Identification and Characterization of AES-135, a Hydroxamic Acid-Based HDAC Inhibitor That Prolongs Survival in an Orthotopic Mouse Model of Pancreatic Cancer

Article abstract of DOI:10.1021/acs.jmedchem.8b01957

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% 1 year survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC is inherently resistant to current treatments, and novel therapies are urgently required. Histone deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC studies, and although there are a few clinical studies investigating combination therapy including HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a three-dimensional coculture model, AES-135 kills low-passage patient-derived tumor spheroids selectively over surrounding cancer-associated fibroblasts and has excellent pharmacokinetic properties in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival significantly, therefore representing a candidate for further preclinical testing.

Full text of DOI:10.1021/acs.jmedchem.8b01957

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Article
Identification and Characterization of AES-135, a Hydroxamic
Acid-based HDAC Inhibitor that Prolongs Survival
in an Orthotopic Mouse Model of Pancreatic Cancer
Andrew E. Shouksmith, Fenil Shah, Michelle L. Grimard, Justyna M. Gawel, Yasir Raouf, Mulu
Geletu, Angelika Berger-Becvar, Elvin D de Araujo, Hema Artee Luchman, William L. Heaton, David
Bakhshinyan, Ashley A. Adile, Chitra Venugopal, Thomas O'Hare, Michael W. Deininger, Sheila
K Singh, Stephen F. Konieczny, Samuel Weiss, Melissa L. Fishel, and Patrick Thomas Gunning
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01957 • Publication Date (Web): 18 Feb 2019
Downloaded from http://pubs.acs.org on February 18, 2019
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Identification and Characterization of AES-135, a Hydroxamic Acid-based HDAC
Inhibitor that Prolongs Survival in an Orthotopic Mouse Model of Pancreatic Cancer
†
ʃ
ʃ
†
†
Andrew E. Shouksmith, Fenil Shah, Michelle L. Grimard, Justyna M. Gawel, Yasir Raouf,
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†
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‡
Mulu Geletu, Angelika Berger-Becvar, Elvin D. de Araujo, H. Artee Luchman, William L.
§
∥
∥
∥
§
Heaton, David Bakhshinyan, Ashley A. Adile, Chitra Venugopal, Thomas O’Hare, Michael
§
∥
^
‡
ʃ,#,*
W. Deininger, Sheila K. Singh, Stephen F. Konieczny, Samuel Weiss, Melissa L. Fishel,
and Patrick T. Gunning†,*
†Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359
Mississauga Road, Mississauga, Ontario, L5L 1C6, Canada
ʃ
Department of Pediatrics, Wells Center for Pediatric Research, IU School of Medicine, 1044 W.
Walnut Street, R4 321, Indianapolis, IN, 46202, USA
#Department of Pharmacology and Toxicology, IU School of Medicine, 1044 W. Walnut Street,
R4 321, Indianapolis, IN, 46202, USA
‡Hotchkiss Brain Institute and Department of Cell Biology and Anatomy, University of Calgary,
Calgary, Alberta, T2N 1N4, Canada
§Huntsman Cancer Institute, Division of Hematology and Hematologic Malignancies, University
of Utah, Salt Lake City, UT, 84112, USA
∥McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Ontario,
L8S 4L8, Canada
^Purdue University, Department of Biological Sciences, West Lafayette, IN, 47907, USA
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Abstract
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% one-year
survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC
is inherently resistant to current treatments and novel therapies are urgently required. Histone
deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC
studies, and although there are a few clinical studies investigating combination therapy including
HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved
for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits
nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a
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D co-culture model, AES-135 kills low passage patient-derived tumor spheroids selectively over
surrounding cancer-associated fibroblasts (CAFs), and has excellent pharmacokinetic properties
in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival
significantly, therefore representing a candidate for further preclinical testing.
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Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with only a
0% one-year survival rate and a 7% five-year survival rate for all stages combined, and is widely
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considered incurable.1-2 It is currently the third leading cause of cancer-related mortality in the
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United States, and is characterized by a complex tumor microenvironment (TME) that is
immunosuppressive and contains myeloid-derived suppressor cells (MDSCs) as well as cancer-
associated fibroblasts (CAFs), heterogeneity within the tumor, and an innate capacity for
metastasis.4-7 Therefore, there is an imminent need for therapies in PDAC, inhibiting novel targets.
Histone/lysine deacetylases (HDACs/KDACs) control post-translational protein
acetylation,8-13 in conjunction with histone acetyltransferases (HATs), which fulfil an antagonistic
role,9, 14 for a large number of substrates; most notably histones. By regulating histone
acetylation/deacetylation, HATs and HDACs play a key indirect role in gene expression.11
Oncogenic HDAC activity has been observed in aggressive human cancers, including pancreatic
cancer.1-2, 15 To date, four small-molecule HDAC inhibitors have been approved by the FDA for
hematological cancer treatment (Cutaneous T-Cell Lymphoma (CTCL), Peripheral T-Cell
Lymphoma (PTCL) and Multiple Myeloma (MM)):8, 11, 13 SAHA (Vorinostat), Romidepsin
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(
depsipeptide-FK228), Belinostat (PXD101), and Panobinostat (LBH-589). Current HDAC
clinical trials in PDAC consist of adjuvant therapies using Vorinostat or Panobinostat in
combination with radiation, surgery or standard-of-care chemotherapy.20-27 Three of the four
HDAC drugs contain an N-hydroxamic acid, which mimics the hydrogen bonds formed by
acetylated lysine substrates; competitively coordinating to the metal ion within the catalytic
8
domain, rendering the HDAC inactive. The catalytic domain is the most structurally conserved
region in the HDAC family primary sequence, and targeting of this domain by small molecules
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often results in the inhibition of more than one HDAC. Despite this, clinical efficacy with pan-
HDAC inhibitors has been observed in select cancer sub-types, but with adverse side effects
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including diarrhea and bone marrow toxicity, observed in patients. HDAC inhibitors, with the
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exception of Romidepsin,28-30 possess a similar linear structural design; with a metal chelating
group (e.g. hydroxamic acid) at one end and a hydrophobic capping group (e.g. a 2-methylindole)
at the other, connected by a linear hydrophobic scaffold, e.g. a benzene ring or an alkyl chain.13, 31
A lack of structural diversity might infer that many of the current clinical candidates are likely to
encounter the same pitfalls in clinical trials.32
Herein, we introduce a small family of novel HDAC inhibitors, including lead compound,
AES-135, which biochemically inhibits HDACs 3, 6, 8 and 11 with IC50 values of 190 – 1100 nM,
and exhibits selective in vitro cytotoxicity in low passage patient-derived pancreatic cancer cells
even in the presence of cancer-associated fibroblast (CAF) cells. AES-135 has other favourable in
vivo properties such as metabolic stability in mouse hepatocytes and bioavailability in µM
concentrations in NSG mice for >10 h (IP injection). AES-135 combines the proven attributes of
an N-hydroxamic acid with a new chemotype for exploration as an HDAC inhibitor.
Results and Discussion
AES-135 was identified as part of a structure-activity relationship (SAR) study designed
to repurpose a class of Signal Transducer and Activator of Transcription 3 (STAT3)-targeting
compounds, including SH-4-54 (1, Table 1), toward HDACs.33-35 Efforts were predominantly
focused on replacing the STAT3 SH2 domain-targeting benzoic acid substituent with an isosteric
36
N-hydroxamic acid for HDAC catalytic domain targeting. A brief SAR around the general
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structure of 1 sought to identify direct-binding nM IC50 HDAC inhibitors. A series of analogs were
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prepared with substitutions made at positions R , R and R (Table 1).
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Table 1. IC50 Values for AES-135 and its Analogs Against HDACs 3, 6, 8 and 11, Evaluated up
to 1 µM (EMSA, n=1)
Substituents
IC50 (µM)
Compound #
R1
R2
R3
HDAC3
HDAC6
HDAC8
HDAC11
AES-135
t-Bu
t-Bu
4-F
3,4-F
3,4-F
2,4-F
2,4-F
4-F
2,3,4,5,6-F
0.654
>1
0.190
0.362
0.188
0.289
0.151
0.230
0.093
0.086
>1
>1
>1
>1
>1
>1
>1
>1
0.636
0.254
0.396
0.288
0.346
0.253
0.304
0.191
2
3
4
5
6
7
8
2-CF
2-CF
2-CF
2-CF
2-CF
2-CF
3
3
3
3
3
3
CF
t-Bu
CF
t-Bu
3
>1
>1
3
>1
>1
CF
3
3
4-F
>1
CF
4-F
2,3,4,5,6-F
>1
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Compounds were synthesized as outlined in Scheme 1. Briefly, anilines 11 – 12 were prepared in
good to excellent yields via reductive amination of benzyl 4-aminobenzoate 10 with different
benzaldehydes under standard conditions. Glycine tert-butyl (t-Bu) ester hydrochloride (13) was
sulfonylated, and the resulting sulfonamides 14 – 17 were benzylated under basic conditions.
Removal of the t-Bu protecting group with diluted trifluoroacetic acid (TFA) furnished the
carboxylic acids 23 – 27 quantitatively. Anilines and carboxylic acids were coupled using
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dichlorotriphenylphosphorane (PPh
3
Cl ) under microwave conditions, and subsequent
2
hydrogenation cleaved the carboxybenzyl group. Chlorination of the benzoic acids using oxalyl
chloride, followed by coupling with O-benzylhydroxylamine, generated the hydroxamate esters,
and the O-benzyl group was removed by hydrogenation.
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Scheme 1: Synthesis of N-hydroxamic acid-based HDAC inhibitors
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(a) BnBr, Cs
iPr
NEt, CH
RT; (f) 11 or 12, PPh
COCl) , THF, DMF, 2 h, 0 °C, N
2
CO
Cl , 16 h, 0 °C-RT, N
Cl , CHCl , 1 h, 100 °C, N
3
, DMF, 24 h, RT; (b) (i) ArCHO, MgSO
4
, THF, 16 h, RT; (ii) NaBH
4
, TFE, 16 h, RT; (c) ArSO
CO H/CHCl (1:3), 3 h,
, 10% Pd/C, THF/MeOH (2:1), 16 h, RT; (h) (i)
2
Cl,
2
2
2
2
; (d) BrCH Ar, Cs CO
2
2
3
, MeCN, 16 h, 50 °C-RT; (e) CF
3
2
3
3
2
3
2
, microwave; (g) H
2
i
(
2
2
; (ii) H
2
NOBn, Pr
2
NEt, THF, 16 h, RT, N .
2
2
The R position of 1, occupied by a pentafluorobenzenesulfonamide, was substituted with less
electron-deficient mono- and di-fluorinated benzene rings to minimize potential nucleophilic
35
1
addition in vivo. The cyclohexyl R group reduced solubility and was susceptible to Phase I
35
) groups.33
oxidation, so this was replaced with less lipophilic t-Bu and trifluoromethyl (CF
3
3
Finally, the R N-methyl group, previously shown to be sensitive to oxidation in mouse
hepatocytes, was substituted with either a pentafluorobenzyl or 2-(trifluoromethyl)benzyl
37
appendage. The prepared library was evaluated for inhibitory activity against select HDACs
representative of groups I (3 and 8), II (6) and IV (11) using an electrophoretic mobility shift assay
(EMSA). In this assay, enzymatic deacetylation of a FAM-labelled peptide substrate is measured
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as a change in the relative fluorescence intensity of the substrate and product following incubation.
In the presence of an inhibitor, deacetylation is impeded, altering the fluorescence intensity of the
product and substrate (a detailed procedure is provided in the supporting information). With the
exception of AES-135, compounds in this library demonstrated selective activity for HDAC
groups II and IV, with limited activity observed against either HDAC3 or HDAC8 (group I). AES-
135 exhibited nanomolar (nM) inhibition of HDACs 3, 6 and 11, with low-µM activity against
HDAC8 (IC50 later confirmed to be 1.10 µM when AES-135 was evaluated up 10 µM against these
targets (Supporting Information, Figure S22 – S25)).
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To explain the observed results, compounds were modelled in silico using AutoDock
Vina/AutoDockTools v4.2.6. Specifically, AES-135 and 6 were chosen as representative ligands
due to their differing HDAC selectivity profiles despite being structurally similar, differing only
3
at the R position (pentafluorobenzyl vs. 2-(trifluoromethyl)benzyl, respectively). These
compounds were docked against eukaryotic, zinc-dependent hHDAC 3, 6, and 8 (PDB: 4A69,
5EDU, and 1T64) (Figure 1). hHDAC11 analysis was not possible, as no crystal structure has
been resolved to date. A detailed description of the experiments performed can be found in the
supporting information.
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Figure 1. Computational modelling/docking studies of AES-135 against hHDAC 3, 6, and 8
(
PDB: 4A69, 5EDU and 1T64, respectively). (Left column) catalytic active site of enzyme,
2
+
Zn (yellow sphere), residues within/around lysine-substrate channel (shown as white ball-and-
stick), catalytic triad residues (shown as colored ball-and-stick). (Centre column) molecular
surface view of channel entrance, low hydrophobicity residues (white), high hydrophobicity
residues (red). (Right column) side view of ligands docked within the active site. Panel
A: hHDAC6 (blue cartoon), AES-135 (magenta), SAHA (green). Panel B: hHDAC3 (green
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cartoon), AES-135 (magenta), 6 (cyan). Panel
35 (magenta), 6 (cyan).
C: hHDAC8
(red
cartoon), AES-
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All three enzymes have a largely hydrophobic surface proximal to the lysine-substrate channel. In
hHDAC6, the lysine tunnel surroundings are largely featureless and flat, while hHDAC3 and 8
contain greater surface topology. While the importance of the metal binding group for HDAC
targeting is critical, the contribution of the capping group to binding and selectivity among the
HDAC isoforms is significant. Increased hydrophobic interaction between the enzyme surface and
the capping group is postulated to greatly increase binding affinity.38-40 These interactions were of
interest when analyzing the in silico binding conformations of AES-135 and 6 to hHDACs 3, 6
and 8 (Figure 1), relative to the in vitro EMSA data shown in Table 1. The docking of AES-135
to hHDAC3, 6 and 8 returned average free energy of binding values (DG
B
) of -8.31 ± 0.08, -8.98
±
0.17, and -8.80 ± 0.13 kcal/mol, while 6 scored -7.31 ± 0.44, -8.64 ± 0.17, and -7.74 ± 0.12
kcal/mol, respectively (Supporting Information, Table S17 – S23). In silico, AES-135 binds more
favorably to all three isoforms than 6, while having greater affinity for hHDAC3/8, yet has more
comparable binding to hHDAC6. Comparing binding to hHDAC 3 and 8 (Figure 1, Panel B and
C), the perfluorinated ring of AES-135 makes significantly more interactions with residues
proximal to the channel, with minimal steric clash compared to the 2-(trifluoromethyl) group of 6,
which appears to occupy poses that unfavourably clash with the HDAC surface.
To confirm AES-135 as the lead candidate, cytotoxicity profiles were determined against a bank
of human cancer cell lines (Supporting Information, Table S1 – S2, Figure S1 – S6).
Encouragingly, AES-135 was shown to be the most promising candidate, with low µM potency
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being observed in multiple brain tumor stem cell glioblastoma lines, MV4-11 and MOLM-13
AML cells, and PC-3 prostate cancer cells. In D425 primary medulloblastoma (MDB) and D458
recurrent MDB cells, nM activity was observed. Patient-derived pancreatic cancer cells were
sensitive to single-digit µM/high nM concentrations of AES-135. Of the lines tested, only chronic
myelogenous leukemia K562 cells were resistant to AES-135. Encouragingly, in MRC9 lung cells
(non-cancerous), AES-135 demonstrated minimal toxicity, identifying a clear therapeutic window.
1
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0
To assess the stability of the pentafluorobenzyl (PFB) ring to biological nucleophiles, a 10 mM
solution of AES-135 in DMSO was mixed with a 100-fold excess of reduced L-glutathione in
HEPES buffer, pH 7.4, and monitored by analytical HPLC at regular intervals. No discernible
reaction was observed, even after 25 h (Supporting Information, Figure S7 – S8). This was
1
9
corroborated by F NMR studies, in which no displacement of fluoride was observed after 16 h
of immersion in 100-fold excess glutathione (Supporting Information, Figure S9).
Given the structural origin of AES-135 from 1, we confirmed that the cytotoxicity observed with
the former was not due to STAT3/5 inhibition. Western blot studies were performed in MDA-MB-
231 breast cancer cells (STAT3-overexpressing) and MV4-11 AML cells (STAT5-
overexpressing). The results showed that AES-135 did not inhibit activation of STAT3 or STAT5
signaling via immunoblotting for Y694 phosphorylation on STAT5b and Y705 phosphorylation
on STAT3 (Supporting Information, Figure S10). In MDA-MB-231 cells, AES-135 returned an
IC50 of 2.72 ± 0.60 µM (n = 4), yet even at 10-fold this concentration, neither total STAT3 nor
pY705 STAT3 was significantly reduced. Similar results were observed in MV4-11 cells, where
AES-135 had an IC50 of 1.88 ± 0.89 µM (n = 4), yet failed to suppress total STAT5b or pY694
STAT5b, even at 10 µM. Evidence from Western blots in pancreatic Pa03C cancer cells and
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multiple BTSC GBM lines was subsequently obtained, further supporting the conclusion that
AES-135 was not a STAT3/5 inhibitor (Supporting Information, Figure S11 – S14).
To confirm that the N-hydroxamic acid group in AES-135 was responsible for HDAC targeting,
the compound was screened against seven metal-dependent HDACs, representing classes I, II and
IV, at a fixed concentration (Table 2). As a negative control, an N-methylhydroxamic acid, 60,
was prepared and assessed in parallel. The synthetic route is described in Scheme 2. Briefly,
starting from O-benzylhydroxylamine (55), Boc-protection of the amino group was followed by
methylation and acid-mediated removal of the Boc group to furnish O-benzyl-N-
methylhydroxylamine (58). Coupling of this compound with 37, followed by hydrogenation, as
previously described, yielded 60.
0
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Scheme 2: Synthesis of N-methylhydroxamic acid 60
(
a) Boc
2
O, THF, 24 h, RT; (b) MeI, NaH (60%), DMF, 24 h, RT; (c) (i) CF
3
CO
2
H/CHCl
3
(1:3), 22 h, RT; (ii) 1M
i
NaOH; (d) (i) (COCl)
2:1), 16 h, RT.
2
, THF, DMF, 2 h, 0 °C, N
2
; (ii) 58, Pr
2
NEt, THF, 16 h, RT, N
2
; (e) H
2
, 10% Pd/C, THF/MeOH
(
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AES-135 and control compound 60 were screened against full recombinant human HDACs 1, 3,
4
, 6, 8, 10 and 11 using an electrophoretic mobility shift assay (EMSA) at 10 µM. AES-135
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inhibited HDACs 3, 6, 8 and 11 (>90%), and showed moderate inhibition of HDACs 1 and 10
(≥70%), with HDAC4 not being affected (<20%). HDAC inhibition was highly sensitive to
modification of the hydroxamic acid motif, with compound 60 demonstrating only modest
inhibition of HDAC11 (64%), but negligible activity against the remaining HDACs (Table 2).
Table 2. Percentage Inhibition of HDACs 1, 3, 4, 6, 8, 10 and 11 by AES-135 and 60 at 10 µM
(
EMSA, n=2)
%-Inhibition
HDAC
AES-135
60
1
3
4
6
8
72
98
15
98
94
70
97
0
0
0
12
0
1
0
0
11
64
To evaluate in vitro stability, AES-135 and 8 were incubated with mouse hepatocytes for 2 h to
assess the rate of intrinsic clearance. The calcium channel blocker Verapamil was used as a positive
6
control. AES-135 reported an intrinsic clearance rate of 36.0 µL/min/10 cells; almost half the rate
6
of Verapamil (63.3 µL/min/10 cells), and a half-life of 38.5 min, which was almost twice that of
Verapamil (21.9 min) (Supporting Information, Table S4, Figure S15 – S20). Derivative 8,
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1
1
possessing a CF
3
in the R position, performed similarly to AES-135 (R = t-Bu), returning a
6
clearance rate of 37.4 µL/min/10 cells and a half-life of 37.1 min, suggesting that the t-Bu group
of AES-135 was not being targeted for oxidation in mouse hepatocytes. The observed stability of
AES-135 supported the in vitro findings that the PFB ring was a relatively stable substituent. A
protein binding study using AES-135 in mouse plasma found that the compound was 99.6% bound
after 6 h incubation. The low recovery (16 – 20%) of AES-135 after this time indicated the
compound to be susceptible to metabolism in plasma (Supporting Information, Table S5). In a
separate study, the experimental LogD7.4 for AES-135 was calculated using a 1-octanol/PBS
system, returning a value of 4.15 (Supporting Information, Table S6).
0
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9
0
To investigate the permeability profile of AES-135 and 8 through the blood brain barrier (BBB),
the compounds were tested using a parallel artificial membrane permeability assay (PAMPA),
which assesses the ability of a compound to cross a lipid-infused artificial membrane, and has been
shown to correlate well with performance in crossing in vivo barriers. Testosterone and the
antimetabolite Methotrexate were used as positive and negative controls, respectively. In this
assay, a permeability coefficient (-Log P
through a lipid membrane, whereas a -Log P
Results from this assay showed that AES-135 and 8 were poorly permeable compounds, returning
Log P values of 7.73 and 7.02, respectively. By comparison, testosterone gave a value of 4.61
and Methotrexate >8.5, where the degree of membrane permeation was below the limit of detection
Supporting Information, Table S7 – S11). The results indicated that AES-135 would be poorly
e
) <6 defined the compound as having high permeability
e
>6 meant the compound had low permeability.
-
e
(
efficacious against cancers surrounded by undamaged membranes, e.g. GBM, despite impressive
in vitro potency.
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AES-135 was also analyzed in a Caco-2 assay to gauge its permeability through a monolayer of
tightly packed epithelial cells; an in vitro model of the human small intestinal membrane. The
Caco-2 cells also express several transporter proteins, e.g. P-glycoprotein (P-gp), and can thus
provide information on the efflux rate of compounds from a cell. Propranolol, Digoxin and
Prazosin were used as controls with low, high and medium efflux rates, respectively. Results from
this assay supported those from the PAMPA, showing AES-135 to have poor permeation through
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0
-6
the monolayer, with an apparent permeability coefficient, Papp (A-B), of 0.27 x 10 cm/s.
Compared to Propranolol and Prazosin, with respective Papp (A-B) values of 15.41 and 19.94 x 10-
6
cm/s, AES-135 was significantly less permeable. In addition, the Papp (B-A) for AES-135 was
-6
1.02 x 10 cm/s, calculating an efflux ratio of 3.83 (Supporting Information, Table S12 – S13).
This suggested that AES-135 was transported out of the cell approximately 4-fold faster than it
was being absorbed, meaning that it would struggle to achieve suitable intestinal absorption in vivo
if administered orally. The data also indicated that, despite potent in vitro activity, AES-135 would
not be efficacious if used to treat cancers requiring penetration of bone marrow, e.g. AML.
To assess the pharmacokinetic properties of AES-135 in vivo, NSG mice were dosed with a single
2
0 mg/Kg intraperitoneal (IP) injection, and blood was taken at 0.5, 1, 2, 4, 8 and 24 h. AES-135
achieved µM concentrations in the blood, reaching Cmax 7,452 ng/mL (10.74 µM) within 30 min,
which was sustained for 8 h, with significant clearance observed only after 24 h (Table 3). In these
mice, AES-135 had a calculated half-life of 5.0 h, with a clearance rate of 0.004 L/h, assuming
bioavailability to be 100% (Supporting Information, Table S14 – S15).
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Table 3. Serum Concentrations of AES-135 Following One Dose at 20 mg/Kg, IP (n=2)
Time (h)
.5
[AES-135] (ng/mL)
7452 ± 1354
0
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5397 ± 1079
5391 ± 2418
3655 ± 400
6392 ± 222
265 ± 29
2
4
In a follow-up study, AES-135 was administered at 10 mg/Kg and 40 mg/Kg, once a day for five
days. Blood was collected from each mouse 5 h following the final injection (Figure 2A).
Encouragingly, the results showed that the blood concentration of AES-135 was dose-dependent,
achieving an average of 323 ng/mL (0.47 µM) with 10 mg/Kg dosing, and 1829 ng/mL (2.64 µM)
with 40 mg/Kg. No visible toxicity associated with either dose, based on percentage weight loss
compared to vehicle control, was observed. This represents an approximate 5.7-fold increase in
blood concentration from quadrupling the dose.
To evaluate AES-135 toxicity in vivo, NSG mice were dosed by IP daily with a range of
concentrations for 4 – 5 days (n=6). Mice were weighed prior to, and following, administration of
the compound and toxicity assessed via weight loss (Figure 2B). At 60 mg/Kg, the mice showed
no significant weight loss, indicating AES-135 to be non-toxic at the highest concentration.
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Figure 2A. Serum concentrations of AES-135 in NSG mice following 10 and 40 mg/Kg injections
daily for 5 days, IP (n=6, ±SD); Figure 2B. Toxicity study with AES-135 in NSG mice
administered over 5 days, IP (n=6, ±SD).
AES-135 was consistently cytotoxic in multiple low-passage patient-derived pancreatic cancer cell
lines, namely Pa03C, Pa02C and Panc10.05 cells (the latter hereto referred as 10.05). IC50 values
were in the low µM range (1 – 4 µM) in monolayer proliferation-based assays of these tumor lines.
The efficacy of AES-135 was also assessed in KPC tumor cells, which are derived from the ‘gold
standard’ genetically engineered mouse model of PDAC (KrasLSL.G12D/+; Trp53R172H/+; Elas-
ER 41
42
Cre ). Interestingly, the tumor cells generated from the KPC genetically engineered PDAC
mouse model were extremely sensitive to AES-135 and had an IC50 of 1.3 µM in the monolayer,
compared to 8.5 µM for the pan-HDAC inhibitor Panobinostat, which was used as a positive
control (Figure 3). KPC mice develop premalignant lesions called Pancreatic Intraepithelial
Neoplasia (PanINs), which progress to visible carcinomas with 100% penetrance, and display a
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morphology similar to that observed in human PDAC. Metastases arise in 80% of KPC mice,
primarily in the liver and lungs; the most common metastatic sites in humans. The KPC tumors
possess intricate genomic rearrangements; a sign of genomic instability, making this one of the
most aggressive PDAC models used in preclinical research. They are notoriously resistant to
standard-of-care therapy; only 12% of tumors demonstrate sensitivity towards gemcitabine.43
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Figure 3. Dose-dependent reduction by A) AES-135 and B) Panobinostat, in cell proliferation in
pancreatic cancer cells in monolayer. Average of at least three experiments + SE.
Next, we evaluated the efficacy of AES-135 in preclinical predictive 3D tumor models of
pancreatic cancer using patient-derived tumor cells as well as CAFs. Pancreatic cancers are
difficult to treat effectively, in part because of the CAFs that surround the tumor and impede access
by chemotherapeutics. Additionally, CAFs facilitate tumor growth through the secretion of growth
factors, e.g. VEGF, IL-6, and TGF-β, promoting invasion and metastasis.47 AES-135 reduced
pancreatic tumor spheroids, even with a protective CAF microenvironment, and showed 5- to 6-
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fold greater selectivity for the tumor cells over the CAFs (Figure 4). Single-digit µM to high nM
potencies were demonstrated in the analogous 3D tumor models, both in reducing tumor area and
intensity (Table 4).
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Figure 4. Dose-dependent reduction in tumor spheroid intensity in patient-derived pancreatic
cancer cells Pa03C (red, n=3, ±SE) and 10.05 (blue, n=3, ±SE); Fold change compares the treated
tumor only spheroids to media control.
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Table 4. IC50 Values for AES-135 in Several Monolayer and 3D Human-Derived PDAC Cell
Lines (n = 3 – 5)
Monolayer PDAC Cells
3D PDAC Cells
Scan Type
Areaa
0
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Cell Line
Pa02C
IC50 (µM)
4.6
Cell Line
Pa03C
IC50 (µM)
1.22
Pa03C
3.4
Pa03C + CAFs
CAF co-culture
Pa03C
Area
1.41
1
0.05
3.9
Area
7.80
Intensityb
KPC
1.3
1.33
Pa03C + CAFs
CAF co-culture
10.05
Intensity
Intensity
Area
1.56
4.50
0.97
10.05 + CAFs
CAF co-culture
Area
0.94
Area
4.70
1
1
0.05
Intensity
Intensity
Intensity
0.60
0.05 + CAFs
0.50
CAF co-culture
3.40
a
2
b
Area: the µ of objects which exceed a minimum tomato red intensity threshold in the well; the sum of all intensity
values for pixels marked as Ch2 objects (i.e. total red OR green fluorescence in the well, after background removal).
Due to the high sensitivity of the KPC cells to AES-135 treatment, we tested the in vivo potency
of AES-135 in a syngeneic orthotopic model. KPC cells were orthotopically implanted in the
pancreas of C57Bl/6 mice, which were subsequently treated with either 50 mg/kg AES-135 or
vehicle control. A murine mouse model was utilized in the in vivo studies due to the role of HDACs
in the modulation of immune cell function.44-45 Furthermore, in lung and renal cell carcinoma
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mouse models, HDAC inhibitor Entinostat (SNDX-275; MS-275) potentiated the effects of PD-1
4
5
inhibition, and this effect was partially mediated by functional inhibition of MDSCs. HDAC
inhibitors would be effective in blockading tumor cell proliferation and cell cycle progression, and
also have immunomodulatory effects.46-47 Mice treated with AES-135 showed significantly
increased survival, with a median survival rate of 36.5 days compared to 29.5 days for the vehicle
mice (Figure 5, p=0.0146). The ability to provide a survival advantage in this aggressive PDAC
model illustrates the potential of AES-135 as a hit-to-lead compound. This effect was only
observed in immunocompetent mice; the equivalent immunodeficient mice showed no obvious
survival advantage (Supporting Information, Figure S21).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
4
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4
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5
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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
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4
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Increased survival of C57Bl/6 mice implanted with KPC tumor cells, following AES-
1
35 treatment. Mice treated with 50 mg/Kg AES-135, IP daily (blue, n=10) exhibited a statistically
significant survival advantage compared to mice treated with vehicle (green, n=10), p = 0.0146
(Log-Rank test). Treatment started on Day 7 with a cycle of 5 days on, 2 days off and continued
until Day 36.
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Conclusion
Several reviews have described the potential of HDAC inhibitors to effectively treat PDAC,48-51
but to date, no compound has been published demonstrating suitable potency and drug-like
properties against this aggressive disease. We have presented a set of structurally novel
hydroxamic acid-containing molecules displaying nM inhibition of HDACs in a target-based
assay. Lead compound, AES-135, demonstrated potent inhibition of HDACs 3, 6, 8 and 11, and
high cytotoxicity in a variety of cancer cell lines, most notably in pancreatic tumor lines. AES-135
was consistently more active in PDAC tumor models, both monolayer and 3D, than STAT
inhibitor 1, even showing single-digit µM IC50 values in the highly aggressive KPC model, which
was superior to the FDA-approved HDAC inhibitor Panobinostat. AES-135 showed an impressive
PK profile in mice, with an in vivo half-life of 5.0 h, prolonged blood concentration above its IC50
value, and unremarkable toxicity, as assessed by a brief study. Subsequent in vivo evaluation in
immunocompetent mice found that AES-135 extended the life of the mice significantly. To
effectively treat pancreatic cancer, single agent therapy has not been effective, therefore
individualized combinations of targeted therapies will be necessary for making therapeutic
advances in this devastating disease. Combination studies including AES-135 in animal models
are crucial in order to determine whether the addition of HDAC inhibitors to standard of care
agents or new combination, such as immune checkpoint inhibitors, will dramatically extend
survival. NMR and X-ray crystallographic studies are ongoing to determine the exact binding
mechanism of AES-135 with HDACs to identify more potent and selective binding agents for
preclinical evaluation.
0
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Experimental Section
Materials and Methods. A 400 MHz Bruker NMR was utilized to obtain H, C, and F
1
13
19
NMR spectra in CDCl
3
(99.8 atom% D), CD
3
CN (99.8 atom% D), or MeOH-d (99.8 atom% D),
4
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
13
19
as indicated ( H at 400 MHz, C at 100 MHz, and F at 54 MHz). Chemical shifts (δ) are reported
in parts per million (ppm), after calibration to residual isotopic solvent, and coupling constants (J)
are reported in Hertz (Hz). Low-resolution mass spectrometry (LRMS) was carried out using a
Waters LC-MS in ESI mode, fitted with a Micromass ZQ MS and an Alliance 2690 LC. High-
resolution mass spectrometry (HRMS) was carried out using an Agilent 6538 UHD Q-TOF MS in
ESI mode with a mass accuracy +/- 1 mDa. Thin Layer Chromatography (TLC) was conducted on
Merck silica gel 60F254 on aluminium sheets. All sheets were dried after use and visualized using
short wave (254 nm) and long wave (365 nm) UV light and/or staining with KMnO . Column
4
chromatography was carried out at room temperature using Biotage Isolera One and Isolera Prime
purification systems, with industry-standard SNAP cartridges loaded with 40–60 µm silica gel
(average pore size 60Å). Semi-preparative HPLC was conducted using a Waters 2487 Dual λ
Absorbance Detector, equipped with a Symmetry® C18 4.6 mm x 150 mm cartridge. Inhibitor
purity was evaluated at room temperature by a Hewlett Packard Series 1100 analytical HPLC
system fitted with a Phenomenex Luna 5.0 µm C18 4.6 mm x 150 mm cartridge, using gradient
mixtures of (A) MilliQ water with 0.1% (v/v) TFA and (B) HPLC-grade acetonitrile. Biologically
evaluated compounds are ≥ 95% chemically pure, as measured by HPLC. Chemicals and solvents
were purchased from Sigma-Aldrich (MilliporeSigma), VWR International, Alfa Aesar, Combi-
Blocks, Caledon Laboratory Chemicals and Promega, and were used as supplied.
General Procedure for the Synthesis of Compounds AES-135, 2 – 8, 36 – 43, and 60.
The benzyl or hydroxamate ester (1.0 equiv.) was dissolved in THF/methanol (2:1) (0.1 M) and
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2
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6
purged with nitrogen. 10% Pd/C (0.04 equiv.) was added after 15 min and the flask was purged
with hydrogen for 10 min. The reaction was allowed to stir at RT under hydrogen. After 16 h, the
reaction was filtered through celite, washing with EtOAc, and concentrated in vacuo. Semi-
preparative HPLC, followed by lyophilization at -50 °C, isolated the target compound.
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
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8
9
0
4
-(N-(4-(tert-butyl)benzyl)-2-((4-fluoro-N-
((perfluorophenyl)methyl)phenyl)sulfonamido)acetamido)-N-hydroxybenzamide
35). Semi-preparative HPLC using acetonitrile/0.1% (v/v) TFA in MilliQ water (0:1 à 1:0, 50
(AES-
1
min à 10 min) eluted the target compound at 42.8 – 44.0 min. The product was suspended in
acetonitrile/MilliQ water (1:3, 4 mL) and lyophilized overnight at -50 °C to give AES-135 as a
1
t
white solid (52.2 mg, 56%); H δ/ppm (400 MHz, CDCl
.61 (s, 2H, CH ), 4.73 (s, 2H, CH ), 6.97 (d, J = 7.8 Hz, 2H, 2 CH), 7.08 (d, J = 7.3 Hz, 2H, 2
CH), 7.14 (t, J = 8.5 Hz, 2H, 2 CH), 7.27 (d, J = 7.9 Hz, 2H, 2 CH), 7.75 (d, J = 7.1 Hz, 2H, 2
3
) 1.29 (s, 9H, Bu), 3.86 (s, 2H, CH
2
),
4
2
2
13
CH), 7.83 – 7.87 (m, 2H, 2 CH), hydroxamic acid NH and OH protons were not observed; C
δ/ppm (100 MHz, CDCl ) 31.1, 34.4, 39.6, 49.2, 53.0, 101.4, 115.9, 116.1, 124.4, 125.4, 128.3,
3
1
28.7, 130.3, 130.4, 132.8, 134.87, 134.91, 143.8, 144.2, 146.8, 150.9, 163.9, 164.8, 166.2, 166.5;
19F δ/ppm (54 MHz, CDCl
) -162.3 (td, J = 6.1 and 20.9 Hz, 2F), -153.9 (t, J = 20.5 Hz, 1F), -
3
1
42.2 (dd, J = 7.0 and 22.5 Hz, 2F), -105.4 to -105.3 (m, 1F); LRMS (ESI+) m/z calcd for
+
+
[
C
33
H
30
F
6
N
3
O
5
S] : 694.67, found: 694.36; calcd for [C33
H
29
F
6
N
3
O
5
SNa] : 716.65, found: 716.34;
+
HRMS (ESI+) m/z calcd for [C33
min (97.9%); HPLC (II) t
H
30
F
6
N
3
O
5
S] : 694.1798, found: 694.1805; HPLC (I) t
R
= 23.55
R
= 38.44 min (99.9%).
4-(N-(4-(tert-butyl)benzyl)-2-((3,4-difluoro-N-(2-
trifluoromethyl)benzyl)phenyl)sulfonamido)acetamido)-N-hydroxybenzamide (2). Semi-
preparative HPLC using acetonitrile/0.1% (v/v) TFA in MilliQ water (0:1 à 1:0, 50 min à 10
(
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min) eluted the target compound at 42.1 – 43.6 min. The product was suspended in
acetonitrile/MilliQ water (1:3, 4 mL) and lyophilized overnight at -50 °C to give 2 as a white solid
1
t
(83.9 mg, 68%); H δ/ppm (400 MHz, CDCl
CH ), 4.74 (s, 2H, CH ), 6.90 (d, J = 7.3 Hz, 2H, 2 CH), 6.94 (d, J = 8.1 Hz, 2H, 2 CH), 7.26 (d, J
8.2 Hz, 2H, 2 CH), 7.30 (d, J = 8.3 Hz, 1H, CH), 7.36 (t, J = 8.3 Hz, 1H, CH), 7.46 (t, J = 7.4
3
) 1.29 (s, 9H, Bu), 3.68 (s, br, 2H, CH
2
), 4.70 (s, 2H,
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
2
=
Hz, 1H, CH), 7.61 – 7.77 (m, 6H, 6 CH), hydroxamic acid NH and OH protons were not observed;
13C δ/ppm (100 MHz, CDCl
3
) 31.3, 34.5, 47.4, 48.0, 53.1, 117.7, 117.87, 117.90, 118.0, 122.7,
1
1
24.87, 124.91, 124.94, 125.0, 125.4, 125.5, 125.75, 125.81, 125.86, 125.92, 128.0, 128.4, 128.5,
1
9
28.65, 128.74, 130.1, 132.5, 132.9, 134.1, 136.5, 143.9, 148.8, 151.0, 151.9, 165.8, 166.0; F
δ/ppm (54 MHz, CDCl
3
) -134.0 (dt, J = 8.5 and 20.7 Hz, 1F), -129.3 to -129.1 (m, 1F), -59.1 (s,
+
3
F); LRMS (ESI+) m/z calcd for [C34
H
33
F N
5 3
O
5
S] : 690.71, found: 690.45; calcd for
+
S]+:
[
C
34
H
32
F
5
N
3
O
5
SNa] : 712.69, found: 712.43; HRMS (ESI+) m/z calcd for [C34
H
33
F
5
N
3
O
5
690.2064, found: 690.2056; HPLC (I) t = 24.65 min (99.9%); HPLC (II) t = 40.23 min (99.9%).
R
R
4-(2-((3,4-difluoro-N-(2-(trifluoromethyl)benzyl)phenyl)sulfonamido)-N-(4-
(
trifluoromethyl)benzyl)acetamido)-N-hydroxybenzamide (3). Semi-preparative HPLC using
acetonitrile/0.1% (v/v) TFA in MilliQ water (0:1 à 1:0, 50 min à 10 min) eluted the target
compound at 37.9 – 39.4 min. The product was suspended in acetonitrile/MilliQ water (1:3, 4 mL)
1
and lyophilized overnight at -50 °C to give 3 as a white solid (82.2 mg, 71%); H δ/ppm (400 MHz,
CDCl
3
) 3.70 (s, 2H, CH
2
), 4.71 (s, 2H, CH
2
), 4.79 (s, 2H, CH ), 6.91 (d, J = 7.6 Hz, 2H, 2 CH),
2
7
.17 (d, J = 7.9 Hz, 2H, 2 CH), 7.29 – 7.33 (m, 1H, CH), 7.37 (t, J = 7.7 Hz, 1H, CH), 7.47 (t, J =
7.7 Hz, 1H, CH), 7.51 (d, J = 8.1 Hz, 2H, 2 CH), 7.61 – 7.76 (m, 6H, 6 CH), 9.19 (s, br, 1H, NH),
13
hydroxamic acid OH proton was not observed; C δ/ppm (100 MHz, CDCl ) 47.4, 48.0, 52.9,
3
1
17.6, 117.8, 117.9, 118.1, 122.6, 122.7, 124.8, 124.87, 124.91, 125.0, 125.3, 125.4, 125.57,
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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
1
1
25.59, 125.63, 125.7, 125.8, 125.9, 128.2, 128.3, 128.5, 128.6, 128.9, 129.0, 130.2, 130.4, 131.2,
1
9
32.6, 133.9, 136.5, 140.0, 143.5, 151.2, 165.2, 166.4; F δ/ppm (54 MHz, CDCl ) -133.9 (dt, J
3
= 8.7 and 20.6 Hz, 1F), -129.0 to -128.9 (m, 1F), -62.6 (s, 3F), -59.0 (s, 3F); LRMS (ESI+) m/z
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
+
+
calcd for [C31
H
24
F
8
N
3
O
5
S] : 702.60, found: 702.36; calcd for [C31
H
23
F
8
N
3
O SNa] : 724.58,
5
+
found: 724.34; HRMS (ESI+) m/z calcd for [C31
(I) t = 23.11 min (99.9%); HPLC (II) t
24
H F
8
N
3
O
5
S] : 702.1308, found: 702.1303; HPLC
R
R
= 37.67 min (99.9%).
4-(N-(4-(tert-butyl)benzyl)-2-((2,4-difluoro-N-(2-
trifluoromethyl)benzyl)phenyl)sulfonamido)acetamido)-N-hydroxybenzamide (4). Semi-
(
preparative HPLC using acetonitrile/0.1% (v/v) TFA in MilliQ water (0:1 à 1:0, 50 min à 10
min) eluted the target compound at 42.0 – 43.7 min. The product was suspended in
acetonitrile/MilliQ water (1:3, 4 mL) and lyophilized overnight at -50 °C to give 4 as a white solid
1
t
(
57.4 mg, 69%); H δ/ppm (400 MHz, CDCl
3
) 1.29 (s, 9H, Bu), 3.72 (s, 2H, CH ), 4.68 (s, 2H,
2
CH ), 4.88 (s, 2H, CH ), 6.91 – 6.98 (m, 6H, 6 CH), 7.24 (d, J = 8.2 Hz, 2H, 2 CH), 7.34 (t, J =
2
2
7.6 Hz, 1H, CH), 7.46 (t, J = 7.5 Hz, 1H, CH), 7.60 – 7.66 (m, 4H, 4 CH), 7.85 – 7.91 (m, 1H,
13
CH), 9.14 (s, br, 1H, NH), hydroxamic acid OH proton was not observed; C δ/ppm (100 MHz,
CDCl
3
) 31.3, 34.5, 47.7, 48.0, 53.0, 105.5, 105.7, 106.0, 111.4, 111.6, 124.7, 125.4, 125.5, 125.7,
125.75, 125.81, 125.9, 127.9, 128.4, 128.5, 128.6, 128.7, 128.75, 128.77, 128.84, 130.1, 131.8,
1
9
1
31.9, 132.5, 133.1, 134.7, 144.0, 150.9, 164.1, 164.6, 165.4; F δ/ppm (54 MHz, CDCl ) -101.3
3
to -101.2 (m, 1F), -100.7 to -100.6 (m, 1F), -59.1 (s, 3F); LRMS (ESI+) m/z calcd for
+
S]+:
[
C
34
H
32
F
5
N
3
O
5
SNa] : 712.69, found: 712.56; HRMS (ESI+) m/z calcd for [C34
H
33
F
5
N
3
O
5
690.2063, found: 690.2056; HPLC (I) t
R
= 24.11 min (95.3%); HPLC (II) t
R
= 39.18 min (98.8%).
4-(2-((2,4-difluoro-N-(2-(trifluoromethyl)benzyl)phenyl)sulfonamido)-N-(4-
(
trifluoromethyl)benzyl)acetamido)-N-hydroxybenzamide (5). Semi-preparative HPLC using
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acetonitrile/0.1% (v/v) TFA in MilliQ water (0:1 à 1:0, 50 min à 10 min) eluted the target
compound at 38.3 – 40.1 min. The product was suspended in acetonitrile/MilliQ water (1:3, 4 mL)
1
and lyophilized overnight at -50 °C to give 5 as a white solid (95.2 mg, 80%); H δ/ppm (400 MHz,
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
CDCl
3
) 3.74 (s, 2H, CH
2
), 4.77 (s, 2H, CH
2
), 4.85 (s, 2H, CH
2
), 6.92 – 6.99 (m, 4H, 4 CH), 7.14
(
d, J = 8.0 Hz, 2H, 2 CH), 7.35 (t, J = 7.6 Hz, 1H, CH), 7.46 (t, J = 7.6 Hz, 1H, CH), 7.49 (d, J =
8.2 Hz, 2H, 2 CH), 7.59 – 7.66 (m, 4H, 4 CH), 7.86 – 7.91 (m, 1H, CH), hydroxamic acid NH and
13
OH protons were not observed; C δ/ppm (100 MHz, CDCl ) 47.6, 48.0, 52.9, 105.4, 105.7, 105.9,
3
1
1
11.45, 111.49, 111.68, 111.71, 122.8, 124.58, 124.62, 124.7, 124.8, 125.3, 125.5, 125.7, 125.78,
25.83, 125.9, 127.3, 128.0, 128.6, 128.7, 128.97, 129.00, 130.2, 131.8, 131.9, 132.0, 132.5, 134.4,
19
140.1, 143.6, 161.6, 164.5, 166.3, 167.1; F δ/ppm (54 MHz, CDCl ) -101.6 to -101.5 (m, 1F), -
3
S]+:
1
7
00.3 to -100.2 (m, 1F), -62.6 (s, 3F), -59.0 (s, 3F); LRMS (ESI+) m/z calcd for [C31
H
24
F
8
N
3
O
5
+
02.60, found: 702.48; calcd for [C31
H
23
F
8
N
3
O
5
SNa] : 724.58, found: 724.40; HRMS (ESI+) m/z
= 22.62 min (99.9%); HPLC
+
calcd for [C31
H
24
F
8
N
3
O
5
S] : 702.1307, found: 702.1303; HPLC (I) t
R
(
II) t = 36.62 min (97.2%).
R
4
-(N-(4-(tert-butyl)benzyl)-2-((4-fluoro-N-(2-
(
trifluoromethyl)benzyl)phenyl)sulfonamido)acetamido)-N-hydroxybenzamide (6). Semi-
preparative HPLC using acetonitrile/0.1% (v/v) TFA in MilliQ water (0:1 à 1:0, 50 min à 10
min) eluted the target compound at 41.0 – 42.5 min. The product was suspended in
acetonitrile/MilliQ water (1:3, 4 mL) and lyophilized overnight at -50 °C to give 6 as a white solid
1
t
(
90.4 mg, 68%); H δ/ppm (400 MHz, CDCl
CH ), 4.75 (s, 2H, CH ), 6.91 (d, J = 8.0 Hz, 2H, 2 CH), 6.94 (d, J = 8.2 Hz, 2H, 2 CH), 7.18 (t, J
8.5 Hz, 2H, 2 CH), 7.25 (d, J = 8.2 Hz, 2H, 2 CH), 7.34 (t, J = 7.6 Hz, 1H, CH), 7.45 (t, J = 7.6
Hz, 1H, CH), 7.62 (t, J = 7.8 Hz, 2H, 2 CH), 7.66 (d, J = 8.0 Hz, 2H, 2 CH), 7.90 – 7.94 (m, 2H,
3
) 1.29 (s, 9H, Bu), 3.69 (s, 2H, CH
2
), 4.69 (s, 2H,
2
2
=
27
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Journal of Medicinal Chemistry
Page 28 of 57
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7
8
9
1
1
1
1
1
1
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1
1
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2
2
2
2
2
2
2
2
2
3
3
3
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3
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3
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
2
CH), 9.14 (s, br, 1H, NH), hydroxamic acid OH proton was not observed; C δ/ppm (100 MHz,
CDCl ) 31.3, 34.5, 47.5, 47.9, 53.0, 116.0, 116.2, 125.5, 125.7, 125.76, 125.81, 125.9, 127.9,
3
128.0, 128.3, 128.5, 128.6, 128.7, 130.0, 130.5, 130.6, 130.7, 132.5, 133.0, 134.45, 134.47, 135.7,
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
19
1
1
35.8, 150.9, 164.0, 166.13, 166.16, 166.5; F δ/ppm (54 MHz, CDCl
3
) -104.84 to -104.77 (m,
+
F), -59.2 (s, 3F); LRMS (ESI+) m/z calcd for [C34
H
34
F
4
N
3
O
5
S] : 672.72, found: 672.50; calcd for
+
S]+:
[C34
H
33
F
4
N
3
O
5
SNa] : 694.70, found: 694.42; HRMS (ESI+) m/z calcd for [C34
H
34
F
4
N
3
O
5
6
72.2157, found: 672.2150; HPLC (I) t
R
= 23.66 min (99.9%); HPLC (II) t
R
= 38.58 min (99.9%).
4
-(2-((4-fluoro-N-(2-(trifluoromethyl)benzyl)phenyl)sulfonamido)-N-(4-
(
trifluoromethyl)benzyl)acetamido)-N-hydroxybenzamide (7). Semi-preparative HPLC using
acetonitrile/0.1% (v/v) TFA in MilliQ water (0:1 à 1:0, 50 min à 10 min) eluted the target
compound at 37.1 – 38.6 min. The product was suspended in acetonitrile/MilliQ water (1:3, 4 mL)
1
and lyophilized overnight at -50 °C to give 7 as a white solid (87.9 mg, 77%); H δ/ppm (400 MHz,
CDCl
7.15 (d, J = 8.5 Hz, 2H, 2 CH), 7.19 (t, J = 8.5 Hz, 2H, 2 CH), 7.35 (t, J = 7.6 Hz, 1H, CH), 7.45
t, J = 7.6 Hz, 1H, CH), 7.49 (d, J = 8.1 Hz, 2H, 2 CH), 7.61 (d, J = 8.5 Hz, 2H, 2 CH), 7.65 (d, J
8.2 Hz, 2H, 2 CH), 7.90 – 7.94 (m, 2H, 2 CH), hydroxamic acid NH and OH protons were not
3
) 3.70 (s, 2H, CH
2
), 4.73 (s, 2H, CH
2
), 4.79 (s, 2H, CH
2
), 6.91 (d, J = 7.6 Hz, 2H, 2 CH),
(
=
1
3
observed; C δ/ppm (100 MHz, CDCl
3
) 47.4, 47.9, 52.9, 116.1, 116.3, 122.7, 125.3, 125.47,
1
1
25.53, 125.57, 125.64, 125.8, 125.85, 125.88, 128.0, 128.3, 128.5, 128.6, 128.9, 128.97, 129.00,
1
9
30.1, 130.4, 130.5, 132.5, 134.2, 135.66, 135.69, 140.1, 143.7, 164.0, 166.55, 166.64; F δ/ppm
(
54 MHz, CDCl
3
) -104.6 to -104.5 (m, 1F), -62.6 (s, 3F), -59.0 (s, 3F); LRMS (ESI+) m/z calcd
+
S]+:
for [C31
H
24
F
7
N
3
O
5
SNa] : 706.59, found: 706.33; HRMS (ESI+) m/z calcd for [C31
H
25
F
7
N
3
O
5
6
84.1399, found: 684.1398; HPLC (I) t
R
= 22.20 min (99.9%); HPLC (II) t
R
= 36.13 min (97.1%).
28
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Page 29 of 57
Journal of Medicinal Chemistry
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4
-(2-((4-fluoro-N-((perfluorophenyl)methyl)phenyl)sulfonamido)-N-(4-
(
trifluoromethyl)benzyl)acetamido)-N-hydroxybenzamide (8). Semi-preparative HPLC using
acetonitrile/0.1% (v/v) TFA in MilliQ water (0:1 à 1:0, 50 min à 10 min) eluted the target
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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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
compound at 37.3 – 37.9 min. The product was suspended in acetonitrile/MilliQ water (1:3, 4 mL)
1
and lyophilized overnight at -50 °C to give 8 as a white solid (14.0 mg, 46%); H δ/ppm (400 MHz,
CDCl
.16 (t, J = 8.5 Hz, 2H, 2 CH), 7.21 (d, J = 7.8 Hz, 2H, 2 CH), 7.52 (d, J = 8.0 Hz, 2H, 2 CH), 7.77
d, J = 7.7 Hz, 2H, 2 CH), 7.84 (dd, J = 5.1 and 8.6 Hz, 2H, 2 CH), hydroxamic acid NH and OH
3
) 3.85 (s, 2H, CH
2
), 4.60 (s, 2H, CH
2
), 4.84 (s, 2H, CH
2
), 7.10 (d, J = 7.8 Hz, 2H, 2 CH),
7
(
1
3
protons were not observed; C δ/ppm (100 MHz, CDCl ) 39.7, 49.3, 53.0, 109.7, 116.1, 116.3,
3
125.6, 125.65, 125.71, 125.74, 128.6, 128.7, 129.00, 129.04, 129.07, 129.09, 130.1, 130.4, 130.5,
19
1
31.1, 133.6, 135.0, 135.1, 140.0, 143.6, 164.1, 165.1, 166.7, 166.8; F δ/ppm (54 MHz, CDCl )
3
-
161.1 (td, J = 7.4 and 21.5 Hz, 2F), -152.5 (t, J = 20.9 Hz, 1F), -141.3 (dd, J = 8.0 and 22.2 Hz,
-
2F), -104.1 to -104.0 (m, 1F), -62.6 (s, 3F); LRMS (ESI-) m/z calcd for [C30
H
19
F
9
N
3
O
5
S] : 704.54,
+
found: 704.35; HRMS (ESI+) m/z calcd for [C30
I) t = 21.96 min (99.9%); HPLC (II) t
21
H F
9
N
3
O
5
S] : 706.1053, found: 706.1044; HPLC
(
R
R
= 35.74 min (99.9%).
4
-(N-(4-(tert-butyl)benzyl)-2-((4-fluoro-N-
((perfluorophenyl)methyl)phenyl)sulfonamido)acetamido)-N-hydroxy-N-methylbenzamide
60). Semi-preparative HPLC using acetonitrile/0.1% (v/v) TFA in MilliQ water (0:1 à 1:0, 50
(
min à 10 min) eluted the target compound at 47.2 – 48.4 min. The product was suspended in
acetonitrile/MilliQ water (1:3, 2 mL) and lyophilized overnight at -50 °C to give 60 as a white
1
t
solid (19.1 mg, 77%); H δ/ppm (400 MHz, CDCl
H, CH ), 4.61 (s, 2H, CH ), 4.75 (s, 2H, CH ), 6.99 (d, J = 8.0 Hz, 2H, 2 CH), 7.09 (d, J = 7.8
Hz, 2H, 2 CH), 7.17 (t, J = 8.6 Hz, 2H, 2 CH), 7.29 (d, J = 8.0 Hz, 2H, 2 CH), 7.57 (d, J = 8.2 Hz,
3
) 1.31 (s, 9H, Bu), 3.42 (s, 3H, CH
3
), 3.87 (s,
2
2
2
2
29
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