Design, synthesis and anticancer activity of piperazine hydroxamates and their histone deacetylase (HDAC) inhibitory activity

Article abstract of DOI:10.1016/j.bmcl.2010.05.020

Six compounds were synthesized with piperazine in linker region and hydroxamate as Zinc Binding Group (ZBG). They were screened against three cancer cell-lines (NCIH460; HCT116; U251). Compounds 5c and 5f with GI₅₀ value of 9.33 ± 1.3 μM and 12.03 ± 4 μM, respectively, were tested for their inhibitory potential on hHDAC8. Compound 5c had IC₅₀ of 33.67 μM. Compounds were also screened for their anticancer activity against HL60 human promyelocytic leukemia cell line due to the presence of pharmacophoric features of RR inhibitors in them. Compound 5c had IC ₅₀ of 0.6 μM at 48 h.

Full text of DOI:10.1016/j.bmcl.2010.05.020

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3906–3910
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and anticancer activity of piperazine hydroxamates and
their histone deacetylase (HDAC) inhibitory activity
Bhadaliya Chetan a, Mahesh Bunha a, Monika Jagrat a, Barij Nayan Sinha a, Philipp Saiko a, Geraldine Graser b,
Thomas Szekeres b, Ganapathy Raman c, Praveen Rajendran c, Dhatchana Moorthy c, Arijit Basu a,
Venkatesan Jayaprakash a,
*
a Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835 215, India
b Department of Medical and Chemical Laboratory Diagnostics, General Hospital of Vienna—Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria
c Orchid Research Laboratories Ltd, Chennai 600 119, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Six compounds were synthesized with piperazine in linker region and hydroxamate as Zinc Binding
Group (ZBG). They were screened against three cancer cell-lines (NCIH460; HCT116; U251). Compounds
Received 23 February 2010
Revised 20 April 2010
Accepted 8 May 2010
Available online 15 May 2010
5c and 5f with GI50 value of 9.33 1.3
lM and 12.03
4
l
M, respectively, were tested for their inhibitory
potential on hHDAC8. Compound 5c had IC50 of 33.67
l
M. Compounds were also screened for their anti-
cancer activity against HL60 human promyelocytic leukemia cell line due to the presence of pharmaco-
phoric features of RR inhibitors in them. Compound 5c had IC50 of 0.6 M at 48 h.
l
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Histone deacetylase inhibitors
Piperazine linker
Hydroxamates
RR pharmacophore
HDAC activity is invariably increased in cancer cells and inhib-
itors of HDAC were proven to be novel class of antiproliferative
agents.1,2 HDAC inhibition has recently been clinically validated
as a new therapeutic strategy for cancer treatment with the FDA
approval of Suberoyl Anilide Hydroxamic Acid (SAHA) for the treat-
ment of cutaneous T cell lymphoma.3 Small molecules inhibitors of
HDAC that are currently in clinical trials for cancer treatment are
MS-275 and NVP-LAQ824 (Fig. 1).
Over-expression of class-I HDAC in certain cancer cells and the
anticancer property of their inhibitors (Pan-HDAC inhibitors) has
been reported by many groups.4 Recently Pan-HDAC inhibitors
have gained appreciation over isoform specific inhibitors for the
treatment of cancer.5 But high expression of only HDAC8 in neuro-
blastoma tumerogenesis demands selective HDAC8 inhibitors.6 We
have recently reported selective inhibitors of hHDAC8 with piper-
azine linker.7 Here, we are reporting six novel hHDAC8 inhibitors
with piperazine linker.
hydrazinecarbodithioate (3) with different aldehydes and ketones.9
Finally, the S-methyl group of 4a–4f upon displacement by the com-
mon intermediate 2 provided the final compound 5a–5f. The inter-
mediates were characterized by means of elemental analysis (for
CHNS, experimental values are within 0.4% variation with that of
calculated values) and FT-IR spectral and the final compounds were
characterized through 1H NMR and FAB-MS spectral data. The struc-
tureandphysicochemicalandspectralcharacteristicsofsynthesized
molecules were presented in Table 1. Experimental procedures were
presented in Supplementary data.
All the six compounds synthesized were tested for anticancer
activity in in vitro cancer cell-line assay based on cell viability using
the dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT).10 The Microculture Tetrazolium Assay is based on met-
abolic reduction of dye MTT to water insoluble blue formazan crystal
by Mictochondrial dehydrogenase enzyme. The formation of forma-
zan complex is directly proportional to number of viable cells. The
compounds were tested against NCIH460, HCT116 and U251 cell
lines11 and the results are presented in Table 2. Except compound
5a all the other five compounds were having mean IG50 value less
The compounds 5a–5f were synthesized by the reactions out-
lined in the Scheme 1. The common intermediate N-hydroxypiper-
azine-1-carboxamide (2) was synthesized in two step using
modified procedures of Grobner and Steinberg by replacing hydra-
zine with piperazine.8 The Schiff’s bases of methyl hydrazinecarbod-
ithioate (4a–4f) were prepared by the condensation of methyl
than or equal to 40
which is having the best mean GI50 value of 9.33 1.3
ies, that was comparable to SAHA (5.03 M). The 5f and 5d were the
next two potent molecules in this series with 12.03 M and
18.33 6.3 M, respectively. Compounds 5a and 5b with bulkier
lM concentration. Compound 5c was the one
lM in the ser-
l
4
l
l
* Corresponding author. Tel.: +91 6512276247.
E-mail address: venkatesanj@bitmesra.ac.in (V. Jayaprakash).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.020

B. Chetan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3906–3910
3907
Figure 1. HDAC inhibitors in clinical trial. Pharmacophoric features of HDAC inhibitors in the title compounds (in box).
groups in the cap region did not exhibited favorable activity profile.
Experimental procedures were presented in Supplementary data.
Compounds 5c and 5f were selected for in vitro enzyme assay
against human HDAC1 and HDAC8 by means of Microplate HDAC
Fluorometric assay.12,13 The assay utilizes acetylated lysine as sub-
strate, which upon deacetylation by HDAC liberates free lysine.
Free lysine upon treatment with developer (Trypsin) produces a
fluorophore, whose intensity can be measured at excitation wave-
length of 360 nm and wavelength of 460 nm. Both the compounds
(5c and 5f) inactivated hHDAC8 preferentially over hHDAC1 and
the results are presented in Table 2. Compound 5c, potent one
potent in the magnitude of 748 and 959-fold, respectively, but
preferentially inhibited hHDAC8. Experimental procedures were
presented in Supplementary data.
Molecular docking studies were performed using the GLIDE pro-
gram (version 5.0, Schrodinger, LLC, New York, 2008) to understand
the interaction of 5c with hHDAC8. The Maestro user interface (ver-
sion 8.5, Schrodinger, LLC, New York, 2008) was employed to set up
and execute the docking protocol and also for analysis of the docking
results. Validation of docking protocol was done by redocking. Hu-
man HDAC8 bound to SAHA (PDB ID: 1T69) was selected for docking
studies and was prepared for docking through protein preparation
wizard, energy minimization has been carried out using OPLS2001
force field. Structures of 5a–5f were sketched using built panel on
amongst two, inhibited hHDAC8 at IC50 33.67
lM and 5f at
43.16 M. Compared with SAHA, compounds 5c and 5f were less
l
Scheme 1. Reagents and conditions: (a) hydroxylamine HCl, K2CO3, ether/water, 4–6 h stirring; (b) piperazine hexahydrate, ethanol, 12 h, reflux; (c) CS2, KOH, water,
isopropanol, 2–3 h stirring at <10 °C; (d) CH3I, 2–3 h stirring at <10 °C; (e) aromatic aldehyde, methanol, 24 h, reflux; (f) intermediate 2 in MeOH or EtOH, 24 h, reflux;
(g) isatin, methanol, 24 h, reflux.

3908
B. Chetan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3906–3910
Table 1
Physicochemical characteristics of synthesized molecules 5a–5f
Code Structure
MF
MW MP (°C)
Yield
(%)
FT-IR
1H NMR (d/ppm)
FAB-MS
(m/z)
(cmꢀ1
)
3223
(v –OH),
1197
(v C@S),
3026
(v –NH),
1562
OH
NH
7.1–8.32 (m, 4H, Ar-H),
2.18–2.82 (m, 8H, piperazine –
CH–),
8.42 (s, 1H, –CS–NH–),
8.63 (s, 1H, –NH–OH),
9.87 (s, 1H, –NH–OH),
10.12 (s, 1H, Isatin NH)
S
N
N
O
NH
N
5a
C14H16N6O3S
348
220 43.5
349 (M+1)
O
(v C@N),
1562
(v C@O)
3153
(v –OH),
1097
(v C@S),
3001
(v –NH),
1552
(v C@N),
1622
N
H
S
NHOH
O
N
N
7.2–8.7 (m, 9H, Ar-H),
2.25–2.7 (m, 8H, piperazine –
CH–),
9.31 (s, 1H, Ar-CH@),
8.97 (s, 1H, –CS–NH–),
9.69 (s, 1H, –NH–OH),
11.85 (s, 1H, –NH–OH)
N
NH
5b
5c
5d
C21H21N5O2S
407
198–200 61.5
280–282 82.7
150 79
408 (M+1)
(v C@O)
6.9–7.47 (m, 4H, Ar-H),
2.78 (s, 8H, piperazine –CH–),
8.50 (s, 1H, Ar-CH@),
9.23 (s, 1H, –CS–NH–),
10.58 (s, 1H, –NH–OH),
10.67 (s, 1H, –NH–OH),
11.53 (s, 1H, Ar-OH)
S
NHOH
O
OH
N
N
N
N
N
N
NH
3238
(v –OH),
C13H17N5O3S
323
324 (M+1)
3236
S
NHOH
O
(v –OH),
1205 (v
C@S),
3008
(v –NH),
1481
Cl
7.2–7.4 (m, 4H, Ar-H),
2.67 (s, 8H, piperazine –CH–),
8.30 (s, 1H, Ar-CH@),
8.96 (s, 1H, –CS–NH–),
9.13 (s, 1H, –NH–OH),
10.25 (s, 1H, –NH–OH)
NH
341(M+),
342 (M+1),
343 (M+2)
C13H16ClN5O2S 341
(v C@N),
1765
(v C@O)
3296
(v –OH),
1253
(v C@S),
3142
(v –NH),
1473
S
NHOH
O
N
N
7.2–7.8 (m, 4H, Ar-H),
2.67 (s, 8H, piperazine –CH–),
8.29 (s, 1H, Ar-CH@),
8.77 (s, 1H, –CS–NH–),
9.33 (s, 1H, –NH–OH),
10.96 (s, 1H, –NH–OH),
2.39 (s, 3H, –CH3)
N
NH
H3C
5e
C14H19N5O2S
321
170–172 52.3
322 (M+1)
(v C@N),
1624
(v C@O)
3230
(v –OH),
1200
(v C@S),
3026
(v –NH),
1453
S
NHOH
N
N
7.2–7.8 (m, 4H, Ar-H),
2.67 (s, 8H, piperazine –CH–),
8.37 (s, 1H, Ar-CH@),
8.94 (s, 1H, –CS–NH–),
9.33 (s, 1H, –NH–OH),
10.30 (s, 1H, –NH–OH)
N
NH
O
Cl
341(M+),
342 (M+1),
343 (M+2)
5f
C13H16ClN5O2S 341
160 83
(v C@N),
1762
(v C@O)
maestro and prepared for docking through Ligprep module (energy
minimized using MMFF force field). GLIDE grid generation wizard
has been used to define the docking space. Docking was performed
using SP (Standard Precision mode) docking protocol. The molecular
docking results are presented in Table 2. ZBG hydroxamate func-
tional group of all the molecules were found to be close to Zn2+ atom
in the active site, and establishes a hydrogen bond with TYR306,
which shows the major and favorable interaction of the ligands with
hHDAC8 (Fig. 2a). Amongst the six molecules docked, compound 5c
was the one with the best Glide and E model score of ꢀ7.67 and
ꢀ94.5, respectively. It exhibited three hydrogen bonding interaction
with HIS142, GLY206 and TYR306 (Fig. 2b). The hydroxamate group
0
is placed near the Zn2+ atom0 with distance of 2.078 ÅA (hydroxamate
carbonylO to Zn) and 1.846 ÅA(hydroxamate hydroxyl0O toZn) which
is close to the measures of SAHA (2.309 and 2.037 ÅA for crystallo-
0
graphic and 2.078 and 2.025 ÅA for redocked). Docking protocol was
presented in Supplementary data.
Present work once again substantiated the suitability of pipera-
zine linker for designing hHDAC selective inhibitors. Further cap
group modification in this series may provide a potent and selec-
tive inhibitor of hHDAC8. Compared with compound 4c reported
earlier,7 the compound 5c has one atom less between hydroxamate

B. Chetan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3906–3910
3909
Table 2
In-vitro cell line assay, hHDAC8 enzyme assay and molecular docking data of compounds 5a–5f
Code
NCIH460 (Lung)
M)
HCT116 (Colon)
M)
U251 (Glioma) (
l
M) MEAN GI50
HDAC8
M)
Molecular docking
HL60 human promyelocytic
leukemia at 48 h ( M)
(
l
(
l
(l
M)
(
l
l
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
Glide
score
E model
score
5a
5b
5c
5d
5e
>100 >100 >100 >100 >100 >100 >100
>100 >100 >100
>100 >100 40.5 16.5
—
—
33.67
—
—
43.16
0.045
ꢀ7.67
ꢀ7.54
ꢀ7.32
ꢀ7.13
ꢀ7.09
ꢀ6.85
ꢀ4.89
ꢀ94.5
ꢀ90.4
ꢀ91.8
ꢀ83.3
ꢀ83.2
ꢀ83.2
ꢀ83
9.5
5.15
30
51
16
8
>100 >100 70
>100 >100 8.6
>100 >100 11.5
>100 >100 65
>100 >100 14.25 >100 >100 9.33 1.3
0.6
62.5
85
51
>100 13.5
>100 27.5
>100 8.5
80
>100 18.33 6.3
60
>100 31
>100 11.6
>100 0.6
>100 >100 36.5 15.8
5f
SAHA
60
100
40
>100 12.03
>100 5.03
4
100
>100 >100 6.5
*
Mean GI50 acceptable range (SAHA = 0.4–8.5 lM) from published and historical data.
Figure 3a. The distance between two carbonyl groups in compounds 4c (green,
7.471 Å) and 5c (colored by atom type, 7.003 Å) are shown.
Figure 2. (a) Alignment of the molecules 5a–5f in the active site, 5c in tube
representation, hydrogen bonds were shown as yellow broken lines; (b) Interaction
of 5c with active site residues in 1t69.
Figure 3b. Docked conformers of compounds 5c (orange), 4c (green) and SAHA
(colored by atom type) were presented.
carbonyl and thiocarbamoyl group. The distance between two car-
bonyl groups in 4c was 7.471 Å, while in 5c was 7.003 Å (Fig. 3a).
At the same time the absence of methylene group between piper-
azine N1 and acyl carbonyl group orient the cap group differently
in the pocket (Fig. 3b). These factors together has kept thiocarba-
moyl N–H at a distance not favouring hydrogen bonding interac-
tion with ASP101, a favorable interaction shown by many HDAC
inhibitors and 4c.7 This may led to the reduced potency of 5c com-
pared with 4c.
5c may act on other targets too. Due to the presence of hydroxy-
urea and salicylaldehyde Schiff base pharmacophores in 5c
(Fig. 3), we suspect that these molecules may also inhibit Ribonu-
cleotide reductase (RR) (Fig. 4). On this background, the com-
pounds 5c was screened against HL60 human promyelocytic
leukemia cell line (ATCC, Manassas, VA, USA), in which over-
expression of RR has been reported. HL60 cells incubated with
increasing concentrations of 5c at 37 °C, Cell counts and IC50 values
were determined after 24, 48, and 72 h using the microcell counter
Also it was observed, higher concentration of 5c was required to
inhibit hHDAC8 (IC50 = 33.67
lM) than that required to inhibit can-
cer cell lines (GI50 = 9.33 1.3
l
M). This suggested that compound

3910
B. Chetan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3906–3910
tral data and the first author was thankful to UGC for Junior
Research Fellowship.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.020.
References and notes
1. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505.
2. Park, J. H.; Jung, Y.; Kim, T. Y.; Kim, S. G.; Jong, H. S.; Lee, J. W.; Kim, D. K.; Lee, J.
S.; Kim, N. K.; Kim, T. Y.; Bang, Y. J. Clin. Cancer Res. 2004, 10, 5271.
3. Duvic, M.; Vu, J. Expert Opin. Invest. Drugs 2007, 16, 1111.
4. Nakagawa, M.; Oda, Y.; Eguchi, T.; Aishima, S.; Yao, T.; Hosoi, F.; Basaki, Y.; Ono,
M.; Kuwano, M.; Tanaka, M.; Tsuneyoshi, M. Oncol. Rep. 2007, 18, 769.
5. Witt, O.; Lindemann, R. Cancer Lett. 2009, 280, 123.
Figure 4. Pharmacophoric features of ribonucleotide reductase inhibitors in 5c. HU:
Hydroxyurea.
6. Oehme, I.; Deubzer, H. E.; Wegener, D.; Pickert, D.; Linke, J. P.; Hero, B.;
Schneider, A. K.; Westermann, F.; Ulrich, S. M.; Deimling, A. V.; Fischer, M.;
Witt, O. Clin. Cancer Res. 2009, 15, 91.
CC-108. Viability of cells was determined by trypan blue exclusion.
7. Chakrabarty, S.; Jasmine; Bhadaliya, C.; Sinha, B. N.; Mahesh, A.; Bai, H.; Blond,
S. Y.; Venkatesan, J. Arch. Pharm. 2010, 343, 167.
8. Ren, S.; Wang, R.; Komatsu, K.; Bonaz-Krause, P.; Zyrianov, Y.; McKenna, C. E.;
Csipke, C.; Tokes, Z. A.; Lien, E. J. J. Med. Chem. 2002, 45, 410.
9. Klayman, D. L.; Bartosevich, J. F.; Griffin, T. S.; Mason, C. J.; Scovill, J. P. J. Med.
Chem. 1979, 22, 855.
Results were calculated as number of viable cells. Compound 5c
was found to have IC50 of 0.6
parable to that of compound 311 having IC50 0.4
l
M at 48 h (Table 2), that was com-
l
M at 48 h.14 This
fact suggests the possible inhibition of Ribonucleotide reductase as
suspected (experimental procedures were presented in Supple-
mentary data). Compound 5c requires further investigation in this
regard for its Ribonucleotide Reductase inhibitory activity that
may provide a novel dual inhibitor of HDAC/RR type.
10. Mosmann, T. J. Immuno. Methods 1983, 65, 55.
11. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose,
C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gray-Goodrich, M.; Campbell, H.;
Mayo, J.; Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757.
12. Wegener, D.; Hildmann, C.; Riester, D.; Schwienhorst, A. Anal. Biochem. 2003,
321, 202.
Acknowledgments
13. Wegener, D.; Wirsching, F.; Riester, D.; Schwienhorst, A. Chem. Biol. 2003, 10,
61.
14. Richardson, D. R.; Milnes, K. Blood 1997, 89, 3025.
The author was grateful to the Head, Sophisticated Analytical
Instrument Facility (SAIF), CDRI Lucknow, India, for providing spec-