Synthesis and Antiproliferative Activity of Nitric Oxide-Donor Largazole Prodrugs

Article abstract of DOI:10.1021/acsmedchemlett.9b00643

The marine natural product Largazole is the most potent Class I HDAC inhibitor identified to date. Since its discovery, many research groups have been attracted by the structural complexity and the peculiar anticancer activity, due to its capability to di

Full text of DOI:10.1021/acsmedchemlett.9b00643

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Letter
Synthesis and Antiproliferative Activity
of Nitric Oxide-Donor Largazole Prodrugs
Matteo Borgini, Claudio Zamperini, Federica Poggialini, Luca
Ferrante, Vincenzo Summa, Maurizio Botta, and Romano Di Fabio
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00643 • Publication Date (Web): 07 Feb 2020
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Synthesis and Antiproliferative Activity of Nitric Oxide-Donor
Largazole Prodrugs
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Matteo Borgini,1 Claudio Zamperini,1,2 Federica Poggialini,1 Luca Ferrante,4 Vincenzo Summa,5,^
Maurizio Botta* 1,2,3, # and Romano Di Fabio* 4,5
1. Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy; 2. Lead
Discovery Siena S.r.l., Castelnuovo Berardenga, 53019 Siena, Italy; 3. Biotechnology College of Science and Technology,
Temple University, BioLife Science Building, Suite 333, 1900 North 12th Street, Philadelphia, Pennsylvania 19122, United
States; 4. Promidis, Via Olgettina 60, 20132 Milano, Italy; 5. IRBM Science Park, Via Pontina Km 30.600, 00070 Pomezia,
Italy. *These two authors share senior authorship.
KEYWORDS: Largazole, HDAC, nitric oxide, cancer, prodrug
ABSTRACT: The marine natural product Largazole is the most potent Class I HDAC inhibitor identified to date. Since its discovery,
many research groups have been attracted by the structural complexity
and the peculiar anticancer activity, due to its capability to
discriminate between tumor cells and normal cells. Herein, we discuss
the synthesis and the in vitro biological profile of hybrid analogues of
Largazole, as dual HDAC inhibitor and nitric oxide (NO) donors,
potentially useful as anti-cancer agents. In particular, the metabolic
stability of the modified thioester moiety of Largazole, bearing the
NO-donor function/s, the in vitro release of NO and the
antiproliferative activity in tumor cell lines are presented.
Largazole is a potent and selective Class-I deacetylase
(HDAC) inhibitor, isolated in 2008 from marine cyanobacteria
Symploca sp.,1 that showed a broad-spectrum growth-inhibitory
activity against epithelial and fibroblastic tumor cell lines and a
remarkable differential cytotoxicity profile over non-
transformed cells.1,2,3 The structure of Largazole is
characterized by the presence of a structurally intriguing planar
16-membered depsipeptide core bearing a metabolically labile
thioester side-chain, which, upon hydrolytic cleavage, liberates
Largazole-thiol, the bioactive HDAC inhibitor species (Figure
1). 4
Largazole was specifically due to the inhibition of HDAC
enzymes targeting Ac-H3 (Lys 9/14).1 More recently, Largazole
was hypothesized to play a relevant role also in the control of
osteogenesis12 and in liver fibrosis.13
O
O
S
S
N
N
NH
O
NH
O
Hydrolysis
HS
N
S
N
S
O
O
O
O
O
C6H13
S
N
N
H
H
Largazole-thiol
O
Largazole
HDACs are a family of epigenetic enzymes that catalyze the
deacetylation of ε-N-Acetyl lysine in H3 and H4 histone tails,
HN
NH
S
S
O
H
O
O
resulting in
a tighter chromatin structure that inhibits
N
NHOH
O
NH
O
transcription. Eighteen different HDAC isoforms have been
identified to date, subdivided into 4 classes (I to IV): class I
(HDAC-1, HDAC-2, HDAC-3, HDAC-8), class IIa (HDAC-4,
HDAC-5, HDAC-7, HDAC-9) class IIb (HDAC-6, HDAC-10),
class III (Sirtuin-1, Sirtuin-2, Sirtuin-3, Sirtuin-5, Sirtuin-6,
Sirtuin-7) and Class IV (HDAC-11). With the only exception of
Sirtuins (class III), the deacetylation of histone proteins is
typically mediated by a Zn2+-dependent mechanism.5,6,7,8,9
The inhibition of HDAC was found to induce cancer cell cycle
arrest and cell death, reduce angiogenesis and modulate
immune response. In particular, the pan-HDAC inhibitor of
class I, II and IV SAHA (suberoylanilide hydroxamic acid,
Vorinostat, Zolinza®),10 depicted in Figure 1, was the first
FDA-approved HDAC inhibitor for the treatment of refractory
primary cutaneous T-cell lymphoma (CTCL).11 Luesch and co-
workers demonstrated that the antiproliferative activity of
HN
O
Vorinostat (SAHA)
O
Romidepsin (FK288)
Figure 1. Chemical structures of Largazole, Largazole-SH and
Vorinostat (SAHA)
In term of structure-activity relationship (SAR) and mode of
action, the X-ray analysis of the co-crystal structure of the
complex HDAC8-Largazole, demonstrated that the 16-
membered depsipeptide core interacted as capping moiety with
the surface rim of the enzyme, while the terminal thiol group,
present in the pendant “warhead” of the macrocycle core,
chelated the catalytic Zn2+-containing catalytic domain of the
enzyme with an ideal coordination geometry.14 In fact, any
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attempt to modify the thiol moiety resulted in the significant
loss of activity.15,16 Largazole shares some structure similarities
with FK288 (Romidepsin, Istodax®, Figure 1),17,18 a Class I
HDAC inhibitor approved in 2009 by FDA for the of treatment
of CTCL. This naturally occurring depsipeptide, upon
metabolic reduction of the disulphide bond, releases the
pharmacologically active species bearing the Zn2+-binding thiol
group.
Over the last decades, many research groups investigated the
anticancer property of nitric oxide (NO) and its capability to
overcome tumor cell resistance to conventional treatments.19
NO is an endogenous and chemically reactive free radical gas,
known as the smallest signaling molecule in living organisms.
It is produced in mammals from amino acid L-arginine, oxygen
and the cofactor tetrahydrobiopterin, by three distinct NO
synthase isozymes, namely: endothelial NOS (eNOS), neuronal
NOS (nNOS) and inducible NOS (iNOS).20 NO is involved in
the regulation of a plethora of physiological and pathological
biochemical pathways in organs and tissues.20,21,22 In particular,
NO plays a key role as neurotransmitter at the synapsis level23
acting as a key a mediator of learning, sleep, and feeding in the
central nervous system. In addition, among other functions, it
controls the vascular tone, regulates gene transcription and
thioester chain, produced one or more equivalents of NO
(Figure 2).
To this aim, compounds 1 and 2 were synthesized and then
sequentially evaluated in terms of capability to release NO in vitro,
metabolic stability and antiproliferative activity in tumor cell lines
with respect to Largazole.
O
S
N
NH
O
Hydrolysis
Largazole-thiol + NO
9
N
S
10
11
12
13
14
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O
O
O
O
S
N
H
NO2
1
O
S
N
NH
Hydrolysis
Largazole-thiol + 2NO
S
N
O
O
O
O
O2N
O
S
N
H
O
O2N
2
Figure 2. Chemical structures of Largazole analogues 1 and 2.
The retrosynthetic analysis of compounds 1 and 2, shown in
Figure 3, suggested the disconnection of the olefin moiety, that
could have then been obtained by cross-metathesis reaction
between the known synthetic Largazole intermediate 3 and the
terminal olefin derivative bearing suitable nitrate group/s.
Compound 3, was prepared by condensation of fragments A and
B, as depicted in Figure 3, following the same synthetic sequence
reported in literature.36 However, to obtain intermediate 3 in
multigram scale, we specifically focused our attention on the
optimization of the synthesis of fragment A (Scheme 1), which was
efficiently prepared in seven synthetic steps and 63 % total yield
from commercially available N-Boc glycine 4. In particular,
intermediate 6, prepared in two steps and 82% yield from 4, was
transformed into the corresponding thiazole derivative with 3-
bromopyruvic acid in THF at 50 °C, to get the corresponding
free amino derivative, which was re-protected as N-Boc,
affording 7 in 95% yield over two steps.
mRNA
translation
and
produces
post-translational
modifications of proteins. Of note, NO shows dichotomous
differential cellular response by distinct concentrations, either
facilitating cancer events, including angiogenesis, apoptosis
and metastasis,23 or, at high concentrations (>200 nM), acting
as a potent an anti-cancer agent.19,23,24,25,26
In the light of these evidence dual NO donors/HDAC
inhibitors emerged over the last years as novel anti-cancer
chemical entities, potentially more efficacious than selective
HDAC inhibitors, owing to the capability of NO to specifically
modulate function of some HDAC isoforms.27 In particular,
class I HDAC 2 has been found to be structurally modified by
direct reaction with NO, either S-nitrosylation or Tyr-nitration
reaction, or shuttled into the cell nucleus through the activation
of protein phosphatase 2A.28,29
The first mixed NO-donor/HDAC inhibitor was reported in
2013, as a compound potentially useful for the treatment of
cardiac hypertrophy and wound healing.30 This novel hybrid
molecule, obtained from the known HDAC inhibitor Entinostat
by the introduction of a constitutive NO-donor furoxan moiety,
showed a similar HDAC inhibitory profile as Entinostat, but an
additive myogenic differentiation activity. Since then, different
hybrid NO-donor/HDAC inhibitor derivatives, potentially
useful as anti-cancer agents, have been reported in literature.31
In particular, hybrids Doxorubicin-NO donors were found to be
active against Doxorubicin-resistant human colon cancer cells
(HT29-dx).32 In addition, interesting results were obtained
joining NO-donor moieties with anticancer platinum
derivatives33 and PepT1 inhibitors.34 Finally, some hybrid
HDAC inhibitor-NO donors exhibited an enhanced cytotoxic
activity compared to HDAC inhibitors.35
Owing to this evidence, our aim was to obtain novel
Largazole derivatives bearing one or more NO-donor functions
at the metabolically labile thioester chain. These novel type of
HDAC inhibitors, exploiting the prodrug character of the
thioester moiety of Largazole, upon enzymatic hydrolysis
would have efficiently released Largazole-thiol, the
pharmacologically active species as HDAC inhibitor, and
depending on the specific functionalization of the aliphatic
O
O
S
S
N
N
NH
O
NH
O
O
R
N
S
N
S
S
O
O
O
O
O
R
N
S
N
H
H
3
S
HO
NH2
O
N
O
N
S
O
COO-t-C4H9
BocHN
B
A
Figure 3. Retrosynthetic approach for the synthesis of NO-donor
Largazole derivatives 1 and 2.
Then, its carboxylic function was smoothly transformed into
the corresponding nitrile group to get compound 8 which was
condensed with -methyl cysteine 10, prepared from cysteine
as reported in literature, affording target fragment A in 94%
yield. The NO-donor thioester olefin derivative 14 was
synthesized as shown in Scheme 2.
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Scheme 1. Optimized synthesis of fragment A
S
O
OH
S
O
a, 91%
H
H
H
b, 90%
c,d, 96%
H
N
N
N
N
Boc
NH2
Boc
NH2
N
Boc
Boc
OH
O
6
7
5
4
6
a, 95%
7
8
9
HS
S
N
NH2
S
S
S
H
N
H
OH
H
f, 94%
CN
e, 93%
N
H2N
+
OH
N
N
N
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N
Boc
Boc
O
Boc
O
O
A
10
8
9
Reagents and conditions: (a) isobutyl chloroformate, N-methyl morpholine, NH4OH, THF, -20 °C, 1 h; (b) Lawesson’s reagent, CH2Cl2,
r.t.,15 h; (c) 3-bromopyruvic acid, THF, 50 °C, 1 h; (d) Boc2O, 1N NaOH, dioxane, r.t., 40 min; (e) TFAA, diisopropylethyl amine, CH2Cl2,
0 °C, 1 h; (f) TEA, MeOH, reflux, 7 h.
In particular, 5-bromovaleric acid 11 was reacted with
AgNO3 in dry CH3CN at 70 °C for 2 h to obtain intermediate
12, which was then transformed into the corresponding
thiocarboxylic acid derivative 13 by Lawesson’s reagent in a
microwave reactor at 60 °C. The final alkylation reaction with
allyl bromide afforded the target olefin derivative 14 in 85%
yield.
Pentenoic acid 15 was protected as p-nitrophenol (PNP) ester.
The following bromination reaction of intermediate 16 afforded
compound 17 in 81% yield, which was easily converted into the
corresponding bis-nitrate compound 18 in the presence of
AgNO3 in CH3CN at 70 °C for 24 h. After the basic hydrolysis
of the ester group, the resulting carboxylic acid 19 was
transformed into the chemically labile thiocarboxylic acid
derivative 20, which was rapidly alkylated with 4-bromo-1-
butene, to obtain compound 21 in 23% yield over two steps
(Scheme 4). The following intermolecular cross-metathesis
reaction with Largazole intermediate 3, performed using the
same reaction conditions set up for the synthesis of compound
1, afforded this time only the side product derived from
homodimeric coupling reaction of the olefin derivative 21.
Due to this unexpected result, to obtain compound 2, the
synthetic approach was significantly modified (Scheme 5).
Scheme 2. Synthesis of the mono-nitrate olefin derivative 14.
O
O
O
a
b
O
OH
O
SH
Br
OH
NO2
NO2
13
12
11
c
O
O
S
NO2
Scheme 4. Synthesis of bis-nitrate olefin 21.
14
O
O
O
Reagents and conditions: (a) AgNO3, dry CH3CN, 70 °C, 1 h. (b)
Lawesson’s reagent, CH2Cl2, 60°C, 10 min, MW. (c) 4-bromo-1-
butene, K2CO3, Acetone, 0 °C to 20°C, 30 min.
a
b
OPNP
Br
OPNP
OH
Br
16
17
15
c
The following key intermolecular cross-metathesis reaction
between intermediate 3 and compound 14, performed with
Grubbs 2nd generation ruthenium-based catalyst (Scheme 3),
gave the target Largazole thioester analogue 1, although only in
26% yield, due to the competitive homodimeric coupling
reaction of the olefin derivative 14, as confirmed by the
isolation of large amount of unreacted olefin derivative 3, after
purification by flash chromatography.
O
O
d
O2N
O2N
O
OPNP
O
OH
O
O
O2N
O2N
18
19
e
O
O
f
O2N
O2N
O
SH
O
S
Scheme 3. Cross-metathesis reaction.
O
O
O2N
O2N
20
21
O
O
S
S
N
N
NH
O
NH
O
a
Reagents and conditions: (a) DIC, DMAP, CH2Cl2, 0 °C to r.t., 1h;
(b) Br2, CCl4, r.t., 30 min; (c) AgNO3, dry CH3CN, 70 °C; 24 h. (d)
NaOH 2N, THF/EtOH, 0 °C, 30 min; (e) Lawesson’s reagent,
CH2Cl2, 60 °C, 15 min, MW; (f) 4-bromo-1-butene, K2CO3,
acetone, 0 °C to r.t., 30 min.
N
S
N
S
14
O
O
O
O
O
N
O
S
N
H
H
NO2
3
1
Reagent and conditions: Grubbs catalyst 2nd generation, dry
CH2Cl2, 90 °C, 16 h.
To this aim, the bis-nitrate carboxylic acid derivative 19 was
transformed into the corresponding S-trityl thiocarboxylic ester
derivative 22.The following de-blocking reaction of the S-trityl
protecting group, with TFA and triethylsilane in CH2Cl2 at room
temperature for 1 h, afforded the corresponding chemically
As far as the synthesis of the corresponding bis-nitrate
derivative 2 is concerned, the same synthetic sequence used
from the preparation of 14 was attempted (Scheme 4). 4-
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labile thiocarboxylic acid 23, which was then rapidly reacted
with the bromo derivative 24, smoothly synthesized from
intermediate 3 by metathesis reaction in 42% yield, to get title
compound 2.
As expected, the NO production was amplified in presence of
increasing concentration of L-cysteine. The NO release was
obviously more abundant for compound 2 than compound 1,
due to the presence of two NO-donor groups.
Once ascertained the capability of both compounds to
efficiently release NO, their antiproliferative activity was
evaluated against U-2OS (human osteosarcoma cell), Caco-2
(human colorectal adenocarcinoma cell) and IMR-32 (human
neuroblastoma cell), using the parent compound Largazole as
internal control. As shown in Table 1, compounds 1 and 2
showed a concentration and time-dependent inhibitory activity
against tumor cell growth. In particular, as per the
antiproliferative activity in the U-2OS cell lines, both
compounds showed a relevant additive effect at 24 h with
respect to the antiproliferative activity of Largazole, whereas,
at 48 h and 72 h, the antiproliferative effect was similar to
Largazole. The greater antiproliferative activity of compound 2
vs compound 1 at 24 h (pEC50 = 6.33 vs 5.71, respectively) is
most likely due to the greater production of NO by compound
2 with respect to compound 1.
5
6
7
8
Scheme 5. Synthesis of compound 2.
O
O2N
O
OH
O
O2N
9
19
10
11
12
13
14
15
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17
18
19
20
21
22
23
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a
O
O
b
O2N
Trt
O2N
O
S
O
SH
O
N
O
O2N
O2N
22
23
O
O
S
S
N
NH
O
NH
O
d
c
2
N
S
N
S
23
O
O
O
O
Br
N
N
Table 1. Cytotoxicity of Largazole, 1 and 2 against U-2OS,
Caco-2, IMR-32 cell line.
H
H
24
3
Reagents and conditions: (a) triphenylmethanethiol, DIC, DMAP,
dry CH2Cl2, 0 °C to r.t., 4 h; (b) TFA, triethylsilane, dry CH2Cl2, 0
°C to r.t., 1 h; (c) 4-bromo-1-butene, Grubbs II, dry CH2Cl2, 90 °C,
16 h; (d) K2CO3, acetone, 0 °C to r.t., 4 h.
Compound#
Cell line Time
24 h
Largazole
4.69±0.12
6.43±0.15
6.48±0.10
>2.0
6.12±0.21
7.84±0.09
7.46±0.16
7.52±0.14
7.91±0.08
1
2
5.71±0.15 6.33±0.10
6.51±0.15 6.13±0.20
6.77±0.09 6.00±0.70
4.95±0.19 5.04±0.24
7.42±0.14 8.25±0.22
8.09±0.16 8.35±0.16
7.82±0.17 7.53±0.12
7.71±0,17 7.21±0,18
7.92±0.06 7.30±0.14
U-2OS
CaCo-2
IMR-32
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
Largazole derivatives 1 and 2 were initially characterized in
terms of capability to release NO, using the Griess method.37 As
shown in Figure 4, in the assay conditions, both compounds
spontaneously
produced
NO.
#
EC50 were determined as described in the Supplementary
Information; they are the average value of n=3 independent
experiments ± SEM.
This general trend is even more evident in the Caco-2 cell line,
in which the improved antiproliferative activity of both
compounds was more pronounced than that shown by
Largazole at 24 h and 48 h and, in part, at 72 h. As anticipated,
compound 2 was more potent than compound 1 at all
timepoints. Conversely, in the IMR-32 cell line the
antiproliferative activity was already evident at 24 h, whereas
the additive effect of compound 1 and 2 with respect to the
parent compound Largazole, was minimal or even absent.
To further explain the additive antiproliferative effect of
compound 1 and 2 vs Largazole, their metabolic stability was
assessed with respect to Largazole in the assay cell medium. As
expected, a rapid hydrolysis of the thioester moiety was
observed (Table 2, Supplementary Information). The chemical
stability of these compounds was evaluated also in HEPES and
DMSO, without observing chemical degradation, hence
confirming the potential use of these compounds as prodrugs.
Finally, the stability in human plasma profile was evaluated
(Table 2, Supplementary Information), detecting the same rapid
hydrolysis of the metabolically labile thioester side chain
reported in literature for Largazole.38,39
Figure 4. NO release assay. Compounds 1 (A) and 2 (B) were
incubated at 0.1 mM concentration in 50 mM phosphate buffer
(pH=7.4) at 37°C, both in the absence and in presence of L-cysteine
(0.5 mM and 5 mM). The yield of NO2 is expressed as % with
respect to the initial concentration of compound at 1 h, 5 h and 24
h, respectively. The reported values are the average of three
-
independent
experiments.
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HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor;
MW, microwave reactor; NO, nitric oxide; cGMP, cyclic
CONCLUSIONS
guanosine
monophosphate;
Trt,
Trityl;
DIC,
N,N′-
diisopropylcarbodiimide; DCM, dichloromethane; DMAP, 4-
Novel Largazole derivatives bearing one and two nitrate
groups at the metabolically labile thioester side chain were
efficiently synthesized. These compounds were endowed with
dual activity profile, as consequence of the rapid liberation in
cell medium of the HDAC inhibitor Largazole-thiol and the
efficient production of NO. When characterized in terms of
cytotoxicity in three different types of tumor cell lines, namely:
U-2OS, Caco-2 and IRM-32, compounds 1 and 2 showed an
additive antiproliferative activity compared to the parent
compound Largazole, effect which was more pronounced in the
U-2OS and Caco-2 cells than in IRM-32.
dimethylaminopyridine;
trifluoroacetic acid
DCE,
1,2-dichloroethane;
TFA,
REFERENCES
(1) Taori, K.; Paul, V. J.; Luesch, H. Structure and Activity of
Largazole, a Potent Antiproliferative Agent from the Floridian
Marine Cyanobacterium Symploca Sp. J. Am. Chem. Soc. 2008,
130, 1806–1807.
2) Yu, M.; Salvador, L. A.; Sy, S. K. B.; Tang, Y.; Singh, R. S. P.;
Chen, Q.-Y.; Liu, Y.; Hong, J.; Derendorf, H.; Luesch, H.
Largazole pharmacokinetics in rats by LC-MS/MS Mar. Drugs
2014, 12, 1623−1640.
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Additional antiproliferative studies are being performed in
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The Supporting Information is available free of charge on the
ACS Publications website http://pubs.acs.org. the synthetic
procedures, the characterization chemical intermediates and
final compounds and biological assay protocols are in details.
AUTHOR INFORMATION
Corresponding Author
*(R.D.F) Phone: +390249491702. Fax: +390249491712. E-
mail: r.difabio@irbm.com
Author Contributions
All authors equally contributed to the preparation of this
manuscript and gave approval to the final version.
Funding Sources
This project was financially supported by the COLLEZIONE
DEI COMPOSTI CHIMICI E CENTRO DI SCREENING–
CNCCS scarl under the PRONAT research project framework.
Notes
# This article is dedicated to the memory of Professor Maurizio
Botta, deceased on August 2nd 2019, who spent his entire life in
science aiming for improving the quality of the life of human
beings.
^
Present address: Department of Pharmacy, University of
Naples “Federico II”, Via D. Montesano 49, 80131 Naples,
Italy.
The authors declare no competing financial interests
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ACKNOWLEDGMENTS
The authors would like to thank prof. Marco Lolli (Università
degli studi di Torino) and prof. Maria Frosini (Università degli
Studi di Siena) for the characterization of compound 1 and 2 in
the in vitro NO release assay.
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ABBREVIATIONS
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Matteo Borgini,Claudio Zamperini,Federica Poggialini,Luca Ferrante,Vincenzo Summa,Maurizio Botta
and Romano Di Fabio
1
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