Synthesis and biological evaluation of Santacruzamate-A based analogues

Article abstract of DOI:10.1016/j.bmc.2017.10.026

Several derivatives of Santacruzamate-A, a natural product that is structurally related to SAHA, were synthesized to explore the potential of carbamates and oxalylamides as novel biasing element for targeting the catalytic site of zinc-dependent histone deacetylases (HDACs). An additional class of Santacruzamate-A derivatives was synthesized to investigate the influence of the cap group and the linker element on HDAC inhibitory activity. All compounds were evaluated in dose response for their in vitro cytotoxic activity in MTT assay in HCT116 cells. HDAC inhibitory activity was evaluated in vitro by western blot analysis for histone hyperacetylation assay and biochemically for representative human HDACs isoforms. Two novel compounds were identified to exhibit potent time dependent anti proliferative activity. However, unlike hydroxamic acid analogues, the tested Santacruzamate-A derivatives showed no noticeable HDAC inhibitory activity. The ethylcarbamate moiety as unusual zinc-binding group displayed no ability to coordinate the zinc ion and thus, presumably, was not able to reproduce known inhibitor-substrate zinc-binding group interactions with the HDAC catalytic site. This study confirmed that the accommodation of the zinc-binding group is deeply critical of the positioning of the linker and the projection of the cap group toward the different surface pockets of the enzyme.

Full text of DOI:10.1016/j.bmc.2017.10.026

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of Santacruzamate-A based
analogues
a
a
b
a,
⇑
Rosario Randino , Patrizia Gazzerro , Ralph Mazitschek , Manuela Rodriquez
a
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy
Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Several derivatives of Santacruzamate-A, a natural product that is structurally related to SAHA, were syn-
thesized to explore the potential of carbamates and oxalylamides as novel biasing element for targeting
the catalytic site of zinc-dependent histone deacetylases (HDACs). An additional class of Santacruzamate-
A derivatives was synthesized to investigate the influence of the cap group and the linker element on
HDAC inhibitory activity. All compounds were evaluated in dose response for their in vitro cytotoxic
activity in MTT assay in HCT116 cells. HDAC inhibitory activity was evaluated in vitro by western blot
analysis for histone hyperacetylation assay and biochemically for representative human HDACs isoforms.
Two novel compounds were identified to exhibit potent time dependent anti proliferative activity.
However, unlike hydroxamic acid analogues, the tested Santacruzamate-A derivatives showed no notice-
able HDAC inhibitory activity. The ethylcarbamate moiety as unusual zinc-binding group displayed no
ability to coordinate the zinc ion and thus, presumably, was not able to reproduce known inhibitor-
substrate zinc-binding group interactions with the HDAC catalytic site. This study confirmed that the
accommodation of the zinc-binding group is deeply critical of the positioning of the linker and the pro-
jection of the cap group toward the different surface pockets of the enzyme.
Received 28 August 2017
Revised 10 October 2017
Accepted 19 October 2017
Available online xxxx
Keywords:
HDAC
Antiproliferation
Tumor progression
Zinc-binding group
Histone acetylation
Ó 2017 Elsevier Ltd. All rights reserved.
1
. Introduction
There is growing evidence that gene expression, governed by
cells. The selective targeting of single HDAC isoform has several
implications in the differentiation, apoptosis, cell cycle regulation,
migration, susceptibility to chemotherapy and angiogenesis.8
,9
epigenetic changes, is crucial for the onset and progression of var-
ious diseases, including cancer.1 Epigenetic alterations can cause
aberrant gene expression, thus leading to disease. These transcrip-
tional changes are potentially reversible by pharmacologically tar-
Natural products, such as the cyclopeptides FK228 [Romidepsin,
,2
Ò
Istodax , 1, FDA approved for the treatment of cutaneous T-cell
1
0–12
lymphoma (CTCL)], Apicidin (2) or FR235222 (3),
are used as
modulators of specific enzymes implicated in cancer cell prolifera-
tion (Fig. 1a) preventing tumorigenesis and cancer progression.
These peptide analogues modulate the acetylation level on his-
tones and non-histone proteins in cells, working as specific histone
deacetylases inhibitors (HDACIs). The first in class FDA approved
HDACI for CTLC is the synthetic pan-HDAC inhibitor SAHA (Vori-
3
geting the enzymes responsible for the epigenetic modifications.
In the past decade dedicated drug development efforts in industry
and academia have been devoted to providing a panel of next-gen-
eration anticancer agents that target the respective epigenetic
4
–7
master regulators responsible for these biological mechanisms.
Among these, epigenetic enzymes, such as histone deacetylases
Ò
nostat, Zolinza , 4, Fig. 1b). All these compounds are able to drive
(
HDACs) or histone acetyltransferases (HATs) are altered in tumor
the silencing or the activation of gene transcription activity at
low nanomolar concentrations; because of their pharmacokinetic
liabilities (chemical and metabolic instability), new solutions in
Abbreviations: AcCN, acetonitrile; BnOH, benzyl alcohol; DMF, N,N-dimethyl-
1
3
terms of structure modification are highly desired.
formamide; DMSO, dimethylsulphoxide; EtOAc, ethyl acetate; EtOH, ethanol;
GABA, -aminobutyric acid; GADPH, glyceraldehyde 3-phosphate dehydrogenase;
HBTU, O-(benzotriazol-1-yl)-N,N,N ,N -tetramethyluronium hexafluorophosphate;
MeOH, methanol; n-hex, n-hexane; NMM, 4-methylmorpholine; SD, standard
deviation; TEA, triethylamine; THF, tetrahydrofuran.
c
Recently the isolation, structure elucidation and HDAC inhibi-
0
0
tory activity of Santacruzamate-A (5a, SCA, Fig. 1b) was reported
14
by Balunas and co-workers. The chemical structure of SCA, a sim-
ple small molecule from a marine Panamanian cyanobacterium,
resembles the SAHA scaffold containing the three canonical
⇑
Corresponding author.
E-mail address: mrodriquez@unisa.it (M. Rodriquez).
https://doi.org/10.1016/j.bmc.2017.10.026
968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Randino R., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.026

2
R. Randino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. (a) Structure of natural HDAC inhibitors. (b) Structure of SAHA, 4 and Santacruzamate-A, 5a and their structure similarities based on HDAC inhibitor pharmacophore
model.
Fig. 2. Chemical structure of class I SCA-like small molecules 5a–e.
structural motifs of HDAC inhibitor pharmacophore model includ-
ing a zinc-binding group (ZBG), an aliphatic linker and a cap group
15–17
(
CAP) for additional enzyme interactions.
Initially, we were
Fig. 3. Chemical structure of class II SCA-like small molecules 6a–h.
intrigued by the ethylcarbamate moiety, as unusual ZBG, reported
to inhibit histone deacetylases in the nanomolar range. Therefore,
we synthesized
a small collection of SCA-analogues herein
provided access to the corresponding carboxylic acid 10 in very
good yield. Coupling with various aromatic amines and subse-
quent one-pot saponification furnished the target compounds
reported as class I SCA-like small molecules (5b–e, Fig. 2) following
2
0
synthetic procedure previously described.14
Then, in an effort to re-evaluate the previously reported anti-
proliferative and HDAC inhibitory activities, we pursued the syn-
thesis of a class II SCA-like small molecules (6a–h) introducing a
6
a–h in good yields and purity (Scheme 1).
terminal oxamic acid as new zinc chelating moiety on
tyric acid-linker (GABA-linker), with different CAP groups (Fig. 3).
c-aminobu-
2.2. Biology
1
8
To evaluate the antiproliferative activity of class I and II SCA
2
. Results and discussion
.1. Chemistry
Class I SCA-like small molecules (5b–e) were obtained accord-
analogous (5a–e and 6a–h), we treated HCT116 colorectal cancer
cells in dose response MTT assay (Table 1). As expected, we found
that SAHA significantly inhibited the viability of HCT116 cells in a
2
dose and time dependent manner after 24 h (IC50
5 lM), 48 h (1
l
M < IC50 < 5 M) and 72 h (IC50 < 1 M) of treatment (Table 1).
l
l
ing to the synthetic procedure reported by Balunas and co-workers
(
HDAC inhibitory activity was tested by western blot analysis using
histone hyperacetylation as well established biomarker (Fig. 4) and
inhibitory activity for various HDAC isoforms was determined in
biochemical assays (data not shown/see Supporting informa-
tion). Consistent with recently published results and contrary to
see also Supporting information, Scheme 1SI).14
Class II SCA-like analogues were synthesized starting from
1
9
21
GABA benzyl ester 8, which was readily converted into corre-
sponding amide-ester 9. The debenzylation of 9 with H and Pd/C
2
Please cite this article in press as: Randino R., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.026

R. Randino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Fig. 4. Representative western blot analysis of total acetyl-histone-H3 (a) and total
acetyl-histon-H4 (b) expression in total protein lysates from HCT116 cells
untreated (DMSO) or treated for 24 h with the compounds (10lM if not otherwise
specified) (mean ± SD). GAPDH served as the loading control. Data are represen-
tative of three different experiments with similar results.
HDAC inhibition enzymatic assay) (entry 1, Table 1 IC50 > 50 lM).
Interestingly, the novel class I SCA-like molecules 5d and 5e
showed potent time dependent antiproliferative activity at both
4
8h and 72h treatment (entry 4 and 5, Table 1). Similar results
were obtained for the class II SCA-like compound 6h, which was
most pronounced after a 24 h (IC50 = 1
Table 1).
To evaluate the role of HDAC-inhibition in the anti-proliferative
effects, the total amount of acetyl-histone-H3 and acetyl-Histone-
H4 were analysed in HCT116 cell line treated with selected mole-
lM) treatment (entry 13,
Scheme 1. Synthesis of class II SCA-like compounds 6a–h. Reagents and conditions:
a) BnOH, SOCl , 3 h, 0 °C to r.t., 80%; (b) ethylchloroxacetate, TEA, dry DMF, 1.5 h, 0
C to r.t., 79%; (c) H , Pd/C, MeOH, r.t., 45 min, 97%; (d) RNH , HBTU, NMM, dry
(
°
2
2
2
2
AcCN, 4 h, r.t.; (e) LiOH, THF/H O (1:1, v/v), 4 h, r.t., 46–70%.
cules (10
lM for 24 h, Fig. 4a, b). Compounds 5d (10 lM) and 6h
Table 1
(10 M) did not significantly increase of histone H3 and H4 acety-
l
MTT Assay results of SCA-like molecules class I and II in HCT116 cell line.
Entry Compounds M)
IC50a,c
4 h
lation compared to vehicle treated cells, while SAHA treatment
strongly increased acetylation of H3 and H4 (Fig. 4).
These data are in agreement with the absence of significant bio-
chemical HDAC inhibitory activity for representative class I and
class II HDAC isoforms (HDAC1,3,6,8) (see Supplementary figure
(
l
2
48 h
72 h
SCA-like Class I compounds
d
1
2
3
4
5
5a, SCA
5b
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.615 ± 0.049
1 < IC50 < 5
5 < IC50 < 10
‘
‘HDAC 16 h preincubation”). Together, these results suggested that
b
5c
5d
5e
n.d.
class I SCA-like molecules exert their cytotoxic activity by an HDAC
independent mechanism. Additional experiments to elucidate the
underlying mode of action of the antiproliferative activity of com-
pounds 5d, 5e, 6h are currently underway.
0.476 ± 0.042
0.825 ± 0.054
SCA-like Class II compounds
6
7
8
9
1
1
1
1
1
6a
6b
6c
6d
6e
6f
>50
>50
n.d.
>50
>50
<50
>50
n.d.
n.d.
>50
n.d.
n.d.
n.d.
>50
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3. Conclusion
0
1
2
3
4
In conclusion, aiming to clarify and better elucidate the SAR of
Santacruzamate-A the putative HDAC inhibitory activity, we pur-
sued the synthesis of natural product SCA 5a, class I (5b–e) and
class II (6a–h) SCA-like analogues. We modified alternatively the
CAP, by using several aromatic amines, and the unusual ethyl-car-
bamate moiety (5b–e) or oxamic acid group (6a–h) as ZBG and
identified class I SCA-like molecules (5d and 5e), and compound
b
6g
n.d.
6h
0,814 ± 0,069
5
d
SAHA
1 < IC50 < 5
0.877 ± 0.019
a
Anti-proliferative activity of compounds 5a–e and 6a–h in HCT116 cell line is
expressed as cell viability. Results are expressed as mean of three independent
experiments ± s.e.m.
b
n.d.: not determined; indicates no relevant cytotoxic activity or registered
6
h, featuring a novel oxamic acid as ZBG, to have potent antiprolif-
values are not fit in a IC50 curve.
c
erative activity in HCT116 cancer cells. However, biochemical pro-
filing, in vitro MTT viability studies and western blot analysis for
histone hyperacetylation in HCT116 cells confirmed that San-
tacruzamate-A and analogues lack HDAC inhibitory activity. These
results may have a structural interpretation based on the absence
in Santacruzamate-A and its derivatives of a moiety involved in
zinc coordination in the HDAC catalytic site. The hydroxamic acid
of SAHA is replaced in Santacruzamate-A by an ethyl-carbamate
moiety devoid of any zinc coordination ability. With this study,
we confirmed that the accommodation of the ZBG into the HDAC
catalytic site is a crucial step of the inhibition process and is finely
Compounds are tested by using serial dilution starting from 50
M.
Control compounds (SAHA e SCA) were tested by using serial dilution starting
from 50 M.
lM, 10 lM, 5
l
M, 1 l
d
l
the original report,22–24 we found that Santacruzamate-A (5a) had
no significant antiproliferative activity in HCT116 cells. Further-
more, we did not observe inhibition of HDAC activity in HCT116
(
using histone acetylation as biomarker) and in biochemical assays
probing individual HDAC isoforms (see Supplementary Material for
Please cite this article in press as: Randino R., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.026

4
R. Randino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
controlled by its zinc coordination ability and by key interactions
with the surrounding protein residues.
(s, 2H), 2.45 (t, J = 7.3 Hz, 3H), 1.90 (p, J = 7.1 Hz, 3H), 1.37 (t, J =
1
3
7.1 Hz, 4H) ppm. C NMR (101 MHz, Methanol d
4
) d: 174.50,
61.47, 159.46, 137.61, 132.39, 129.86, 129.53, 129.18, 67.33,
3.80, 40.04, 32.29, 25.32, 14.23 ppm. HRMS (ESI-Q-TOF) m/z [M
1
6
4
. Experimental section
+
5
+H] Calcd. for C15H20NO 294.1336; Found 294.1345.
4.1. Chemistry
4.1.1.3. 4-(2-Ethoxy-2-oxoacetamido) butanoic acid, 10. In an oven
dried round bottom flask, to a solution of 10 (300 mg, 1.03 mmol)
in 20 mL of MeOH, catalytic amount of Pd/C was added. After three
vacuum/H (1 atm) atmosphere cycling operations, the reaction
2
was stirred at room temperature for 45 min till starting material
disappearing. The mixture was then filtered (attention: the residue
Pd may be pyrophoric) by Celite and the solvent evaporated in vacuo
to furnish compound 11 as white solid (197 mg, 97%). 1H NMR
All reagents were purchased from Sigma-Aldrich (Milan, Italy)
in the highest available purity and were used as received. All reac-
tions involving air or moisture sensitive reagents were carried out
under a dry nitrogen atmosphere using dry solvents. Dry DMF,
AcCN and MeOH were purchased and used without further distil-
lation. When necessary, compounds were dried in vacuo over
P O or by azeotropic removal of water with toluene under
2 5
reduced pressure. Reaction temperatures were measured exter-
nally; reactions were monitored by TLC on Merck silica gel plates
(
400 MHz, Methanol d
4
) d: 4.33 (q, J = 7.1 Hz, 2H), 3.34 (t, J = 7.0
Hz, 2H), 2.36 (t, J = 7.4 Hz, 2H), 1.87 (p, J = 7.2 Hz, 2H), 1.36 (t, J =
1
3
7
4
.1 Hz, 3H) ppm.
0.12, 32.20, 25.34, 14.22 ppm. HRMS (ESI-Q-TOF) m/z [M+H]
204.0866; Found 204.0861.
4
C NMR (101 MHz, Methanol d ) d: 63.80,
(
4
0.25 mm) and visualized by UV light, KMnO , p-anisaldehyde, or
+
ninhydrin solutions and drying. Flash chromatography was per-
formed on Merck silica gel 60 (particle size: 0.040–0.063 mm)
and the solvents employed were of analytical grade. Yields refer
to chromatographically and spectroscopically (1H and 13C NMR)
pure compounds. NMR spectra were generally recorded at room
temperature, on Bruker Avance series 400 and 300 spectrometer.
Chemical shifts (d) are reported in ppm relatively to the residual
8 5
Calcd. for C H14NO
4
6
4
6
.1.2. General procedure for the synthesis of class II SCA-like molecules
a–h
.1.2.1. 2-Oxo-2-((4-oxo-4-(phenethylamino)butyl)amino)acetic acid,
a. In an oven dried round bottom flask, to a ice cold solution of 11
(
20 mg, 0.098 mmol) in 1 mL of dry AcCN, HBTU (48 mg, 0.13
mmol) and NMM (38.0 L, 0.34 mmol) were added. After 5 min
magnetic stirring, phenylethylamine (11.9 mg, 0.098 mmol, 12
L) was added at 0 °C and then the mixture was stirred at room
temperature over night under N dry atmosphere. After that per-
iod, the mixture was monitored via TLC and at the end of the reac-
tion the mixture was washed with twice with NH Clss, Na
NaClss and extracted with DCM. The organic phase were collected
and dried under Na SO , and filtered. The crude was directly dis-
solved in H O/THF mixture (1:1 v/v, 1.6 mL) then LiOH added
47 mg, 1.96 mmol) and the reaction kept stirred for additional
3 3 2
solvent peak (CHCl , d: 7.26, 13CDCl , d: 77.0; CD HOD, d: 3.35,
l
1
3CD OD, d: 49.0) and the multiplicity of each signal is designated
3
by the following abbreviations: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad; app, apparent. Coupling constants
J) are quoted in Hz. High resolution mass spectra (HRMS) were
recorded on a high resolution mass spectrometer equipped by elec-
trospray (ESI) and nanospray sources, and a quadrupole-time of
flight hybrid analyser, coupled with capillary UPLC system
l
2
(
4
2
CO3ss,
2
4
(
Q-TOF Premier/nanoAquity, Waters) in positive mode, and either
+
2
protonated molecular ions [M+H] were used for empirical formula
confirmation, unless otherwise stated.
(
1
4 h. After this period, the mixture was acidified up to pH 7 with
HCl 2 N and then extracted twice with EtOAc. The organic fractions
were collected, dried under Na SO and filtered. Purification by
column chromatography (DCM/MeOH 95:5) furnished compound
4
4
.1.1. Synthetic procedures
.1.1.1. Benzyl 4-aminobutanoate hydrochloride, 8. To a ice-cold
2
4
solution of benzyl alcohol (5 mL) and GABA 7 (516 mg, 5 mmol),
SOCl (547 L, 7.5 mmol) was added dropwise and the reaction
stirred at room temperature for 3 h under N dry atmosphere. After
that period the uncoloured solution passed through a pale yellow
solution. The solvent was evaporated up to the formation of a
white solid. Crystallization of the crude in EtOH/n-hex (1:1, v/v)
at 4 °C furnished the pure hydrochloride 9 (920 mg, 80%).
1
6
a as a white solid (16 mg, 60%). H NMR (400 MHz, Methanol d
4
)
2
l
d: 7.29 (t, J = 7.3 Hz, 1H), 7.26–7.17 (m, 2H), 3.42 (t, J = 7.4 Hz, 2H),
2
3
1
1
3
C
.27 (t, J = 6.9 Hz, 2H), 2.81 (t, J = 7.3 Hz, 2H), 2.21 (t, J = 7.4 Hz, 2H),
.83 (p, J = 7.1 Hz, 2H) ppm. C NMR (101 MHz, Methanol d ) d:
4
13
75.22, 159.11, 140.51, 129.81, 129.47, 127.34, 41.96, 40.22,
+
6.50, 34.30, 26.30. HRMS (ESI-Q-TOF) m/z [M+H] Calcd. for
1
H
H
14 19
N
2
O
4
279.1339; Found 279.1349.
NMR (400 MHz, Deuterium Oxide) d: 7.48 (d, J = 3.7 Hz, 3H), 5.20
(
s, 2H), 3.05 (t, J = 7.8 Hz, 2H), 2.58 (t, J = 7.3 Hz, 2H), 1.99 (p, J =
4
.1.2.2.
2-Oxo-2-((4-oxo-4-((3-phenylpropyl)amino)butyl)amino)
1
3
7
1
3
C
.4 Hz, 2H) ppm.
C NMR (101 MHz, Deuterium Oxide) d:
acetic acid, 6b. The synthesis of compound 6b was carried out fol-
lowing the general procedure for the synthesis of class II SCA-like
molecules using 11 (20 mg, 0.098 mmol) in 1 mL dry AcCN and
HBTU (48.3 mg, 0.13 mmol), NMM (38.0
lphenylamine (14 L, 0.098 mmol), LiOH (47 mg, 1.96 mmol) in
H O/THF mixture (1:1 v/v, 1.6 mL) furnishing final product 6b as
2
a white solid (16 mg, 55%). H (400 MHz, Methanol d ) d: 7.27 (t,
4
J = 7.4 Hz, 1H), 7.24–7.13 (m, 2H), 3.35–3.29 (m, 3H), 3.21 (t, J =
7.1 Hz, 2H), 2.65 (t, J = 7.7 Hz, 2H), 2.24 (t, J = 7.4 Hz, 2H), 1.85
74.72, 135.57, 128.88, 128.79, 128.71, 128.38, 67.19, 38.70,
+
0.76, 22.03 ppm. HRMS (ESI-Q-TOF) m/z [M+H] Calcd. for
11 2
H16NO 194.1176; Found 194.1170.
lL, 0.34 mmol), propy-
l
4
.1.1.2. Benzyl 4-(2-ethoxy-2-oxoacetamido) butanoate, 9. In an oven
dried round bottom flask, to an ice-cold DMF solution (5 mL) of 9
300 mg, 1.31 mmol) ethylchloroxacetate (146 L, 1.31 mmol)
and TEA (548 L, 3.93 mmol) were added and the reaction stirred
under N dry atmosphere for 30 min at 0 °C and then the mixture
1
(
l
l
1
3
2
4
(dp, J = 15.9, 7.3 Hz, 4H) ppm. C NMR (101 MHz, Methanol d )
was allowed to reach room temperature for an additional 1 h. The
reaction was monitored by TLC up to starting material disappeared.
The solvent was removed in vacuo and the mixture washed with
NaClss and extracted with EtOAc. The organic fractions were col-
d: 175.26, 166.68, 159.12, 142.99, 129.40, 126.88, 40.27, 40.10,
34.31, 34.21, 32.21, 26.32 ppm. HRMS (ESI-Q-TOF) m/z [M+H]+
21 2 4
Calcd. for C15H N O 293.1496; Found 293.1488.
lected, dried under Na
by column chromatography (n-hex/EtOAc 7:3) to furnish 10 as yel-
lowish oil (304 mg, 79%). H NMR (400 MHz, Methanol d
2
SO
4
and filtered. The crude was purified
4.1.2.3. 2-((4-(Benzylamino)-4-oxobutyl)amino)-2-oxoacetic acid, 6c.
The synthesis of compound 6c was carried out following the gen-
eral procedure for the synthesis of class II SCA-like molecules using
11 (20 mg, 0.098 mmol) in 1 mL dry AcCN and HBTU (48.3 mg,
1
4
) d: 7.36
(
dd, J = 13.4, 3.8 Hz, 4H), 5.14 (s, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.36
Please cite this article in press as: Randino R., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.026

R. Randino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
0
0
.13 mmol) e NMM (38.0
.098 mmol), LiOH (47 mg, 1.96 mmol) in H
l
L, 0.34 mmol), benzylamine (11
l
L,
phenylbutan-2-amine (16
mmol) in H O/THF mixture (1:1 v/v, 1.6 mL) furnishing final pro-
duct 6g as a white solid (19 mg, 63%). H NMR (400 MHz,
Methanol d ) d: 4.33 (q, J = 7.1 Hz, 2H), 3.34 (t, J = 7.0 Hz, 2H),
2.36 (t, J = 7.4 Hz, 2H), 1.87 (p, J = 7.2 Hz, 2H), 1.36 (t, J = 7.1 Hz,
lL, 0.098 mmol), LiOH (47 mg, 1.96
2
O/THF mixture (1:1
2
1
v/v, 1.6 mL) furnishing final product 6c as a white solid (18 mg,
7
3
ppm. C NMR (101 MHz, Methanol d
1
ppm. HRMS (ESI-Q-TOF) m/z [M+H] Calcd. for
1
0%). H NMR (400 MHz, Methanol d
4
) d: 7.47–7.11 (m, 4H),
4
.35–3.30 (m, 3H), 2.30 (t, J = 7.5 Hz, 2H), 1.89 (p, J = 7.1 Hz, 2H)
1
3
13
4
) d: 175.14, 161.96, 159.13,
3H) ppm.
32.20, 25.34, 14.22 ppm. HRMS (ESI-Q-TOF) m/z [M+H] Calcd.
for C16 307.1652; Found 307,1661.
4
C NMR (101 MHz, Methanol d ) d: 63.80, 40.12,
+
39.96, 129.54, 128.56, 128.20, 128.15, 44.14, 40.27, 34.27, 26.31
+
C
13
17
H N
2
O
4
23 2 4
H N O
2
65.1183; Found 265.1176.
4
.1.2.4. 2-((4-((3-Hydroxyphenethyl)amino)-4-oxobutyl)amino)-2-
4.1.2.8. 2-Oxo-2-((4-oxo-4-(phenylamino)butyl)amino)acetic acid,
6h. The synthesis of compound 6h was carried out following the
general procedure for the synthesis of class II SCA-like molecules
using 11 (20 mg, 0.098 mmol) in 1 mL dry AcCN and HBTU (48.3
oxoacetic acid, 6d. The synthesis of compound 6d was carried
out following the general procedure for the synthesis of class II
SCA-like molecules using 11 (40 mg, 0.196 mmol) in 2 mL dry
AcCN and HBTU (97.08 mg, 0,256 mmol) e NMM (75
mmol), 3-(2-aminoethyl)phenol hydrochloride (33 mg, 0.196
mmol), LiOH (94 mg, 3.92 mmol) in H O/THF mixture (1:1 v/v,
.2 mL) furnishing final product 6d as a white solid (27 mg,
l
L, 0.686
mg, 0.13 mmol) e NMM (38.0
0.098 mmol), LiOH (47 mg, 1.96 mmol) in H
v/v, 1.6 mL) furnishing final product 6h as a white solid (17 mg,
l
L, 0.34 mmol), aniline (7.5
lL,
2
O/THF mixture (1:1
2
1
3
4
6
6
4
68%). H NMR (400 MHz, Methanol d ) d: 7.53 (d, J = 8.0 Hz, 2H),
1
4
6%). H NMR (400 MHz, Methanol d ) d: 7.08 (t, J = 7.8 Hz, 1H),
7.29 (t, J = 7.8 Hz, 2H), 7.08 (t, J = 7.4 Hz, 1H), 3.37 (d, J = 6.9 Hz,
.65 (dt, J = 15.1, 7.9 Hz, 2H), 3.38 (t, J = 7.4 Hz, 2H), 3.26 (t, J =
.9 Hz, 2H), 2.71 (t, J = 7.3 Hz, 2H), 2.19 (t, J = 7.4 Hz, 2H), 1.81
2H), 2.41 (t, J = 7.4 Hz, 2H), 1.94 (p, J = 7.1 Hz, 2H) ppm. 13C NMR
(101 MHz, Methanol d
4
) d: 173.66, 161.93, 159.17, 139.81,
13
(
p, J = 7.2 Hz, 2H) ppm.
4
C NMR (101 MHz, Methanol d ) d:
129.75, 125.15, 125.08, 121.32, 121.27, 40.27, 35.13, 26.13 ppm.
+
1
1
75.23, 161.95, 159.11, 158.55, 141.98, 130.45, 121.00, 116.65,
15 2 4
HRMS (ESI-Q-TOF) m/z [M+H] Calcd. for C12H N O 251.1026;
14.27, 41.92, 40.22, 36.45, 34.33, 34.30, 26.31 ppm. HRMS
Found 251, 1019.
+
(
ESI-Q-TOF) m/z [M+H] Calcd. for C14
H
19
2
N O
5
295.1288; Found
2
95.1296.
4.2. Biology
4.1.2.5.
2-((4-((3-Fluorophenethyl)amino)-4-oxobutyl)amino)-2-
oxoacetic acid, 6e. The synthesis of compound 6e was carried out
following the general procedure for the synthesis of class II SCA-
like molecules using 11 (20 mg, 0.098 mmol) in 1 mL dry AcCN
4.2.1. Cell cultures, treatments and cell viability assay
Human colorectal cancer cell (CRC) lines HCT116 and DLD1
were obtained from the Interlab Cell Line Collection (IST, Genoa,
Italy) and grown in McCoy’s 5A and RPMI medium respectively,
and HBTU (48.3 mg, 0.13 mmol) e NMM (38.0
fluorophenylethylamine (33 mg, 0.196 mmol), LiOH (47 mg, 1.96
mmol) in H O/THF mixture (1:1 v/v, 1.6 mL) furnishing final pro-
duct 6e as a white solid (20 mg, 70%). H NMR (400 MHz,
Methanol d ) d: 7.28 (q, J = 7.4 Hz, 1H), 7.03 (d, J = 7.5 Hz, 1H),
.00–6.88 (m, 2H), 3.41 (t, J = 7.1 Hz, 2H), 3.25 (t, J = 6.7 Hz, 2H),
.80 (t, J = 7.1 Hz, 2H), 2.19 (t, J = 7.3 Hz, 2H), 1.81 (p, J = 6.8 Hz,
lL, 0.34 mmol), 3-
2
at 37 °C in a 5% CO atmosphere. Cells were seeded at a density
5
2
of 1 ꢀ 10 cells/well and exposed to increasing concentrations of
compounds. To evaluate cell viability the colorimetric MTT (3-
(4,5 di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
metabolic activity assay was performed as described previously.25
Briefly, MTT stock solution (5 mg/mL in PBS, Sigma) was added to
1
4
7
2
2
1
1
4
1
3
H) ppm. C NMR (101 MHz, Methanol d
4
) d: 175.26, 165.55,
2
each well and incubated for 4 h at 37 °C in humidified CO . The for-
63.13, 161.96, 159.10, 143.42, 143.35, 131.16, 131.07, 125.74,
25.71, 116.57, 116.36, 114.12, 114.10, 113.91, 113.88, 41.59,
0.20, 36.16, 34.31, 34.26, 26.29 ppm. HRMS (ESI-Q-TOF) m/z [M
mazan crystals were solubilized with acidic isopropanol (0.1 N HCl
in absolute isopropanol). MTT conversion to formazan by metabol-
ically viable cells was monitored by spectrophotometer at an opti-
cal density of 595 nm. Each data point represents the average of
three separate experiments in triplicate.
+
+H] Calcd. for C14
2
H18FN O
4
297.1245; Found 297.1253.
4.1.2.6. 2-((4-((2-(1H-Indol-3-yl)ethyl)amino)-4-oxobutyl)amino)-2-
oxoacetic acid, 6f. The synthesis of compound 6f was carried out
following the general procedure for the synthesis of class II SCA-
like molecules using 11 (20 mg, 0.098 mmol) in 1 mL dry AcCN
4.2.2. Western blot analysis
Cells were seeded in 60-mm dishes, scraped and washed with
ice-cold phosphate buffer saline (PBS). After treatments at indi-
cated time points, western blot analysis was performed as previ-
and HBTU (48.3 mg, 0.13 mmol) e NMM (38.0
tryptamine (15.7 mg, 0.098 mmol), LiOH (47 mg, 1.96 mmol) in
O/THF mixture (1:1 v/v, 1.6 mL) furnishing final product 6f as
lL, 0.34 mmol),
2
6
H
2
ously described. Briefly, total protein extracts were obtained
through lysis in buffer A (50 mM Tris–HCl pH 8.0 buffer containing
150 mM NaCl, 1% Nonidet P-40, 2 mg/mL aprotinin, 1 mg/mL pep-
1
a white solid (20 mg, 65%). H NMR (400 MHz, Methanol d
4
) d:
7
6
2
.55 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.11–7.03 (m, 2H),
.99 (t, J = 7.4 Hz, 1H), 3.47 (t, J = 7.2 Hz, 2H), 3.24 (t, J = 6.9 Hz,
H), 2.94 (t, J = 7.2 Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 1.80 (p, J = 7.1
3 4
statin, 2 mg/mL leupeptin, 1 mM Na VO ). Protein concentration
was determined by the Bradford assay using bovine serum albu-
min as standard. About 30 mg of proteins were loaded on 15%
SDS–polyacrylamide gels under reducing conditions. After SDS–
PAGE, proteins were transferred to nitrocellulose membranes that
were blocked with 5% milk (Bio-Rad Laboratories, Inc.) and incu-
bated with specific antibodies, anti-acetyl-Histone H4 (Santa Cruz
Biotechnology), anti-acetyl-Histone H3 (Millipore) and GAPDH
(Cell Signaling). Filters were washed and incubated with horserad-
ish peroxidase-conjugated secondary antibodies. Membranes were
stained using a chemoluminescence system (ECL-Amersham Bio-
sciences, Glattbrugg, CH) and then exposed to X-ray film (Kodak,
Rochester, NY).
1
3
Hz, 2H) ppm. C NMR (101 MHz, Methanol d
4
) d: 175.22, 161.94,
59.07, 138.15, 128.81, 123.59, 123.52, 123.43, 122.29, 119.56,
19.27, 113.25, 112.27, 112.21, 41.42, 41.32, 40.23, 34.36, 26.27,
6.20 ppm. HRMS (ESI-Q-TOF) m/z [M+H] Calcd. for C16H N O
20 3 4
18.1448; Found 318.1454.
1
1
2
3
+
4.1.2.7. 2-Oxo-2-((4-oxo-4-((4-phenylbutan-2-yl)amino)butyl)amino)
acetic acid, 6g. The synthesis of compound 6g was carried out fol-
lowing the general procedure for the synthesis of class II SCA-like
molecules using 11 (20 mg, 0.098 mmol) in 1 mL dry AcCN and
HBTU (48.3 mg, 0.13 mmol) e NMM (38.0
lL, 0.34 mmol), 4-
Please cite this article in press as: Randino R., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.026

6
R. Randino et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9.
Zhu P, Martin E, Mengwasser J, Schlag P, Janssen KP, Göttlicher M. Cancer Cell.
004;5:455–463.
A. Supplementary data
2
1
0. Randino R, Cini E, D’Ursi AM, Petricci E, Rodriquez M. Pharmacologyonline.
2014;3:203–208.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2017.10.026.
11. Rodriquez M, Terracciano S, Cini E, et al. Angew Chem Int Ed. 2006;45:423–427.
12. Rodriquez M, Bruno I, Cini E, Marchetti M, Taddei M, Gomez-Paloma L. J Org
Chem. 2006;71:103–107.
13. Randino R, Moronese I, Cini E, et al. Curr Top Med Chem. 2017;17:441–459.
14. Pavlik CM, Wong CYB, Ononye S, et al. J Nat Prod. 2013;76:2026–2033.
15. Chan CT, Qi J, Smith W, et al. Cancer Res. 2014;74:7475–7486.
References
1.
2.
3.
Lim PS, Li J, Holloway AF, Rao S. Immunology. 2013;139:285–293.
Abu-Remaileh M, Bender S, Raddatz G, et al. Cancer Res. 2015;75:2120–2130.
Giannini G, Cabri W, Fattorusso C, Rodriquez M. Future Med Chem.
16. Bowers AA, West N, Newkirk TL, et al. Org Lett. 2009;11:1301–1304.
17. Bowers A, West N, Taunton J, Schreiber SL, Bradner JE, Williams RM. J Am Chem
Soc. 2008;130:11219–11222.
2
012;4:1439–1460.
Nagaraju M, Deepthi EG, Ashwini C, et al. Bioorg Med Chem Lett.
012;22:4314–4317.
Roell D, Rösler TW, Degen S, Matusch R, Baniahmad A. Chem Biol Drug Des.
011;77:450–459.
18. Rose NR, Ng SS, Mecinovi c´ J, et al. J Med Chem. 2008;51:7053–7056.
19. Kimura H, Sampei S, Matsuoka D, et al. Bioorg Med Chem. 2016;24:2251–2256.
20. Beaulieu PL, Cameron DR, Ferland JM, et al. J Med Chem. 1999;42:1757–1766.
21. Bradner JE, West N, Grachan ML, et al. Nat Chem Biol. 2010;6:238–243.
22. Liu Q, Lu W, Ma M, et al. RSC Adv. 2015;5:1109–1112.
23. Gromek SM, Maxwell AT, West AM, et al. Bioorg Med Chem.
2016;24:5183–5196.
24. Krieger V, Hamacher A, Gertzen CGW, et al. J Med Chem. 2017;60:5493–5506.
25. Gazzerro P, Malfitano AM, Proto MC, et al. Oncol Rep. 2010;23:171–175.
26. Proto MC, Gazzerro P, Di Croce L, et al. J Cell Physiol. 2012;227:250–258.
4.
5.
6.
7.
8.
2
2
Rodriquez M, Cini E, Petricci E, D’Ursi AM, Saturnino C, Randino R.
Pharmacologyonline. 2014;3:209–215.
Randino R, Cini E, Taddei M, Rodriquez M. Pharmacologyonline.
2
014;3:184–189.
Haberland M, Montgomery RL, Olson EN. Nat Rev Gen. 2009;10:32–42.
Please cite this article in press as: Randino R., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.10.026