Property based optimization of δ-lactam HDAC inhibitors for metabolic stability

Article abstract of DOI:10.1016/j.bmcl.2010.08.117

The novel δ-lactam based HDAC inhibitor, KBH-A118 (3) shows a good HDAC enzyme and cancer cell growth inhibitory activities but has undesirable pharmacokinetics profiles because of instability in mouse liver microsome. To improve metabolic stability, various analogues were prepared with substituents on aromatic ring of cap group and various chain lengths between the cap group and δ-lactam core. The newly prepared analogues showed moderate to potent in vitro activities. Among them six compounds (8a, 8e, 8j, 8n, 8t, and 8v) were evaluated on mouse liver microsome assay and it turned out that the microsomal stabilities were dependent on lipophilicity and the number of the rotatable bonds. Finally, the animal pharmacokinetic profiles of 8e displayed improving oral exposure and oral bioavailability.

Full text of DOI:10.1016/j.bmcl.2010.08.117

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6808–6811
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Property based optimization of d-lactam HDAC inhibitors for metabolic stability
Hong Chul Yoon a, Eunhyun Choi a, Jung Eun Park a, Misun Cho a, Jeong Jea Seo a, Soo Jin Oh b,
a,c,
Jong Soon Kang b, Hwan Mook Kim b, Song-Kyu Park b, Kiho Lee b, , Gyoonhee Han
⇑
⇑
a Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seodaemun-gu, 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 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:
Received 6 June 2010
Revised 3 August 2010
Accepted 24 August 2010
Available online 17 September 2010
The novel d-lactam based HDAC inhibitor, KBH-A118 (3) shows a good HDAC enzyme and cancer cell
growth inhibitory activities but has undesirable pharmacokinetics profiles because of instability in mouse
liver microsome. To improve metabolic stability, various analogues were prepared with substituents on
aromatic ring of cap group and various chain lengths between the cap group and d-lactam core. The
newly prepared analogues showed moderate to potent in vitro activities. Among them six compounds
(8a, 8e, 8j, 8n, 8t, and 8v) were evaluated on mouse liver microsome assay and it turned out that the
microsomal stabilities were dependent on lipophilicity and the number of the rotatable bonds. Finally,
the animal pharmacokinetic profiles of 8e displayed improving oral exposure and oral bioavailability.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Histone deacetylase
Inhibitor
In vitro ADME
Optimization
Lactam
Histones are the main components of chromatin and play an
important role in DNA packing. Tails and globular domain of
histones can be occurred post-translational modification, such as
acetylation, phosphorylation, methylation, ubiquitination, and
sumoylation. These post-translational modification changes DNA–
histone interaction and binding-transcription factors.1 Histone
acetylation and deacetylation involved in chromatin remodeling
and epigenetic gene expression by histone acetyltransferase
(HAT) and histone deacetylase (HDAC). HAT lead to acetylation of
histone lysine moieties and loose of DNA–histone interaction, this
result recruits additional transcriptional proteins and initiates
transcription. The other way, HDAC induces DNA–histone conden-
sation and suppresses gene expression through deacetylation of
We have designed and prepared the series of novel d-lactam
based HDAC inhibitors containing zinc binding moiety and cap
group. Hydroxamic acid was introduced as a zinc binding group
for strong inhibition of HDAC.8 The cap groups were important
for the enzyme inhibition depending on their hydrophobicity.
And the carbon chain length connected cap group and d-lactam
core also affect HDAC inhibition while larger the bond length gave
higher inhibition of HDAC enzymes. The prepared d-lactam ana-
logues have evaluated their anti-proliferative activities in vitro
and in vivo levels.9,10 Among these HDAC inhibitors, KBH-A118
(3) showed a good HDAC enzyme inhibitory activity and in vitro
and in vivo tumor growth inhibitory activities. For the use of oral
purpose, however, the pharmacokinetic (PK) profiles of KBH-
A118 (3) were not desirable. Thus, we here report optimization
e-amino group in lysines residues located the nearby N-terminal
of histone proteins. In tumor cells, HDACs cause down-regulation
of tumor suppressor genes and they HDAC have been considered
as promising targets for anti-cancer chemotherapy.2–4
For the structural features, HDAC inhibitors are classified as
hydroxamic acid, benzamide, cyclic peptide, and short chain fatty
acid.5 Recently, many HDAC inhibitors are developed clinically
for monotherapy or combination with other anti-cancer drugs.
Among them, SAHA (ZolinzaÒ, Vorinostat)6 and depsipeptide (Isto-
daxÒ, Romidepsin)7 are approved US FDA for treatment of cutane-
ous T-cell lymphoma (CTCL) in 2006 and 2009, respectively (Fig. 1).
O
H
N
OH
H
O
N
H
N
O
H
N
S
O
SAHA (1)
HN
S
O
O
O
O
N
O
NH
O
OH
N
H
Depsipeptide (2)
⇑
Corresponding authors. Tel.: +82 43 240 6522; fax: +82 43 240 6529 (K.L.); tel.:
+82 2 2123 2882; fax: +82 2 362 7265 (G.H.).
KBH-A118 (3)
E-mail addresses: kiholee@kribb.re.kr (K. Lee), gyoonhee@yonsei.ac.kr (G. Han).
Figure 1. Synthetic HDAC inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.117

H. C. Yoon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6808–6811
6809
O
O
process to improve the ADME profiles using various in vitro screen-
ing tools.
Pharmacokinetic profiles of KBH-A118 (3) showed that it has a
high systemic clearance (CL = 2.99 l/h/kg), a short half-life (t1/2
+
N
H
n R
HO
OMe
5
4
=
1.29 h) in intravenous administration (Table 4). For the oral admin-
istration, it was absorbed rapidly (Tmax = 15 min) but eliminated
very fast (t1/2 = 9 min). High oral clearance lowered the oral expo-
sure and resulted in poor oral bioavailability (F = 1.0%). The poor
oral exposure of KBH-A118 (3) may be explained by poor absorp-
tion (low Cmax) or poor metabolic stability (high CL). The ADME
profiles of KBH-A118 (3) could be examined by caco-2 cell
permeability assay and microsomal stability assay (Table 1).
a
O
O
MeO
N
n R
6
KBH-A118 (3) showed
a good caco-2 cell permeability at
23.9 Â 10À6 cm/s, and this result also explained the rapid oral
absorption (Tmax = 15 min). For the liver microsomal stability,
KBH-A118 (3) was very unstable and remaining only 9% both with
and without NADPH in mouse liver microsome assay. These results
indicated that KBH-A118 was rapidly degraded in non-oxidative
metabolic pathway.11 Thus, the metabolic instability of KBH-
A118 (3) accounts for the poor pharmacokinetic profiles and it is
the major concern on improvement. Therefore, the new d-lactam
analogues were synthesized and evaluated on their microsomal
stability. These analogues contained meta- or para-substituents
on the aromatic cap group for blocking NIH shift, and had various
carbon chain numbers between cap group and d-lactam core for
evaluating their lipophilicity.12
b
O
O
MeO
N
n R
7
c
O
O
HOHN
N
n R
The Scheme 1 showed preparation of new d-lactam analogues.
Secondary amine 4 was obtained by N-alkylation of synthetic or
commercial amines with 4-bromo-1-butene. Amines 4 were cou-
pled with monoacid 5 using EDC and to afford amides 6. d-Lactams
7 were prepared by ring closing metastasis using 2–3 mol % of
Grubb’s catalyst (I) and the methyl ester was altered to hydroxamic
acids 8 with KONH2 in MeOH.
First, in vitro activities of the prepared d-lactam analogues were
evaluated on their HDAC and cancer cell growth inhibitory activi-
ties using PC-3 (prostate cancer), MDA-MB-231 (breast cancer),
ACHN (renal cancer) NUGC-3 (gastric cancer), HCT-15 (colon can-
cer), NCI-H23 (lung cancer) cell lines. In general, most of com-
pounds showed HDAC enzyme inhibitory activities and cancer
cell growth inhibitory activities. For HDAC inhibition, the activity
8a n=1 R=3-Cl-Ph
8b n=1 R=3-CN-Ph
8c n=1 R=4-CN-Ph
8d n=1 R=4-t-Bu-Ph
8e n=1 R=2-F, 4-Br-Ph
8f n=1 R=6-Bromonaphtyl 8o n=2 R=3-CF3-Ph
8j n=2 R=2-F-Ph
8k n=2 R=2-Cl-Ph
8l n=2 R=4-Cl-Ph
8m n=2 R=3-Br-Ph
8n n=2 R=4-Br-Ph
8r n=2 R=4-t-Bu-Ph
8s n=2 R=2-Naphtyl
8t n=3 R=4-t-Bu-Ph
8u n=3 R=1-Bromonaphtyl
8v n=4 R=4-Me-Ph
8w n=4 R=4-F-Ph
8g n=2 R=3-Me-Ph
8x n=4 R=4-Cl-Ph
8y n=4 R=4-Br-Ph
8p n=2 R=4-CF3-Ph
8h n=2 R=4-Me-Ph
8q n=2 R=4-NO2-Ph
8i n=2 R=3-OMe-Ph
Scheme 1. Reagents and conditions: (a) EDC, DMAP, CH2Cl2; (b) Grubb’s catalyst
(I), CH2Cl2; (c) KONH2 (1.7 M in MeOH), MeOH, 0 °C.
correlation between HDAC inhibition and cancer cell growth inhi-
bition of these prepared analogues is relatively high for NCI-H23
and is fairly low for MDA-MB-231. It can be explained that HCI-
H2313 showed the more sensitive to up-relegating p21WAF1/CIP1
by HDAC inhibitors, compared to MDA-MB-231.14
Next, we performed microsomal stability assay for evaluating
the substituent effect of cap group. The highly lipophilic com-
pounds showed high metabolic clearance. Through in silico profiles
using PreADME program,15 we selected 8a, 8e, 8j, 8n, 8t, and 8v,
which have similar c log P16,17 to KBH-A118 (2.18 0.5) and a di-
verse substituent and various number of rotatable bond18 for met-
abolic stability evaluation. This assay determined by incubation
of the analogues ranges from 0.05 to 0.55 lM for IC50. Most of
meta- or para-substituted analogues are active and relatively bulky
groups (8r) are more active than small substituent (8h). The cyano
groups (8b and 8c) and nitro (8q) on the aromatic cap group dem-
onstrated relatively weak activities. Cancer cell growth inhibition
was influenced by HDAC enzyme inhibitory activities; compounds
which have showed lower 0.10
lM of IC50 show good GI50 (0.3–
10 M) and the rest analogues have moderate activity (Table 2).
l
Mechanism of cancer cell growth arrest by HDAC inhibitors is quite
complicated but it is known that up-regulating of p21WAF1/CIP1
expression is the most common in many HDAC inhibitors.10 The
1 lM of analogues with mouse liver microsome (1 mg protein/
ml) for 30 min at 37 °C. The metabolic stabilities of 8a, 8e, 8j,
8n, 8t, and 8v using mouse liver microsome were displayed in
Table 3. c Log P values of the compounds correlated well with their
microsomal stabilities. Higher c log P than 2.67 resulted in poor
metabolic stability (8t and 8v). And the higher number of rotatable
bond may also result in the negative impact on microsomal stabil-
ity by increasing lipophilicity. Among the six analogues, 8e and 8n
are equally stable in microsome and showed a good in vitro activ-
ity, but 8e displayed better in silico parameters, such as the num-
ber of rotatable bonds and c log P12, and these can be more useful
for future design for the further PK optimization.
Table 1
In vitro ADME profiles of KBH-A118 and other reference compounds
Compound
Caco-2 cell
Remaining at 60 mina (%)
Papp  106 (cm/s)
ÀNADPH
+NADPH
Buffer
97.3
KBH-A118
Negative ctrl
Positive ctrl
23.9
8.5
8.7
100.8d
1.2e
1.5 0.1b
29.6 0.8c
96.2d
86.8e
a
Microsomal stability was determined by incubating 1 lM of test compounds
with female ICR mouse liver microsomes (0.5 mg protein/mL) for 60 min at 37 °C.
b
Ranitidine.
Metoprolol.
Atenolol.
Chlorpromazine.
Finally, 8e19 was evaluated in mouse pharmacokinetic model
and it showed an improved PK profiling compared to KBH-A118.
c
d
e
Half-life had a fivefold increased at 6.2 h compared to 1.29 h (t1/2
,

6810
H. C. Yoon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6808–6811
Table 2
KBH-A118) and plasma exposure rise two times at intravenous
(2587.7 h ng/ml vs 1671.7 h ng/ml) and ten times at oral adminis-
tration (732.3 h ng/ml vs 64.4 h ng/ml). The oral bioavailability of
8e had been remarkably increased to 14.1% compared to 1% of
KBH-A118 (Table 4). The systemic clearance of 8e, however, was
slightly increased to 3.9 l/h/kg compared to 2.99 of KBH-A118. This
result indicated that non-oxidative phase I mechanism such as
phase II conjugation may be involved in metabolic degradation of
these compounds.10 It turns out that improving microsomal stabil-
ity is an appropriate to improve in vivo pharmacokinetic profiles in
d-lactam based HDAC inhibitors. The metabolic stability of the
compounds depends on their physical chemical properties such
as, c log P and the number of rotatable bonds.
In conclusion, KBH-A118 showed a good HDAC enzyme and
cancer cell growth inhibitory activities, but its pharmacokinetics
profiles was very poor because of instability in mouse liver micro-
some. The microsomal instability caused a low oral exposure, high
clearance and resulted in a poor oral bioavailability. For improving
metabolic stability, meta- or para-substituents were introduced on
aromatic ring of cap group, for blocking NIH shift and reduced car-
bon chain length, to decrease lipophilicity of the compounds. The
newly prepared analogues also showed a good HDAC enzyme
inhibitory activities and cancer cell growth inhibitory activities.
Based on the microsomal stability screening, 8e was selected for
animal pharmacokinetic experiment, which showed the similar
in vitro activity profiles to KBH-A118. As a result, the pharmacoki-
netic profiles of 8e displayed a long half-life (6.2 h), a good oral
exposure and a moderate oral bioavailability (F = 14.1%).
HDAC enzyme and growth inhibition by d-lactam analogues, KBH-A118 (3) and SAHA
(1)
Compound
IC50
(
l
M)a
GI50 (l
M)a
HDAC PC-3 MDA-MB-231 ACHN NUGC-3 HCT-1 NCI-H23
1
3
0.11
0.21
0.36
0.31
0.23
0.05
0.31
0.13
0.23
0.55
0.46
0.52
0.61
0.31
0.30
0.13
0.03
0.12
0.67
0.05
0.10
0.12
0.03
0.24
0.50
0.38
0.26
2.69 2.00
3.25 2.03
4.65 3.58
8.48 4.18
>10 8.17
5.19 2.84
5.81 3.12
2.61 2.31
>10 4.04
6.34 2.84
4.84 3.03
7.07 3.02
7.25 3.91
1.51 0.92
0.81 0.87
3.41 1.87
2.47 1.90
4.82 1.81
>10 >10
0.50 1.13
0.39 0.27
1.13 >10
0.30 0.56
2.53 3.05
>10 >10
4.15 2.56
4.81 3.54
4.22
1.89
4.48
NT
NT
3.00
NT
2.94
3.19
3.92
NT
2.49
5.40
6.22
>10
2.34
5.99
2.96
NT
NT
NT
NT
NT
NT
5.56
7.06
6.09
6.50
2.12
1.83
NT
8a
8b
8c
8d
8e
8f
8g
8h
8i
NT
NT
NT
NT
>10
2.94
5.37
1.10
4.41
4.00
3.96
3.60
4.21
1.66
1.25
0.87
2.35
1.70
>10
0.52
0.31
1.66
0.53
2.94
>10
NT
NT
NT
4.55
7.60
4.47
8.71
1.28
0.30
NT
3.57
4.26
3.78
5.93
1.06
0.82
NT
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
8y
2.64
NT
1.70
NT
3.97
NT
>10
2.27
0.38
1.72
0.46
2.49
9.53
2.46
2.39
>10
1.10
0.36
2.81
0.79
2.13
9.02
1.79
2.01
>10
0.99
0.43
2.00
0.49
2.79
>10
2.88
2.73
3.30
3.19
a
Values are means of a minimum of three independent experiments. NT: not
tested.
Acknowledgments
This research was supported by National Research Foundation
(2009-0092966), Korea Research WCU grant (R31-2008-000-
10086-0) and Brain Korea 21 project, Republic of Korea.
Table 3
The microsomal stability of d-lactam analogues
Remaining at 30 mina (%)
In silico parametersb
Supplementary data
Compound
ÀNADPH
+NADPH
Buffer
Rotatable bond #
c log P
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.117.
8a
8e
8j
8n
8t
8v
40.3
55.0
52.7
56.6
8.0
31.3
31.7
45.3
29.6
3.3
102.7
102.3
103.8
103.9
103.2
99.3
5
5
6
6
7
8
1.61
1.90
1.48
2.02
3.13
2.67
References and notes
5.7
96.6
4.0
0.3
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b
PreADME program, BMDRC.
c
Positive control.
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Table 4
Pharmacokinetics profiles of KBH-A118 and 8e in Balb/c mouse
Parameter*
Unit
KBH-A118
PO
8e
IV
IV
PO
Dose
Tmax
Cmax
(mg/kg)
(h)
5
—
—
20
10
—
—
2587.7
3.9
0.8
6.2
—
20
0.25
196.8
64.4
—
—
0.15
1
0.25
1387.3
732.3
—
—
—
(ng/ml)
(h ng/ml)
(l/h/kg)
(l/kg)
(h)
**
AUC0-inf
1671.7
2.99
0.27
1.29
CL
Vss
t1/2
F
(%)
14.1
*
PK parameters were based on mean plasma concentration-time profiles of three
15. BMDRC, PreADMET 2.0, Bmdrc, Seoul, Korea, 2007.
16. Kerns, E. H.; Di, L. Drug Discovery Today 2003, 8, 316.
17. Wang, J.; Skolnik, S. Chem. Biodivers. 2009, 6, 1887.
18. Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.
J. Med. Chem. 2002, 45, 2615.
animals per time points. PK parameters were calculated by noncompartmental
analysis using PK Solutions 2.0 (Summit Research Services, Montrose, CO, USA).
**
AUC after iv administration was calculated from 0 to infinity, whereas the oral
AUC was calculated from 0 to 5 h.

H. C. Yoon et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6808–6811
6811
19. 3-(1-(4-Bromo-2-fluorobenzyl)-2-oxo-1,2,5,6-tetrahydropyridin-3-yl)-N-
hydroxypropanamide (8e): 1H NMR (500 MHz, DMSO-d6) d 10.36 (s, 1H), 8.69
(s, 1H), 7.54 (dd, 1H, JA = 9.5 Hz, JB = 2.0 Hz), 7.40 (dd, 1H, JA = 8.0 Hz,
JB = 2.0 Hz), 7.24 (t, 1H, J = 8.0 Hz), 6.38 (br t, 1H, J = 4.0 Hz), 4.53 (s, 2H),
3.31 (d, 2H, J = 7.0 Hz), 2.39 (t, 2H, J = 7.0 Hz), 2.08 (br q, 2H, JA = 7.5 Hz,
JB = 6.5 Hz), 2.08 (t, 2H, J = 8.0 Hz).