Structure and property based design, synthesis and biological evaluation of γ-lactam based HDAC inhibitors: Part II

Article abstract of DOI:10.1016/j.bmcl.2012.04.045

Histone deacetylases (HDACs) are involved in post-translational modification and epi-genetic expression, and have been the intriguing targets for treatment of cancer. In previous study, we reported synthesis and the biological preliminary results of γ-lactam based HDAC inhibitors. Based on the previous results, smaller γ-lactam core HDAC inhibitors are more active than the corresponding series of larger δ-lactam based analogues and the hydrophobic and bulky cap groups are required for better potency which decreased microsomal stability. Thus, γ-lactam analogues with methoxy, trifluoromethyl groups of ortho-, meta-, para-positions of cap group were prepared and evaluated their biological potency. Among them, trifluoromethyl analogues, which have larger lipophilicity, showed better HDAC inhibitory activity than other analogues. In overall, lipophilicity leads to increase hydrophobic interaction between surface of HDAC active site and HDAC inhibitor, improves HDAC inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2012.04.045

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4189–4192
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure and property based design, synthesis and biological evaluation
of c-lactam based HDAC inhibitors: Part II
Chulho Lee a, Eunhyun Choi a, Misun Cho a, Boah Lee a, Soo Jin Oh b, Song-Kyu Park c, Kiho Lee c,
Hwan Mook Kim d, Gyoonhee Han a,e,
⇑
a Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
b Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Yangcheong, Ochang, Cheongwon, Chungbuk 363-883, Republic of Korea
c College of Pharmacy, Korea University, Yeongi, Chungnam 339-700, Republic of Korea
d College of Pharmacy, Gachon University of Medicine and Science, Incheon 406-799, Republic of Korea
e Department of Biomedical Sciences (WCU Program), Yonsei University, Seodaemun-gu, Seoul 120-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylases (HDACs) are involved in post-translational modification and epi-genetic expression,
Received 25 January 2012
Revised 16 March 2012
Accepted 3 April 2012
Available online 21 April 2012
and have been the intriguing targets for treatment of cancer. In previous study, we reported synthesis and
the biological preliminary results of
ler -lactam core HDAC inhibitors are more active than the corresponding series of larger d-lactam based
analogues and the hydrophobic and bulky cap groups are required for better potency which decreased
microsomal stability. Thus, -lactam analogues with methoxy, trifluoromethyl groups of ortho-, meta-,
c-lactam based HDAC inhibitors. Based on the previous results, smal-
c
c
Keywords:
Histone deacetylases
Inhibitor
Docking
Property
para-positions of cap group were prepared and evaluated their biological potency. Among them, trifluo-
romethyl analogues, which have larger lipophilicity, showed better HDAC inhibitory activity than other
analogues. In overall, lipophilicity leads to increase hydrophobic interaction between surface of HDAC
active site and HDAC inhibitor, improves HDAC inhibitory activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Optimization
Post-translational modifications are inheritable changes in gene
expression without changes in DNA sequences.1 Chromatin com-
prises DNA–histone complex and undergo structural changes by
post-translational modifications which include methylation, acety-
lation, phosphorylation and ubiquitination.2 Among the modifica-
tion above, histone acetylation has been widely studied and
known to have various roles for transcriptional regulation and
epi-genetic gene expression.3 Histone deacetylase (HDAC) are cat-
(vorinostat, ZolinzaÒ, 1)8 and depsipeptide (Romidepsin, IstodaxÒ,
2)9 were approved by US FDA for the treatment of cutaneous T-cell
lymphoma (CTCL) in 2006 and 2009, respectively (Fig. 1).
We reported novel series of c-lactam based HDAC inhibitors (3)
and their anti-cancer evaluation, which are composed with zinc
binder, core group and cap group.10 Hydroxamic acids were intro-
duced as zinc binder for chelation of zinc ion in active site of HDAC
enzymes.
c-Lactam is the core linked to diverse substituent cap
alytic enzymes to remove acetyl moiety from
e
-amino group in
group by carbon chain. In our previous publication, a smaller
lysine residues of histone.4 HDACs regulate gene expression by
changing of chromatin structure. Deacetylation of histone leads
to increase the electrostatic charge of histone and enhance the
interaction between DNA and histone. Therefore, the structure of
chromatin becomes compact and blocks the access of transcription
factors to DNA template, and finally gene expression is sup-
pressed.5 In addition, HDACs catalyze the deacetylation of
sequence-specific DNA binding transcriptional factors.6 The abnor-
mal transcriptional regulations by HDACs are related with carcino-
genesis.7 Based on these rationales, HDACs have been considered
as promising targets for anti-cancer treatment and many HDAC
inhibitors have been reported. Among these inhibitors, SAHA
⇑
Corresponding author. Tel.: +82 2 2123 2882; fax: +82 2 362 7265.
E-mail address: gyoonhee@yonsei.ac.kr (G. Han).
Figure 1. Structure of HDAC inhibitors.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.045

4190
C. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4189–4192
c
-lactam core showed better fit than larger d-lactam core into nar-
SAHA. c-Lactam HDAC inhibitors bind into the narrow tubular
row hydrophobic pocket of HDAC active site and showed better
pocket of HDAC active site; hydroxamic acid interacts with Zn2+
which is located at the end of the interior active site, and cap
groups are located outside of the HDAC active site. Hydroxamic
acid of 9l,18 the most active compound in these analogues, inter-
inhibition profiles of HDACs.11 In this study, we designed and syn-
thesized c-lactam HDAC inhibitor analogues with diverse carbon
chain linker and various substituents on cap groups to improve
HDAC inhibition profiles. We prepared 1–3 carbon chain linker be-
acts with His145 and His146 by hydrogen bonding, and
c-lactam
tween
c
-lactam core and cap group with methoxy, trifluoromethyl
core has interaction with two aromatic side chains of
p–p
substituents of ortho-, meta-, para-positions of cap groups. These
modifications of analogues led to better potency, while the meta-
bolic stability slightly decreased.
Phe155 and Phe210. On the other hand, ortho-methoxy benzyl ana-
logue, 9b, showed the poorest HDAC inhibitory activity, because of
short carbon chain linker and o-methoxy substituent in cap group.
1-Carbon chain linker of 9b is not enough long to bind into narrow
tubular pocket of HDAC active site and ortho-methoxy substituent
hindered to enter deep into HDAC active site. Therefore, 9b could
not enter into HDAC active site enough and it was difficult to che-
late with Zn2+. The 2-carbon chain analogues showed proper fitting
in active site and they showed better HDAC inhibitory activities
than other analogues.
The Scheme 1 showed preparation of c-lactam analogues. Com-
mercial benzyl alkyl halide 4 is used as the starting material, sec-
ondary amine 5 was synthesized by N-alkylation with allyl
amine. Secondary amine 5 was coupled with monoacid 6 by
EDC-mediated coupling and to afford amide 7.
c-Lactam 8 was
prepared by ring closing method using 2–3 mol% of Grubb0s 2nd
catalyst and the methyl ester was converted to hydroxamic acid
9 by KONH2 in MeOH.
All
c-lactam based HDAC analogues also evaluated their cell
We evaluated all prepared
c
-lactam analogues on HDAC en-
growth inhibitory activities using six cancer cell lines; PC-3 (pros-
tate cancer), MDA-MB-231 (breast cancer), ACHN (renal cancer),
HCT-15 (colon cancer), NCI-H23 (non-small lung cancer) and
NUGC-3 (gastric cancer). In vitro anti-cancer activity showed aver-
zyme inhibitory activity (Table 1). HDAC Flourescent Activity Assay
kit was used for evaluation of HDAC inhibitory activity.12 All of the
analogues have 0.07–0.45
lM of IC50 values in HDAC inhibitory
activity. One exception, 9b, showed over 10
l
M of IC50 value and
age 1.9–3.57 lM of GI50 value in six cancer cell line and HDAC
it could be explained through docking study. Among these ana-
logues, the analogues with 2 carbon chain length (9g–l) showed
better HDAC inhibitory activity at 0.07–0.19 lM compare to 1 or
inhibitory activity and tumor cell growth inhibitory activity are
mostly related. Most of these analogues showed good cancer cell
growth inhibitory activities. These analogues showed the best
3-carbon analogues. 3-Carbon chain length analogues (9m–o)
showed medium level of HDAC inhibitory activity at 0.13–
activity at 1.9
(MDA-MB-231) and at 2.25
(NUGC-3). And they showed poor activity at 3.48 and 3.57 l
l
M of average GI50 in breast cancer cell lines
M of average GI50 in gastric cell lines
M of
l
0.27
l
M in the range of IC50 whereas 1-carbon chain length ones
M in
(9a–f) showed the poorest HDAC inhibition at 0.12–0.45
l
average IC50 in prostate cell lines (PC-3) and human colon cancer
the range of IC50. These results can be explained using the Discov-
ery Studio program13 to perform a docking study with human
HDAC-2 (PDB code 3MAX14). Figure 2 shows binding modes of
the HDAC active site and HDAC inhibitors based on the docking
study.15,16 Compounds 9b and 9l have a similar binding mode with
cell lines (HCT-15), respectively.
There are two different substituents on cap group in this paper.
Most of trifluoromethyl analogues showed better HDAC inhibitory
activity (0.07–0.26
lM) than methoxy analogues (0.13–0.45 lM)
or non-substituted analogues. One of important interactions
a
O
O
n
X
N
nH
R
R
HO
O
4
5
6
O
O
b
c
nN
O
R
7
O
O
O
O
d
nN
nN
R
R
OH
O
N
H
8
9
9a
9i
n=1 R = H
n=2 R = 3-methoxy
n=2 R = 4-methoxy
n=2 R = 3-CF3
9j
9b n=1 R = 2-methoxy
9c
9d
9e
9f
9k
9l
n=1 R = 3-methoxy
n=1 R = 4-methoxy
n=1 R = 3-CF3
n=2 R = 4-CF3
9m
9n
9o
n=3 R = 2-methoxy
n=3 R = 3-methoxy
n=3 R = 4-methoxy
n=1 R = 4-CF3
9g
9h
n=2 R = H
n=2 R = 2-methoxy
Scheme 1. Synthesis of
c-lactam based HDAC inhibitor. Reagents and conditions: (a) allylamine, DIPEA, acetonitrile, 0 °C; (b) EDC, DMAP, methylene chloride; (c) Grubb’s 2nd
catalyst, methylene chloride; (d) KONH2 (1.7 M in MeOH), MeOH, 0 °C.

C. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4189–4192
4191
Table 1
HDAC enzyme and cancer cell growth Inhibition by c-lactam analogues
a
a
Compound
n
R
IC50
(lM)
GI50
ACHN
(
l
M)
HDAC
PC-3
MDA-MB-231
HCT-15
NCI-H23
NUGC-3
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
H
0.12
>10
4.43
3.56
3.89
3.12
0.88
4.36
4.43
3.51
2.59
3.17
2.77
2.38
3.51
2.59
7
0.99
6.03
1.77
1.63
0.22
3.07
0.99
1.5
1.88
1.08
2.17
1.17
1.5
1.08
>10
5.37
>10
2.82
1.72
0.54
4.54
5.37
>10
>10
>10
6.01
2.2
4.29
6.13
1.97
1.64
0.68
6.64
4.29
4.49
1.17
4.02
1.11
1.12
4.49
1.17
2.51
3.05
NT
>10
2-Methoxy
3-Methoxy
4-Methoxy
3-CF3
4-CF3
H
2-Methoxy
3-Methoxy
4-Methoxy
3-CF3
0.38
0.45
0.12
0.26
0.12
0.13
0.16
0.19
0.12
0.07
0.13
0.16
0.27
0.19
4.43
2.48
0.67
4.29
1.08
2.53
2.48
2.38
1.17
2.48
2.53
2.48
4.75
2.49
1.75
1.11
0.16
3.23
NT
2.02
3.93
1.42
0.92
1.08
2.02
3.93
5.48
2.25
4-CF3
9m
9n
9o
Average
2-Methoxy
3-Methoxy
4-Methoxy
>10
>10
>10
3.57
1.88
2.56
1.9
3.48
a
Values are means of a minimum of three independent experiments. NT: not tested.
than trifluoromethyl or non-substituent analogues. Trifluoro-
methyl analogues showed larger logD value than methoxy and
non-substituent analogues. Methoxy and non-substituent ana-
logues have similar logD value. Three analogues of 2-carbon chain
linker were compared. (9g, 9j, 9l) These analogues have 0.12, 0.19
and 0.07
methyl analogue (9l) showed the best HDAC inhibitory activity
(0.07 M) in three analogues, and methoxy analogue (9j) showed
lM of IC50 for HDAC inhibition, respectively. Trifluoro-
l
slightly lower HDAC inhibitory activity than non-substituent (9g)
(0.19 vs 0.12, IC50). This difference in HDAC inhibitory activity
could be explained with physicochemical properties. Three ana-
logues have similar values of tPSA (78.87 and 69.64) and number
of rotational bond (6 and 7). However, 9l, the best active analogue,
has significantly larger value of logD than other analogues. LogD
value of 9l is 1.50, it is about three times larger than logD value
of 9g (0.59) or 9j (0.54). Therefore, 9l is the most lipophilic com-
pound and it lead to the highest HDAC inhibitory activity at
Figure 2. Docking study of c-lactam core HDAC inhibitors in human HDAC-2 active
site: 9b (blue), 9l (green) and SAHA (purple).
0.07
induced increase hydrophobic interaction at surface of HDAC en-
zyme, and increase lipophilicity of -lactam based HDAC analogues
lM of IC50. Increasing values of logD with lipophilic cap group
c
between HDAC active site and HDAC inhibitor is hydrophobic
interaction at the surface of HDAC active site. Hydrophobic interac-
tion could be predicted by physicochemical properties. The num-
ber of rotational bond, topological polar surface area (tPSA),
lipophilicity at pH 7.4 (logD) values are calculated using PreADME
program17 (Table 2). In these results, methoxy analogues showed
larger number of rotational bond by one, and larger tPSA value
lead to improve HDAC enzyme inhibitory activity.
Compound 9l, the most lipophilic compound, showed the most
potent HDAC inhibition profiles. Increasing lipophilicity leads to an
increase in HDAC inhibition activity while it leads to a decrease in
microsomal stability. Three analogues with 2-carbon chain linkers
were selected to confirm the metabolic stability of c-lactam HDAC
inhibitors (9g, 9j, 9l). The most lipophilic compound 9l showed a
slight decrease, but remained moderately stable in the mouse liver
microsome (Table 3).
Table 2
In conclusion, we prepared
c-lactam based HDAC inhibitors
Physicochemical properties of c-lactam based analogues
which have 1–3 carbon chain linker and methoxy and trifluoro-
methyl substituent in ortho-, meta-, para-position of cap group.
Compound
Rotatable bond number
tPSA
logD (pH 7.4)
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
5
6
6
6
5
5
6
7
7
7
6
6
8
8
8
69.64
78.87
78.87
78.87
69.64
69.64
69.64
78.87
78.87
78.87
69.64
69.64
78.87
78.87
78.87
0.43
0.37
0.42
0.39
1.39
1.35
0.59
0.53
0.58
0.54
1.54
1.5
Table 3
The metabolic stability of 9g, 9j, 9l
Compound
% Remaining at 30 mina
+NADPH
ꢀNADPH
82.9
98.3
75.9
106.2
Buffer
9g
9j
9l
85.5
92.0
73.9
0.1
96.7
101.6
101.6
107.7
Positive controlb
0.98
1.03
0.99
a
Test compounds (1 lM) were incubated with 0.5 mg/ml of pooled mouse liver
microsomes in the presence or absence of NADPH for 30 min at 37 °C.
b
Buspirone.

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C. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4189–4192
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.
045. These data include MOL files and InChiKeys of the most
important compounds described in this article.
18. N-Hydroxy-3-{2-oxo-1-[2-(4-trifluoromethyl-phenyl)-ethyl]-2,5-dihydro-1H-
pyrrol-3-yl}-propionamide (9l): 1H NMR (DMSO-d6) d 7.55–7.47 (m, 4H), 6.85
(s, 1H), 3.88 (s, 2H), 3.73 (t, 2H, J = 7.3 Hz), 3.00 (t, 2H, J = 7.2 Hz), 2.55 (t, 2H,
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