Development of novel Asf1-H3/H4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.11.067

The histone chaperone anti-silencing function 1 (Asf1) has emerged as a promising target for therapeutic intervention for multiple cancers (Cell 2006, 127, 458). Asf1 is involved in the packaging of the eukaryotic genome into chromatin, which is essential

Full text of DOI:10.1016/j.bmcl.2014.11.067

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Development of novel Asf1-H3/H4 inhibitors
Greg F. Miknis, Sarah J. Stevens, Luke E. Smith, David A. Ostrov, Mair E.A.
Churchill
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http://dx.doi.org/10.1016/j.bmcl.2014.11.067
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Bioorganic & Medicinal Chemistry Letters
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Please cite this article as: Miknis, G.F., Stevens, S.J., Smith, L.E., Ostrov, D.A., Churchill, M.E.A., Development
of novel Asf1-H3/H4 inhibitors, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/
j.bmcl.2014.11.067
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Development of novel Asf1-H3/H4 Inhibitors
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Greg F. Miknisa, Sarah J. Stevensa, Luke E. Smith b, David A. Ostrovc , and Mair E.A. Churchillb

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Development of novel Asf1-H3/H4 inhibitors
Greg F. Miknisa*, Sarah J. Stevensa, 0F Luke E. Smithb, David A. Ostrovc, Mair E.A. Churchillb
aColorado Center for Drug Discovery, Colorado State University, Department of Chemistry, Fort Collins, CO 80523-1872, USA
b Department of Pharmacology and the Program in Structural Biology and Biochemistry, University of Colorado School of Medicine, Aurora, CO 80045, USA.
cDepartment of Pathology, Immunology and laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610-3633, USA
Corresponding author. Tel.: 970-491-8601; e-mail: Greg.Miknis@Colostate.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The histone chaperone anti-silencing function 1 (Asf1) has emerged as a promising target for
therapeutic intervention for multiple cancers.1 Asf1 is involved in the packaging of the
eukaryotic genome into chromatin, which is essential for normal growth, development, and
differentiation, as this regulates all nuclear processes that use DNA as a substrate. Starting from
a collection of HTS leads, we identified a series of N-acyl hydrazones as novel inhibitors of the
Asf-histone H3/H4 interaction. These compounds represent the first example of inhibitors
capable of disrupting the Asf1-H3/H4 complex.
Keywords:
Asf1
Histone chaperone
hydrazone
H3/H4
2009 Elsevier Ltd. All rights reserved.
Drug discovery
Introduction
Recently crystal structures of Asf1 from Saccharomyces
cerevisiae and human Asf1a in complex with a dimer of H3/H4
were reported.6, 7
Interestingly, as depicted in Figure 1, the
4F
5F
The histone chaperone anti-silencing function one (Asf1) has
emerged as a promising target for therapeutic intervention for
structures revealed one molecule of Asf1 binds to the H3/H4
dimer via a small pocket on the concave surface of the Asf1 that
binds the leucine-arginine-isoleuucine (leu-arg-ile) Ig-like region
at the histone N-terminus that is highly conserved among
eukaryotes.
multiple cancers.1,2, 3,4
Asf1 is involved in the packaging of the
1F
2
3F
eukaryotic genome into chromatin. Correct chromatin structure is
essential for normal growth, development, and differentiation, as
this regulates all nuclear processes that use DNA as a substrate.
Asf1 binds directly to H3/H4 dimers and promotes acetylation of
histone H3 lysine 56,5 which has been positively linked to
genomic stability, DNA replication and repair, whereas decreased
acetylation sensitizes cells to DNA damaging agents. Therefore,
identification of compounds that disrupt this process may
represent a new therapeutic approach. The goal of our program
was to identify small molecules that are capable of disrupting the
Asf1-H3/H4 interaction in order to validate the biological target.
Our search for inhibitors began by conducting virtually
screening a 139,735 compound library (the National Cancer
Institute Developmental Therapeutics Program [NCI–DTP] 2007
plated set) to identify candidates that may interact with residues
on Asf1 that participate in binding H3/H4. More than 800 Å2 of
surface area is buried at the H3/Asf1 interface whereas
approximately 400 Å2 of surface area is buried at the H4/Asf1
interface.8 Asf1 Tyr112 contributes a significant fraction of
surface area buried at the interface with H3 compared to other
residues. Spheres depicting the sites of potential ligand atoms
were selected within 8 Å of Asf1 Tyr112 using PDB code 2HUE.
Molecular docking of each compound in the library (using
DOCK6.5, UCSF) to this selected site resulted in a ranked list of
compounds predicted to bind Asf1 and prevent H3/H4
interactions. Samples of the top scoring compounds (0.03%)
were obtained from the NCI–DTP, further testing identified 6
examples which demonstrated promising binding activity in an
Asf1-H3/H4 ELISA assay.7F9 The set of HTS lead compounds was
further refined to two chemical series exemplified by 1
(NSC23925) and 2 (DTP-35), both of which became the focus of
medicinal chemistry efforts (Figure 2).
Figure 1. Ribbon diagram of Asf-1/ histones H3/H4 complex along
with expanded view of purported binding interaction of Asf-1
(purple) with Leu-Arg-Ile of histone H3 (blue).

spectra). Interestingly, reduction of amide 8 followed by Boc
deprotection appeared to provide the opposite diastereomer
(9,Threo). Additional conformation of the structural assignment
was later obtained by comparing NMR data with data recently
reported by Duan et al.11
Figure 2. Lead series from HTS
In addition to preparing an authentic sample of 1, we prepared
a series of closely related amide analogs (e.g. 10-15) designed to
explore the role of the amino alcohol moiety and the results
summarized in Figure 4.
Based on analysis of drug-like properties, initial medicinal
chemistry efforts were carried out on the quinolone series 1.
These compounds have been reported to possess antiviral8F10 and
Figure 4. Summary of cytotoxicity activity of quinolone
analogs9
more recently have been reported to possess MDR activity.11
The
9
F
proposed binding orientation of quinolone 1 in the Asf1 binding
pocket is shown in Figure 3.
Figure 3. Proposed
binding orientation of
quinolone derivative 1
docked into AsF-1
binding pocket
The original NCI sample (DTP-37), as well as the synthetic
variants 1 and 9 appeared to be potently inhibit cell viability
Preparation of 1 (NSC23925) has been reported previously.12
Although we investigated this approach, we opted to develop an
alternative route shown in Scheme 1. Pfitzinger reaction of isatin
3 with acetephenones 4 under basic conditions provided the
quinolone carboxylic 5 which was converted to the hydroxamide
6. Lithiation of the Boc-piperidine 7 using conditions develop by
(Figure 4).11
F
However, all closely related amide-containing
analogs 10-15 were inactive suggesting the hydroxyl-piperidine
group is essential for activity. Unfortunately, in subsequent
binding studies with Asf1 using two different versions of the
Amplified Luminescent Proximity Homogeneous Assay
(ALPHA)9 compound 1 and analogs were shown not to bind to
untagged Asf1 (data not shown). Based on this result, we felt the
previously observed cytotoxic effect was most likely not due to
disruption of Asf1-H3/H4 complex, and this series was halted.
Beak et.al10 13
followed by condensation with the hydroxamic ester
F
afforded the amino ketone 8.
We next turned our attention to exploring the - lactam-based
series exemplified by 2 (DTP-35) since binding data suggested
the compound acted upon Asf1. Azetidinones have been reported
to display a wide variety of biological activities.14,15 During QC
evaluation of the sample from NCI, (LCMS, and 1H NMR) we
became concerned regarding the chemical identity. For example,
-lactam compounds as 2 are reported to be prepared via a
Staudinger reaction involving N-acyl hydrazines and
chloroketene.16 However, after repeated attempts we were
unsuccessful in preparing an azetidinone.
Upon closer
inspection, we discovered the original NCI material appeared to
be comprised predominately of an N-acyl hydrazone 17, not the
-lactam 2 (Equation 1).
This finding was confirmed through independent synthesis of
the -chloro keto N-acyl hydrazone 17 from hydrazone 16.
Scheme 1. Preparation of quinolone derivatives
1
Comparison of H NMR spectrum of 17with samples obtained
from the NCI collection confirmed the structural assignment.12F17
Fortuitously we discovered by simply changing the sequence
of the subsequent reduction and Boc deprotection steps, we were
able prepare both diastereomers. For example removal of the
Boc protecting group from 8 followed by amide reduction
afforded an authentic sample of DTP-37 that was identical to the
sample provided by NCI (as judged by comparing proton NMR
Having serendipitously uncovered a new chemotype of Asf1
inhibitors, we explored a small set of N-acyl hydrazone
derivatives as chemical probes that could be used to further
validate the biological mechanism of action of the target.

Hydrazones have been reported to exhibit a wide variety of
biological activity13 18
although their use as inhibitors of histone
We also explored modification to the hydrazone substituent.
Keeping R1= phenyl acetamide, we explored replacement or
modification to the vanillin motif. Dramatic changes in potency
were observed when modifications were made (Table 2),
although few compounds showed improved potency.
F
chaperones is novel. Moreover, we felt the ease of synthesis
would allow rapid analog preparation thus expediting exploration
of SAR (Scheme 2). It was anticipated that once we had
identified more potent derivatives it might be possible to replace
the hydrazone portion therefore improving the overall drug-like
nature of the compounds.14F19
Table 2. SAR of Phenyl acetamide derivatives
Scheme 2. Proposed optimization of N-acyl hydrazones
Our approach to explore SAR included dividing the molecule
into three sections, which included the carboxylic acid (R1), the
hydrazone linker and the group derived from aldehydes/ketones
(R2). N-acyl hydrazones were readily prepared using known
chemistry and all compounds were evaluated for Asf1-H3/H4
binding. Results from a variety of 3-bromovanillin derivatives
are shown in Table 1.
Table 1. SAR of 3-bromovanillin derivatives
Interestingly, a significant boost in potency was observed in a
series of 2-hydroxy substituted compounds exemplified by
compounds 46 and 47. However, upon further analysis we
discovered compounds these compounds were acting as potent
metal scavengers, leading to false positive results. Similar
compounds have been reported to be potent metal chelators.15F20
Lastly, efforts were also directed at modification or
replacement of the hydrazine linkage (48-52, Figure 5).
Surprisingly, all attempts to modify or remove the acyl hydrazine
moiety resulted in significant loss of activity. Only N-
methylation of the hydrazine 52 was tolerated. Interestingly, this
outcome is similar to reports regarding other N-acyl hydrazone
derivatives. 21, 22
For reasons that are unexplained at this time, the
16F
17F
acyl hydrazone appears to required for biological activity.
Figure 5. Summary of hydrazine modifications
The biological activity of the synthesized -chloro ketone
hydrazone 17 recapitulated the biological activity observed with
the original NCI sample. As expected, removal of the -chloro
group 18, resulted in a significant loss of potency. However we
discovered potency could be retained by incorporating a phenyl
group (20) or other bulky substituent (e.g. 21). Incorporation of
non-aromatic groups such as the piperidine 25 or pyran 26 was
ineffective. Attempts to incorporate branched groups or inclusion
of solubilizing groups (eg. 33) did not improve potency.
Conclusion Starting from a computational high throughput
screen of the NCI compound collection, a series of N-acyl
hydrazones that appear to inhibit the Asf1-H3/H4 interaction has
been discovered.
This is the first reported example of
compounds that potentially inhibit Asf-1/histone interactions.

Acknowledgements. Program was supported through a grant
from the State of Colorado Economic Development BDEGP
Infrastructure grant (G.F.M), and a pilot grant from the
University of Colorado Cancer Center (M.E.A.C.).
overnight incubation. After overnight incubation, culture medium
in each well was replaced by 1mL of fresh drug medium
(medium without 10% FBS and drug dissolved as 24 μM). After
1 h drug treatment, 100 μL of FBS was added to each well.
Trypan blue exclusion was used to quantify cell density after
additional 24-hour treatment. Following data were reported as:
Cell viability (%) = [cell density (drug-treated sample) / cell
density (control sample – with 100%DMSO only)]*100.
ALPHA assay:The ALPHA assays with biotin-labeled H3/H4
(50 nM) and either His-tagged human Asf1a (1-155 aa) at 50 nM
or GST-tagged human Asf1a (1-155 aa) at 50 nM were
performed according to manufacturers instructions. Briefly,
H3/H4 dimer and Asf1 were incubated together for 15 min at
room temperature. The compounds (at 100 µM or lower
concentrations in 2 % DMSO) were then added and incubated for
30 min. The appropriate donor and acceptor beads were then
added, and the 96-well tray incubated at 4 °C overnight in the
dark. The fluorescence intensity of each sample was then
measured using an EnVision Plate Reader (Perkin Elmer). The
values for IC50 or % inhibition at 100 µM compound were
determined from curves fitted using Graphpad Prism software.
10 Kim, K.H.; Hansch, C.; Fukunaga, J.Y.; Edward E. Steller,
E.E. and Jow P.Y.C,; J. Med Chem, 1979, Vol. 22, No. 4. 366-
391
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2 Das, C.; Tyler, J.K.; Churchill, M. E.A., Trends in Biochemical
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Research, 2011, 39 (13), 5449.
4 Corpet, A.; De Koning, L.; Toedling, J.; Savignoni, A.;
Berger, F.; Lamaitre, C.; O’Sullivan, R.J.; Karlseder, J.;
Barillot, E.; Asselain, B.; Sastre-Garau, X.; Almouzni, G.;
EMBO J, 2011, 30 (3), 480
5 Das, C.; Lucia, M.S.; Hansen, K.C.; Tyler, J.K.; Nature, 2009,
459 (7243), 113
6 English, C.M.;Adkins, M.W; Carson, J.J;, Churchill M.E.E.A.;
Cell, 2006, 127(3), 495-508
7 Natsume, R.; Eitoku, M.;Yusuke Akai, Y.; Sano, N.; Horikoshi,
M.; Senda, T.; Nature, 2007, Vol 446, 338.
8 The atomic coordinates for H3/H4 from PDB code 2HUE were
extracted to permit small molecule docking to a structural pocket
on the surface of Asf1. The site for molecular docking was
selected using DMS (UCSF) to generate a molecular surface, and
SPHGEN_CPP to define spheres that represent sites of potential
ligand atoms. Spheres within 8 Å of Asf1 position 112 (Tyr)
were selected as the site for molecular docking. Grid-based
scoring implemented in DOCK6.5 was used. This was
accomplished using a scoring grid extending 5 Å in 3 dimensions
from the selected spheres. DOCK6.5 was used to screen drug-
like compounds (National Cancer Institute Developmental
Therapeutics Program NCI plated 2007, 139,735 compounds,
http://zinc.docking.org/catalogs/ncip). Each compound was
docked in 1,000 orientations and scored for hydrophobic (van der
Waals score) and electrostatic interactions (electrostatic score) at
the UF High Performance Computing Center by parallel
processing using 8 cpu. Compounds were ranked based on
overall Energy Score (van der Waals score + electrostatic score).
The top 40 scoring compounds (out of 103, 539 drug-like small
molecules screened by molecular docking) were obtained from
the NCI DTP to measure effects on Asf1 activity.
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17 Diagnostic proton NMR signals for the acyl hydrazone was the
methylene group which appears in the NMR as a singlet. In
comparison, the beta lactam protons appear as a doublet of
doublets due to germinal coupling. See for example ref 15 or
Upadhyay, A., Srivastava, S. K.; and Srivastava, S.D.; Synthetic
Communications, 2011, 41: 2544–2556.
9 Biological evaluation of compounds was carried out using
several assays. ELISA assay:50 µL H3/H4 dimer at 1 μg/mL in
PBS-T (phosphate buffered saline and Tween 20) was used to
coat the wells of a PVC microtiter plate and left at 4°C overnight.
The wells were washed 3 times with 200 µL PBS-T. They were
then blocked by incubating with 200 μL blocking buffer (5% non
fat dry milk in PBS-T) for at least 2 h at room temperature,
followed by 3 washes with PBS-T. The globular core domain of
Asf1 at 5 µM was incubated with each compound at 100 µM (or
various concentrations) for 1 h at 4°C. After diluting samples
with buffer (10 mM Tris pH 7.9, 1 mM DTT), 50 µL of each
sample was applied to the microtitre plate, and incubated for 1 h
at room temperature, prior to washing 3 times with PBS-T. The
presence of Asf1 was evaluated using the primary Asf1 antibody
(sc-166482; Santa Cruz) and HRPT-conjugated secondary
antibody following the manufactures protocol. 50 uL TMB
(3,3’,5,5’-tetramethylbenzidine) was added to each well, and
incubated for 15-30 min, prior to addition of stop solution (2 M
H2SO4). The optical density was measured at 450 nm and Asf1
binding to H3/H4 was analyzed using Graphpad Prism software.
Cell viability: MDA-MB-231 cells were cultured to reach 80%
confluence. Cells were harvested, suspended in medium (1.5 x
105 cells/mL) and seeded into 12 well plates (1 ml/ well) for
18 For a review of acyl hydrazone biological activity see Padmini,
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Table 1. SAR of phenyl acetamide derivatives

Table 2. SAR of vanillin derivatives