Discovery of a New Isoxazole-3-hydroxamate-Based Histone Deacetylase 6 Inhibitor SS-208 with Antitumor Activity in Syngeneic Melanoma Mouse Models

Article abstract of DOI:10.1021/acs.jmedchem.9b00946

Isoxazole is a five-membered heterocycle that is widely used in drug discovery endeavors. Here, we report the design, synthesis, and structural and biological characterization of SS-208, a novel HDAC6-selective inhibitor containing the isoxazole-3-hydroxamate moiety as a zinc-binding group as well as a hydrophobic linker. A crystal structure of the Danio rerio HDAC6/SS-208 complex reveals a bidentate coordination of the active-site zinc ion that differs from the preferred monodentate coordination observed for HDAC6 complexes with phenylhydroxamate-based inhibitors. While SS-208 has minimal effects on the viability of murine SM1 melanoma cells in vitro, it significantly reduced in vivo tumor growth in a murine SM1 syngeneic melanoma mouse model. These findings suggest that the antitumor activity of SS-208 is mainly mediated by immune-related antitumor activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and the enhanced ratio of M1 and M2 macrophages in the tumor microenvironment.

Full text of DOI:10.1021/acs.jmedchem.9b00946

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Article
Discovery of a New Isoxazole-3-hydroxamate Based
Histone Deacetylase 6 Inhibitor SS-208 with Anti-
tumor Activity in Syngeneic Melanoma Mouse Models
Sida Shen, Melissa Hadley, Kseniya Ustinova, Ji#í Pavlí#ek, Tessa Knox, Satish
Noonepalle, Mauricio Temotheo Tavares, Chad A. Zimprich, GuiPing Zhang,
Matthew B. Robers, Cyril Barinka, ALAN KOZIKOWSKI, and Alejandro Villagra
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00946 • Publication Date (Web): 15 Aug 2019
Downloaded from pubs.acs.org on August 16, 2019
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Discovery of a New Isoxazole-3-hydroxamate Based Histone Deacetylase 6 Inhibitor
SS-208 with Anti-tumor Activity in Syngeneic Melanoma Mouse Models
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Sida Shen3,‡,†, Melissa Hadley , Kseniya Ustinova , Jiri Pavlicek , Tessa Knox , Satish
1,‡
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Noonepalle , Mauricio T. Tavares , Chad A. Zimprich , Guiping Zhang , Matthew B.
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,2
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Robers , Cyril Bařinka , Alan P. Kozikowski* , Alejandro Villagra*
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Department of Biochemistry and Molecular Medicine, The George Washington
University, Washington, District of Columbia 20052, United States.
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StarWise Therapeutics LLC, University Research Park, Inc., Madison, Wisconsin
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3719, United States.
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Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,
University of Illinois at Chicago, Chicago, Illinois 60612, United States.
4
Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of
Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic.
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Promega Corporation, Madison, Wisconsin 53711, United States.
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Bontac Bio-Engineering (Shenzhen) Co., Ltd, Shenzhen, Guangdong 518102, China.
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KEYWORDS: Ames-negative, catalytic domain 2, acetylated -tubulin, non-cytotoxic,
immunoregulation
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ABSTRACT
Isoxazole is a five-membered heterocycle that is widely used in drug discovery
endeavors. Here, we report the design, synthesis, and structural and biological
characterization of SS-208, a novel HDAC6-selective inhibitor containing the isoxazole-
3-hydroxamate moiety as a zinc-binding group as well as a hydrophobic linker. A crystal
structure of the Danio rerio HDAC6/SS-208 complex reveals a bidentate coordination of
the active-site zinc ion that differs from the preferred monodentate coordination observed
for HDAC6 complexes with phenylhydroxamate-based inhibitors. While SS-208 has
minimal effects on the viability of murine SM1 melanoma cells in vitro, it significantly
reduced in vivo tumor growth in a murine SM1 syngeneic melanoma mouse model. These
findings suggest the antitumor activity of SS-208 is mainly mediated by immune-related
antitumor activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and
the enhanced ratio of M1 and M2 macrophages in the tumor microenvironment.
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INTRODUCTION
Malignant melanoma represents the most aggressive and the deadliest form of
cutaneous cancer with increasing incidence, high metastasis, rapid disease progression,
poor prognosis, and high mortality.1, 2 Patients with metastatic melanoma have poor
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prognosis with a 5-year survival rate of less than 10%. A few targeted agent therapies have
recently become available for metastatic melanoma. For instance, BRAF inhibitors (i.e.,
dabrafenib) and MEK1/2 inhibitors (trametinib) have been approved as monotherapy or
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combined approach for the treatment of melanoma with mutation. However, the clinical
benefit of these therapies is limited due to the rapid development of resistance associated
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with the activation of alternative pro-oncogenic pathways. Within the last five years,
immunotherapy has revolutionized the treatment of patients with advanced-stage
melanoma. Antibodies that block the negative co-stimulatory receptors CTLA-4
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, 6
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(ipilimumab) , and PD-1 (nivolumab and pembrolizumab) have been approved based
on significantly longer overall survival in clinical trials compared to other therapies. The
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objective response rates of these immune checkpoint blockades are around 20-30%, while
primary, adaptive, and acquired resistance to immunotherapy is common and may be
related to the lack of recognition by T-cells.11, 12 Novel aiming to overcome these
limitations, especially on the resistance mechanisms originated in other components of the
tumor microenvironment (TME) are still highly needed to improve currently available
therapeutics.13-16
Histone deacetylases (HDACs) are a family of proteins involved in the epigenetic
regulation of target genes through deacetylating lysine ε-amino groups on histone tails to
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promote a status of DNA condensation and transcriptional silencing. Furthermore,
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HDACs have been found to modify other non-histone proteins, and these modifications
can influence a variety of cellular functions without modifying the acetylation of the
chromatin acetylation status.18-20 HDACs have been proven to be promising therapeutic
targets for the treatment of cancer based on the successful launch of five HDAC inhibitors
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(vorinostat, romidepsin, panobinostat, belinostat, and chidamide) which are relatively non-
selective.21, 22 In general, broad-spectrum HDAC inhibitors (pan-HDACis) are involved in
chromosome remodeling, cell cycle arrest, apoptosis, and cytotoxicity in transformed
cancer cells.23, 24 In addition to these effects, there is growing evidence to demonstrate that
the treatment with HDACis increases differentiation antigen expression, enhances MHC
class I and II surface expression, and elevates immunogenicity in terms of increased
expression of CD25, CD40, or CD80.25, 26 It has been reported that pan-HDACis can
upregulate the expression of PD-L1 and PD-L2 in human and murine melanoma cells.27
Therefore, pan-HDACis show ability to possess anti-tumor activity through inhibiting
28
proliferation and improving immune responses. However, phase I/II clinical trials of pan-
HDACis (vorinostat, panobinostat, and quisinostat) with melanoma patients only exhibited
limited efficacy and tolerability as single agents, while hematological toxicity, fatigue,
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nausea, and laboratory abnormalities occurred as frequent adverse events. Important
concerns regarding the toxicity of pan-HDACis are rising since their broad inhibition may
result in unwanted off-target effects that eventually impair their clinical profile. Thus, there
is an emerging interest in the development and investigation of selective HDACis to better
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understand the molecular mechanisms mediating the anti-tumor effects. So far, there are
few subtype-selective or isoform-selective inhibitors that are non-cytotoxic and better
tolerated, and that effectively impair tumor growth in preclinical models.30-35
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Eighteen known mammalian HDACs have been identified, which can be classified
into four subgroups according to their homology to yeast enzymes: class I (HDAC1, 2, 3,
and 8), class IIa (HDAC4, 5, 7, and 9), class IIb (HDAC6 and HDAC10), class III or
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sirtuins (SIRT 1−7), and class IV (HDAC11). Class I, II, and IV HDACs require Zn
zinc ion) as a cofactor to exert their hydrolytic activity and are also referred to as the
conventional HDACs. On the other hand, SIRT 1−7 are dependent on nicotinamide adenine
(
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dinucleotide for their activity. In comparison with other HDAC family members, HDAC6
is relatively unique. It contains two tandem deacetylase catalytic domains (CD1 and CD2)
and is primarily localized to the cytosol, and its preferred substrates include a variety of
non-histone proteins, such as α-tubulin, cortactin, HSP-90, and HSF-1.38, 39 Moreover, only
40
CD2 exhibits catalytic activity with broad specificity. Intriguingly, according to a patent
survey based on the recent efforts on the synthesis and applications of HDACis from 2013
to 2017, most patents focused on the discovery of new HDAC6 inhibitors (HDAC6is).41
42
Up to date, two partially selective HDAC6is, ricolinostat (ACY-1215) and citarinostat
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(
ACY-241), have been advanced into clinical trials for different types of cancer, such as
multiple myeloma, lymphoma, breast cancer, and melanoma, through monotherapy or a
combination approach. Moreover, selective HDAC6is have been considered as potential
therapeutic agents for the treatment of various neurological disorders.44-46
HDAC6 promotes the proliferation and metastasis of melanoma cells, and knockdown
of HDAC6 decreases proliferation and induces G1 cell cycle arrest of melanoma cells.28, 47
Moreover, it was found that HDAC6 increases the protein level of tyrosine-protein
phosphatase non-receptor type 1 (PTPN1), which is responsible for promoting
proliferation, colony formation, and migration while decreasing apoptosis of melanoma
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cells through activating extracellular signal-regulated kinase 1/2 (ERK1/2). A preclinical
study of ricolinostat showed that this partially selective HDAC6i impaired proliferation
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and induced apoptosis of BRAF-mutant melanoma A375 cells. Notably, the combination
treatment of BRAF inhibitor vemurafenib and ricolinostat displayed a synergetic effect in
BRAF-mutant melanoma cells partially through induction of endoplasmic reticulum stress
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and inactivation of ERK. In addition to the effects on survival, we have found that
HDAC6 is a modulator of the expression of specific tumor-associated antigens, MHC class
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I, and costimulatory molecules in melanoma. In previous works, we reported that either
selective pharmacological inhibition or genetic abrogation of HDAC6 plays a critical role
in the immune checkpoint blockade by down-regulating the expression of PD-L1, which
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exhibits an opposite effect relative to class I and pan-HDACis. Also, we recently found
that the combination of a selective HDAC6i, nexturastat A (NextA), and PD-1 immune
blockade resulted in a significant improvement in anti-tumor immune responses leading to
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further reduction of tumor growth compared to monotherapy. Taken together, all the
preclinical and clinical stage studies indicate that HDAC6 inhibition shows potential to
become a promising approach for melanoma therapy. However, neither ricolinostat nor
citarinostat can be considered to be highly selective HDAC6is due to their only about 10-
fold enzymic selectivity against the class I isoform, HDAC1 (Figure 1).52, 53 Therefore,
there is a need for the advancement of drug candidates with higher selectivity and good
pharmacologic attributes.
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Cap
Linker
ZBG
O
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OH
N
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H
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O
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N
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OH
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Cl
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N
ricolinostat (ACY-1215)
HDAC6 (IC50) = 4.7 nM
HDAC1 (IC50) = 58 nM
SI (HDAC1/6) = 12-fold
citarinostat (ACY-241)
HDAC6 (IC50) = 2.6 nM
HDAC1 (IC50) = 35 nM
SI (HDAC1/6) = 13-fold
tubastatin A
HDAC6 (IC50) = 15 nM
HDAC1 (IC50) = 16,400 nM
SI (HDAC1/6) = 1,093-fold
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S
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O
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Cl
nexturastat A
SW-100
HDAC6 (IC50) = 2.3 nM
HDAC1 (IC50) = 5,230 nM
SI (HDAC1/6) = 2,273-fold
HPB
HDAC6 (IC50) = 31 nM
HDAC1 (IC50) = 1,130 nM
SI (HDAC1/6) = 36-fold
bavarostat
HDAC6 (IC50) = 60 nM
HDAC1 (IC50) >1,000 nM
SI (HDAC1/6) > 16-fold
benzothiophene 7a
HDAC6 (IC50) = 14 nM
HDAC1 (IC50) = 7,500 nM
SI (HDAC1/6) = 535-fold
HDAC6 (IC50) = 5.02 nM
HDAC1 (IC50) = 3,020 nM
SI (HDAC1/6) = 601-fold
O
O
O
O
S
O
O
OH
OH
OH
O
N
OH
N
N
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H
N
OH
N
H
N
H
N
S
H
H
N
N
H
N
O
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F
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MeO
benzimidazole 23d
HDAC6 (IC50) = 0.81 nM
HDAC1 (IC50) = 8,960 nM
5-aroylindole 6
quinoline 13
tubathian 3l
HDAC6 (IC50) = 8.2 nM
HDAC1 (IC50) = 12,000 nM
SI (HDAC1/6) = 1463-fold
phenothiazine 7i
HDAC6 (IC50) = 3.9 nM
HDAC1 (IC50) = 2,190 nM
SI (HDAC1/6) = 561-fold
HDAC6 (IC50) = 0.29 nM
HDAC1 (IC50) = 9,550 nM
SI (HDAC1/6) = 32,931-fold
HDAC6 (IC50) = 5.0 nM
HDAC1 (IC50) = 2,690 nM
SI (HDAC1/6) = 538-fold
SI (HDAC1/6) = 11,061-fold
Figure 1. The structures of recent published HDAC6 inhibitors.
A typical HDACi structure contains a zinc-binding group (ZBG) that coordinates the
active-site Zn2+ at the deep bottom of the substrate pocket, a linker to occupy the
hydrophobic channel, and a capping group (cap) to interact with residues at the rim of the
catalytic cavity. Recent results suggest that the connecting unit (CU) region that links the
cap with the linker and ZBG may strongly impact the ligand’s selectivity towards
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HDAC6. The phenylhydroxamate moiety has been recognized as a useful ZBG as well
as a hydrophobic linker in the discovery of selective HDAC6is (Figure 1).31, 34, 55-64 The
majority of recent advancements has concentrated on the development of hydroxamate
analogs bearing a phenyl or pyrimidyl ring.65, 66 Meanwhile, minimal effort has gone into
developing linkers containing five-membered heteroaromatics such as thiazole and
oxazole.67, 68 Isoxazole is a five-membered heterocycle which contains two heteroatoms,
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one oxygen and one nitrogen atom, in adjacent positions. Two double bonds contribute to
the unsaturated character of the molecule. The structural features of isoxazole make it
suitable for multiple non-covalent interactions, including hydrogen bonding (N and O as
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acceptors) and π-π stacking. We note that the isoxazole moiety is a central part of multiple
compounds aimed at a wide spectrum of biological targets, and the inclusion of isoxazole
in a structure may contribute to its increased efficacy, decreased toxicity, and improved
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pharmacokinetic profile. Herein, we report on the discovery of SS-208, a novel isoxazole-
-hydroxamate-based, selective HDAC6i, its structural characterization in a complex with
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the catalytic domain 2 of Danio rerio HDAC6 (drHDAC6), and in vivo efficacy results in
a syngeneic mouse model of melanoma.
RESULTS AND DISCUSSION
Chemistry. In our prior work, we identified an isoxazole-3-hydroxamate-based hit 1a,
which demonstrates potent HDAC6 inhibitory activity and modest potency against
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HDAC1, a member of class I HDACs, unlike its corresponding alkylhydroxamate (1b)
and phenylhydroxamate (1c) analogs. Moreover, the compound 1d incorporating the
reverse direction of the amide CU dramatically loses selectivity (Figure 2).71-73 However,
the non-ideal physicochemical properties of 1a (TPSA = 168.82, MW = 457.44, calculated
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by SwissADME ) and short half-lives (t ) in microsomal stability studies (mouse: 16
1/2
min; human: 85 min, determined by Pharmaron, Inc., Irvine, CA) constitute an obstacle for
its advance into in vivo studies. Notably, 1a was incubated with two strains of Salmonella
typhimurium (TA97A and TA1537) by Pharmaron in the presence and absence of
mammalian microsomal enzymes (S9 mix) to evaluate the mutagenicity potential of the
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hydroxamate moiety. The results in Table S1 indicate that 1a is Ames-negative under the
testing conditions, which encouraged us to pursue further structural tuning in order to
discover more druggable isoxazole-3-hydroxamate-based HDAC6is.
OH
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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
NH
O
O
O
OH
BocHN
N
N
N
H
H
BocHN
N
O
O
N
H
O
N
1a
1b
HDAC6 (IC50) = 6.0 nM
HDAC1 (IC50) = 16,900 nM
SI (HDAC1/6) = 2,817-fold
HDAC6 (IC50) = 13.8 nM
HDAC1 (IC50) = 3.2 nM
SI (HDAC1/6) = 0.23-fold
O
O
OH
NH
N
H
BocHN
N
O
H
O
N
N
N
O
HN
OH
S
O
1c
1d
HDAC6 (IC50) = 0.33 nM
HDAC1 (IC50) = 436 nM
SI (HDAC1/6) = 1,321-fold
HDAC6 (IC50) = 31 nM
HDAC1 (IC50) = 307 nM
SI (HDAC1/6) = 9.9-fold
Figure 2. The structures of isoxazole-3-hydroxamate HDAC inhibitors 1a-d.
To improve the physicochemical properties of this series of compounds, we started our
optimization by replacing the Boc-protected phenylisoxazole cap with simple monocyclic
and bicyclic aromatic rings (7a-d), while keeping the same ethylamide and isoxazole-3-
hydroxamate moieties to maintain close structural similarity to 1a. The synthetic routes to
analogs 7a-d shown in Scheme 1 started from 3-butyn-1-ol (2a) and led to the preparation
of the same 1-amino-3-butyne intermediate 4 through a Mitsunobu reaction using
phthalimide/Ph P/DEAD. Subsequent deprotection and acylation with the appropriate acyl
3
chlorides provided the alkyne intermediates 5a-d. The acetylenic moieties in 5a-d were
transformed into the isoxazole esters 6a-d by 1,3-dipolar cycloaddition with a nitrile oxide
generated in situ from ethyl 2-chloro-2(hydroximino)acetate and a base under microwave
conditions.76 These esters upon treatment with an aqueous NH OH/NaOH solution
2
efficiently afforded the desired hydroxamic acids 7a-d bearing aromatic caps.
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O
a
b
c
N
HO
H2N
O
2a
3
4
O
O
O
O
OEt
NHOH
O
d
e
O
O
R1
N
N
N
H
R1
R1
N
N
H
H
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3
4
5
6
7
8
9
0
5
a, Phenyl
5b, 3,4-Dichlorophenyl
c, 2-Naphthyl
5d, Biphenyl-3-yl
6a, Phenyl
6b, 3,4-Dichlorophenyl
6c, 2-Naphthyl
7a, Phenyl
7b, 3,4-Dicholorophenyl
7c, 2-Naphthyl
5
6d, Biphenyl-3-yl
7d, Biphenyl-3-yl
Scheme 1. The synthetic route to compounds 7a-d. (a) phthalimide, Ph P, DEAD, THF, 0
3
°
°
C to rt, yield: 93%; (b) hydrazine monohydrate, MeOH, rt; (c) ArCOCl, TEA, DCM, 0
C, yields: 52-90% over two steps; (d) ethyl 2-chloro-2-(hydroxyimino)acetate, NaHCO3,
EtOAc, 100 °C (MW), yields: 49-78%; (e) NH OH (aq., 50%), NaOH, THF/MeOH (1:1),
2
0
°C to rt, yields: 10-43%.
Given the wide rim of the HDAC6 substrate pocket, aryl caps bearing a sterically bulky
moiety are usually preferred to enhance inhibitor affinity and specificity via interactions
with the residues forming the surface.55, 64, 77, 78 Therefore, we further investigated the
replacement of the cap and amide CU in 1a with bicyclic and tricyclic caps. To prepare the
indole-capped analogue 11a illustrated in Scheme 2, 1,3-dipolar cycloaddition of 5-hexyn-
1
-ol (2b) and ethyl 2-chloro-2(hydroximino)acetate was conducted to establish the key
isoxazole ester 8a from the beginning. Subsequently, 8a underwent bromination with
CBr /Ph P, N-alkylation under Cs CO /DMF conditions, and transformation to the desired
4
3
2
3
hydroxamate analog 11a in the presence of an aqueous NH OH/NaOH solution. The
2
synthetic routes to 11b-c started from 4-chloroaniline (12), which was first converted to
the N-phenyl amides 13a-b by treating with 3-chloropropanoyl chloride and 3,3-
dimethylacryloyl chloride, respectively. Subsequently, intramolecular Friedel−Crafts
cyclization under AlCl conditions followed by reduction of the intermediate lactam with
3
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63, 79
LiAlH afforded the tetrahydroquinolines 14a-b.
Reductive amination of 14a-b and
4
the aldehyde intermediate 9b which was prepared from 8a by PCC oxidation, provided the
corresponding esters 10b-c. In the end, the desired hydroxamate analogs 11b-c were
obtained using the same standard procedure as above. The synthetic route to the
dibenz[b,f]azepine-capped analog 11d began with the dichlorination of 15 using
NCS/silica gel80 followed by N-alkylation with 1,4-dibromobutane under NaH/DMF
conditions and alkynation with sodium acetylide to provide the key dibenz[b,f]azepine-
substituted alkyne 18. Then the building block 18 underwent 1,3-dipolar cycloaddition,
and the desired isoxazole-3-hydroxamate 11d was then obtained in the usual manner.
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
O
Cl
OEt
OEt
c
Cl
N
N
N
O
Br
O
d
b
e
O
O
9a
9b
10a
O
O
a
OEt
O
Cl
NHOH
OEt
HO
N
Cl
N
HO
N
N
2b
O
O
11a
8a
Cl
Cl
O
g
O
O
O
f
i
N
H
Cl
N
H
OEt
NHOH
Cl
Cl
Cl
9b, h
d
1
3a
14a
14b
N
N
NH2
O
N
N
Cl
j
Cl
R
12
R
R
R
10b, R = H
N
H
11b, R = H
1c, R = CH3
N
O
H
10c, R = CH3
1
1
3b
k
l
Cl
Cl
Br
m
Cl
Cl
a
Cl
Cl
N
N
N
N
H
H
15
16
17
18
O
Cl
O
Cl
Cl
OEt
d
Cl
NHOH
N
N
N
N
O
O
10d
11d
Scheme 2. The synthetic routes to compounds 11a-d. (a) ethyl 2-chloro-2-
hydroxyimino)acetate, NaHCO , EtOAc, 100 °C (MW), yields: 67-87%; (b) CBr , Ph P,
(
3
4
3
DCM, rt, yield: 89%; (c) 5,6-dichloro-1H-indole, Cs CO , DMF, 80 °C, 80%; (d) NH OH
2
3
2
(
aq., 50%), NaOH, THF/MeOH (1:1), 0 °C to rt, yields: 34-94%; (e) PCC, DCM, rt, yield:
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2
2
2
2
2
2
3
3
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3
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3
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4
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4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
8%; (f) 3-chloropropanoyl chloride, acetone, reflux, yield: 54%; (g) i) AlCl , 140 °C
3
(neat), ii) LiAlH , THF, 0 °C to reflux, yield: 70% over two steps; (h) 9b, NaBH(OAc) ,
4
3
AcOH/EtOH, rt, yields: 42-58%; (i) 3,3-dimethylacryloyl chloride, CHCl , reflux, yield:
3
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
4
8%; (j) i) AlCl , toluene, 80 °C, ii) LiAlH , THF, 0 °C to reflux, yield: 55% over two
3 4
steps; (k) NCS, silica gel, CHCl , rt, yield: 74%; (l) 1,4-dibromobutane, NaH, DMF, rt,
3
yield: 43%; (m) sodium acetylide, xylene/DMF, 40 °C, yield: 25%.
As a 3,4-dichlorophenyl cap on 7b retained HDAC6 potency and selectivity over HDAC1
(data are shown in Table 1), we further synthesized compounds 20a-c in order to evaluate
the effects of the amide CU and isoxazole-based ZBG. To prepare the analog 20a which
contains an ether linker in place of the amide group, the synthetic route shown in Scheme
3
was followed. 1,3-Dipolar cycloaddition between 4-pentyn-1-ol (2c) and ethyl 2-chloro-
2-(hydroximino)acetate generated the isoxazole ring (compound 8b). This intermediate
underwent bromination with CBr /Ph P followed by O-alkylation with 3,4-dichlorophenol
4
3
under Cs CO /DMF conditions. The synthesis was completed as above to afford the desired
2
3
hydroxamate 20a. To synthesize analog 20b which only contains an alkyl chain rather than
an amide group, the α,β-unsaturated carboxylic acid in the starting material 21 was first
transformed to a bromoalkyl intermediate 22 through LiAlH reduction and bromination
4
with CBr /Ph P. Compound 22 was further converted to the nitrile 23 with NaCN.
4
3
Subsequent hydrolysis under basic condition and borane reduction provided the butanol
4. The alkyne 25 was obtained through bromination and alkynation under similar
2
conditions as described above and underwent 1,3-dipolar cycloaddition and conversion to
the hydroxamic acid 20b. The synthetic route to the benzyl hydroxamate analog 20c started
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from Cu(OAc) -catalyzed one-pot conversion of 3,4-dichlorobenzaldehyde (26) into 3,4-
2
8
1
dichlorobenzamide (27) through a Beckmann-type rearrangement. Subsequent reductive
N-alkylation of the amide intermediate 27 using TFA/Et SiH with methyl 4-
3
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
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7
8
9
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2
3
4
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0
82
formylbenzoate led to the generation of the corresponding ester 19c, which upon standard
treatment afforded the desired hydroxamate 20c.
O
O
O
O
O
O
OEt
NHOH
a
OEt
b
OEt
c
d
HO
N
N
Cl
Cl
O
Cl
Cl
O
N
N
HO
Br
O
O
2c
8b
9c
19a
20a
O
e
Cl
Cl
f
Cl
g
Cl
Cl
OH
h
Cl
Cl
Br
CN
OH
Cl
21
22
24
23
O
O
O
OEt
NHOH
Cl
Cl
a
d
N
N
Cl
Cl
Cl
Cl
O
25
19b
20b
O
O
O
Cl
Cl
Cl
Cl
Cl
Cl
CHO
i
j
d
Cl
Cl
N
N
NH2
H
H
O
O
OMe
NHOH
26
27
19c
20c
Scheme 3. The synthetic routes to compounds 20a-c. (a) ethyl 2-chloro-2-
(hydroxyimino)acetate, NaHCO , EtOAc, 100 °C (MW), yields: 58%-96%; (b) CBr , Ph P,
3
4
3
DCM, rt, yield: 83%; (c) 3,4-dichlorophenol, Cs CO , DMF, 80 °C, yield: 80%; (d)
2
3
NH OH (aq., 50%), NaOH, THF/MeOH (1:1), 0 °C to rt, yields: 30-40%; for 20c, yield:
2
22% over three steps; (e) i) LiAlH , THF, 0 °C to reflux, ii) CBr , Ph P, DCM, 0 °C to rt,
4
4
3
yield: 11% over two steps; (f) NaCN, DMSO, 100 °C, yield: 72%; (g) i) aq. NaOH, EtOH,
reflux, ii) BH -THF, THF, 0 °C to rt, yield: 88% over two steps; (h) i) CBr , Ph P, DCM,
3
4
3
rt, ii) sodium acetylide, DMF, 40 °C, yield: 39% over two steps; (i) NH OH·HCl, K CO ,
2
2
3
Cu(OAc) , H O, reflux; (j) TFA, Et SiH, methyl 4-formylbenzoate, toluene, reflux.
2
2
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6
HDAC Isoform Inhibition. To evaluate isoform potency and selectivity of all new analogs
(7a-d, 11a-d, and 20a-c), we first prescreened their inhibitory activity against human
HDAC6 and HDAC1 in vitro (performed by Reaction Biology Corp, Malvern, PA). As
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
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0
1
2
3
4
5
6
7
8
9
0
illustrated in Table 1, the replacement of the phenylisoxazole cap of the parent compound
1
a with phenyl rings as in compounds 7a-b led to a 12.5-fold to 32-fold decrease in
inhibitory potency for HDAC6, while micromolar inhibition values against HDAC1
remained constant. Compounds 7c-d, in which the cap moiety was replaced with a naphthyl
or biphenyl group, retained low nanomolar inhibitory potency for HDAC6 with a
concomitant decrease of the HDAC1/HDAC6 selectivity index. Notably, this replacement
significantly decreased the number of heavy atoms and TPSA values compared to the hit
1
a (Table S2) and improved the ligand efficiency (LE) values beyond 0.4 while keeping
the lipophilic ligand efficiency (LipE) values above 5.0 (Table 1).83, 84 Incorporation of
sterically bulky caps into the molecule to form the bicyclic- or tricyclic-capped analogs
1
1a-d resulted in a significant decrease in potency, and none of them delivered meaningful
ligand efficiency values (LE and LipE). Replacement of the amide moiety in 7b with an
ether (20a) or alkyl chain (20b) was also disadvantageous, as it resulted in a 3-fold to 26-
fold decrease in HDAC6 potency, suggesting that the amide moiety is necessary for
retaining high potency in this class. Compared to 7b, its corresponding phenylhydroxamate
analog 20c demonstrated about 3-fold improvement in HDAC6 potency (IC = 28 nM)
50
and similar ligand efficiency values. However, the isozyme selectivity significantly fell to
around 7-fold, and the observed trend replicates finding for the 1a and 1c pair (Figure 2).
Based on the evaluation results of ligand efficiency and maintained modest potency against
HDAC1, we chose compound 7b, named SS-208, for further investigation.
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To fully evaluate the advantage of isoxazole-3-hydroxamates relative to the typical
phenylhydroxamate-based HDAC6 inhibitors, we assessed SS-208, 20c, and NextA
against Class I HDACs 1 and 8, Class IIa HDACs 4, 5, 7, and 9, Class IIb HDAC6, and
Class IV HDAC11 in-house. The HDAC profiling against Class IIa and Class IV isoforms
shows that SS-208 only exhibited micromolar potency against all these isoforms. In the
side-by-side comparison with the selective HDAC6i, NextA, SS-208 exhibited similarly
low nanomolar HDAC6 inhibitory activity with better selectivity over all the other tested
HDAC isoforms (Table 2).
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. HDAC1 and HDAC6 enzymatic evaluation of analogs 7a-d, 11a-d, and 20a-ca
IC50 (µM)
Cmpd
ae
cap
CU
ZBG
SIb
LEc LipEd
HDAC1 HDAC6
O
O
BocHN
1
N
H
16.9
> 30
31.5
3.03
0.006
0.19
2817 0.35
> 155 0.47
418 0.45
294 0.47
6.2
5.8
5.1
5.7
O
O
O
N
N
N
NHOH
O
N
O
O
7
a
N
H
NHOH
Cl
Cl
O
O
7b
N
H
0.075
0.010
NHOH
O
O
7
c
N
H
O
O
N
N
NHOH
O
O
7d
N
H
NHOH
2.04
> 30
11.2
> 30
0.024
4.36
0.69
1.99
85
> 7
16
0.41
0.31
0.36
5.7
2.0
3.3
2.1
O
O
Cl
Cl
N
1
1a
N
H
O
O
O
N
N
N
NHOH
O
N
1
1b
NHOH
Cl
O
N
1
1c
NHOH
> 15 0.31
Cl
O
N
11d
0a
20b
0c
O
N
NHOH
> 30
> 30
> 30
1.35
0.20
1.98
> 22 0.27
> 152 0.45
> 15 0.38
1.3
3.9
2.5
Cl
Cl
Cl
O
O
O
2
O
O
N
N
NHOH
Cl
Cl
O
NHOH
Cl
Cl
O
O
2
N
H
NHOH
NHOH
0.20
0.028
7.2
0.48
4.8
Cl
Bu
H
NextAf
TSAg
N
N
2.86
0.005
0.002
572 0.46
4.4 0.55
5.7
6.4
O
-
-
-
0.009
a
IC values are the mean of two experiments obtained from curve-fitting of a 10-point
5
0
enzymatic assay starting from 30 µM with 3-fold serial dilution against HDAC1 and
b
HDAC6 (Reaction Biology Corp, Malvern, PA). SI: HDAC6 selectivity over HDAC1.
c
d
LE: ligand efficiency = 1.4*pIC /number of heavy atoms. LipE: lipophilic ligand
5
0
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2
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
e
efficiency = pIC – clog P . IC values were extracted from the article by Gaisina, I. N.
5
0
o/w
50
f
et al., ChemMedChem 2016, 11, 81-92. IC values were extracted from the article by
5
0
g
Kozikowski, A. P. et al., ACS Chem. Neurosci. 2019, 10, 1679-1695. TSA: trichostatin A.
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 2. HDAC profiles of the 3,4-dichlorophenyl capped compounds SS-208 and 20c, as
well as NextAa
Cmpd
SS-208 (7b)
20c
NextA
O
O
Cl
Cl
Cl
Cl
OH
OH
H
N
N
H
O
H
N
N
H
H
N
Structure
N
O
O
N
HN OH
O
O
Isoform
HDAC1
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
HDAC9
HDAC11
IC (µM)
1.39 ± 0.73
19.5 ± 4.2
6.91 ± 0.29
0.012 ± 0.002
8.34 ± 0.71
1.23 ± 0.59
38.2 ± 5.1
SIb
116
1625
576
1
695
103
3183
427
IC (µM)
SI
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IC (µM)
0.36 ± 0.12
14.8 ± 1.7
6.62 ± 2.66
0.004 ± 0.002
2.43 ± 0.30
1.54 ± 0.76
2.0 ± 0.77
SI
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0.034 ± 0.003
3.53 ± 0.13
3.12 ± 0.48
0.002 ± 0.0003
0.50 ± 0.008
0.92 ± 0.15
3.07 ± 0.17
1.05 ± 0.11
1765
1560
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460
1535
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3700
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500
2650
5.12 ± 1.29
10.6 ± 2.2
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IC values are the mean of two experiments ± SEM obtained from curve-fitting of a 10-
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point enzymatic assay starting from 100 µM with 3-fold serial dilution against HDAC
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isoform. SI: HDAC6 selectivity index over other HDAC isoforms.
Crystallization Studies. The recently published co-crystal structures of
phenylhydroxamate-based HDAC6is in complex with drHDAC6 displayed an unusual
monodentate phenylhydroxamate-Zn2+ coordination geometry, while a bidentate
coordination is generally observed for inhibitors that possess either flexible aliphatic
linkers or aromatic linkers lacking a cap.85, 86 The recent findings demonstrated that the
caps attached to the phenylhydroxamate affect the coordination between the hydroxamates
2+
and the catalytic Zn to be bidentate or monodentate, which is also crucial to stabilize the
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monodentate coordination mode that is slightly less stable (0.5 kcal/mol) than the bidentate
coordination mode.85, 87 Moreover, the key residues H463, P464, F583, and L712 create
an L1-loop pocket to provide a rigid binding site for hydrophobic caps. Recent research
further supports that the interaction between ligand and L1-loop pocket is critical for
maintaining high selectivity against class I HDACs.87
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To investigate the molecular basis of interactions between SS-208 and HDAC6, we solved
the crystal structure of the drHDAC6-CD2/SS-208 complex to the ultra-high resolution
limit of 1.15 Å. Binding of SS-208 to HDAC6 did not cause any major structural
rearrangement of the enzyme as documented by the RMSD of 0.15 Å for 300 Cα atoms
between the drHDAC6/SS-208 complex and the unliganded enzyme (PDB code: 5EEM).88
The hydroxamate functional group of SS-208 coordinates the active-site zinc ion in a
canonical bidentate fashion, forming a typical five-membered chelate complex with
2
+
interatomic distances of 2.1 Å between the Zn and each of the hydroxamate C=O and
N-O groups (Figure 3A). Moreover, the hydroxamate C=O group accepts a hydrogen bond
from the hydroxyl group of Y745 (2.7 Å), and the N-O- group forms hydrogen bonds with
the side chains of H573 (2.6 Å) and H574 (2.5 Å and 2.9 Å). The isoxazole ring and the
distal ethylene part of the linker are positioned within van der Waals distances from the
side chains of F583, F643, and L712. The C=O group of the amide moiety establishes an
interaction with S531 in a distance of 3.3 Å, and the N-H group engages in a water-
mediated interaction with H614 as observed in the binding with other HDAC6is in the
87
literature. Finally, the capping dichlorophenyl ring packs against the hydrophobic patch
at the entrance of the internal tunnel, the L1-loop pocket, formed by side chains of H463,
P464, F583, and L712 (Figure 3B).87
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It is interesting to note that the bidentate zinc ion coordination reported here differs from
the preferred monodentate coordination observed in drHDAC6 complexes where a
phenylhydroxamate group is capped by a hydrophobic moiety of an inhibitor.40, 85, 87 In our
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complex, the center of the isoxazole ring is shifted by 0.5 Å towards the active-site Zn2+,
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and this shift stems from the bidentate coordination of the Zn by the hydroxamate moiety
(Figure 3C). This shift is likely facilitated by the presence of a “long” four-atom linker
between the isoxazole ring and the capping dichlorophenyl moiety, allowing thus for the
more energetically favorable bidentate hydroxamate coordination while simultaneously
preserving hydrophobic interactions between the cap and the L1-loop pocket.87
Figure 3. Crystal structure of the drHDAC6-CD2/SS-208 complex. (A) Detail view of the
substrate tunnel of HDAC6 in complex with SS-208. Fo-Fc maps (green mesh) for SS-208
are contoured at 4.0 σ and the inhibitor and selected HDAC6 residues are shown in stick
representation with atoms colored grey (carbon), red (oxygen), blue (nitrogen), and green
(chlorine). The active-site zinc ion and water molecules are shown as orange and red
spheres, respectively. H-bonds and salt bridges are shown as dashed lines. (B) Interactions
between the capping dichlorophenyl moiety and the “L1-loop pocket” formed by side
chains of H463, P464, F583, and L712 (colored blue). The surface of HDAC6 is shown in
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semi-transparent surface representation. (C) Superposition of SS-208 and DDK-115 (PDB
code: 6DVL) in the substrate tunnel of HDAC6. Carbon atoms are colored green and pink
for SS-208 and DDK-115, respectively. The active-site zinc ion is coordinated in a
bidentate fashion by SS-208, while monodentate coordination is observed for DDK-115,
resulting in the 0.5 Å shift of the SS-208 ring closer towards the zinc ion.
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HDAC target engagement measurement in HEK293 cells. We further assessed the
potency and selectivity of SS-208 in live cells using NanoBERT target engagement assay
to investigate its binding characteristics with HDAC1 and HDAC6 within intact cells.89
These results performed in HEK293 are summarized in Figure 4, and suggest that SS-208
retains modest activity against HDAC1 in cells (IC = 6.67 µM), while the measured
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potency of SS-208 against the α-tubulin preferring deacetylase domain of HDAC6, the
catalytic domain 2 (IC = 0.5 µM), indicates SS-208 retains about 13-fold selectivity over
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HDAC1 in cells (418-fold enzymatic selectivity over HDAC1 in Table 1) which is
comparable with our recent observation on another highly selective HDAC6i, SW-100,
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tested under the same conditions. SW-100 showed an IC value of 2.3 nM against
50
HDAC6 and 2273-fold selectivity over HDAC1 in the enzymatic assay, while it exhibited
IC of 0.10 µM against HDAC6-CD2 and 46-fold selectivity over HDAC1 in the cellular
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target engagement assay. Vorinostat (SAHA), tested as positive control, demonstrated to
be potent and non-selective towards HDAC6-CD2 (IC = 0.24 µM) as well as HDAC1
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(
IC = 0.12 µM) (Figure 4).
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Figure 4. SS-208 selectively inhibits HDAC6-CD2 in cells. HDAC1 and HDAC6
(catalytic domain 2) NanoBRET target engagement assay for compound SS-208 in
HEK293. IC values are the mean of four technical replicates ± SD obtained from curve-
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fitting of a 12-point engagement assay starting from a concentration of 20 µM with 3-fold
serial dilution. SAHA was used as a positive control.
Initial ADMET properties. To evaluate the potential mutagenicity of this hydroxamate-
based compound, SS-208 was incubated with two strains of Salmonella typhimurium
(TA98 and TA1537) in the presence and absence of mammalian microsomal enzymes (S9
mix). SS-208 did not induce ≥ 2-fold increases (for tester strain TA98) or ≥ 3-fold increases
(for tester strain TA1537) in the mean number of revertant colonies at any dose levels when
compared to the concurrent negative/solvent control, both in the presence and absence of
the S9 mix, thus supporting the lack of mutagenicity of SS-208 under the conditions of the
Ames assay (Table S3). No significant inhibition was observed in the hERG assay up to
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0 µM (Figure S1). The liver microsomal stability is shown in Table 3 (half-lives of 37
min and 135 min in mouse and human microsomes, respectively). This constitutes an
improvement relative to the original compound 1a. Moreover, half-lives of 22 min and 135
min in mouse and human hepatocytes were determined, respectively.
Table 3. Initial ADMET profiling of compound SS-208.a
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hERG test (IC , µM)
HEK293 cells > 30
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Liver microsomal stability
(t min, with NADPH)
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Hepatocytes stability (t min)
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aAll the ADMET assays were conducted by Pharmaron, Inc., Irvine, CA.
Inhibitory potency of SS-208 is comparable to NextA and TubA in cancer cells. As
mentioned before, several selective HDAC6is are currently available for pre-clinical
research. Among them, NextA and tubastatin A (TubA) were reported by our group.55, 56
Although these two compounds have shown outstanding in vivo anti-tumor effects in
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numerous syngeneic tumor models, they were found to be Ames-positive (data are not
shown). We thus wanted to compare the Ames-negative HDAC6i SS-208 with these two
previously reported HDAC6is. We performed an initial screening of the in vitro activity of
SS-208 by evaluating the acetylation status of α-tubulin (Ac-α-Tubulin), the best
characterized physiological substrate of HDAC6. As expected, SS-208 increased Ac-α-
Tubulin levels in SM1 murine melanoma (Figure 5A) and WM164 human melanoma
(Figure 5B). Additionally, SS-208 had a negligible effect on the levels of acetylated histone
H3 (Ac-H3) in SM1 murine melanoma cells, and these findings are comparable to results
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of experiments where NextA was used (Figure 5C).
We previously observed that the selective HDAC6is NextA and TubA had similar in vitro
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HDAC inhibitory potency across different human and murine cancer cell lines. A similar
outcome was observed with SS-208, which was also comparable to the above mentioned
selective HDAC6is when tested in human PC3, human 5637, human T24, and mouse SM1
cell lines (Figure 5D). We also observed that SS-208 induced minimal cell death in the
concentration range evaluated above, and these results were also comparable to NextA and
TubA (Figure 5E). Importantly, we have reported that the in vivo antitumor effect of
selective HDAC6is is mainly mediated by their role as modulators of anti-tumor immune
responses and that their direct cytotoxicity towards cancer cells is minimal at
concentrations lower than 5 μM.28 Supporting these previously reported findings, we
observed that SS-208 has marginal effects on viability, cytotoxicity, and apoptosis in
murine SM1 melanoma cells evaluated by the multiplexed ApotoxTM assay (Figure 5F).
The absence of cytotoxic effects in cells treated with these selective HDAC6is is in sharp
contrast to the effects observed with other pan-HDACis such as panobinostat (LBH589 in
Figure 5F), among others, therefore removing one of the most critical side effects of non-
selective HDACis or other cytotoxic anticancer drugs.
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Figure 5. SS-208 activity is similar to other selective HDAC6is in cancer cells in vitro. (A)
SS-208 treatment of SM1 murine melanoma cells resulted in increased Ac-α-Tubulin
similar to other HDAC6is NextA and TubA. (B) The activity of HDAC6 inhibition with
SS-208 was dose-dependent as indicated by Western-blot analysis of Ac-α-Tubulin in
WM164 human melanoma cells. (C) SM1 melanoma cells were treated with SS-208,
NextA, or LBH589 for 24 h. The levels of Ac-H3 were evaluated by Western-blot analysis.
(D) SS-208, NextA, and TubA exhibited similar inhibition of deacetylase activity when
tested in a panel of cell lines of various tissue origins. (E) Similarly, HDAC6is did not
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induce cell death across the range of concentrations tested. (F) Multiplexed assay indicated
that pan-HDACi, LBH589 exhibited apoptosis and cytotoxicity at concentrations as low as
100 nM, while the HDAC6is tested did not affect cell viability up to 10 µM. HDAC6is also
did not induce apoptosis and cytotoxicity compared to LBH589.
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SS-208 reduces the expression of PD-L1 in melanoma cells. Our group has previously
reported that the genetic and pharmacological targeting of HDAC6 affected the expression
of the immunosuppressive molecule PD-L1 (CD274). Abrogation of HDAC6 activity
results in inactivation of the STAT3 pathways upon treatment with HDAC6is or
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knockdown of HDAC6. To further evaluate if SS-208 affects the processes mentioned
above, we evaluated the activation of the STAT3 pathway by assessing the phosphorylation
of STAT3 after treatment with SS-208 (5 µM). Western blot results illustrated in Figure
6A demonstrated that SS-208 efficiently reduced IL-6-mediated Y705 phosphorylation of
STAT3 and the subsequent downregulation of the PD-L1 expression. HDAC6 inhibition
was evidenced by the increased levels of Ac-α-Tubulin. Further verification by qRT-PCR
indicated that IL-6 significantly increased CD274 gene expression by at least 2-fold
compared to the untreated condition, and SS-208 effectively negated IL-6 induced CD274
gene expression suggesting that this HDAC6i regulates PD-L1 expression at the mRNA
level rather than through post-translational modifications at the protein level (Figure 6B).
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Figure 6. Immunomodulatory role of SS-208 by decreasing the expression of PD-L1 in
melanoma cells. (A) SM1 murine melanoma cells pre-treated with 5 μM of SS-208
followed by IL-6 treatment for overnight effectively reduced the expression of
immunosuppressive molecule PD-L1 at the protein level. (B) SS-208 decreased IL-6
mediated STAT3 activation measured as Y705 phosphorylation and subsequently PD-L1
gene expression measured by qRT-PCR.
SS-208 impairs melanoma tumor growth in immunocompetent mice. The in vivo
antitumor effect of selective HDAC6is has been extensively investigated.35, 50, 51
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we wanted to evaluate whether SS-208 could also impair tumor growth in a syngeneic
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murine model with a functional immune system. To evaluate this possibility, we injected
immunogenic murine SM1 melanoma cells subcutaneously in the flank of C57BL/6 mice
and subsequently treated them with SS-208 (25 mg/kg, ip administration) or vehicle after
one-week post tumor engraftment. As observed previously for other HDAC6is, the tumor
growth was significantly reduced after treatment with SS-208 (Figure 7A-B), suggesting
that this Ames-negative selective HDAC6i also exerts its anti-tumor effect by modulating
the host immune system. To test this hypothesis, we evaluated the composition of different
cellular components of the TME. Although we did not observe a significant infiltration of
cytotoxic T cells (CD8+), their presence in the TME was slightly superior to the vehicle
control (Figure 7C). Among other screened tumor-infiltrating lymphocytes (TILs), the
infiltration of CD4+ T cells did not change (Figure 7D). However, the infiltration of natural
killer T cells (NKT) was significantly upregulated in the SS-208 treatment arm (Figure
7E).
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The anti-tumoral M1 macrophage phenotype has been reported to be associated with better
prognosis in several malignancies, and there is an active search for agents improving their
infiltration in tumors90 On the other hand, tumor infiltration of pro-tumoral M2
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macrophages is directly associated with a bad prognosis in cancer. In previous studies
from our group, we observed a significant increase in the ratio of M1 and M2 macrophages
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in tumors treated with selective HDAC6is but not with other non-selective inhibitors. As
expected, this outcome was also observed in the melanomas of the mice treated with SS-
2
08 (Figure 7F-H), confirming that this compound possesses similar immunological
properties as previously investigated NextA.48
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Figure 7. In vivo anti-tumor activity of SS-208 is mediated through modulation of tumor
microenvironment. (A) SS-208 treatment (25 mg/kg, ip) significantly decreased the tumor
growth compared to the control group in SM1 murine melanoma syngeneic model. (B)
Tumor growth curves of individual animals in control and SS-208 treatment groups.
Animals receiving SS-208 treatment consistently showed lower tumor volume during the
course of study compared to control groups. The decrease in tumor volume in the SS-208
treatment group was associated with infiltration of intra-tumoral CD8+ T-cells (C), CD4+
T-cells (D), and significant increase in NKT-cells (E). SS-208 treatment was associated
with significant increase in anti-tumor M1 macrophages (F), M2 macrophages (G), and the
ratio of M1/M2 (H) which is an indication of effective anti-tumor immunity.
CONCLUSIONS.
We have designed and synthesized a series of HDAC6 inhibitors bearing isoxazole-3-
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hydroxamate instead of phenylhydroxamate as ZBG. By evaluating their inhibitory
activities at HDAC isoforms, we found that: (a) the amide moiety is an important
constituent of the connecting unit to retain high HDAC6 potency; (b) bulky caps, like
napththyl and biphenyl groups, result in better HDAC6 activity but lower selectivity versus
HDAC1; (c) the replacement of the corresponding phenylhydroxamate leads to the
significant potency improvement against HDAC6 but the loss of hundred-fold HDAC1
selectivity. We have thus identified a novel potent and selective HDAC6i, namely SS-208.
The X-ray structure analysis of the crystallized complex of SS-208 with catalytic domain
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from Danio rerio HDAC6 demonstrates that: (a) the hydroxamate moiety coordinates in
a distinct bidentate manner with the active-site zinc ion, forming a typical five-membered
chelate complex, in contrast to the preferred monodentate coordination observed in most
HDAC6 complexes with phenylhydroxamate-based inhibitors; (b) direct and indirect
engagements are observed between the key amide moiety and amino acid residues in the
active site that may be responsible for the ligand’s nanomolar HDAC6 potency; (c) the
interactions between the 3,4-dichlorophenyl cap and the L1-loop pocket is critical for high
selectivity against HDAC1. Both in vitro and in vivo ADMET profiling and
pharmacological observation suggest that: (a) SS-208 shows Ames-negative results under
the testing conditions and no significant inhibition in the hERG assay, and is more
metabolically stable relative to the original compound 1a; (b) In murine and human
melanoma cells, SS-208 induces minimal cell death and exhibits marginal effects on
viability, cytotoxicity, and apoptosis, while significantly increases the levels of Ac-α-
Tubulin; (c) Furthermore, SS-208 efficiently reduces the IL-6-mediated Y705
phosphorylation of STAT3 and downregulates the expression of PD-L1 at the mRNA
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levels in melanoma cells; (d) Finally, SS-208 impairs tumor growth in a murine SM1
syngeneic melanoma mouse model which is mainly mediated by immune-related antitumor
activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and the
enhanced ratio of M1 and M2 macrophages in the TME.
Experimental section.
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Chemistry
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General information. H and C NMR spectra were obtained on 400/101 and 500/126
MHz Bruker spectrometers, except where noted otherwise, using the solvent residual peak
as the internal reference (chemical shifts: CDCl , δ 7.26/77.0; DMSO-d , 2.50/39.52;
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acetone-d , 2.05/29.84 and 206.26). The following abbreviations for multiplicities were
6
used: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, dd = double
doublet, dt = double triplet, ddd = double-double doublet, and br s = broad singlet. TLC
plates (Merck silica gel 60 F , 250 μm thickness) were used to monitor reaction progress,
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and spots were visualized under UV (254 nm). High-resolution mass spectrometry
HRMS) was carried out on a Shimadzu IT-TOF instrument under the following
(
conditions: column, ACE 3AQ (50 × 2.1 mm, id); mobile phase, 5 – 100%
acetonitrile/water containing 0.1% formic acid at a flow rate of 0.5 mL/min for 4 min. Flash
chromatography was performed on a Combi-Flash Rf system (Teledyne ISCO) with silica
gel cartridges. Preparative HPLC was used in the purification of all final compounds using
a Shimadzu preparative LC under the following conditions: column, ACE 5AQ (150 × 21.2
mm, id); mobile phase: 5 – 100% acetonitrile/water containing 0.05% TFA at a flow rate
of 17 mL/min for 30 min; UV detection at 254 and 280 nm. Analytical HPLC was used to
determine the purity of all final products using an Agilent 1260 series instrument under the
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