Design, Synthesis, and Biological Evaluation of Imidazopyrazinone Derivatives as Antagonists of Inhibitor of Apoptosis Proteins (IAPs)

Article abstract of DOI:10.1002/bkcs.12271

Apoptosis inhibitor (IAP) proteins are overexpressed in many cancers and implicated in tumor growth, so the development of antagonist that disrupts with the binding of IAP to their partner protein is a promising therapeutic strategy. In an effort to incre

Full text of DOI:10.1002/bkcs.12271

Article
DOI: 10.1002/bkcs.12271
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J. Kim et al.
KOREAN CHEMICAL SOCIETY
Design, Synthesis, and Biological Evaluation of Imidazopyrazinone
Derivatives as Antagonists of Inhibitor of Apoptosis Proteins (IAPs)
Jisook Kim ,† Inhwan Bae
,
‡,§ Jiyoung Song,‡ Younghoon Kim,‡ Younggil Ahn,‡
§,
Hyun-Ju Park,† Ha Hyung Kim,¶, and Dae Kyong Kim
*
*
†School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea
‡Hanmi Research Center, Hanmi Pharm. Co. Ltd., Gyeonggi-Do 18469, Republic of Korea
§Department of Environmental and Health Chemistry, College of Pharmacy, Chung-Ang University,
Seoul 06974, Republic of Korea. *E-mail: kimdk@cau.ac.kr
¶Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, Seoul 06974,
Republic of Korea. *E-mail: hahyung@cau.ac.kr
Received November 18, 2020, Accepted March 8, 2021
Apoptosis inhibitor (IAP) proteins are overexpressed in many cancers and implicated in tumor growth, so
the development of antagonist that disrupts with the binding of IAP to their partner protein is a promising
therapeutic strategy. In an effort to increase cellular activity and improve favorable drug-like properties,
we newly designed and synthesized monovalent analogues based on imidazopyrazinone structure of 9.
Optimization of cellular potency led to the identification of 17, which showed increase of submicromolar
activity (GI50 = 234 nM) and caspase-3 activation (6.3-fold) in MDA-MB-231 breast cancer cells. These
findings clearly show the potential for 17 as a promising monovalent antagonist for the development of an
effective anticancer treatment.
Keywords: Imidazopyrazinone, Inhibitors of apoptosis proteins antagonist, Second mitochondrial activa-
tor of caspases, Apoptosis
Introduction
binding
interaction
is
Ala(P1)-Val(P2)-Pro(P3)-Ile
(P4) tetramer, 1, and the variety of modified monovalent
antagonists have been developed.
The development of an IAP antagonists, called a mono-
mers or monovalents, are based on the Amgen-IAP, 2 publi-
shed in 2004 by Amgen’s research group and the structure
Programmed cell death or apoptosis is closely related to
inhibitory of apoptosis protein (IAP) and regulates cell
death. These apoptosis inhibiting proteins are known as
proteins involved in the regulation of various signaling
pathways. Some of the baculovirus inhibitor of apoptosis
protein repeat (BIR) domains of IAP bind to caspases,
which are proteases that induce and regulate apoptosis of
cells, thereby inhibiting cell death.1,2 In addition, it has
been revealed that inhibitory of apoptosis protein plays an
additional role in addition to the negative feedback of apo-
ptosis, including cell differentiation, cell motility, migra-
tion, invasion, and metastasis.3–6 Therefore, the
development of new IAP antagonists can serve the potential
of new therapeutic options, and can provide an opportunity
to increase clinical efficacy and resolve refractory and resis-
tance causes through combination therapy with approval
agents.7–9
of Novartis’ LBW242,
3
announced in 2007
(Figure 1).10,11 The key sequence, Ala-Val-Pro-Ile (AVPI)
had no cell permeability, therefore, Amgen-IAP, 2 with
modification of Ile(P4), and LBW242, 3 through cycliza-
tion between Pro(P3) and Ile(P4) developed as the IAP
antagonists with cellular activities. However, these antago-
nists have an unmet need for new IAP antagonists with
improved activity due to low cellular activity. In this paper,
we investigated the structure–activity relationship of a
novel imidazopyrazinone-based derivatives and character-
ized its pharmacokinetics and pharmacodynamics as a
potent SMAC mimetics.
Through the previous study of the structure of the second
mitochondrial activator of caspases (SMAC) protein that
induces apoptosis mechanism in vivo, various studies on
the natural IAP-binding amino acid sequence that can bind
to the BIR3 and BIR2 domains of IAP have been con-
ducted. Based on the results of research on the binding
structure of SMAC and BIR domains, it was found that the
key amino acid sequence of SMAC participating in the
Experimental
General Information. Reagents and solvents were pur-
chased from commercial sources and were used without
purification. Reactions were monitored by thin-layer chro-
matography. All purifications were proceed flash chroma-
tography system (ISCO, combiflash Rf+; Lincoln, NE,
USA). H NMR and 13C NMR spectra were recorded on
1
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Figure 1. Structures of AVPI and monovalent IAP antagonists.
1
Bruker, Avance DPX 300 (300 MHz for H, 75 MHz for
cells with a recombinant vector prepared from pET28a vec-
tor (Novagen) using standard DNA cloning process and
polymerase chain reaction (PCR) method (see Sambrook &
Russell, Molecular cloning, Chapter 1. Third edition).
13C; Seattle, WA, USA). Mass spectra were obtained on
Waters Aquity UPLC/QTOF. High-performance liquid
chromatography was used Agilent, 1200 series using a
Gemini-NX C18 4.6 × 150 mm, 3 μm column eluting with
a mixture of acetonitrile and Methanol (80:20 [vol/vol %])
containing pH 7.0 buffer (25 mM Na2HPO4, pH 7.0 with
Caspase Activation Assay. The cells were treated with a
solution of the analysis samples and media (1:1) in an
amount of l00 μL/well using Caspase-Glo™ 3/7 Assay Kit
(Cat#. 08090; Promega, USA). After the cells were incu-
bated at 37 ꢀC for 2 h, luminescence was measured using
Infinite™ M1000 multireader (Tecan).
H3PO4) with
a
15 min gradient of 55–40% and
Phenomenex Kinetex C18, 4.6 × 100 mm, 2.6 μm column
eluting with a mixture of acetonitrile and Methanol (80:20
[vol/vol %]) containing pH 7.5 buffer (50 mM ammonium
formate, pH 7.5 with ammonia) with a 20 min gradient of
55–15%. Liquid chromatography–mass spectrometry results
were obtained on Waters QTOF (SYNAPT G2) with
electrospray ionization.
Cell Growth Inhibition Assay. MDA-MB-231 breast can-
cer cells (ATCC # HTB-26) were plated in 96-well plates
at a density of 1.0 × 104 to 1.5 × 104 cells/well. After 24 h,
the cells were treated with test compounds at a concentra-
tion of 0 μM, 0.5 μM, l μM, and 5 μM, respectively, for
12 h to caspase 3/7 and for 24 h to caspase 9. Then, media
were removed and the cells were washed two times with
phosphate-buffered saline at 4 ꢀC. The half maximal
growth inhibition concentration (GI50) was determined
using GraphPad Prism (GraphPad Software; San Diego,
CA, USA).
BIR3 Binding Affinity Assay. X-chromosome-linked
inhibitors of apoptosis proteins (XIAP) baculovirus inhibi-
tors of apoptosis proteins repeat (BIR) prediluted by
1.25 μM was placed into a black, round-bottom 96-well
plate at 5 μL/well, and 4F (AbuRPF-K(5-Fam)-NH2)
prediluted by 0.0625 μM was added thereto at 10 μL/well
under a dark condition. At the time, XIAP BIR is a 241–
356th amino acid residue of human XIAP protein, which
was prepared by transforming Escherichia coli BL21(DE3)
Animal Studies. The Sprague-Dawley rat (SD rat)
received imidazopyrazinone derivatives either by intra-
venous (iv) injection (1 mg/kg) in the tail vein, or by
oral (po) administration (3 mg/kg). The pharmacokinetic
parameters (area under curve (AUC)0–24, AUCinf, and
half-life) of imidazopyrazinone derivatives were calcu-
lated by noncompartment assay via WinNonLin®
(Certara, NJ, USA). Cmax and Tmax were obtained
directly from the observed data. Bioavailability
(BA) was calculated as follows: BA (%) = (AUC0–24
× Doseiv)/(AUC0–24 iv × Dosepo) × 100.
po
The SD male rat (8 weeks age, n = 3/group) were
obtained from the Koatech, Inc. (Pyeongtaek, Korea).
The experimental processes on animals, and animals
care were carried out in accordance to study protocol
approved by the Hanmi Research Center Institutional
Animal Care and Use Committee as Animal Ethics
Committee.
Results and Discussion
Previously reported LBW242, 3 derives a new 5–6 mem-
bered bicyclic template by changing AVPI’s Proline and
Isoleucine cyclization and Valine to cyclohexylglycine
(Chg). However, LBW242, 3 exhibits moderate binding
Figure 2. General design method of compound 9.
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out to propose potent and cell-permeable peptidomimetics.
In order to improve cellular activity, we designed a new
derivative 9 with the insertion of the carbonyl groups in
LBW242 to reduce flexibility and to introduce a new
R. Most monovalent structures induce an “opposite U-
shaped” binding conformation, which bind to the IAP BIR3
domain through its hydrophobic interaction with Trp323.
Additionally, the structure of the R2-introduced compounds
was designed as an “opposite Y-shaped” binding conforma-
tion to have π-π stacking interaction with Tyr324 to
improve binding affinity (Figure 2).
The general synthetic procedure for the preparation of
structure 9 is shown in Scheme 1. Commercially available
dimethylacet aldehyde, 4 was coupled with R1-NH2 via
reductive amination to give 5 with a good yield. The sec-
ondary amine 5 was coupled to unnatural amino acid with
R2-group using 1-[Bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
(HATU) amide coupling reaction, which, after Fmoc-
deprotection with piperidine, yielded 6. The 7 was obtained
through coupling and deprotection in the same way as Fmoc-
Gly-OH. The primary amine 7 was coupled to Fmoc-
Chg-OH, which, after cyclized with formic acid using
sealed tube gave 8. At this time, it should be noted that if
Scheme 1. Synthesis of the general compound 9. Reagents and
conditions: (a) R1-NH2, NaCNBH3, MeOH, rt., 12 h; (b) Fmoc-
amino acid, HATU, DIPEA, DCM, rt., 4 h; (c) 10% piperidine/
DCM, rt., 2 h; (d) Fmoc-Gly-OH, EDCI, HOBT, DIPEA, DCM,
rt., 12 h; (e) 10% piperidine/DCM, rt., 2 h; (f) Fmoc-Chg-OH,
EDCI, HOBT, DIPEA, DCM, rt., 4 h; (g) formic acid, 80 ꢀC, 3 h;
(h) 10% piperidine/DCM, rt., 2 h; (i) Boc-MeAla-OH, EDCI,
HOBT, DIPEA, DCM, rt., 4 h; (j) 4 M HCl/dioxane, rt., 2 h.
ꢀ
the external reaction temperature exceeds 100 C or less
than 60 ꢀC, undesirable compounds are obtained. The
8 was sequentially deprotected against Fmoc and coupled
with Boc-MeAla-OH, followed by deprotection of Boc
using 4 M HCl/Dioxane to yield 9.
affinity, and peptides like this have a common drawback,
that is, poor cell permeability, similar to other peptidic
compounds. Therefore, extensive modifications using
reduction or insertion of other binding partner were carried
Table 1. Cell growth inhibitory activity of compounds 10–24 against MDA-MB-231 and Hs27 cell lines.
GI50 (nM)
Cpd.
R1
R2
MDA-MB-231
Hs27
2
-
-
276
868
274
2758
657
639
283
1076
234
818
379
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Benzyl
Phenethyl
Phenylpropyl
4-fluorophenthyl
4-fluorophenthyl
Phenethyl
Phenethyl
Phenethyl
Phenethyl
Phenethyl
Phenethyl
Benzyl
4-chlorobenzyl
4-methoxybenzyl
4-methoxyphenethyl
Benzyl
Benzyl
Benzyl
Benzyl
4-methoxybenzyl
4-hydroxybenzyl
4-methoxybenzyl
4-methylbenzyl
1H-indol-3-ylmethyl
Naphthalene-1-ylmethyl
Naphthalene-2-ylmethyl
Naphthalene-2-ylmethyl
Naphthalene-2-ylmethyl
Naphthalene-2-ylmethyl
Naphthalene-2-ylmethyl
170
1940
2774
4584
2008
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Table 2. Microsomal stability of 17 and 20.
Remaining % of parent molecule at 60 min
Human Dog Rat
22
24
affinity assay was performed to obtain Ki values of 315 and
356 nM for compound 2 and compound 11, respectively.
These increased activities were thought to have various
interactions with Trp323 or Tyr324 of the BIR3 domain by
the introduction of the R2 group. In order to identify other
substituents of phenethyl length, 13 and 14 having
4-fluorophenthyl were tested, but lower cell line activity
than 11 was confirmed. The phenethyl was fixed on R1-
position, and various substituents for R2 were evaluated. As
a result of comparing compounds 15–20, it was possible to
confirm 17 (GI50 = 234 nM) and 20 (GI50 = 170 nM),
which are more potential than 11 (GI50 = 274 nM). There-
fore, naphthalene-2-ylmethyl, which is R2, which has the
best activity, was immobilized and compounds 21–24 in
which R1 was substituted were tested. However, all of these
compounds 21–24 showed cellular activity of more than
1000 nM. Therefore, among these compounds, sequential
evaluation was performed on 17 and 20. To derive lead
compound, 17 and 20 having good cellular activity were
tested in microsomal stability and CYP isozyme. Both com-
pounds 17 and 20 showed moderate microsomal stability as
the lead compound criteria (Table 2).
In CYP inhibition assay, 17 showed strong inhibition of
CYP3A4 and weak four major CYP isozymes, whereas 20
strongly inhibited CYP3A4 and moderate two major CYP
isozymes (Table 3). These results indicate that 17 has a
lower risk of drug–drug interactions than 20, although both
showed strong inhibition of 3A4.
According to similar CYP inhibition and microsomal sta-
bility results, caspase-3 activation assay was performed in
MDA-MB-231 cells with 1 μM concentration of com-
pounds to select lead compound. As results of the assay,
2 increased 5.6-fold activity compared to the control group,
whereas compounds 17 and 20 increased 6.3-fold and
3.8-fold, respectively. Therefore, 17 was nominated as the
lead compound for developing compound and the follow-
ing evaluation was conducted.
Cpd.
17
20
32
46
9
25
Table 3. In vitro evaluation of 17 and 20 in various CYP
isozymes.
CYP isozymes (IC50, μM)
Cpd.
1A2
2.29
>50.0
39.38
2C9
2C19
2D6
3A4
P.C.a
17
42.18
42.18
19.68
>50.0
>50.0
23.78
>50.0
>50.0
36.82
<0.0062
<0.62
<0.62
20
a P.C., positive control; Furafylline (1A2), sulfaphenazole (2C9),
tranylcypromine (2C19), Qunidine (2D6), Ketoconazole (3A4).
This privileged structure 9 was analyzed by solution-
1
phase 2D-NMR spectroscopy (1H-1H COESY and H-1H
NOESY) to conform on accurate structure of 5–6 bicyclic
template and chirality of junction proton.12 Compounds
10–23 were prepared according to the same synthetic route
as Scheme 1. More detailed synthetic methods and condi-
tions of representative compound 17 are shown in
Supporting Information.
We newly designed and synthesized monovalent IAP
antagonists based on imidazopyrazinone, and these com-
pounds were assessed for MDA-MB-231 breast cancer cells
and Hs27 foreskin normal cells. All of compounds 10–24
did not show cellular activities with more than >10 000 nM
to Hs27 (Table 1
). In addition, 3 as reference compound showed the cel-
lular activity of over 10 000 nM against MDA-MB-231. In
order to determine the optimized chain length of R1, the
cell growth inhibition of compounds 10–12, which the sim-
plest R2 is benzyl, were compared. As a result of compari-
son using 2 as a control material, 11 into which a
phenethyl group was introduced showed the most similar
activity. In order to confirm the binding ability of the active
compounds to XIAP-BIR3 was compared, BIR3 binding
We subsequently conducted an in vivo pharmacokinetic
study using the SD rat model (Table 4). Oral administra-
tion of 3 mg/kg dose was found that the absolute bioavail-
ability was 1.8% after oral administration, while the
values of AUC0–24, Cmax, and half-life were 10.0 ng h/
mL, 1.7 ng/mL, and 56.6 h, respectively. Following
1 mg/kg iv dose of 17, Clearance (Cl) was 5.2 mL/min/
Table 4. Pharmacokinetic parameters of compound 17 following a single intravenous (1 mg/kg) or oral (3 mg/kg) administration in SD
rat (n = 3).
Route Dosea (mg/kg) AUC0–24 (ng h/mL) C0 (ng/mL) Cmax (ng/mL) Tmax (h)
t1/2 (h)
8.6 Æ 0.5 64.6 Æ 8.7
1.7 Æ 0.8 2.0 Æ 0.0 56.6 Æ 53.7 3114 Æ 2078 141.7 Æ 109.0
Vd (L/kg)
Cl (L/h*kg) BA (%)
iv.
1
3
189.2 Æ 14.2
10.0 Æ 3.2
473.3 Æ 39.0
-
-
5.2 Æ 0.4
-
1.8
po.
-
a As amount of free form.
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kg, the t1/2 was 8.6 h, and the volume of distribution (Vd)
was 64.6 L/kg. Altogether, our results showed that 17
presented moderate clearance and high distribution rates
in SD rat by iv dosing. Therefore, it is necessary to inves-
tigate whether the dosing regimen is iv bolus or iv infu-
sion depending on the convenience of administration with
combination therapy.
Conflict of Interest. The authors declare no conflicts of
interest.
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
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In summary, we developed a synthesis of a novel series of
SMAC mimetics using a imidazopyrazinone-based struc-
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