Potent and orally efficacious bisthiazole-based histone deacetylase inhibitors

Article abstract of DOI:10.1021/ml400470s

Inspired by the thiazole-thiazoline cap group in natural product largazole, a series of structurally simplified bisthiazole-based histone deacetylase inhibitors were prepared and evaluated. Compound 8f was evaluated in vivo in an experimental autoimmune encephalomyelitis (EAE) model and found to be orally efficacious in ameliorating clinical symptoms of EAE mice.

Full text of DOI:10.1021/ml400470s

Letter
pubs.acs.org/acsmedchemlett
Potent and Orally Efficacious Bisthiazole-Based Histone Deacetylase
Inhibitors
Fei Chen,† Hui Chai,† Ming-Bo Su,† Yang-Ming Zhang, Jia Li,* Xin Xie,* and Fa-Jun Nan*
Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, 189
Guoshoujing Road, Shanghai, 201203, China
S
* Supporting Information
ABSTRACT: Inspired by the thiazole−thiazoline cap group
in natural product largazole, a series of structurally simplified
bisthiazole-based histone deacetylase inhibitors were prepared
and evaluated. Compound 8f was evaluated in vivo in an
experimental autoimmune encephalomyelitis (EAE) model
and found to be orally efficacious in ameliorating clinical
symptoms of EAE mice.
KEYWORDS: Histone deacetylase inhibitors, largazole, bisthiazole
recise control of gene expression is important for many
Pcellular processes including cell survival, apoptosis, and
proliferation. Histone deacetylases (HDACs) and histone
acetyltransferases (HATs) are two functionally opposing
enzymes that control gene transcription via regulation of
histone lysine acetylation. Histone acetylation neutralizes
positive charges on lysine residues and disrupts nucleosome
structure, allowing unfolding of associated DNA and binding of
transcription factors, resulting in changes of gene expression.1
HDACs have been validated as targets for cancer chemo-
therapy.2−5 Of the 18 known human HDAC isoforms, which
are divided into zinc-dependent classic HDACs (class I, II, and
IV enzymes) and NAD+-dependent class III enzymes
(sirtuins),6,7 evidence suggests that class I HDACs are most
relevant to cancer growth.8,9
HDAC inhibitors (HDACis) block the deacetylation action
of HDACs, resulting in hyperacetylation of histones and
thereby affecting gene expression.10 Currently reported small-
molecule HDACis can be categorized into short-chain fatty
acids, hydroxamic acids, cyclic tetrapeptides/depsipeptides,
electrophilic ketones, and benzamides.11−13 A typical HDACi
has a three-motif pharmacophoric model consisting of a
recognition cap group, a hydrophobic linker, and a zinc-binding
group (ZBG).14 To date, two HDACis, vorinostat (SAHA) and
romidepsin, have been approved by the FDA to treat cutaneous
T cell lymphoma.15,16 In addition, the pro-apoptotic activity of
HDACis holds potential for treatment of inflammatory and
autoimmune diseases.17
high demand. HDACis have been shown to inhibit
proinflammatory cytokine expression.21,22 HDACi trichostatin
(TSA) was reported to alleviate clinical symptoms of EAE, an
animal model for MS.23 In our previous work, HDACi valproic
acid (VPA) was demonstrated to induce apoptosis in
pathogenic T cells and ameliorate pathogenesis in EAE
mice.24 Accordingly, the idea of repurposing VPA for MS
therapy has elicited interest.25
Largazole (1a, Figure 1) is a depsipeptide natural product
isolated from the cyanobacterium Symploca sp. by Luesch and
co-workers in 2008.26 As a novel natural HDACi with potent
antiproliferative activity and selectivity for cancer cells, it has
attracted significant interest. To date more than ten groups
have reported its total synthesis and SAR studies.27−46
We previously reported that C7-demethyl bisthiazole
largazole analogue 2a (Figure 1) has significantly decreased
HDAC inhibitory activity and antiproliferative activity against
HCT-116 colon cancer cells compared to largazole.33 de Lera
et al. have since reported that C7-demethyl largazole (1c) is
equipotent to largazole,34 indicating that the C7-methyl is
unimportant for activity; they also reported the potent activity
of 2a against NB4 cells. Williams et al. have demonstrated that
related thiazole-thiazole analogue 2b displays intermediate
HDAC inhibitory activity compared to largazole thiol.31 These
results inspired us to design a series of simplified analogues of
2a that retains the bisthiazole cap group while cleaving across
the β-hydroxy and thiazole fragments. The synthesis of ring-
opened analogues 3a−3d and 4a−4d eventually culminated in
the discovery of structurally simple bisthiazole-based HDACis
8a−8t (Figure 1).
Multiple sclerosis (MS), one of the foremost causes of
nontraumatic neurological disability in young adults, is a
representative autoimmune disease, which induces inflamma-
tory demyelination of the central nervous system (CNS).18−20
Because of the limited understanding of the pathogenesis of
MS, there remain many difficulties in treating MS. The
development of novel therapeutic targets and agents is in
Received: November 17, 2013
Accepted: April 4, 2014
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
Figure 1. Structures of synthesized ring-opened analogues.
a
Scheme 1. Synthesis of Ring-Opened Analogues
a
Reagents and conditions: (a) Lawesson’s reagent, DME, room temperature, 12 h. (b) BrCH2COCOOEt, KHCO3, TFAA, 2,6-lutidine, DME, room
temperature, 10 h. (c) LiOH, MeOH/H2O, room temperature, 8 h. (d) EDCI, HOBt, i-Pr2NEt, DMF, room temperature, 12 h. (e) Grubbs’ second
generation catalyst, toluene, reflux, 8 h. (f) TFA, Et3SiH, CH2Cl2, room temperature, 2 h. (g) Et3N, TFAA, THF, room temperature, 2 h. (h) Et3N,
MeOH, 50 °C, 12 h. (i) NH2OH·HCl, KOH, MeOH, room temperature, 2 h. (j) Zn, dry THF, reflux, 2 h. (k) Pd(OAc)2, PPh3, toluene, reflux, 8 h.
(l) NaOH, MeOH/H2O, reflux, 1 h.
To access 3a−3d, the thiazole−thiazole moiety was
constructed by a modified Hantzsch procedure (route 1,
Scheme 1).47 The thiazoline−thiazole moiety in 4a−4d was
accessed via condensation of cyanothiazole with 15.48 Side
chain installation via olefin cross-metathesis then afforded ring-
opened analogues 3a−3d and 4a−4d.27
With ring-opened analogues 3a−3d and 4a−4d in hand, we
assessed their inhibitory activities against human HDAC1, 3,
B
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ACS Medicinal Chemistry Letters
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Table 1. Inhibitory Activity against Human HDACs
d
IC50 (μM)
compd
R1
R2
HDAC1
0.0137
HDAC3
HDAC6
11.50
a
1a
n-C7H15CO
0.2450
0.0034
16.8
b
1b
H
0.0012
0.049
14.7
>30
a
2a
n-C7H15CO
2.0
b
2b
H
0.077
0.085
c
3a
3b
3c
3d
4a
4b
4c
4d
H
n-C7H15CO
1.90 0.25
0.09 0.02
2.60 0.45
0.12 0.02
2.83 0.34
0.06 0.01
3.56 0.44
0.11 0.02
NA
H
H
3.71 0.27
NA
Me
Me
H
n-C7H15CO
H
4.32 0.33
c
c
c
n-C7H15CO
NA
NA
NA
c
c
H
H
16.82 1.0
NA
NA
c
c
c
Me
Me
n-C7H15CO
NA
NA
NA
c
c
H
13.86 2.2
NA
NA
a
b
c
d
Data reported in our previous work.33 Data reported by Williams et al.31 No inhibitory activity observed at 20 μg/mL. Values are expressed as
the mean SD, and the test was performed in triplicate.
and 6 (Table 1). Unexpectedly, the thiazoline−thiazole
analogues (4a−4d) displayed significantly decreased inhibitory
activity, while the thiazole−thiazole analogues (3a−3d)
retained moderate activity. Compound 3b in particular was
nearly equipotent to the thiol form of C7-demethyl bisthiazole
largazole 2b, indicating that the thiazole−thiazole moiety might
be a suitable cap group for a HDAC inhibitor.
A series of HDACis was then synthesized containing the
thiazole−thiazole cap group and classic hydroxamic acid ZBG,
as found in SAHA (5a−5f, Figure 1). To assess the potential
impact of side chain length on activity,30,49 amines 19 with
variant linker length were condensed with acids prepared from
esters 18a−18b to afford respective amides 20a−20f. Upon
treatment with hydroxylamine, compounds 5a−5f were rapidly
accessed (route 2, Scheme 1). Disappointingly, most of these
hydroxamic acid analogues showed significantly reduced
inhibitory activity compared to compound 3b (Table 2).
hydoxamic acids 6a,6b were afforded (route 3, Scheme 1).
Negishi coupling of methyl 2-bromothiazole-4-carboxylates 24
with 2-thiazole-zinc bromide, prepared by treating 2-bromo-
thiazole 23 with activated zinc dust, afforded 2,2′-bisthiazole
25,50 which was hydrolyzed to the corresponding acid (route 4,
Scheme 1). Condensation of amine 21 with this acid and
subsequent treatment with hydroxylamine afforded compound
7 directly. Analogues 6a,6b and 7 displayed improved
inhibitory activity compared with 5a−5f (Table 2). Interest-
ingly, compound 7, which contains a 2,2′-bisthiazole moiety
instead of a 2,4′-bisthiazole moiety, showed a 2-fold increase in
activity compared to 6a.
Encouraged by the comparable inhibitory activity of
compound 7 and its facile synthetic accessibility, a series of
2,2′-bisthiazole-based hydroxamic acids 8a−8n were synthe-
sized (route 4, Scheme 1). The preparation of compounds 8o−
8t is additionally described in the Supporting Information. This
approach allowed for rapid diversification of the substituent on
the left thiazole ring (alkyl, aryl, and alkenyl, Table 3) and
efficient synthesis of a small focused 2,2′-bisthiazole-based
library.
Table 2. Inhibitory Activity against Human HDACs
b
IC50 (μM)
compd
R1
HDAC1
HDAC3
HDAC6
Pleasingly, most of the R1 substituents were well tolerated
(Table 3). Among compounds 8a−8t, 8s was found to be the
most potent HDAC1 inhibitor with IC50 as low as 0.01 μM.
The isopropyl group in 8s might contribute to key hydrophobic
interactions with the protein surface. In contrast, compound 8p,
which lacks a hydrophobic substitute on the left thiazole,
showed much reduced activity. Moreover, compound 8f not
only displayed potent activity against nine HDACs except
HDAC7 and HDAC9 (Table S1, Supporting Information) but
also showed excellent pharmacokinetic profiles characterized by
high Cmax (7.04 μg/mL) and AUC0‑t (12.07 μg·h/mL) and high
absolute bioavailability (F% = 118.7%) in mice (20 mg/kg,
P.O., Table S2, Supporting Information). We thus went on to
evaluate its efficacy in vivo.
Compound 8f induced hyperdeacetylation of histone H3 and
H4 and apoptosis of T cells in vitro. The cellular histone
acetylation levels after treatment with 8f were determined using
mouse embryonic fibroblast (MEF) cells. The cells were
cultured with 8f (0.3, 1, or 3 μM) for 6 h, and cell lysates were
subjected to Western blot analysis. As shown in Figure 2a,b,
resting levels of acetylated histone H3 and H4 were low in
MEF cells. When treated with 8f, acetylation levels of histone
H3 and H4 increased significantly in a dose-dependent manner.
a
a
a
5a
5b
5c
5d
5e
5f
H
NA
NA
NA
a
a
a
a
a
H
9.69 2.5
3.17 0.45
1.64 0.13
NA
NA
NA
NA
NA
H
2.59 0.49
a
a
Me
Me
Me
H
NA
NA
2.66 0.71
2.45 0.31
0.45 0.11
0.24 0.03
0.21 0.03
2.22 0.41
1.58 0.30
0.27 0.06
0.15 0.04
6a
6b
7
2.24 0.32
1.19 0.25
1.10 0.06
Me
H
0.11 0.02
a
b
No inhibitory activity observed at 20 μg/mL. Values are expressed
as the mean SD, and the test was performed in triplicate.
While compounds 5c, 5e, and 5f showed moderate inhibition
to HDAC1 and 3, compounds 5a and 5d displayed no
inhibitory activity. These results were consistent with previous
reports regarding the importance of the trans double bond
linker in largazole analogues.30
Further structural simplification, featuring replacement of the
chiral L-valine moiety with a chained carbon linker, was
achieved by condensation of amine 21 with 2,4′-bisthiazole-4-
carboxylic acids obtained by hydrolysis of 10a,10b to give
intermediates 22a,22b. Upon treatment with hydroxylamine,
C
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markers revealed that 8f enhanced apoptosis in both CD4+ and
CD8+ T cells after activation in vitro (Figure 2d).
Table 3. Inhibitory Activity against Human HDACs
Compound 8f Ameliorates Clinical Symptoms of EAE
Mice. HDACis have been shown to ameliorate inflammation in
several autoimmune animal models,54,55 including EAE.23 To
examine the in vivo effect of 8f, EAE was induced in C57BL/6
mice by immunization with MOG35−55 peptide. Compound 8f
(10 mg/kg) was given b.i.d. via oral administration from day 3
or day 15 following immunization until the end of the
experiment. PBS was used as a vehicle control. Compound 8f
treatment led to decreased incidence, reduced peak severity,
and decreased cumulative clinical score of EAE when given
from day 3 (Figure 3a and Table S2, Supporting Information).
a
Values are expressed as the mean SD, and the test was performed
in triplicate.
Figure 3. Compound 8f alleviates clinical symptoms of EAE. (a,b)
Clinical score of EAE mice treated with 8f (n = 10) or PBS (n = 10)
b.i.d. by oral administration on the day indicated by the arrow. Data
are mean
SEM, ***p < 0.001 (two-way ANOVA test). (c,d)
Representative H&E or Luxol fast blue stained spinal cord sections
isolated from naive, 8f, or vehicle-treated EAE mice on day 22
postimmunization. Boxed areas in left columns are presented enlarged
on the right. (e,f) Quantitative analysis of the number of spinal cord
infiltrates in the sections presented in panel c and the amount of
demyelination presented in panel d. Data are mean SEM; ***p <
0.001 versus naive group; ###p < 0.001 versus vehicle control
(Student’s t-test).
Figure 2. Compound 8f induces histone acetylation in MEF cells and
apoptosis in T cells. (a,b) MEF cells were treated with 8f or VPA for 6
h and then lysed for Western blot analysis to detect the acetylation
level of histone H3 and H4. Quantification of histone acetylation was
analyzed with Quantity One software (Bio-Rad). (c,d) Mouse
lymphocytes were stimulated in vitro and treated with 8f for 36 h.
Apoptosis was detected by Annexin V and PI staining, and the
percentages of apoptotic cells (Annexin V single positive) were
analyzed by flow cytometry.
More interestingly, when given after the onset of the disease
(day 15), 8f (10 mg/kg) was still able to reduce the severity of
EAE (Figure 3b and Table S2, Supporting Information),
indicating both a therapeutic benefit and preventative effect of
this drug.
Compound 8f Ameliorates CNS Pathology and
Leukocytes Infiltration in EAE Mice. Histological examina-
tion of spinal cords was performed at day 22 postimmunization.
A dramatic decrease in leukocyte infiltration in spinal cord was
observed with 8f treatment (Figures 3c,e). In vehicle-treated
EAE mice, Luxol fast blue staining showed multiple widespread
areas of myelin damage in white matter regions; demyelination
was greatly ameliorated in 8f-treated EAE mice (Figures 3d,f).
Leukocyte infiltration was further assessed by immunofluor-
escent staining of the spinal cord sections with anti-CD45
antibody; 8f treatment significantly reduced the number of
CD45+ cells in the spinal cords (Figure S1a, Supporting
Information).
Notably, at a concentration of 3 μM, 8f displayed better
inhibitory effects than VPA at 4 mM.
We next examined the effect of 8f on apoptosis induction in
lymphocytes; HDAC enzymes play important roles in cell
survival and proliferation, and HDACis have been shown to
induce apoptosis in several cell types.51−53 Naive lymphocytes
were isolated from mouse spleen and activated with anti-CD3
antibody or LPS, respectively, and cultured with various
concentrations of 8f for 36 h. Activated splenocytes displayed
a relatively high apoptosis level (∼40% for T cells) when
cultured in vitro. Compound 8f treatment further enhanced
apoptosis in a dose-dependent way in T cells (∼75%), but not
in B cells (Figure 2c). Subpopulation analysis with cell surface
Leukocyte infiltration into the CNS was isolated and
quantified by flow cytometry at day 18 postimmunization.
Results confirmed that the total CNS infiltrates (Figure S1b,
D
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Supporting Information) and CD4+ T cells accumulated in
CNS (Figure S1c, Supporting Information) both decreased
with 8f treatment. IL-17-producing Th17 and IFN-γ-producing
Th1 are the main pathogenic CD4+ T effector cells in EAE.
With surface staining of CD4 and intracellular staining of IL-17
and IFN-γ, the number of both Th17 and Th1 cells was
observed to decrease significantly in the CNS of 8f-treated EAE
mice (Figures S1c,d, Supporting Information).
In conclusion, a series of bisthiazole-based hydroxamic acids
evolved from natural product largazole were synthesized as
potent HDAC inhibitors. Several compounds exhibited low
micromolar to midnanomolar IC50 values in in vitro HDAC
inhibition assay. Compound 8f was evaluated in vivo in an EAE
model and showed efficacy in ameliorating clinical symptoms of
EAE mice when administered orally. These bisthiazole-based
HDACis are under further investigation for treatment of
autoimmune diseases and cancer, and the results will be
reported in due course.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic details and characterization data for all compounds
reported in this letter. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*(F.-J.N.) E-mail: fjnan@mail.shcnc.ac.cn.
*(X.X.) E-mail: xxie@mail.shcnc.ac.cn.
*(J.L.) E-mail: jli@mail.shcnc.ac.cn.
Author Contributions
†F.C., H.C. and M.-B.S. contributed equally to this work.
Funding
This work was supported by grants from The National Natural
Science Foundation of China (30725049, 81021062, and
81072652), Chinese Academy of Sciences (XDA01040301),
Ministry of Science and Technology of China
(2009CB940900), and Shanghai Science and Technology
Committee (No. 11431921102).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
DME, ethylene glycol dimethyl ether; DMF, N, N-dimethyl-
formamide; EAE, experimental autoimmune encephalomyelitis;
EDCI, 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hy-
drochloride; IC50, half maximal inhibitory concentration;
TFA, trifluoroacetic acid; TFAA, trifluoroacetic anhydride;
HOBt, 1-hydroxybenztriazole; THF, tetrahydrofuran; Trt,
triphenylmethyl
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K.; Gray, S. G.; Imitola, J.; Duran, G.; Assaf, B.; Langley, B.; Khoury, S.
J.; Stephanopoulos, G.; De Girolami, U.; Ratan, R. R.; Ferrante, R. J.;
Dangond, F. Transcriptional therapy with the histone deacetylase
inhibitor trichostatin A ameliorates experimental autoimmune
encephalomyelitis. J. Neuroimmunol. 2005, 164 (1−2), 10−21.
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antiepileptic drug valproic acid restores T cell homeostasis and
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