New Potent DOT1L Inhibitors for in Vivo Evaluation in Mouse

Article abstract of DOI:10.1021/acsmedchemlett.9b00452

In MLL-rearranged cancer cells, disruptor of telomeric silencing 1-like protein (DOT1L) is aberrantly recruited to ectopic loci leading to local hypermethylation of H3K79 and consequently misexpression of leukemogenic genes. A structure-guided optimization of a HTS hit led to the discovery of DOT1L inhibitors with subnanomolar potency, allowing testing of the therapeutic principle of DOT1L inhibition in a preclinical mouse tumor xenograft model. Compounds displaying good exposure in mouse and nanomolar inhibition of target gene expression in cells were obtained and tested in vivo.

Full text of DOI:10.1021/acsmedchemlett.9b00452

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
New Potent DOT1L Inhibitors for in Vivo Evaluation in Mouse
́
́
̈
Frederic Stauffer,* Andreas Weiss, Clemens Scheufler, Henrik Mobitz, Christian Ragot, Kim S. Beyer,
Keith Calkins, Daniel Guthy, Michael Kiffe, Bernard Van Eerdenbrugh, Ralph Tiedt,
and Christoph Gaul
Novartis Institutes for Biomedical Research, 4056 Basel, Switzerland
S
* Supporting Information
ABSTRACT: In MLL-rearranged cancer cells, disruptor of
telomeric silencing 1-like protein (DOT1L) is aberrantly
recruited to ectopic loci leading to local hypermethylation of
H3K79 and consequently misexpression of leukemogenic
genes. A structure-guided optimization of a HTS hit led to the
discovery of DOT1L inhibitors with subnanomolar potency,
allowing testing of the therapeutic principle of DOT1L
inhibition in a preclinical mouse tumor xenograft model.
Compounds displaying good exposure in mouse and nano-
molar inhibition of target gene expression in cells were
obtained and tested in vivo.
KEYWORDS: Dot1L, lysine histone methyltransferase, inhibitor, HTS hit, structure-based design
inhibitor candidate that has been investigated in clinical trials
hromosomal rearrangements of the MLL (MLL1,
CKMT2A) gene can lead to a variety of fusion proteins to date, albeit with limited success.10
We applied several approaches to discover new DOT1L
that cause an aggressive form of acute leukemia. In these fusion
proteins, the N-terminal portion of MLL is preserved, while
the C-terminal part is replaced by a portion of another protein,
leading to novel oncogenic functionalities.1−4 A key concept in
the current model of leukemic transformation by MLL fusions
is the aberrant maintenance of a stem cell-like state through
persistent expression of genes such as HOXA9 and MEIS1. At
least a subset of MLL fusion proteins, including those with the
frequently observed fusion partners AF4, AF9, and AF10,
depend on DOT1L as a cofactor for driving expression of these
critical genes.1−4 DOT1L is the only known histone 3 lysine 79
(H3K79) methyltransferase, and the H3K79me2 mark is
broadly associated with active transcription.5 In mouse models
of acute lymphoblastic or myeloid leukemia caused by MLL-
AF4 or MLL-AF9, aberrantly increased H3K79me2 was
detected at target genes of those fusion proteins.6,7 Genetic
inactivation of DOT1L led to downregulation of target genes
and loss of the leukemic phenotype with minimal effects on
global gene expression.7 Accordingly, the DOT1L inhibitor
EPZ004777 demonstrated selective activity against MLL-
rearranged leukemia models.8 A related, further optimized
DOT1L inhibitor named pinometostat (EPZ-5676) was
subsequently shown to produce robust antitumor activity
against an MV4-11 xenograft model in rats.9 This S-adenosyl
methionine (SAM) competitive agent, which carries the
adenosyl portion of the SAM cofactor, is not suitable for oral
absorption and needs to be administered as a continuous
infusion in order to achieve sufficient exposure and sustained
target inhibition. Pinometostat is also the only DOT1L
inhibitors that overcome the PK limitations of SAM-related
compounds,11−13 including high throughput screening (HTS)
with a coupled luminescence-based readout for detection of
the methyltransferase product SAH that yielded compound 1
(Table 1), a single micromolar hit (IC50 SPA DOT1L = 8.5 μM).
In a similar manner as described for a fragment-based
screening hit,13 compound 1 is not binding in the SAM
binding pocket but in an adjacent induced pocket formed by
engaging the SAM pocket lid loop (Figure 1). Despite no
overlap with the SAM cofactor binding pocket, compound 1
and SAM have mutually exclusive binding, leading to an
apparent SAM competitive inhibition for this new series.
The sulfonamide group of our ligand is a key contributor to
the potency of the ligand by forming three direct hydrogen-
bonds with DOT1L, but at the same time it brings a large
contribution to its polar surface area and a desolvation penalty
upon binding. We envisioned replacing the sulfonamide by the
more hydrophobic methylsulfone. The acceptor capability of
such compounds is unchanged while the donors are now
polarized C−H. Despite being weaker hydrogen-bond donors,
they are also paying less energetic contribution to their
desolvation. We observed in general a loss of around 3-fold in
potency for sulfonamide to methylsulfone match pairs. In order
to favor the binding conformation and to desymmetrize the
benzhydrylamino moiety, we introduced a nitrogen at the
Received: October 1, 2019
Accepted: November 25, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00452
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Letter
Table 1. DOT1L Inhibitors 1 to 11
IC50 SPA DOT1L
compound
R1
R2
R3
X
[μM]
b
1
2
3
4
5
6
−NO2
−NO2
pyrimidin-2-ylamino
pyrimidin-2-ylamino
pyrimidin-2-ylamino
phenyl
phenyl
−NH2
−Me
−Me
−Me
−Me
−Me
8.5
a
b
3-chlorophenyl/2-pyridyl 2-pyridyl/3-chlorophenyl
2-pyridyl
3-chlorphenyl
2-pyridyl
2-pyridyl
4.6
b
3-chlorophenyl
2-pyridyl
3-chloro-2-fluorophenyl
3-chloro-2-fluorophenyl
0.27
b
22.8
b
0.097
0.0039
b
(5-carbamoylpyrimidin-2-yl)amino
c
0.0021
0.016
b
7
8
9
pyrimidin-2-ylamino
(4,6-dimethoxy-1,3,5-triazin-2-yl)amino
(4,6-dimethoxy-1,3,5-triazin-2-yl)amino
3-fluoro-2-pyridyl
3-fluoro-2-pyridyl
3-chloro-2-pyridyl
3-chloro-2-fluorophenyl
3-chloro-2-fluorophenyl
2,2-difluorobenz[d][1,3]dioxole-4-
yl
2,2-difluorobenz[d][1,3]dioxole-4-
yl
2,2-difluorobenz[d][1,3]dioxole-4-
yl
−Me
−Me
−Me
c
0.00059
c
0.00019
c
10
11
(4-methoxy-6-(piperazin-1-yl)-1,3,5-triazin-2-yl)
amino
(4-amino-6-methoxy-1,3,5-triazin-2-yl)amino
3-chloro-2-pyridyl
3-chloro-2-pyridyl
−Me
0.00011
c
−NH2
0.00017
a
b
c
Racemate. SPA format as described.13 SPA high sensitivity format modified to increase sensitivity using 0.05 nM nM DOT1L as described.13
stacking of Phe131 with the central phenyl ring of compound
3. The ligand is engaging three backbone hydrogen-bond
interactions with Ser311: one pseudo H-bond with a polarized
C−H as well as a dual donor and acceptor interaction with the
methylsulfone group. The methylsulfone is also donor for the
Ser269 backbone carbonyl. The chlorophenyl is filling a
hydrophobic pocket (Leu143, Met147, Val267, Ser269,
Tyr311) and the pyridyl a hydrophobic channel (Phe131,
Tyr136, S140, Val169) partitioned by Val144, Phe239, and
Asn241 (Figure 2).
Figure 1. Overlay of X-ray crystal structures of Dot1L with SAM (pdb
1nw3) and 1 (undisclosed structure). Cartoon representation of
Dot1L (gray) with the flexible loop 126−140 shown in light blue for
SAM and red for 1; stick models of ligands 1 (red) and SAM (blue).
The residues Phe129 and Pro130 that form the roof of the induced
pocket are shown as sticks.
position 2 of one of the phenyl groups to allow an
intramolecular hydrogen-bond interaction. Further, we deco-
rated the second phenyl at position 3 with a chloro group in
compound 2 (IC50 SPA DOT1L = 4.6 μM) to map the extension
of this pocket on one side of the phenyl.
One undesired feature of compound 2 was still the nitro
group. It was replaced with a 2-pyrimidinylamino moiety that
interacted more efficiently with Asn214 by forming a bidentate
hydrogen-bond interaction and inserted between Pro130 and
Phe243. At the same time, it opened a new growth vector. The
more active (S)-enantiomer compound 3 had submicromolar
activity (IC50 SPA DOT1L = 270 nM) and, as one would expect
based on the binding mode of this series, the (R)-enantiomer
was inactive (compound 4, IC50 SPA DOT1L = 22.8 μM). A key
stabilizing interaction of the lid loop rearrangement is the π−π
Figure 2. Detailed interactions of 3 with Dot1L (pdb 6TE6). Amino
acid side chains engaging in key interactions with the ligand are
illustrated as sticks, and polar contacts are highlighted as dotted red
lines. The binding mode of 3 is driven by the stacking of aromatic
groups and three key polar contacts with Asn241, Ser311 and Ser269.
The Asp161 does not engage the ligand. Compared to 1, the
pyrimidine moiety of 3 increases the stacking contacts with Phe243
and Phe131-Pro130, and the intramolecular hydrogen bond of the
pyridine with the central aniline preorganizes the bound conforma-
tion.
B
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One concern with compound 3 was the high clearance
measured in rat liver microsomes (Clint = 553 μL × min−1
×
mg−1). Adding to the 3-chlorophenyl moiety a fluoro
substituent at position 2 led to compound 5 with a 2-fold
improvement of potency (IC50 SPA DOT1L = 97 nM) and
significant reduction of intrinsic clearance (Clint = 50 μL ×
min−1 × mg−1), possibly by steric shielding of the benzylic
metabolic weak spot and/or an electronic effect.
A further improvement in potency was achieved by
introducing an aminocarbonyl substituent at position 4 of
the pyrimidine (compound 6). In addition to its electron
withdrawing effect, this group extended the stacking capability
of the pyrimidine with Pro130 and Phe243 (IC50 SPA DOT1L
=
2.1 nM). This low nanomolar inhibitor of DOT1L was tested
Figure 3. Mouse PK single vs multiple doses of 8 indicative of a
cytochrome P450 induction.
in cellular assays11 to assess the ability to inhibit the
dimethylation of H3K79 in HeLa cells (ED50 H3K79me2 Elisa
=
300 nM) and HOXA9 gene expression in Molm-13 cells (ED50
interaction with the Asn241 carbonyl (Figure 4 A). Compound
10 was shown to interact with DOT1L by SPR with slow
kinetics (t1/2 > 5 h). Selectivity testing for compound 10
against a limited panel of histone methyl transferases showed
no inhibition below 5 μM (Table S4).
Compound 10 was formulated as solution (Solutol SH15/
saline 10/90%) and could be administered at 300 mg/kg
allowing to reach 4 to 8 μM in blood over 24 h after a single
administration and no loss of exposure upon multiple daily
administrations, but rather the expected accumulation (Figure
s1). Unfortunately, this compound was not tolerated at such a
high dose by tumor xenograft bearing mice, and at a 6-fold
reduced dose, the tumor growth as well as the HOXA9
reporter gene mRNA were reduced only by less than half as
compared to control animals.
= 4500 nM). The compound showed a suitable PK
HOXA9 RGA
profile in mouse and rat with an oral bioavailability of 26% and
36%, respectively, at 3 mg/kg (Tables s1 and s2). At a maximal
dose of 280 mg/kg p.o., the blood exposure was above 10 μM
for 8 h. Treating mice bearing MV4-11 subcutaneous xenograft
tumors for 23 days at a dose of 200 mg/kg twice daily lead to
no tumor growth inhibition. While global H3K79 dimethyla-
tion levels in tumor were reduced by 50%, no impact on
expression of the MLL-AF4 target genes HOXA9 and MEIS1
was observed. We concluded from this experiment that we
needed to improve the exposure/potency ratio significantly to
reach efficacy after oral dosing.
An increase in potency was achieved by introducing a
halogen such as fluorine at position 3 of the pyridyl group
(compound 7, IC50 SPA DOT1L = 16 nM). Replacement of the
pyrimidine with the more electron deficient triazine to allow
extension of the stacking capability by adjunction of two
methoxy groups provided a subnanomolar inhibitior in our
biochemical assay (compound 8, IC50 SPA DOT1L = 0.59 nM)
that translated in potent cell activity (ED50 H3K79me2 Elisa = 43
nM). Further fine-tuning of the van der Waals contacts by
replacing the 3-fluoropyridyl group with a 3-chloropyridyl
group and the 2-fluoro-3-chlorophenyl group with a 2,2-
difluorobenzo[d][1,3]dioxole-4-yl group provided compound
9 (IC50 SPA DOT1L = 0.19 nM, ED50 H3K79me2 Elisa = 12 nM, and
ED50 HOXA9 RGA = 170 nM). While both compounds 8 and 9
showed an excellent blood exposure after a single dose,
multiple dosing uncovered an issue of cytochrome P450
(Cyp450) 3A4 isoform induction leading to reduced exposure
over time (e.g., compound 8 at 100 mg/kg: AUC0−12 = 216 h
× μM at day 1 vs AUC0−12 = 47 h × μM at day 20, Figure 3).
Induction was confirmed by rodent and human Cyp450
hepatocyte functional assays.
CYP450 3A4 isoform induction of compound 9 could also
be mitigated by restoring the initial sulfonamide instead of the
methylsulfonyl group (Figure 4B) and by changing one
methoxy group for an amino group (compound 11,
IC50 SPA DOT1L = 0.17 nM, ED50 H3K79me2 Elisa = 2.9 nM and
ED50
= 30 nM; log DpH 7.4 = 3.5). Similarly to
HOXA9 RGA
compound 10, the logD was decreased as compared to
compound 9. However, with a substantially increased PSA and
hydrogen bond donor number, the compound lost its oral
bioavailability and had to be administered subcutaneously as a
solution (20% Kolliphor HS 15/80% saline by volume). At a
dose of 50 mg/kg twice daily, exposure was maintained after 7
days. Mice bearing subcutaneous MV4-11 tumor xenografts
were treated for 20 days at 75 mg/kg once or twice daily by
subcutaneous injection. While once daily administration did
not lead to tumor growth inhibition, 73% growth inhibition
was measured in the twice daily treated group (Figure 5A)
with only temporary body weight loss in the beginning of
treatment (Figure 5B). While both treatment regimens
resulted in very strong inhibition of global H3K79
dimethylation level in tumor (Figure 5C), the efficacious
regimen was superior in reducing the mRNA expression of the
target genes HOXA9 (25% in qd regimen vs 53% in bid
regimen) and MEIS1 (53% vs 74%) (Figure 5D)
Compound 9 was modified by replacing one methoxy group
with an ionizable piperazine that conferred a logD (pH 7.4)
decrease superior to 1.4 and a further increase in potency
(compound 10, IC50 SPA DOT1L = 0.11 nM, ED50 H3K79me2 Elisa
=
1.7 nM and ED50 = 33 nM; logDpH 7.4 = 3.3).
HOXA9 RGA
Cocrystallization of compound 10 showed that the binding
mode is similar to the one of compound 3 with optimized van
der Waals contacts and extended stacking between Phe243 and
Pro130. The 2,2-difluorobenzo[d][1,3]dioxole-4-yl group is
shapewise nicely complementary to the hydrophobic pocket.
However, the hydrogen-bond pattern with Asn241 was altered
in comparison to compound 3 with only the NH donor at
adequate distance to form an efficient hydrogen-bond
Compound 11 was synthesized starting with the con-
densation of the commercially available 2,2-difluorobenzo[d]-
[1,3]dioxole-4-carbaldehyde with (R)-2-methylpropane-2-sul-
finamide catalyzed by tetraethoxytitanium (Scheme 1).
Commercially available 2-bromo-3-chloro-pyridine was reacted
with isopropylmagnesium bromide to generate the Grignard
reagent to stereoselectively14 engage with intermediate 12 to
yield after careful flash chromatography the desired diaster-
C
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ACS Medicinal Chemistry Letters
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Figure 4. (A) Detailed interactions of 10 with Dot1L (pdb 6TEL).
Amino acid side chains engaging in key interactions with the ligand
are illustrated as sticks, polar contacts are highlighted as dotted red
lines. Compared to 3, the piperazine triazine moiety of 10 increases
the stacking contacts with Phe243 and Phe129-Pro130 and the
hydrophobic pocket of the benzhydrylamino moiety is filled more
efficiently. (B) Detailed interactions of 11 with Dot1L (pdb 6TEN).
Amino acid side chains engaging in key interactions with the ligand
are illustrated as sticks, and polar contacts are highlighted as dotted
red lines. Compared to 10, the amino methoxy triazine moiety of 11
makes efficient stacking contacts with Phe243 and Phe129-Pro130
and the reintroduction of the sulfonamide as hydrogen bond donor
shortens the ligand distance with Ser269 and Ser311 carbonyl groups.
Figure 5. (A) Volume of MV4-11 xenograft tumors upon treatment
with 11 or vehicle control. SEM, standard error of the mean; s.c.,
subcutaneous injection; qd, once daily; bid, twice daily. (B) Body
weight of xenograft-bearing NOD-SCID mice. (C) Global
H3K79me2 normalized by total H3, measured by ELISA. All values
as percent of vehicle mean. SD, standard deviation. (D) Relative
mRNA expression of HOXA9 or MEIS1 determined by qPCR. All
values as fold vehicle mean.
eoisomer 13. After acid-catalyzed deprotection, amine 14 was
reacted in an aromatic nucleophilic substitution with the
commercially available 4-fluoro-3-nitrobenzenesulfonamide.
Reduction of the nitro group of intermediate 15 generated
the aniline used to selectively displace the first chloro
substituent of the commercially available dichloro-methoxy-
triazine to provide intermediate 16 that was submitted to a
final aromatic nucleophilic substitution with ammonia to yield
compound 11.
In conclusion, we progressed toward the aspired goal of
making DOT1L inhibitors with improved rodent PK proper-
ties compared to EPZ-5676.15 Further optimization is still
required to generate a viable clinical candidate from the series
described here. The limited efficacy obtained with compound
11 illustrates the difficulty to achieve a sustained and very
profound level of DOT1L inhibiton in vivo needed in order to
effectively suppress MLL-fusion tumor growth. When admin-
istering EPZ-5676 subcutaneously at or slightly beyond the
maximally tolerated dose in the same tumor model, no
antitumor efficacy was obtained (data not shown).
Subcutaneous administration of compound 11 was ad-
equately delivering a steady concentration of inhibitor in mice
that allowed exploring a maximally tolerated dose that led to
deep and sustained target inhibition. However, even this level
of DOT1L inhibition was not able to fully control growth of
the MV4-11 tumor model. The apparent disconnect between
substantial reduction of global H3K79 dimethylation and less
marked downstream pharmacodynamic responses, namely
D
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a
Scheme 1. Synthetic Route to Compounds 11
ACKNOWLEDGMENTS
■
́
The authors thank Flavia Adler, Celine Be, Alexandra Buhles,
Patrick Graff, Aude Izaac, Julia Klopp, Elke Koch, Corinne
Marx, Kerstin Pollehn, Paul Westwood, and Aurelie Winter-
halter for excellent technical assistance, and Anton Kessler,
Martin Klumpp and Kim Twesten for HTS assay.
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a
Reagents and conditions: (a) (R)-2-methylpropane-2-sulfinamide,
Ti(OEt)4, THF, 45 °C, 2 h, yield 92%; (b) (i) 2-bromo-3-
chloropyridine, iPrMgBr, THF, −55 °C to rt, 2 h; (ii) to 12, −50
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00452.
■
S
PK compound 10 Figure S1, selectivity HMT panel
Table S4, summary of reported activities, HTS assay
principle, and experimental procedures (PDF)
(11) Chen, C.; Zhu, H.; Stauffer, F.; Caravatti, G.; Vollmer, S.;
Machauer, R.; Holzer, P.; Mobitz, H.; Scheufler, C.; Klumpp, M.;
Tiedt, R.; Beyer, K. S.; Calkins, K.; Guthy, D.; Kiffe, M.; Zhang, J.;
Gaul, C. Discovery of Novel Dot1L Inhibitors through a Structure-
Based Fragmentation Approach. ACS Med. Chem. Lett. 2016, 7, 735−
740.
AUTHOR INFORMATION
Corresponding Author
*E-mail: frederic.stauffer@novartis.com.
ORCID
■
(12) Mobitz, H.; Machauer, R.; Holzer, P.; Vaupel, A.; Stauffer, F.;
Ragot, C.; Caravatti, G.; Scheufler, C.; Fernandez, C.; Hommel, U.;
Tiedt, R.; Beyer, K. S.; Chen, C.; Zhu, H.; Gaul, C. Discovery of
Potent, Selective, and Structurally Novel Dot1L Inhibitors by a
Fragment Linking Approach. ACS Med. Chem. Lett. 2017, 8, 338−
343.
(13) Scheufler, C.; Mobitz, H.; Gaul, C.; Ragot, C.; Be, C.;
Fernandez, C.; Beyer, K. S.; Tiedt, R.; Stauffer, F. Optimization of a
Fragment-Based Screening Hit toward Potent DOT1L Inhibitors
́
́
Frederic Stauffer: 0000-0003-0918-5147
Christoph Gaul: 0000-0001-9162-553X
Notes
The authors declare the following competing financial
interest(s): Authors are shareholders of Novartis and/or
employees of Novartis.
PyMol was used for structural visualization and figure
preparation.17
E
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