Synthesis, enzymatic inhibition, and cancer cell growth inhibition of novel δ-lactam-based histone deacetylase (HDAC) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.04.091

δ-Lactam-based hydroxamic acids, inhibitors of histone deacetylase (HDAC), have been synthesized via ring closure metathesis of key diene intermediates followed by conversion to hydroxamic acid analogues. The hydroxamic acids 12a, 12b, and 17c showed potent inhibitory activity in HDAC enzyme assay. The hydroxamic acid 12b exhibited growth inhibitory activity on five human tumor cell lines, showing good sensitivity on the MDA-MB-231 breast tumor cell.

Full text of DOI:10.1016/j.bmcl.2006.04.091

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4068–4070
Synthesis, enzymatic inhibition, and cancer cell growth inhibition
of novel d-lactam-based histone deacetylase (HDAC) inhibitors
Hwan Mook Kim,a, Kiho Lee,a, Bum Woo Park,a Dong Kyu Ryu,a Kangjeon Kim,a
Chang Woo Lee,a Song-Kyu Park,a,* Jung Whan Han,b Hee Yoon Lee,c
Hyun Yong Leed and Gyoonhee Hand,*
aKorea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305-333, Republic of Korea
bCollege of Pharmacy, Sung Kyun Kwan University, Suwon 440-746, Republic of Korea
cDepartment of Chemistry, Korea Advanced Institute of Science and Technology, Yuseong, Daejeon 305-701, Republic of Korea
dDepartment of Biotechnology, Yonsei University, Seodaemun-gu, Seoul 120-740, Republic of Korea
Received 23 March 2006; revised 20 April 2006; accepted 28 April 2006
Available online 24 May 2006
Abstract—d-Lactam-based hydroxamic acids, inhibitors of histone deacetylase (HDAC), have been synthesized via ring closure
metathesis of key diene intermediates followed by conversion to hydroxamic acid analogues. The hydroxamic acids 12a, 12b,
and 17c showed potent inhibitory activity in HDAC enzyme assay. The hydroxamic acid 12b exhibited growth inhibitory activity
on five human tumor cell lines, showing good sensitivity on the MDA-MB-231 breast tumor cell.
Ó 2006 Elsevier Ltd. All rights reserved.
O
O
O
Histone deacetylase (HDAC) and histone acetyltransfer-
ase (HAT) are involved in the co-regulation of chroma-
tin remodeling and the functional regulation of gene
transcription.1 HDAC catalyzes deacetylation of e-ami-
no group in lysines located near the N-terminal of core
histone proteins. Abnormal recruitment of HDAC is
related to carcinogenesis.2 Thus, the identification of po-
tent HDAC inhibitors has been considered as a very
intriguing approach for the development of cancer che-
motherapy.3 A number of natural and synthetic HDAC
inhibitors have shown an anti-proliferative activity on
tumor cells. Among them, trichostatin A (TSA, 1),4
apicidin (4),5 trapoxin B (TPX, 5),6 and FK-2287 were
classified as natural substances, and suberoylanilide
hydroxamic acid (SAHA, 2)8 and other TSA or
SAHA-like analogues were reported as synthetic HDAC
inhibitors.9
O
O
H
N
OH HO
OH
OH
N
H
N
H
N
H
N
H
O
N
Trichostatin A (1)
SAHA (2)
CBHA (3)
O
O
O
O
O
HN
NHHN
O
HN
O
NHHN
O
O
O
O
Apicidin (4)
Trapoxin B (5)
N
OMe
Especially, CBHA (3) with the aromatic linker demon-
strated toleration of the aromatic ring and the importance
of the proper orientation of the linker.10 Based on the
structures of the analogues 1–3, we designed and synthe-
sized novel HDAC inhibitors that incorporate structural
features of d-lactam ring as a linker domain, which is
expected to endow the inhibitors with the control of the
dihedral angle between surface recognition and metal
binding region, and the decreased entropy penalty due
to the flexible alkyl chain linker. Herein, we describe our
results on the synthesis, enzyme inhibition, and cancer cell
growth inhibition of novel d-lactam-based HDAC
inhibitors.
Intensive efforts have recently been focused on the mod-
ifications of the aromatic ring moiety and the aliphatic
linker region present in the molecules.
Keywords: d-Lactam; Ring closure metathesis; Histone deacetylase;
Anticancer.
*
Corresponding authors. Tel.: +82 42 860 4689; fax: +82 42 860 4605
(S-K.P.), tel.: +82 2 2123 2882; fax: +82 2 362 7265 (G.H.); e-mail
addresses: spark123@kribb.re.kr; gyoonhee@yonsei.ac.kr
These authors contributed equally to this work.
Schemes 1–3 outline the preparation of d-lactam ana-
logues.11 Diester 6 was prepared by literature proce-
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.091

H. M. Kim et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4068–4070
4069
For N-alkyl analogues, 2,4-dimethoxybenzylamine was
alkylated with 4-bromo-1-butene, followed by coupling
with 7 to give amide 13 (Scheme 3). Compound 13
was then converted to d-lactam 14 in the presence of
Grubb’s catalyst (I). 2,4-Dimethoxybenzyl (DMB)
group was removed in acidic condition and the interme-
diate 15 was methylated with KHMDS/MeI (16a) or
allylated with KHMDS/allyl bromide (16b). Esters
16a, 16b, and 14 were converted to hydroxamates 17a,
17b, and 17c,14 respectively, in the same conditions as
Scheme 2.
a
b
O
O
HO
O
O
O
O
O
6
7
Scheme 1. Reagents: (a) LiOH, THF–H2O; (b) CSA, MeOH.
a
HO
+
O
H2N
HN
O
O
n
n
9a n = 1
9b n = 2
7
We have evaluated the HDAC inhibitory activities of
the newly prepared d-lactam analogues on partially
purified HDAC enzyme obtained from HeLa cell lysate
and their anti-proliferative effects using PC-3 cells as
well.15
8a n = 1
8b n = 2
b
O
O
O
OH
N
OMe
OMe
H
d
c
N
O
N
O
Among propionic hydroxamates, compounds 12a, 12b,
17b, and 17c were active in the HDAC enzyme assay.
The activity of 12b with two carbon extended phenyl
group was two times more than that of 12a with one car-
bon extended phenyl group. Compounds 17a–c with
methyl, allyl, and DMB groups for their surface recogni-
tion exhibited a molecular size-dependent activity
against HDAC: 17a with a methyl group was not active
and 17b with an allyl group showed a moderate activity,
whereas 17c with an aromatic substituent DMB group
showed a potent inhibitory activity against HDAC.
These analogues showed some growth inhibitory activi-
ties on the PC-3 cell line (Table 1).
N
O
n
n
n
10a n = 1
10b n = 2
12a n = 1
12b n = 2
11a n = 1
11b n = 2
Scheme 2. Reagents and condition: (a) 8a ! 9a: 4-bromo-1-butene,
hunig base, reflux; (b) EDC, DMAP; (c) Grubb’s catalyst (I); (d)
KONH2.
O
O
OMe
OMe
MeO
H2N
OMe
a
b
c
N
O
N
O
Growth inhibitory activities of all the active HDAC
inhibitors (12a, 12b, and 17c) were evaluated in 6 human
tumor cell lines. Growth inhibition (GI50) measured by
SRB assay16 of these 3 HDAC inhibitors and the tumor
cell line types are listed in Table 2. With a similar pat-
tern to the enzyme inhibition, 12b exhibited growth
inhibitory activity on five human tumor cell lines, while
12a was active only on the MDA-MB-231 cell line. 17c
with a DMB group exhibited similar inhibitory activity
to that of 12b. In addition, 12b and 17c showed more
sensitive inhibitory activity on the MDA-MB-231 breast
tumor cell line.
MeO
OMe
MeO
OMe
13
14
f
d
O
O
O
OH
N
OMe
OMe
H
f
e
N
R
O
N
R
O
N
O
H
15
17a R = Me
17b R = Allyl
17c R = DMB
16a R = Me
16b R = Allyl
We have efficiently prepared novel d-lactam-based
HDAC inhibitors using a ring closure metathesis and
evaluated their HDAC inhibitory activities and
Scheme 3. Reagents and condition: (a) 4-bromo-1-butene, hunig base,
reflux; (b) 7, EDC, DMAP; (c) Grubb’s catalyst (I); (d) TFA, reflux; (e)
KHMDS, MeI or allyl bromide; (f) KONH2.
Table 1. HDAC enzyme and growth inhibition by d-lactam analogues
and SAHA (2)a
dures.12a,12b Hydrolysis by treatment of LiOH followed
by mono-esterification with camphor sulfonic acid
(CSA) gave monoacid 7 (Scheme 1).
Compound
IC50 (lM)
GI50 (lM)
PC-3
Enzymea
12a
12b
0.67
0.35
NA
4.27
0.50
0.11
NA
8.58
NT
Commercial amines 8a and 8b were alkylated with
4-bromo-1-butene and the resulted 9a and 9b were
reacted with monoacid 7 to give amides 10a and 10b,
respectively (Scheme 2). Upon treatment of catalytic
2–3 mol % of Grubb’s catalyst (I),13 unsaturated
d-lactams 11a and 11b were obtained and these methyl
esters were converted to hydroxamic acids 12a and
12b14 with KONH2 in MeOH, respectively.
17a
17b
NA
4.55
0.89
17c
SAHA (2)
NA, not active.
NT, not tested.
a Values are means of a minimum of three experiments.

4070
H. M. Kim et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4068–4070
Table 2. GI50 and tissue type for cells treated with 12a, 12b, and 17ca
added via cannular at room temperature under Ar
atmosphere. EDC (1.3 equiv) and DMAP (0.3 equiv) were
added and the mixture was stirred for 8 h at room
temperature. After usual work-ups, 10a, obtained by flash
chromatography, was dissolved in methylene chloride
(0.01 M) and the mixture was gassed by Ar bubbling for
30 min. Grubb’s catalyst (Type I, 0.02 equiv) was added to
the reaction mixture and stirred for 12 h in dark. The
mixture was concentrated in vacuo and 11a was obtained
by flash chromatography. Compound 11a was dissolved in
methanol (0.5 M solution) and then NH2OK (1.7 M
suspension in methanol, 5.0 equiv) was added at 0°C.
The mixture was stirred for 20 h at room temperature.
10% HCl was added to pH 2–3 and the mixture was
concentrated in vacuo. White solid formed was removed
by filtration, eluting with methanol/chloroform (1:9).
Compound 12a was obtained by flash chromatography.
Compounds 12b and 17c were obtained following similar
methods, respectively.
Cell line
Tissue
Colon
Growth inhibitionb (lM)
12b 17c SAHA
8.16 7.98 0.82
12a
HCT-15
LOX-IMVI
PC-3
>10
Melanoma >10
5.84
8.58
7.76
1.62
6.63 1.39
4.55 0.89
9.58 0.73
2.67 0.66
Prostate
Kidney
>10
>10
ACHN
MDA-MB-231 Breast
NCI-H23 Lung
4.26
>10
>10
>10
0.92
a Values are means of a minimum of three experiments.
b Growth inhibition was measured by SRB (sulforhodamine B) assay.
anti-proliferative effects. The propionic hydroxamic
acids (12b and 17c) have good enzyme inhibitory and
cell growth inhibitory activities and showed the most
potent growth inhibitory activity to MDA-MB-231
among 6 human tumor cell lines. Further structure–
activity relationships of zinc binder and chain unit size
from hydroxamic acid or hydrophobic aromatic group
to the core d-lactam will be reported in due course.
12. (a) Kagan, J.; Tolentino, L.; Ettlinger, M. C. J. Org.
Chem. 1975, 40, 3085; (b) Samarat, A.; Fargeas, V.;
Villieras, J.; Lebreton, J.; Amri, H. Tetrahedron Lett.
2001, 42, 1273.
13. (a) Fu, G. C.; Grubbs, R. H. J. Am. Chem. Soc. 1992, 114,
7324; (b) Fu, G. C.; Nguyen, S. T.; Grubbs, R. H. J. Am.
Chem. Soc. 1993, 115, 9856; (c) Huwe, C. M.; Kiehl, O. C.;
Blechert, S. Synlett 1996, 65; (d) Rutjes, F. P.; Schoe-
maker, H. E. Tetrahedron Lett. 1997, 38, 677; (e) Miller, S.
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Acknowledgments
This research was supported by a Grant (0405-
NS01-0704-0001) of the Korean Health 21 R&D Project,
Ministry of Health and Wellfare, and the Ministry of
Commerce, Industry and Energy, the Republic of Korea.
14. N-Hydroxy-3-(2-oxo-1-phenethyl-1,2,5,6-tetrahydro-pyri-
din-3-yl)- propionamide (12b): 1H NMR (CDCl3)
d
7.29–7.18 (m, 5H), 6.40 (br t, 1H), 3.62 (t,
J = 7.2 Hz, 2H), 3.19 (t, J = 7.1 Hz, 2H), 2.85 (t,
J = 7.1 Hz, 2H), 2.54–2.44 (m, 2H), 2.18–2.15 (m,
References and notes
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d 169.72, 165.29, 138.95,
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136.14, 133.66, 128.89, 128.51, 126.45, 49.29, 46.34,
34.17, 23.70; ESI (m/z) 289.1 (MH+); HRMS (m/z)
(MH+) calcd for C16H20N2O3 289.1539; found
289.1547.
tetrahydro-pyridin-3-yl]-N-
3-[1-(2,4-Dimethoxy-benzyl)-2-oxo-1,2,5,6-
hydroxy-propionamide
(17c): 1H NMR (CDCl3) d 7.12 (d, J = 9.0 Hz, 1H),
6.43–6.33 (m, 3H), 4.51 (s, 2H), 3.75 (s, 3H), 3.74 (s,
3H), 3.27 (t, J = 6.9 Hz, 2H), 2.55 (m, 2H), 2.38 (m,
2H), 2.22 (m, 2H); 13C NMR (CDCl3) d 170.1, 165.4,
160.2, 158.5, 135.8, 133.5, 130.4, 117.5, 104.2, 98.3,
55.3, 44.9, 44.6, 32.8, 27.1, 23.8; ESI (m/z) 357.5
(MNa+); HRMS (m/z) (MH+) calcd for C17H22N2O5
335.1602; found 335.1609.
15. HDAC assay: HDAC fluorescent activity assays using a
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PA), which contains an acetylated lysine side chain, were
performed according to manufacturer’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 fluores-
cence was measured using a microplate spectrofluorometer
(LS 50B, Perkin-Elmer) with excitation at 360 nm and
emission at 460 nm.
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11. General procedures: 7 (1.2 equiv) was dissolved in meth-
ylene chloride and then 9a in methylene chloride was