Concise Synthesis of 1,4-Benzoquinone-Based Natural Products as Mitochondrial Complex I Substrates and Substrate-Based Inhibitors

Article abstract of DOI:10.1002/cmdc.202000307

A short, efficient one-step synthesis of 2-methyl-5-(3-methyl-2-butenyl)-1,4-benzoquinone, a natural product from Pyrola media is described. The synthesis is based on a direct late C?H functionalization of the quinone scaffold. The formation of the natural product was confirmed by means of 2D-NMR spectroscopy. Additional derivatives were synthesized and tested alongside the natural product as potential substrate and substrate-based inhibitors of mitochondrial complex I (MCI). The structure-activity relationship study led to the discovery of 3-methylbuteneoxide-1,4-anthraquinone (1 i), an inhibitor with an IC₅₀ of 5 μM against MCI. The identified molecule showed high selectivity for MCI when tested against other quinone-converting enzymes, including succinate dehydrogenase, and the Na (+)-translocating NADH:quinone oxidoreductase. Moreover, the identified inhibitor was also active in cell-based proliferation assays. Therefore, 1 i can be considered as a novel chemical probe for MCI.

Full text of DOI:10.1002/cmdc.202000307

Accepted Article
Title: Concise synthesis of 1,4-benzoquinone based natural products
as mitochondrial complex I substrates and substrate-based
inhibitors
Authors: Hamid Nasiri, Zhenyu Han, Heike Ange, Yihan Qin, Dennis
Stegmann, Karina Tuz, Andrea Marco Amati, Günter Fritz,
Oscar Juarez, Dana Lashley, Iris Bischoff, and Robert Fürst
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000307
Link to VoR: https://doi.org/10.1002/cmdc.202000307
01/2020

10.1002/cmdc.202000307
ChemMedChem
Concise synthesis of 1,4-benzoquinone based natural products as mitochondrial
complex I substrates and substrate-based inhibitors
Zhenyu Han,[a] Dr. Heike Angerer,[b,c] Dr. Iris Bischoff,[d] Yihan Qin,[a] Dennis Stegmann,[e] Prof.
Karina Tuz,[f] Andrea Marco Amati,[g] PD Dr. Günter Fritz,[e] Prof. Oscar Juarez,[f] Prof. Robert Fürst,[d]
Prof. Dana Lashley,*[a] and Dr. Hamid R. Nasiri*[e]
[a] Z. Han, Y. Qin, Prof. D. Lashley
Department of Chemistry William & Mary
Williamsburg, VA 23185 (USA)
E-mail: DLashley@wm.edu
[b] Dr. H. Angerer
Medical School Institute of Biochemistry II
Structural Bioenergetics Group
Goethe University
60590 Frankfurt am Main (Germany)
[c] Dr. H Angerer
Centre for Biomolecular Magnetic Resonance
Institute for Biophysical Chemistry
Goethe University 60590 Frankfurt am Main (Germany)
[d] Dr. Iris Bischoff, Prof. Robert Fürst
Institute of Pharmaceutical Biology
Goethe University 60438 Frankfurt am Main (Germany)
[e] D. Stegmann, PD Dr. G. Fritz, Dr. H. Nasiri
Department of Cellular Microbiology
University Hohenheim 70599 Stuttgart (Germany)
E-mail: Hamid.Nasiri@uni-hohenheim.de
[f] Prof. K. Tuz, Prof. O. Juarez
Department of Biological Sciences
Illinois Institute of Technology Chicago, IL 60616 (USA)
[g] Dr. A.M. Amati
Department of Chemistry and Biochemistry
University of Bern CH-3012 Bern (Switzerland)
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
Keywords
Natural products; 1,4-benzoquinone; one-step synthesis; chemical probe; late CH-functionalization;
substrate-based inhibitors
Abstract
A short, efficient one-step synthesis of 2-Methyl-5-(3-methyl-2-butenyl)-1,4-benzoquinone, a natural
product from Pyrola media is described. The synthesis is based on a direct late CH-functionalization
of the quinone scaffold. The formation of the natural product was confirmed by means of 2D-NMR
spectroscopy. Additional derivatives were synthesized and tested alongside the natural product as
potential substrate and substrate-based inhibitors of mitochondrial complex I (MCI). The structure-
activity relationship study led to the discovery of 3-methylbuteneoxide-1,4-anthraquinone (1i) an
inhibitor with an IC50 of 5 M against MCI. The identified molecule showed high selectivity for MCI
when tested against other quinone-converting enzymes including cytochrome bo3 oxidase, succinate
dehydrogenase and the Na (+)-translocating NADH:quinone oxidoreductase. Moreover, the identified
inhibitor was also active in cell-based proliferation assay. Therefore, 1i can be considered as a novel
chemical probe for MCI.
Introduction
Quinones are ubiquitous motifs present in natural products, pharmaceuticals and materials. Natural
products containing the 1,4-benzoquinone scaffold exhibit a wide range of biological activities.1–5
Examples of this class of molecules are shown in Figure 1. 2-Methyl-5-(3-methyl-2-butenyl)-1,4-
benzoquinone, 1 is a natural product first isolated by Burnett et al. in 1968 from the leaves of Pyrola
media.1 Coenzyme Q1 2 is a substrate of various oxidoreductases, including the mitochondrial complex
I (MCI).2 Another example is blattellaquinone 3, which has been identified as the sex pheromone of
the German cockroach Blattella germanica3 and primin 4 is a natural product isolated from Primula
obconica and exhibits significant antiprotozoal activity.4 Lastly, the benzoquinone (-)-perezone 5,
found in Pseudopterogorgia rigida, has shown promising cytotoxicity toward human tumor cell lines.5
2
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
Figure 1. Examples of natural products containing the 1,4-benzoquinone structure.
In light of the interesting biological properties of 1,4-benzoquinone containing natural products, an
easy access via a straight and efficient synthesis is needed. We had recently reported on the concise
synthesis of primin 4.6 In this present work, we describe the synthesis and biological testing of the
natural product 1 and its derivatives.
Several synthetic strategies have been reported, but challenges remain in the introduction of isoprenoid
side chains onto quinonoid motifs via C-C bond formation. Fujiwara et al. developed a method to
functionalize quinones with alkyl- and arylboronic acid. This method is excellent for saturated side-
chains, but suffers from low yields when employing vinylboronic acids.7 Alternative methods aimed
to introduce isoprenoid side chains suffer from lengthy multi-step sequences,8 toxic reagents,9 and low
yields.10 Thus, a direct functionalization is still challenging due to the unique electronic properties of
quinones.11 Herein, we report two direct functionalization methodologies, via organozinc reagents and
via decarboxylation, to access the natural product 1 and its analogues in a quantity that is sufficient
for biological testing.
Due to the structural similarity to the natural substrate 2, the natural product 1 was subsequently tested
as a potential substrate of mitochondrial complex I (MCI) in a biochemical assay. MCI, also known
3
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
as NADH:ubiquinone oxidoreductase, is a multi-subunit membrane protein with a total mass of
approximately 1 MDa. It couples the electron transfer from NADH to ubiquinone with a simultaneous
translocation of protons and contributes to the formation of the proton motive force, the basis for ATP
synthesis in mitochondria. The access pathway and the binding site of the substrate are still topics
under investigation.12 Chemical probes have been shown to be useful in the structural evaluation of
quinone converting enzymes.13–17 Moreover, inhibitors targeting the substrate binding site of MCI
have the potential to be developed into new antitumor agents.18 BAY-179, BAY-87-2243 (Bayer) and
IACS-010759 (MD Anderson) are examples of MCI inhibitors in clinical studies. 19–22
Results and discussion
Synthesis of natural product 1 and its derivatives 1d-1h
We envisioned to synthesize natural product 1 via a late-stage modification of the quinone scaffold.
This goal was achieved by the introduction of an isoprenyl side chain via a direct C-H functionalization
using an organozinc reagent that was described previously by Oliveira et al.23 This reaction is
regioselective for 1,4-addition and works for 1,4-benzoquinones, 1,4-naphtoquinones24 and 1,4-
anthraquinones. The reaction was unsuccessful for 1,2-napthoquinones and the coupling is limited to
allylic halides (see SI Schemes S7-S9). This method can also be employed for the introduction of an
isoprenyl group to the beta position of --unsaturated ketones, specifically those with aromatic
substituents (chalcones), as was shown previously by Zhao et al.25
Starting from commercially available 2-methyl-1,4-benzoquinone 6a, prenyl bromide 7 was added
dropwise to the reaction mixture in the presence of zinc granules. The prenylated natural product 1
was formed in a single reaction step in 39% isolated yield (Scheme 1). The reaction was carried out
overnight at ambient pressure, moderate temperature, and with an inexpensive starting material.23
O
O
Br
Zn,
7
+
byproducts
THF, 65 °C, 24 h
O
O
6a
1 (39%)
Scheme 1.
Synthesis of the natural product 1, a prenylated 2-methyl-1,4-benzoquinone.
4
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
A 1,1-ADEQUATE experiment was conducted to determine the correct constitution of the natural
product 1. 1,1-ADEQUATE is a powerful analytical method for the structural elucidation of
structurally closely related natural product isomers.6 This 2D NMR experiment establishes
1
1
correlations of adjacent neighboring carbons via out-and-back JCH and JCC couplings, and thus
provides the identity of a proton and carbons that are exclusively two bonds away.26 As highlighted in
Figure 2a, the experiment provides an unambiguous assignment of quinone 1. Protons H7 and H8
yield distinguishable chemical shifts due to the unique electronic environment in methyl and prenyl
groups. Therefore, the chemical shifts of C2 and C5 were first identified via the two-bond correlations
with H7 and H8, respectively (see SI Figure S1). Similarly, the coupling of C2 and H3, and that of
C5 and H6 lead to the identification of quinonoid H3 (6.60 ppm) and H6 (6.51 ppm). The exclusive
correlations of H3-C4 and H6-C1, along with the absence of H3-C1 and H6-C4 couplings, support the
final 2,5- structural assignment of 1.
Additionally, Heteronuclear Multiple Bond Correlation (HMBC) NMR, a gradient variant with
improved sensitivity, was demonstrated to show exclusive three bond correlations between protons
and carbons in our quinonoid system. The HMBC spectrum of quinone 1 with 8 Hz couplings
demonstrated correlations between C1-H3 and C4-H6 (Figure 2b). This three-bond correlation pattern
was also evidenced between C4-H8 coupling. Spectra acquired with coupling constants of 6 Hz and
10 Hz also exhibited the same coupling patterns as observed in 8 Hz without additional correlations
greater or less than three bonds in distance. Together with the early structural confirmation with the
1,1-ADEQUATE experiment, this result demonstrated that HMBC can detect exclusive three-bond
couplings within 6-10 Hz range.
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
Figure 2.
NMR characterization of natural product 1 and its regioisomers 1b and 1c: (a) Section
of 1,1-ADEQUATE spectrum of the natural product 1 showing H3-C4 and H6-C1 correlations and
additional proton-carbon two bond couplings exist in this system. (b) Section of HMBC NMR
1
spectrum of the natural product 1 showing exclusive three bond couplings. (c) Section of H NMR
spectrum for byproducts from Scheme 1, a 1:1.4 mixture of regioisomers 1c and 1b, demonstrating
unique quinonoid proton shift for 2,3-regioisomers.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
During the prenylation of 6a (Scheme 1) three regioisomers 1, 1b, and 1c were formed in a ratio of
4.7:1.4:1.0, respectively. It was foreseeable that the 2,3-regioisomer 1c would be the most minor
product, due to steric interactions of the prenyl side chain with the methyl group. Isomer 1b has also
been reported as a natural product isolated from the stems and leaves of Gunnera perpensa and has
shown significant antimicrobial activity against Staphylococcus epidermidis.27 Isomers 1b and 1c
1
yielded a largely inseparable mixture. In the H-NMR spectrum, the quinonoid protons of the 2,3-
isomer 1c at C5 and C6 appeared as an overlapping singlet at approximately 6.7 ppm. In contrast, its
isomer 1b yielded two quinonoid proton signals between 6.4 and 6.6 ppm (Figure 2c).
Using the same synthetic pathway, additional derivatives of the natural product were synthesized for
a structure-activity relationship study (Scheme 2). Further derivatization to replace the C2 methyl
group was carried out with 2-tert-butyl-1,4-benzoquinone 6b and 1,4-anthraquinone 6c as starting
materials (Scheme 2a). Quinones 1d and 1e were synthesized from 6b and isolated by column
chromatography in a combined yield of 69%. As expected, due to the steric hindrance of the tert-butyl
group, the formation of the 2,3-regioisomer was not observed. The structures of the 2,5-isomer 1d and
the 2,6-isomer 1e were characterized by HMBC with 8 Hz coupling (see SI Figure S2). Quinone 1f
with an extended aromatic ring was synthesized in 62% yield, starting from 6c. The observation also
suggested the applicability of this reaction across 1,4-quinones with different substituents and
electronic properties.
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
O
O
(a)
Br
Zn,
R
7
R
THF, 65 °C, 24 h
O
O
6b-6c
1d-1f
6b R = C(CH3)3
6c R = (CH)2C6H5
O
O
2
6
5
O
O
1d 2,5-isomer (28%)
1e 2,6-isomer (41%)
1f (62%)
O
O
(b)
mCPBA (3.0 equiv)
1
O
DCM, rt, 3h
1g (45%)
O
O
4-methylpentanoic acid,
AgNO3, K2S2O8
(c)
6a
CH3CN/H2O (1:2)
50 °C, 1 h
1h (17%)
Scheme 2.
Synthesis of derivatives of the natural product following a substrate-based inhibitor
design: the synthesis of (a) prenylated 2-tert-butyl-1,4-benzoquinones 1d-1e, prenylated 1,4-
anthraquinone 1f, (b) epoxide 1g and (c) benzoquinone 1h with saturated sidechain.
Further derivatization of the natural product was carried out, which resulted in compounds 1g and 1h
by a modification of the side chain (Scheme 2b-2c). A direct epoxidation of quinone 1 was conducted
to investigate the potential effect of the epoxide group on the enzymatic activity. Previously, the
Weitz-Scheffer epoxidation of -unsaturated carbonyls was achieved on the quinonoid ring of 1,4-
naphthoquinone with H2O2.13 In 1,4-benzoquinone, however, the two double bonds on the quinonoid
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
ring are highly deactivated towards electrophilic attack. Therefore, it is impossible to epoxidize the
quinone double bonds of benzoquinones at either position unless one employs a multi-step synthesis
involving a harsh retro Diels-Alder cleavage.28 The addition of 3 equivalents of mCPBA to the natural
product 1 resulted in the epoxidation of the prenyl side chain and the formation of racemic epoxidation
product 1g in 45% isolated yield.
Next, derivative 1h with a saturated isopentyl side chain was synthesized in a single step, an outcome
which could not be afforded by employing organozinc reagents. The Hunsdiecker decarboxylation
reaction2,13,29 was utilized, and the desired products were synthesized in 43% combined yield for all
regioisomers. After heating in acetonitrile/water = 1:2 mixture, the 2,5-isomer 1h was isolated via
column chromatography in 17% isolated yield. The 2,6- and 2,3-regioisomers of this quinone were
afforded as byproducts in an inseparable mix. The mechanism of this reaction involves the generation
of radicals, the subsequent nucleophilic radical Michael addition to the quinone, and an in-situ
oxidation step.6 It is worth noting that molecules 1d-1h were synthesized in the laboratory for the very
first time.
Biological Data
The natural product 1 and the derivatives 1d-1h were tested in replicates against isolated
bovine heart mitochondrial membranes (BHM) containing mitochondrial complex I (MCI). BHM
were pre-incubated with compounds, and NADH:quinone catalytic activity was determined by
monitoring the rate of absorbance change at 340 nm due to NADH oxidation after the addition of Q1
and compounds 1 and 1h, respectively. The substrate of MCI Q1, the natural product 1 and the
derivative 1h were tested at different concentrations for Michaelis-Menten kinetics in both the
presence and the absence of a highly potent MCI quinone-site inhibitor 2-decyl-4-quinazolinylamine
(DQA) (Figure 3a). DQA sensitive activity values directly correlate to the real physiological turnover
activity of MCI at the quinone site. After fitting of the DQA-sensitive curves using the bioinformatics
tool Graph Pad Prism,30 Q1 showed a maximal turnover rate (Vmax) of 0.45 U/mg and a Km value of
9.8 µM, which is in line with the literature31. Compounds 1 and 1h were recognized as substrates for
mammalian mitochondrial complex I and showed Vmax values of 0.20 and 0.22 U/mg, respectively.
Compounds 1 and 1h showed Km values in the same range of Q1 indicating similar affinity to MCI.
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
Figure 3.
(a) Michaelis-Menten kinetics. Specific complex I NADH:substrate activities of
bovine heart mitochondrial membranes were determined in dependence of substrate concentration.
NADH:Q1 kinetics were compared to kinetics of compounds 1 and 1h that show turnover by complex
I with half-maximal rate of Q1. DQA, 2-decyl-4-quinazolinylamine (highly potent complex I
inhibitor). (b) [inhibitor] vs. normalized response and the corresponding IC50 values of complex I
NADH:Q1 inhibition by compounds 1g and 1f.
In determining the half-maximal inhibitory concentration (IC50) of potential MCI inhibitors
compounds 1g and 1f were tested in the presence of 80 µM Q1 with increasing concentrations
(Figure 3b). After numerical fitting of the curves, it was found that compounds 1g and 1f were able
to inhibit the mammalian MCI with IC50 values of 238 and 8.9 µM, respectively.
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
As shown in Figure 3b for inhibitor 1f, the solubility limit is reached at higher concentration of 80
µM. Compounds 1d and 1e were neither substrates nor inhibitors.
Substrate-based inhibitor design and testing of 1i
Based on the encouraging results of the enzymatic testing a new molecule 1i was designed by
combining both structural features of the identified inhibitors 1g and 1f, namely an extended aromatic
ring and an epoxy group in the side chain (Figure 4). Furthermore, as a result of the introduction of
the epoxy-group it was expected that 1i would have an increased solubility compared to compound 1f.
O
O
O
O
O
1g
1f
MW = 206.2
MW = 276.3
cal. clogP = 1.68
IC50 = 238 mM
cal. clogP = 4.61
IC50 = 8.9 mM
O
O
O
MW = 292.3
cal. clogP = 3.58
1i
mCPBA (3.0 equiv)
DCM, rt, 3h
1f
1i (27%)
Figure 4.
Design and Synthesis of 1i, combining both structural features of the identified
inhibitors 1g and 1f.
Compound 1i was synthesized by the epoxidation of 1f in 27% isolated yield. The solubility of
compound 1i was measured experimentally by an absorbance-based solubility assay32 and compared
to 1f (Figure 5). First, the maximum peaks of the absorbance spectra for both compounds were
determined (1f λ = 275 nm; 1i λ = 415 nm). Next, the saturation point for both compounds was
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
calculated by plotting the absorbance against the concentration. The outcome of the solubility testing,
highlighted in Figure 5, confirmed the increased solubility of 1i over 1f.
Figure 5. Solubility testing of 1f and 1i in buffer (20mM Tris-H2SO4, 50mM Na2SO4, 0.05% DDM,
50µg/ml BSA) in replicates.
Compound 1i was tested in the MCI enzymatic assay and showed an improved inhibitory effect with
an IC50 of 5.2 µM (Figure 6a). The new improved inhibitor 1i was further characterized by testing its
selectivity for MCI over other quinone-converting enzymes. For this purpose, 1i was tested at
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
increased concentration against three off-target oxidases (Figure 6b). The outcome showed that for 1i
MCI is the primary target, with no activity against other oxidases.
Figure 6. Enzymatic testing of 1i a) Inhibition of MCI by 1i, b) selectivity of 1i for MCI over
cytochrome bo3 oxidase, succinate dehydrogenase and the Na (+)-translocating NADH:quinone
oxidoreductase.
Small molecules targeting the MCI have the potential to be further developed to anticancer agents. In
order to assess the antiproliferative capacity of the identified inhibitors, 1f, 1g and 1i were tested
against solid tumor cell lines. For the selected MCI inhibitors cell-based testing was performed against
HeLa (cervix carcinoma), HepG2 (hepatic cancer), MDA-MB-231 (breast cancer) and PC3 (prostate
cancer) cells in 96-well plates. The well-known anticancer drug doxorubicin and the MCI inhibitor
rotenone were used as controls. The concentration-response curves and the respective IC50 values are
highlighted in Figure 7.
13
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
Figure 7. Cellular testing of 1f, 1g and 1i in four different cancer cell lines. 2.5x103 HeLa cells, 1x104
HepG2 cells, 6x103 MDA-MB-231 cells and 4x103 PC3 cells on 96-well plates were treated with the
indicated concentrations of MCI inhibitors, doxorubicin or rotenone 24 h post-seeding. Doxorubicin
and the MCI inhibitor rotenone served as controls. After 72 h of incubation the cells were stained with
crystal violet, air dried and subsequently DNA-bound crystal violet was dissolved using acetic acid to
determine cell proliferation at 590 nm using a plate reader. IC50 values were determined using
GraphPad Prism. HeLa (n=4-6), HepG2 (n=3-7), PC3 (n=3-8) and MDA-MB-231 (n=4-8).
Based on the cell-based testing, the identified MCI inhibitors 1f and 1i demonstrated antiproliferative
activities against all tested cancer cell lines in low µM range (3-9 µM). No antiproliferative activity
was observed for compound 1g at the tested concentration range. The outcome of the cellular testing
is in line with the determined MCI inhibitory potencies of the tested compounds. The trend of MCI
inhibition (1i IC50 = 5.2 < 1f IC50 = 8.9 < 1g IC50 = 238) is also reflected in the cellular findings.
Finally, the stability of the new inhibitor in solution was investigated using an NMR spectroscopy-
based approach (see SI Figure S3). Inhibitor 1i is stable in solution for at least 20 weeks when stored
at -20°C and for 9 weeks when stored at room temperature. At +40 °C, 1i decomposes within 1-1.5
weeks as a result of epoxide opening.
14
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
In light of these results, we deduce that 1i can be considered as a novel chemical probe for MCI. With
the discovery of this molecule, we aim to contribute to “target 2035”, an initiative which was defined
by scientists in academia and industry (in particular Bayer) to develop small-molecule probes for every
protein in the human proteome.33
The outcome of the enzymatic testing confirmed our hypothesis that, firstly, the natural product
resembles the structure of the substrate and hence is accepted and converted as a substrate. Secondly,
it confirmed that the precise modifications, and the synthesis of just a handful of compounds allowed
the transformation of a substrate into an inhibitor.
Conclusions
In summary, we described the synthesis of the natural product 1 in a single reaction step. By employing
this synthesis pathway, further derivates of the natural product were designed, synthesized and tested
as substrates and substrate-based inhibitors against MCI. Due to the structural similarities with the
quinone substrate of MCI, the natural product 1 was accepted and converted by the enzyme as an
artificial substrate. Consequently, by pursuing the strategy of substrate-based inhibitor design, 1i was
discovered as a novel and selective inhibitor of the multi-subunit membrane protein MCI with
antiproliferative activity against selected cancer cell lines.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank PD. Dr. Volker Zickermann, Dr. Jürgen Graf and Prof. Dr. Christoph von Ballmoos for the
fruitful discussions and critical review of this manuscript. We thank Prof. Deborah C. Bebout for her
continuing support and guidance. We thank Anna Mosch and Yvonne Jung for their technical support.
Many thanks to Dana S. Nasiri for the inspiration for this study. This work was supported by the Roy
R. Charles Center for Academic Excellence and the Reves Center for International Studies’ Faculty
Fellows Program.
15
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
TOC
We report the design and synthesis of natural product derived mitochondrial complex I (MCI)
substrates and substrate-based inhibitors bearing the 1,4-benzoquinone scaffold. Precise structural
modifications led to the discovery of a novel, selective chemical probe for MCI with antiproliferative
activity against selected cancer cell lines. Our study provides valuable insights on the rational design
of natural product substrates and substrate-based inhibitors as small-molecule probes for clinically
relevant membrane proteins.
Notes and references
1
2
A. R. Burnett and R. H. Thomson, J. Chem. Soc. C Org., 1968, 857–860.
H. Angerer, H. R. Nasiri, V. Niedergesäß, S. Kerscher, H. Schwalbe and U. Brandt, in
Biochimica et Biophysica Acta - Bioenergetics, 2012.
3
4
S. Nojima, C. Schal, F. X. Webster, R. G. Santangelo and W. L. Roelofs, Science (80-. )., 2005,
307, 1104–1106.
D. Tasdemir, R. Brun, V. Yardley, S. G. Franzblau and P. Rüedi, Chem. Biodivers., 2006, 3,
16
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
1230–1237.
5
P. A. Abreu, D. V. Wilke, A. J. Araujo, J. D. B. Marinho-Filho, E. G. Ferreira, C. M. R. Ribeiro,
L. S. Pinheiro, J. W. Amorim, A. L. Valverde, R. A. Epifanio, L. V. Costa-Lotufo and P. C.
Jimenez, Rev. Bras. Farmacogn., 2015, 25, 634–640.
6
7
H. R. Nasiri, J. Ferner, C. Tükek, R. Krishnathas and H. Schwalbe, Tetrahedron Lett., 2015, 56,
2231–2233.
Y. Fujiwara, V. Domingo, I. B. Seiple, R. Gianatassio, M. Del Bel and P. S. Baran, J. Am. Chem.
Soc., 2011, 133, 3292–3295.
8
S. Shan, Huaxue Tongbao, 2007, 70, 155–156.
9
R. N. Khanna and P. K. Singh, Synth. Commun., 1990, 20, 1743–1749.
J. T. Koehn, D. C. Crick and D. C. Crans, ACS Omega, 2018, 3, 14889–14901.
Y. Wang, S. Zhu and L.-H. Zou, European J. Org. Chem., 2019, 2019, 2179–2201.
K. Parey, C. Wirth, J. Vonck and V. Zickermann, Curr. Opin. Struct. Biol., 2020, 63, 1–9.
H. R. Nasiri, M. G. Madej, R. Panisch, M. Lafontaine, J. W. Bats, C. R. D. Lancaster and H.
Schwalbe, J. Med. Chem., 2013, 56, 9530–9541.
10
11
12
13
14
H. Palsdottir, C. G. Lojero, B. L. Trumpower and C. Hunte, J. Biol. Chem., 2003, 278, 31303–
31311.
15
16
M. Murai, S. Murakami, T. Ito and H. Miyoshi, Biochemistry, 2015, 54, 2739–2746.
H. R. Bridges, A. J. Y. Jones, M. N. Pollak and J. Hirst, Biochem. J., ,
DOI:10.1042/BJ20140620.
17
18
19
U. Fendel, M. A. Tocilescu, S. Kerscher and U. Brandt, Biochim. Biophys. Acta - Bioenerg.,
2008, 1777, 660–665.
R. Vatrinet, L. Iommarini, I. Kurelac, M. De Luise, G. Gasparre and A. M. Porcelli, Int. J.
Biochem. Cell Biol., 2015, 63, 41–45.
P. Ellinghaus, I. Heisler, K. Unterschemmann, M. Haerter, H. Beck, S. Greschat, A. Ehrmann,
H. Summer, I. Flamme, F. Oehme, K. Thierauch, M. Michels, H. Hess-Stumpp and K.
Ziegelbauer, Cancer Med., 2013, 2, 611–624.
20
21
H. V Vangapandu, B. Alston, J. Morse, M. L. Ayres, W. G. Wierda, M. J. Keating, J. R.
Marszalek and V. Gandhi, Oncotarget, 2018, 9, 24980–24991.
J. R. Molina, Y. Sun, M. Protopopova, S. Gera, M. Bandi, C. Bristow, T. McAfoos, P.
Morlacchi, J. Ackroyd, A.-N. A. Agip, G. Al-Atrash, J. Asara, J. Bardenhagen, C. C. Carrillo,
C. Carroll, E. Chang, S. Ciurea, J. B. Cross, B. Czako, A. Deem, N. Daver, J. F. de Groot, J.-
17
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000307
ChemMedChem
W. Dong, N. Feng, G. Gao, J. Gay, M. G. Do, J. Greer, V. Giuliani, J. Han, L. Han, V. K. Henry,
J. Hirst, S. Huang, Y. Jiang, Z. Kang, T. Khor, S. Konoplev, Y.-H. Lin, G. Liu, A. Lodi, T.
Lofton, H. Ma, M. Mahendra, P. Matre, R. Mullinax, M. Peoples, A. Petrocchi, J. Rodriguez-
Canale, R. Serreli, T. Shi, M. Smith, Y. Tabe, J. Theroff, S. Tiziani, Q. Xu, Q. Zhang, F. Muller,
R. A. DePinho, C. Toniatti, G. F. Draetta, T. P. Heffernan, M. Konopleva, P. Jones, M. E. Di
Francesco and J. R. Marszalek, Nat. Med., 2018, 24, 1036–1046.
22
A. Naguib, G. Mathew, C. R. Reczek, K. Watrud, A. Ambrico, T. Herzka, I. C. Salas, M. F.
Lee, N. El-Amine, W. Zheng, M. E. Di Francesco, J. R. Marszalek, D. J. Pappin, N. S. Chandel
and L. C. Trotman, Cell Rep., 2018, 23, 58–67.
23
24
25
26
R. A. de Oliveira, F. Carazza and M. O. da S. Pereira, Synth. Commun., 1997, 27, 1743–1749.
K. Inami, Y. Okayama, M. Harada, M. Morita and M. Mochizuki, Heterocycles, 2017, 94, 865.
L.-M. Zhao, S.-Q. Zhang, F. Dou and R. Sun, Org. Lett., 2013, 15, 5154–5157.
G. E. Martin, in Annual Reports on NMR Spectroscopy, ed. G. A. Webb, Academic Press, 2011,
vol. 74, pp. 215–291.
27
S. E. Drewes, F. Khan, S. F. van Vuuren and A. M. Viljoen, Phytochemistry, 2005, 66, 1812–
1816.
28
29
K. Alder, F. H. Flock and H. Beumling, Chem. Ber., 1960, 93, 1896–1899.
M. G. Madej, H. R. Nasiri, N. S. Hilgendorff, H. Schwalbe, G. Unden and C. R. D. Lancaster,
Biochemistry, , DOI:10.1021/bi0618161.
30
31
32
33
GraphPad Prism version 7.00 Wind. GraphPad Software, La Jolla Calif. USA,
www.graphpad.com).
R. Fato, E. Estornell, S. Di Bernardo, F. Pallotti, G. P. Castelli and G. Lenaz, Biochemistry, ,
DOI:10.1021/bi9516034.
I. Elamri, M. Radloff, K. F. Hohmann, V. D. Nimbarte, H. R. Nasiri, M. Bolte, S. Safarian, H.
Michel and H. Schwalbe, ChemMedChem, , DOI:10.1002/cmdc.201900707.
A. Mullard, Nat. Rev. Drug Discov., 2019, 18, 733–736.
18
This article is protected by copyright. All rights reserved.