Property-based optimization of hydroxamate-based γ-lactam HDAC inhibitors to improve their metabolic stability and pharmacokinetic profiles

Article abstract of DOI:10.1021/jm3009376

Hydroxamate-based HDAC inhibitors have promising anticancer activities but metabolic instability and poor pharmacokinetics leading to poor in vivo results. QSAR and PK studies of HDAC inhibitors showed that a γ-lactam core and a modified cap group, includ

Full text of DOI:10.1021/jm3009376

Brief Article
pubs.acs.org/jmc
Property-Based Optimization of Hydroxamate-Based γ‑Lactam HDAC
Inhibitors to Improve Their Metabolic Stability and Pharmacokinetic
Profiles
Eunhyun Choi,†,‡,▽ Chulho Lee,‡,▽ Misun Cho,‡ Jeong Jea Seo,‡ Jee Sun Yang,‡ Soo Jin Oh,§ Kiho Lee,∥
Song-Kyu Park,∥ Hwan Mook Kim,⊥ Ho Jeung Kwon,‡ and Gyoonhee Han*,‡,#
†Severance Integrative Research Institute for Cerebral & Cardiovascular Disease, Yonsei University Health System, 250 Seongsanno,
Seodaemun-Gu, Seoul 120-752, Republic of Korea
‡Translational Research Center for Protein Function Control, Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul
120-749, Republic of Korea
§Bioevaluation Center, Korea Research Institute of Bioscience and Biotechnology, Yangcheong, Ochang, Cheongwon, Chungbuk
363-883, Republic of Korea
∥College of Pharmacy, Korea University, Yeongi, Chungnam 339-700, Republic of Korea
⊥College of Pharmacy, Gachon University of Medicine and Science, Incheon 406-799, Republic of Korea
#Department of Biomedical Sciences (WCU Program), Yonsei University, Seodaemun-gu, Seoul 120-749, Republic of Korea
S
* Supporting Information
ABSTRACT: Hydroxamate-based HDAC inhibitors have promising anticancer activities but metabolic instability and poor
pharmacokinetics leading to poor in vivo results. QSAR and PK studies of HDAC inhibitors showed that a γ-lactam core and a
modified cap group, including halo, alkyl, and alkoxy groups with various carbon chain linkers, improved HDAC inhibition and
metabolic stability. The biological properties of the γ-lactam HDAC inhibitors were evaluated; the compound designated 8f had
potent anticancer activity and high oral bioavailability.
INTRODUCTION
■
Histone deacetylases (HDACs) and histone acetyltransferases
(HATs) are involved in chromatin remodeling and tran-
scription.1 HATs activate transcription by releasing chromatin
from histones, whereas HDACs lead to compaction of
chromatin structures and suppression of gene expression.2
Silencing of tumor suppressor genes, such as p21 and p53,
represses apoptosis.3,4 HDACs are known to be related to
tumorigenesis and cell differentiation by virtue of their gene
silencing ability. Thus, HDAC inhibitors are considered
intriguing anticancer agents.5,6 Hydroxamate-based HDAC
inhibitors are well-known for their potent anticancer activity.
The hydroxamate group, a controversial warhead, is often
introduced as a potent zinc chelator for strong binding with
metal in the active site of enzymes.7 Nevertheless, only
suberoylanilide hydroxamic acid (SAHA, Zolinza, Vorinostat)8
(1) has been approved by the U.S. Food and Drug
Administration. Other hydroxamate-based HDAC inhibitors
including trichostatin A (TSA)9 (2) (Figure 1) have been
developed but remain in clinical trials because of poor
metabolic stability and pharmacokinetic (PK) profiles. Hydrox-
amate is easily metabolized through hydrolysis and glucur-
onidation by liver microsomes, but it results in PK problems.10
It is reported that SAHA has a short half-life of about 21−58
min when administered intravenously.11 Despite these
metabolic instabilities, hydroxamate-based HDAC inhibitors
are still a promising anticancer candidate. We searched for
Figure 1. Chemical structures of hydroxamate-based HDAC
inhibitors.
hydroxamate-based HDAC inhibitors with improved PK
profiles using the property optimization process.
We designed and synthesized novel lactam-based HDAC
inhibitors (3) composed of a cap group, lactam core, and
hydroxamic acid.3−5,12−17 These analogues had good HDAC
enzyme inhibitory and tumor growth inhibitory activities both
in vitro and in vivo. Quantitative structure−activity relationship
(QSAR) and docking studies demonstrated that the active site
of the HDAC enzyme is a narrow pocket; a zinc ion in the
interior active pocket interacts with the hydroxamic acid. The
entrance of the active site is important in the hydrophobic
interaction.18 PK studies and metabolic stability studies of δ-
lactam-based HDAC inhibitors have shown that these
Received: July 3, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm3009376 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
analogues are metabolically unstable.4,14 Hydroxamic acid, a
good zinc chelator, is easily hydrolyzed, and the hydrophobic
cap group can be oxidized by liver microsomes. These reactions
result in poor metabolic stability and eventually lead to poor
PK profiles of the δ-lactam-based HDAC inhibitors. Our
previous studies indicated that lipophilicity plays an important
role in HDAC inhibition as well as PK profile. On the basis of
these results, we changed the δ-lactam core to a γ-lactam core
to reduce the core size and lipophilicity, and various
substituents were introduced on the cap group to increase
the metabolic stability by blocking the NIH shift.
selective inhibition against HDAC-6 of the HDAC isoforms;12
however, the crystal structure of HDAC-6 is not yet available.
Thus, human HDAC-2, the second most potent isoform, was
used for the docking study. 8d, 8f, and 8i with 4-fluoro, 4-
bromo, and 4-t-butyl on the cap group, respectively, were used
for the docking studies. The results show that γ-lactam-based
HDAC inhibitors have a similar binding mode and orientation
as SAHA in the active site. The hydroxamic acid chelated with
the zinc ion and formed hydrogen bonds with His 145 and His
146. The γ-lactam core is located in the hydrophobic narrow
pocket, stabilized by the π−π interaction between Phe 155 and
Phe 210 (Figure 2).
RESULTS AND DISCUSSION
■
The synthetic process for producing γ-lactam analogues is
shown in Scheme 1. EDC-mediated coupling with monoacid
a
Scheme 1. Synthesis of γ-Lactam-Based HDAC Inhibitors
Figure 2. Docking simulation of 8d (sky blue), 8f (pink), 8i (yellow),
and SAHA (green) in human HDAC-2 active site (PDB 3MAX).
The potency of the synthesized γ-lactam-based analogues was
evaluated for HDAC inhibition and in cancer cell growth
inhibition assays. The HDAC Fluorescent Activity Assay kit21
and SRB assay were used on six human cancer cell lines: PC-3
(prostate), MDA-MB-231 (breast), ACHN (renal), HCT-15
(colon), NCI-H23 (nonsmall lung), and NUGC-3 (gastric)
(Table 1). The γ-lactam inhibitors showed promising HDAC
a
Reagents: (a) EDC, DMAP, CH2Cl2; (b) Grubbs’ catalyst (2nd
generation), CH2Cl2, reflux; (c) KONH2 (1.7 M in MeOH), MeOH, 0
°C.
Table 1. Inhibition of HDAC Enzyme Activity and Cancer
Cell Growth by γ-Lactam Analogues
(5) with secondary amines (4), which were synthesized by N-
alkylation or reductive amination, produced amides (6) with
good yields. Metathesis reaction of amides with 2−3 mol % of
Grubbs’ catalyst (2nd generation) produced the γ-lactam rings
(7) in high yields. Hydroxamic acid analogues (8) were
obtained by reacting the esters of the γ-lactam with KONH2 in
MeOH at low temperature. The synthesized γ-lactam-based
analogues contained one to four carbons between the cap
group and γ-lactam core. Halo, alkyl, and alkoxy moieties were
introduced as the cap groups in the ortho, meta, and para
positions, respectively.
HDACs are classified into 11 isoforms. Among them, four
isoforms have their X-ray structures in the Protein Data Bank
(PDB): HDAC-2 (3MAX19), HDAC-3 (4A69), HDAC-7
(3C0Z), and HDAC-8 (1T69). These enzymes commonly
have a narrow pocket and zinc ion in their active site. HDAC
active sites are conserved structures, but they have slightly
different shapes depending on the isoform. For example, the
active site of HDAC-3 is shallower than that of the other
isoforms and HDAC-2 has more room below the zinc. We
performed docking simulations with human HDAC-2 using the
Discovery Studio program.20 On the basis of our previous
study, the γ-lactam-based HDAC inhibitors had the most
a
IC50
a
(μM)
GI50 (μM)
MDA-
compd HDAC PC-3 MB-231 ACHN
HCT- NCI-
15 H23 NUGC-3
SAHA
8d
0.11
0.06
0.11
0.1
2.69
5.31
2.94
1.83
7.92
>10
8.69
>10
2.00
1.94
0.6
4.22
5.99
0.41
0.86
7.75
>10
5.7
2.49
2.34
4.67
3.46
0.42
4.08
2.32
4.39
7.8
2.94
6.06
NT
4.07
1.65
0.57
>10
>10
>10
>10
8f
8i
0.69
3.6
0.72
4.12
>10
6.57
>10
8l
0.16
0.15
1.08
1.47
8m
5.38
3.18
4.97
8n
8o
8.96
Average
8a−m
8n−v
8w-d′
8e′−f′
all
0.19
0.42
0.42
0.44
0.34
4.62
4.14
3.48
6.09
4.40
1.99
2.76
2.21
4.15
2.50
3.33
3.60
3.33
3.73
3.45
4.72
4.25
5.10
8.54
5.13
2.50
2.55
2.52
6.25
2.96
4.28
3.38
2.20
3.53
3.44
a
Values are the means of a minimum of three independent
experiments. NT: not tested.
B
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Journal of Medicinal Chemistry
Brief Article
inhibitory activity, exhibiting a range of IC50 values from 0.04−
1.47 μM. Most of these analogues also showed good cancer cell
growth inhibitory activities (GI50 = 0.22−10 μM). Of the six
cancer cell lines, MDA-MB-231 was the most sensitive (average
GI50 = 2.50 μM) to the γ-lactam inhibitors, whereas HCT-15
was the least sensitive (average GI50 = 5.13 μM). The
structure−activity relationship revealed that the length of the
carbon chain between the γ-lactam core and hydroxamate
affected the HDAC inhibitory activity and cancer cell growth
inhibition. In general, compounds with the two-carbon linker
were more active than the other compounds (8a−m, 8w−f′).
With the exception of the weak activity of 8n−o with electron
withdrawing group (NO2), the average IC50 value of
compounds with the two-carbon linker (8p−v) was 0.17 μM.
The two-carbon linker provides a proper fit into the active site,
leading to better HDAC inhibition profiles.17 The functional
groups on the cap group also affected HDAC inhibitory activity
and metabolic stability. The substituents of the cap group
conferred resistance to oxidation of the hydrophobic cap group
by blocking the NIH shift. The substituents on the meta- and
para- position (8b and 8c) had better HDAC inhibitory activity
than the ortho- position (8a). In addition, bulky and
hydrophobic cap groups induced better HDAC inhibitory
activity. Of the analogues synthesized, those with a nitro (8n−
o) substituent attached on the cap group exhibited weak
activity against HDAC and on cancer cell growth inhibition,
whereas those with a cyano (8l−m) group had strong HDAC
inhibitory activity but weak activity for cancer cell growth
inhibition. It appears that the lone pair electrons located on the
cyano group substituent may have an adverse effect on cancer
cell growth inhibition activity.
log D values and number of rotational bonds to confirm these
observations. We also evaluated their ADME profiles with
microsomal stability assays and pharmacokinetic studies. Four
analogues, 8d, 8f, 8i, and 8k, had one-carbon chain linkers, a
small rotatable bond number (5), and the same tPSA (69.64) in
common but had a wide range of lipophilicity. The other three
analogues, 8v, 8d′, and 8f′, had the same tPSA (69.64), similar
log D values, and from one to four carbon chain linkers. These
also showed good inhibitory activity against HDAC and cancer
cell growth.
The positive results of the parallel artificial membrane
permeability assay (PAMPA) experiment indicated that most of
these compounds have a good absorption rate (Table 3). An
Table 3. PAMPA Permeability and Metabolic Stability of γ-
Lactam HDAC Inhibitors
a
d
PAMPA
% remaining at 30 min
b
c
compd
Pe (nm/s)
R (%) −NADPH +NADPH
8d
8f
19.31 9.46
10.16 1.42
21.95 3.70
28.32 3.84
65.29 26.07
15.04 11.96
4.94 0.98
24.28
22.47
21.77
21.23
20.15
25.35
28.11
18.27
12.13
91.4
100.0
79.0
87.7
84
8i
58.2
8k
8v
8d′
8f′
40.55
51.01
57.63
34.65
35.71
38.06
35.32
24.94
e
g
h
negative control
15.06 0.29
96.2
100.8
f
h
h
positive control
63.62 4.05
86.8
1.2
a
b
c
The final concentration was 200 μM. Passive diffusion. Mass
d
retention. Microsomal stability was determined by incubating 1 μM
of the test compounds with female ICR mouse liver microsomes (0.5
e
f
mg protein/mL) for 30 min at 37 °C. Ranitidine. Metoprolol.
Physicochemical properties have an effect on metabolic
stability, which is an important factor in predicting absorption,
distribution, metabolism, and excretion (ADME) profiles. The
number of rotatable bonds, topological polar surface area
(tPSA), and log D values of the γ-lactam analogues were
predicted with the PreADME program22 (Table 2). As the
g
h
Atenolol. Chlorpromazine.
increase of lipophilicity generally leads to an increase in
membrane permeability. However, compounds 8d′ and 8f′,
which have long carbon chain linkers (n = 3 and 4, respectively)
and high log D values (2.71 and 2.16, respectively), showed low
permeability. It appears that too high a lipophilicity leads to
adhesion between the compounds and membrane. Metabolic
stability using mouse liver microsomes showed that lipophilicity
is related to metabolic stability. Highly lipophilic compounds
such as 8i, 8k, and 8f′ were easily degraded in the mouse liver
microsome with or without NADPH, whereas 8d and 8f, with
low lipophilicity, showed good metabolic stability (Table 3).
This is mainly caused by the hydrolysis and glucuronidation of
the hydroxamic acid.14 On the basis of these results, low
lipophilic compounds are more stable in liver microsomes but
less active in HDAC inhibition than high lipophilic compounds.
The in vivo pharmacokinetic studies showed that lipophilicity
and microsomal stability influence pharmacokinetic profiles
(Table 4). The low metabolic stability of 8i resulted in low oral
exposure (738.6 h·ng/mL) and oral bioavailability (10.7%).
However, the low lipophilic compounds 8d and 8f had similar
Cmax values (2302 and 2707.5 ng/mL, respectively) after oral
administration, but 8f was about three times higher than 8d for
oral exposure (3108.0 h/ng/mL compared to 1178.9 h/ng/
mL). The low lipophilicity of 8d lowered its cellular membrane
permeability (caco-2 cell 7.8 × 106 cm/s) and led to low oral
exposure. Overall, compound 8f had the best oral bioavailability
(44.6%) and other PK characteristics.
Table 2. Physicochemical Properties of the γ-Lactam
Analogues
compd
n
R
rotatable bond no.
tPSA
log D
8d
8f
1
1
1
1
1
1
2
2
2
3
4
4-F
5
5
5
5
6
6
6
6
6
7
8
69.64
69.94
69.64
69.64
93.43
93.43
115.46
115.46
69.64
69.64
69.64
0.5714
1.2620
2.0950
2.3962
0.2357
0.1968
0.6331
0.5942
2.2477
2.7017
2.1665
4-Br
8i
4-t-Bu
4-bi-CF3
3-CN
4-CN
3-NO2
4-NO2
4-t-Bu
4-t-Bu
4-Cl
8k
8l
8m
8n
8o
8v
8d′
8f′
carbon chain length increased, the number of rotatable bonds
and the lipophilicity increased. Most of the analogues had a
similar tPSA of 69.64, but the cyano and nitro analogues (8l−
o) had a large tPSA of 93.43 and 115.46, respectively. Larger
tPSA values probably have relatively smaller effects on cancer
cell growth inhibition due to the poor membrane permeability.
The higher lipophilicity of the analogues causes metabolic
instability in mouse liver microsomes, which leads to poor
ADME profiles.4 We selected some compounds with a range of
In conclusion, most of the synthetic γ-lactam-based HDAC
inhibitors were designed to improve metabolic stability. The γ-
C
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Journal of Medicinal Chemistry
Brief Article
Table 4. Pharmacokinetic Profiles of 8d, 8f, and 8i in Balb/c Mouse
8d
8f
8i
a
parameters
unit
IV
10
PO
IV
10
PO
IV
10
PO
dose
Tmax
Cmax
mg/kg
h
30
30
20
0.25
0.15
0.25
ng/mL
h·ng/mL
L/h/kg
L/kg
h
2302
1178.9
2707.5
3108.0
461.7
738.6
b
AUC0−inf
1284.9
7.8
2325.2
4.3
3440.3
2.9
CL
Vss
t1/2
F
1.8
4.1
1.6
2.6
1.1
2.8
3.1
7.8
13.6
%
30.6
44.6
10.7
a
PK parameters were based on the mean plasma concentration−time profiles of three animals per time point. PK parameters were calculated by
b
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.
1.7 M KONH2 (0.9 mL, 1.50 mmol) in anhydrous methanol (3.0 mL)
was stirred at 0 °C for 12 h. The mixture was concentrated in vacuo to
yield the crude product, which was purified by column chromatog-
raphy (silica gel, 5% methanol in dichloromethane), yielding 8f as an
orange solid (20 mg, 20%). 1H NMR (500 MHz, DMSO-d6) 10.43 (s,
1H), 8.71 (s, 1H), 7.52 (d, 2H, J = 8.5 Hz), 7.14 (d, 2H, J = 8.5 Hz),
6.83 (s, 1H), 4.51 (s, 2H), 3.80 (s, 2H), 2.40 (t, 2H, J = 6.7 Hz), 2.21
(t, 2H, J = 7.5 Hz). 13C NMR (125 MHz, DMSO-d6) δ 171.17, 168.89,
138.08, 137.89, 137.28, 132.17, 130.45, 120.98, 50.96, 45.35, 30.79,
22.20. ESI (m/z) 339, 341 (MH+), 361, 363 (MNa+). HRMS (FAB+)
calcd for C14H15BrN2O3 (MH+) 339.0344, found 339.0345. HPLC (tR:
purity = 9.52 min, 98.55%).
lactam core was introduced to reduce lipophilicity; the smaller
core provides better inhibition profiles by fitting in the narrow
binding pocket of HDAC. The diverse substituents on the cap
group were also introduced to block the NIH shift and improve
their microsomal stability. These HDAC inhibitors showed
good in vitro antitumor activities. The analogues with a one-
carbon linker between the γ-lactam core and cap group were
selected for their improved microsomal stability to reduce their
lipophilicity and number of rotatable bonds. Too low a
lipophilicity led to poor cell permeability and oral absorption.
Metabolic blockers on the bulky and hydrophobic cap group
are also an important parameter for metabolic stability.
Compound 8f shows both promising HDAC inhibitory activity
and good PK characteristics.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed information on the biological data, analytical data, and
the HPLC method for final compound 8 and recommended
compound characterization checklise (XLS). This material is
available free of charge via the Internet at http://pubs.acs.org.
EXPERIMENTAL SECTION
■
General Procedure for the Synthesis of 6−8. All chemicals,
anhydrous solvents, and other solvents were purchased from
commercial suppliers. The purity of final compounds, determined by
Agilent analytic column eclipse-XDB-C18 (250 mm × 4.6 mm, 5 μm,
or 150 mm × 4.6 mm, 5 μm) at a flow rate of 0.3 mL/min on Shimazu
HPLC 2010 instruments using a isocratic elution of 90% CH3CN/
H2O 0.1% formic acid or a gradient elution of 20−80% CH3CN in
0.1% aqueous formic acid over 30 min, was found to be >95%.
4-[Allyl-(4-bromo-benzyl)-carbamoyl]-pent-4-enoic Acid Methyl
Ester (6f). A mixture of 4f (400 mg, 1.77 mmol), monoacid (5, 460
mg, 2.12 mmol), EDC (602 mg, 2.30 mmol), and DMAP (89 mg, 0.53
mmol) in anhydrous dichloromethane (5 mL) was stirred at room
temperature for 12 h under Ar atmosphere. The mixture was quenched
by 10% HCl (aq) at pH 1 and extracted with dichloromethane (10 mL
× 2). The organic phase was washed with sodium bicarbonate (aq)
and brine, dried over sodium sulfate, and then filtered and
concentrated in vacuo to yield the crude product, which was purified
by column chromatography (silica gel, 1:2 ethyl acetate/hexane),
AUTHOR INFORMATION
Corresponding Author
*Phone: +82 2 2123 2882. Fax: +82 2 362 7265. E-mail:
gyoonhee@yonsei.ac.kr.
■
Author Contributions
▽These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by National Research Foundation
(2012-0000884), Korea Research WCU grant (R31-2008-000-
10086-0), and Brain Korea 21 project, Republic of Korea.
1
yielding 6f as a colorless oil (430 mg, 69%). H NMR (300 MHz,
CDCl3) δ 7.33−7.17 (m, 4H), 5.68 (br s, 1H), 5.10−4.98 (m, 4H),
3.62 (s, 3H), 3.37 (s, 2H), 2.57 (dd, 4H, JA = 8.3 Hz, JB = 5.4 Hz), 2.26
(s, 2H).
ABBREVIATIONS USED
■
HDAC, histone deacetylase; HAT, histone acetyltransferase;
QSAR, quantitative structure−activity relationship; tPSA,
topological polar surface area; EDC, N-(3-dimethylaminoprop-
yl)-N′-ethylcarbodiimide hydrochloride; DMAP, 4-(N,N-
dimethylamino)pyridine; PK, pharmacokinetics; ADME, ab-
sorption, distribution, metabolism, and excretion
3-[1-(4-Bromo-benzyl)-2-oxo-2,5-dihydro-1H-pyrrol-3-yl]-pro-
pionic Acid Methyl Ester (7f). A mixture of 6f (400 mg, 1.2 mmol)
and 2nd generation Grubbs’ catalyst (108 mg, 0.12 mmol) in
anhydrous dichloromethane (136 mL) was stirred at reflux for 12 h
under an Ar atmosphere in the dark. The mixture was concentrated in
vacuo to yield the crude product, which was purified by column
chromatography (silica gel, 1:1 ethyl acetate/hexane), yielding 9f as a
dark-gray oil (100 mg, 27%). 1H NMR (300 MHz, CDCl3) δ 7.29 (d,
2H, J = 8.4 Hz), 6.97 (d, 2H, J = 8.1 Hz), 6.57 (s, 1H), 4.43 (s, 2H),
3.62 (s, 3H), 3.53 (s, 2H), 2.50 (s, 2H), 2.45 (s, 2H).
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Journal of Medicinal Chemistry
Brief Article
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