixture was allowed to cool. LCMS showed a single product

peak. The reaction mixture was diluted with 150 mL of water, the

solid was collected by ﬁltration, and the ﬁlter cake was dried under

vacuum in air on a sinter funnel to furnish the title compound, 750

mg, as a colorless solid (yield quant). MS (ESI⁺) m/z: 254[M + H]⁺.

tert-Butyl 4-(2H-1,3-benzodioxol-5-yl)-5-bromo-2-methyl-7H-

pyrrolo[2,3-d]pyrimidine-7-carboxylate (53). 52 (750 mg 2.96

mmol) was dissolved in anhydrous DMF under N₂and cooled to 0

°C. N-bromosuccinamide (600 mg 3.35 mmol, 1.1 equiv) was then

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concentration). The cells were pelleted after 16hrs and stored at −80

°C.

with Refmac5⁵²software from the CCP4 package,⁵³alternating with

manual rebuilding using the program COOT.⁵⁴Compounds were

modeled in the surplus peaks in the diﬀerence Fourier electron

density maps. Topology ﬁles for the ligands were created by

ProDrug,⁵⁵also from within the CCP4 package. Reﬁnement statistics

for each new structure reported in this paper are in Table S2.

Protein Kinase Assays. A number of diﬀerent assay formats were

used to measure inhibition of diﬀerent kinases by compounds

described in this paper.

ADP Hunter Plus (DiscoverX) was used to measure the activity of

the protein kinases DYRK1A, PAK4, Pim1, Aurora A, and DYRK2.

A TR-FRET assay was used to measure the activity of the protein

kinases DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4, CDK9, and

CLK1.

A similar protein puriﬁcation protocol was used for all constructs.

Cell pellets were resuspended in lysis buﬀer [50 mM N-(2-

hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) pH7.5,

500 mM NaCl, 5% glycerol containing ethylenediaminetetraacetic

acid (EDTA)-free protease inhibitor cocktail (Roche) and DNase I

(Sigma)] and lysed by mechanical homogenization. The lysate was

centrifuged at 20,000 rpm for 60 mins, 4 °C. The clariﬁed lysate was

applied to a 1 mL HiTrap chelating column (Cytiva) charged with

Ni²⁺pre-equilibrated with lysis buﬀer. After washing the column, the

protein of interest was eluted using a series of step gradients of lysis

buﬀer + 500 mM imidazole. Sodium dodecyl sulfate polyacrylamide

gel electrophoresis (SDS-PAGE) analysis conﬁrmed the fractions of

interest which were pooled and concentrated using an Amicon Ultra

30 kDa MWCO concentrator (Merck Millipore). The sample was

applied to a HiLoad 16/60 Superdex 75 pg size exclusion

chromatography column (Cytiva) pre-equilibrated in 50 mM

HEPES pH7.5, 500 mM NaCl, and 5 mM DTT. Samples were

analyzed by SDS-PAGE and concentrated using an Amicon 30 kDa

MWCO concentrator (Merck Millipore) to 18 mg/mL. The puriﬁed

protein was aliquoted and snap-frozen prior to being stored at −80

°C.

If required, the N-terminal His-tag was removed by incubation

overnight at 4 °C, with AcTEV protease (ThermoFisher) at 1 U/3 μg

puriﬁed protein after aﬃnity chromatography. A repeated aﬃnity

puriﬁcation resulted in the protein of interest in the nonabsorbed

fraction before size exclusion chromatography was performed.

Fragment Screen. All protein NMR spectra were measured on a

600 MHz Bruker AVANCE III spectrometer ﬁtted with a cryoprobe.

Ligand-observed fragment screening was performed with 500 μM of

each compound, 10 μM Dyrk1a (produced from His-TEV-

DYRK1A(127−485) with the tag removed) in 20 mM HEPES pH

7.4, and 15 μM NaCl, in mixtures of six compounds per sample as

described previously.^35,36

Crystallization of Dyrk1a. Crystal structures of diﬀerent ligands

complexed to Dyrk1a were obtained using the following protein

constructsAMPNP; compounds 3, 8, 10, and 34 with His-TEV-

DYRK1A (aa127-485); compounds 1 and 6 with His-Thrombin-

DYRK1A (148-479-Ala-Ala); and all others with His-Thrombin

-DYRK1A (aa127-485).

For all compounds other than 34, crystals were prepared by

replacement of AMPNP from Dyrk1a/AMPNP crystals. Puriﬁed

Dyrk1a protein (all constructs) at ∼15 mg/mL concentration was

mixed with fourfold excess (molar ratio) of AMPNP in vapor

diﬀusion experiments. The best crystals were obtained from a solution

containing 0.1 M MES buﬀer at pH 6.5, 12% Peg3350, and 0.2 M

MgCl₂at 20 °C. To replace the AMPNP, the crystals were soaked for

∼24 h in a crystallization solution containing 4−10 mM ligand (ﬁnal

DMSO concentration of 10−20%).

The K_dfor binding of compounds to kinases was determined in an

11-point threefold serial dilution in the KINOMEscan assay available

at DiscoveRx (now Euroﬁns).

A radiometric assay was used to measure the activity of the protein

kinase GSK3-β.

The inhibition of TTK was measured at CEREP (now Euroﬁns).

ADP Hunter Plus Assay. The ADP Hunter Plus assay measures the

accumulation of ADP. Protein kinase assays were performed in 96-

well black plates (corning #3686) at 30 °C in a ﬁnal volume of 20 μL

in 50 mM Hepes, pH 7.4, 20 mM NaCl, 10 mM MgCl₂, 1 mM

EGTA, 0.01 mg/mL BGG, 0.02% Tween 20, and 2% DMSO. The

order of the addition of reagents to the assay plate was 5 μL of

compound/8% DMSO, 10 μL of kinase/substrate mix, and 5 μL of

ATP. Final concentrations and assay conditions used are described in

Table S3. Following incubation, 5 μL of Reagent A was added,

followed by 10 μL of Reagent B and plate incubated for a further 20

min at 30 °C. Finally, 5 μL of the stop reagent was added, and the

plate was read on a Molecular Devices ﬂexstation plate reader using

Ex/Em 530/590 nm in conjunction with a 585 nm cutoﬀ dichroic

mirror.

TR-FRET Assay. The TR-FRET assay was performed on a TECAN

Evo robotic platform. The compound at 5% DMSO was added to

reaction buﬀer (50 mM Hepes pH7.4, 1 mM EGTA, 10 mM MgCl₂;

2 mM DTT & 0.01% Tween20) containing 20 ng/μL DYRK1A

Kinase (Carna Biosciences #04−130), 100 μM Ulight-MBP substrate

(PerkinElmer #TRF109L), and 10 μM ATP cosubstrate (Sigma-

Aldrich #A2383) and incubated for 40 min at room temperature. The

kinase reaction was stopped by adding 10 mM EDTA (Sigma-Aldrich

#E7889) and 1 nM anti-phospho-MBP (PerkinElmer #TRF0201L)

and incubated for 1 h minimum at room temperature. Plates were

read on a multimode reader (PerkinElmer Envision 2400) with an

excitation wavelength of 337 nm and emission wavelengths of 620 nm

for the donor and 665 nm for the acceptor. A similar format was used

for other TR-FRET kinase inhibition assays.

Radiometric Assay. The radiometric assay for glycogen synthase

3β (GSK3β) inhibition was performed in 96-well plates (Greiner;

VWR #651201) at 30 °C for 40 min in a ﬁnal volume of 50 μL where

reagents were added in the following order: 1 μL of compound/100%

DMSO, 39 μL of kinase/substrate mix, and 10 μL of ATP/³³P ATP

mix. Final concentrations used were 39 nM GSK3β (Merck #14−

306), 16.5 μM GSK3βtide substrate (YRRAAVPPSPSLSRHSSPHQ-

(pS)EDEEE), 10 μM ATP, 0.5μCi ³³P ATP, 50 mM Hepes, pH 7.5,

15 mM Mg(C₂H₃O₂)₂, 0.01 mg/mL bovine serum albumin (BSA),

and 2% DMSO. Following incubation, the assay was stopped by the

addition of 50 μL of 50 mM phosphoric acid solution and 90 μL of

assay reaction transferred to a prewetted ﬁlter plate, washed three

times with 100 μL of 50 mM phosphoric acid solution, dried, and

counted in the presence of 50 μL of scintillation ﬂuid on a Trilux plate

reader.

Crystals of the complex of Dyrk1a with 34 were obtained from a

protein solution at ∼15 mg/ml mixed with approximately fourfold

excess of 34 (ﬁnal DMSO concentration of 8%) that had been

incubated overnight. Crystals appeared from vapor diﬀusion experi-

ments, with the best crystals obtained from conditions containing 0.1

M Hepes buﬀer at pH 7.5, 20% Peg4 k, and 0.2 M MgCl₂, also at 20

°C.

Crystallographic Data Collection. Crystals were cryoprotected

in a solution of crystallization buﬀer enriched in glycerol up to 20−

25% and ﬂash frozen in liquid nitrogen. Table S1 summarizes the data

collection statistics, with data obtained from ESRF (beamline id23-1)

or DLS (i02, i03, and i04 beamlines). Data were processed and scaled

with DENZO/Scalepack package⁴⁹or XDS⁵⁰software.

Structure Determination. Structures of Dyrk1a complexes were

solved by molecular replacement (MolRep⁵¹) using the coordinates

from PDB code 2VX3³¹as a model.

The reﬁned structure of the relevant protein construct (stripped of

the compound) was used as a starting model for solving (by molecular

replacement) subsequent structures of the complexes with ligands.

After structure solution, several cycles of reﬁnement were conducted

DYRK1A (pS520) Cell Assay. U2OS cells were transiently

transfected with a plasmid expressing full length wild-type DYRK1A

(pcDNA3.1). After 5 h, compounds were added, and cells were

incubated for a further 5 h. Cells were lysed, and equal amounts of

protein were separated by SDS-PAGE and transferred to the

poly(vinylidene diﬂuoride) membrane. Membranes were probed for

pS520 DYRK1A (custom rabbit-polyclonal antibody immunized with

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the synthetic peptide SNSGRARpSDPTHQHR, Eurogentec; 1:500 in

5% BSA-TBST) and detected with anti-rabbit IgG-HRP (7074, CST;

1:3000) and then quantiﬁed on an electrochemical luminescence

camera (BioRad). Membranes were subsequently stripped and

reprobed with a total anti-DYRK1A antibody (H0000-1859, Abnova;

1:1000 in 5% BSA-TBST) and quantiﬁed as mentioned above.

Caco2Papp Assay. The Caco-2 cell line was cultured in

adapted media and used between 21 and 30 days post seeding. The

compound was incubated at 20 μM in the apical chamber in HBSS

buﬀer containing 0.1% BSA. Samples (duplicate) were collected at 30

and 90 min in the basolateral chamber and analyzed by HPLC-LC/

MS/MS. The apparent permeability was calculated as follows: Papp

(cm/s) = K·Vr/A·60, where V_ris the volume of the receiver chamber

(mL), A is the surface area of the membrane (cm²), and K is the slope

of the cumulated absorbed fraction as a function of time (min⁻¹).

Metabolic Stability (Mouse/Rat/Human). Pooled liver micro-

somes from mouse (CD-1), rat (Sprague−Dawley), and human liver

microsomes (mixed gender) are used with a ﬁnal microsomal protein

concentration of 0.3 mg/mL. The compound (0.1 μM) was tested in

duplicate. After preincubation with pooled liver microsomes (10 min,

37 °C), the reaction is initiated by adding a reduced nicotinamide

adenine dinucleotide phosphate-generating system and incubated in a

37 °C shaking water bath. Samples were collected at diﬀerent time

points (0, 7, 17, 30, and 45 min), and the reaction was stopped by a

mixture of 20 nM antipyrine and 0.1% formic acid in acetonitrile/

methanol/water (35/35/30, v/v/v). Samples were analyzed by

HPLC-MS/MS.

current amplitude at −80 mV. The percentage of inhibition was

determined as compared to the baseline.

In Vivo Eﬃcacy Studies. Female nude mice (Crl/NU(NCr)-

Foxn1nu) were purchased from Charles River Laboratories. Xenograft

studies were undertaken by Charles River Laboratories Discovery

Services, North Carolina, and accredited by the Association for

Assessment and Accreditation of Laboratory Animal Care Interna-

tional. The U87MG tumor line was maintained by serial SC

transplantation in female athymic nude mice. Tumor fragments,

approximately 1 mm³each, were implanted SC into the right ﬂank of

each animal and allowed to grow toward a target size of 100−150

mm³. On day 1 of the study, tumors were randomized into treatment

groups before compound administration. For tumor PK studies, the

tumors were allowed to reach a target volume of 300 mm³prior to

randomization.

Compound 34 was formulated in 60 mM HCl and 20%

hydroxypropyl-β-cyclodextrin and administered by oral gavage once

daily for 21 days. The tumor size was measured with electronic

calipers, and the tumor volume was calculated according to the

formula ((width × width) × length)/2. Body weight was measured

daily for the ﬁrst 5 days and then twice weekly thereafter. The study

endpoint was deﬁned as a tumor volume of 2000 mm³or D28,

whichever came ﬁrst. Each animal was euthanized when its tumor

reached the endpoint, and the last day of the study was D28.

The time to endpoint (TTE) for each mouse was calculated from

the following equation: TTE = log 10 (endpoint volume) − b/m

where b is the intercept and m is the slope of the line obtained by

linear regression of a log-transformed tumor growth data set. The data

set is composed of the ﬁrst observation that exceeded the study

endpoint volume and the three consecutive observations that

immediately preceded the attainment of the endpoint volume.

Animals that did not reach endpoint were euthanized at the end of

the study and assigned a TTE value equal to the last day of the study

(D28). Animals determined to have died from treatment-related (TR)

causes were assigned a TTE value equal to the day of death. If the

animal died from nontreatment-related causes, it was excluded from

the TTE calculations.

The hepatic blood clearance was extrapolated using the following

equation

Cl_int× LW × sc × Q_h× f_u

Cl_H(mL/min) =

(Cl_int× LW × sc × f_u) + Q_h

where LW is the liver weight (g), sc is the scaling factor (g protein/g

liver), Q_his the hepatic blood ﬂow (mL/min), and f_uis the blood

unbound fraction (1 by default), and the intrinsic clearance (Cl_int) is

as follows

0.693

Cl_int(μL/min /mg protein) =

The treatment outcome is evaluated by TGD, which is deﬁned as

the increase in the median TTE in a treatment group compared to the

control group: TGD = T − C expressed in days or as a percentage of

the median TTE of the control group: % TGD = (T − C)/C × 100

where T = median TTE for a treatment group and C = median TTE

for the control group.

t_1/2× protein concentration

Plasma protein Binding (Mouse/Human) Assay. Each pool of

mouse and human plasma was spiked with the compound to obtain a

ﬁnal concentration of 5 μM. Dialysis was performed (in triplicate)

using 300 μL of each plasma spiked (donor chamber) and 500 μL of

phosphate buﬀer pH 7.4 (receiver chamber). All replicates were

incubated for 3 h at 37 °C using rapid equilibrium dialysis (device-

Pierce) under orbital stirring. All plasma and buﬀer samples (40 μL)

were analyzed with the HPLC-LC-MS/MS method.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The extent of plasma protein binding in each sample was calculated

as follows

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00024.

f_u(%) = C_u/C_pl× 100

Supporting ﬁgures, tables and data: Compound 34 target

engagement with DYRK1A demonstrated in nano-BRET

assay; comparison of the conformation of equivalent

leucine residues in the kinase hinge of CLK1 and Dyrk1a

structures; in vivo PK characterisation of compound 34

in mice; compound 34 exhibits activity against U87MG

glioblastoma cells in vitro; comparison of binding pose

of 34 with recently published inhibitors of DYRK1A;

crystallographic data collection; reﬁnement statistics;

ﬁnal concentrations of reagents in the kinase inhibition

assays; kinase proﬁling of 34; brain penetrance of 34; in

vivo PK; complete list of kinome screen data; LC

chromatograms, and 1H NMR spectra of key com-

pounds (PDF)

Where f_uis the unbound fraction expressed as a percentage, C_plis

the concentration of the compound in the plasma at the end of

dialysis, and C_uis the concentration of the unbound compound in the

buﬀer. The unbound fraction (f_u%) was determined as the mean of

the unbound fraction calculated from the triplicate.

hERG Assay. The eﬀect of compounds on hERG activity was

studied at room temperature (22 2 °C) on hERG currents in HEK-

293 cells stably expressing the hERG potassium channel. Cells were

maintained at a potential of −80 mV, and hERG currents were

activated by cell depolarization (+30 mV, 1 s) followed by cell

repolarization (40 mV, 1 s). Cells were incubated with DMSO 0.1% in

extracellular solution for 10 min (baseline) followed by three

increasing consecutive concentrations of the compound (1, 3, and

10 μmol/L, n = 4) until the steady state was reached. The hERG tail

current amplitude was determined by the diﬀerence between the tail

current amplitude upon repolarization to −40 mV and the base

Molecular formula strings (CSV)

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AUTHOR INFORMATION

ABBREVIATIONS

■

DYRK, dual-speciﬁcity tyrosine-regulated kinase; CMGC,

cyclin-dependent, mitogen-activated, glycogen synthase, and

CDC-like; EGFR, epidermal growth factor; GLI, glioma-

associated oncogene; Mcl-1, myeloid cell leukemia 1; Bcl-2, B-

cell lymphoma 2; EGCG, epigallocatechin gallate; PDB,

protein databank; PK, pharmacokinetic; PD, pharmacody-

namic; PAK4, p21-associated kinase 4; ADP, adenosine

diphosphate; TR-FRET, time-resolved ﬂuorescence energy

transfer; GSK3β, glycogen synthase 3β; ADPNP, 5′-adenylyl-

β,γ-imidodiphosphate; NMR, nuclear magnetic resonance;

HAC, heavy atom count; ATP, adenosine triphosphate; LE,

ligand eﬃciency; CDK9, cyclin-dependent kinase 9; CLK,

CDC-like kinase; hERG, human ether-a-go-go-related gene;

HIPK2, homeodomain interacting protein kinase 2; SCID,

severe combined immunodeﬁciency; po, per os; SAR,

structure−activity relationship

Corresponding Author

Roderick E. Hubbard − Vernalis (R&D) Ltd., Cambridge

CB21 6GB, U.K.; orcid.org/0000-0002-8233-7461;

Email: r.hubbard@vernalis.com

Authors

David Lee Walmsley − Vernalis (R&D) Ltd., Cambridge

CB21 6GB, U.K.

James B. Murray − Vernalis (R&D) Ltd., Cambridge CB21

6GB, U.K.; orcid.org/0000-0003-1007-8218

Pawel Dokurno − Vernalis (R&D) Ltd., Cambridge CB21

6GB, U.K.; orcid.org/0000-0002-7332-8889

Andrew J. Massey − Vernalis (R&D) Ltd., Cambridge CB21

6GB, U.K.; orcid.org/0000-0002-0276-2573

Karen Benwell − Vernalis (R&D) Ltd., Cambridge CB21

6GB, U.K.

Andrea Fiumana − Vernalis (R&D) Ltd., Cambridge CB21

6GB, U.K.

Nicolas Foloppe − Vernalis (R&D) Ltd., Cambridge CB21

6GB, U.K.

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Didier Demarles − Technologie Servier, Orleans 45000,

France

Mike Burbridge − Institut de Recherches Servier, Croissy-sur-

Seine 78290, France

Francisco Cruzalegui − Institut de Recherches Servier, Croissy-

sur-Seine 78290, France; Present Address: Francisco

Cruzalegui:Evotec (France) SAS 195 Route d’Espagne

31036 Toulouse, France E-mail: cruzalegui.francisco@

orange.fr

Andras Kotschy − Servier Research Institute of Medicinal

Chemistry, Budapest H-1031, Hungary; orcid.org/0000-

0002-7675-3864

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00024

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

The authors thank co-workers at the Analytical Division of the

Servier Research Institute of Medicinal Chemistry for

providing the detailed chemical analysis of the compounds

and fromVernalis, and we thank Hayley Angove for assistance

with the assay protocols.

The X-ray structures of compounds complexed with Dyrk1a

reported in this paper have been deposited in the PDB with the

following codes. ADPNP: 7A4O, 1: 7A4R, 2: 7A4S, 3: 7A4W;

4: 7A4Z, 5: 7A51, 6: 7A52, 7: 7A53, 8: 7A55, 10: 7A5B, 16:

7A5D, 24: 7A5L, and 34: 7A5N. The authors will release the

atomic coordinates and experimental data upon article

publication.

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Journal of Medicinal Chemistry

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Article

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Article Doi

Fragment-Derived Selective Inhibitors of Dual-Specificity Kinases DYRK1A and DYRK1B

DOI: 10.1021/acs.jmedchem.1c00024

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