Modification of cap group in δ-lactam-based histone deacetylase (HDAC) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.09.034

Novel δ-lactam-based HDAC inhibitors which have various substituted benzyl, bi-aromatic cap groups were prepared using ring closure metathesis reaction, and evaluated their HDAC inhibitory activities and anti-proliferative effects. Among prepared analogue

Full text of DOI:10.1016/j.bmcl.2007.09.034

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6234–6238
Modification of cap group in d-lactam-based histone
deacetylase (HDAC) inhibitors
Hwan Mook Kim,a, Sung Hee Hong,b, Myung Sook Kim,b Chang Woo Lee,a
Jong Soon Kang,a Kiho Lee,a Song-Kyu Park,a Jeung Whan Han,c Hee Yoon Lee,d
Yongseok Choi,e Ho Jeung Kwonb and Gyoonhee Hanb,*
aKorea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305-333, Republic of Korea
bDepartment of Biotechnology, Yonsei University, Seodaemun-gu, Seoul 120-740, Republic of Korea
cCollege of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
dDepartment of Chemistry, Korea Advanced Institute of Science and Technology, Yuseong, Daejeon 305-701, Republic of Korea
eSchool of Biosciences and Biotechnology, Korea University, Seongbuk-gu, Seoul 136-713, Republic of Korea
Received 4 July 2007; revised 3 September 2007; accepted 5 September 2007
Available online 11 September 2007
Abstract—Novel d-lactam-based HDAC inhibitors which have various substituted benzyl, bi-aromatic cap groups were prepared
using ring closure metathesis reaction, and evaluated their HDAC inhibitory activities and anti-proliferative effects. Among pre-
pared analogues, 11m and 11o have very strong HDAC enzymatic inhibition and showed the most potent growth inhibitory activity
to five human tumor cell lines including PC-3, ACHN, NUGC-3, HCT-15, and MBA-MB-231 tumor cell lines. Compounds 11m
and 11o also showed good tumor growth inhibition of MDA-MB-231 cells in in vivo xenograft model. Structure–activity relation-
ship study using docking model explained the significance of hydrophobic aromatic cap groups for their in vitro activities.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Histone deacetylase (HDAC) and histone acetyltransfer-
ase (HAT) are involved in chromatin remodeling and epi-
genetic regulation of genes.1 HDAC catalyzes
deacetylation of e-amino group in lysines located near
the N-terminal of core histone proteins. Abnormal
recruitment of HDAC is related to carcinogenesis,2 and
the control of HDAC enzymes has been considered as a
very intriguing target for anti-cancer chemotherapy.3 A
number of natural and synthetic HDAC inhibitors have
been reported (Fig. 1); trichostatin A (TSA, 1),4 apicidin
(4),5 trapoxin B (TPX, 5),6 and FK-228.7 And suberoy-
lanilide hydroxamic acid (SAHA, 2)8 was approved for
the treatment of cutaneous T-cell lymphoma (CTCL).
d-lactam ring in a linker domain between surface recog-
nition cap group and zinc binding hydroxamate region.9
We found that the compounds with two methylene car-
bon units between zinc binding hydroxamates and d-lac-
tam core showed potent HDAC inhibitory activity but
others showed no activity at all. And the length between
d-lactam core and hydrophobic cap groups is tolerable
to the inhibitory activities.
Herein, we report the results of the cap group modifica-
tion approaches of d-lactam-based HDAC inhibitors;
synthesis, HDAC inhibition, and in vitro and in vivo
cancer cell growth inhibition.10
We reported that design, synthesis, and biological eval-
uation of novel d-lactam core HDAC inhibitors
(5, Fig. 2), which incorporate structural features of
Scheme 1 outlines the preparation of d-lactam ana-
logues.9 Commercial amines 6 were alkylated with
4-bromo-1-butene in SN2 manner and resulted in sec-
ondary amines 7. Compound 7 were reacted with
monoacid 8 to give amides 9. Upon treatment of cat-
alytic 2–3 mol % of Grubb’s catalyst (I), unsaturated
cyclic d-lactams 10 were obtained. These methyl
esters of 10 were converted to hydroxamic acids 11
with KONH2 in MeOH, which have various cap
groups on d-lactam nitrogen.
Keywords: Histone deacetylase; HDAC; Anticancer chemotherapy;
Enzyme inhibitor; Growth inhibition; In vivo xenograft model; Doc-
king model.
*
Corresponding author. Tel.: +82 2 2123 2882; fax: +82 2 362 7265;
e-mail: gyoonhee@yonsei.ac.kr
These authors contributed equally to this work.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.034

H. M. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6234–6238
6235
O
O
O
H
N
OH
OH
N
N
H
H
O
N
Trichostatin A (1)
SAHA (2)
O
O
O
O
O
NH
H
N
N
NH
H
N
N
O
O
H
H
H
H
N
N
O
O
O
O
Apicidin (3)
Trapoxin B (4)
N
OMe
Figure 1. The natural and synthetic HDAC inhibitors.
esters of 13 were converted to hydroxamates 14a, 14b,
and 14c, respectively, in the same conditions as
Scheme 1.
δ-Lactam
H
N
N
HO
n
Cap group
Zinc-binding
Hydroxamate
O
O
We have evaluated the HDAC inhibitory activities of
the newly prepared analogues on partially purified
HDAC enzyme obtained from HeLa cell lysate
(Table 1).11 Among prepared compounds, the substi-
tuted benzyl analogues 11a–i (n = 1 in Scheme 1), meth-
oxy-substituted analogues (11d–f) are more active than
methyl analogues (11a–c) in the HDAC enzyme assay.
For the position of benzyl group, 4-substituted methoxy
benzyl analogue (11f) showed the best HDAC inhibitory
activity at 0.44 lM of IC50 value among these ana-
logues. 4-Fluoro analogue (11g) and 4-bromo analogue
(11h) showed the same range of activities to 4-methoxy
analogue (11f), and 4-nitro analogue (11i) is better in
inhibitory activities. Surprisingly, 2-naphthyl analogue
(11j) did not show any activity while its chain elongated
analogues (n = 2, 11m; n = 3, 11o)12 showed very strong
inhibitory activity at 37 and 30 nM of IC50, respectively.
1-Naphthyl analogues (11l, 11q) are less active than
2-naphthyl analogues. 4-Biphenyl analogues (11k, 11n,
and 11p) also showed less active inhibitory activities at
0.1–0.2 lM of IC50s. 4-N-Acyl and sulfonylated aryl
analogues (14a–c) showed moderate inhibitory activities
ranging from 0.5 to 0.8 lM of IC50. Among these pre-
pared analogues, 2-naphthyl analogues (11m and 11o)
are the best compounds in HDAC inhibition assay.
δ-Lactam base HDAC inhibitor (5)
Figure 2. Structural features of d-lactam-based HDAC inhibitor.
H2N
n
a
R
N
n
OMe
HO
R
H
+
O
O
6
7
8
O
n
N
b
c
R
MeO
O
9
H
N
d
N
MeO
N
HO
n
n
O
O
O
O
R
R
10
11a n=1, R=
11b n=1, R=
11c n=1, R=
11d n=1, R=
11e n=1, R=
11f n=1, R=
o
m
p
o
m
p
p
p
-CH3; 11i n=1, R=
-CH3; 11j n=1, 2-naphthyl
-CH3; 11k n=2, R= p
p
-NO2
-Ph
Growth inhibitory activities of all the active HDAC
inhibitors (11a–q and 14a–c) were evaluated on five
human tumor cell lines using PC-3 (prostate cancer),
ACHN (renal cancer), NUGC-3 (gastric cancer),
HCT-15 (colon cancer), and MDA-MB-231 (breast can-
cer) cell lines. Cell growth inhibition of the tested ana-
logues was measured by SRB assay13 and the obtained
GI50 values of the types of tumor cell lines compared
with SAHA are summarized in Table 1. Overall growth
inhibitory activities were related to their HDAC inhibi-
tory activities. The most potent analogues 11m and 11o
exhibited very strong growth inhibitory activities to five
human tumor cell lines at 0.28–1.18 lM. For the ana-
logues with comparable HDAC inhibitory activity, 11i,
11n, and 11q showed very comparable growth inhibitory
-OCH3; 11l n=2, 1-naphthyl
-OCH3; 11m n=2, 2-naphthyl
-OCH3; 11n n=3, R= -Ph
11o n=3, 2-naphthyl
11p n=4, R= -Ph
11q n=4, 1-naphthyl
p
11g n=1, R=
11h n=1, R=
-F;
-Br;
p
Scheme 1. Reagents: (a) 4-bromo-1-butene, Hunig base, reflux; (b)
EDC, DMAP; (c) Grubb’s catalyst (I); (d) KONH2.
4-N-acyl and sulfonylated aryl analogues were prepared
from 4-nitro compound 10i (Scheme 2). Compound 10i
was reduced to amino compound 12 with zinc and 12
was acylated with acyl chloride or tosyl chloride to give
amide 13a and 13b or sulfonamide 13c. The methyl

6236
H. M. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6234–6238
NH2
b
NO2
MeO
N
a
MeO
N
O
O
O
O
12
10i
H
H
N
N
R
R
H
N
N
MeO
N
c
HO
O
O
O
O
14a R= COCH3 (Ac)
13a R= COCH3 (Ac)
14b R= COPh (Bz)
14c R= SO2tol (Ts)
13b R= COPh (Bz)
13c R= SO2tol (Ts)
Scheme 2. Reagents: (a) Zn, AcOH–MeOH, reflux; (b) RCOCl, TEA, DMAP; (c) KONH2.
Table 1. HDAC enzyme and growth inhibition by d-lactam analogues and SAHA (2)a
Compound
IC50 (lM)a HDAC
GI50 (lM)a
PC-3
MDA-MB-231
NUGC-3
HCT-15
ACHN
11a
11b
11c
11d
11e
11f
11g
1h
2.97
1.90
2.10
0.86
1.94
0.44
0.56
0.45
0.23
NA
NA
NA
8.10
NA
NA
7.08
NT
4.92
8.12
1.43
NA
3.92
3.86
NT
NT
NT
NT
NT
NT
NT
NT
NT
1.11
NT
1.65
3.26
0.37
3.70
0.79
4.20
2.01
NT
NA
NT
2.79
NA
NA
8.51
NA
9.03
6.89
9.96
1.72
NA
NT
NA
4.38
5.00
NA
7.60
1.67
NT
3.15
3.01
NT
1.63
0.47
NT
2.48
5.21
NT
11i
11j
11k
11l
11m
11n
11o
11p
11q
14a
14b
14c
2
0.23
0.32
0.037
0.27
0.030
0.20
0.11
0.82
0.51
0.59
0.11
0.53
2.58
0.61
3.23
0.30
2.95
1.69
NA
NA
1.80
2.69
0.28
2.53
0.28
1.04
0.56
1.28
1.47
NT
1.00
5.43
1.16
3.19
0.53
4.56
1.67
NT
1.18
6.62
1.18
2.49
0.46
2.67
1.82
NT
6.03
0.56
2.00
NA
NA
2.49
NA
0.37
2.22
NA, GI50 > 10 lM; NT, not tested.
a Values are means of a minimum of three independent experiments.
activities to SAHA but 11k showed very strong anti-pro-
liferative activity comparable to the most potent ana-
logues 11m and 11o. The analogues which have
moderate HDAC inhibitory activity (over 0.5 lM of
IC50) showed poor growth inhibitory activity to five hu-
man tumor cell lines, while some compounds were active
on the PC-3 and MDA-MB-231 cell lines. Thus, these
new analogues showed better sensitivity on the MDA-
MB-231 breast tumor cell line and PC-3 prostate tumor
cell line.
the group without treatment. Thus, both compound
11m and SAHA displayed anti-tumor growth activities
and 11m appeared to be better than SAHA in inhibition
of the growth of tumor.
In order to understand the binding mode of these inhib-
itors, we examined a docking model of human HDAC-1
catalytic core based on the crystal structure of a bacte-
rial HDAC homologue (HDLP, PDB code 1C3R) and
docked compound 11m within the site using the pro-
gram Discover (Fig. 3).15 The hydroxamic acid moiety
of 11m chelates to the catalytic Zn2+ ion bound in the
active site and the d-lactam ring was bounded in the
tubular hydrophobic pocket. Furthermore, longer
2-naphthyl hydrophobic cap group gives the extra sta-
bilization energy by hydrophobic interaction. Although
HDLP has a large deletion at entrance to the active site,
the docking data suggest that the chain length between
hydrophobic cap group and d-lactam ring and the size
The most promising inhibitor 11m was selected to fur-
ther evaluate in vivo tumor growth inhibitory activity
and the results are summarized in Table 2.14 In this
model, compound 11m and SAHA were administered
daily (ip, 30 mg/kg) to nude mice after the sizes of
MDA-MB-231 human breast xenografts reached 50–
60 mm3. Compound 11m and SAHA inhibited 51%
and 39% in tumor growth, respectively, compared to

H. M. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6234–6238
6237
Table 2. Inhibition of the growth of the MDA-MB-231 human breast
tumor xenograft by compound 11m and SAHA (2)a
2. (a) Kouzarides, T. Curr. Opin. Genet. Dev. 1999, 9, 40; (b)
Archer, S. Y.; Hodin, R. A. Curr. Opin. Genet. Dev. 1999,
9, 171.
Ipb
Compound
3. (a) Saunders, N. A.; Popa, C.; Serewko, M. M.; Jones, S.
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4. Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. J. Biol.
Chem. 1990, 265, 17174.
11m
2
Dose (mg/kg)
% Inhibition
% Body weight to vehicle
30
30
51*
94
39**
100
5. Han, J. W.; Ahn, S. H.; Park, S. H.; Wang, S. Y.; Bae, G.
U.; Seo, D. W.; Kwon, H. K.; Hong, S.; Lee, H. Y.; Lee,
Y. W.; Lee, H. W. Cancer Res. 2000, 60, 6068.
6. Kijima, M.; Yoshida, M.; Sugita, K.; Horinouchi, S.;
Beppu, T. J. Biol. Chem. 1993, 268, 22429.
7. Ueda, H.; Manda, T.; Matsumoto, S.; Mukumoto, S.;
Nishigaki, F.; Kawamura, I.; Shimomura, K. J. Antibiot.
1994, 47, 315.
a Nude mice (six per group) were treated.
b 11m and SAHA (2) were administered (ip) after the size of tumors
reached to 50–60 mm3.
* P < 0.05.
** P < 0.01.
8. Butler, L. M.; Agus, D. B.; Scher, H. I.; Higgins, B.; Rose,
A.; Cordon-Cardo, C.; Thaler, H. T.; Rifkind, R. A.;
Marks, P. A.; Richon, V. M. Cancer Res. 2000, 60, 5165.
9. (a) Kim, H. M.; Lee, K.; Park, B. W.; Ryu, D. K.; Kim,
K.; Lee, C. W.; Park, S.-K.; Han, J. W.; Lee, H. Y.; Han,
G. Bioorg. Med. Chem. Lett. 2006, 16, 4068; (b) Kim, H.
M.; Ryu, D. K.; Choi, Y.; Park, B. W.; Lee, K.; Han, S.
B.; Lee, C.-W.; Kang, M.-R.; Kang, J. S.; Boovanahalli, S.
K.; Park, S.-K.; Han, J. W.; Chun, T.-G.; Lee, H.-Y.;
Nam, K.-Y.; Choi, E. H.; Han, G. J. Med. Chem. 2007, 50,
2737.
10. For QSAR study of various cap groups, see: Juvale, D.
C.; Kulkarni, V. V.; Deokar, H. S.; Wash, N. K.;
Padhye, S. B.; Kulkarrni, V. M. Org. Biomol. Chem
2006, 4, 2858.
11. HDAC inhibition assay: HDAC fluorescent activity
assays using a Fluror de LysTM Substrate (Biomol,
Plymouth Meeting, PA), which contains an acetylated
lysine side chain, were performed according to manu-
facturer’s instructions. In brief, HeLa nuclear extracts,
which were used as an HDAC enzyme source, were
incubated at 25 ꢁC with 250 mM of Fluror de LysTM
Substrate and various concentrations of each sample.
Reactions were stopped after 20 min with Fluror de
LysTM Developer and fluorescence was measured
using a microplate spectrofluorometer (LS 50B, Per-
kin-Elmer) with excitation at 360 nm and emission at
460 nm.
12. N-hydroxy-3-(1-(2-(naphthalen-2-yl)ethyl)-2-oxo-1,2,5,6-
tetrahydropyridin-3-yl)propanamide (11m). 1H NMR
(CDCl3) d 7.72 (br, 3H), 7.59 (s, 1H, 7.26–7.38 (br, 3H),
6.27 (br, 1H), 3.60 (br, 2H), 2.41–3.04 (m, 8H), 1.98 (br,
2H); 13C NMR (CDCl3) d 170.14, 164.92, 136.35, 135.57,
133.19, 133.13, 131.82, 127.77, 127.28, 127.16, 127.06,
126.84, 125.71, 125.08, 77.42, 77.19, 77.00, 76.57, 49.06,
46.04, 33.95, 32.25, 26.94, 23.30; ESI (m/z) 338 (M+) 361
(M+Na+).
Figure 3. The docked orientations for compounds 11m bound to the
HDAC1 catalytic core of HDLP.
of cap group are quite important to form the flexible
conformation in binding pocket.
Collectively, we have prepared novel d-lactam-based
HDAC inhibitors which have various substituted
benzyl, bi-aromatic cap groups with 1–4 carbon length
between d-lactam and cap group, and evaluated their
HDAC inhibitory activities and anti-proliferative
effects. The 2-naphthyl analogues (11m and 11o) have
very strong enzymatic and showed the most potent
growth inhibitory activity to five human tumor cell lines.
Further structure–activity relationship study using
docking model explained the significance of hydropho-
bic aromatic cap groups for their in vitro activities.
3-(1-(3-(1-Bromonaphthalen-2-yl)propyl)-2-oxo-1,2,5,6-
tetrahydropyridin-3-yl)-N-hydroxypropanamide (11o). 1H
NMR (CDCl3)d 7.73 (br, 3H), 7.57–7.59(d, 1H,
J = 6.0 Hz), 7.38 (br, 2H), 7.24–7.31 (m, 1H), 6.27 (br,
1H), 3.38–3.43 (br, 2H), 3.20–3.25 (br, 2H), 2.14–2.74 (m,
8H), 1.88 (br, 2H); 13C NMR (CDCl3) d 170.29, 165.31,
139.034, 135.83, 133.51, 133.43, 131.94, 127.89, 127.51,
127.36, 127.10, 126.27, 125.87 125.12, 46.902, 45.45, 33.28,
32.76, 29.07, 27.23, 23.69; ESI (m/z) 353 (M+) 375
(M+Na+).
Acknowledgments
This research was partly supported by a grant from
KRIBB Research Initiative program, a Grant (0405-
NS01-0704-0001) of the Korean Health 21 R&D Pro-
ject, Ministry of Health and Welfare, and the Brain
Korea 21 project the Republic of Korea.
13. (a) Papazisis, K. T.; Geromichalos, G. D.; Dimitriadis, K.
A.; Kortsaris, A. H. J. Immunol. Methods 1997, 208, 151;
(b) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.;
McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.;
Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
107.
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H. M. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6234–6238
14. In vivo tumor growth inhibition experiment. An equal
volume of the MDA-MB-231 human breast cancer cells
was injected sc in the right flank of each BALB/c nude
mouse. When the size of the tumors reached 50–60 mm3,
compounds were intraperitoneally administered daily to
the nude mice for 15 days. Tumor length and width
were periodically measured until the end of the exper-
iment and tumor volume was calculated using the
following formula: Tumor volume = Length · (width)2 ·
p/6.
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