Discovery of Selective Small-Molecule Inhibitors for the ENL YEATS Domain

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Discovery of Selective Small-Molecule Inhibitors for the ENL YEATS

Domain

Xinyu R. Ma,^△Longxia Xu,^△Shiqing Xu, Brianna J. Klein, Hongkuan Wang, Sukant Das, Kuai Li,

Kai S. Yang, Sana Sohail, Andrew Chapman, Tatiana G. Kutateladze, Xiaobing Shi, Wenshe Ray Liu,*

and Hong Wen *

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ABSTRACT: Eleven-nineteen leukemia (ENL) protein is a

histone acetylation reader essential for disease maintenance in

acute leukemias, in particular, the mixed-lineage leukemia (MLL)-

rearranged leukemia. In this study, we carried out high-throughput

screening of a small-molecule library to identify inhibitors for the

ENL YEATS domain. Structure−activity relationship studies of the

hits and structure-based inhibitor design led to two compounds, 11

and 24, with IC₅₀values below 100 nM in inhibiting the ENL−

acetyl-H3 interaction. Both compounds, and their precursor

compound 7, displayed strong selectivity toward the ENL

YEATS domain over all other human YEATS domains. Moreover,

7 exhibited on-target inhibition of ENL in cultured cells and a

synergistic eﬀect with the bromodomain and extraterminal domain inhibitor JQ1 in killing leukemia cells. Together, we have

developed selective chemical probes for the ENL YEATS domain, providing the basis for further medicinal chemistry-based

optimization to advance both basic and translational research of ENL.

for disease maintenance in acute leukemias, in particular, the

mixed-lineage leukemia (MLL)-rearranged leukemia.^14,22De-

pletion of ENL or disrupting the interaction between its

YEATS domain and acetylated histones suppresses leukemia

progression. In addition, hotspot ENL YEATS domain

mutations were found in Wilms’ tumor patients.^23,24We

showed that the reader function of the ENL YEATS domain is

indispensable for the gain-of-function mutations in the

oncogenesis of Wilms’ tumor.²⁵Together, all these studies

suggest that the YEATS domain of ENL is an attractive

therapeutic target.

The acetyllysine-binding pocket of the ENL YEATS domain

is a long and narrow hydrophobic channel, making it a

potentially good target for developing small-molecule inhib-

itors.¹⁴Indeed, recent publications of acetyllysine competitive

small compounds and peptide-mimic chemical probes

demonstrate that the ENL YEATS domain is pharmacologi-

cally tractable.²⁶⁻³²The peptide-mimic chemical probes

showed slightly higher potency to the ENL YEATS domain

INTRODUCTION

■

Post-translational modiﬁcations of histones play an important

role in the epigenetic regulation of gene expression. These

modiﬁcations serve as binding sites to recruit reader proteins,

which in turn transduce the epigenetic signals into downstream

functional outcomes.^1,2In addition to small compounds that

modulate enzymatic activities of the histone-modifying

enzymes, perturbations of reader−histone interactions also

provide attractive therapeutic potentials. One such example is

the BET bromodomain inhibitor.^3,4Bromodomains are known

as readers of histone acetylation.⁵Recent studies from our

laboratories and others have identiﬁed the YEATS domains as

a new family of epigenetic readers that bind to not only histone

acetylation but also other types of acylations such as

crotonylation.⁶⁻¹⁶

The YEATS domain, named after its ﬁve founding members

(Yaf9, ENL, AF9, Taf14, and Sas5), is evolutionarily conserved

from yeast to humans.¹⁷The human genome encodes four

YEATS domain-containing proteins ENL, AF9, YEATS2, and

GAS41 that all associate with chromatin-associated protein

complexes.^18,19ENL and AF9 are paralogues that share a

similar protein structure including a highly conserved YEATS

domain. Both ENL and AF9 are subunits of the super

elongation complex and the complex of the histone H3K79

methyltransferase DOT1L, but mutually exclusive.^20,21We and

others previously showed that ENL, but not AF9, is required

Received: February 28, 2021

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Figure 1. Structural modeling of the ﬁve initial HTS hits, 1−5, and an amine analogue 6 with the ENL YEATS domain. (A) Chemical structures of

compounds 1−6 (left) and their IC₅₀values in inhibiting the His-ENL−H3K9ac interaction in the AlphaScreen assay (right). (B) Structural model

showing binding of 1−5 to the ENL YEATS domain. Modeling was based on the crystal structure of the ENL YEATS domain (PDB entry: 5J9S).

1−5 are shown in stick representation, and the ENL YEATS domain is shown in a contoured surface structure. Atoms in ENL are colored in gray,

compound 1 in green, 2 in hot pink, 3 in yellow, 4 in cyan, and 5 in orange. (C) Modeled interaction of 2 with ENL. The Cα atoms of 2 are

colored in orange and two hydrogen bonds (dashed lines) to S58 and Y78 of ENL are colored in yellow. Acetyllysine in the H3K27ac ligand in the

original crystal structure is colored in hot pink and its two hydrogen bonds with E58 and Y78 are shown for comparison. (D) Modeled interaction

of 6 with ENL. The Cα atoms of 6 are colored in orange. The amine in 6 shows a salt-bridge interaction with E26 in ENL.

than other YEATS domains, largely due to interactions outside

of the acetyllysine binding pocket.³⁰In contrast, the small

molecule ENL inhibitors reported so far failed to distinguish

ENL from its close paralogue AF9. In addition, none of these

small-molecule compounds showed signiﬁcant impact on ENL-

dependent leukemia cell growth, suggesting that development

of potent, selective ENL YEATS-domain inhibitors is in great

need. Here, we report the discovery of small-molecule

compounds that exhibit preferential binding to ENL compared

to AF9 and other YEATS-domain proteins. Two compounds,

11 and 24, displayed IC₅₀values below 100 nM in inhibiting

the ENL−acetyl-H3 interaction in vitro. In leukemia cells,

compound 7 reduced ENL target gene expression and

suppressed leukemia cell growth. In addition, 7 exhibited a

synergistic eﬀect with the BET bromodomain inhibitor JQ1 in

killing leukemia cells. Our study provided valuable selective

ENL chemical probes and potential leads for further medicinal

chemistry-based optimization to advance both basic and

translational research of ENL.

10 μg/mL. This assay system was further evaluated in a high-

throughput setting in 384-well plates. Interplate variations were

measured between two separate plates and on two separate

days, yielding robust and highly reproducible results with a

high signal/background (S/B) ratio (39.02), low coeﬃcient of

variation (3.5%), and an excellent Z′ factor (0.92) (Supporting

Information, Figure S1D). Dimethyl sulfoxide (DMSO)

tolerance of the assay (0.1−1%) indicated that the

AlphaScreen signals were maintained at 95 and 85% in the

presence of 0.1 and 0.5% DMSO, respectively. We also set up a

counter assay using a biotin-14× His peptide to eliminate

compounds that interfere with AlphaScreen assay components.

Together, these data demonstrate that the AlphaScreen assay

we developed is suitable for HTS of ENL inhibitors, with

superior sensitivity and reproducibility.

After adapting the AlphaScreen-based HTS system to an

automated format for ENL (100 nM His-ENL, 10 nM biotin-

H3K9ac, 0.1% DMSO, and 2.5 μg/mL AlphaScreen beads), we

proceeded to screen a small-molecule library of 66,625

compounds with diverse chemical scaﬀolds. Nonfragment

compounds were screened at a concentration of 10 μM, and

fragment-based compounds were screened at a concentration

of 50 μM. In the primary screen, we obtained 4648 hits with

above 50% inhibition. Conﬁrmation and counter assays yielded

524 compounds with above 60% inhibition of the His-ENL−

H3K9ac interaction and below 20% inhibition of the counter

screen. We then subjected the top 100 compounds to full

dose−response curve validation and obtained 37 compounds

with IC₅₀values below 5 μM, including 8 compounds with

IC₅₀below 1 μM (Supporting Information, Table S1).

RESULTS

■

High-Throughput Library Screen for ENL YEATS-

Domain Inhibitors. In order to identify small-molecule

inhibitors for the YEATS domain of ENL, we ﬁrst established

an AlphaScreen assay system for high-throughput screening

(HTS) of small-molecule compounds. In this assay system,

two analytes, a 6× His-tagged ENL YEATS domain (His-

ENL) and a biotin-H3K9ac peptide (histone H3 residues 1−

21 with an acetylation at Lys 9) were immobilized on

PerkinElmer Ni²⁺-chelating acceptor and streptavidin donor

beads, respectively (Supporting Information, Figure S1A).

Protein and peptide dose−response assays determined optimal

concentrations of His-ENL and biotin-H3K9ac to be 100 and

30 nM, respectively (Supporting Information, Figures S1B,C).

We also determined the optimized beads concentration to be

Structure-Based Inhibitor Design and Structure−

Activity Relationship Studies. Among the top eight hits

that have an IC₅₀value below 1 μM (Supporting Information,

Table S1), ﬁve, named as 1−5, are structurally similar and

share a same pharmacophore [1,2,4]triazolo[4,3-a]pyridine-6-

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Scheme 1. Synthesis of Compounds 6−19

Table 1. Chemical Structures and IC₅₀Values of 7−28

amide, suggesting a preferential binding of this pharmacophore

to the ENL YEATS domain (Figure 1A and Supporting

Information, Figure S2). All these ﬁve compounds also contain

an aryl substituent at the amide nitrogen side, allowing them to

be generally deﬁned as N,C-diarylamides. To understand how

these compounds interact with ENL, we performed docking

analysis using an existing crystal structure of the ENL YEATS

domain (the PDB entry: 5J9S). The results showed that all ﬁve

compounds ﬁt nicely to the acetyllysine binding channel of

ENL (Figure 1B). The compounds are bound to the ENL

YEATS domain with a similar orientation as an acetyllysine in

a native histone ligand. Similar to the acetyllysine side chain

amide, the amide in 1−5 is poised to form two hydrogen

bonds with S58 and Y78. Although the two aromatic rings can

ﬂip to bind either side of the channel, both potentially form π-

stacking and van der Waals interactions with residues F28,

H56, F59, Y78, and F81 in ENL for preferential binding

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Scheme 2. Synthesis of Compounds 20−28

(Figure 1C). The modeling analysis also indicated that 1−5

occupy almost fully the acetyllysine binding channel of ENL.

Since ENL has relatively ﬂat interfaces on the two sides of

the acetyllysine binding channel, there is a little space for

chemical maneuvers of 1−5 for improved binding. However,

we noticed that E26, a residue at the edge of the acetyllysine

binding channel can potentially ﬂip its side chain toward the

acetyllysine binding channel to interact with a ligand such as 2

(Figure 1C). We deemed that by adding a positively charged

amine or amidine to 2, it is possible that a salt-bridge

interaction with E26 can be introduced for strong binding to

ENL. Therefore, we synthesized compound 6 (Scheme 1A)

and tested its inhibition of the interaction between His-ENL

and biotin-H3K9ac. The determined IC₅₀value for 6 was 0.63

μM, which is similar to that for 2 (Figure 1A and Supporting

Information, Figure S2). Since the introduction of an amine

makes the compound more favorable to dissolve in water, the

salt-bridge interaction may compensate the energy loss due to

desolvation when 6 binds ENL, resulting in no improvement.

Indeed, in the modeled structure, 6 interacts with ENL similar

to 2 except that it engages E26 for a salt-bridge interaction

(Figure 1D). We have also attempted to cocrystalize ENL with

6 for crystal structure determination, but unfortunately, it has

not been successful.

Encouraged by the results from 6, we expanded the scope of

substitution groups on both sides of the amide bond of hit

compounds for a comprehensive structure−activity relation-

ship (SAR) study. A major focus was to maintain a positive

charged amine, amidine, or guanidine as in 6 but tune the ENL

binding as well as lower the energy loss due to desolvation by

adding diﬀerent alkyl substituents to the amine, amidine, or

guanidine. The inhibition potency of all compounds was ﬁrst

tested at 1 and 0.1 μM. Promising compounds were then

subjected to a more accurate AlphaScreen assay for IC₅₀

determination. We ﬁrst started with replacing the primary

amine of 6 with diﬀerent kinds of tertiary amines through

reductive amination of key aldehyde intermediate 46 (Scheme

1B), which resulted in 7−11 (Table 1 and Supporting

Information, Figure S2). IC₅₀measurement showed that

compounds tended to be more potent as the ring size of the

substitutional groups on the tertiary amine decreased. Among

them, 11 that has a four-membered ring azetidine moiety

exhibited the most potency with an IC₅₀value as 51 nM in

inhibiting the interaction between His-ENL and biotin-

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H3K9ac. These results suggest that the azetidine ring assists

the binding to ENL. Further modiﬁcations were then

introduced to 11 to aﬀord 12−14 with diﬀerent alkyl groups

on the 2′ position of azetidine, which we wished to increase the

electron density on the N atom and enhance the interaction

between azetidine and Glu26. However, these compounds

displayed lower potency than 11. Given that Glu26 is located

at a loop area with much conformational ﬂexibility, we moved

the azetidine moiety from the para to meta position aﬀording

15 but did not result in any increase of potency. We also

attempted to increase the rigidity of the molecule by adding a

methyl group to the benzylic carbon aﬀording 16. However, it

greatly reduced the inhibition potency. We also substituted the

triazolopyridine moiety with similar heterocycles to aﬀord 17−

19, but none of these compounds outcompeted 11 (Table 1,

Scheme 1C and Supporting Information, Figure S2).

In addition to amine derivatives, we also designed a series of

amidine derivatives based on compound 6 to aﬀord 20−23.

These compounds were synthesized from corresponding nitrile

intermediates, followed by acid-catalyzed ethanolysis and then

ammonolysis (Scheme 2A). However, none of these

compounds showed improved potency. Although an amidine

or guanidine tend to form a stronger salt bridge with a

carboxylate than an amine, it may have a higher desolvation

energy than an amine, contributing to weak binding to ENL.

For this reason, we focused the synthesis of additional amidine

and guanidine derivatives 24−28 that have higher hydro-

phobicity than 20−23. These compounds were synthesized by

directly reacting 6 with corresponding N-heterocycle building

blocks, except for compound 27, which was made through 5-

ﬂuoro-2-aminopyridine due to the inadequate reactivity of 5-

ﬂuoro-2-chloropyridine in the reaction with 6 (Scheme 2B).

Among them, 24 exhibited an IC₅₀value as 85 nM. 11 and 24

are the two most potent compounds in our compound series.

We further evaluated their binding to ENL using the surface

plasmon resonance (SPR) analysis. His-ENL was immobilized

on dextran-coated Au chips through 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDCI)/N-hydroxy succi-

nimide (NHS) coupling, followed by ﬂow-through of a buﬀer

containing diﬀerent concentrations of 11 and 24. The

responses in sensorgrams were ﬁtted to the Langmuir 1:1

binding kinetics model to obtain both association and

dissociation rate constants, from which K_dvalues were then

determined (Supporting Information, Figure S3A). Compared

to the kinetics of typical small molecule−protein interactions,

both association and dissociation of 11 and 24 toward ENL are

relatively slow (association: 1800 and 1600 M⁻¹·s⁻¹;

dissociation: 8.3 × 10⁻⁵and 7.0 × 10⁻⁵s⁻¹, respectively).

Their determined K_dvalues by SPR were 45 and 46 nM,

respectively. As far as we know, 11 and 24 are the two most

potent inhibitors for ENL that have so far been developed.

Small-Molecule Inhibitors Occupy the Same Binding

Site of the ENL YEATS Domain as the Histone Acyl-

lysine. To study the molecular basis of 7, 11, and 24 binding

to ENL, we attempted to cocrystalize the ENL YEATS domain

with these compounds, but it was not successful. We then

modeled these compounds to the acetyllysine binding pocket

of the ENL YEATS domain by docking analysis. 7 and 11 were

docked in their protonated form, while 24 was docked in the

neutral form, given that it is less likely to be much protonated

under physiological pH. In the modeled structures (Figure

2A−D), all three compounds interact with ENL similar to 6

(Figure 1D). The amide forms two hydrogen bonds with S58

Figure 2. Compounds 7, 11, and 24 and their docking models bound

to the ENL YEATS domain. (A) Chemical structures of compounds

7, 11, and 24 and their IC₅₀values in inhibiting the His-ENL−

H3K9ac interaction in the AlphaScreen assay. (B−D) Molecular

docking models of compounds 7 (B), 11 (C), and 24 (D) bound to

the ENL YEATS domain. Modeling was based on the crystal structure

of the ENL YEATS domain (PDB: 5J9S). Compounds are shown in

stick representation, and the ENL YEATS domain is shown as a

cartoon in gray. Compound-interacting residues of ENL are

highlighted and shown in stick representation.

and Y78. The triazolopyridine ring was involved in π-stacking

interactions with H56 in a parallel conﬁguration and with Y78

in a T-shaped conﬁguration. The phenyl group was also

involved in π-stacking interactions with F28 and F59, both in a

T-shaped conﬁguration. Importantly, the amine of 7, the

azetidine of 11, and the guanidine of 24 are all within 3 Å to

E26, suggesting a common salt bridge or hydrogen bond

interaction that stabilizes their interactions with ENL (Figure

2B−D).

To experimentally validate that 7 binds to the acyl-lysine

binding pocket of ENL, we compared the binding of 7 and a

H3K27cr peptide (histone H3 residues 22−31 with a

crotonylation at K27) to ENL by nuclear magnetic resonance

(NMR) spectroscopy.³³We expressed ¹⁵N-labeled His-ENL

and recorded its ¹H, ¹⁵N heteronuclear single quantum

coherence (HSQC) spectra, while 7 or the H3K27cr peptide

was titrated into the sample (Figure 3 and Supporting

Information, Figure S3B). As expected, the H3K27cr peptide

induced large chemical shift perturbations (CSPs) in the ENL

YEATS domain, which were in the intermediate to fast

exchange regime on the NMR timescale. Addition of 7 caused

CSPs in the intermediate to slow exchange regime, indicating

Figure 3. Compound 7 and the H3K27cr peptide occupy the same

binding site of the ENL YEATS domain. Superimposed ¹H,¹⁵N

HSQC spectra of His-ENL collected as H3K27cr (H3 residues 22−

31, left) or 7 (right) was added stepwise. Spectra are color-coded

according to the protein/ligand molar ratios.

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Figure 4. Compounds 7, 11, and 24 are highly speciﬁc to ENL over other YEATS domains. (A) Peptide pull-downs of ENL, AF9, YEATS2, and

GAS41 with the indicated acylated histone peptides with or without 1, 7, 11, and 24. Unmodiﬁed histone peptides were used as negative controls

to the acylated peptides and DMSO as a negative control to compound treatment. (B) AlphaScreen measurement of IC₅₀of 1, 7, 11, and 24 in

inhibition of YEATS domains binding to the corresponding acylated histone peptides as in (A). Data represent mean standard errors of the mean

(SEM), n = 4.

that the ENL YEATS domain binds to 7 tighter than to the

H3K27cr peptide. An overall similar pattern of CSPs observed

in both experiments suggest that 7 and the H3K27cr peptide

occupy the same binding site in the ENL YEATS domain.

Compounds 7, 11, and 24 Are Highly Selective

toward the ENL YEATS Domain over All Other Human

YEATS Domains. To determine whether the small-molecule

inhibitors are selective toward ENL among the four human

YEATS domains, we assessed 7, 11, and 24 in their inhibition

of ENL, AF9, GAS41, and YEATS2 in peptide pull-down

assays. We also included the original hit 1 in the assays for

comparison. We used the H3K9ac peptide for AF9 and ENL,

the H3K27ac peptide for GAS41, and the H3K27cr peptide for

YEATS2 as these peptides are the preferred ligands of the

corresponding YEATS domains.^7,11,14,16We found 1 μM of 11

and 24 and 5 μM of 1 and 7 strongly inhibited the binding of

ENL to H3K9ac, whereas at even a 20 μM concentration, none

of these compounds showed notable inhibition to AF9,

GAS41, or YEATS2 binding to their corresponding acylated

histone peptides (Figure 4A). We further measured the IC₅₀

values of 1, 7, 11, and 24 in their inhibition of the binding of

four human YEATS domains to their preferred histone peptide

ligands using AlphaScreen assays. All four compounds

displayed preferential inhibition of ENL over the other three

YEATS domains. Compound 1 showed ∼4-fold higher

potency toward ENL than AF9, whereas no detectable

inhibition was measured for the YEATS2 or GAS41 YEATS

domain. Compounds 7, 11, and 24 exhibited even higher

speciﬁcity to ENL. Particularly, the IC₅₀value of 11 to ENL

was ∼20-fold lower than that to AF9 (ENL IC₅₀51 nM and

AF9 IC₅₀984 nM) (Figure 4B). As the previously reported

small-molecule ENL inhibitors are not able to diﬀerentiate

between ENL and AF9, our compounds provide promising

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Figure 5. Compound 7 exhibits on-target eﬀect of ENL inhibition in MLL-rearranged leukemia cell lines. (A) 7 inhibits leukemia cell growth. Cell

growth inhibition of 7 at various concentrations in MV4-11, MOLM13, and U2OS cells. Survived cells were calculated as % relative to DMSO-

treated cells. (B,C) CETSAs in MV4-11 (B) and MOLM13 (C) cells treated with 20 μM 7 at the indicated temperatures. β-Actin was used as a

loading control. (D) qRT-PCR analysis of HOXA9 and MYC gene expression in MOLM13 cells treated with 7 or the DMSO negative control.

(E,F) 7 shows a synergistic eﬀect with JQ1 in MOLM13 cells. (E) 3D synergy distribution of 7 and JQ1. MOLM13 cells were treated with

indicated doses of 7 and JQ1 or DMSO for 6 days. Surviving cells were calculated as % relative to DMSO-treated cells. Synergistic interactions were

analyzed using the Combeneﬁt software. (F) GI₅₀of 7 when cotreated with indicated concentrations of JQ1. Data in (A,D) are shown as mean

SEM, n = 3, two-tailed Student’s t-test, ns, not signiﬁcant, *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001. Data in (E,F) represent mean

SEM, n = 3.

scaﬀolds for further development of ENL-speciﬁc inhibitors for

the study of ENL biology and for disease intervention.

protein for comparison. Compared to the DMSO-treated cells,

we detected higher abundance of soluble ENL proteins in cells

treated with 7, indicating that 7 bound and stabilized ENL

proteins.³⁴In contrast, the thermal stability of GAS41 proteins

showed little or no diﬀerence between DMSO and 7 treatment

(Figure 5B,C). Similarly, 7 stabilized ENL but not AF9

proteins in HeLa cells (Supporting Information, Figure S4C).

The CETSA results suggest speciﬁc engagement of ENL with 7

in living cells.

We also evaluated the expression of two ENL target genes,

HOXA9 and MYC, in MOLM13 cells. Compound 7 eﬀectively

suppressed HOXA9 gene expression at as low as 2.5 μM of

drug concentration. At 10 μM, it suppressed ∼80% of the

expression of both HOXA9 and MYC genes, comparable to the

levels of gene suppression in ENL knock-down cells (Figure

5D and Supporting Information, S4D), suggesting potent and

on-target eﬀect of the ENL inhibitor.

Compound 7 Exhibits a Synergistic Eﬀect with JQ1.

Previously we found that CRISPR/Cas9-mediated ENL

knockout sensitized leukemia cells to JQ1, an eﬀective

inhibitor of BET bromodomain proteins including BRD4.^4,14

An intriguing question was whether 7 has any synergy with

JQ1 in killing leukemia cells. To answer this question, we

carried out combinatory treatment of MV4-11 and MOLM13

cells with series of concentrations of 7 (0 to 20 μM) and JQ1

(0−200 nM). In both cell lines, we observed synergistic eﬀect

between ENL inhibition and JQ1 (Figure 5E and Supporting

Compound 7 Inhibits the Endogenous ENL Protein in

MLL-Rearranged Cell Lines. To explore the small molecule

ENL inhibitors we developed in biological applications, we ﬁrst

analyzed their cellular eﬀects in MV4-11 and MOLM13 cells,

two MLL-rearranged cell lines whose growth is dependent on

ENL.^14,22We screened 15 compounds with in vitro IC₅₀values

lower than 2 μM, and we found 7 as the most potent

compound in cell growth inhibition (Supporting Information,

Figure S4A). The discrepancy between in vitro IC₅₀and

cellular eﬃcacy is not due to cell permeability as 7 was

comparable to 11 in the standard Caco-2 permeability assay

(Supporting Information, Figure S4B). Compound 7 exhibited

∼40% inhibition of MOLM13 cell growth at 5 μM and 80%

inhibition at 10 μM concentrations, while around double

amounts of the compound were needed to achieve similar

levels of inhibition in MV4-11 cells (Figure 5A). In contrast,

U2OS cells, an ENL-independent cell line, showed little or no

response to the treatment with 7, even at 50 μM.

Next, we asked whether the growth inhibition eﬀect was

caused by on-target inhibition of the endogenous ENL protein.

In this regard, we carried out the cellular thermal shift assay

(CETSA) to evaluate thermal stability of the ENL protein in

MV4-11 and MOLM13 cells treated with 7. As the AF9

protein is undetectable in these cells with commercial

antibodies, we evaluated thermal stability of the GAS41

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Information, Figure S4E).³⁵Particularly, at a concentration of

200 nM of JQ1, the GI₅₀of compound 7 in MOLM13 cells

was reduced from 3.64 to 1.34 μM (Figure 5F). Together,

these results demonstrate therapeutic potentials of the ENL

inhibitors for future exploration in disease treatment.

1000-fold lower over GAS41, providing a good lead for future

drug development.

The selectivity of our compounds to ENL over AF9 is

intriguing, given that the AF9 YEATS domain has a 10-fold

higher aﬃnity than ENL YEATS in acyl-lysine binding. The

YEATS domains of AF9 and ENL share a high degree of

structural similarity.^11,14It is not clear what interactions

contribute to the ENL selectivity. By comparing the modeled

structures of compounds 1, 7, 11, and 24 docked to the

acetyllysine binding pocket of the ENL and AF9 YEATS

domains, we observed that the triazolopyridine pharmacophore

of these compounds adopts conformations to form stronger

π−π interactions with the H56 residue in ENL than in the AF9

YEATS domain. When bound with the ENL YEATS domain,

the distances between the triazole rings and the imidazole rings

range from 3.4 to 3.6 Å and their dihedral angles range from 20

to 22°, whereas in the case of AF9 YEATS domain, the

distances between them increase to 4.3−4.6 Å and their

dihedral angles also increase to a range of 28−37°, both

leading to weaker π−π interactions compared to those in the

ENL YEATS domain (Supporting Information, Figure S5).

Additionally, the salt-bridge interaction in the case of 7 and 11

and the hydrogen bond for 24 with E26 in the ENL YEATS

domain may also contribute to selectivity. Further structural

study of YEATS domains in complex with these compounds

will provide insights to guide future development of more

potent and selective ENL YEATS-domain inhibitors.

In leukemia cells, our synthesized compound 7 exhibited

clear on-target cellular eﬀects in reducing ENL target gene

expression and suppressing leukemia cell growth. In addition,

consistent with previous results of genetic ENL ablation, 7

exhibited a synergistic eﬀect with the BET bromodomain

inhibitor JQ1 in killing leukemia cells. The cellular eﬀects of

our compounds are superior to all reported ENL inhibitors.

Overall, our study provides valuable selective ENL small-

molecule inhibitors that can serve as potential leads for further

medicinal chemistry-based optimization to advance both basic

and translational research of ENL. It also provides a molecular

platform for the development of more complicated, multifunc-

tional probes for applications such as visualization or targeted

degradation in cells.

DISCUSSION

■

The YEATS domain is a newly identiﬁed family of histone

acylation readers. The four human YEATS domain-containing

proteins, ENL, AF9, YEATS2, and GAS41, are subunits of

protein complexes involved in chromatin and transcription

regulation.^18,19The evolutionally conserved histone-reading

function of the YEATS domains is essential for the

functionality of all the YEATS domain proteins in both yeasts

and humans.⁶⁻¹⁶Dysregulation of the YEATS domain-

containing proteins has been associated with various human

diseases, including cancers. We and others showed that ENL

and particularly, its YEATS domain is essential for disease

maintenance and progression of acute leukemias.^14,22Recently,

we also found that the reader function of the ENL YEATS

domain is indispensable for the aberrant gene activation and

tumorigenesis caused by the gain-of-function ENL YEATS

domain mutations identiﬁed in Wilms’ tumor patients.²⁵In

addition, YEATS2 and GAS41 are frequently ampliﬁed in

various types of human cancers.³⁶⁻³⁸All these studies suggest

that the YEATS domains are promising drug targets, and

therefore, targeting the YEATS domains may provide a novel

therapeutic approach for a broad spectrum of human cancers.

Developing YEATS-domain inhibitors has been a research

focus of the epigenetic reader ﬁeld in recent years. The initial

eﬀorts were focused on targeting the YEATS domain of ENL

because of great therapeutic potentials. Both small-molecule

chemical compounds and peptide-mimic probes have recently

been developed as acetyllysine competitive inhibitors of the

ENL YEATS domain.²⁶⁻³²However, target selectivity has

been a big challenge because the YEATS domains share high

structural similarity, especially between ENL and its close

homologue AF9. The few small-molecule ENL inhibitors

reported so far have poor speciﬁcity that fail to distinguish

ENL from AF9. The peptide-mimic chemical probes

developed by the Li group showed slightly higher potency to

the ENL YEATS domain than other YEATS domains, largely

due to interactions outside of the acetyllysine binding pocket.³⁰

These results suggest that targeting both the acyl-lysine-

binding pocket and additional proximal sites outside of the

binding pocket might be a good approach to develop speciﬁc

inhibitors. Indeed, based on this concept, the Li group has

recently developed a conformationally preorganized cyclo-

peptide that showed a 38-fold higher binding aﬃnity toward

AF9 YEATS over ENL.²⁹

CONCLUSIONS

■

In this study, we carried out HTS of a small-molecule library of

>66,000 compounds against the ENL YEATS domain and

identiﬁed a series of hit molecules that share a [1,2,4]triazolo-

[4,3-a]pyridine-6-amide pharmacophore and a common N,C-

diarylamide scaﬀold. By introducing a potential salt-bridge

interaction with E26 in ENL, we were able to generate

compounds with IC₅₀and K_dvalues less than 100 nM.

Importantly, our compounds outcompeted the previously

reported ENL inhibitors by showing high selectivity toward

ENL over AF9, the close paralogue of ENL. Furthermore,

compound 7 exhibited on-target eﬀect in inhibiting ENL target

gene expression and leukemia cell growth. Our ENL-speciﬁc

YEATS-domain inhibitors provide the basis for development

of potent ENL-speciﬁc chemical probes in the future.

Despite the success in developing peptide-mimic chemical

probes speciﬁc to AF9, ENL-speciﬁc inhibitors were still

lacking. Because ENL, but not AF9, is essential for MLL-

rearranged acute leukemias and ENL mutant Wilms’ tumors, it

is in urgent need to develop ENL speciﬁc inhibitors for further

drug development. In our study, through HTS, we identiﬁed

compound 1, which showed a 4-fold preference toward ENL

over AF9 YEATS. After several rounds of structure-based

inhibitor design and SAR studies, we were able to develop

several compounds with much better selectivity. In particular,

the IC₅₀value of compound 11 to ENL was ∼20-fold lower

over AF9, ∼360-fold lower over YEATS2, and more than

EXPERIMENTAL SECTION

■

Materials. The biotinylated histone peptides used in the

AlphaScreen assay, H3 (aa 1−22, ARTKQTARKSTGGKAPRKQ-

LAT), H3K9ac (aa 1−22, ARTKQTARK(ac)STGGKAPR-KQLAT),

H3 (aa 21−44, ATKAARKSAPATGGVKKPHRYRPG), H3K27ac

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(aa 21−44, ATKAARK(ac)SAPSTGGVKKPHRYRPG), H3K27cr

(aa 21−44, ATKAARK(cro)SAPS-TGGVKKPHRYRPG), and the

biotin-14× His peptide used in counter assay were purchased from

CPC Scientiﬁc. Anti-ENL (14893S) antibody was from Cell

Signaling, anti-GAS41 (sc-393708) and anti-glutathione-S-transferase

(GST) (sc-459) antibodies were from Santa Cruz, and anti-AF9

(HPA001824) and anti-β-actin (A1978) antibody were from Sigma.

Human cell lines MV4-11, MOLM13, U2OS, and HeLa cells were

purchased from ATCC.

Protein Expression and Puriﬁcation. The complementary

DNA (cDNA) encoding sequences of four human YEATS domains:

ENL (aa 1−145), AF9 (aa 1−145), full-length GAS41, and YEATS2

(aa 201−332) were cloned in pGEX-6P-1 and pET19b expression

vectors, respectively. The His-tagged YEATS proteins were expressed

in Escherichia coli Rosetta-2 (DE3) pLysS cells in the presence of 0.2

mM isopropyl-β-^D-1-thiogalactopyranoside (IPTG) for 18 h at 16 °C.

The His-tagged YEATS proteins were puriﬁed using Ni−nitrilotri-

acetic acid (NTA) resins following the manufacturer’s instructions.

The eluted protein was dialyzed in a buﬀer containing 50 mM N-(2-

hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES, pH 7.4),

100 mM NaCl, and 20% glycerol to remove imidazole. Proteins were

adjusted to 0.5 mg/mL, aliquoted, and stored at −80 °C. Each batch

of puriﬁed protein was tested in AlphaScreen assay conditions

discussed as follows. The GST-tagged proteins used in peptide pull-

down assays were expressed in the same way and puriﬁed using

glutathione sepharose resins (GE Healthcare).

AlphaScreen Assay Setup. The AlphaScreen assay was carried

out in 384-well plates. Manual assay setup was performed in a 30 μL

reaction in α-reaction buﬀer (50 mM HEPES pH 7.4, 100 mM NaCl,

0.1% bovine serum albumin, and 0.05% CHAPS) with ﬁnal

concentrations of 100 nM His-ENL YEATS, 30 nM biotin-H3K9ac,

and 10 μg/mL of AlphaScreen-donor and -acceptor beads. During the

automation step, we were able to reduce the assay volume to 20 μL

per well while maintaining the quality and robustness of the assay,

with optimal ﬁnal concentrations of His-ENL YEATS (100 nM),

biotin-H3K9ac (10 nM), DMSO (0.1%), and AlphaScreen-beads (2.5

μg/mL). The protein, peptide, and compounds were mixed and

incubated for 1 h at room temperature before adding the α beads.

AlphaScreen-signals were detected by an EnVision microplate reader

equipped with an Alpha-laser (PerkinElmer).

HTS Using AlphaScreen. High-throughput screening was

performed at the Texas Screening Alliance for Cancer Therapeutics

(TxSACT) facility. A total of 66,625 compounds screened were from

Maybridge HitFinder Set (14,080), Chembridge Diversity Set

(12,900), Chembridge Kinase Set (11,250), Chembridge Fragment

Library (4000), ChemDiv Fragment Collection (14,143), Legacy

Collection (2092), MicroSource Spectrum Collection (2000),

LOPAC Collection (1275), Selleck Kinase and Bioactive Collection

(2260), NCI Diversity (1595), NCI Mechanistic collection (820),

and NCI natural products (210). In the primary HTS, fragments were

screened at 50 μM and nonfragment compounds were screened at 10

μM. After the single-shot screen and hits triage, 990 hits were picked

for conﬁrmation assay and counter assay with biotin-14× His peptide.

IC₅₀Determination with the AlphaScreen Assay. The

AlphaScreen assay conditions are essentially the same as the one

used in high-throughput screening. The protein concentrations of

AF9, Gas41, and YEATS2 are 30, 100, and 100 nM, respectively, and

the peptide concentrations are 30 nM. All assays have been validated

using protein and peptide competitors. For IC₅₀determination,

compounds were subjected to eight threefold serial dilutions for a

total of nine concentrations ranging from 50 μM to 8 nM for dose−

response-curve AlphaScreen assays. IC₅₀values were determined from

the plot using nonlinear regression of variable slope (four parameters)

and curve ﬁtting performed using GraphPad Prism.

H3K9ac deleted from the complexes. Structures of the YEATS

domains were then preprocessed in MGLTools 1.5.6 to remove water

molecules and add polar hydrogens. The grid box was set to be

centered at coordinates (x = 27.352, y = −42.139, z = 3.0) for ENL

and at coordinates (x = 52.734, y = 10.522, z = −11.134) for AF9,

with a size of 40 × 40 × 40 npts, which is big enough to contain the

binding channel and surrounding amino acid residues. The Glu26

residue of the ENL YEATS domain was set to be ﬂexible. The target

compounds were then docked in the grid box. The conformations

with lowest binding energies were converted to PDB ﬁles for

visualization.

Compound Synthesis. All reagents and solvents for synthesis

were purchased from commercial sources and used without

puriﬁcation. All glassware were ﬂame-dried prior to use. Thin-layer

chromatography (TLC) was carried out on aluminum plates coated

with 60 F254 silica gel. TLC plates were visualized under UV light

(254 or 365 nm) or stained with 5% phosphomolybdic acid. Normal-

phase column chromatography was carried out using a Yamazen

Smart Flash AKROS system. Analytical reverse-phase high-pressure

liquid chromatography (RP-HPLC) was carried out on a Shimadzu

LC20 HPLC system with an analytical C18 column. Semipreparative

HPLC was carried out on the same system with a semipreparative

C18 column. The mobile phases were H₂O with 0.1% formic acid (A)

and acetonitrile with 0.1% formic acid (B) if not mentioned

otherwise. NMR spectra were recorded on a Bruker AVANCE Neo

400 MHz or Varian INOVA 300 MHz spectrometer in speciﬁed

deuterated solvents. High-resolution electrospray ionization mass

spectrometry (HRMS-ESI) was carried out on a Thermo Scientiﬁc Q

Exactive Focus system. The purities of compounds were conﬁrmed by

NMR and analytical HPLC-UV as ≥95%.

tert-Butyl (4-(([1,2,4]Triazolo[4,3-a]pyridine-6-carboxamido)-

methyl)benzyl)carbamate (41). To a solution of 39 (1 mmol, 163

mg) and 40 (1 mmol, 236 mg) in dry dimethylformamide (DMF, 5

mL) were added DIPEA (2 mmol, 258 mg) and EDCI (1.2 mmol,

230 mg). The resulting solution was stirred at room temperature

overnight. Then, the solution was diluted with EtOAc (50 mL) and

washed with saturated NaHCO₃solution (2 × 50 mL), 1 M HCl (2 ×

50 mL), and saturated brine (50 mL). The organic layers were then

dried with anhydrous Na₂SO₄and then concentrated. The residue

was puriﬁed by column chromatography (silica gel, 10% MeOH/

DCM as the eluent) to yield 41 as a light-yellow solid (250 mg, 66%).

(4-(([1,2,4]Triazolo[4,3-a]pyridine-6-carboxamido)methyl)-

phenyl)methanamine Hydrochloride (6). To a solution of 41 (0.5

mmol, 190 mg) in 5 mL of 1,4-dioxane was added 10 mL of 4 M HCl

solution in 1,4-dioxane. The resulting solution was stirred at room

temperature for 2 h. Then, the reaction mixture was concentrated to

1

dryness in vacuo to yield 6 as a light-yellow solid (150 mg, 95%). H

NMR (400 MHz, deuterium oxide): δ 9.49 (s, 1H), 9.24 (s, 1H), 8.29

(dt, J = 9.7, 1.6 Hz, 1H), 8.10 (d, J = 9.6 Hz, 1H), 7.52−7.42 (m,

4H), 4.66 (s, 2H), 4.19 (s, 2H). ¹³C NMR (101 MHz, D₂O): δ 165.4,

145.4, 138.5, 137.8, 134.1, 131.8, 129.2, 128.0, 127.4, 125.1, 112.1,

43.5, 42.7. ESI-HRMS (m/z): calcd for C₁₅H₁₆N₅O (M + H)⁺,

282.1349; found, 282.1344.

4-(1,3-Dioxolan-2-yl)benzonitrile (43). To a solution of 42 (38

mmol, 5.0 g) and ethylene glycol (76 mmol, 4.2 mL) in toluene (50

mL) was added pyridinium p-toluenesulfonate (4 mmol, 0.96 g). The

resulting solution was heated to reﬂux with a Dean−Stark trap for 4 h.

The resulting solution was then concentrated in vacuo, and the residue

was then puriﬁed by column chromatography (silica gel, 10% EtOAc/

1

hexanes as the eluent) to yield 43 as a white solid (5.25 g, 79%). H

NMR (300 MHz, chloroform-d): δ 7.67 (d, J = 8.3 Hz, 2H), 7.58 (d, J

= 8.3 Hz, 2H), 5.84 (s, 1H), 4.17−3.99 (m, 4H).

(4-(1,3-Dioxolan-2-yl)phenyl)methanamine (44). LiAlH₄(0.99 g,

26 mmol) was suspended in dry tetrahydrofuran (THF, 50 mL) and

was cooled under 0 °C. A solution of 43 (4.5 g, 25.7 mmol) in dry

THF (50 mL) was added dropwise to the LiAlH₄suspension at the

same temperature. After the addition, the reaction mixture was

warmed up to room temperature and stirred for 4 h. Water (3 mL)

was added dropwise, followed by 2 M aqueous NaOH solution (3

mL) and then, water (3 mL). The precipitate was ﬁltered and washed

Modeling of Inhibitors Bound with ENL and AF9 YEATS

Domains. Molecular docking of target compounds was carried out

using AutoDock 4.³⁹The initial conformations of target compounds

were ﬁrst generated and MM2 minimized by PerkinElmer Chem3D

software. Structures of the ENL and AF9 YEATS domains were

obtained from PDB 5J9S and 4TMP, respectively, with H3K27ac and

I

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with THF. The combined ﬁltrate was then dried with anhydrous

Na₂SO₄and evaporated in vacuo. The residue was then puriﬁed by

column chromatography (silica gel, 100% EtOAc as the eluent) to

yield 44 as a colorless oil to a white solid (3.0 g, 67%). ¹H NMR (300

MHz, chloroform-d): δ 7.44 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz,

2H), 5.78 (s, 1H), 4.15−3.95 (m, 4H), 3.85 (s, 2H).

N-(4-(1,3-Dioxolan-2-yl)benzyl)-[1,2,4]triazolo[4,3-a]pyridine-6-

carboxamide (45). To a solution of 44 (2 mmol, 358 mg) and 39 (2

mmol, 326 mg) in DMF (10 mL) were added DIPEA (4 mmol, 516

mg) and EDCI (2.4 mmol, 460 mg). The resulting solution was then

stirred at room temperature overnight. The reaction mixture was then

diluted with EtOAc (50 mL) and then washed with saturated

NaHCO₃solution (2 × 50 mL), 1 M HCl (2 × 50 mL), and brine

(50 mL). The organic layers were dried with anhydrous Na₂SO₄and

concentrated in vacuo. The residue was then puriﬁed by column

chromatography (silica gel, 20% methanol/EtOAc as the eluent) to

yield 45 as a white solid (520 mg, 80%). ¹H NMR (400 MHz,

DMSO-d₆): δ 9.36 (d, J = 0.8 Hz, 1H), 9.25 (t, J = 5.9 Hz, 1H), 9.14

(t, J = 1.4 Hz, 1H), 7.90−7.76 (m, 2H), 7.44−7.33 (m, 4H), 5.70 (s,

1H), 4.52 (d, J = 5.8 Hz, 2H), 4.09−3.88 (m, 4H).

23.7. ESI-HRMS (m/z): calcd for C₂₀H₂₄N₅O (M + H)⁺, 350.1975;

found, 350.1970.

N-(4-(Morpholinomethyl)benzyl)-[1,2,4]triazolo[4,3-a]pyridine-

6-carboxamide (9). To a solution of 46 (35 mg, 0.12 mmol) in THF

(3 mL) were added morpholine (21 mg, 0.24 mmol) and Ti(OⁱPr)₄

(0.36 mmol, 102 mg). The mixture was stirred at room temperature

for 10 min. Then, NaBH(OAc)₃(0.36 mmol, 76 mg) was added and

the mixture was reﬂuxed under N₂. The reaction was monitored by

TLC. Small portions of additional NaBH(OAc)₃were added to drive

the reaction to completion. Upon complete consumption of 46, the

reaction mixture was diluted with saturated NaHCO₃solution (20

mL) and the precipitate was ﬁltered. The ﬁltrate was then extracted by

EtOAc (2 × 20 mL). The combined organic layers were dried with

anhydrous Na₂SO₄and concentrated in vacuo. The residue was then

puriﬁed by column chromatography (silica gel, 20% methanol/EtOAc

as the eluent) to give 9 as a white solid (15 mg, 43%). ¹H NMR (400

MHz, DMSO-d₆): δ 9.37 (s, 1H), 9.22 (t, J = 5.9 Hz, 1H), 9.14 (t, J =

1.4 Hz, 1H), 7.87−7.75 (m, 2H), 7.32−7.24 (m, 4H), 4.49 (d, J = 5.8

Hz, 2H), 3.55 (t, J = 4.7 Hz, 4H), 3.42 (s, 2H), 2.37−2.27 (m, 4H).

¹³C NMR (101 MHz, DMSO): δ 163.5, 148.4, 137.8, 137.6, 136.5,

129.0, 127.3, 126.8, 126.4, 121.1, 114.4, 66.2, 62.1, 53.1, 42.5. ESI-

HRMS (m/z): calcd for C₁₉H₂₂N₅O₂(M + H)⁺, 352.1768; found,

352.1764.

N-(4-Formylbenzyl)-[1,2,4]triazolo[4,3-a]pyridine-6-carboxa-

mide (46). To a solution of 45 (520 mg, 1.6 mmol) in 1,4-dioxane (5

mL) was added 5 mL of 4 M HCl solution in 1,4-dioxane. The

resulting solution was stirred for 2 h and concentrated in vacuo. The

residue was suspended in H₂O and basiﬁed with saturated NaHCO₃

solution. The precipitation was ﬁltered and dried to yield 46 as a

N-(4-(Pyrrolidin-1-ylmethyl)benzyl)-[1,2,4]triazolo[4,3-a]-

pyridine-6-carboxamide (10). To a solution of 46 (35 mg, 0.12

mmol) in THF (3 mL) were added pyrrolidine (17 mg, 0.24 mmol)

and Ti(OⁱPr)₄(0.36 mmol, 102 mg). The mixture was stirred at room

temperature for 10 min. Then, NaBH(OAc)₃(0.36 mmol, 76 mg)

was added and the mixture was reﬂuxed under N₂. The reaction was

monitored by TLC. Small portions of NaBH(OAc)₃were added to

drive the reaction to completion. Upon complete consumption of 46,

the reaction mixture was diluted with saturated NaHCO₃solution (20

mL) and the precipitate was ﬁltered. The ﬁltrate was then extracted by

EtOAc (2 × 20 mL). The combined organic layers were dried with

anhydrous Na₂SO₄and concentrated in vacuo. The residue was then

puriﬁed by column chromatography (silica gel, 20% methanol/EtOAc

1

yellowish solid (390 mg, 87%). H NMR (400 MHz, DMSO-d₆): δ

9.99 (s, 1H), 9.42−9.31 (m, 2H), 9.16 (t, J = 1.4 Hz, 1H), 7.89 (d, J =

8.1 Hz, 2H), 7.87−7.78 (m, 2H), 7.57 (d, J = 7.9 Hz, 2H), 4.61 (d, J

= 5.9 Hz, 2H).

N-(4-((Dimethylamino)methyl)benzyl)-[1,2,4]triazolo[4,3-a]-

pyridine-6-carboxamide (7). To a solution of 46 (35 mg, 0.12 mmol)

in THF (3 mL) was added a solution of dimethylamine in THF (1 M,

0.25 mL) and Ti(OⁱPr)₄(0.36 mmol, 102 mg). The mixture was

stirred at room temperature for 10 min. Then, NaBH(OAc)₃(0.36

mmol, 76 mg) was added, and the mixture was reﬂuxed under N₂.

The reaction was monitored by TLC. Small additional portions of

NaBH(OAc)₃were added to drive the reaction to completion. Upon

complete consumption of 46, the reaction mixture was diluted with

saturated NaHCO₃solution (20 mL), and the precipitate was ﬁltered.

The ﬁltrate was then extracted by EtOAc (2 × 20 mL). The combined

organic layers were dried with anhydrous Na₂SO₄and concentrated in

vacuo. The residue was then puriﬁed by column chromatography

(silica gel, 20% methanol/EtOAc as the eluent) to give 7 as a white

solid (11 mg, 30%). ¹H NMR (400 MHz, methanol-d₄): δ 9.29 (d, J =

0.8 Hz, 1H), 9.09 (t, J = 1.4 Hz, 1H), 7.91−7.76 (m, 2H), 7.57−7.44

(m, 4H), 4.65 (s, 2H), 4.29 (s, 2H), 2.83 (s, 6H). ¹³C NMR (101

MHz, DMSO): δ 164.1, 148.8, 141.0, 138.1, 131.6, 129.5, 128.1,

127.4, 127.0, 121.5, 114.9, 59.5, 42.9, 41.8. ESI-HRMS (m/z): calcd

for C₁₇H₂₀N₅O (M + H)⁺, 310.1662; found, 310.1654.

N-(4-(Piperidin-1-ylmethyl)benzyl)-[1,2,4]triazolo[4,3-a]pyridine-

6-carboxamide (8). To a solution of 46 (35 mg, 0.12 mmol) in THF

(3 mL) were added piperidine (20 mg, 0.24 mmol) and Ti(OⁱPr)₄

(0.36 mmol, 102 mg). The mixture was stirred at room temperature

for 10 min. Then, NaBH(OAc)₃(0.36 mmol, 76 mg) was added, and

the mixture was reﬂuxed under N₂. The reaction was monitored by

TLC. Small portions of NaBH(OAc)₃were added to drive the

reaction to completion. Upon complete consumption of 46, the

reaction mixture was diluted with saturated NaHCO₃solution (20

mL) and the precipitate was ﬁltered. The ﬁltrate was then extracted by

EtOAc (2 × 20 mL). The combined organic layers were dried with

anhydrous Na₂SO₄and concentrated in vacuo. The residue was then

puriﬁed by column chromatography (silica gel, 20% methanol/EtOAc

as the eluent) to give 8 as a white solid (12 mg, 28%). ¹H NMR (400

MHz, methanol-d₄): δ 9.26 (d, J = 0.8 Hz, 1H), 9.04 (t, J = 1.4 Hz,

1H), 7.87−7.76 (m, 2H), 7.38−7.29 (m, 4H), 4.60 (s, 2H), 3.50 (s,

2H), 2.37 (t, J = 5.1 Hz, 4H), 1.58 (p, J = 5.6 Hz, 4H), 1.45 (p, J = 5.6

Hz, 2H). ¹³C NMR (101 MHz, MeOD): δ 164.8, 149.0, 137.5, 137.5,

135.9, 129.9, 127.4, 127.3, 126.4, 122.5, 114.2, 62.9, 53.8, 43.1, 25.0,

1

as the eluent) to give 10 as a white solid (15 mg, 38%). H NMR

(300 MHz, methanol-d₄): δ 9.28 (s, 1H), 9.07 (d, J = 1.6 Hz, 1H),

7.86 (dd, J = 9.7, 1.7 Hz, 1H), 7.79 (d, J = 9.7 Hz, 1H), 7.41 (s, 4H),

4.61 (s, 2H), 3.94 (s, 2H), 3.02−2.76 (m, 4H), 1.97−1.88 (m, 4H).

¹³C NMR (75 MHz, CD₃OD): δ 164.8, 149.0, 138.8, 137.5, 134.0,

129.7, 127.7, 127.4, 126.4, 122.4, 114.2, 58.8, 53.4, 43.0, 22.6. ESI-

HRMS (m/z): calcd for C₁₉H₂₂N₅O (M + H)⁺, 336.1819 (M + H);

found, 336.1810.

N-(4-(Azetidin-1-ylmethyl)benzyl)-[1,2,4]triazolo[4,3-a]pyridine-

6-carboxamide (11). To a solution of 46 (35 mg, 0.12 mmol) in

THF (3 mL) were added azetidine hydrochloride (22 mg, 0.24

mmol) and Ti(OⁱPr)₄(0.36 mmol, 102 mg). The mixture was stirred

at room temperature for 10 min. Then, NaBH(OAc)₃(0.36 mmol, 76

mg) was added and the mixture was reﬂuxed under N₂. The reaction

was monitored by TLC. Small portions of additional NaBH(OAc)₃

were added to drive the reaction to completion. Upon complete

consumption of 46, the reaction mixture was diluted with saturated

NaHCO₃solution (20 mL) and the precipitate was ﬁltered. The

ﬁltrate was then extracted by EtOAc (2 × 20 mL). The combined

organic layers were dried with anhydrous Na₂SO₄and concentrated in

vacuo. The residue was then puriﬁed by column chromatography

(silica gel, 20% methanol/EtOAc as the eluent) to give 11 as a white

solid (15 mg, 40%). ¹H NMR (400 MHz, methanol-d₄): δ 9.29 (d, J =

0.8 Hz, 1H), 9.09 (t, J = 1.4 Hz, 1H), 7.91−7.76 (m, 2H), 7.52−7.42

(m, 4H), 4.63 (s, 2H), 4.30 (s, 2H), 4.06 (t, J = 8.1 Hz, 4H), 2.47 (p,

J = 8.1 Hz, 2H). ¹³C NMR (101 MHz, MeOD): δ 166.3, 150.4, 141.7,

138.9, 131.2, 130.9, 129.5, 128.8, 127.8, 123.7, 115.6, 59.2, 55.2, 44.3,

17.2. ESI-HRMS (m/z): calcd for C₁₈H₂₀N₅O (M + H)⁺, 322.1662;

found, 322.1654.

N-(4-((2-Methylazetidin-1-yl)methyl)benzyl)-[1,2,4]triazolo[4,3-

a]pyridine-6-carboxamide (12). To a solution of 46 (58 mg, 0.2

mmol) in DMF (1 mL) were added 2-methylazetidine hydrochloride

(43 mg, 0.4 mmol) and Ti(OⁱPr)₄(0.6 mmol, 170 mg). The mixture

was stirred at room temperature for 10 min. Then, NaBH(OAc)₃(0.6

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mmol, 127 mg) was added, and the mixture was heated to 70−75 °C

for 24 h under N₂. The reaction mixture was diluted with saturated

NaHCO₃solution (20 mL), and the precipitate was ﬁltered. The

ﬁltrate was then extracted by EtOAc (2 × 30 mL). The combined

organic layers were dried with anhydrous Na₂SO₄and concentrated in

vacuo. The residue was then puriﬁed by column chromatography

(silica gel, 20% methanol/EtOAc as the eluent) to give 12 as a white

solid (58 mg, 88%). ¹H NMR (400 MHz, methanol-d₄): δ 9.22 (d, J =

0.8 Hz, 1H), 9.02 (t, J = 1.4 Hz, 1H), 7.80 (dd, J = 9.6, 1.6 Hz, 1H),

7.71 (dt, J = 9.6, 1.0 Hz, 1H), 7.31−7.20 (m, 4H), 4.54 (s, 2H), 3.58

(d, J = 12.4 Hz, 1H), 3.46 (d, J = 12.4 Hz, 1H), 3.35−3.27 (m, 1H),

3.24−3.15 (m, 1H), 2.89 (dt, J = 9.8, 7.7 Hz, 1H), 2.04 (dtd, J = 10.2,

7.8, 2.3 Hz, 1H), 1.72 (tt, J = 10.0, 8.5 Hz, 1H), 0.97 (d, J = 6.2 Hz,

3H). ¹³C NMR (101 MHz, CD₃OD): δ 167.0, 151.2, 139.8, 139.7,

138.3, 131.5, 129.7, 129.6, 128.6, 124.7, 116.4, 64.8, 63.7, 53.1, 45.3,

27.4, 22.1. ESI-HRMS (m/z): calcd for C₁₉H₂₂N₅O (M + H)⁺,

336.1819; found, 336.1814.

N-(4-((2-Isopropylazetidin-1-yl)methyl)benzyl)-[1,2,4]triazolo-

[4,3-a]pyridine-6-carboxamide (13). To a solution of 46 (58 mg, 0.2

mmol) in DMF (1 mL) were added 2-isopropylazetidine hydro-

chloride (54 mg, 0.4 mmol) and Ti(OⁱPr)₄(0.6 mmol, 170 mg). The

mixture was stirred at room temperature for 10 min. Then,

NaBH(OAc)₃(0.6 mmol, 127 mg) was added, and the mixture was

heated to 70−75 °C for 24 h under N₂. The reaction mixture was

diluted with saturated NaHCO₃solution (20 mL), and the precipitate

was ﬁltered. The ﬁltrate was then extracted by EtOAc (2 × 30 mL).

The combined organic layers were dried with anhydrous Na₂SO₄and

concentrated in vacuo. The residue was then puriﬁed by column

chromatography (silica gel, 20% methanol/EtOAc as the eluent) to

give 13 as a white solid (61 mg, 84%). ¹H NMR (400 MHz,

methanol-d₄): δ 9.29 (dt, J = 1.9, 0.9 Hz, 1H), 9.08 (ddt, J = 4.2, 2.8,

1.4 Hz, 1H), 7.91−7.77 (m, 2H), 7.46−7.33 (m, 4H), 4.62 (s, 2H),

4.18−4.03 (m, 1H), 3.90−3.65 (m, 1H), 3.43 (s, 1H), 3.39−3.34 (m,

1H), 3.22 (d, J = 8.8 Hz, 1H), 2.25 (d, J = 10.6 Hz, 1H), 2.07−1.95

(m, 1H), 1.85 (dd, J = 14.8, 8.4 Hz, 1H), 0.95 (dd, J = 6.7, 0.8 Hz,

3H), 0.85 (dd, J = 6.7, 2.8 Hz, 3H). ESI-HRMS (m/z): calcd for

C₂₁H₂₅N₅O (M + H)⁺, 364.2132; found, 364.2128.

N-(4-((2-(tert-Butyl)azetidin-1-yl)methyl)benzyl)-[1,2,4]triazolo-

[4,3-a]pyridine-6-carboxamide (14). To a solution of 46 (48 mg,

0.165 mmol) in DMF (1 mL) were added 2-tert-butyl azetidine

hydrochloride (50 mg, 0.33 mmol) and Ti(OⁱPr)₄(0.495 mmol, 141

mg). The mixture was stirred at room temperature for 10 min. Then,

NaBH(OAc)₃(0.495 mmol, 105 mg) was added, and the mixture was

heated to 70−75 °C for 24 h under N₂. The reaction mixture was

diluted with saturated NaHCO₃solution (20 mL), and the precipitate

was ﬁltered. The ﬁltrate was then extracted by EtOAc (2 × 30 mL).

The combined organic layers were dried with anhydrous Na₂SO₄and

concentrated in vacuo. The residue was then puriﬁed by column

chromatography (silica gel, 20% methanol/EtOAc as the eluent) to

give 14 as a white solid (50 mg, 80%). ¹H NMR (400 MHz,

methanol-d₄): δ 9.30 (d, J = 0.8 Hz, 1H), 9.07 (t, J = 1.4 Hz, 1H),

7.92−7.79 (m, 2H), 7.40−7.29 (m, 4H), 4.61 (s, 2H), 3.95 (d, J =

12.9 Hz, 1H), 3.47−3.37 (m, 1H), 3.19−2.98 (m, 2H), 2.76 (d, J =

9.0 Hz, 1H), 2.01−1.85 (m, 2H), 0.93 (s, 9H). ESI-HRMS (m/z):

calcd for C₂₂H₂₇N₅O (M + H)⁺, 378.2288; found, 378.2286.

2H). ESI-HRMS (m/z): calcd for C₁₈H₂₀N₅O (M + H)⁺, 322.1662;

found, 322.1656.

Methyl 4-(1-Aminoethyl)benzoate (49). Methyl 4-acetylbenzoate

(500 mg, 2.8 mmol), ammonium acetate (1.29 g, 16.8 mmol), and

sodium cyanoborohydrate (263 mg, 4.2 mmol) were dissolved in 10

mL of methanol, and the solution was stirred at room temperature for

16 h. The reaction mixture was concentrated and acidiﬁed with 2 M

HCl (5 mL) and then extracted with DCM. The aqueous layer was

basiﬁed with solid NaHCO₃and extracted with DCM (2 × 30 mL).

The combined DCM layers were dried over Na₂SO₄and

concentrated. The residue was used without further puriﬁcation.

Methyl 4-(1-((tert-Butoxycarbonyl)amino)ethyl)benzoate (50).

Methyl 4-(1-aminoethyl)benzoate 49 (250 mg, 1.39 mmol) was

dissolved in DCM (5 mL), and Boc anhydride (348 mg, 1.6 mmol),

DIPEA (0.5 mL, 2.7 mmol), and 4-dimethylaminopyridine (DMAP)

(17 mg, 0.139 mmol) were added and stirred for overnight. The

reaction was washed with water and extracted with ethyl acetate (2 ×

20 mL). The combined organic layers were dried over Na₂SO₄,

concentrated, and puriﬁed by silica gel column chromatography (20%

EtOAc/hexane) to yield 50 as a white solid (300 mg, 77%).

tert-Butyl (1-(4-(Hydroxymethyl)phenyl)ethyl)carbamate (51).

Methyl 4-(1-((tert-butoxycarbonyl)amino)ethyl)benzoate 50 (0.3 g,

1.0 mmol) was dissolved in THF (3 mL), and the solution cooled to

below −5 °C in an ice/salt bath. LiAlH₄(2 M in THF, 1 mL) was

added dropwise over 10 min. Upon completion of addition, the

reaction was stirred at 0 °C for 75 min. Water (0.16 mL) was added

dropwise, followed by 2 M aqueous NaOH solution (0.16 mL) and

then, water (0.16 mL). The suspension was stirred for 15 min and

then diluted with EtOAc (15 mL). The mixture was dried over

Na₂SO₄and ﬁltered, and the resulting ﬁltrate was concentrated in

vacuo to aﬀord the title compound, which was used without further

puriﬁcation (215 mg, 80%).

tert-Butyl (1-(4-(Chloromethyl)phenyl)ethyl)carbamate (52). To

a stirred solution of 51 (200 mg, 0.8 mmol) in DCM (5 mL) were

added methanesulfonyl chloride (108 mg, 0.95 mmol) and triethyl-

amine (0.23 mL, 1.6 mmol). The solution was stirred for 16 h at room

temperature and then washed with water and brine. After separation,

the organic phase was dried over Na₂SO₄, ﬁltered, and concentrated.

The residue was puriﬁed by silica gel column chromatography

(EtOAc/hexane 0 to 80%) to yield 52 as a white solid (100 mg, 46%

yield).

tert-Butyl (1-(4-(Azetidin-1-ylmethyl)phenyl)ethyl)carbamate

(53). To a stirred solution of 52 (100 mg, 0.37 mmol) in acetonitrile

(5 mL) were added azetidine hydrochloride (41 mg, 0.44 mmol) and

DIPEA (0.2 mL, 1.1 mmol). The solution was stirred at 80 °C for 16

h. The reaction mixture was diluted with water and extracted with

DCM. The DCM layers were dried over Na₂SO₄and concentrated to

yield the crude product, which was used without further puriﬁcation

(100 mg).

1-(4-(Azetidin-1-ylmethyl)phenyl)ethan-1-amine (54). To a

stirred solution of 53 (100 mg, 0.3 mmol), 4 M HCl in 1,4-dioxane

(0.5 mL, 1.8 mmol) was added and stirred at room temperature for 1

h. The reaction mixture was concentrated to yield 54 as an oﬀ-white

solid. ESI-HRMS (m/z): calcd for C₁₂H₁₉N₂(M + H)⁺, 191.1543;

found, 191.1539.

N-(3-(Azetidin-1-ylmethyl)benzyl)-[1,2,4]triazolo[4,3-a]pyridine-

6-carboxamide (15). To a solution of 47 (22 mg, 0.125 mmol) and

39 (27 mg, 0.14 mmol) in DMF (0.4 mL) were added 2-(1H-

benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuorophosphate

(HBTU) (52 mg, 0.14 mmol) and DIPEA (33 mg, 0.25 mmol).

The resulting solution was then stirred at room temperature

overnight. The reaction mixture then diluted with EtOAc (30 mL)

and then washed with saturated NaHCO₃solution (2 × 5 mL) and

brine (5 mL). The organic layers were dried with anhydrous Na₂SO₄

and concentrated in vacuo. The residue was then puriﬁed by column

chromatography (silica gel, 20% methanol/EtOAc as the eluent) to

yield 15 as a white solid (25 mg, 62%). ¹H NMR (400 MHz,

methanol-d₄): δ 9.30 (d, J = 0.9 Hz, 1H), 9.09 (t, J = 1.4 Hz, 1H),

7.94−7.79 (m, 2H), 7.38−7.30 (m, 3H), 7.24 (d, J = 1.0 Hz, 1H),

4.63 (s, 2H), 3.63 (s, 2H), 3.31 (t, J = 7.2 Hz, 4H), 2.21−2.07 (m,

N-(1-(4-(Azetidin-1-ylmethyl)phenyl)ethyl)-[1,2,4]triazolo[4,3-a]-

pyridine-6-carboxamide (16). To a stirred solution of 39 (50 mg, 0.3

mmol) and 54 (58 mg, 0.3 mmol) in DMF (1 mL) were added

HBTU (136 mg, 0.36 mmol) and DIPEA (0.1 mL, 0.6 mmol). The

solution was stirred at room temperature for 16 h. The reaction

mixture was diluted with water and extracted with DCM. The DCM

layers were dried over Na₂SO₄and concentrated to give the crude

product, which was puriﬁed by ﬂash chromatography to yield

1

compound 16 as an oﬀ-white solid (30 mg, 34%). H NMR (400

MHz, DMSO-d₆): δ 9.37 (s, 1H), 9.13 (s, 1H), 8.99 (d, J = 7.9 Hz,

1H), 7.90−7.72 (m, 2H), 7.32 (d, J = 7.5 Hz, 2H), 7.21 (d, J = 7.8

Hz, 2H), 5.14 (dd, J = 13.9, 7.1 Hz, 1H), 3.47 (s, 2H), 3.08 (t, J = 6.8

Hz, 4H), 1.94 (dd, J = 13.4, 6.9 Hz, 2H), 1.47 (d, J = 7.0 Hz, 3H).

ESI-HRMS (m/z): calcd for C₁₉H₂₂N₅O (M + H)⁺, 336.1819; found,

336.1808.

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N-(4-(Azetidin-1-ylmethyl)benzyl)-[1,2,4]triazolo[4,3-a]pyridine-

7-carboxamide (17). To a solution of 55 (44 mg, 0.25 mmol) and 56

(45 mg, 0.275 mmol) in DMF (1 mL) were added HBTU (104 mg,

0.275 mmol) and DIPEA (65 mg, 0.5 mmol). The resulting solution

was then stirred at room temperature overnight. The reaction mixture

then diluted with EtOAc (30 mL) and then washed with saturated

NaHCO₃solution (2 × 5 mL) and brine (5 mL). The organic layers

were dried with anhydrous Na₂SO₄and concentrated in vacuo. The

residue was then puriﬁed by column chromatography (silica gel, 20%

methanol/EtOAc as the eluent) to yield 17 as a white solid (52 mg,

65%). ¹H NMR (400 MHz, methanol-d₄): δ 9.27 (s, 1H), 8.58 (d, J =

7.2 Hz, 1H), 8.26 (s, 1H), 7.44 (d, J = 7.3 Hz, 1H), 7.37 (d, J = 7.8

Hz, 2H), 7.29 (d, J = 7.1 Hz, 2H), 4.61 (s, 2H), 3.61 (s, 2H), 3.32−

3.26 (m, 4H), 2.12 (p, J = 7.2 Hz, 2H). ESI-HRMS (m/z): calcd for

C₁₈H₂₀N₅O (M + H)⁺, 322.1662; found, 322.1653.

DCM (20 mL), and washed with saturated NaHCO₃solution (2 × 20

mL), 1 M HCl (2 × 20 mL), and brine. The combined DCM layer

was dried over Na₂SO₄and concentrated in vacuo. The residue was

puriﬁed by ﬂash chromatography (0−10% MeOH/DCM) to yield 63

1

as a pale-yellow solid (58 mg, 52%). H NMR (300 MHz, DMSO-

d₆): δ 9.37 (s, 1H), 9.29 (t, J = 5.7 Hz, 1H), 9.15 (t, J = 1.2 Hz, 1H),

7.90−7.66 (m, 2H), 7.43−7.23 (m, 4H), 4.50 (d, J = 5.8 Hz, 2H),

4.01 (s, 2H).

N-(4-(2-Amino-2-iminoethyl)benzyl)-[1,2,4]triazolo[4,3-a]-

pyridine-6-carboxamide (20). To 1.5 mL of absolute EtOH was

dropwise added 1 mL of acetylchloride under N₂at 0 °C. The

solution was stirred at 0 °C for 5 min before a solution of 63 (20 mg,

0.069 mmol) in absolute EtOH (0.5 mL) was added. The reaction

mixture was stirred at room temperature for 36 h. The reaction

mixture was then evaporated to dryness under high vacuum. To the

residue was added a 7 M NH₃solution in methanol (1 mL). The

reaction mixture was then stirred overnight and concentrated in vacuo.

The residue was dissolved in 1 M HCl solution, washed with EtOAc

to remove residual 63, and then evaporated to dryness to yield 20 as

N-(4-(Azetidin-1-ylmethyl)benzyl)-[1,2,4]triazolo[1,5-a]pyridine-

6-carboxamide (18). To a solution of 55 (44 mg, 0.25 mmol) and 57

(45 mg, 0.275 mmol) in DMF (1 mL) were added HBTU (104 mg,

0.275 mmol) and DIPEA (65 mg, 0.5 mmol). The resulting solution

was then stirred at room temperature overnight. The reaction mixture

was then diluted with EtOAc (30 mL) and then washed with

saturated NaHCO₃solution (2 × 5 mL) and brine (5 mL). The

organic layers were dried with anhydrous Na₂SO₄and concentrated in

vacuo. The residue was then puriﬁed by column chromatography

(silica gel, 20% methanol/EtOAc as the eluent) to yield 18 as a white

1

its hydrochloride salt (17 mg, 71%). H NMR (300 MHz, D₂O): δ

9.47 (s, 1H), 9.22 (s, 1H), 8.24 (dt, J = 9.6, 1.8 Hz, 1H), 8.08 (d, J =

9.6 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 1H), 4.65

(s, 2H), 3.89 (s, 2H). ESI-HRMS (m/z): calcd for C₁₆H₁₇N₆O (M +

H)⁺, 309.1458; found, 309.1451.

tert-Butyl (3-(Hydroxymethyl)-4-methylbenzyl)carbamate (65).

2-Methyl-5-cyanobenzoic acid 64 (5 mmol, 0.81 g) was dissolved in

anhydrous THF (15 mL). A solution of LiAlH₄in THF (1.0 M, 20

mL) was added dropwise to the solution under N₂at 0 °C. After

completion of addition, the reaction mixture was heated to reﬂux

overnight. The solution was then cooled to room temperature and

then to 0 °C. Water (5 mL) was added dropwise, followed by 2 M

NaOH solution (5 mL). After stirring for another 10 min, the mixture

was ﬁltered over Celite. To the ﬁltrate was added Boc₂O (5 mmol,

1.09 g), and the solution was stirred at room temperature for 4 h. The

solution was then concentrated in vacuo to yield crude 65 as a yellow

oil (0.75 g, 60%). ¹H NMR (300 MHz, chloroform-d): δ 7.28 (s, 1H),

7.16−7.09 (m, 2H), 4.84 (br s, 1H), 4.68 (d, J = 5.2 Hz, 2H), 4.27 (d,

J = 5.9 Hz, 2H), 2.32 (s, 3H), 1.66 (s, 1H), 1.45 (s, 9H).

1

solid (49 mg, 61%). H NMR (400 MHz, methanol-d₄): δ 9.34 (s,

1H), 8.52 (d, J = 1.4 Hz, 1H), 8.14 (dd, J = 9.3, 1.9 Hz, 1H), 7.86 (d,

J = 9.3 Hz, 1H), 7.38 (d, J = 7.7 Hz, 2H), 7.30 (d, J = 7.3 Hz, 2H),

4.62 (s, 2H), 3.65 (s, 2H), 3.36 (d, J = 4.2 Hz, 4H), 2.14 (p, J = 7.2

Hz, 2H). ESI-HRMS (m/z): calcd for C₁₈H₂₀N₅O (M + H)⁺,

322.1662; found, 322.1655.

N-(4-(Azetidin-1-ylmethyl)benzyl)-2-methylpyrazolo[1,5-a]-

pyrimidine-6-carboxamide (19). To a solution of 55 (44 mg, 0.25

mmol) and 58 (49 mg, 0.275 mmol) in DMF (1 mL) were added

HBTU (104 mg, 0.275 mmol) and DIPEA (65 mg, 0.5 mmol). The

resulting solution was then stirred at room temperature overnight.

The reaction mixture was then diluted with EtOAc (30 mL) and then

washed with saturated NaHCO₃solution (2 × 5 mL) and brine (5

mL). The organic layers were dried with anhydrous Na₂SO₄and

concentrated in vacuo. The residue was then puriﬁed by column

chromatography (silica gel, 20% methanol/EtOAc as the eluent) to

yield 19 as a white solid (49 mg, 59%). ¹H NMR (400 MHz,

methanol-d₄): δ 9.29 (d, J = 2.2 Hz, 1H), 8.88 (d, J = 2.2 Hz, 1H),

7.38 (d, J = 7.9 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 6.59 (s, 1H), 4.61

(s, 2H), 3.62 (s, 2H), 3.29 (d, J = 7.2 Hz, 4H), 2.53 (s, 3H), 2.13 (p, J

= 7.2 Hz, 2H). ESI-HRMS (m/z): calcd for C₁₉H₂₂N₅O (M + H)⁺,

336.1819; found, 336.1812.

tert-Butyl (4-(Bromomethyl)benzyl)carbamate (60). To a sol-

ution of 59 (238 mg, 1 mmol) in DCM (2 mL) was added

triphenylphosphine (316 mg, 1.2 mmol). Then, carbon tetrabromide

(400 mg, 1.2 mmol) was added in portions in an ice−water bath. The

reaction was left in the ice−water bath for another 3 h. Then, the

reaction mixture was ﬁltered and concentrated in vacuo. The residue

was puriﬁed by column chromatography (10:1 hexanes/EtOAc) to

yield 60 as a white solid (252 mg, 83%).

tert-Butyl (3-(Bromomethyl)-4-methylbenzyl)carbamate (66).

To a solution of 65 (1.2 mmol, 300 mg) in DCM (10 mL) was

added triphenylphosphine (1.44 mmol, 380 mg). Then, carbon

tetrabromide (1.44 mmol, 480 mg) was added in portions at 0 °C.

The reaction was stirred at 0 °C for 3 h. Then, the reaction mixture

was ﬁltered and concentrated in vacuo. The residue was puriﬁed by

column chromatography (hexanes/EtOAc = 10:1) to yield 66 as a

1

white solid (284 mg, 76%). H NMR (300 MHz, chloroform-d): δ

7.23 (s, 1H), 7.19−7.13 (m, 2H), 4.81 (br s, 1H), 4.59 (s, 2H), 4.27

(d, J = 5.9 Hz, 2H), 2.40 (s, 3H), 1.46 (s, 9H).

tert-Butyl (3-(Cyanomethyl)-4-methylbenzyl)carbamate (67). To

a solution of 66 (156 mg, 0.5 mmol) in 5 mL of DMF was added

NaCN (50 mg, 1 mmol). The reaction mixture was then stirred at 60

°C for 4 h. The reaction mixture was cooled and diluted with water

and extracted with DCM. The combined DCM layers were dried over

Na₂SO₄and then concentrated to yield 67 as a white solid, which was

used without further puriﬁcation (78 mg, 60%).

tert-Butyl (4-(Cyanomethyl)benzyl)carbamate (61). To a solution

of 60 (150 mg, 0.5 mmol) in 2 mL of DMF was added NaCN (50 mg,

1 mmol). The reaction mixture was then stirred at 60 °C for 4 h. The

reaction mixture was cooled and diluted with water and extracted with

DCM. Combined DCM layers were dried over Na₂SO₄and then

concentrated to yield 61 as a white solid, which was used without

further puriﬁcation (96 mg, 78%).

N-(4-(Cyanomethyl)benzyl)-[1,2,4]triazolo[4,3-a]pyridine-6-car-

boxamide (63). To a solution of 61 (96 mg, 0.39 mmol) in 1 mL of

1,4-dioxane was added 4 M HCl solution in dioxane (4 mL). The

reaction mixture was stirred at room temperature for 1 h and then

concentrated to dryness in vacuo. The residue was dissolved in 2 mL

of DMF, to which were added 39 (64 mg, 0.39 mmol), DIPEA (155

mg, 1.2 mmol), and HBTU (175 mg, 0.46 mmol). The reaction

mixture was stirred at room temperature overnight, then diluted with

N-(3-(Cyanomethyl)-4-methylbenzyl)-[1,2,4]triazolo[4,3-a]-

pyridine-6-carboxamide (69). To a solution of 67 (78 mg, 0.3

mmol) in 1 mL of 1,4-dioxane was added 4 M HCl solution in

dioxane (4 mL). The reaction mixture was stirred at room

temperature for 1 h and then concentrated to dryness in vacuo. The

residue was dissolved in 2 mL of DMF, to which was added 39 (49

mg, 0.3 mmol), DIPEA (116 mg, 0.9 mmol), and HBTU (137 mg,

0.36 mmol). The reaction mixture was stirred at room temperature

overnight, then diluted with DCM (20 mL), and washed with

saturated NaHCO₃solution (2 × 20 mL), 1 M HCl (2 × 20 mL) and

brine. The combined DCM layer was dried over Na₂SO₄and

concentrated in vacuo. The residue was puriﬁed by ﬂash

chromatography (0−10% MeOH/DCM) to yield 69 as a white

1

solid (46 mg, 50%). H NMR (300 MHz, DMSO-d₆): δ 9.37 (t, J =

0.7 Hz, 1H), 9.24 (t, J = 5.9 Hz, 1H), 9.14 (s, 1H), 7.88−7.76 (m,

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2H), 7.32 (s, 1H), 7.25−7.18 (m, 2H), 4.47 (d, J = 5.9 Hz, 2H), 3.98

(s, 2H), 2.27 (s, 3H).

residue was added a 7 M NH₃solution in methanol (1 mL). The

reaction mixture was then stirred overnight and concentrated in vacuo.

The residue was dissolved in 1 M HCl solution and washed with

EtOAc to remove residual 75 and then evaporated to dryness to yield

22 as its hydrochloride salt (10 mg, 47%). ¹H NMR (300 MHz,

D₂O): δ 9.46 (s, 1H), 9.22 (s, 1H), 8.22 (dd, J = 9.6, 1.5 Hz, 1H),

8.06 (d, J = 9.6 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.33−7.14 (m, 2H),

4.63 (s, 2H), 3.93 (s, 2H). ESI-HRMS (m/z): calcd for C₁₆H₁₆FN₆O

(M + H)⁺, 327.1364; found, 327.1352.

tert-Butyl (4-Fluoro-3-(hydroxymethyl)benzyl)carbamate (77). 2-

Fluoro-5-cyanobenzoic acid 76 (5 mmol, 0.83 g) was dissolved in

anhydrous THF (15 mL). A solution of LiAlH₄in THF (1.0 M, 20

mL) was added dropwise to the solution under N₂at 0 °C. After

completion of addition, the reaction mixture was heated to reﬂux

overnight. The solution was then cooled to room temperature and

then to 0 °C. Water (5 mL) was added dropwise, followed by addition

of 2 M NaOH solution (5 mL). After stirring for another 10 min, the

mixture was ﬁltered over Celite. To the ﬁltrate was added Boc₂O (5

mmol, 1.09 g), and the solution was stirred at room temperature for 4

h. The solution was then concentrated in vacuo to yield crude 77 as a

yellow oil (0.81 g, 59%).

N-(3-(2-Amino-2-iminoethyl)-4-methylbenzyl)-[1,2,4]triazolo-

[4,3-a]pyridine-6-carboxamide (21). To 1.5 mL of absolute EtOH

was dropwise added 1 mL of acetylchloride under N₂at 0 °C. The

solution was stirred at 0 °C for 5 min before a solution of 69 (20 mg,

0.066 mmol) in absolute EtOH (0.5 mL) was added. The reaction

mixture was stirred at room temperature for 36 h. The reaction

mixture was then evaporated to dryness under high vacuum. To the

residue was added a 7 M NH₃solution in methanol (1 mL). The

reaction mixture was then stirred overnight and concentrated in vacuo.

The residue was dissolved in 1 M HCl solution and washed with

EtOAc to remove residual 69, then evaporated to dryness to yield 21

as its hydrochloride salt (14 mg, 60%). ¹H NMR (300 MHz, D₂O): δ

9.46 (s, 1H), 9.22 (s, 1H), 8.33 (d, J = 9.3 Hz, 1H), 8.08 (d, J = 9.2

Hz, 1H), 7.31−7.13 (m, 3H), 4.50 (s, 2H), 3.83 (s, 2H), 2.14 (s, 3H).

ESI-HRMS (m/z): calcd for C₁₇H₁₉N₆O (M + H)⁺, 323.1615; found,

323.1611.

tert-Butyl (3-Fluoro-4-(hydroxymethyl)benzyl)carbamate (71). 2-

Fluoro-4-cyanobenzoic acid 70 (5 mmol, 0.83 g) was dissolved in

anhydrous THF (15 mL). A solution of LiAlH₄in THF (1.0 M, 20

mL) was added dropwise to the above solution under N₂at 0 °C.

After completion of addition, the reaction mixture was heated to

reﬂux overnight. The solution was then cooled to room temperature

and then to 0 °C. Water (5 mL) was added dropwise, followed by 2

M NaOH solution (5 mL). After stirring for another 10 min, the

mixture was ﬁltered over Celite. To the ﬁltrate was added Boc₂O (5

mmol, 1.09 g) and the solution was stirred at room temperature for 4

h. The solution was then concentrated in vacuo to yield crude 71 as a

yellow oil (0.71 g, 55%).

tert-Butyl (4-(Bromomethyl)-3-ﬂuorobenzyl)carbamate (72). To

a solution of 71 (1.2 mmol, 306 mg) in DCM (10 mL) was added

triphenylphosphine (1.44 mmol, 380 mg). Then, carbon tetrabromide

(1.44 mmol, 480 mg) was added in portions at 0 °C. The reaction was

stirred at 0 °C for 3 h. Then, the reaction mixture was ﬁltered and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (hexanes/EtOAc = 10:1) to yield 72 as a white

solid (250 mg, 66%). ¹H NMR (300 MHz, chloroform-d): δ 7.34 (t, J

= 7.8 Hz, 1H), 7.13−6.95 (m, 2H), 4.88 (br s, 1H), 4.50 (s, 2H), 4.30

(d, J = 6.2 Hz, 2H), 1.46 (s, 9H).

tert-Butyl (4-(Cyanomethyl)-3-ﬂuorobenzyl)carbamate (73). To

a solution of 72 (159 mg, 0.5 mmol) in 5 mL of DMF was added

NaCN (50 mg, 1 mmol). The reaction mixture was then stirred under

60 °C for 4 h. The reaction mixture was cooled and diluted with water

and extracted with DCM. The combined DCM layers were dried over

Na₂SO₄and then concentrated to yield 73 as a white solid, which was

used without further puriﬁcation (85 mg, 64%).

tert-Butyl (3-(Bromomethyl)-4-ﬂuorobenzyl)carbamate (78). To

a solution of 77 (1.2 mmol, 306 mg) in DCM (10 mL) was added

triphenylphosphine (1.44 mmol, 380 mg). Then, carbon tetrabromide

(1.44 mmol, 480 mg) was added in portions at 0 °C. The reaction was

stirred at 0 °C for 3 h. Then, the reaction mixture was ﬁltered and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (hexanes/EtOAc = 10:1) to yield 78 as a white

1

solid (234 mg, 62%). H NMR (300 MHz, chloroform-d): δ 7.34−

7.27 (m, 1H), 7.25−7.18 (m, 1H), 7.05−6.96 (m, 1H), 4.87 (br s,

1H), 4.49 (s, 2H), 4.29 (d, J = 6.2 Hz, 2H), 1.46 (s, 9H).

tert-Butyl (3-(Cyanomethyl)-4-ﬂuorobenzyl)carbamate (79). To

a solution of 78 (159 mg, 0.5 mmol) in 5 mL of DMF was added

NaCN (50 mg, 1 mmol). The reaction mixture was then stirred under

60 °C for 4 h. The reaction mixture was cooled and diluted with water

and extracted with DCM. The combined DCM layers were dried over

Na₂SO₄and then concentrated to yield 79 as a white solid, which was

used without further puriﬁcation (79 mg, 60%).

N-(3-(Cyanomethyl)-4-ﬂuorobenzyl)-[1,2,4]triazolo[4,3-a]-

pyridine-6-carboxamide (81). To a solution of 79 (79 mg, 0.3

mmol) in 1 mL of 1,4-dioxane was added 4 M HCl solution in

dioxane (4 mL). The reaction mixture was stirred at room

temperature for 1 h and then concentrated to dryness in vacuo. The

residue was dissolved in 2 mL of DMF, to which were added 39 (49

mg, 0.3 mmol), DIPEA (116 mg, 0.9 mmol), and HBTU (137 mg,

0.36 mmol). The reaction mixture was stirred at room temperature

overnight, then diluted with DCM (20 mL), and washed with

saturated NaHCO₃solution (2 × 20 mL), 1 M HCl (2 × 20 mL), and

brine. The combined DCM layer was dried over Na₂SO₄and

concentrated in vacuo. The residue was puriﬁed by ﬂash

chromatography (0−10% MeOH/DCM) to yield 81 as a white

solid (57 mg, 62%). ¹H NMR (300 MHz, DMSO-d₆): δ 9.37 (s, 1H),

9.29 (t, J = 5.6 Hz, 1H), 9.14 (s, 1H), 7.85 (d, J = 9.6 Hz, 1H), 7.78

(dd, J = 9.7, 1.5 Hz, 1H), 7.48−7.20 (m, 3H), 4.50 (d, J = 6.1 Hz,

2H), 4.05 (s, 2H).

N-(4-(Cyanomethyl)-3-ﬂuorobenzyl)-[1,2,4]triazolo[4,3-a]-

pyridine-6-carboxamide (75). To a solution of 73 (79 mg, 0.3

mmol) in 1 mL of 1,4-dioxane was added 4 M HCl solution in

dioxane (4 mL). The reaction mixture was stirred at room

temperature for 1 h and then concentrated to dryness in vacuo. The

residue was dissolved in 2 mL of DMF, to which were added 39 (49

mg, 0.3 mmol), DIPEA (116 mg, 0.9 mmol), and HBTU (137 mg,

0.36 mmol). The reaction mixture was stirred at room temperature

overnight, then diluted with DCM (20 mL), and washed with

saturated NaHCO₃solution (2 × 20 mL), 1 M HCl (2 × 20 mL), and

brine. The combined DCM layer was dried over Na₂SO₄and

concentrated in vacuo. The residue was puriﬁed by ﬂash

chromatography (0−10% MeOH/DCM) to yield 75 as a white

N-(4-(2-Amino-2-iminoethyl)-3-ﬂuorobenzyl)-[1,2,4]triazolo[4,3-

a]pyridine-6-carboxamide (23). To 1.5 mL of absolute EtOH was

dropwise added 1 mL of acetylchloride under N₂at 0 °C. The

solution was stirred at 0 °C for 5 min before a solution of 81 (20 mg,

0.065 mmol) in absolution EtOH (0.5 mL) was added. The reaction

mixture was stirred at room temperature for 36 h. The reaction

mixture was then evaporated to dryness under high vacuum. To the

residue was added a 7 M NH₃solution in methanol (1 mL). The

reaction mixture was then stirred overnight and concentrated in vacuo.

The residue was dissolved in 1 M HCl solution, washed with EtOAc

to remove residual 81, and then evaporated to dryness to yield 23 as

1

solid (51 mg, 55%). H NMR (300 MHz, methanol-d₄): δ 9.29 (s,

1H), 9.09 (s, 1H), 7.83 (t, J = 8.7 Hz, 2H), 7.46 (t, J = 7.8 Hz, 1H),

7.34−7.16 (m, 2H), 4.63 (s, 2H), 3.92 (s, 2H).

N-(4-(2-Amino-2-iminoethyl)-3-ﬂuorobenzyl)-[1,2,4]triazolo[4,3-

a]pyridine-6-carboxamide (22). To 1.5 mL of absolute EtOH was

dropwise added 1 mL of acetylchloride under N₂at 0 °C. The

solution was stirred at 0 °C for 5 min before a solution of 75 (20 mg,

0.065 mmol) in absolute EtOH (0.5 mL) was added. The reaction

mixture was stirred at room temperature for 36 h. The reaction

mixture was then evaporated to dryness under high vacuum. To the

1

its hydrochloride salt (13 mg, 60%). H NMR (300 MHz, D₂O): δ

9.51 (s, 1H), 9.27 (s, 1H), 8.35 (dd, J = 9.6, 1.6 Hz, 1H), 8.13 (d, J =

9.6 Hz, 1H), 7.50−7.34 (m, 2H), 7.27−7.10 (m, 1H), 4.62 (s, 2H),

M

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3.91 (s, 2H). ESI-HRMS (m/z): calcd for C₁₆H₁₆FN₆O (M + H)⁺,

327.1364; found, 327.1359.

anhydrous Na₂SO₄and concentrated. The residue was then puriﬁed

by ﬂash chromatography to provide 27 as a pale-yellow solid (38 mg,

56%). H NMR (400 MHz, DMSO-d₆): δ 9.36 (d, J = 0.8 Hz, 1H),

1

N-(4-(((5-Fluoropyrimidin-2-yl)amino)methyl)benzyl)-[1,2,4]-

triazolo[4,3-a]pyridine-6-carboxamide (24). To a stirred solution of

2-chloro-5-ﬂuoropyrimidine (24 mg, 0.18 mmol) and amine 6 (48

mg. 0.15 mmol) in ethanol (1 mL) was added DIPEA (0.1 mL, 0.57

mmol), and the reaction mixture was heated to 75 °C for 36 h. The

reaction was concentrated and puriﬁed by ﬂash chromatography

(silica gel, 5% methanol/EtOAc as the eluent) to yield 24 as a light-

9.21 (t, J = 5.9 Hz, 1H), 9.13 (t, J = 1.4 Hz, 1H), 7.90 (d, J = 3.1 Hz,

1H), 7.87−7.73 (m, 2H), 7.33 (td, J = 8.8, 3.1 Hz, 1H), 7.28 (s, 4H),

7.04 (t, J = 6.0 Hz, 1H), 6.51 (dd, J = 9.1, 3.6 Hz, 1H), 4.47 (d, J =

5.9 Hz, 2H), 4.40 (d, J = 6.0 Hz, 2H). ¹³C NMR (101 MHz, DMSO):

δ 163.5, 155.7, 153.7, 151.3, 148.4, 139.2, 137.6, 137.4, 133.5, 133.3,

127.3, 127.2, 126.7, 126.5, 125.1, 124.9, 121.1, 114.4, 108.8, 108.7,

44.4, 42.6. ESI-HRMS (m/z): calcd for C₂₀H₁₇FN₆O (M + H)⁺,

377.1521; found, 377.1520.

N-(4-(((3,6-Diﬂuoropyridin-2-yl)amino)methyl)benzyl)-[1,2,4]-

triazolo[4,3-a]pyridine-6-carboxamide (28). To a stirred solution of

2,3,6-triﬂuoropyridine (32 mg, 0.24 mmol) and amine 6 (63 mg, 0.2

mmol) in N-methyl-2-pyrrolidone (0.5 mL) was added DIPEA (0.1

mL, 0.57 mmol), and the reaction mixture was heated to 90 °C

overnight. The reaction mixture was cooled, then diluted with EtOAc

(25 mL), and then washed with brine (2 × 5 mL). The organic layers

were dried with anhydrous Na₂SO₄and concentrated in vacuo. The

residue was then puriﬁed by column chromatography (silica gel, 5%

methanol/EtOAc as the eluent) to yield 28 as a light-yellow solid (30

mg, 38%). ¹H NMR (400 MHz, methanol-d₄): δ 9.27 (d, J = 1.0 Hz,

1H), 9.04 (q, J = 1.4 Hz, 1H), 7.90−7.76 (m, 2H), 7.41−7.25 (m,

5H), 6.02 (dtd, J = 8.2, 3.1, 2.6, 1.3 Hz, 1H), 4.59 (d, J = 1.1 Hz, 2H),

4.57 (s, 2H). ESI-HRMS (m/z): calcd for C₂₀H₁₇F₂N₆O (M + H)⁺,

395.1426; found, 395.1426.

SPR Assay. All SPR experiments were performed on a Biosensing

BI-4500 instrument with 1× PBS with 0.1% DMSO as the running

buﬀer and a ﬂow rate at 60 μL/min. His-ENL YEATS was

immobilized through EDCI/NHS coupling on CM dextran-coated

sensor chips (Biosensing). The sensor chip was activated by ﬂowing

200 μL of 0.05 M NHS plus 0.2 M EDCI solution through the

surface. Then, 200 μL of 6 μM His-ENL YEATS in 10 mM NaOAc/

HOAc and 150 mM NaCl, pH 5.5, was injected and ﬂowed through

the activated surface. Then, 100 μL of 1 M ethanolamine pH 7.8

solution was injected to block the remaining activated ester on the

surface. 11 and 24 were dissolved in the running buﬀer and subjected

to a twofold serial dilution for a total of four concentrations ranging

from 5.6 to 0.7 μM (for 24, 5.4 to 0.675 μM). For each cycle, 350 μL

of the compound solution was injected and ﬂowed through the

surface, followed by a 600 s delay for dissociation. Prior to the ﬁrst

cycle, 350 μL of the running buﬀer was injected for baseline

calibration. A control ﬂow channel was set up in parallel without His-

ENL YEATS immobilization to subtract nonspeciﬁc binding signals.

The data analysis was performed on the kinetic analysis software

provided by Biosensing Instrument Inc. and ﬁtted into the Langmuir

1:1 binding model.

1

yellow solid (22 mg, 39%). H NMR (400 MHz, DMSO-d₆): δ 9.36

(s, 1H), 9.21 (t, J = 5.9 Hz, 1H), 9.12 (t, J = 1.4 Hz, 1H), 8.32 (d, J =

1.0 Hz, 2H), 7.86−7.73 (m, 3H), 7.26 (s, 4H), 4.46 (d, J = 5.9 Hz,

2H), 4.43 (d, J = 6.4 Hz, 2H). ¹³C NMR (101 MHz, DMSO): δ

163.9, 159.9, 153.3, 150.9, 148.8, 146.1, 145.8, 139.4, 138.0, 137.8,

127.8, 127.5, 127.2, 126.9, 121.6, 114.9, 44.7, 43.0. ESI-HRMS (m/z):

calcd for C₁₉H₁₇FN₇O (M + H)⁺, 378.1473; found, 378.1465.

N-(4-(((5-Isopropylpyrimidin-2-yl)amino)methyl)benzyl)-[1,2,4]-

triazolo[4,3-a]pyridine-6-carboxamide (25). To a stirred solution of

amine 6 (50 mg, 0.17 mmol) and 2-chloro-5-isopropylpyrimidine (15

mg, 0.17 mmol) in ethanol (2 mL) was added DIPEA (68 mg, 0.53

mmol) and heated to 80 °C for 48 h. The reaction mixture was

concentrated and puriﬁed by ﬂash chromatography (0−10%

methanol/DCM) to yield 25 as an oﬀ-white solid (10 mg, 18%).

¹H NMR (400 MHz, DMSO-d₆): δ 9.35 (d, J = 0.8 Hz, 1H), 9.21 (t, J

= 5.8 Hz, 1H), 9.13 (t, J = 1.4 Hz, 1H), 8.16 (s, 2H), 7.86−7.73 (m,

2H), 7.49 (t, J = 6.4 Hz, 1H), 7.26 (s, 4H), 4.49−4.40 (m, 4H), 2.71

(hept, J = 6.9 Hz, 1H), 1.15 (d, J = 6.9 Hz, 7H). ¹³C NMR (101

MHz, DMSO-d₆): δ 163.9, 161.7, 156.5, 148.8, 139.9, 138.0, 137.7,

129.2, 127.7, 127.5, 127.2, 127.0, 121.6, 114.9, 44.3, 43.0, 28.7, 24.0.

ESI-HRMS (m/z): calcd for C₂₂H₂₃N₇ONa (M + Na)⁺, 424.1856;

found, 424.1849.

N-(4-(((4,5-Dihydro-1H-imidazol-2-yl)amino)methyl)benzyl)-

[1,2,4]triazolo[4,3-a]pyridine-6-carboxamide (26). To a stirred

solution of amine 6 (95 mg, 0.3 mmol) and 82 (70 mg, 0.6 mmol)

in DMF (2 mL) was added triethylamine (91 mg, 0.9 mmol) and

heated to 100 °C for 16 h. The reaction mixture was concentrated

under reduced pressure. Water was added to the crude product, and

the solid was ﬁltered and concentrated under reduced pressure to

provide the crude compound 26 (20 mg, 19%). The crude compound

was puriﬁed by RP-HPLC (HPLC gradient: 0−70 min: 95% A to 50%

1

A). H NMR (300 MHz, DMSO-d₆): δ 9.38 (s, 1H), 9.28 (s, 1H),

9.14 (s, 1H), 8.67 (s, 1H), 7.82 (q, J = 9.7 Hz, 2H), 7.46−7.18 (m,

4H), 4.49 (d, J = 5.9 Hz, 2H), 4.35 (d, J = 5.9 Hz, 2H), 3.60 (s, 4H).

ESI-HRMS (m/z): calcd for C₁₈H₂₀N₇O (M + H)⁺, 350.1724; found,

350.1724.

tert-Butyl (4-(((5-Fluoropyridin-2-yl)amino)methyl)benzyl)-

carbamate (84). To a stirred solution of 83 (1 mmol, 300 mg) in

5 mL of DMF were added 60 (1 mmol, 112 mg) and K₂CO₃(1

mmol, 138 mg). The reaction mixture was stirred at room

temperature overnight. The reaction mixture was diluted with water

(25 mL) and then extracted with DCM (2 × 20 mL). The organic

layer was dried over anhydrous Na₂SO₄and concentrated. The

residue was then puriﬁed by ﬂash chromatography to yield 84 as a

white solid (100 mg, 30%). ¹H NMR (400 MHz, CDCl₃): δ 7.96 (d, J

= 3.0 Hz, 1H), 7.31 (d, J = 7.9 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H), 7.19

(ddd, J = 9.0, 7.9, 3.0 Hz, 1H), 6.32 (dd, J = 9.1, 3.4 Hz, 1H), 4.84 (s,

2H), 4.46 (d, J = 5.6 Hz, 2H), 4.30 (d, J = 6.0 Hz, 2H), 1.46 (s, 9H).

N-(4-(Aminomethyl)benzyl)-5-ﬂuoropyridin-2-amine Dihydro-

chloride (85). To a stirred solution of 84 (0.3 mmol, 100 mg) in

1,4-dioxane (1 mL) was added a 4 M HCl solution in dioxane (4

mL). The solution was stirred at room temperature for 1 h. The

reaction mixture was then concentrated in vacuo to yield 85 as a white

solid (85 mg, 95%), which was used without further puriﬁcation.

N-(4-(((5-Fluoropyridin-2-yl)amino)methyl)benzyl)-[1,2,4]-

triazolo[4,3-a]pyridine-6-carboxamide (27). To a stirred solution of

85 (54 mg, 0.18 mmol) and 39 (32 mg, 0.20 mmol) in DMF (2 mL)

were added DIPEA (0.15 mL, 0.8 mmol) and HBTU (80 mg, 0.22

mmol). The solution was then stirred at room temperature overnight.

The reaction mixture was then diluted with water (20 mL) and

extracted with EtOAc (2 × 20 mL). The organic layer was dried over

NMR Experiments for the ENL YEATS Domain. For NMR

experiments, the YEATS domain of ENL (aa 1−148) was expressed

as a C-terminal, uncleavable 6× His fusion protein (plasmid was a

generous gift from Oleg Fedorov). The ¹⁵N-labeled protein was

expressed in E. coli Rosetta-2 (DE3) pLysS cells grown in NH₄Cl

(Sigma-Aldrich) minimal media. After induction with IPTG (ﬁnal

concentration 0.5 mM) (Gold Biotechnology) for 18 h at 16 °C, the

cells were harvested via centrifugation and lysed by sonication. The

uniformly ¹⁵N-labeled YEATS domain was incubated with Ni−NTA

resins (Thermo Fisher Scientiﬁc), washed and eluted with imidazole.

The protein was further puriﬁed by size exclusion chromatography,

concentrated (Millipore) and stored at −80 °C.

The NMR experiments were performed at 298 K on a Varian

1

INOVA 600 MHz spectrometer. The H,¹⁵N HSQC spectra of a 0.2

mM uniformly ¹⁵N-labeled YEATS domain (25−50 mM Tris-HCl pH

7.5 buﬀer, supplemented with 150 mM NaCl, 0.2 mM tris(2-

carboxyethyl)phosphine and 10% D₂O) were collected in the

presence of an increasing amount of the H3K27cr (aa 22−31)

peptide (synthesized by Synpeptide) or compound 7. NMR data were

processed and analyzed with NMRPipe and NMRDraw, as previously

described.³³

Competitive Peptide Pull-Down Assay. Compounds at

indicated concentrations were mixed with 2 μg of GST-fused

N

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YEATS proteins in 300 μL of the binding buﬀer (50 mM Tris-HCl

pH 7.5, 250 mM NaCl, 0.1% NP-40, 1 mM phenylmethylsulfonyl

ﬂuoride) and rotated at 4 °C for 1 h. Then, 0.5 μg of biotinylated

histone peptides with diﬀerent modiﬁcations were added and

incubated for 4 h. Streptavidin magnetic beads (Amersham) were

added to the mixture, and the mixture was incubated for 1 h with

rotation. The beads were then washed 3 times and analyzed using

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) and western blotting.

IC₅₀Determination in the Inhibition of YEATS Domains. To

assess the speciﬁcity of 7, 11, and 24, their IC₅₀values in inhibition of

the four human YEATS domain proteins binding to targeted histone

peptides were determined in AlphaScreen assays. The compounds

were subjected to 12 threefold serial dilutions for a total of 13

concentrations ranging from 54 μM to 0.1 nM for the dose response

curve. IC₅₀values were determined from the plot using nonlinear

regression of variable slopes (four parameters) and curve ﬁtting

performed by the GraphPad Prism software.

Cell Growth Inhibition Assay. Human leukemia MV4-11 and

MOLM13 cells were maintained in RPMI-1640 (Cellgro) supple-

mented with 10% fetal bovine serum (Sigma). Human U2OS cells

were maintained in Dulbecco’s modiﬁed Eagle medium (DMEM,

Cellgro) supplemented with 10% fetal bovine serum. A total of 5000

cells were seeded in a 96-well plate in 100 μL of the medium, treated

with DMSO or compounds at indicated concentrations for 3 days.

Cell viability was measured using the CellTiter-Glo luminescent cell

viability assay kit (Promega) according to the manufacturer’s

instructions. Surviviving cells were calculated as % relative to

DMSO-treated cells.

RNA Extraction and qRT-PCR. Total RNA was extracted using

the RNeasy plus kit (Qiagen) and reverse-transcribed using an iScript

cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR (qRT-

PCR) analyses were performed as described previously using

PowerUp SYBR Green PCR Master Mix and the Bio-Rad CFX96

real-time PCR detection system. Gene expressions were calculated

following normalization to glyceraldehyde 3-phosphate dehydrogen-

ase (GAPDH) levels using the comparative C_t(cycle threshold)

method.

normalized to no compound controls. GraphPad Prism 9 was used

for regression analysis for IC₅₀determination.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00367.

■

sı

Overview of screen assay development, IC₅₀of top

compounds, SPR and NMR data, cell growth inhibition,

cell permeability assay, CETSA result of 7 in HeLa,

qRT-PCR data, synergistic eﬀect of 7 and JQ1 in MV4-

11, molecular docking models of compounds bound to

AF9 and ENL YEATS domains, HPLC traces for lead

compounds, and table of the structure and IC₅₀of

compounds from HTS with IC₅₀below 5 μM (PDF)

Molecular formula strings (CSV)

Docking models (ZIP)

AUTHOR INFORMATION

Corresponding Authors

■

Wenshe Ray Liu − Texas A&M Drug Discovery Laboratory,

Department of Chemistry, Department of Biochemistry and

Biophysics, and Department of Molecular and Cellular

Medicine, College of Medicine, Texas A&M University,

College Station, Texas 77843, United States; Institute of

Biosciences and Technology and Department of Translational

Medical Sciences, College of Medicine, Texas A&M

University, Houston, Texas 77030, United States;

orcid.org/0000-0002-7078-6534; Email: wliu@

chem.tamu.edu

Hong Wen − Department of Epigenetics, Van Andel Institute,

Grand Rapids, Michigan 49503, United States; orcid.org/

0000-0001-8739-4572; Email: Hong.Wen@vai.org

Cellular Thermal Shift Assay.³⁴MOLM13, MV4-11, and HeLa

cells were incubated with 20 μM of compound 7 for 5 h. The cells

were then collected and washed with PBS 3 times. The cell pellets

were resuspended in PBS-containing protease inhibitors and aliquoted

into PCR microtubes (approximately 3 million cells in 54 μL). Cells

were heated at indicated temperatures for 3 min in a thermal cycler

(Bio-Rad) and then incubated at room temperature for 2 min. A total

of 6 μL of 10× cell lysis buﬀer (8% NP-40, 50% glycerol, and 10 mM

dithiothreitol) was added to each sample before subjecting to three

freeze−thaw cycles by liquid nitrogen and 37 °C water bath

incubations to lyse the cells. The cell lysates were centrifuged at

13,000 rpm at 4 °C for 10 min, and the supernatants were analyzed

using SDS-PAGE and western blotting.

Caco-2 Cell Permeability Assay. The cell permeability assay was

performed at Charles River Labs to measure the rate of ﬂux of a

compound across polarized Caco-2 cell monolayers. Ranitidine,

talinolol, and warfarin were used as controls. All compounds were

tested at 10 μM with 2 h incubation in triplicate. P_app(apparent

permeability) in apical to basolateral (A−B) direction was calculated

and used to predict the in vivo absorption of a compound. P_app> 1 ×

10⁻⁶cm/s is considered as higher permeability.

Authors

Xinyu R. Ma − Texas A&M Drug Discovery Laboratory,

Department of Chemistry, Texas A&M University, College

Station, Texas 77843, United States

Longxia Xu − Department of Epigenetics, Van Andel Institute,

Grand Rapids, Michigan 49503, United States

Shiqing Xu − Texas A&M Drug Discovery Laboratory,

Department of Chemistry, Texas A&M University, College

Station, Texas 77843, United States

Brianna J. Klein − Department of Pharmacology, University of

Colorado School of Medicine, Aurora, Colorado 80045,

United States

Hongkuan Wang − Department of Epigenetics, Van Andel

Institute, Grand Rapids, Michigan 49503, United States

Sukant Das − Texas A&M Drug Discovery Laboratory,

Department of Chemistry, Texas A&M University, College

Station, Texas 77843, United States

Kuai Li − Department of Epigenetics, Van Andel Institute,

Grand Rapids, Michigan 49503, United States

Kai S. Yang − Texas A&M Drug Discovery Laboratory,

Department of Chemistry, Texas A&M University, College

Station, Texas 77843, United States; orcid.org/0000-

0002-1890-4169

Sana Sohail − Department of Epigenetics, Van Andel Institute,

Grand Rapids, Michigan 49503, United States

Andrew Chapman − Texas A&M Drug Discovery Laboratory,

Department of Chemistry, Texas A&M University, College

Station, Texas 77843, United States

Combinatorial Treatment of Compound 7 and JQ1.

MOLM13 or MV4-11 cells were treated with DMSO, compound 7,

JQ1, or a combination of compounds 7 and JQ1 at indicated

concentrations for 6 days. Cell viability was measured using the

CellTiter-Glo luminescent cell viability assay kit (Promega).

Surviviving cells were calculated as % relative to DMSO-treated

cells. Synergistic interactions were analyzed and visualized using the

Combeneﬁt software.³⁵

Quantiﬁcation and Statistical Analysis. All AlphaScreen assays

were performed in 4−6 replicates. Raw signal readings were

O

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Tatiana G. Kutateladze − Department of Pharmacology,

University of Colorado School of Medicine, Aurora, Colorado

80045, United States; orcid.org/0000-0001-7375-6990

Xiaobing Shi − Department of Epigenetics, Van Andel

Institute, Grand Rapids, Michigan 49503, United States

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00367

Author Contributions

^△X.R.M. and L.X. contributed equally. Conceptualization,

X.S., W.R.L., and H.W.; methodology, T.G.K., W.R.L., and

H.W.; investigation, X.R.M., L.X., S.X., B.J.K., H.W., S.D., K.L.,

K.S.Y., S.S., and A.C.; writingoriginal draft, X.R.M., L.X.,

B.J.K., W.R.L., and H.W.; writingreview & editing, S.X.,

T.G.K., and X.S.; funding acquisition, X.S., T.G.K., W.R.L., and

H.W.; supervision, T.G.K., X.S., W.R.L., and H.W.

(8) Hsu, C.-C.; Zhao, D.; Shi, J.; Peng, D.; Guan, H.; Li, Y.; Huang,

Y.; Wen, H.; Li, W.; Li, H.; Shi, X. Gas41 links histone acetylation to

H2A.Z deposition and maintenance of embryonic stem cell identity.

Cell Discovery 2018, 4, 28.

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̂

Bourriquen, G.; Bridgers, J. B.; Zhang, J.; Strahl, B. D.; Coté, J.;

Kutateladze, T. G. Yaf9 subunit of the NuA4 and SWR1 complexes

targets histone H3K27ac through its YEATS domain. Nucleic Acids

Res. 2018, 46, 421−430.

Notes

The authors declare no competing ﬁnancial interest.

(10) Li, Y.; Sabari, B. R.; Panchenko, T.; Wen, H.; Zhao, D.; Guan,

H.; Wan, L.; Huang, H.; Tang, Z.; Zhao, Y.; Roeder, R. G.; Shi, X.;

Allis, C. D.; Li, H. Molecular coupling of histone crotonylation and

active transcription by AF9 YEATS domain. Mol. Cell 2016, 62, 181−

193.

ACKNOWLEDGMENTS

■

This work was supported by funds from NIH grants

(GM135671 and CA252707) to T.G.K., the Cancer

Prevention and Research Institute of Texas (RP160237) to

X.S., Welch Foundation (A-1715), the Texas A&M X Grant

Program, and the Texas A&M College of Science Strategic

Transformative Research Program to W.R.L., and NIH grant

(CA255506) and the Thomas and Garretta Newhof/Prein &

Newhorf Research Fund to H.W.

(11) Li, Y.; Wen, H.; Xi, Y.; Tanaka, K.; Wang, H.; Peng, D.; Ren, Y.;

Jin, Q.; Dent, S. Y. R.; Li, W.; Li, H.; Shi, X. AF9 YEATS domain links

histone acetylation to DOT1L-mediated H3K79 methylation. Cell

2014, 159, 558−571.

(12) Mi, W.; Guan, H.; Lyu, J.; Zhao, D.; Xi, Y.; Jiang, S.; Andrews,

F. H.; Wang, X.; Gagea, M.; Wen, H.; Tora, L.; Dent, S. Y. R.;

Kutateladze, T. G.; Li, W.; Li, H.; Shi, X. YEATS2 links histone

acetylation to tumorigenesis of non-small cell lung cancer. Nat.

Commun. 2017, 8, 1088.

(13) Shanle, E. K.; Andrews, F. H.; Meriesh, H.; McDaniel, S. L.;

Dronamraju, R.; DiFiore, J. V.; Jha, D.; Wozniak, G. G.; Bridgers, J.

B.; Kerschner, J. L.; Krajewski, K.; Martín, G. M.; Morrison, A. J.;

Kutateladze, T. G.; Strahl, B. D. Association of Taf14 with acetylated

histone H3 directs gene transcription and the DNA damage response.

Genes Dev. 2015, 29, 1795−1800.

(14) Wan, L.; Wen, H.; Li, Y.; Lyu, J.; Xi, Y.; Hoshii, T.; Joseph, J.

K.; Wang, X.; Loh, Y.-H. E.; Erb, M. A.; Souza, A. L.; Bradner, J. E.;

Shen, L.; Li, W.; Li, H.; Allis, C. D.; Armstrong, S. A.; Shi, X. ENL

links histone acetylation to oncogenic gene expression in acute

myeloid leukaemia. Nature 2017, 543, 265−269.

(15) Zhang, Q.; Zeng, L.; Zhao, C.; Ju, Y.; Konuma, T.; Zhou, M.-

M. Structural insights into histone crotonyl-lysine recognition by the

AF9 YEATS domain. Structure 2016, 24, 1606−1612.

(16) Zhao, D.; Guan, H.; Zhao, S.; Mi, W.; Wen, H.; Li, Y.; Zhao, Y.;

Allis, C. D.; Shi, X.; Li, H. YEATS2 is a selective histone crotonylation

reader. Cell Res. 2016, 26, 629−632.

(17) Le Masson, I.; Yu, D. Y.; Jensen, K.; Chevalier, A.;

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novel NuA4 histone acetyltransferase subunit, is required for the

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ABBREVIATIONS

■

AF9, ALL1-fused gene from chromosome 9 protein; BET,

bromodomain and extraterminal domain; CETSA, cellular

thermal shift assay; CSP, chemical shift perturbations; DOT1L,

disruptor of telomeric silencing 1-like; ENL, eleven-nineteen-

leukemia protein; GAS41, glioma-ampliﬁed sequence 41; GI₅₀,

half growth inhibition concentration; HTS, high-throughput

screening; MLL, mixed-lineage leukemia; PTM, post-transla-

tional modiﬁcation; SEC, super elongation complex; SEM,

standard errors of the mean; SPR, surface plasmon resonance;

YEATS domain, Yaf9, ENL, AF9, Taf14, and Sas5 domain;

YEATS2, YEATS domain-containing protein 2

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Discovery of Selective Small-Molecule Inhibitors for the ENL YEATS Domain

DOI: 10.1021/acs.jmedchem.1c00367

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