Structure-activity relationships of furazano[3,4-b]pyrazines as mitochondrial uncouplers

Article abstract of DOI:10.1016/j.bmcl.2015.06.040

Chemical mitochondrial uncouplers are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. These uncouplers have potential

Full text of DOI:10.1016/j.bmcl.2015.06.040

Bioorganic & Medicinal Chemistry Letters 25 (2015) 4858–4861
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of furazano[3,4-b]pyrazines
as mitochondrial uncouplers
Brandon M. Kenwood a, , Joseph A. Calderone b, , Evan P. Taddeo a, Kyle L. Hoehn a,c,d,e,f, Webster L. Santos b,
⇑
a Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
b Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061, USA
c Department of Medicine, University of Virginia, Charlottesville VA 22908, USA
d Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA
e Emily Couric Clinical Cancer Center, University of Virginia, Charlottesville, VA 22908, USA
f Department of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW 2052, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical mitochondrial uncouplers are lipophilic weak acids that transport protons into the mitochon-
drial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation
from ATP production. These uncouplers have potential for the treatment of diseases such as obesity,
Parkinson’s disease, and aging. We have previously identified a novel mitochondrial protonophore,
named BAM15, which stimulates mitochondrial respiration across a broad dosing range compared to car-
bonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Herein, we report our investigations on the
structure–activity relationship profile of BAM15. Our studies demonstrate the importance of the furazan,
pyrazine, and aniline rings as well as pKa in maintaining its effective protonophore activity.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 20 May 2015
Revised 9 June 2015
Accepted 11 June 2015
Available online 16 June 2015
Keywords:
Mitochondrial uncoupler
Mitochondrial bioenergetics
Protonophore
Structure–activity relationships
Pyrazines
Mitochondrial uncouplers or protonophores are small molecule
organic compounds, typically lipophilic weak acids, that utilize the
pH gradient in the mitochondria to shuttle protons from the inner
membrane space to the mitochondrial matrix.1 Their uncoupling
activity is dependent on the efficiency in which they shuttle pro-
tons across the lipid bilayer of the mitochondrial inner membrane,2
which is determined by the diffusion of the uncoupler into and out
of a phospholipid bilayer and the efficiency by which the protono-
phore transports protons in the intermembrane space and releases
them into the mitochondrial matrix. The net result is the dissipa-
tion of a proton gradient across the mitochondrial inner membrane
that is associated with an increase in energy expenditure, a phe-
nomenon that has implications in several disease states. For exam-
ple, mitochondrial uncouplers have therapeutic potential as an
anti-obesity drugs1,2 or as treatments of disorders linked to mito-
chondrial oxidative stress such Parkinson’s disease,3 insulin resis-
tance,4,5 aging,6 heart failure,7 and ischemia-reperfusion injury.8
A number of mitochondrial uncouplers have been reported
(Fig. 1). Among these, 2,4-dinitrophenol (DNP) was temporarily
used for weight loss, but was removed from the market by the
FDA because of its narrow therapeutic window.9 Carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP) is widely used
a
potent protonophore;10,11 however, it has off-target effects such
as the depolarization of the plasma membrane.12–14 Other mito-
chondrial uncouplers such as bupivacaine and ellipticine have been
disclosed.15,16 The properties of an ideal mitochondrial uncoupler
includes exclusive selectivity for the mitochondria and wide ther-
apeutic window. To date, there is a paucity of such compounds.
Due to our interest in this area, we developed a phenotypic
assay to identify compounds that meet these criteria, and reported
the discovery of BAM15, which stimulates mitochondrial respira-
tion across a broad dosing range compared to FCCP.17 Herein, we
disclose the structure–activity relationship studies of BAM15.
Our investigations suggest the pivotal role of the furazan, pyrazine,
and aniline in the scaffold as well as the substituents in the aniline
ring. Further, our studies indicate the pKa of BAM15 is important in
the ability to transport protons from the mitochondrial inner
membrane to the matrix.
The diffusion of uncouplers in and out of the mitochondrial
inner membrane is determined by the energy barrier required to
permeate the hydrophobic membrane core.18 Hence, lipophilicity
of the compound plays a significant role in its ability to accumulate
in the membrane and also to penetrate through the membrane.19,20
To be effective as a mitochondrial protonophore, the uncoupler
⇑
Corresponding author.
E-mail address: santosw@vt.edu (W.L. Santos).
Denotes equal contribution.
http://dx.doi.org/10.1016/j.bmcl.2015.06.040
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

B. M. Kenwood et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4858–4861
4859
OH
Me
Me
NO2
H
N
N
F
F
N
HN
HN
N
N
N
H
N
N
Cl
Cl
NH2
F
O
a
Me
NO2
DNP
+
Me
Ellipticine
Bupivacaine
F
H
N
N
CN
CN
N
O
N
1
H
N
N
HN
N
F
F3CO
F
F
FCCP
H2N
H2N
BAM15
N
Br
HN
HN
a
N
N
O
+
O
N
F
Figure 1. Small molecule protonophores.
must transport hydrogen ions (H+) across the inner mitochondrial
membrane. Two characteristics are necessary to facilitate this
transport: (1) an ionizable proton that is donated in the matrix19
and (2) a large surface area that functions to delocalize the result-
ing charge.21,22 These are properties that are shared among uncou-
plers shown in Figure 1.
2
Scheme 1. Synthesis of compounds 1 and 2. Reagents and conditions: (a) 10 mol %
Pd(dba)2, 20 mol % BINAP, 4 equiv NaOtBu, toluene, reflux, 13–25%.
We started our investigation by determining the importance of
the aniline, pyrazine, and furazan rings of BAM15 (Fig. 2). The syn-
thesis of these compoundsis shownin Scheme1. Followingstandard
Buchwald–Hartwig coupling conditions, 2,3-dichloropyrazine and
2-fluoroaniline were coupled in the presence of Pd(dba)2 under
reflux to afford compound 1.23 Compound 2 was achieved in a sim-
ilar manner while 3 was commercially available. Once the impor-
tance of each ring system was established, a series of substitutions
were placed on the pyrazine ring via nucleophilic aromatic substitu-
tion of 5,6-dichlorofurazano[3,4-b]pyrazine with a variety of anili-
nes or with alcohol (Scheme 2).24,25 It should be noted that N,N-
diethylaniline was crucial for the reactions with anilines.
With the key compounds available (obtained either by chemical
synthesis or bought commercially), we determined their protono-
phoric effect by stimulating respiration in cells. Changes in cellular
respiration following uncoupler treatment were measured in intact
L6 myoblasts using a Seahorse XF-24 Flux Analyzer as previously
described.17 In this assay, mitochondrial uncoupling activity was
determined as a function of oxygen consumption rate (OCR). As
shown in Figure 3, OCR increased as a function of BAM15 concen-
tration. At the outset, we investigated the importance of the fura-
zan (1), pyrazine (2), and aniline (3) rings. Our studies suggest that
removal of each of these moieties individually resulted in complete
loss in the ability to stimulate respiration in concentrations up to
Cl
Cl
N
N
N
O
O
N
N
N
N
a
R
R
O
O
N
3
5a = CH3CH2-
5b = CF3CH2-
5c = Ph-
5d = (2-F)Ph-
5e = (2-I)Ph-
b
4a = CH3CH2CH2-
4b = CH3(CH2)2CH2-
4c = CH3(CH2)4CH2-
4d = Ph
H
N
N
N
N
Ar
Ar
O
N
N
H
4e = (4-F)Ph-
4f = (3-F)Ph-
4g = (2,4-diF)Ph-
4h = (4-Cl)Ph-
4i = 2-pyridyl-
Scheme 2. Synthesis of compounds 4a–i and 5a–e. Reagents and conditions: (a)
alcohol, K2CO3, acetonitrile, 53–96%; (b) alkylamine or substituted aniline, (2 equiv)
N,N-diethylaniline, acetonitrile, reflux, 6–60%.
10 lM (data not shown). Hence, we focused our attention with
5,6-disubstituted furazano[3,4-b]pyrazines and performed a struc-
ture–activity profile. As shown in Table 1, substitution of the ani-
line ring with short propyl to much larger butyl or hexyl amines
pyrazine
H
Ar =
N
N
N
N
Ar
Ar
O
F
N
H
N
Figure 3. Changes in cellular oxygen consumption rate relative to DMSO control for
BAM15 and compounds 4e and 4f. Each data point is from 5 to 6 replicates.
furazan
H
N
N
N
aniline
Ar
Ar
H2N
H2N
N
N
N
O
N
H
or various alkyl ethers resulted in molecules with a poor capacity
to increase cellular respiration (entries 1–5). For reference, in L6
myoblasts BAM15 has an EC50 value of 270 nM that reaches a max-
imum respiration of 215% above basal levels (Fig. 3). Removal of
the fluorine atom of BAM15 to generate compound 4d significantly
decreased its uncoupling ability (entry 7). On the one hand, moving
N
H
1
N
N
Ar
Ar
3
O
2
N
N
H
Figure 2. Three regions of BAM15.

4860
B. M. Kenwood et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4858–4861
Table 1
(4h) afforded similar activity as BAM15 although the maximum
Activity of furazano[3,4-b]pyrazines
oxygen consumption level decreased slightly (compare entry 6 vs
11). To determine the role of aniline nitrogen linker to the pyrazine
ring, we synthesized various ether linkages. Phenyl (5c), 2-fluo-
rophenyl (5d), and 2-iodophenyl (5e) ethers were found to be inac-
tive (entries 12–14). Our results indicate the possibility of the key
role of nitrogen for its protonophoric activity to either affect the
protonation state of the scaffold and its ability to transport a pro-
ton across the membrane.
R
R
N
N
N
N
O
a
Entry
1
Cmpd
R
EC50
(lM)
Max% basalb
98.0 2.8c
H
N
4a
—
*
To understand the potential mechanism by which BAM15
transports protons from the inner membrane space to the mito-
chondrial matrix, we determined the pKa of BAM15. Because
BAM15 was insoluble in water at required concentrations of the
assay, a water-methanol mixture was used (see Supporting infor-
mation). Titrations using UV spectroscopic methods and Yasuda-
Shedlovsky extrapolation, the pKa of BAM15 was determined to
be 7.56 0.08. As the pH in the inner membrane space is 6.8,1
BAM15 is expected to be protonated and allow it to shuttle a pro-
ton to the mitochondrial matrix where the pH is significantly
increased to 8.0. We suspect that this pH differential as well as
the lipophilic nature of BAM15 (cLogP = 6.52) facilitates the effi-
cient transfer of protons across the membrane.
In summary, our investigations demonstrate the importance of
the furazan, pyrazine, and aniline rings in the BAM15 pharma-
cophore as deletion of these rings led to completely inactive com-
pounds. Further, both the nitrogen and phenyl ring of the ‘aniline’
moiety are essential to the uncoupling activity, suggesting an
essential structural role in the protonation of BAM15. Finally, our
studies indicate the importance of pKa in the ability to transport
protons across the membrane, which is affected by halogens such
as fluorine atoms. Efforts to fully elucidate the mechanism by
which BAM15 transports protons as well as further development
of molecules towards improved uncoupling capacity is currently
underway.
H
116.0 1.9c
160.2 8.2c
N
2
3
4b
4c
—
—
*
H
N
*
O
4
5
5a
5b
—
—
100.0 0.6c
96.3 2.2c
*
O
F3C
*
*
H
N
6
7
8
BAM15
4d
0.27
—
214.8 3.0
172.8 3.0c
204.0 3.5
F
H
N
*
H
N
*
*
4e
0.46
F
H
N
*
9
4f
0.18
220.8 4.6
F
H
N
10
11
4g
4h
0.04
0.27
222.6 6.4
190.4 4.3
F
F
H
N
Acknowledgments
*
Cl
N
We acknowledge financial support by the Department of
Chemistry at Virginia Tech and the Department of Pharmacology
at the University of Virginia.
H
N
d
*
*
*
12
12
13
4i
—
—
O
O
5c
5d
—
—
98.3 0.5c
93.0 2.6c
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.06.
040.
F
O
*
14
5e
—
98.8 0.5c
References and notes
I
1. Harper, J. A.; Dickinson, K.; Brand, M. D. Obes. Rev. 2001, 2, 255.
2. Tseng, Y. H.; Cypess, A. M.; Kahn, C. R. Nat. Rev. Drug Disc. 2010, 9, 465.
3. Wu, Y.-N.; Munhall, A. C.; Johnson, S. W. Brain Res. 2011, 1395, 86.
4. Anderson, E. J.; Lustig, M. E.; Boyle, K. E.; Woodlief, T. L.; Kane, D. A.; Lin, C. T.;
Price, J. W., 3rd; Kang, L.; Rabinovitch, P. S.; Szeto, H. H.; Houmard, J. A.;
Cortright, R. N.; Wasserman, D. H.; Neufer, P. D. J. Clin. Invest. 2009, 119, 573.
5. Hoehn, K. L.; Salmon, A. B.; Hohnen-Behrens, C.; Turner, N.; Hoy, A. J.; Maghzal,
G. J.; Stocker, R.; Van Remmen, H.; Kraegen, E. W.; Cooney, G. J.; Richardson, A.
R.; James, D. E. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 17787.
6. Caldeira da Silva, C. C.; Cerqueira, F. M.; Barbosa, L. F.; Medeiros, M. H.;
Kowaltowski, A. J. Aging Cell 2008, 7, 552.
a
Cellular oxygen consumption rates following a dose response of 0.01–10 lM
were normalized from 0% to 100% and subjected to nonlinear regression analysis to
obtain EC50 and 95% confidence intervals.
b
The maximum percentage of basal (100%) respiration that could be stimulated
using uncoupler concentrations from 0.01 to 10
l
M.
The maximum oxygen consumption rate was not obtained using uncoupler
concentrations of 0.01–10 M. The highest oxygen consumption rate using
c
l
uncoupler concentrations from 0.01 to 10
lM are reported.
d
Compound is insoluble.
7. Brennan, J. P.; Southworth, R.; Medina, R. A.; Davidson, S. M.; Duchen, M. R.;
Shattock, M. J. Cardiovasc. Res. 2006, 72, 313.
8. Korde, A. S.; Pettigrew, L. C.; Craddock, S. D.; Maragos, W. F. J. Neurochem. 2005,
94, 1676.
9. Grundlingh, J.; Dargan, P. I.; El-Zanfaly, M.; Wood, D. M. J. Med. Toxicol. 2011, 7,
205.
the position of the fluorine atom to the para (4e) position
decreased EC50 value (entry 8). On the other hand, placement of
the fluorine atom to the meta position (4f) improved the activity
to 180 nM (entry 9). These results indicate the importance of the
fluorine atom around the ring. Remarkably, fluorination on the
ortho and para positions in 4g improved the EC50 value to 40 nM
(entry 10). Introduction of a chlorine moiety on the 4-position
10. Benz, R.; McLaughlin, S. Biophys. J. 1983, 41, 381.
11. Kasianowicz, J.; Benz, R.; McLaughlin, S. J. Membr. Biol. 1984, 82, 179.
12. Park, K. S.; Jo, I.; Pak, K.; Bae, S. W.; Rhim, H.; Suh, S. H.; Park, J.; Zhu, H.; So, I.;
Kim, K. W. Pflugers Arch. 2002, 443, 344.
13. Juthberg, S. K.; Brismar, T. Cell. Mol. Neurobiol. 1997, 17, 367.

B. M. Kenwood et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4858–4861
4861
14. Buckler, K. J.; Vaughan-Jones, R. D. J. Physiol. 1998, 513, 819.
15. Sun, X.; Garlid, K. D. J. Biol. Chem. 1992, 267, 19147.
19. Miyoshi, H.; Tsujishita, H.; Tokutake, N.; Fujita, T. Biochim. Biophys. Acta 1990,
1016, 99.
16. Schwaller, M.-A.; Allard, B.; Lescot, E.; Moreau, F. J. Biol. Chem. 1995, 270,
22709.
20. Martineau, L. C. J. Theor. Biol. 2012, 303, 33.
21. Skulachev, V. P.; Sharaf, A. A.; Liberman, E. A. Nature 1967, 216, 718.
22. Escher, B. I.; Hunziker, R.; Schwarzenbach, R. P.; Westall, J. C. Environ. Sci.
Technol. 1999, 33, 560.
23. Xie, X.; Zhang, T. Y.; Zhang, Z. J. Org. Chem. 2006, 71, 6522.
24. Starchenkov, I. B.; Andrianov, V. G. Chem. Heterocycl. Compd. 1997, 33, 1219.
17. Kenwood, B. M.; Weaver, J. L.; Bajwa, A.; Poon, I. K.; Byrne, F. L.; Murrow, B. A.;
Calderone, J. A.; Huang, L.; Divakaruni, A. S.; Tomsig, J. L.; Okabe, K.; Lo, R. H.;
Cameron Coleman, G.; Columbus, L.; Yan, Z.; Saucerman, J. J.; Smith, J. S.;
Holmes, J. W.; Lynch, K. R.; Ravichandran, K. S.; Uchiyama, S.; Santos, W. L.;
Rogers, G. W.; Okusa, M. D.; Bayliss, D. A.; Hoehn, K. L. Mol. Met. 2014, 3, 114.
18. Spycher, S.; Smejtek, P.; Netzeva, T. I.; Escher, B. I. Chem. Res. Toxicol. 2008, 21,
911.
´
25. Fernández, E.; Garcıa-Ochoa, S.; Huss, S.; Mallo, A.; Bueno, J. M.; Micheli, F.;
Paio, A.; Piga, E.; Zarantonello, P. Tetrahedron Lett. 2002, 43, 4741.