Discovery of Novel Dot1L Inhibitors through a Structure-Based Fragmentation Approach

Article abstract of DOI:10.1021/acsmedchemlett.6b00167

Oncogenic MLL fusion proteins aberrantly recruit Dot1L, a histone methyltransferase, to ectopic loci, leading to local hypermethylation of H3K79 and misexpression of HoxA genes driving MLL-rearranged leukemias. Inhibition of the methyltransferase activity of Dot1L in this setting is predicted to reverse aberrant H3K79 methylation, leading to repression of leukemogenic genes and tumor growth inhibition. In the context of our Dot1L drug discovery program, high-throughput screening led to the identification of 2, a weak Dot1L inhibitor with an unprecedented, induced pocket binding mode. A medicinal chemistry campaign, strongly guided by structure-based consideration and ligand-based morphing, enabled the discovery of 12 and 13, potent, selective, and structurally completely novel Dot1L inhibitors.

Full text of DOI:10.1021/acsmedchemlett.6b00167

Subscriber access provided by UNIVERSITY OF LEEDS
Letter
The Discovery of Novel Dot1L Inhibitors through
a Structure-Based Fragmentation Approach
Chao Chen, Hugh Zhu, Frederic Stauffer, Giorgio Caravatti, Susanne Vollmer, Rainer
Machauer, Philipp Holzer, Henrik Moebitz, Clemens Scheufler, Martin Klumpp, Ralph Tiedt,
Kim S Beyer, Keith Calkins, Daniel Guthy, Michael Kiffe, Jeff Zhang, and Christoph Gaul
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00167 • Publication Date (Web): 01 Jun 2016
Downloaded from http://pubs.acs.org on June 5, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Medicinal Chemistry Letters is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
The Discovery of Novel Dot1L Inhibitors through a Structure-Based
Fragmentation Approach
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chao Chen,† Hugh Zhu,† Frédéric Stauffer,‡ Giorgio Caravatti,‡ Susanne Vollmer,‡ Rainer Ma-
chauer,‡ Philipp Holzer,‡ Henrik Möbitz,‡ Clemens Scheufler,‡ Martin Klumpp,‡ Ralph Tiedt,‡
Kim S. Beyer,‡ Keith Calkins,‡ Daniel Guthy,‡ Michael Kiffe,‡ Jeff Zhang,† and Christoph Gaul*‡
‡Novartis Institutes for Biomedical Research, Basel, Switzerland
†Novartis Institutes for Biomedical Research, Shanghai, China
Dot1L, protein lysine methyltransferase, inhibitor, mixed lineage leukemia, protein structure-based design
ABSTRACT: Oncogenic MLL fusion proteins aberrantly recruit Dot1L, a histone methyltransferase, to ectopic loci, leading
to local hypermethylation of H3K79 and misexpression of HoxA genes driving MLL-rearranged leukemias. Inhibition of
the methyltransferase activity of Dot1L in this setting is predicted to reverse aberrant H3K79 methylation, leading to re-
pression of leukemogenic genes and tumor growth inhibition. In the context of our Dot1L drug discovery program, high-
throughput screening led to the identification of 2, a weak Dot1L inhibitor with an unprecedented, induced pocket bind-
ing mode. A medicinal chemistry campaign, strongly guided by structure-based consideration and ligand-based morph-
ing, enabled the discovery of 12 and 13, potent, selective, and structurally completely novel Dot1L inhibitors.
Protein lysine methyltransferases (PKMT) are a class of
51 enzymes which catalyze the transfer of a methyl group
from co-factor S-adenosylmethionine (SAM) to the lysine
ε-amino group of proteins.1–3 Typically, PKMTs mono-, di-
, or tri-methylate lysine residues of histone proteins,
thereby controlling chromatin structure and transcrip-
tional accessibility of genes. However, a number of other
non-histone substrate proteins have also been identified.4
reverse ectopic H3K79 methylation, leading to repression
of leukemogenic genes (HoxA9, Meis1) and tumor growth
inhibition.13 The recent quest for Dot1L inhibitors has
been spearheaded by Epizyme and culminated in the dis-
covery of EPZ-5676, a SAM-competitive, nucleoside-
containing Dot1L inhibitor, which is currently being eval-
uated in MLL patients in Phase 1b clinical trials.14 The
agent is administered by uninterrupted, continuous intra-
venous (i.v.) infusion due to its physicochemical proper-
ties.15 Other research groups, based at Baylor College and
the Structural Genomics Consortium, have identified ad-
ditional Dot1L inhibitors structurally related to EPZ-
5676.16–19
Dot1L is a 165 kD protein comprised of 1537 amino ac-
ids. The catalytic domain of Dot1L, residing in the N-
terminal 400 amino acids, is structurally distinct to the
SET-domain containing protein lysine methyltransferases
and clusters more closely with the catalytic domain of a
related class of proteins, the protein arginine methyl-
transferases (PRMT).3,5 Dot1L is the only known enzyme
to methylate lysine 79 of histone 3 (H3K79), with the
H3K79me2 mark being associated with active transcrip-
tion.6–8 Under physiological conditions, Dot1L is critical
for normal hematopoiesis,9,10 however, misdirected cata-
lytic activity is believed to be causative for a subset of
acute leukemias.11,12 Several oncogenic fusion proteins
including MLL-ENL, MLL-AF4 and MLL-AF9 aberrantly
recruit Dot1L to ectopic loci, leading to local hypermeth-
ylation of H3K79 and misexpression of genes (including
HoxA) which drive the leukemic phenotype. Inhibition of
the methyltransferase activity of Dot1L in MLL-rearranged
leukemias (mixed lineage leukemia, MLL) is predicted to
Herein, we describe the discovery of SAM-competitive,
structurally novel Dot1L inhibitors which bind to an in-
duced pocket adjacent to the SAM binding site. The com-
pounds display cellular potency and molecular properties
suitable for further optimization.
A variety of approaches to discover chemical starting
points in the Dot1L program were taken and included
virtual, biophysical, and biochemical screens. In our high-
throughput screening (HTS) campaign, a luminescence-
based coupled assay was used,20 which had been adapted
for measuring Dot1L-catalyzed nucleosome methylation
and miniaturized for allowing screening of the Novartis
Compound Collection in 1536-well plates (primary hit rat
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 9
0.47%, threshold 50% inhibition at 26.7 uM compound onto the Ser164-Gly165 backbone amide. The tetrazole
1
2
3
4
5
6
7
8
concentration). For IC50 and Ki determinations, a scintilla-
tion proximity assay (SPA) format was employed as a
more direct readout, quantifying enzyme activity via the
forms a hydrogen bond with Gln168 and the tetrazole-
phenoxy motif engages the flexible loop through hydro-
phobic contacts, while the p-chlorophenyl substituent
slides between the Asn241-containing loop and an α-helix,
occupying a similar position as the tert-Bu group of EPZ-
5676 (Figure 2C).
3
amount of the transfer of H-labelled methyl group from
SAM to biotinylated nucleosomes. Experiments described
herein were carried out at SAM concentration equal to KM
while nucleosome concentration was in excess relative to
KM (Supporting Information). In the HTS, a commercial
compound sample, assigned to structure 1, was identified
as an inhibitor of Dot1L (IC50 = 14 ꢀM) (Figure 1). Analysis
of the screening sample revealed that the active principle
is a regioisomeric impurity, compound 2 (IC50 = 4.4 ꢀM),
whereas purified 1 had no inhibitory activity up to 100
ꢀM.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Protein structure-guided optimization of HTS hit 1.
Figure 2. (A) X-ray cocrystal structure of Dot1L (grey) with 2
(blue) (PDB code 5drt). Amino acid side chains engaged in
key interactions with the ligand are illustrated as sticks. Key
polar interactions of Dot1L and 2 are shown as red dotted red
lines, the tetrahedral coordination of the two waters ligating
Asp161 as grey dotted lines. (B) Comparison of the flexible
loop (amino acids 126-140) in Dot1L bound to 2 (blue, protein
grey) (PDB code 5drt) and SAM (green) (PDB code 3qow).
(C) Overlay of ligands 2 (blue) (PDB 5drt) and EPZ-5676
(green) (PDB code 4hra) bound to Dot1L in the same view as
2A.
The crystal complex structure with compound 2 was
solved using the catalytic domain of Dot1L (Figure 2, PDB
code 5drt). To our surprise, 2 binds to a newly formed
pocket adjacent to the SAM binding site, inducing a fun-
damentally different conformation of Dot1L compared to
the conformation of SAM-bound Dot1L (Figure 2B).21
A
flexible loop comprised of amino acids 126-140, which
forms the “lid” of the SAM binding pocket in the SAM-
bound state, is completely rearranged, partially disor-
dered and forms a part of the induced binding pocket of
2. We and others subsequently observed that only SAM
and SAH are able to induce a closed lid (e.g. PDB code
3uwp), whereas inhibitors rearrange and often engage the
flexible loop.16,22 Other residues, such as Leu143, Met147,
Phe239 and Tyr312, also undergo conformational shifts
compared to the SAM-bound complex and are forming
the bottom lining of the binding pocket of 2. Remarkably,
2 forms five direct hydrogen bonds with Dot1L (Figure
2A). The central urea NH’s are coordinating Asp161,
whose side chain rotation is cushioned by crystal waters.
The interaction of the urea carbonyl with Asn241 varies
between 3.2 and 3.8 Å across several cogeneric cocrystal
structures. The urea of 2 interacts with Dot1L in a compa-
rable fashion to the urea-containing Dot1L inhibitor EPZ-
4777.16 The second hydrazide amide forms a hydrogen
bond to Gly163 perpendicular to the urea plane and stacks
Structural analysis of the Dot1L-compound 2 complex
revealed a large hydrophobic pocket, which is only par-
tially filled by the p-chlorophenyl substituent. Structure-
based chemistry optimization led to the discovery of the
significantly more potent compound 3 (IC50 = 20 nM) in
which the p-chlorophenyl substituent was replaced by an
N-arylated indole system (Figure 1). The indole core is
deeply buried in a hydrophobic cleft while the N-phenyl
moiety engages in a face-edge interaction with Phe131
thereby further stabilizing the flexible loop of Dot1L (Fig-
ure 3, PDB code 5dry). The o-Cl substituent does not only
pre-organize the bioactive conformation of 3, but also fills
a hydrophobic nook of Dot1L.
ACS Paragon Plus Environment

Page 3 of 9
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Figure 3. Overlay of X-ray cocrystal structures of Dot1L with
2 (ligand blue, protein grey) (PDB code 5drt) and 3 (ligand
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
green, parts of the protein green) (PDB code 5dry). Promi-
nent amino acid residues are illustrated as sticks.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Fragmentation of 3 to urea 4. Discovery of urea replacements 5-9.
Inhibitor 3 shows low permeability as assessed in the
parallel artificial membrane permeation (PAMPA) assay,
which might not be surprising, considering its number of
hydrogen bond acceptors and donors (Supporting Infor-
mation). It is likely that the weak flux of 3 contributes to
the lack of oral bioavailability in rats and weak cellular
activity.
complementary to the hydrophobic pocket formed by the
Asn241-containing loop and the α-helix. Importantly, the
carbonyl group of amino-pyrimidone 7 and the N-methyl
group of diamino-pyrimidine 6 interact with Dot1L in
distinct ways: The N-methyl group of 6 does not undergo
any specific contacts with Dot1L, but rather plays a role in
pre-organizing the ligand by pointing into the N-aryl
moiety (van der Waals contact). In contrast, the carbonyl
group of 7 engages in polar interactions with Asn241 and
the backbone NH of Tyr136, thereby tightly stabilizing the
flexible loop of Dot1L (Figure 5). Consequently, potential
growth vectors in ortho- and meta-position of the N-
pyridyl moiety of pyrimidone 7 are blocked by the loop,
whereas they are open for pyrimidine 6 (Figure 5).
Considering the poor physicochemical properties, the
complex functional group arrangement and high molecu-
lar weight (MW = 521), we regarded 3 unsuitable for fur-
ther stepwise optimization. Therefore, we decided to pur-
sue a more radical fragmentation approach in which the
entire aryl-tetrazole system was eliminated leading to
urea 4, which showed residual biochemical potency (IC50
= 40 ꢀM) (Figure 4). Compound 4 was viewed a more
suitable starting point for additional chemistry activities.
Initial work focused on the identification of urea re-
placements with the goal to reduce the risk of low solubil-
ity and poor permeability often associated with ureas. A
series of functional groups were discovered as urea
isosters capable of coordinating the central Asp161 side
chain of Dot1L. Diamino-pyrimidines 5 and 6 (in their
protonated forms), amino-pyrimidone 7, amino-
pyridazine 8 (with its polarized C-H group) and amino-
imidazolone 9 inhibit Dot1L with improved potency com-
pared to urea 4 (Figure 4). It is worth noting that the me-
thyl group in the 2-position of the indole and the inser-
tion of a nitrogen atom into the N-phenyl substituent
typically contribute to the potency by a 5-fold reduction
of the IC50. Remarkably, the N-methyl group of 6 increas-
es inhibitory activity against Dot1L by almost 20-fold over
5, possibly through a pre-organization effect (discussed
below).
The optimization opportunities for urea replacements 6
and 7 were evaluated in detail. In line with our structural
understanding and using molecular modeling, the dia-
mino-pyrimidine of 6 and the amino-pyrimidone of 7 un-
dergo polar interactions with Asp161 (Figure 5). The N-
aryl 2-methylindole core of both analogs is shape-
Figure 5. Overlay of X-ray cocrystal structures of Dot1L with
6 (ligand green, parts of the protein green) and 7 (ligand
blue, protein grey) bound to Dot1L.23 Amino acid side chains
engaging in key interactions with the ligands are illustrated
as sticks, polar contacts are highlighted as dotted red lines.
ACS Paragon Plus Environment

Page 5 of 9
ACS Medicinal Chemistry Letters
Growth vectors (solid red arrow) open in ortho and meta-
Chemistry optimization continued on both structural
1
2
3
4
5
6
7
8
position for 6, but blocked for 7 due to tighter engagement of
the flexible loop.
subtypes exemplified by compounds 10 (“meta”) and 11
(“ortho”). A number of opportunities were discovered to
further improve potency. For example, the 2-methyl in-
dole core of 10 can be replaced by a 2-chloro benzothio-
phene core, leading to the highly potent Dot1L inhibitor
12 (IC50 = 1.8 nM) (Table 1). Along different lines, the aro-
matic stacking capacity of 11 can be dramatically im-
proved by substituting the outer phenyl group of 11 by an
aza-benzimidazole group, resulting in 400-fold more po-
tent compound 13 (IC50 = 0.4 nM). Both compounds 12
and 13 are characterized by long on-target residence times
as assessed by surface plasmon resonance experiments (τ
= 45 min for 12 and >240 min for 13 (the detection limit of
our internal SPR assay). Most importantly, 12 and 13 po-
tently suppress H3K79 dimethylation (IC50 = 23 nM and 16
nM, respectively), the direct product of the Dot1L-
catalyzed reaction, as well as the activity of the HoxA9
promoter (IC50 = 384 nM and 340 nM, respectively) in
cellular systems (Table 1). We speculate that the signifi-
cantly higher SAM concentration in cells (≈ 200 ꢀM)24
compared to our biochemical assay (200 nM) is contrib-
uting to the considerable cell-enzyme IC50 shift. Finally,
both compounds effectively inhibit proliferation of the
human MLL-rearranged leukemia cell line MV4-11 carry-
ing the oncogenic MLL-AF4 fusion (IC50 = 85 nM and 128
nM, respectively) (Table 1). Importantly, 12 and 13 display
a favorable selectivity profile against a panel of 22 PKMTs
and PRMTs with no inhibitory activity up to 50 ꢀM.
Both growth vectors, extruding from either the ortho-
or the meta-position were explored for 6. Guided by
abovementioned protein-structural considerations, the
direct attachment of a pyridone in meta-position, leading
to compound 10 (Figure 6), turned out to have a benefi-
cial impact on potency (IC50 = 10 nM). Growing 6 from the
ortho-position was best achieved with an aryloxy group
while eliminating the Cl substituent in the “other” ortho-
position, as e.g. in compound 11 (IC50 = 150 nM) (Figure
6).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Optimization of 6 by growing from the meta-
position, leading to 10, or from the ortho-position, leading to
11.
Analysis of the X-ray cocrystal structures of 10 and 11
bound to Dot1L revealed some noteworthy aspects (Figure
7, PDB codes 5dsx and 5dt2): both compounds stack tightly
with Phe243 and engage the flexible loop through their
diaryl and diaryl ether groups, respectively. Furthermore,
the pyridone carbonyl of 10 forms a hydrogen bond to the
side-chain NH2 of Asn241. The N-phenyl group of 11
moves deep into the hydrophobic pocket, taking the place
of the o-Cl substituent of 10.
Table 1. Biochemical, biophysical and cellular charac-
terization of lead compounds 12 and 13
12
1.4
13
0.4
IC50 (Dot1L SPA) [nM]
Ki (Dot1L SPA) [nM]
0.36
43
0.08
>240
16
τ (Dot1L SPR) [min]
IC50 (HeLa, H3K79me2 ELISA) [nM]
IC50 (Molm-13, HoxA9 RGA) [nM]
IC50 (MV4-11, prolif.) [nM]
23
384
85
340
128
All data are the results of at least two assay runs with the
mean value reported. The coefficient of variation was less
than 60% in all cases. Biochemical IC50 values were deter-
mined at KM for SAM. Ki values were determined by applying
the Morrison tight binding model (Supporting Information).
Figure 7. Overlay of ligands 10 (ligand blue, protein grey)
(PDB code 5dsx) and 11 (ligand green, parts of the protein
green) (PDB code 5dt2) bound to Dot1L based on X-ray co-
crystal structures. Both aromatic extensions engage Phe243
and the Pro130-Phe131 motif of the flexible loop.
By virtue of its in vitro potency and selectivity as well as
good molecular properties (e.g. improved permeability vs
3, Supporting Information), 12 was evaluated in PK exper-
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 9
iments in rats (Table 2). After intravenous bolus admin-
aReagents and conditions: (a) Cu, quinoline, 2 h, 150 °C,
1
2
3
4
5
6
7
istration (1 mg/kg), the compound showed high total
blood clearance, high volume of distribution and a mod-
erate half-life. After oral administration in suspension (15
mg/kg), blood levels were detected up to 24 h. The maxi-
mum total concentration (ca. 225 nM) was reached after
3.5 h, total exposure was 2370 nM·h. Oral bioavailability at
15 mg/kg was medium, reaching 40%.
81%, (b) Br2, CHCl3, 30 h, 60 °C, 93%, (c) NCS,
TMPMgCl*LiCl, THF, toluene, -75 °C, 80%, (d) RaNi, H2,
EtOH, rt, 96%, (e) 24, p-TsOH*H2O, DMF, 15 h, 80 °C,
66%, (f) 20, Na2CO3 2 M aq, Pd(amphos)Cl2, MeCN, 30
min, 80 °C, 25%, (g) 22, Na2CO3
PdCl2(dppf)*DCM, DME, 80 °C, 68%, (h) NaNO2, KI, HCl
M, H2O, MeCN, °C to rt, 36%, (i)
2
M
aq,
4
5
8
9
Bis(pinacolato)diboron, KOAc, PdCl2(dppf)*DCM, diox-
ane, 10 h, 110 °C, 49%, (j) Bis(pinacolato)diboron, KOAc,
PdCl2(dppf)*DCM, dioxane, 3 h, 100 °C, 80%, (k) Boc2O,
DMAP, DCM, rt, 98%.
Table 2. Pharmacokinetic parameters of 12 in male
Sprague Dawley rats following IV and PO dosing
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1 mg/kg
iva
15 mg/kg
pob
In conclusion, we have discovered potent, selective and
orally bioavailable Dot1L inhibitors which have a novel
binding mode and are structurally unique. Future work
on further improving potency and in vivo exposure based
on lead compounds 12 and 13 is warranted.
Cl [mL/min/kg]
Vd [L/kg]
84
10
n.a.
n.a.
n.a.
2370
225
40
t1/2 [h]
1.7
ASSOCIATED CONTENT
AUC0-inf [nM·h]
Cmax [nM]
BAV [%]
396
n.a.
n.a.
PyMol was used for structural visualization and figure prepa-
ration.25 Synthetic procedures, compound characterization
and assay protocols. This material is available free of charge
via the Internet at http://pubs.acs.org.
aNMP:PEG200 (30:70), bMEPC5:Water (10:90).
AUTHOR INFORMATION
Compound 12 can be conveniently prepared from
Corresponding Author
commercially
available
5-nitrobenzo[b]thiophene-2-
*E-mail: christoph.gaul@novartis.com
carboxylic acid 14 (Scheme 1). Thermal decarboxylation,
followed by bromination and a lithiation-chlorination
sequence, yielded intermediate 15. Nitro reduction to 16
and nucleophilic aromatic substitution with chloro-
pyrimidine 24 delivered key intermediate 17, suitable for
late state derivatization in the 3-position of the benzothi-
ophene core. Suzuki coupling of 17 with the customized
boronic ester 20 produced lead compound 12 in a conver-
gent fashion (Scheme 1).
Author Contributions
The manuscript was written through contributions of all
authors. / All authors have given approval to the final version
of the manuscript.
ACKNOWLEDGMENT
We are grateful to our medicinal chemistry colleagues Ross
Strang, Christian Ragot, Rainer Tschan, Michael Hediger,
and Stephan Kläusler for their scientific contributions. We
thank Kim Twesten and Anton Kessler for developing and
running the high-throughput screening assay, and
acknowledge supply of Dot1L enzyme and nucleosome sub-
strate by the laboratories of Lukas Leder, Dirk Erdmann and
Kehao Zhao, respectively. Jidong Zhu is thanked for valuable
and inspiring discussions on target biology, Steffen Renner
for in silico hit list analysis and Justin Gu for verification of
screening hits in an orthogonal LC-MS assay. We thank Paul
Westwood, Kerstin Pollehn, Shin Numao, Claudio Thoma
and Andreas Theuer for developing and supporting project-
related biophysical, biochemical and cellular assays. The
authors would like to acknowledge Elke Koch, Céline Be,
Aurelie Winterhalter, Aude Izaac, Julia Klopp and Patrick
Graff for their contributions to the protein structural work.
The crystallographic experiments were performed on the
X10SA beamline at the Swiss Light Source, Paul Scherrer In-
stitut, Villigen, Switzerland supported by the team of Expose
GmbH. We thank Sandrine Desrayaud and her team for per-
forming the rat PK studies as well as Bernard Faller and his
team for contributing to the in vitro ADME studies.
Scheme 1. Synthetic route to compound 12.a
REFERENCES
ACS Paragon Plus Environment

Page 7 of 9
ACS Medicinal Chemistry Letters
(1) Copeland, R. A.; Solomon, M. E.; Richon, V. M. Protein Me-
thyltransferases as a Target Class for Drug Discovery. Nat. Rev.
Drug Discov. 2009, 8, 724–732.
C. H.; Bradner, J. E.; Schapira, M. Catalytic Site Remodelling of
the Dot1L Methyltransferase by Selective Inhibitors. Nat. Com-
mun. 2012, 3, 1288-1298.
1
2
3
4
5
6
7
8
9
(2) Copeland, R. A. Molecular Pathways: Protein Methyltrans-
ferases in Cancer. Clin. Cancer Res. 2013, 19, 6344–6350.
(3) Richon, V. M.; Johnston, D.; Sneeringer, C. J.; Jin, L.; Majer,
C. R.; Elliston, K.; Jerva, L. F.; Scott, M. P.; Copeland, R. A.
Chemogenetic Analysis of Human Protein Methyltransferases.
Chem. Biol. Drug Des. 2011, 78, 199–210.
(4) Hamamoto, R.; Saloura, V.; Nakamura, Y. Critical Roles of
Non-Histone Protein Lysine Methylation in Human Tumorigen-
esis. Nat. Rev. Cancer 2015, 15, 110–124.
(17) Anglin, J. L.; Deng, L.; Yao, Y.; Cai, G.; Liu, Z.; Jiang, H.;
Cheng, G.; Chen, P.; Dong, S.; Song, Y. Synthesis and Structure-
Activity Relationship Investigation of Adenosine-Containing
Inhibitors of Histone Methyltransferase Dot1L. J. Med. Chem.
2012, 55, 8066–8074.
(18) Yao, Y.; Chen, P.; Diao, J.; Cheng, G.; Deng, L.; Anglin, J.
L.; Prasad, B. V. V.; Song, Y. Selective Inhibitors of Histone Me-
thyltransferase Dot1L: Design, Synthesis, and Crystallographic
Studies. J. Am. Chem. Soc. 2011, 133, 16746–16749.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(5) Min, J.; Feng, Q.; Li, Z.; Zhang, Y.; Xu, R.-M. Structure of
the Catalytic Domain of Human Dot1L, a Non-SET Domain Nu-
cleosomal Histone Methyltransferase. Cell 2003, 112, 711–723.
(6) Nguyen, A. T.; Zhang, Y. The Diverse Functions of Dot1
and H3K79 Methylation. Genes Dev. 2011, 25, 1345–1358.
(7) Steger, D. J.; Lefterova, M. I.; Ying, L.; Stonestrom, A. J.;
Schupp, M.; Zhuo, D.; Vakoc, A. L.; Kim, J.-E.; Chen, J.; Lazar, M.
A.; Blobel, G. A.; Vakoc, C. R. Dot1L/KMT4 Recruitment and
H3K79 Methylation Are Ubiquitously Coupled with Gene Tran-
scription in Mammalian Cells. Mol. Cell. Biol. 2008, 28, 2825–
2839.
(8) Barski, A.; Cuddapah, S.; Cui, K.; Roh, T.-Y.; Schones, D. E.;
Wang, Z.; Wei, G.; Chepelev, I.; Zhao, K. High-Resolution Profil-
ing of Histone Methylations in the Human Genome. Cell 2007,
129, 823–837.
(9) Jo, S. Y.; Granowicz, E. M.; Maillard, I.; Thomas, D.; Hess, J.
L. Requirement for Dot1L in Murine Postnatal Hematopoiesis
and Leukemogenesis by MLL Translocation. Blood 2011, 117,
4759–4768.
(10) Feng, Y.; Yang, Y.; Ortega, M. M.; Copeland, J. N.; Zhang,
M.; Jacob, J. B.; Fields, T. A.; Vivian, J. L.; Fields, P. E. Early
Mammalian Erythropoiesis Requires the Dot1L Methyltransfer-
ase. Blood 2010, 116, 4483–4491.
(11) Bernt, K. M.; Zhu, N.; Sinha, A. U.; Vempati, S.; Faber, J.;
Krivtsov, A. V.; Feng, Z.; Punt, N.; Daigle, A.; Bullinger, L.; Pol-
lock, R. M.; Richon, V. M.; Kung, A. L.; Armstrong, S. A. MLL-
Rearranged Leukemia Is Dependent on Aberrant H3K79 Methyl-
ation by Dot1L. Cancer Cell 2011, 20, 66–78.
(19) Deng, L.; Zhang, L.; Yao, Y.; Wang, C.; Redell, M. S.; Dong,
S.; Song, Y. Synthesis, Activity and Metabolic Stability of Non-
Ribose Containing Inhibitors of Histone Methyltransferase
Dot1L. Med. Chem. Commun. 2013, 4, 822–826.
(20) Ibáňez, G.; McBean, J. L.; Astudillo, Y. M.; Luo, M. An En-
zyme-Coupled Ultrasensitive Luminescence Assay for Protein
Methyltransferases. Anal. Biochem. 2010, 401, 203–210.
(21) A related protein-structural rearrangement of Dot1L is de-
scribed in a manuscript co-submitted with this manuscript:
Scheufler, C.; Be, C.; Fernandez, C.; Moebitz, H.; Ragot, C.; Bey-
er, K.; Gaul, C. Stauffer, F. Optimization Towards Potent Dot1L
Inhibitors of a Fragment-Based Screen Hit Containing a 2,6-
Dichlorophenyl Hydrophobic Anchor and Interacting in an In-
duced Binding Pocket. ACS Med. Chem. Lett. 2016, 7, submitted.
(22) Anglin, J. L.; Song, Y. A. Medicinal Chemistry Perspective
for Targeting Histone H3 Lysine-79 Methyltransferase Dot1L. J.
Med. Chem. 2013, 56, 8972–8983.
(23) Clemens Scheufler, personal communication
(24) Bennett, B. D.; Kimball, E. H.; Gao, M.; Osterhout, R.; an
Dien, S. J.; Rabinowitz, J. D. Absolute Metabolite Concentrations
and Implied Enzyme Active Site Occupancy in Escherichia coli.
Nat. Chem. Biol. 2009, 5, 593-599.
(25) The PyMOL Molecular Graphics System, Version 1.5.0.4
Schrödinger, LLC.
(12) Slany, R. K. The Molecular Biology of Mixed Lineage Leu-
kemia. Haematologica 2009, 94, 984–993.
(13) Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.; Majer, C. R.;
Sneeringer, C. J.; Song, J.; Johnston, L. D.; Scott, M. P.; Smith, J.
J.; Xiao, Y.; Jin, L.; Kuntz, K. W.; Chesworth, R.; Moyer, M. P.;
Bernt, K. M.; Tseng, J. C.; Kung, A. L.; Armstrong, S. A.;
Copeland, R. A.; Richon, V. M.; Pollock, R. M. Selective Killing of
Mixed Lineage Leukemia Cells by a Potent Small-Molecule Dot1L
Inhibitor. Cancer Cell 2011, 20, 53–65.
(14) Daigle, S. R.; Olhava, E. J.; Therkelsen, C. A.;
Basavapathruni, A.; Jin, L.; Boriack-Sjodin, P. A.; Allain, C. J.;
Klaus, C. R.; Raimondi, A.; Scott, M. P.; Waters, N. J.; Chesworth,
R.; Moyer, M. P.; Copeland, R. A.; Richon, V. M.; Pollock, R. M.
Potent Inhibition of Dot1L as Treatment of MLL-Fusion Leuke-
mia. Blood 2013, 122, 1017–1025.
(15) Basavapathruni, A.; Olhava, E. J.; Daigle, S. R.; Therkelsen,
C. A.; Jin, L.; Boriack-Sjodin, P. A.; Allain, C. J.; Klaus, C. R.; Rai-
mondi, A.; Scott, M. P.; Dovletoglou, A.; Richon, V. M.; Pollock,
R. M.; Copeland, R. A.; Moyer, M. P.; Chesworth, R.; Pearson, P.
G.; Waters, N. J. Nonclinical Pharmacokinetics and Metabolism
of EPZ-5676, a Novel Dot1L Histone Methyltransferase Inhibitor.
Biopharm. Drug Dispos.2014, 35, 237-252.
(16) Yu, W.; Chory, E. J.; Wernimont, A. K.; Tempel, W.; Scop-
ton, A.; Federation, A.; Marineau, J. J.; Qi, J.; Barsyte-Lovejoy, D.;
Yi, J.; Marcellus, R.; Iacob, R. E.; Engen, J. R.; Griffin, C.; Aman,
A.; Wienholds, E.; Li, F.; Pineda, J.; Estiu, G.; Shatseva, T.; Hajian,
T.; Al-Awar, R.; Dick, J. E.; Vedadi, M.; Brown, P. J.; Arrowsmith,
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
ACS Paragon Plus Environment

Page 9 of 9
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment