Discovery of cytochrome bc1 complex inhibitors inspired by the natural product karrikinolide

Article abstract of DOI:10.1039/c6ra19424a

The cytochrome bc₁ complex (cyt bc₁ or complex III) is a promising target of numerous antibiotics and fungicides. With an aim to indentify new lead structures for the bc₁ complex, a series of novel inhibitors were discovered from the natural product karrikinolide for the first time. Extensive screening results suggested variable inhibitory activities of these compounds against succinate-cytochrome reductase [SCR, a mixture of respiratory complex II (SQR) and complex III (the bc₁ complex)], implying the essential role of a 4-substituted phenyl group for the high potency. Exceptionally, compound 12g showed excellent inhibition potency having an IC₅₀ value in the sub-micromolar range, demonstrating its higher potency than the commercial control amisulbrom by over two orders of magnitude. Further experiments inferred that these newly prepared compounds mainly target the bc₁ complex. Seemingly, this work has presented a new lead scaffold for further development of bc₁ complex inhibitors.

Full text of DOI:10.1039/c6ra19424a

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Discovery of cytochrome bc1 complex inhibitors inspired by the
natural product Karrikinolide
Cheng Chen,b Qiong-You Wu*a Lian-Ying Shan,a Bei Zhang,a Francis Verpoortb and Guang-Fu
Yang*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
The cytochrome bc1 complex (cyt bc1 or complex III) is a promising target of numerous antibiotics and fungicides. With an
aim to indentify new lead structures for the bc1 complex, a series of novel inhibitors were discovered from the natural
product Karrikinolide for the first time. Extensive screening results suggested variable inhibitory activities of these
compounds against succinate-cytochrome reductase [SCR, a mixture of respiratory complex II (SQR) and complex III (the
bc1 complex)], implying the essential role of a 4-substituted phenyl group for the high potency. Exceptionally, compound
12g showed excellent inhibition potency having an IC50 value in the sub-micromolar range, demonstrating its higher
potency than the commerical control Amisulbrom by over two orders of magnitude. Further experiments inferred that
these newly prepared compounds mainly target the bc1 complex. Seemingly, this work has presented a new lead scaffold
for further development of bc1 complex inhibitors.
www.rsc.org/
attracts growing attention.
1. Introduction
Normally, natural products show a broad range of interesting and
useful biological activities. More significantly, they demonstrate
Oxidative phosphorylation and the tricarboxylic acid (or Krebs) cycle,
two bioprocesses that are coupled with electron transport and
proton pump flow, constitute the highly effective mitochondrial
respiration. In animals and bacteria, the oxidative phosphorylation
system comprises five multiprotein complexes (complexes I, II, III,
IV, and V) and two mobile electron carriers (ubiquinone and
cytochrome c) embedded in the lipid bilayer of the mitochondrial
inner membrane.1 Among them, cytochrome bc1 complex (EC
1.10.2.2, bc1) is an essential component of the cellular respiratory
chain and plays a pivotal role in the life cycle. The bc1 complex
catalyzes the electron transfer from ubiquinol (hydroxyquinone,
QH2) to a water-soluble cytochrome c (cyt c) and couples this
electron transfer to the translocation of protons across the
membrane, generating a proton gradient and membrane potential
for ATP synthesis.2-5 In case the electron transport process of the
bc1 complex is disturbed or disrupted, the cellular respiration will be
blocked, resulting in cell death. Therefore, the bc1 complex is one of
the most attractive targets for the development of pharmaceuticals
and agrochemicals. Over the past years, pronounced attention has
been shown for the development of cytochrome bc1 complex
inhibitors,6-17 and our group has also been engaged extensively in
this area.1, 18-23 Although numerous inhibitors have been identified,
the discovery of novel inhibitors with entirely new scaffolds still
excellent environmental compatibility and could be readily
biodegraded.24-26 However, limited activities and adverse photo and
chemical stabilities of these compounds often indulge the use of
their synthetic analogues instead,27 which usually serve as ideal
candidates for the development of medicines and pesticides.28, 29
Through structural modifications, these analogues could give rise to
novel structures with improved biological activities, new modes of
action, and increased safety towards living beings and the
environment. Consequently, structural modifications inspired from
natural products are an ideal approach to identify new lead
molecules with useful functionalities.30
To date, notable examples of the successful utilization of natural
products as commercial inhibitors of the cytochrome bc1 complex
are analogues of the naturally occurring β-methoxyacrylate
strobilurin A.31-33 In addition, structural optimizations based on
Antimycin A,34-37 Neopeltolide,38-44 Crocacin D45-48 have also been
extensively reported. Despite enormous efforts devoted to the
discovery of natural product-derived inhibitors, there is still a high
demand for identification of new lead structures from natural
products which contain totally different structures from existing
inhibitors. Karrikinolide, a natural product bearing a 2H-furo[2,3-
c]pyran-2-one motif, captured our attention due to its molecular
simplicity, structural stability, novel skeleton, as well as diverse
bioactivities including its incredible potency for promoting seed
germination and seedling development of crop plants,49-62 and
antibacterial activities.63-66 It has been demonstrated that
substituents on the C-3 position played a significant role on the
a.Key Laboratory of Pesticide & Chemical Biology, College of Chemistry, Central
China Normal University, Wuhan 430079, P. R. China. Fax: +86-27-67867141; Tel:
+86-27-67867706 ; E-mails: qywu@mail.ccnu.edu.cn; gfyang@mail.ccnu.edu.cn.
b.State Key Laboratory of Advanced Technology for Materials Synthesis and
Processing, Wuhan University of Technology, Wuhan 430070, P.R. China.
67-69
biological activities of the corresponding compounds.55,
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O
2
O
CHO
3
O
1
O
4
5
7
O
6
O
O
OH
3
Karrikinolide
HO
HO
OH
O
R
O
O
O
2
3
Scheme 1 Karrikinolide and the designed molecules.
O
Br
O
2
1
O
1
4
5
7
Therefore, our goal was to synthesize a multitude of Karrikinolide
derivatives by introducing diverse functional groups on the C-3
position with the aim to adjust the global molecular
O
6
O
Target
4
Molecules
physicochemical characteristics (Scheme 1). Afterwards, as
a
continuation of our research on the development of novel
cytochrome bc1 complex inhibitors, the novel Karrikinolide
derivatives were extensively screened on their inhibiting
performance towards the cytochrome bc1 complex. Hence, it was
discovered, for the first time, that some of these compounds exhibit
high inhibitory activities.
Scheme 2 Synthetic route to Karrikinolide and the designed molecules.
Pd(PPh3)4 in refluxing THF provided the key intermediate 2.
Thereafter two reaction directions are possible using 2 - it would
either form the aldehyde 3, which could subsequently undergo a
reductive deoxygenation to give the natural product Karrikinolide,
or would be brominated to the corresponding bromide 4, which
2. Results and discussion
could be further functionalized to
derivatives.
a variety of Karrikinolide
It has been well-documented that Karrikinolide was originally
isolated from plant smoke.49, 50 However, it is insufficient to obtain
this compound merely in this way. Consequently, several research
groups developed their respective total synthesis of Karrikinolide
e.g. via the intramolecular Wittig reaction with thioketone, Ti-
crossed aldol addition and Cu(II)-catalyzed lactonization.70-74 Among
these approaches, Goddard-Borger’s protocol achieved the target
Karrikinolide in relatively higher yield.71 In particular, this approach
takes advantage of readily available D-xylose (1) to construct the
2H-furo[2,3-c]pyran-2-one 2 which, we realized, would allow us to
investigate the effect of modifications at this position on the activity
of Karrikinolide analogues (Scheme 2). As depicted in Scheme 2, the
key intermediate 2 in our envisioned approach could be obtained
from D-xylose (1) after a few steps. Afterwards, 2 could be
converted to the corresponding aldehyde 3, which could be further
transformed to the natural product Karrikinolide. Alternatively, 2
could react with NBS to give the bromide 4, which would undergo
palladium-catalyzed cross coupling reactions for formation of our
designed target molecules.
Succinate-cytochrome reductase (SCR) is composed of
respiratory complex II (SQR) and complex III (the bc1 complex), and
are believed to form a complex II-complex III supercomplex.75
Inhibitory activities of the newly prepared compounds against SCR
were tested at a concentration of 100 μM and listed in Table 1. The
IC50 values of the compounds with inhibition activities higher than
50% were further determined. The first and foremost, the inhibitory
activity of Karrikinolide was tested against SCR. Unfortunately, an
inhibition ratio of 13% was obtained at the concentration of 100 μM
(entry 1). Meanwhile, low inhibition rates of compounds 2-4
demonstrated that the less bulky groups such as H, -CHO and Br
were unfavorable (entries 2-4). To our delight, when a phenyl group
was employed as R, an inhibition rate of 52% was observed (entry
5). This result inspired us to conduct further structural
optimizations via implementation of different substituents on the
phenyl ring. Therefore, substituted phenyl groups were then
introduced for R. The results implied that employment of a
substituent at the para-position of the phenyl ring resulted in
significant improvement of the inhibitory activities in most cases
(entries 6-13). Apparently, alkyl substituents bearing hydrophobic
linear chains are superior to other functional groups such as
The detailed synthetic procedure for the synthesis of
Karrikinolide and its analogues could be described in Scheme 3. The
first step is the straightforward generation of compound 5 from 1,
followed by the regioselective tritylation of 5 to give the alcohol 6.
Oxidation of 6 afforded the ketone 7, which was transformed to the
desired (Z)-olefin 8 in high yield by utilizing the Horner-Wadsworth-
Emmons methodology. Treatment of 8 with aqueous trifluoroacetic
acid resulted in rapid formation of the butenolide 9 in excellent
yield via hydrolysis, lactonisation and rearrangement. Afterwards, 9
was treated with acetic anhydride in pyridine to exclusively yield
the α-diacetate 10, which went through an elimination to give 11 in
the presence of triethylamine. Indeed, treatment of 11 with
halogen and ethoxyl. Notably,
a sub-micromolar inhibitor,
compound 12g with 4-n-butylphenyl as R, was identified as being
over 100-fold more active compared to the commercial control
Amisulbrom (IC50 = 0.737 μM for entry 11 vs. IC50 = 93.0 μM for
entry 22). Replacing the n-butyl group at the para-position on the
phenyl ring with sterically hindered alkyl substituents reduced the
potency of the resulting compounds, as observed for the i-butyl 12h
and t-butyl 12i derivatives. The i-butyl analogue 12h (IC50 = 1.1 μM)
demonstrated comparable inhibitory activity in comparison to the
counterpart n-butyl inhibitor, while the t-butyl analogue (IC50 = 10.7
2 | J. Name., 2012, 00, 1-3
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μM) was at least 10 times less potent than the n-butyl-bearing Table 1. Inhibitory activities of the synthesized compounds against
derivative. In addition, a substituent at either the meta- or ortho- porcine SCR.
position of the phenyl ring didn’t significantly affect the activities
O
R
(entries 14-15), with respect to the 49% and 46% inhibition rates of
compounds 12j and 12k in which
a 3-methoxyl or 2-chloro
O
substituent was introduced, respectively. Furthermore, poly-
substituted phenyls were also evaluated. It appears that poly-
substitution on the phenyl ring is detrimental to the inhibition
potency. Compounds 12l-12o manifested inhibition rates ranging
from 25-47% at the concentration of 100 μM (entries 16-19).
Compounds of which the phenyl ring is replaced with a heterocyclic
ring (either furyl or pyridyl), compounds 12p and 12q respectively,
produced relatively poor levels of inhibition activities,
demonstrating inhibition potency of less than 10% (entries 20-21).
O
Inhibition ratio
(100 μM) /
IC50 (μM)
Entry
Compound No.
R
1
Karrikinolide
2
-Me
13%a
2
-H
<10%a
<10%a
3
3
-CHO
4
4
-Br
21%a
OH
HO
TrO
5
12a
12b
12c
12d
12e
12f
C6H5-
52%a
O
O
26.90 ± 0.16b
3.55 ± 0.11b
50%a
9.39 ± 0.12 b
8.50 ± 0.13 b
0.737 ± 0.011 b
1.10 ± 0.20 b
10.71 ± 0.10 b
49%a
46%a
47%a
41%a
30%a
HO
HO
OH
6
4-Cl-C6H4-
4-CF3O-C6H4-
4-EtO-C6H4-
4-iPrO- C6H4-
4-nPr- C6H4-
4-nBu-C6H4-
4-iBu-C6H4-
4-tBu-C6H4-
3-MeO-C6H4-
2-Cl-C6H4-
2-C12H7-
O
O
O
(a)
(b)
7
O
O
O
HO
HO
8
6
1
5
85% in two steps
9
TrO
O
10
11
12
13
14
15
16
17
18
19
TrO
O
O
(e)
80%
HO
(d)
(c)
12g
12h
12i
O
O
OH
O
O
75%
74%
O
O
COOEt
O
9
7
12j
8
O
12k
12l
O
O
(g)
(h)
(f)
O
12m
12n
12o
2,4-di-Cl-C6H3-
4-Me-2-Cl-C6H3-
3,5-di-F-C6H3-
OAc
OAc
80%
AcO
O
O
11
25%a
10
91% in two steps
<10%a
O
20
21
12p
O
O
Me
CHO
N
O
(j)
<10%a
(i)
90%
O
12q
81%
22
Amisulbrom
93.0 ± 1.3 b
O
O
a
O
Karrikinolide
The inhibition ratio was determined at the concentration of 100
μM; b IC50 values.
2
3
O
O
R
Br
Since SCR is composed of respiratory complex II (SQR) and
complex III (cyt bc1), it is of significant interest and importance to
further determine whether the synthesized compounds are
inhibitors targeting SQR or the bc1 complex. As listed in Table 2, we
compared the activities of four representative compounds (12c, 12e,
12g and 12h) against SCR, SQR and cyt bc1. The results indicated
that all these compounds exhibited limited activity against SQR at a
concentration of 10 μM. Moreover, their inhibitory activities against
SCR and the bc1 complex were comparable at the same
concentration. Therefore, it can be concluded from the above
results that the target compounds in this article have been
confirmed as inhibitors of the bc1 complex.
O
O
(l)
(k)
75%
O
O
12, 70-92%
4
Reagents and conditions: (a) (1) CuSO4, H2SO4, acetone, rt,
12 h, (2) aq. HCl, rt, 15 h; (b) (1) Et3N,CH2Cl2, rt, 1 h, (2)
CPh3Cl, rt,
3 h; (c) Ac2O, DMSO, rt, 22 h; (d) NaH,
(EtO)2POCH2CO2Et, THF, -10 oC, 1.5 h; (e) CF3COOH, H2O, rt,
30 min; (f) Ac2O, C5H5N, rt, 2 h; (g) Et3N, CH2Cl2, 30 min; (h)
Pd(PPh3)4, THF, reflux, 48 h; (i) (1) POCl3, DMF, 50 oC, (2) sat.
.
aq. NaHCO3; (j) tBuNH2 BH3, AlCl3, CH2Cl2, reflux; (k) NBS,
MeOH, rt, 20 min; (l) R-B(OH)2, Pd(OAc)2, SPhos, K3PO4,
toluene, reflux.
Scheme 3 The designed synthetic route of Karrikinolide and its
analogues.
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Table 2. The inhibition effect of the selected inhibitors against porcine SCR, SQR and cyt bc1.
Journal Name
Compound No.
IC50 (µM), SCR
3.55 ± 0.11
9.39 ± 0.12
0.737 ± 0.011
1.10 ± 0.20
I% (10 μM), SCR
I% (10 μM), SQR
I% (10 μM), cyt bc1
Selectivity
cyt bc1
12c
12e
12g
12h
76%
50%
82%
73%
14%
<10%
39%
26%
66%
46%
83%
67%
cyt bc1
cyt bc1
cyt bc1
c]pyran-4,7-diyl diacetate (10),71 2-oxo-4,5-dihydro-2H-furo[2,3-
c]pyran-4-yl acetate (11),71 and Karrikinolide71 were prepared
according to literature reports.
3. Conclusion
A new series of 2H-furo[2,3-c]pyran-2-one derivatives were
designed and synthesized via structural modifications from the
natural product Karrikinolide. It is worth mentioning that this is
the first time to discover bc1 complex inhibitors based on
Karrikinolide. The bioassay results indicated that these newly
prepared compounds exhibited varied inhibition against SCR, and
a 4-substituted phenyl group as the R substituent is paramount for
maintaining high potency. Excitingly, compound 12g showed
inhibition potency at the sub-micromolar level, which
demonstrated over 100-fold higher activity than amisulbrom, a
commercial inhibitor of the bc1 complex. Further evaluation
against the respective SQR and the bc1 complex indicated that
these compounds are inhibitors of the bc1 complex. Consequently,
a totally new skeleton that is different from existing bc1 complex
inhibitors was discovered, which could provide a new lead for
further development of bc1 complex inhibitors.
4.2. General procedure for synthesis of compounds 12a-12q
Inside an argon-filled glove box, Pd(OAc)2 (0.03 mmol), 2-
dicyclohexylphosphino-2',6'-dimethoxybiphenyl (Sphos, 0.075
mmol), compound 4 (1.0 mmol), K3PO4 (2.0 mmol), a boronic acid
(1.50 mmol) and toluene (5 mL) were added to an oven-dried
Schlenk tube. The Schlenk tube was taken out of the glove box and
the mixture was heated to reflux under argon atmosphere for 24 h.
The reaction mixture was cooled down to room temperature, and
the solvent was removed in vacuo. The residue was purified by
silica gel flash column chromatography to afford the desired
products 12a-12q.
4.3 Analytical data for Karrikinolide, compounds 2-4 and 12a-12q
3-Methyl-2H-furo[2,3-c]pyran-2-one (Karrikinolide).71 White solid:
m.p. 118.5-119.8 °C. Isolated yield: 81%. 1H-NMR (500 MHz, CDCl3)
δ 7.44 (s, 1H), 7.32 (d, J = 5.1 Hz, 1H), 6.51 (d, J = 5.1 Hz, 1H), 1.93
(s, 3H). 13C-NMR (125 MHz, CDCl3) δ 171.21, 147.96, 142.29,
139.70, 126.75, 103.43, 100.38, 7.67.
4. Experimental section
2H-furo[2,3-c]pyran-2-one (2).71 White solid: m.p. 106.8-
4.1 General considerations
1
1H-NMR spectra were recorded on a VARIAN Mercury-Plus 600 or
400 spectrometer or a Bruker Avance 500 spectrometer in CDCl3
or (CD3)2CO with TMS as the internal reference. 13C-NMR spectra
were recorded in CDCl3 or (CD3)2CO on a VARIAN Mercury-Plus 600
(150 MHz) or 400 (100 MHz) spectrometer or a Bruker Avance 500
(125 MHz) spectrometer. The following abbreviations are used to
designate multiplicities: s = singlet, d = doublet, dd = doublet of
108.0 °C. Isolated yield: 80%. H-NMR (500 MHz, CDCl3) δ 7.57 (s,
1H), 7.44 (d, J = 4.3 Hz, 1H), 6.67 (d, J = 4.2 Hz, 1H), 5.41 (s, 1H).
13C-NMR (125 MHz, CDCl3) δ 170.46, 149.11, 144.88, 143.34,
128.02, 104.76, 90.96.
2-Oxo-2H-furo[2,3-c]pyran-3-carbaldehyde (3).71 White solid:
m.p. 216-217.5 °C. Isolated yield: 90%. 1H-NMR (500 MHz, CDCl3) δ
9.92 (s, 1H), 8.07 (s, 1H), 7.94 (d, J = 2.3 Hz, 1H), 7.74 (d, J = 2.5 Hz,
1H). 13C-NMR (125 MHz, CDCl3) δ 185.50, 168.01, 153.69, 146.82,
142.97, 133.48, 107.95, 100.66.
3-Bromo-2H-furo[2,3-c]pyran-2-one (4).72 Light brown oil.
Isolated yield: 75%. 1H-NMR (500 MHz, CDCl3) δ 7.72-7.50 (m, 2H),
6.67 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ 165.80, 150.23, 142.36,
142.26, 128.47, 103.86, 82.91.
doublets,
MS was taken on an Agilent 6520 Accurate-Mass Q-TOF
instrument or a Bruker Daltonics microTOF-QII instrument.
t = triplet, q = quartet, m = multiplet. HR-
Melting points were taken on a Buchi B-545 melting point
apparatus and are uncorrected. Reagents including D-xylose (1)
and solvents were commercially available and treated with
standard methods before use. Besides, 2H-furo[2,3-c]pyran-2-one
(2),71 2-oxo-2H-furo[2,3-c]pyran-3-carbaldehyde (3),71 3-bromo-
3-Phenyl-2H-furo[2,3-c]pyran-2-one (12a). White solid: m.p.
1
90.0-91.1 °C. Isolated yield: 85%. H-NMR (500 MHz, CDCl3) δ 7.74
(4)72
,
5-(hydroxymethyl)-2,2-
(d, J = 6.9 Hz, 2H), 7.63 (s, 1H), 7.50-7.41 (m, 3H), 7.34-7.29 (m,
1H), 6.99 (d, J = 3.6 Hz, 1H). 13C-NMR (125 MHz, CDCl3) δ 168.77,
149.24, 142.40, 138.47, 130.89, 128.77, 128.37, 127.56, 127.03,
104.50, 103.54. HRMS (ESI): m/z calcd. for C13H9O3 [M+H]+:
213.05462; Found 213.05475.
2H-furo[2,3-c]pyran-2-one
dimethyltetrahydrofuro[2,3-d][1,3]dioxol-6-ol (5),76 2,2-dimethyl-
5-((trityloxy)methyl)tetrahydrofuro[2,3-d][1,3]dioxol-6-ol
(6),77
2,2-dimethyl-5-((trityloxy)methyl)dihydrofuro[2,3-d][1,3]dioxol-
6(3aH)-one (Z)-ethyl 2-(2,2-dimethyl-5-
(7),77
3-(4-Chlorophenyl)-2H-furo[2,3-c]pyran-2-one (12b). White solid:
m.p. 176.1-177.4 °C. Isolated yield: 90%. 1H-NMR [400 MHz,
(CD3)2CO] δ 8.08 (s, 1H), 7.88 (d, J = 5.5 Hz, 1H), 7.85-7.77 (m, 2H),
((trityloxy)methyl)furo[2,3-d][1,3]dioxol-6(3aH,5H,6aH)-ylidene)
acetate (8),71 4,7-dihydroxy-4,5,7,7a-tetrahydro-2H-furo[2,3-
c]pyran-2-one
(9),71
2-oxo-4,5,7,7a-tetrahydro-2H-furo[2,3-
4 | J. Name., 2012, 00, 1-3
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7.48-7.40 (m, 2H), 7.26 (d, J = 5.5 Hz, 1H). 13C-NMR [100 MHz,
(CD3)2CO] δ 168.33, 151.48, 142.61, 139.47, 132.62, 130.77,
130.31, 129.15, 128.81, 104.95, 101.04. HRMS (ESI): m/z calcd. for
C13H8ClO3 [M+H]+: 247.01565; Found 247.01583.
128.10, 127.94, 126.73, 125.70, 104.52, 103.56, 34.68, 31.25.
HRMS (ESI): m/z calcd. for C17H17O3 [M+H]+: 269.11722; Found
269.11756.
3-(3-Methoxyphenyl)-2H-furo[2,3-c]pyran-2-one (12j). White
1
3-(4-(Trifluoromethoxy)phenyl)-2H-furo[2,3-c]pyran-2-one (12c).
White solid: m.p. 111.9-113.3 °C. Isolated yield: 83%. 1H-NMR (500
MHz, CDCl3) δ 7.78 (d, J = 7.4 Hz, 2H), 7.67 (s, 1H), 7.52 (d, J = 3.5
Hz, 1H), 7.29 (d, J = 7.9 Hz, 2H), 6.97 (d, J = 3.9 Hz, 1H). 13C-NMR
(125 MHz, CDCl3) δ 168.55, 149.67, 148.24, 142.32, 138.76, 129.70,
128.79, 128.38, 121.28, 120.49 (q, J = 257.4 Hz), 104.29, 102.15.
HRMS (ESI): m/z calcd. for C14H8F3O4 [M+H]+: 297.03692; Found
297.03758.
solid: m.p. 117.1-118.4 °C. Isolated yield: 92%. H-NMR (500 MHz,
CDCl3) δ 7.64 (s, 1H), 7.47 (d, J = 3.8 Hz, 1H), 7.35 (s, 2H), 7.31 (s,
1H), 7.00 (d, J = 4.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 3.86 (s, 3H).
13C-NMR (125 MHz, CDCl3) δ 168.64, 159.90, 149.27, 142.37,
138.60, 132.18, 129.72, 128.41, 119.41, 113.32, 112.61, 104.59,
103.36, 55.34. HRMS (ESI): m/z calcd. for C14H11O4 [M+H]+:
243.06519; Found 243.06553.
3-(2-Chlorophenyl)-2H-furo[2,3-c]pyran-2-one (12k). White solid:
m.p. 171.4-172.8 °C. Isolated yield: 92%. 1H-NMR (500 MHz, CDCl3)
δ 7.69 (s, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.48 (s, 1H), 7.47 (d, J = 7.6
Hz, 1H), 7.37-7.28 (m, 2H), 6.65 (d, J = 4.5 Hz, 1H). 13C-NMR (125
MHz, CDCl3) δ 168.67, 148.82, 142.41, 141.05, 133.43, 131.68,
130.02, 129.40, 129.17, 128.73, 126.85, 105.82, 102.05. HRMS
(APCI): m/z calcd. for C13H8ClO3 [M+H]+: 247.01565; Found:
247.01563.
3-(4-Ethoxyphenyl)-2H-furo[2,3-c]pyran-2-one (12d). White solid:
m.p. 141.9-143.4 °C. Isolated yield: 82%. 1H-NMR (500 MHz, CDCl3)
δ 7.68 (d, J = 8.1 Hz, 2H), 7.58 (s, 1H), 7.41 (d, J = 4.9 Hz, 1H), 6.96
(d, J = 8.0 Hz, 2H), 6.93 (d, J = 5.0 Hz, 1H), 4.07 (q, J = 6.5 Hz, 2H),
1.43 (t, J = 6.6 Hz, 3H). 13C-NMR (125 MHz, CDCl3) δ 169.00, 158.39,
148.78, 142.36, 137.11, 128.28, 127.86, 123.17, 114.79, 104.41,
103.50, 63.53, 14.80. HRMS (ESI): m/z calcd. for C15H13O4 [M+H]+:
257.08084; Found 257.08099.
3-(Naphthalen-2-yl)-2H-furo[2,3-c]pyran-2-one (12l). White solid:
m.p. 148.1-149.6 °C. Isolated yield: 81%. 1H-NMR (500 MHz, CDCl3)
δ 8.27 (s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.83 (d, J = 7.3 Hz, 2H), 7.65
(s, 1H), 7.49 (d, J = 3.1 Hz, 3H), 7.09 (d, J = 4.5 Hz, 1H). 13C-NMR
(125 MHz, CDCl3) δ 168.80, 149.28, 142.44, 138.54, 133.46, 132.53,
128.41, 128.36, 128.31, 128.17, 127.64, 126.37, 126.22, 126.18,
124.62, 104.61, 103.51. HRMS (ESI): m/z calcd. for C17H11O3
[M+H]+: 263.07027; Found 263.07052.
3-(4-Isopropoxyphenyl)-2H-furo[2,3-c]pyran-2-one (12e). White
1
solid: m.p. 115.4-116.8 °C. Isolated yield: 78%. H-NMR [600 MHz,
(CD3)2CO] δ 8.00 (s, 1H), 7.80 (d, J = 5.5 Hz, 1H), 7.76 (d, J = 8.7 Hz,
2H), 7.21 (d, J = 5.5 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 4.74-4.65 (m,
1H), 1.32 (d, J = 6.0 Hz, 6H). 13C-NMR [150 MHz, (CD3)2CO] δ
169.18, 158.02, 150.97, 142.93, 138.06, 129.56, 128.97, 124.15,
116.52, 105.02, 102.67, 70.21, 22.22. HRMS (ESI): m/z calcd. for
C16H15O4 [M+H]+: 271.09649; Found 271.09696.
3-(2,4-Dichlorophenyl)-2H-furo[2,3-c]pyran-2-one (12m). White
1
3-(4-Propylphenyl)-2H-furo[2,3-c]pyran-2-one (12f). White solid:
m.p. 112.3-113.8 °C. Isolated yield: 86%. 1H-NMR (500 MHz, CDCl3)
δ 7.66 (d, J = 7.5 Hz, 2H), 7.60 (s, 1H), 7.44 (d, J = 5.3 Hz, 1H), 7.26-
7.24 (m, 2H), 6.97 (d, J = 5.4 Hz, 1H), 2.61 (t, J = 7.4 Hz, 2H), 1.73-
1.64 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H). 13C-NMR (125 MHz, CDCl3) δ
168.89, 148.95, 142.40, 142.31, 137.89, 128.86, 128.16, 128.09,
126.88, 104.53, 103.68, 37.87, 24.48, 13.82. HRMS (APCI): m/z
calcd. for C16H15O3 [M+H]+: 255.10157; Found 255.10161.
solid: m.p. 164.1-165.4 °C. Isolated yield: 77%. H-NMR (500 MHz,
CDCl3) δ 7.71 (s, 1H), 7.51 (d, J = 4.5 Hz, 1H), 7.49 (s, 1H), 7.47 (d, J
= 8.3 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 6.64 (d, J = 4.7 Hz, 1H). 13C-
NMR (125 MHz, CDCl3) δ 168.44, 149.06, 142.39, 141.17, 134.62,
134.06, 132.44, 129.86, 129.00, 127.85, 127.31, 105.80, 100.97.
HRMS (ESI): m/z calcd. for C13H7Cl2O3 [M+H]+: 280.97668; Found
280.97689.
3-(2-Chloro-4-methylphenyl)-2H-furo[2,3-c]pyran-2-one (12n).
White solid: m.p. 174.1-175.4 °C. Isolated yield: 83%. 1H-NMR [600
MHz, (CD3)2CO] δ 8.10 (s, 1H), 7.85 (d, J = 5.3 Hz, 1H), 7.40 (d, J =
7.9 Hz, 1H), 7.38 (s, 1H), 7.24 (d, J = 7.7 Hz, 1H), 6.78 (d, J = 5.4 Hz,
1H), 2.39 (s, 3H). 13C-NMR [150 MHz, (CD3)2CO] δ 162.63, 145.11,
137.02, 135.93, 134.82, 127.79, 126.42, 124.94, 124.22, 122.58,
121.30, 99.84, 95.72, 14.83. HRMS (ESI): m/z calcd. for C14H10ClO3
[M+H]+: 261.03130; Found 261.03413.
3-(4-Butylphenyl)-2H-furo[2,3-c]pyran-2-one (12g). White solid:
m.p. 90.1-91.4 °C. Isolated yield: 87%. 1H-NMR (500 MHz, CDCl3) δ
7.66 (d, J = 7.1 Hz, 2H), 7.60 (s, 1H), 7.44 (d, J = 4.9 Hz, 1H), 7.25 (s,
2H), 6.97 (d, J = 4.7 Hz, 1H), 2.64 (t, J = 7.3 Hz, 2H), 1.66-1.58 (m,
2H), 1.41-1.33 (m, 2H), 0.94 (t, J = 7.1 Hz, 3H). 13C-NMR (125 MHz,
CDCl3) δ 168.88, 148.95, 142.53, 142.40, 137.88, 128.81, 128.12,
128.08, 126.89, 104.52, 103.67, 35.47, 33.53, 22.33, 13.92. HRMS
(ESI): m/z calcd. for C17H17O3 [M+H]+: 269.11722; Found 269.11757.
3-(4-Isobutylphenyl)-2H-furo[2,3-c]pyran-2-one (12h). White
solid: m.p. 99.3-100.2 °C. Isolated yield: 75%. 1H-NMR (500 MHz,
CDCl3) δ 7.66 (d, J = 6.8 Hz, 2H), 7.60 (s, 1H), 7.47-7.42 (m, 1H),
7.22 (d, J = 6.9 Hz, 2H), 6.98 (d, J = 3.9 Hz, 1H), 2.50 (d, J = 6.4 Hz,
2H), 1.96-1.84 (m, 1H), 0.92 (d, J = 5.2 Hz, 6H). 13C-NMR (125 MHz,
CDCl3) δ 168.91, 148.96, 142.40, 141.33, 137.89, 129.50, 128.19,
128.10, 126.73, 104.55, 103.67, 45.25, 30.22, 22.37. HRMS (APCI):
m/z calcd. for C17H17O3 [M+H]+: 269.11722; Found 269.11703.
3-(4-(Tert-butyl)phenyl)-2H-furo[2,3-c]pyran-2-one (12i). White
3-(3,5-Difluorophenyl)-2H-furo[2,3-c]pyran-2-one (12o). White
1
solid: m.p. 156.0-157.4 °C. Isolated yield: 78%. H-NMR (500 MHz,
CDCl3) δ 7.73 (s, 1H), 7.59 (d, J = 4.2 Hz, 1H), 7.33 (d, J = 6.1 Hz, 2H),
7.03 (d, J = 4.2 Hz, 1H), 6.76 (dd, J = 7.6, 7.4 Hz, 1H). 13C-NMR (125
MHz, CDCl3) δ 167.95, 163.29 (dd, J = 247.9, 13.0 Hz), 150.11,
142.19, 139.37, 133.97 (t, J = 11.1 Hz), 129.32, 109.61 (dd, J = 20.1,
6.5 Hz), 104.39, 102.82 (t, J = 25.2 Hz), 101.21. HRMS (ESI): m/z
calcd. for C13H7F2O3 [M+H]+: 249.03578; Found 249.03609.
3-(Furan-3-yl)-2H-furo[2,3-c]pyran-2-one (12p). Pale yellow oil.
1
Isolated yield: 72%. H-NMR (500 MHz, CDCl3) δ 8.07 (s, 1H), 7.58
1
solid: m.p. 154.1-155.2 °C. Isolated yield: 85%. H-NMR (500 MHz,
(s, 1H), 7.51 (s, 1H), 7.45 (d, J = 4.8 Hz, 1H), 6.80 (d, J = 4.8 Hz, 1H),
6.78 (s, 1H). 13C-NMR (125 MHz, CDCl3) δ 168.48, 148.74, 143.33,
142.64, 140.88, 136.10, 128.06, 115.69, 107.84, 104.33, 97.28.
CDCl3) δ 7.68 (d, J = 7.3 Hz, 2H), 7.61 (s, 1H), 7.47 (d, J = 7.3 Hz, 2H),
7.44 (d, J = 5.3 Hz, 1H), 6.98 (d, J = 5.0 Hz, 1H), 1.35 (s, 9H). 13C-
NMR (125 MHz, CDCl3) δ 168.90, 150.67, 148.96, 142.41, 137.96,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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Journal Name
HRMS (ESI): m/z calcd. for C11H7O4 [M+H]+: 203.03389; Found
203.03338.
5.
6.
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C13H10NO3 [M+H]+: 228.06552, Found 228.06548.
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4.4 Enzyme assay
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The overall activity of SCR (both SQR and cyt bc1) was determined
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SQR in SCR was selectively determined by using succinate and
dichlorophenolindophenol (DCIP) as substrates, while that of cyt
bc1 in SCR was determined by using decylubiquinol (DBH2) and
13.
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cytochrome c as substrates.
The preparation of SCR from the
porcine heart was essential as reported,1 and the preparation of
DBH2 from DB was carried out according to the procedure
described in a previous publication.75 To obtain the IC50 values of
the compounds, all reactions were initiated by the addition of
enzyme and monitored continuously by following the absorbance
change at certain wavelengths on a PerkineElmer Lambda 45
spectrophotometer equipped with a magnetic stirrer at 23 °C.78
The activity of SCR was measured by monitoring the increase of
cytochrome c at 550 nm, by using the extinction coefficient of 18.5
mM-1 cm-1. The activity of SQR was measured by monitoring the
decrease of 2,6-dichlorophenolindophenol (DCIP) at 600 nm, by
using the extinction coefficient of 21 mM-1 cm-1. The reaction
mixture may be scaled down to 1.8 mL with final concentrations of
PBS (pH 7.4), 100 mM; EDTA, 0.3 mM; succinate, 20 mM; oxidized
cytochrome c, 60 mM (or DCIP, 53 mM); and appropriate amounts
of enzyme to start the reaction. Moreover, the activity of cyt bc1 in
catalyzing the oxidation of DBH2 by cytochrome c was measured
by monitoring the increase of cytochrome c at 550 nm, and
assayed in 100 mM PBS (pH 7.4), 0.3 mM EDTA, 750 μM lauryl
maltoside (n-dodecyl-b-D-maltoside), 100 μM DBH2, 100 μM
oxidized cytochrome c, and an appropriate amount of SCR. The
detailed description for performing the enzyme assay could be
referred to our previous publications.1, 18-23
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,
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22.
23.
24.
25.
26.
27.
5. Acknowledgement
This research was supported by the National Key Technologies
R&D Program (2014BAD23B01）and the National Natural Science
Foundation of China (Nos. 21272091, 21332004, 21472063).
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DOI: 10.1039/C6RA19424A
Graphical Abstract
Discovery of cytochrome bc1 complex inhibitors inspired by the
natural product Karrikinolide
Cheng Chen,b Qiong-You Wu*a, Lian-Ying Shan,a Bei Zhang,a Francis Verpoortb and
Guang-Fu Yang*a
Novel and potent inhibitors targeting the cytochrome bc1 complex were discovered
from the natural product Karrikinolide for the first time.