Discovery of Potent 2-Aryl-6,7-dihydro-5 H-pyrrolo[1,2- a]imidazoles as WDR5-WIN-Site Inhibitors Using Fragment-Based Methods and Structure-Based Design

Article abstract of DOI:10.1021/acs.jmedchem.8b00375

WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5H-pyrrolo[1,2-a]imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer.

Full text of DOI:10.1021/acs.jmedchem.8b00375

Subscriber access provided by TUFTS UNIV
Article
Discovery of Potent 2-Aryl-6,7-Dihydro-5H-
Pyrrolo[1,2-a]imidazoles as WDR5 WIN-site Inhibitors Using
Fragment-Based Methods and Structure-Based Design
Feng Wang, Kyu Ok Jeon, James M. Salovich, Jonathan D Macdonald, Joseph Alvarado, Rocco D. Gogliotti,
Jason Phan, Edward T Olejniczak, Qi Sun, Shidong Wang, DeMarco V. Camper, Joannes P. Yuh, J. Grace
Shaw, Jiqing Sai, Olivia W. Rossanese, William P Tansey, Shaun R. Stauffer, and Stephen W. Fesik
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00375 • Publication Date (Web): 11 Jun 2018
Downloaded from http://pubs.acs.org on June 12, 2018
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 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 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.
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 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Discovery of Potent 2-Aryl-6,7-Dihydro-5H-
Pyrrolo[1,2-a]imidazoles as WDR5 WIN-site
Inhibitors Using Fragment-Based Methods and
Structure-Based Design
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
‡
§
‡§
§
§
§
Feng Wang, Kyu Ok Jeon, James M. Salovich, Jonathan D. Macdonald, Joseph Alvarado,
§
§
§
§
§
Rocco D. Gogliotti, Jason Phan, Edward T. Olejniczak, Qi Sun, Shidong Wang, DeMarco
§
§
§
§
§
⊥
Camper, Joannes P. Yuh, J. Grace Shaw, Jiqing Sai, Olivia W. Rossanese, William P. Tansey,
Shaun R. Stauffer,#,║ and Stephen W. Fesik §,#,║,*
§
#
║
Department of Biochemistry, Department of Pharmacology, Department of Chemistry, and
⊥Department of Cell and Developmental Biology Vanderbilt University, Nashville, Tennessee
37232.
KEYWORDS: WDR5, fragment screening, structure-based design, mixed-lineage leukemia
ABSTRACT: WDR5 is a chromatin regulatory scaffold protein overexpressed in various cancers
and a potential epigenetic drug target for the treatment of mixed lineage leukemia. Here we
describe the discovery of potent and selective WDR5 WIN-site inhibitors using fragment-based
1
ACS Paragon Plus Environment

Page 3 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
methods and structure-based design. NMR-based screening of a large fragment library identified
several chemically distinct hit series that bind to the WIN-site within WDR5. Members of a 6,7-
dihydro-5H-pyrrolo[1,2-a]imidazole fragment class were expanded using a structure-based design
approach to arrive at lead compounds with dissociation constants < 10 nM and micromolar cellular
activity against an AML leukemia cell line. These compounds represent starting points for the
discovery of clinically useful WDR5 inhibitors for the treatment of cancer.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
WDR5 is part of a large family of WD40 repeat proteins1-3 that functions as a ubiquitous
scaffold protein in multiple complexes found in epigenetic machinery and chromatin regulation.4
5
Based on its role in cell cycle regulation and reported prognostic expression level disease outcome
relationships, WDR5 has become an increasingly important target of interest as a potential
6
7
treatment strategy for a number of cancers, including mixed-lineage leukemia, neuroblastoma,
8
9
10
11
breast, bladder, pancreatic, and colorectal cancer. WDR5 shares over 90% sequence identity
among all vertebrates and consists of a characteristic circular barrel shaped 7-bladed b-propeller
structure, with each blade interconnected through four stranded antiparallel b-sheets. Two major
binding sites operate as the interfaces for complex formation. Mixed lineage leukemia 1 (MLL1)
is one of the most well studied protein-protein interaction partners of WDR5 and belongs to a well
characterized SET1 family of histone methyltransferases (HMTs), that mediates histone H3 Lysine
4
(H3K4) methylation and binds to WDR5 via a conserved arginine containing motif called the
“
WIN” or WDR5 interaction motif.12-14 Pediatric acute leukemia cases that harbor MLL1-
rearrangements are caused by the chromosomal translocation of a single allele in the 11q23 MLL1
gene to one of more than 70 different partner genes and are associated with a poor prognosis.15-17
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The most common fusion partners AF4, AF9, and ENL, account for more 70% of all diagnosed
MLL leukemias which manifest as either acute lymphoid or acute myeloid leukemias.17,18
However, fusion partners lack the C-terminal catalytic SET domain with the proper required WIN-
motif and thus are devoid of histone methyltransferase activity. Interestingly, early reports
demonstrated that the wild-type MLL allele works in concert with the MLL fusion allele and is
critical for leukemogenesis and maintenance of MLL-AF9 transformed cells.19,20 Consequently,
strategies targeting the wild-type MLL complex and specifically WDR5 at the WIN-site have been
pursued as a therapeutic strategy for the treatment of MLL harboring leukemia. Understanding the
contributing factors of the MLL fusion protein and the wild-type MLL continues to be a highly
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
active area of research and debate. In addition to MLL1, other SET1 family members include
MLL2-4, SETd1A, and SETd1B. The full catalytic activity of MLL1 and SETd1A depends on a
core complex that includes WDR5, RbBP5, ASH2L, and Dpy30 (WRAD complex). Within the
WRAD complex, RbBP5 binds to the opposite side of WDR5 near the C-terminus at a shallow
hydrophobic cleft between blades 5 and 6. This interface of WDR5 is electropositive in nature and
is involved in additional important protein-protein interactions, including the MOF/HAT complex
via KANSL2,22 and a direct interaction with MYC via its MbIIIb motif.23 Peptidomimetic
compounds designed to mimic the MLL peptide residues within the WIN-site, such as MM-
26
4
0124,25 and recently MM-589 (1, Figure 1), have been shown to bind to WDR5 with low
nanomolar affinity at the WIN site, selectively inhibit MLL1 methyltransferase activity and induce
leukemia cell growth arrest in MLL-r harboring cells. The findings described with 1 indicate that
targeting the WDR5 WIN-site as a means to impact MLL-r leukemia as well as other cancer types
dependent on WDR5 may be a viable path forward.
3
ACS Paragon Plus Environment

Page 5 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
N
HN
HN
O
HN
O
O
O
HN
O
N
H
N
H
NH
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
N
F3C
O
HN
N
H
NH
1
(MM-589)
2 (OICR-9429)
WDR5 K = 64 nM
WDR5 K < 1 nM
i
i
Figure 1. Structures and properties of representative WDR5 WIN-site binders.
The first reported non-peptidomimetic class of small molecule WIN-site inhibitors of
WDR5, represented by OICR-9429 (2, Figure 1),27,28 maintains 64 nM binding affinity to WDR5
and was shown to demonstrate reduced viability of primary human AML cells bearing C/EBPα
mutations (GI50 ~5 µM). A series of analogs structurally related to 2 was recently described with
29
improved binding affinity and promising HMT inhibition, but these compounds have similar
limitations in their effects on cellular proliferation. Collectively, these results suggest that targeting
the WIN-site of WDR5 may serve as a new therapeutic approach for the treatment of leukemias
and other cancer types that depend on WDR5.
In an effort to discover more potent non-peptidic small molecules that inhibit WDR5 via
the WIN-site, we pursued a fragment-based approach to identify novel hits and used structure-
based design to optimize these hits for binding to WDR5. Our NMR-based fragment screen yielded
multiple classes of hits. To determine how fragment hits bind to WDR5, we obtained crystal
structures of the fragments bound to WDR5. On the basis of their potential drug-likeness and
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ligand efficiency, the most promising series of compounds were chosen for subsequent structure-
based optimization. Optimized compounds were evaluated in cells harboring MLL-rearrangements
for their anti-proliferative activity and for their functional inhibition of MLL1 methylation activity
of nucleosomes in the presence of the purified WRAD MLL1 complex. This work resulted in the
discovery of potent non-peptidic inhibitors of WDR5.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RESULTS AND DISCUSSION
1
5
Hit Identification. Recombinant, N-labeled WDR5 (residues 22-334) was used to screen our
1
15
fragment library (>13,800 compounds) by recording SOFAST H- N HMQC spectra of WDR5.
15
An initial HMQC spectrum of uniformly N-labeled WDR5 in complex with an unlabeled MLL1
1
0mer peptide Ac-ARTEVHLRKS-NH was obtained (Figure 2A), which showed peak shifts
2
corresponding to amino acids in regions specific for the WIN-site of WDR5. To conduct the
screen, the WDR5 protein was incubated with mixtures of 12 fragments. Fragment mixtures that
caused similar peak shifts as the MLL1 peptide were identified as MLL WIN-site hits as illustrated
in Figure 2B for a representative fragment mixture. Deconvolution of the mixtures by screening
individual compounds generated 47 hits (0.34% hit rate), which can be divided into 12 classes
based on their chemical scaffolds. Of the initial hits, 14 fragments that induced the largest chemical
shift perturbations were selected for affinity determination by fluorescence polarization assay.
Four fragments showed K values lower than 0.5 mM, and the other 10 were between 0.5 to 1 mM.
i
Measurable fragment hits demonstrated ligand efficiency indices (LE) of between 0.20–0.39. A
representative list of fragments is shown in Figure 3 and the remaining hits can be found in
Supplemental Figure S1. Not surprisingly, many of the hits contained an arginine mimetic. For
example, fragment hits F-2 through F-4 bear a basic amidine motif (calculated pK ~11) with NMR
b
based affinities between ~550–850 µM. The phenoxy ethers (F-2 and F-3) exhibited slightly
5
ACS Paragon Plus Environment

Page 7 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
improved affinities over biaryl F-4. Fragment hits F-1, F-5, and F-6 represent a distinct and
interesting class and share a common bicyclic imidazole containing core. Fragment F-1 possesses
a K
expected to have inherently weaker basicity relative to the amidine class (calculated pK
.9). The dihydro-pyrrolo-imidazole fragment F-1 was particularly interesting to us for two
reasons: 1) F-1 represented the most potent member of the imidazole and amidine class and 2) F-
maintained excellent ligand efficiency with no inherent H-bond donors. Therefore, based on
i
value of 323 µM, and a relatively high LE of 0.34. Interestingly, the cyclic imidazole class is
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
’s 5.4–
5
1
structural novelty as a putative amidine mimetic and with potentially superior drug-like properties
versus traditional amidine mimetics (e.g. reduced H-bond count and weak basicity), we prioritized
fragment series F-1 for our initial structure-based design optimization campaign.
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
15
Figure 2. H- N HMQC spectra of WDR5 with (red) and without (black) added ligand: A) WIN-
peptide and B) representative fragment mixture. The NMR sample contained 2 mg/mL (~60 µM)
15N-labeled protein and 12-compound mixture of 670 µM of each ligand.
X-ray Structure of Fragment Co-Complex F-1 and WDR5. Figure 4A depicts an X-ray co-
crystal structure of the fragment F-1 bound to WDR5. The cyclic imidazole group of F-1 binds to
WDR5 deeply in the central S2 pocket, where it normally accommodates the MLL1 peptide R3765
13
2
guanidine side chain. It is noted that the sp -pyrrolidine moiety is not as puckered as observed in
7
ACS Paragon Plus Environment

Page 9 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the energy-minimized unbound ligand structure due to limitations of the X-ray diffraction
resolution and the Ligand Building and Optimization Workbench (eLBOW) program, which is
used for generating non-standard ligands in Phenix. A WDR5 X-ray co-crystal structure with an
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MLL bound peptide with the major sub-pockets labeled is shown for reference in Figure 4B.12,30
As previously shown, this S2 interaction greatly contributes to the total binding affinity. A
comparison of the F-1/WDR5 and MLL1-peptide arginine side-chain interactions with WDR5 is
shown in Figures 4C and 4D. In the case of F-1, the cyclic imidazole group stacks with the aromatic
ring of F133 and F263, and the N3 imidazole nitrogen of F-1 appears to interact with the backbone
C=O of C261 through an H-bond with an inter-atomic distance of 2.8 angstroms (Figure 4C).
These interactions drive complex formation. The phenyl group of F-1 exits the S2 channel, towards
the solvent and upper rim region, which can be utilized as a handle to grow the fragment toward
sub-pockets within the central cavity that are normally occupied by the MLL1 peptide.
N
HN
O
HN
O
N
H N
H N
Cl
2
2
F-1
F-2
F-3
K = 323 µM
K = 589 µM
K = 651 µM
i
i
i
LE = 0.35
LE = 0.38
LE = 0.36
HN
N
N
OH
O
H N
S
N
N
2
F-4
F-5
F-6
K = 843 µM
K > 1000 µM
K > 1000 µM
i
i
i
LE = 0.28
Figure 3. Representative hit fragments that bind to WDR5 at the MLL-site with putative arginine
(S2) mimics in blue. K
i
values were obtained by Fluorescence Polarization Assay.
8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. X-ray co-crystal structure of fragment F-1 bound to WDR5 (PDB: 6D9X) and the
reference MLL1 peptide bound to WDR5 (PDB: 3EG6) showing respective unoccupied and
occupied pockets: A) F-1 left panel top view surface depiction; B) right panel top view MLL1
peptide surface depiction (only Ac-ARA-NH
2
residues shown, yellow capped sticks); C) F-1 lower
left panel, key residues and hydrogen bonding within S2 pocket (orange capped sticks); D) lower
right panel MLL1 peptide (only Ac-ARA-NH
2
shown) key residues and hydrogen bonding within
S2 pocket (blue capped sticks).
9
ACS Paragon Plus Environment

Page 11 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
S5 and S4 Pocket Optimization. Based on the bound structure of the F-1 fragment hit, our initial
efforts focused on growing from the pendant phenyl to explore occupation of the nearby upper rim
3
0
S5 or S4 pockets of the WIN-site (Figure 4A). Both pockets are hydrophobic in nature and based
on earlier studies involving peptidomimetics, these pockets contribute significantly to the WIN-
peptide WDR5 interaction.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Optimization/SAR of aryl substitution pattern.
Ar
N
N
3
a
b
Cmpd
Ar Structure
FPA K
i
(µM)
LE
F-1
323
8.44
61
0.34
0.35
0.36
0.30
0.33
ND
3a
3
b
3
c
118
3
d
29.7
>125
64.3
1.12
85.2
64.0
117
3
e
3f
0.26
0.37
0.31
0.34
0.34
3
3
g
h
3
i
3
j
1
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3k
109
0.30
ND
3
l
>125
aK
values represent the average of two or more independent determinations conducted in replicate
i
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
(CV < 0.3). Ligand efficiency index, LE = 1.4*pK
i
/ HAC.
A high throughput FPA binding assay using MLL-derived fluorescent probes, similar to
6
that reported by Wang and co-workers, was adopted to assess the binding affinity of analogs
relative to F-1. Previously reported MLL peptides, including tripeptide Ac-ART-NH were utilized
2
as initial controls and the data were fit to standard four-parameter curve fit (see Experimental and
SI for further details). Independent experiments (n of 2 or more) were performed in replicate to
generate the final average K
i
value. Each assay plate performed with an average Z’ value >0.5 and
the average K values reported fall within a 30% variance. The SAR and ligand efficiency (LE)
i
metrics of the resulting compounds can be found in Table 1. Our efforts focused on amide, ether,
and biaryl analogs using either the meta or para position to expand into the neighboring S5 and
S4 pockets. As shown in Table 1, a simple para substituted biaryl 3a led to a ~40-fold increase in
affinity with a K of 8.4 µM. The difluoro congener 3g, gave a further 7.5-fold boost in affinity,
i
representing the most potent analog from this initial effort. Ligand efficiencies for both 3a and 3g
were well maintained. Aryl ethers designed to probe the S5 and S4 pockets, including the para
congeners 3b, 3d, 3e, and 3f demonstrated a preference for small, branched hydrophobic
substituents (e.g. 3d i-PrO K
tolerated, were not as favorable (MeO 3j, i-PrO 3k, K
efficiency in the case of 3k. N-anilido acetamides 3c and 3l were unproductive as polar moieties;
i
= 29.7 µM, LE = 0.33). Similar meta-substituted analogs, although
i
> 100 µM) leading to an erosion of ligand
1
1
ACS Paragon Plus Environment

Page 13 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
however, bicyclic heterocycles 3h and 3i were moderately effective, and N-methyl indoline 3h
was sufficiently soluble and potent to obtain a subsequent X-ray co-crystal structure bound to
WDR5. As shown in Figure 5, we achieved partial occupation of the S4 pocket, in which the N-1
methyl group of 3h was in close proximity to A47 and A65. Importantly, the indoline ring
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
backbone was found to point away from the S5 and S4 pockets with potential sp carbon vectors
toward the unoccupied S1 or S7 pockets.
Figure 5. X-ray co-crystal structure of fragment analog 3h (magenta capped sticks) bound to
WDR5 with surface display and unoccupied S1 and S7 pockets labeled along with S4 pocket A47
and A65 residues highlighted (atom color capped sticks) (PDB: 6DAI).
S7 Pocket Optimization. In an effort to further improve binding affinity, we wished to incorporate
an amide or similar polar bioisostere from the pendant phenyl to engage the WIN-site in additional
backbone hydrogen bonding interactions while also providing a linker and vector to reach the
neighboring S7 and potentially S1 pockets (Figure 4A).
1
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Optimization/SAR of meta-aminomethyl amides.c,d
R1
N
O
N
R2
N
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
b
Cmpd
R
R
FPA K
i
LE
a
(µM)
4a
H
7.21
0.29
4b
H
H
7.54
0.29
0.32
4
c
0.665
4
d
H
2.60
0.31
4
e
H
H
3.13
17.6
0.29
0.26
4
f
4
g
H
H
H
H
H
0.118
0.690
0.095
0.184
3.91
0.35
0.30
0.36
0.34
0.31
4
h
4
4
i
j
4
k
4
l
H
H
H
4.13
1.38
0.31
0.33
0.33
4
m
4
n
0.924
4o
CH
3
0.473
0.503
0.219
1.26
0.32
0.30
0.30
0.26
0.29
4
p
-i-Pr
c
4
4
q
2
-CH -c-Pr
d
r
-CH
2
-c-Bu
c
4
s
-CH
2
-c-Pr
0.480
1
3
ACS Paragon Plus Environment

Page 15 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
K
i
values represent the average of two or more independent determinations conducted in replicate
b
c
d
(
CV < 0.3). Ligand efficiency index, LE = 1.4*pK / HAC. c-Pr = cyclopropyl, c-Bu =
i
cyclobutyl.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In addition to the well conserved hydrogen bonding interactions found between key S2
residues and the arginine side chain of reported MLL and H3 peptide complexes, an additional
conserved water-mediated hydrogen bond can be found with the backbone NH of C261. Multiple
docking studies suggested this interaction could be targeted directly from F-1 while providing a
suitable linker to reach the S7 pocket. Thus, based upon our docking studies, we pursued a series
of tolyl amides from the meta-position of F-1 rather than the para-position, which initially
appeared to have a more appropriate vector, due to the capacity to participate in a direct H-bond
with C261 while also reaching the S7 pocket.
SAR results from a survey of meta-tolyl amides (both secondary and tertiary) are shown in
Table 2. Docking suggested that benzamide 4a might engage C261 as predicted and occupy the S7
pocket; a region of WDR5 partially occupied by the MLL1 peptide H3769 imidazole side-chain
which is defined by the aromatic residues F133, F149, and Y191. Inhibitor 4a afforded modest
inhibition of WDR5 with a K
observed level of overall binding affinity was deemed encouraging considering the lack of success
from simple meta-substituted analogs explored previously (Table 1, meta examples K > 100 µM,
i
= 7.21 µM and although this resulted in a slight loss in LE, the
i
e.g. 3l). With this finding in mind, an X-ray co-crystal structure of 4a bound to WDR5 was
obtained to confirm the binding orientation (Figure 6).
1
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. X-ray co-crystal structure of 4a bound to WDR5 (PDB: 6DAK): A) 4a (blue carbon
capped sticks) with semi-transparent surface display with key S7 residues and unoccupied pockets
highlighted; B) 4a engaged in bidentate-type H-bond interaction with C261 (white carbon capped
sticks).
The structure revealed that 4a is indeed bound in an orientation with the amide engaging
in a direct H-bond with C261. The C261 backbone C=O N-(3) imidazole nitrogen and the 4a amide
C=O C261 NH interaction maintain interatomic distances of 2.6–2.9 Å (Figure 6B). The
unsubstituted benzamide phenyl ring of 4a was found to be nearly co-planar with the amide and
was readily accommodated in a groove defined by the S7 site defined by K259 and Y260 residues
between the blade 5 and 6 loop structure. Further exploration of the secondary amide SAR
indicates that the saturated cyclohexyl amide 4b was similarly tolerated. Secondary benzamide
SAR of the meta and para positions within mono or disubstituted congeners (4c–e, 4h–j) exhibited
a preference for a 3,4-disubstitution pattern affording the first fragment-based analog with an
affinity below 100 nM (4i, K
i
= 95 nM). Notably the LE index significantly increased within this
series of analogs vs. 4a, suggesting a favorable interaction between the ligand and protein,
1
5
ACS Paragon Plus Environment

Page 17 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
particularly for inhibitors 4c and 4i (LE >0.31). Unsubstituted pyridyl (4k–4m) and 5-methyl
substituted pyridyl analogs (4n) were 2–7 fold more potent vs. phenyl 4a. Acetamide 4f displayed
weaker affinity vs. 4a (2.4-fold). Interestingly, within the acetamide backbone, the 3,4-dichloro
derivative 4g provided a significant, more than two orders of magnitude boost in affinity (~150-
fold). Tertiary amides were also explored in an attempt to develop a vector towards the neighboring
S1 and S4 pockets. Methyl (4o) and isopropyl (4p) congeners afforded only slight improvements
in potency (less than 2-fold). Larger cycloalkyl methyl derivatives (4q–r) were also examined,
with cyclopropyl methyl 4q retaining a favorable LE and overall 3-fold improvement vs. 4c. In an
attempt to reduce lipophilicity, the 2-methoxy pyridyl analog 4s was found to be tolerated and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
retained binding affinity (K < 500 nM).
i
In an effort to understand the preferred bound orientation of the cyclopropyl methyl of 4q,
an X-ray co-crystal structure of this inhibitor was obtained when bound to WDR5. From this
structure, we hoped to further define a strategy to access one of the three upper rim pockets;
potentially S1, S5, or S4. Figure 7 depicts a solvent accessible surface displaying 4q bound to
WDR5 at the WIN-site. Two key structural features are noted in this structure relative to that of
secondary benzamide 4a. First, the 3-methoxybenzamide ring binds in an orthogonal conformation
now optimally filling the S7 pocket through favorable contacts with F133, F149, and P173.
Secondly, the cyclopropyl methyl does not appear to reach any of the targeted neighboring pockets,
but rather reinforces the low-energy orthogonal benzamide conformation and engages in contacts
with neighboring Y260. This result is consistent with the SAR and indicated that an alternative
approach would be required to target the remaining upper rim pockets.
1
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. X-ray co-crystal structure of 4q bound to WDR5 (grey surface display) revealing
orthogonal benzamide conformation filling S7 pocket and unoccupied S1, S5, and S4 pockets
(
PDB: 6DAR).
Combining S4 and S7 Binding Elements. In an effort to target the nearest S4 pocket, we revisited
series 3 (Table 1) and specifically the crystal structure involving 3h to apply a mix-and-match
strategy in an attempt to co-occupy the S7 and S4 pockets through additional substitution of the
central phenyl core. Results from this approach for a series of 2-substituted-1-benzyl-acetamides
are found in Table 3.
ꢀ
1
7
ACS Paragon Plus Environment

Page 19 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 3. Optimization/SAR of 3,4-disubstituted amides.c
3
R3
2
R1
N
O
1
N
R2
N
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
b
F
R
R
R
FPA K
i
LE
a
(µM)
5a
-CH
-CH
-CH
-CH
2
2
2
2
-c-Pr
-c-Pr
-c-Pr
-c-Pr
3-OMe
2-OMe
1.46
0.25
0.29
0.31
0.30
0.32
0.30
0.31
0.32
0.33
0.32
0.35
0.31
0.37
0.29
0.32
0.33
5
b
0.148
5c
2-OEt
0.0425
0.0276
0.0761
0.0228
0.105
5
d
2-O-i-Pr
2-O-i-Pr
2-O-c-Bu
2-O-c-Bu
5
e
f
H
5
-CH
2
-c-Pr
5
5
g
H
H
H
H
H
h
2-N(CH
2-N(CH
3
3
)
)
2
2
0.185
5
i
0.0519
0.0207
0.00989
0.0131
0.00282
0.0289
0.00353
0.0164
5j
(
1,2)
5
k
2-O-i-Pr
5l
-CH
2
-c-Pr
2-O-i-Pr
5
m
H
2-O-c-Bu
2-O-c-Bu
2-O-c-Bu
2-O-c-Bu
5n
-CH
2
2
-c-Pr
5
5
o
-CH
-c-Pr
p
H
1
8
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aK
values represent the average of two or more independent determinations conducted in replicate
i
b
c
(
CV < 0.3). Ligand efficiency index, LE = 1.4*pK
i
/ HAC. c-Pr = cyclopropyl, i-Pr = isopropyl,
c-Bu = cyclobutyl.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Employing the prior SAR from the first series (Table 1), methoxy substitution at either the
3
-position (5a, 7-fold loss vs. 4q) or 2-position (5b) confirmed a preference for a 1,2 disubstitution
pattern as predicted (5b K = 148 nM) based on the structure involving 3h. In the context of the
i
tertiary amide 5b, ether homologs and small cycloalkyl derivatives were examined at the 2-
position in order to more optimally fill the S4 pocket. Results from this study demonstrated an
orderly improvement in affinity from 5c (O-Et, 3.5-fold improved vs. 5b) to 5d (O-i-Pr, 5.5-fold
improved vs. 5b), demonstrating the first examples of inhibitors within the F-1 series with
affinities less than 100 nM. O-Cyclobutyl derivative 5f was comparable in affinity to 5d. Within
the secondary amide series, a similar trend was noted favoring O-cyclopropyl (5e K
i
= 76 nM vs.
5g K =105 nM). Alternate 2-amino dialkyl derivatives designed to fill the proximal S4 pocket
i
were also examined and were generally less favored. For example, N,N’-dimethyl 5h was 2.4-fold
less potent relative to O-isopropyl congener 5e. Hybrid analogs of the homologated 3,4-
dichlorophenyl acetamide identified within series 4 (e.g. 4g K = 118 nM) furnished secondary
i
amides 3i–3k, and 3m. Addition of S4 binding within the acetamide series proved to be additive.
Once again, small alkyl and cycloalkyl groups were preferred with affinity maintained below 100
nM. Interestingly, O-cyclobutyl exhibited the highest affinity within the dichloroacetamide series
and the overall highest LE (secondary amide 5m K = 2.8 nM). Conformational analysis indicated
i
that the additional strain within the tertiary acetamide was likely contributing to the divergent SAR
observed upon cyclopropyl methyl introduction (e.g. 5m vs 5n, 10-fold loss). Returning to the
1
9
ACS Paragon Plus Environment

Page 21 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
benzamide 3,4-disubstituted series (5o-5p), inhibitor 5o demonstrated the expected additive SAR
of the tertiary amide, affording an inhibitor with single digit nanomolar potency, comparable to
5m.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Further Optimization via the Incorporation of Conformational Constraints. Based on the
counterproductive results obtained from the tertiary acetamide class, lack of direct access to S4
pocket from the amide, and the general steric congestion of 1,2-disubstituted systems within series
5
, we designed a series of cyclic constraints intended to simultaneously occupy the S4 pocket and
restrict the benzylic methylene closer to the bioactive conformation in order to optimally position
the amide toward the S7 pocket. Our basic strategy is outlined in Figure 8 with the bound
conformation of 4a shown for reference.
ring
S4/S5
4
a bound
conformation
Figure 8. Strategy constraining the bioactive conformation 4a.
Select inhibitors and resulting profiles from this optimization campaign are shown below
in Figure 9. Using this strategy, compound 6a was designed and synthesized resulting in a ~50-
fold improvement in binding affinity based on the K determination relative to the direct acyclic
i
comparator 4g. Moreover, benzamide indane 6b resulted in a ~20-fold improvement in affinity
relative to comparator 4i. Based on the level of enhanced potency achieved at this juncture and the
2
0
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
reported and confirmed K
FPA assay was expected to have an IC50 and resulting K
~1 nM). To circumvent this issue we developed a stable Anti-His TR-FRET assay to allow for
improved sensitivity below the nanomolar level in order to rank compounds based on potency (see
d
of the FITC-10mer peptide developed by the Wang laboratory, the
i
limit near 0.5-1 nM (10mer-Thr probe
K
d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental and SI for details). The FPA and TR-FRET determined K
i
values for series 6 analogs
are shown in Figure 9 for comparison.
N
O
O
N
N
HN
N
HN
O
HN
N
N
Cl
OCH3
OCH3
Cl
6
a
6b
FPA K = 4.94 ± 0.79 nM
6c
FPA K = 9.36 ± 1.97 nM
TR-FRET K = 26.8 ± 1.78 nM
FPA K = 2.51 ± 0.62 nM
i
i
i
TR-FRET K = 2.66 ± 0.43 nM
TR-FRET K = 8.37 ± 1.85 nM
i
i
i
LE = 0.40
LE = 0.38
LE = 0.35
MLL1 HMT IC50 = 9.27 ± 0.43 µM
d MV4;11 GI50 = 8.89 ± 0.55 µM
MLL1 HMT IC50 = 12.0 ± 0.5 µM
7d MV4;11 GI50 = 17.2 ± 3.5 µM
MLL1 HMT IC50 > 20 µM
7d MV4;11 GI50 = 22.3 ± 4.7 µM
7
O
N
O
N
HN
HN
N
N
OCH3
N
H CO
3
6
d
6e
FPA K = 65.1 ± 5.86 nM
FPA K < 1.00 nM
i
i
TR-FRET K = 182 ± 38.1 nM
TR-FRET K = 0.902 ± 0.162 nM
i
i
LE = 0.34
LE = 0.40
MLL1 HMT IC50 > 20 µM
d MV4;11 GI50 > 30 µM
MLL1 HMT IC50 = 3.20 ± 0.13 µM
7d MV4;11 GI50 = 7.25 ± 1.9 µM
7
Figure 9. Indane and tetrahydronaphthalene constrained analogs profile summary.
Expansion of the indane core to a tetrahydronaphthalene resulted in a modest 2–3-fold loss
in affinity. Introduction of a 2-substituted pyridine ring in the S7 as before gave an inhibitor with
2
1
ACS Paragon Plus Environment

Page 23 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
modest affinity suggesting that alternate sites for pyridine ring incorporation will be necessary in
order to modulate the physicochemical properties while maintaining potency. The pseudo-
symmetrical benzamide 6e was the most potent inhibitor from this effort, bottoming out the prior
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
FPA assay and giving a K
i
of 0.90 nM in our TR-FRET assay. Inhibitor 6e represents an overall
~360,000-fold improvement in affinity relative to starting fragment F-1. In an effort to develop
refined heterocyclic design elements within S7 and confirm the binding orientation and
stereochemistry of the indane ring system, an X-ray co-crystal structure of 6b bound to WDR5
was determined. Shown in Figure 10 is a solvent accessible surface of 6b within the WIN binding
site that highlights the amino acid residues of the S4 and S7 pockets.
Figure 10. X-ray co-crystal structure surface display of 6b bound to WDR5 at the WIN-site. The
residues that form the S7 and S4 pockets are labeled (PDB: 6DAS).
Structure 6b confirmed the pre-oriented R-configuration for the benzylic benzamide as well
as favorable interactions within S7 between the 3-methoxy-4-methyl groups and the F149, P173,
2
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and Y191 residues. In the S4 pocket the indane displayed favorable interactions with L321 and
I305 residues.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Biological Activity. Since the WDR5-MLL1 interaction requires the full WRAD complex to
achieve robust H3K4 histone methyl transferase (HMT) activity, compounds that target this
interaction are expected to inhibit methylation activity. Assessment of WRAD-mediated MLL1
H3K4 methylation thus serves as a useful orthogonal and functional measure of PPI inhibition of
a native WDR5 containing multi-protein complex. To this end, potent compounds from series 6
were profiled for their functional inhibition of the WRAD mediated MLL1 H3K4 histone methyl
transferase (HMT) activity. HMT activity studies were performed at Reaction Biology Corp. using
purified recombinant human MLL1 complex (MLL1 aa 3745-3969 plus WRAD), purified HeLa
oligonucleosomes as substrate, and S-adenosyl-L-[methyl-3H]methionine (SAM) as the
3
1
methylation cofactor. MLL1 HMT inhibition potency rank order tracked with affinity rank order
6e > 6a > 6b >> 6c-6d); even though weak potency was observed with IC50 values in the
micromolar range. A large functional to biochemical shift of ~2800-fold (HMT IC50 / TR-FRET
(
i
K ) based on the TR-FRET competition assay was observed.
Compounds from the bicyclic series were also profiled for their cellular anti-proliferative
activity using human acute leukemia MV4-11 cancer cells. Similarly, a significant shift was
observed in a 7-day anti-proliferative growth inhibition assay, with measured GI50 values of 7 µM
or greater (rank order 6e ~ 6a > 6b > 6c-6d). Importantly, compound 6e was found to have
reasonable intrinsic permeability in a MDR1 expressing MDCK cell line (A-B Papp = 9.6 * 10-6
cm/sec) with no apparent active efflux (B-A/A-B ratio = 1.22), indicating that cellular penetration
2
3
ACS Paragon Plus Environment

Page 25 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
32
is unlikely a barrier that would account for the weak cellular activity observed. Based on the
suboptimal functional inhibition and weak cellular activity demonstrated by analogs 6a–6e, we
concluded that compounds with much better binding affinity will be required in order to achieve
adequate functional and cellular inhibition of the fully intact multimeric WDR5-containing protein
complex from this imidazole-based series of inhibitors. The origin of the functional and cellular
shifts observed is not fully understood at this time; however, the permeability exhibited by 6e and
the comparable cellular and functional activity across the examples tested suggest perhaps the
disconnect is more likely related to the artificial nature of the equilibrium binding assays and or a
property inherent to the scaffold. For example, the FPA and TR-FRET binding assays are
conducted using purified non-complexed WDR5. In contrast, in the HMT and cellular context the
inhibitor ligand must compete against the multi-valent WRAD complex in HMT, as well as
WRAD and additional protein partners within the full cellular milieu. Secondly, it conceivable that
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
physicochemical properties, such as basicity of the S2 cyclic imidazole for example, may not be
fully optimal for performing in a more native context or reaching WDR5 efficiently within the
nuclear target organelle. Recently, Wang and co-workers described a series of highly potent and
basic guanidine containing S2 peptidomimetics which achieve sub-nanomolar affinity with
2
6
functional HMT inhibition below 100 nM. Interestingly, they also exhibit a large functional to
biochemical shift (MLL1 IC50 / K ~300–600); however, the shift is somewhat lower than that
i
observed for the imidazole S2 system described here.
Chemical Synthesis. Synthesis of final compounds in Tables 1 and 2 began from 2-(2-
oxopyrrolidin-1-yl)acetamide 7 (Scheme 1). Cyclization using phosphoryl bromide or chloride
gave key cyclic haloimidazole intermediates 8a–b. Treatment of 8a or 8b under Suzuki cross-
2
4
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
coupling conditions using catalytic PdCl
2
(dppf).CH
2
Cl
2
in a binary acetonitrile-water solvent
mixture with aqueous K CO affords 3a-3l or precursors 9–10. Deprotection of 9a afforded benzyl
2
3
amine 9b in quantitative yield. Acylation readily afforded final compounds 4a–4n. In order to
obtain tertiary amides 4o-4s reductive amination was performed using aldehyde 10. The resulting
intermediate amides were then treated with sodium hydride in DMF with various alkylating agents
allowing access to tertiary amides 4o-4s.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Synthesis of precursor 8 and series 3-4.
H N
2
O
a
N
b,c
N
O
X
Ar
N
N
N
7
8a (X = Cl)
3a-3l
8
b (X = Br)
d
g
N
N
N
N
NHR
CHO
9a (R = Boc)
9b (R = H)
1
0
e
f
h,f
i
N
N
N
N
R1
NH
O
N
R2
R2
O
1
4a-4n
4o-4s (R = alkyl)
(a) POBr3, 70 ˚C, 1 h or POCl
ArBpin, CH CN, aq. K CO , 95 ˚C, 1–16h; (c) 3e-3f phenol alkylation: RX, CsCO
h or 3c, 3l aniline acetylation: acetyl chloride, CH Cl , rt, 2 h; (d) b using 3-(N-Boc-
3
, 85 ˚C, 15 h (65–70%); (b) PdCl
2
(dppf)CH
2
Cl
2
, ArB(OH)
2
or
3
2
3
3
, DMF, rt, 12
2
2
2
5
ACS Paragon Plus Environment

Page 27 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
aminomethyl)phenylboronic acid (96%); (e) TFA, CH
2
Cl
2
(quant.); (f) R COCl, Et
3
N, CH
2
Cl , rt,
2
2
2
h or R CO
2
H, HATU, DMF, rt, 16–40 h; (g) b using 3-formylphenylboronic acid (47%); (h)
1
1
R NH
2
, NaBH(OAc)
3
, HOAc, DCE, rt, 2 h; (i) NaH, R X, DMF, 0 ˚C to rt, 1 h.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of compounds in Table 3 begins similarly from bromide 8b (Scheme 2). Suzuki cross-
coupling with aldehydes 11 affords biaryl intermediates of type 12. Reductive amination and
acylation affords specified compounds of series 5. Alternatively, nitriles 14 were utilized to afford,
after coupling and reduction, primary amines of type 15. Final acylation proceeds smoothly to give
the remaining specified analogs 5.
Scheme 2. Synthesis of series 5 analogs.
R3
R3
N
N
a
b
Br Bpin
N
N
CHO
CHO
8
b
11
12
R3
c
R3
N
N
N
N
R1
R1
NH
N
1
3
R2
5
5
a-5d, 5f 5e, 5g-5k
l, 5n-5p 5m, 5p
O
(From 12) (From 15)
c
R3 d,e
R3
N
Bpin
8
b
N
CN
NH2
1
4
15
1
(a) PdCl
2
(dppf)CH
2
Cl
2
, ArB(OH)
2
or ArBpin, CH
3
CN, aq. K
2
CO
3
, 95 ˚C, 1–16 h; (b) R NH
2
,
2
2
NaBH(OAc)
3
, HOAc, DCE, rt, 2 h; (c) R COCl, Et
3
N, CH Cl , rt, 2 h or R CO
2
2
2
H, HATU, DMF,
2
6
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 77
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
rt, 16–40 h; (d) PdCl
2
(dppf)CH
2
Cl
2
, ArB(OH)
2
or ArBpin, CH
3
CN, aq. K
2
CO , 95 ˚C, 1–16 h; (e)
3
LiAlH , THF, 0–60 ˚C.
4
Synthesis of compounds in the indane and tetrahydronaphthalene series employed an
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ellman sulfinamide reduction strategy to set the benzylic stereochemistry.34,35 Based upon the
structural information provided from X-ray complexes 4a and 4q the R-configuration was targeted
via the appropriate auxiliary.
Scheme 3. Synthesis of 6a–6e.
N
a
N
Br
Bpin
N
n
N
n
O
8
b
O
1
1
6a (n = 1)
6b (n = 2)
17a (n = 1)
17b (n = 2)
N
N
b,c
N
n
d
N
n
HN
H N
2
1
1
8a (n = 1)
8b (n = 2)
S
19a (n = 1)
19b (n = 2)
bis HCl
O
N
N
n
e
HN
O
6a-6e
R
(
a) PdCl
2
(dppf)CH
2
Cl
2
, ArB(OH)
2
or ArBpin, CH
3
CN, aq. K
2
CO , 95 ˚C, 1–16 h; (b) (R)-(+)-2-
3
methyl-2-propanesulfinamide, Ti(OEt)
4
THF, reflux, 16 h; then (c) NaBH
4
, THF, -60 ˚C to rt (58-
2
2
6
2%); (d) 4.0 M HCl, MeOH, rt (95-97%); (e) R COCl, Et
3
N, CH
2
Cl
2
, rt, 2 h or R CO
2
H, HATU,
DMF, rt, 16–40 h.
2
7
ACS Paragon Plus Environment

Page 29 of 77
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The construction of the target compounds proceeds with the pinacol boryl indanone (16a)
or tetralone (16b) and a Suzuki cross-coupling using 8b to install the S2 cyclic dihydro-pyrrolo-
imidazole moiety resulting in 17a and 17b. Condensation of each ketone with (R)-(+)-2-methyl-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
-propanesulfinamide using Ti(OEt) afforded the chiral sulfinamides, which upon reduction in
4
the same pot, gave diastereomers 18a and 18b in good yield and high purity (>97% diastereomeric
excess by NMR) after column chromatography. Subsequent deprotection and acylation furnished
the desired constrained enantiopure analogs 6a–6e.
CONCLUSION
In summary, we have discovered potent WIN-site WDR5 inhibitors using a fragment-based
approach. Optimization of the fragment hits was rapidly achieved utilizing multiple co-crystal
structures to guide design resulting in several potent inhibitors. The imidazole inhibitors displayed
single digit nanomolar inhibition displacing WIN peptides from binding to WDR5, demonstrate
dose-dependent inhibition of H3K4 methylation activity, and display moderate growth inhibition
against an MLL-r harboring cell line. The most potent inhibitor described (6e), represents an
overall ~360,000-fold improvement in affinity relative to starting fragment F-1. The key dihydro-
pyrrolo-imidazole that binds in the S2 pocket lacks an intrinsic hydrogen bond donor, maintains
less than five rotatable bonds, and displays a favorable ligand efficiency and molecular weight
profile (LE > 0.35, MW < 450). This work highlights the power of unbiased fragment-based
screening and the resulting identification of a novel dihydro-pyrrolo-imidazole substructure as an
arginine side-chain mimetic for the S2 channel. Prior WDR5 WIN-site inhibitors such as 1 and 2
are currently limited to S2 binders bearing either highly basic guanidines or piperidine
substructures; both of which potentially face challenges associated with cell permeability and/or
2
8
ACS Paragon Plus Environment