Synthesis and Pharmacological Evaluation of Selective Histone Deacetylase 6 Inhibitors in Melanoma Models

Article abstract of DOI:10.1021/acsmedchemlett.7b00223

Only a handful of therapies offer significant improvement in the overall survival in cases of melanoma, a cancer whose incidence has continued to rise in the past 30 years. In our effort to identify potent and isoform-selective histone deacetylase (HDAC) inhibitors as a therapeutic approach to melanoma, a series of new HDAC6 inhibitors based on the nexturastat A scaffold were prepared. The new analogues 4d, 4e, and 7b bearing added hydrophilic substituents, so as to establish additional hydrogen bonding on the rim of the HDAC6 catalytic pocket, exhibit improved potency against HDAC6 and retain selectivity over HDAC1. Compound 4d exhibits antiproliferative effects on several types of melanoma and lymphoma cells. Further studies indicates that 4d selectively increases acetylated tubulin levels in vitro and elicits an immune response through down-regulating cytokine IL-10. A preliminary in vivo efficacy study indicates that 4d possesses improved capability to inhibit melanoma tumor growth and that this effect is based on the regulation of inflammatory and immune responses.

Full text of DOI:10.1021/acsmedchemlett.7b00223

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Letter
Synthesis and Pharmacological Evaluation of Selective
Histone Deacetylase 6 Inhibitors in Melanoma Models
Mauricio Temotheo Tavares, Sida Shen, Tessa Knox, Melissa Hadley,
Zsofia Kutil, Cyril Barinka, Alejandro Villagra, and Alan P. Kozikowski
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00223 • Publication Date (Web): 05 Sep 2017
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Maurício T. Tavares,†║ Sida Shen,†║ Tessa Knox,‡ Melissa Hadley,‡ Zsófia Kutil,§ Cyril Bařinka,§
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Alejandro Villagra,‡* and Alan P. Kozikowski#*
† Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, IL,
60612, United States.
‡ Department of Biochemistry and Molecular Medicine, The George Washington University, Washington, DC, 20052, United
States.
§ Institute of Biotechnology, Academy of Sciences of the Czech Republic, BIOCEV, Prumyslova 595, 252 50 Vestec, Czech
Republic.
# StarWise Therapeutics LLC. University Research Park, Inc., 510 Charmany Drive, Madison, Wisconsin, 53719, United
States.
║These authors contributed equally to this work.
KEYWORDS: HDAC6 inhibitors, Nexturastat A, Melanoma, Inflammatory response, Immune response
ABSTRACT: Only a handful of therapies offer significant improvement in the overall survival in cases of melanoma, a cancer whose
incidence has continued to rise in the past 30 years. In our effort to identify potent and isoform-selective histone deacetylase (HDAC)
inhibitors as a therapeutic approach to melanoma, a series of new HDAC 6 inhibitors based on the nexturastat A scaffold were
prepared. The new analogs 4d, 4e, and 7b bearing added hydrophilic substituents, so as to establish additional hydrogen bonding on
the rim of the HDAC6 catalytic pocket, exhibit improved potency against HDAC6 and retain selectivity over HDAC1. Compound
4d exhibits anti-proliferative effects on several types of melanoma and lymphoma cells. Further studies indicates that 4d selectively
increases acetylated tubulin levels in vitro and elicits an immune response through down-regulating cytokine IL-10. A preliminary in
vivo efficacy study indicates that 4d possesses improved capability to inhibit melanoma tumor growth, and that this effect is based
on the regulation of inflammatory and immune responses.
Melanoma is a common type of skin cancer that is potentially
lethal, and whose incidence has doubled over the past 30 years.
Despite the progress made in the understanding of the cell biol-
ogy, genetics, and immunology of melanoma, the outcome for
patients with advanced-stage disease has remained modest with
a median survival period ranging from 12 to 24 months and with
an overall survival rate at 5 years of less than 20%.1 A few ad-
vancements have recently been achieved for metastatic mela-
noma with mutation-based targeted therapies such as the
dabrafenib and trametinib for the treatment of melanoma with
BRAFV600E or BRAFV600K mutations2, and with immune check-
point blockade [e.g., ipilimumab (CTLA-4) and pembroli-
zumab (PD-1)].3 However, primary nonresponse and acquired
resistance to therapy remain challenges and require the devel-
opment of novel treatment approaches.4 One of the recent ad-
vances in cancer treatment focuses on the role of epigenetic
modifiers in the regulation of immuno-modulatory pathways.5
Among these, histone deacetylases (HDACs) are attractive tar-
gets due to the availability of several marketed, broad-spectrum
inhibitors of these zinc-containing enzymes. Several pan-
HDAC inhibitors (HDACis) such as vorinostat, panobinostat,
and quisinostat have recently been tested in Phase I or early
Phase II trials for melanoma, yet most of these show limited
efficacy and tolerability as single agents (Figure 1), with hema-
tological toxicity, fatigue, nausea, and laboratory abnormalities
occurring as frequent adverse effects.6 Significant wider con-
cerns regarding pan-HDACis are also rising since their broad
activity may cause unwanted off-target effects that may impair
their tolerability in clinical use. Since “one-size-fits-all” ap-
proaches have been dominating the design of new HDACis, the
relevance of targeting one specific HDAC isoform has not been
well established. In our prior work, we have demonstrated that
pan-HDACis possess anti-tumor activity through direct cyto-
toxicity and improved immune responses.7 There is now grow-
ing interest in developing isozyme-selective HDACis that
maintain beneficial effects but exhibit reduced toxicity or dele-
terious effects compared to broad-spectrum inhibitors.8
HDACs are a family of proteins responsible for catalyzing
the hydrolysis of acetylated lysine residues in histones to pro-
vide free lysine residues.9 There are 18 known mammalian
HDACs, which are divided into 4 classes, based on their se-
quence similarity to yeast homologs: class I (HDAC1, 2, 3, and
8), class IIa (HDAC4, 5, 7, and 9), class IIb (HDAC6 and 10),
class III (SIRT1-7), and class IV (HDAC11).10-12 Among these
isoforms, HDAC6 has received particular attention in the last
10 years due to its relative uniqueness within its family. Unlike
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its related family members, HDAC6 contains two tandem pro- rapid reductive amination with the appropriate amines to pro-
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tein deacetylase catalytic domains (CD1 and CD2), primarily
within the cytosol rather than the nucleus, and has no apparent
role in the post-translational modification of histone proteins,
but rather is involved in regulating the acetylation status of α-
tubulin, cortactin, HSP-90, HSF-1, and other non-histone pro-
teins.13 We previously identified a potent and highly selective
HDAC6i named nexturastat A (NexA, Figure 1), which pre-
sents low micromolar anti-proliferative activity in vitro against
a panel of human melanoma cell lines including both mutant
and wild type NRAS/BRAF.14 Further experiments demon-
strated that treatment with NexA in vivo resulted in both im-
paired tumor growth and increased tumor-specific immuno-
genic signals, which are characteristics highly desired in anti-
cancer therapies.7,15
vide intermediates 5a-c, followed by reaction with phenyl iso-
cyanate or isopropyl chloroformate to generate compounds 6a-
c. Further transformation to the hydroxamic acids 7a-c was ef-
ficiently performed as above. To synthesize the analog 7d bear-
ing a phenyl linker instead of a benzyl linker (Scheme 1), me-
thyl 4-aminobenzoate (9) underwent reductive alkylation to
give the intermediate ester 5d, which upon treatment with 3-
nitrophenyl isocyanate afforded the urea intermediate 10. This
compound was subjected to a two-step procedure consisting of
hydrogenation and reductive alkylation using zinc-modified cy-
anoborohydride16 and aqueous formaldehyde to provide the es-
ter 6d in high yield, which gave the hydroxamate product 7d in
the usual manner.
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Scheme 1. Synthesis of analogs 4a-e, 7a-d, 8, and 14a-d.a
Herein, we report further structure-activity relationship
(SAR) studies in a series of compounds based on the NexA scaf-
fold bearing a urea as cap, a benzyl linker, and a hydroxamate
moiety as zinc-binding group (ZBG). Furthermore, we provide
evidence that these NexA analogs, while displaying only mod-
est anti-proliferative effects on melanoma cell lines, possess an
improved capability to inhibit tumor growth in a melanoma
xenograft model, and that this effect is based on the regulation
of inflammatory and immune responses rather than direct cyto-
toxicity.
Figure 1. Structures of nexturastat A, and hydroxamate based HDACis
in clinical trials for melanoma.
In general, HDACis contain three main motifs: a cap group
that interacts with the surface of the enzyme, a linker group that
occupies a hydrophobic channel, and a ZBG that coordinates
with the zinc ion (Zn2+) at the bottom of the catalytic pocket
(Figure 1). Previously, we reported that a side chain, attached
to the urea nitrogen proximal to the benzyl linker, plays a sig-
nificant role for improving both HDAC6 selectivity and po-
tency.14 Thus, to explore additional modifications in the cap re-
gion, we initially designed and synthesized analogs 4a-e with
phenyl replaced by amine-substituted phenyl and by different
nitrogen heterocycles, while the n-butyl-substituted urea motif
as present in NexA was retained (Scheme 1). The synthesis of
these compounds began with the two-step reductive amination
of aldehyde 1 with n-butylamine to generate a common ester
intermediate 2. The reaction between 2 and appropriate phenyl
carbamates afforded the urea derivatives 3a-e.14 The final prod-
ucts 4a-e were obtained by reaction of these precursors with
aqueous hydroxylamine under basic conditions.
aReagents and conditions: a) i. amine, EtOH, reflux, 2 h; ii. NaBH4,
MeOH, 0 °C-r.t., 2 h; b) N-substituted carbamic acid phenyl esters,
TEA, THF, reflux, 2 h; c) NH2OH (50 wt. % in H2O), NaOH,
THF/MeOH, 0 °C, 15 min. d) phenyl isocyanate or 3-nitrophenyl iso-
cyanate, DCM, r.t., overnight; e) i-PrOCOCl, DIPEA, DCM, 0 °C-r.t.,
1 h; f) 1N NaOH, THF/MeOH, r.t., overnight; g) i. H2, 10% Pd/C,
MeOH, r.t., 1 h; ii. aq. CH2O, NaCNBH3, ZnCl2, MeOH, r.t., 3 h. h) i.
.
N2H4 H2O, 5% Pd/C, 0 °C, 1 h; ii. formic acid, EDCI, THF, 0 °C, 3 h;
i) H2, 10% Pd/C, MeOH, r.t., 1 h; j) i. Boc-glycine or Boc-L-alanine,
HATU, DIPEA, THF, r.t., 16 h; ii. TFA, DCM, r.t., 1 h; k) NH2OH (50
wt. % in H2O), EtOH, 80 °C, 3 h.
To explore alternative ZBGs, the non-hydroxamate analogs 8
and 14a-d were synthesized (Scheme 1). The carboxylic acid
analog 8 was directly obtained from the ester 6b by basic hy-
drolysis. To synthesize the retro-hydroxamate 14a, 4-nitroben-
zaldehyde (11) underwent reductive amination and then reac-
tion with phenyl isocyanate to produce the urea intermediate
13a. This compound was partially hydrogenated with hydrazine
To investigate the effect of the structure of the alkyl side
chain attached to the proximal urea nitrogen, analogs 7a-c bear-
ing isobutyl, 4-hydroxybutyl, and phenethyl substituents were
synthesized (Scheme 1). Methyl 4-formylbenzoate 1 underwent
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catalyzed by 5% Pd/C, and the resulting arylhydroxylamine was
Table 1. HDAC inhibitory activity of NexA analogs 4a-e, 7a-
e, 8, and 14a-d.a
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2
3
4
5
6
7
8
condensed with formic acid to afford the final product 14a. The
aniline intermediate 13b was obtained by standard catalytic hy-
drogenation from its nitro precursor 13a and was acylated with
Boc-glycine or Boc-L-alanine under HATU condition. Depro-
tection with TFA provided the analogs 14b-c. To synthesize
compound 14d, 4-formylbenzonitrile (15) was first converted
to the intermediate 13c via reductive amination and reaction
with phenyl isocyanate. Upon treatment with aqueous hydrox-
ylamine under reflux, compound 13c gave the final product
14d.
HDAC isoform inhibitory activ-
ity (IC50, nM)
Selectivity
Index
Compd.
HDAC1
HDAC6
HDAC1/6
NexAb
4a
4b
4c
4d
4e
7a
7b
7c
3,020 ± 740
14,400 ± 1,500
7,540 ± 145
2,740 ± 85
721 ± 1
5 ± 0.06
74 ± 4
43 ± 4
600
194
176
456
450
355
511
3350
107
>17
-
9
6 ± 1
1.6 ± 0.2
1.7 ± 0.2
12 ± 0.4
0.87 ± 0.66
53 ± 11
1,790 ± 60
N.A.d
10
11
12
13
14
15
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Inhibitory activity of new analogs was initially evaluated
against the HDAC1 and 6 isoforms (Table 1), and the four most
potent and selective compounds were further tested against
HDAC8 (Table S1). As is apparent from the data shown in Ta-
ble 1, replacement of the aryl cap with a non-aromatic hetero-
cycle as in compound 4a led to a significant decrease in potency
at both HDAC1 and HDAC6 (>14000 and 74 nM, respectively)
compared to NexA, suggesting that the interaction of the aryl
cap and the enzyme surface is necessary for potent binding. Ad-
ditionally, the increased flexibility and rotational freedom avail-
able to 4a would result in a greater loss of configurational en-
tropy upon protein binding compared with a more rigid scaf-
fold, and lead to a penalty in its potency. Heteroaromatic rings
or an amine-substituted phenyl group as cap groups (com-
pounds 4b-e) were beneficial for maintaining excellent potency
except in compound 4b, which displayed an ~8 fold decrease in
activity compared to NexA. We assume that the additional ni-
trogen groups present in the indazole ring of 4d or in the dime-
thylaniline moiety of 4e contribute to binding through engage-
ment in additional hydrogen bonds with amino acids on the rim
of the pocket.
604 ± 17
6,130 ± 240
2,913 ± 929
5,740 ± 530
>30,000
7d
8
N.D.c
>30,000
1,025 ± 253
>29
-
-
-
11
14a
14b
14c
14d
SAHA
N.D.c
N.A.d
N.D.c
N.D.c
31 ± 12
N.A.d
N.A.d
2.8 ± 2.34
aIC50 values displayed are the mean of two experiments ± standard de-
viation obtained from curve fitting of a 10-point enzyme assay starting
from a 30 µM concentration of each analog with 3-fold serial dilution.
Values are extracted from fitting dose−response curves to the data
points. bReference 14. cNot determined. dNo activity.
Although three-dimensional structures of many HDACs have
been reported, no crystal structures of HDAC6 catalytic do-
mains were available until last year. Compared to most HDAC
isoforms, HDAC6 is unique as it contains tandem deacetylase
catalytic domains (CD) designated CD1 and CD2. Two groups
respectively reported the crystal structures of both catalytic do-
mains of HDAC6 in complex with several substrates and inhib-
itors, which provided mechanistic insights into the catalytic
mechanism, substrate specificity, and inhibitor selectivity of
HDAC6.20,21 Early studies indicated that both domains are cat-
alytically active toward histone substrates, with only CD2 ex-
hibiting tubulin deacetylase activity,22,23 whereas subsequent
studies suggested that only CD2 is catalytically active.24 It was
demonstrated that different point mutations in the sequence en-
coding CD1 do not result in compromised deacetylation activity
on α-tubulin. CD2 active sites are highly conserved and feature
the typical narrow hydrophobic channel formed by residues
Pro464, Gly582, Phe583, Phe643, and Leu712. The Zn2+ ion is
coordinated by Asp612, His614, and Asp705 in CD2.20,21 Thus,
we mainly focused our attention on CD2 and carried out molec-
ular modeling studies using the available CD2-NexA complex
as template (PDB entry: 5G0I).
Our previous SAR study on NexA has demonstrated the im-
portance of a lipophilic alkyl chain on the proximal urea nitro-
gen for HDAC activity. The isobutyl analog 7a retained both
high potency at HDAC6 (IC50 = 12 nM) and excellent selectiv-
ity over HDAC1, suggesting a certain tolerance for bulky alkyl
chains in this position. Additionally, the introduction of a hy-
droxyl group at the end of the n-butyl chain (compound 7b) sig-
nificantly improved potency against HDAC6. This finding sug-
gests that an additional hydrogen bonding interaction may be
established, thereby enhancing the interaction with the rim of
the cavity. Deletion of one nitrogen and of the carbonyl group
of the urea moiety together with the attachment of an electron-
withdrawing group (an isopropoxycarbonyl group, compound
7c) as a side chain in lieu of butyl resulted in decreased HDAC6
activity (IC50 = 53 nM). Lastly, shortening the linker from ben-
zyl to phenyl (compound 10c) resulted in a ~300-fold decrease
in potency compared to 4e. We further tested the inhibitory ac-
tivity of 4c-e and 7b at HDAC8, and compounds 4d, 4e, and 7b
displayed more than 500 fold selectivity over this class I iso-
form (Table S1).
Docking simulations were performed for the selected com-
pounds 4d, 7b, and 14a. The best-scored poses of each com-
pound as well as the published conformation of NexA bound to
CD2 are presented in Figure 2. The docking poses of com-
pounds 4d and 7b revealed a binding mode quite consistent with
that of NexA in which only the hydroxamate C=O group en-
gages in coordination with Zn2+ and binding is further rein-
forced by hydrogen bonds between NH and Gly582 of the back-
bone; C=O and Tyr745; and OH and His573, respectively. For
compound 14a, both OH and formyl groups of the retro-hydrox-
amate engage in bi-chelation with Zn2+, and in additional hy-
drogen bonds with His573 and Tyr745, respectively. The resi-
dues Phe583 and Phe643 located in the hydrophobic channel
The carboxylic acid 8, in contrast to the related hydroxamate
7b which displayed subnanomolar potency at HDAC6, did not
show any activity at concentrations up to 30 μM. We further
evaluated compounds 14a-d bearing non-hydroxamate ZBGs,
which were chosen from a variety of such groups appearing in
recent publications.17–19 Only the retro-hydroxamate analog 14a
showed low-micromolar potency at HDAC6, which may pro-
vide an opportunity to further refine HDAC6is with this alter-
native ZBG.
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engage in a π-stacking interaction with the benzyl linker for all based HDAC specificity. As is apparent from Figure 3B, acet-
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3
4
5
6
7
8
three compounds, which is consistent with the π-stacking ar-
rangement observed for NexA. One of the nitrogen atoms in the
indazole ring of compound 4d and the oxygen atom in the hy-
droxybutyl chain of compound 7b engage in hydrogen bonding
interactions with the carbonyl groups of Ala641 and Leu712
backbones, respectively, which could be responsible for the im-
proved HDAC6 activity of these compounds. Additionally, an
overlay of the structures of NexA and its analogs indicates that
the proximal urea nitrogen plays a significant role in keeping
the structural features related to the tetrahedral steric configura-
tion of these compounds favoring the interaction (Figure S1).
ylated α-tubulin levels increased dramatically upon treatment
with NexA and 4c-e at each concentration compared to the non-
treatment conditions, although we didn’t observe obvious ef-
fects in the MTS cell line assays. Moreover, the acetylation sta-
tus of H3 remained unaltered or increased slightly in the pres-
ence of all HDAC6is at all tested concentrations.
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Figure 3. Western blot illustrating tubulin acetylation and histone acet-
ylation levels in WM795 melanoma cells following 24 h treatment with
increasing concentrations of NexA and analogs 4c-e. The selective
HDAC6 inhibitor tubastatin A and the pan-HDAC inhibitor LBH-589
were included on each blot as positive controls. Protein extracts were
prepared and subjected to SDS-PAGE and immunoblotting with α-
HDAC6, α-tubulin, α-acetylated tubulin, α-histone 3, and α-acetylated
H3 specific antibodies. This figure is representative of three independ-
ent experiments.
Melanomas are highly immunogenic and often heavily infil-
trated by various types of immune cells. In these cancers, the
immune system fails to eradicate the tumor cells which is usu-
ally related to negative regulation by tumor-generated immuno-
suppressive cytokines, in particular interleukin-10 (IL-10).26,27
IL-10 is produced by tumor-associated macrophages and tu-
mor-related lymphocytes associated with early stages of tumor
evolution, and its levels are elevated in serum obtained from
patients with later stages of melanoma.28 IL-10 is generally ac-
cepted as a major immunosuppressive cytokine, and its expres-
sion was reported for several types of cancers but not in adjacent
non-malignant tissue of the patients.29-31 We previously re-
ported that the treatment of antigen-presenting cells (APCs)
such as macrophages and dendritic cells with selective
HDAC6is in vitro and in vivo improved T-cell activation via
diminished production of a major immunosuppressive cytokine,
IL-10. This effect was not observed in experiments performed
using pan-HDACis.32,33 Thus, our next step was to determine the
effect of treatment with compound 4d on the production of IL-
10 in macrophages after stimulation with lipopolysaccharide
(LPS). Interestingly, the treatment of primary peritoneal elicited
macrophages (PEMs) isolated from C57BL/6 mice and murine
macrophage RAW264.7 cells with 3 µM of 4d down-regulated
the production of IL-10 after 24 h treatment (Figure 4A). Out-
come observed previously when using other HDAC6is.32 Addi-
tionally, an initial metabolism assessment showed that com-
pound 4d was metabolically stable both in human (t1/2 = 408
min) and mouse (t1/2 = 239 min) liver microsomes, which is ben-
eficial for animal studies in the next stage (Figure S3).
Figure 2. (A) Crystal structure of the complex of NexA (green) with
HDAC6 CD2 [PBD entry: 5G0I]. (B) Binding interaction of 4d (green)
with HDAC6 CD2 (∆Gexp = -10.3 kcal/mol). (C) Binding interaction of
7b (green) with HDAC6 CD2 (∆Gexp = -11.0 kcal/mol). (D) Binding
interaction of 14a (green) with HDAC6 CD2 (∆Gexp = -8.9 kcal/mol).
We previously reported that NexA exhibits micromolar anti-
proliferative activities against human melanoma cell lines bear-
ing different mutations, and found that HDACis possessed anti-
cancer effects both through direct cytotoxicity and improved
immune responses.7,14 To investigate the effect of our newly de-
veloped compounds in cells, MTS proliferation assays using the
selected analogs 4c-e were conducted in different types of mel-
anoma and lymphoma cell lines, including the murine SM1,
B16, and FcMCL cell lines which have been widely used in the
in vitro and in vivo screening of HDACis and other epigenetic
modifiers in syngeneic models aiming to study anti-tumor im-
mune responses (Figure S2).7,25 Notably, compounds 4c and 4d
displayed consistent modest anti-proliferative effects on these
cell lines at concentrations increasing from 1 µM to 15 µM,
while compound 4e displayed more obvious anti-proliferative
effects in murine FcMCL lymphoma cells and murine B16 mel-
anoma cells at lower concentrations.
To this end, C57BL/6 mice bearing B16-F10-luc melanoma
tumors were treated with 4d (20 mg/kg) for 22 days, resulting
in 100% survival rates and significant reduction of tumor vol-
umes compared to the vehicle group (40% survival) (Figure 4B
and 4C). Therefore, the experiment continued with the same
dosage until the last animal in the control group died (or the
tumor reached 2500 mm3). Moreover, an improved ability of 4d
to inhibit tumor growth was observed in comparison with an-
other selective HDAC6i, tubastatin A (Figure 4C). In contrast,
no significant effect on the growth of B16-F10 melanoma tu-
mors was observed in immunodeficient (SCID) mice after treat-
ment with compound 4d for 20 days (Figure 4D). This result
indicates that the mechanism for the compound’s action is the
regulation of inflammatory and immune responses. These in
Acetylated α-tubulin is an important physiological substrate
for HDAC6 and is not deacetylated by other zinc-containing
HDACs. In contrast, HDAC6 does not influence the acetylation
status of histone 3 (H3), which is mainly deacetylated by the
class I HDACs. The analysis of these two substrates indicates
whether HDAC6 is selectively inhibited in the concentration
range used in the anti-proliferation experiments. Thus, we fur-
ther measured the selective effects of these compounds on the
acetylation status of both H3 and α-tubulin in human WM795
melanoma cells with the same dose range to determine cell-
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vivo data have been partly disclosed in our previous publica-
tion.7
1
A.V.: e-mail: avillagra@email.gwu.edu. Phone: (+1) 202 994
9547;
A.P.K.: e-mail: AKozikowski@starwisetrx.com. Phone: (+1) 312
996 7577.
2
3
4
5
6
7
8
9
This work was supported by the NIH (NS079183, to A.P.K.;
CA184612 and MRF CDA grant award, to A.V.), and by the CAS
(RVO: 86652036), project BIOCEV (CZ.1.05/1.1.00/02.0109)
from the ERDF, and CSF to C.B. (15-19640S).
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The authors declare no competing financial interests.
The authors gratefully acknowledge Dr. Werner Tueckmantel for
reviewing the article and providing comments.
Figure 4. (A) Murine peritoneal elicited macrophages (PEM) (left) and
murine RAW264.7 macrophage cells (right) were treated with LPS (1
µg/mL) or LPS plus 3 µM of compound 4d for 24 hours. Supernatants
were then collected, and the production of IL-10 was determined by
ELISA. ***p<0.001 as compared to the untreated cells. (B-D) In vivo
tumor growth of C57BL/6 mice injected subcutaneously with B16-
F10-luc WT cells. Mice were treated by intraperitoneal injection daily
with the compound 4d (20 mg/kg). Survival (B) and tumor growth (C)
were monitored throughout the experiment. (D) In vivo tumor growth
of B16-F10 WT melanoma cells in immunodeficient SCID mice treated
with 4d compared with control vehicle treatment.
CTLA-4, cytotoxic T lymphocyte associated protein 4; PD-1, pro-
grammed cell death protein 1; HDAC, histone deacetylase;
HDACi, histone deacetylase inhibitor; SIRT, sirtuin; CD, catalytic
domain; HSP-90, heat shock protein 90; HSF-1, heat shock factor
1; NexA, Nexturastat A; NRAS, neuroblastoma RAS gene; BRAF,
B-Raf proto-oncogene; SAR, structure-activity relationship; ZBG,
zinc-binding group; DIPEA, N,N-diisopropylethylamine; HATU,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyri-
dinium 3-oxide hexafluorophosphate; TFA, trifluoroacetic acid;
EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; PDB,
Protein Data Bank; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; H3, his-
tone 3; APC, antigen-presenting cell; LPS, lipopolysaccharide;
PEM, peritoneal elicited macrophage; IL-10, interleukin 10;
ELISA, enzyme-linked immunosorbent assay; SCID, severe com-
bined immunodeficiency.
In conclusion, fourteen new NexA derivatives with different
caps, linkers, and ZBGs were designed, synthesized, and ini-
tially evaluated in class I and class IIb HDACs. Several hydrox-
amate-based analogs exhibited improved potency against
HDAC6 compared to NexA while maintaining excellent selec-
tivity over HDAC1 and 8. The selectivity of 4c, 4d, and 4e was
further verified in melanoma cells in terms of increasing levels
of acetylated tubulin rather than levels of acetylated histone.
Moreover, the analog 4d exhibited modest in vitro anti-prolif-
erative effects in different types of melanoma cells and human
lymphoma cells, but significantly down-regulated the produc-
tion of the immunosuppressive cytokine IL-10 in macrophages.
The in vivo efficacy study of the metabolically stable HDAC6
inhibitor 4d demonstrated improved capability to inhibit tumor
growth in melanoma models through the regulation of inflam-
matory and immune responses. While Ames activity does not
typically constitute a go/no go decision in advancing cancer
drugs (the hydroxamate-based HDAC inhibitors vorinostat and
panobinostat are Ames active), a lack of Ames activity would
be of benefit in terms of avoiding mutagenic events that may
lead to the generation of secondary tumors.34 As such, the lead
compound 4d will be further evaluated in mutagenicity assays,
as well as profiled in other standard ADMET assays. Moreover,
we plan follow-up mechanistic studies to investigate HDAC6i-
induced regulation of other immunosuppressive cytokines in
melanoma cancer models.
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Figure 1. Structures of nexturastat A, and hydroxamate based HDACis in clinical trials for melanoma.
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Scheme 1. Synthesis of analogs 4a-e, 7a-d, 8, and 14a-d.a
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Figure 2. (A) Crystal structure of the complex of NexA (green) with HDAC6 CD2 [PBD entry: 5G0I]. (B)
Binding interaction of 4d (green) with HDAC6 CD2 (∆Gexp = ꢀ10.3 kcal/mol). (C) Binding interacꢀtion of 7b
(green) with HDAC6 CD2 (∆Gexp = ꢀ11.0 kcal/mol). (D) Binding interaction of 14a (green) with HDAC6 CD2
(∆Gexp = ꢀ8.9 kcal/mol).
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Figure 3. Western blot illustrating tubulin acetylation and histone acetylation levels in WM795 melanoma
cells following 24 h treatment with increasing concentrations of NexA and analogs 4c-e. The selective
HDAC6 inhibitor tubastatin A and the panꢀHDAC inhibitor LBHꢀ589 were included on each blot as positive
controls. Protein extracts were prepared and subjected to SDSꢀPAGE and immunoblotting with αꢀHDAC6, αꢀ
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representative of three independent experiments.
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Figure 4. (A) Murine peritoneal elicited macrophages (PEM) (left) and murine RAW264.7 macrophage cells
(right) were treated with LPS (1 µg/mL) or LPS plus 3 µM of compound 4d for 24 hours. Supernatants were
then collected, and the production of ILꢀ10 was determined by ELISA. ***p<0.001 as compared to the
untreated cells. (BꢀD) In vivo tumor growth of C57BL/6 mice injected subcutaneously with B16ꢀF10ꢀluc WT
cells. Mice were treated by intraperitoneal injection daily with the compound 4d (20 mg/kg). Survival (B)
and tumor growth (C) were monitored throughout the experiment. (D) In vivo tumor growth of B16ꢀF10 WT
melanoma cells in immunodeficient SCID mice treated with 4d compared with control vehicle treatment.
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