N-Phenylbenzamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore

Article abstract of DOI:10.1002/cmdc.201500545

Persistent opening of the mitochondrial permeability transition pore (PTP), an inner membrane channel, leads to mitochondrial dysfunction and renders the PTP a therapeutic target for a host of life-threatening diseases. Herein, we report our effort toward identifying small-molecule inhibitors of this target through structure-activity relationship optimization studies, which led to the identification of several potent analogues around the N-phenylbenzamide compound series identified by high-throughput screening. In particular, compound 4 (3-(benzyloxy)-5-chloro-N-(4-(piperidin-1-ylmethyl)phenyl)benzamide) displayed noteworthy inhibitory activity in the mitochondrial swelling assay (EC₅₀=280 nm), poor-to-very-good physicochemical as well as in vitro pharmacokinetic properties, and conferred very high calcium retention capacity to mitochondria. From the data, we believe compound 4 in this series represents a promising lead for the development of PTP inhibitors of pharmacological relevance.

Full text of DOI:10.1002/cmdc.201500545

DOI: 10.1002/cmdc.201500545
Communications
N-Phenylbenzamides as Potent Inhibitors of the
Mitochondrial Permeability Transition Pore
+
[a]
+ [b]
[a]
[d]
ˇ
˙
Sudeshna Roy ,* Justina Sileikyte , Benjamin Neuenswander, Michael P. Hedrick,
Thomas D. Y. Chung,[e] Jeffrey AubØ,[a] Frank J. Schoenen,*[a] Michael A. Forte,*[c] and
Paolo Bernardi*[b]
Persistent opening of the mitochondrial permeability transition
pore (PTP), an inner membrane channel, leads to mitochondrial
dysfunction and renders the PTP a therapeutic target for
a host of life-threatening diseases. Herein, we report our effort
toward identifying small-molecule inhibitors of this target
through structure–activity relationship optimization studies,
which led to the identification of several potent analogues
around the N-phenylbenzamide compound series identified by
high-throughput screening. In particular, compound 4 (3-(ben-
zyloxy)-5-chloro-N-(4-(piperidin-1-ylmethyl)phenyl)benzamide)
displayed noteworthy inhibitory activity in the mitochondrial
swelling assay (EC50 =280 nm), poor-to-very-good physico-
chemical as well as in vitro pharmacokinetic properties, and
conferred very high calcium retention capacity to mitochon-
dria. From the data, we believe compound 4 in this series rep-
resents a promising lead for the development of PTP inhibitors
of pharmacological relevance.
ty of human pathologies.[1] Persistent opening of pore causes
depolarization, release of matrix Ca2+, cessation of oxidative
phosphorylation, and can result in mitochondrial swelling and
rupture of outer mitochondrial membrane (OMM) culminating
in cell death.[2] The molecular nature of the PTP has been clari-
fied only recently. Traditional models of PTP involving com-
plexes of OMM and IMM proteins have been discounted on
the basis of genetic studies showing the PTP can still form in
the absence of each of these proteins.[3–4] Other recent studies
have suggested that dimers of the FOF1 ATP synthase (F-ATP
synthase) are able to form channels with properties expected
for the PTP.[5] The precise nature of the molecular transition of
this complex from a Mg2+-dependent ATP synthetic device
into a Ca2+-dependent pore has yet to be determined. While
progress has been made on the molecular identity of PTP, our
ability to treat diseases in which inappropriate activation of
the PTP plays a role has been limited to the use of cyclospori-
ne A (CsA) and its analogues, Debio 025 and NIM811, which
act on the pore through their matrix receptor and PTP modula-
tor cyclophilin D (CyPD).[6]
It has been appreciated for some time that the mitochondrial
permeability transition pore (PTP), a channel of the inner mito-
chondrial membrane (IMM), can inappropriately open in a varie-
In vivo treatment has proven effective in models of collagen
VI muscular dystrophy[7–11] and provided encouraging results in
a pilot trial on patients affected by Ullrich congenital muscular
dystrophy and Bethlem myopathy.[12] The limit of cyclophilin in-
hibitors is that their target, CyPD, modulates the pore indirect-
ly, as shown by the fact that the PTP can still open when CyPD
has been genetically ablated.[13–16] Therefore, we[17] as well as
others[18] have carried out programs aimed at identifying novel
PTP inhibitors through the unbiased screening of compound li-
braries. Here, we report a novel series of potent small-molecule
inhibitors of the PTP that can be used as investigative tools,
and possibly, developed into therapeutics for PTP-based dis-
eases.
[a] Dr. S. Roy,++ + B. Neuenswander, Prof. J. AubØ,++ Dr. F. J. Schoenen
University of Kansas Specialized Chemistry Center
2034 Becker Drive, Lawrence, KS 66049 (USA)
E-mail: s.roy@unc.edu
schoenen@ku.edu
+
ˇ
[b] Dr. J. Sileikyte˙, Prof. P. Bernardi
CNR Neuroscience Institute and Department of Biomedical Sciences
University of Padova, Via Ugo Bassi 58/B, Padova, 35131 (Italy)
E-mail: bernardi@bio.unipd.it
[c] Prof. M. A. Forte
Vollum Institute, Oregon Health & Science University
3181 S.W. Sam Jackson Park Road, Portland, OR 97239 (USA)
E-mail: forte@ohsu.edu
A high-throughput screen of the US National Institutes of
Health Molecular Libraries Small Molecule Repository (MLSMR)
collection of 363827 compounds revealed benzamides 1 and 2
(Figure 1), among several other hits (data not presented), that
were chosen as starting points for structure–activity relation-
ship (SAR) studies based on their biological activity and physi-
cochemical properties.[19] The two keys assays that were used
to identify the hits were: (i) Ca2+-induced mitochondrial swel-
ling, a light-scattering-based assay that depends on PTP open-
ing that allowed us to identify the inhibitors and assess their
concentration–response, and (ii) rhodamine 123 (Rh123)
uptake, a counter screen assay that allowed us to identify com-
pounds that prevent Ca2+ uptake by interfering with develop-
[d] Dr. M. P. Hedrick
Conrad Prebys Center for Chemical Genomics
Sanford Burnham Prebys Medical Discovery Institute
10901 N. Torrey Pines Road, La Jolla, CA 92037 (USA)
[e] Dr. T. D. Y. Chung
Office of Translation to Practice, Mayo Clinic
Rochester, MN 55905 (USA)
++] Current address: Division of Chemical Biology and Medicinal Chemistry and
the Center for Integrative Chemical Biology and Drug Discovery, UNC Eshel-
man School of Pharmacy, University of North Carolina, Chapel Hill, NC
27599 (USA)
[
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500545.
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Figure 1. Probes 3 and 4 derived from screening hits 1 and 2.
ment or maintenance of the IMM potential, thereby preventing
mitochondrial swelling by preventing Ca2+ entry rather than
by inhibiting the PTP. Indeed, Ca2+ uptake through the selec-
tive mitochondrial Ca2+ uniporter in respiring mitochondria is
driven by the Ca2+ electrochemical gradient, which does not
form if the test compound inhibits respiration and/or has un-
coupling properties.
During the hit validation and SAR optimization stages of the
project, in addition to the mitochondrial swelling and Rh123
uptake assays, we used the Ca2+ retention capacity (CRC)
assay, a sensitive method that allows one to define precisely
the effect of inhibitors on the Ca2+-dependent propensity of
the pore to open.[20–21] To the best of our knowledge, this is
the first report of the inhibitory activity of N-phenylbenzamide
compounds toward the PTP.
Scheme 1. General procedure for synthesis of the benzamide analogues. Re-
agents and conditions: a) 1. K2CO3, DMF, rt, 24 h; 2. 10m aq. KOH, MeOH,
508C, 5 h (50–80% yield); b) PyBOP, i-Pr2NEt, DMF, mW, 20–30 min (40–70%
yield).
The benzamide analogues were assembled in a two-step
process (Scheme 1). 3-Hydroxybenzoic acid derivatives 5 were
treated with either substituted or unsubstituted benzyl bro-
mide along with potassium carbonate in DMF for 24 h to give
the corresponding benzyl 3-(benzyloxy)benzoate derivatives.
These benzoate esters were then saponified with 10m potassi-
um hydroxide in methanol under reflux conditions to yield cor-
responding carboxylic acids 6 in 50–80% yield. Subsequent
in Table 1. These studies revealed that replacement of the
pendant piperidine with piperizines (14 and 15) in the eastern
region was well-tolerated and afforded compounds with simi-
lar potency at preventing mitochondrial swelling that in-
creased the CRC of mitochondria as well. All other changes
failed to afford compounds with any significant improvement
in activity compared with 3 and 4 (Figure 1 and Table 2). As
noted in Table 1, most of the analogues showed activity in the
Rh123 uptake assay, suggestive of interference with the IMM,
an untoward side effect. The strategy to replace the chloro
with a fluoro substituent in the western aryl ring resulted in
analogues 9 and 10 with a much-improved profile in the coun-
ter screen and CRC assays.
benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluoro-
phosphate (PyBOP)-mediated amide coupling reactions with
various substituted anilines 7 in the presence of Hünig’s base
and DMF under microwave conditions afforded the corre-
sponding analogues (8) of the benzamide series.
An SAR campaign was carried out around the hit molecules,
1 and 2, that led to the identification of a couple of very
potent analogues, 3 and 4, out of 82 analogues that were
screened.[22] Results for a representative SAR study that was
mainly focused on investigating the 6-membered heterocyclic
pendant attached to the eastern aryl ring (12–15) as well as on
studying the effect of halogen substitution around the western
half of the structure (9–11 and 16–20) have been summarized
Clearly compounds 3 and 4 (Figure 1and Table 2) demon-
strated the best activity in the swelling and CRC assays. Of the
two compounds, 4 showed slightly superior activity and hence
was chosen for extensive biological characterization using a va-
riety of established in vitro assays. First, we examined PTP-de-
pendent swelling in isolated mouse liver mitochondria follow-
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Table 1. Structure–activity relationship optimization of representative analogues.[a]
Compd
CRC/CRC0
at 12.5 mm
EC50 [mm]
mitochondrial swelling
Compd
CRC/CRC0
at 12.5 mm
EC50 [mm]
mitochondrial swelling
Rh123 uptake
Rh123 uptake
9
10.8Æ0.6
13.3Æ1.0
2.26Æ0.11
8.81Æ0.39
14.1Æ1.1
16.1Æ1.5
18.3Æ1.3
0.521Æ0.046
0.372Æ0.028
3.60Æ0.17
58.6Æ2.9
>100.00
75.7Æ1.8
>100
16
17
18
19
6.50Æ1.20
2.37Æ0.41
5.91Æ0.38
2.59Æ0.54
10.1Æ0.8
1.19Æ0.33
4.00Æ0.66
1.24Æ0.39
1.39Æ0.17
0.49Æ0.08
0.073Æ0.007
0.149Æ0.009
23.2Æ1.3
>100
10
11
12
13
14
15
24.7Æ1.5
0.403Æ0.031
0.423Æ0.028
0.286Æ0.027
0.206Æ0.021
>100
49.6Æ4.3
42.6Æ3.1
46.4Æ3.2
20
28.6Æ1.1
>100
>100
GNX-865
Cyclosporin A
7.67Æ0.35
4.40Æ0.24
[a] Data are the averageÆSEM of ꢀ3 experiments.
Table 2. Comparison of activity and physicochemical properties, and summary of in vitro pharmacology of compounds 3 and 4.
Parameter
Comparative data
Assessment
Result
compd 3
compd 4
compd 3
compd 4
CRC/CRC0 at 12.5 mm
17.2Æ0.6
19.5Æ1.7
Aqueous solubility in 1xPBS
26.8/7.0/0.2
[61.6/16.1/0.5]
83
22.8/22.6/0.64
[52.5/52.1/1.5]
91
pH 5.0/6.2/7.4 mgmLÀ1 [mm][a]
Chemical stability with 5”DTT
%parent remaining after 8 h[a]
Aqueous stability (1:1 PBS/acetonitrile)
% parent remaining after 48 h[b]
Plasma-protein binding [% bound for
human; mouse (1 mm/10 mm)][a]
Plasma stability (% remaining
after 3 h) human; mouse[a]
EC50 [mm]
mitochondrial swelling
EC50 [mm]
Rh123 uptake
MW[c] [Da]
0.398Æ0.025
0.280Æ0.024
36.5Æ1.82
24.5Æ1.12
89.3
93.4
434.9
41.5
6.2
2
434.9
41.5
6.4
2
99.0/99.0;
99.0/98.8
44; 57
99.2/99.2;
99.3/98.3
39; 35
tPSA[c]
cLogP[c]
HBA[d]
PAMPA permeability, Pe (”10À6 cmsÀ1
)
1281/1425/1276
381
1300/1339/und
113
Donor pH 5.0/6.2/7.4 Acceptor pH 7.4[a]
BBB PAMPA Permeability, Pe (”10À6 cmsÀ1
Donor pH 7.4 Acceptor pH 7.4[a]
)
HBD[d]
2
2
Hepatic microsome stability (% remaining after 1 h)
human; mouse (+NADPH/-NADPH)[a]
Toxicity towards Fa2N-4 immortalized
human hepatocytes LC50 [mm][a]
65/75;
9.6/80
30
61/67;
30/74
32
Heavy atoms[e]
LE[f]
31
31
0.29
0.30
[a] Data collected by David B. Terry at the Conrad Prebys Sanford Burnham Medical Research Institute. [b] Data collected by Patrick Porubsky at the Univer-
sity of Kansas Analysis, Purification, and Compound Management Core, Specialized Chemistry Center. [c] Molecular weight (MW), topological polar surface
area (tPSA) lipophilicity (cLogP) data were generated using CambridgeSoft ChemBioDraw, version 12. [d] Hydrogen-bond acceptors (HBA) and hydrogen-
bond donors (HBD); data were calculated using SYBYL 8.0, Tripos Associates, St. Louis, MO (USA), 2010. [e] Data was calculated using Marvin 15.3.23.0,
2015, ChemAxon. [f] Ligand efficiency (LE).
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Figure 2. Effect of 4 on the permeability transition pore (PTP) and cell viability. (A) Full symbols: 4 prevents mitochondrial swelling induced with 50 mm Ca2+
in a concentration-dependent manner; open symbols: interference with Rh123 uptake upon treatment with compound 4; (B) Concentration response of 4-to-
solvent CRC ratios of wild type (squares) and CyPD knock-out (triangles) mitochondria; (C) respiratory control ratios (RCR) of 4 or solvent-treated mouse liver
mitochondria; (D) Effect of 4 with known chemical inducers of the PTP. Mitochondria were supplemented with 10 mm Ca2+ only, traces (a); 10 mm Ca2+ and
with, as indicated, 7 mm PhAsO, 2 mm diamide, 7 mm Cu(OP)2 or 2 mm N-ethylmaleimide traces (b)–(d); in traces (c) and (d) 3.125 mm CsA or 4, respectively,
were also present; (A)–(D) assays were performed on isolated mouse liver mitochondria. (E) 4-to-solvent CRC ratios of permeabilized HeLa cells (0.8 million/
condition). (F) Oxygen-consumption rates (OCR) of HeLa cells; treatments were made as indicated. (G) Interference with HeLa cell proliferation after 24 h treat-
ment with indicated concentration of 4. Data are a representative (D, F) and an average ÆSEM of ꢀ4 experiments.
ing uptake of Ca2+ (50 mm). In a full concentration–response
study employing concentrations ranging from 12.2 nm to
1.56 mm, inhibition of swelling was demonstrated with an EC50
value of 0.280Æ0.024 mm (Figure 2A), which is in the same
order of magnitude as for standard PTP inhibitors CsA and
GNX-865,[18] a cinnamic anilide identified in a high-throughput
screen similar to the one employed here (Table 1).
to open in response to 4 in CyPD-null mouse liver mitochon-
dria, which lack the mitochondrial CsA binding site. We ob-
served a sevenfold increase in CRC in these mitochondria
(which are already partially desensitized due to the absence of
CyPD), suggesting that benzamides have a different molecular
target. Maximal PTP inhibition by 4, as assessed by both mito-
chondrial swelling and CRC assays, occurred at concentrations
higher than those observed with diarylisoxazole-3-carboxa-
mides, the other class of inhibitors that was identified in the
high-throughput screen.[17]
We next tested the CRC, which allows quantification of the
amount of Ca2+ necessary to open the pore. At 12.5 mm, a com-
pound-to-solvent CRC ratio of 19 was generated, the highest
reported in the literature to date (Figure 2B). We also observed
that the maximum CRC ratios of isolated mouse liver mito-
chondria treated with 4 are about four times higher than ones
treated with CsA, which implied that the compounds might be
acting on different biological targets. To test this hypothesis,
we investigated the threshold Ca2+ load required for the PTP
We also tested whether compound 4 is protective against
known inducers of the PTP that trigger pore opening by induc-
ing oxidative stress. Isolated mouse liver mitochondria were
loaded with 10 mm Ca2+ (which is not able to induce PTP
opening per se, traces a in Figure 2D) and then challenged
with reagents that modify distinct classes of redox-sensitive
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thiols (-SH) (traces b–d in Figure 2D), which affect the PTP sen-
sitivity to Ca2+ and trigger pore opening: (i) matrix -SH groups
that react with diamide and phenylarsine oxide (PhAsO),[23]
(ii) inner membrane external thiols that react with copper(II)
bis(1,10-phenanthroline) complex [Cu(OP)2], and (iii) outer
membrane N-ethylmaleimide (NEM)-reactive thiols.[24–25] In all
cases, the PTP transition from the closed to open conformation
was delayed by 3.12 mm CsA (traces c in Figure 2D) and pro-
hibited by the same concentration of 4 (traces d in Figure 2D),
as assessed in mitochondrial swelling assays (Figure 2D).
Therefore, these inhibitors are effective in preventing PTP
opening regardless of the methods used to induce it.
strated moderate plasma stability and very high plasma pro-
tein binding. A parallel artificial membrane permeability assay
(PAMPA) was used as an in vitro model for passive transport,
and blood–brain barrier (BBB) permeability was used to predict
central nervous system (CNS) penetration. Compounds 3 and 4
demonstrated very good permeability in both of these assays.
Metabolic liability was apparent for both 3 and 4 after 1 h ex-
posure, especially in mouse liver microsomes in the presence
of NADPH. The key analogues showed some degree of toxicity
towards Fa2N-4 immortalized human hepatocytes, having LC50
values of 30 and 32 mm with 75-fold and 114-fold selectivity
compared to the EC50 values for the mitochondrial swelling for
3 and 4, respectively. Activity of these key analogues in the
Rh123 uptake and cytotoxicity assays suggests a possible
trend, which will be monitored during future studies.
In all the above-mentioned studies, murine mitochondria
were used for identifying and optimizing PTP inhibitors. How-
ever, due to the existence of species-specific PTP regula-
tion,[26–28] we deemed it essential to test whether an inhibitory
effect could be also detected in human mitochondria. As dem-
onstrated in Figure 2E, compound 4 induced a concentration-
dependent increase in the CRC of permeabilized HeLa cells.
It has been suggested that the PTP forms from a unique
conformation of dimers (or higher oligomeric forms) of F-ATP
synthase.[5] In light of these findings, we investigated whether
4 also affects ATP synthesis, which would be potentially an un-
desirable side effect. Mitochondrial respiration was measured
both in isolated mouse liver mitochondria and in intact HeLa
cells in the presence or absence of 4. No significant differences
were observed in respiratory control ratios, FCCP-stimulated,
and oligomycin-sensitive respiration in either isolated mouse
liver mitochondria (Figure 2C and data not shown) or HeLa
cells (Figure 2F) at low concentrations of 4 (i.e., ꢁ10 mm). A
decrease in the IMM potential (Figure 2A) and respiratory con-
trol ratio (Figure 2C) in isolated mouse liver mitochondria (re-
flecting a decreased ability to generate ATP) and of oxygen-
consumption rate in HeLa cells was observed only at higher
concentrations of 4. These findings confirm that compound 4
shows no effect on ATP synthesis and HeLa cell proliferation at
low concentrations (5 mm and ꢁ10 mm, respectively). However,
at concentrations above 10 mm, it might cause cellular toxicity,
as confirmed in HeLa (Figure 2G) and Fa2N-4 cells (last entry in
Table 2).
In summary, SAR optimization studies around the N-phenyl-
benzamide scaffold led to the discovery of potent inhibitors of
the PTP conferring mitochondria with a very high CRC, which
is a robust measure of inhibition of the PTP. Compound 4 con-
fers a CRC ratio of 19 (the highest reported for a PTP inhibitor
to date) and showed promising inhibition of swelling, with an
EC50 value of 280 nm. We carried out biological characterization
of the PTP through a series of in vitro assays and found that
compound 4 was protective against both Ca2+- and oxidative-
stress-triggered pore opening, and that it inhibits both the
mouse and human PTP. Moreover, we found that the biological
target for this compound series is not CyPD, and that no inhib-
ition of F-ATP synthase is observed at concentrations that fully
inhibit the PTP. Higher concentration (>10 mm) of compound 4
showed interference with the IMM potential and cytotoxicity.
Overall, this compound series, represented by compounds 3
and 4, possesses a promising in vitro pharmacological profile,
poor-to-good aqueous solubility (pH-dependent), and good
permeability. Future studies will involve additional optimization
in order to decrease compound toxicity and provide analogues
suitable for in vivo testing for efficacy in relevant disease
models.
Acknowledgements
We next examined the physicochemical and in vitro pharma-
cokinetic properties of analogues 3 and 4 (Figure 1 and
Table 2). Analogues 3 and 4 displayed promising physicochem-
ical parameters, possessing a desirable number of hydrogen-
bond donors and acceptors, decreased topological polar sur-
face area, a molecular weight of less than 500 Da and moder-
ately favorable ligand efficiency. Although the cLogP values
were generally high for 3 and 4 (above 6), analogues 14 and
15 had much decreased cLogP values around 4.9.
These key analogues were characterized further for in vitro
pharmacology to create a baseline profile for future structure–
property relationship (SPR) optimization efforts. Overall, ana-
logues 3 and 4 demonstrated poor-to-very-good in vitro phar-
macokinetic features, having poor-to-good aqueous solubility
(pH-dependent) and good chemical stability in the presence of
excess dithiothreitol (DTT), confirming that these inhibitors do
not possess reactive functionality. Compounds 3 and 4 demon-
The authors gratefully acknowledge funding from the US Nation-
al Institutes of Health (NIH) and Telethon–Italy. Chemistry efforts
at the University of Kansas Specialized Chemistry Centerwere sup-
ported by NIH grant U54HG005031awarded to J. AubØ. Support
for the University of Kansas NMR instrumentation was provided
by an NIH Shared Instrumentation Grant (S10RR024664) and
a US National Science Foundation (NSF) Major Research Instru-
mentation Grant (0320648). The authors thank Patrick Porubsky
(University of Kansas) for compound management, and for aque-
ous and chemical stability data. Initial assay validation, high-
throughput screening, and hit confirmation efforts at the Conrad
Prebys Center for Chemical Genomics were supported by NIH
grant U54HG005033 awarded to J.C. Reed. Funding for the bio-
logical assays was supported by NIH grants R03A033978 award-
ed to M. Forte, P. Bernardi and U54HG005031-05S1 awarded to J.
AubØ, and by Telethon–Italy grant GGP14037 to P. Bernardi.
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Keywords: calcium retention capacity
mitochondrial swelling · N-phenylbenzamides · permeability
·
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·
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Received: November 20, 2015
Published online on December 23, 2015
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