Design, Synthesis, and Biological Testing of Novel ... - ACS Publications

Subscriber access provided by Mississippi State University Libraries

Article

Design, Synthesis and Biological Testing of Novel Naphthoquinones as Substrate-Based Inhibitors of the Quinol:Fumarate Reductase from Wolinella succinogenes Hamid Reza Nasiri, M Gregor Madej, Robin Panisch, Michael Lafontaine, Jan W. Bats, C. Roy D. Lancaster, and Harald Schwalbe J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Nov 2013 Downloaded from http://pubs.acs.org on November 25, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Design, Synthesis and Biological Testing of Novel Naphthoquinones as Substrate-Based Inhibitors of the Quinol:Fumarate Reductase from Wolinella succinogenes Hamid Reza Nasiri,*,†,

M. Gregor Madej,‡,┴ Robin Panisch,§ Michael Lafontaine, # Jan W.

Bats,† C. Roy D. Lancaster,‡,# and Harald Schwalbe*,† † Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Straße 7, D60438 Frankfurt am Main, Germany ‡ Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Cluster of Excellence Frankfurt “Macromolecular Complexes”, Max-von-Laue-Strasse 3, D-60438 Frankfurt am Main, Germany § Institute of Inorganic and Analytical Chemistry, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Str. 7, D-60438 Frankfurt am Main, Germany # Department of Structural Biology, Center of Human and Molecular Biology, Saarland University, Faculty of Medicine, Building 60, D-66421 Homburg, Germany

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 41

ABSTRACT

Novel naphthoquinones were designed, synthesized and tested as substrate-based inhibitors against the membrane-embedded protein quinol:fumarate reductase (QFR) from Wolinella succinogenes, a target closely related to QFRs from the human pathogens Helicobacter pylori and Campylobacter jejuni. For a better understanding of the hitherto structurally unexplored substrate binding pocket, a structure-activity relationship (SAR) study was carried out. Analogues of lawsone (2-hydroxy-1,4-naphthoquinone 3a) were synthesized that vary in length and size of the alkyl side chains (3b-k). A combined study on the prototropic tautomerism of 2-hydroxy-1,4-naphthoquinones series indicated that the 1,4-tautomer is the more stable and biologically relevant isomer and that the presence of the hydroxyl group is crucial for inhibition. Furthermore, 2-bromine-1,4-naphthoquinones (4a-c) and 2-methoxy1,4-naphthoquinones (5a-b) series were also discovered as novel and potent inhibitors. Compounds 4a and 4b showed IC50-values in low µM range in the primary assay and no activity in the counter DT-diaphorase assay.


Page 3 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction The human pathogens Helicobacter pylori and Campylobacter jejuni1 colonize mammalian intestines, causing peptic ulcers, gastric atrophy, gastric MALT (mucosa-associated lymphoid tissue) lymphoma,2 and are associated with the development of gastric adeno-carcinoma, the world’s second leading cause of cancer-related death.3 Quinol:fumarate reductase (QFR) is essential in H. pylori for the colonization of murine stomach,1 and therefore it has been considered to be a potential drug target.4 Analogous results have been obtained for the QFR from C. jejuni.5 The QFR enzyme belongs to the superfamily of succinate:quinone oxidoreductases (SQORs)6 consisting of membrane-embedded protein complexes. SQORs couple the two-electron reduction of fumarate to succinate to the two-electron oxidation of quinol to quinone, the terminal reaction of fumarate respiration, a form of anaerobic respiration replacing oxygen by fumarate as the terminal electron acceptor; as well as the reverse reaction, the oxidation of succinate by quinone as part of the citric acid cycle (succinate:quinone reductase, SQRs). The QFRs from epsilon-proteobacteria7 like H. pylori and C. jejuni or the non-pathogen Wolinella succinogenes differ from those found in other Gram-negative bacteria (e.g. Escherichia coli) in that the membrane spanning domain consists of one large poly-peptide that binds two heme groups instead of two small polypeptides with no heme bound in the case of E. coli QFR. The two heme groups support transmembrane electron transfer8-10 and thereby allow the quinone binding-site to be oriented to the opposite side of membrane compared to E. coli QFR.11 The anaerobic epsilon-proteobacterium W. succinogenes has been particularly well studied.12 The crystal structure of QFR from W. succinogenes was solved initially at 2.2 Å8 and later improved to 1.78 Å resolution.10 However, despite the high-resolution crystal structure of this membrane complex, the exact organization of the quinone substrate binding-site is still elusive. Substances such as 2thenoyltrifluoroacetone

(TTFA)

and

3’-methyl-5,6-dihydro-2-methyl-1,4-oxathin-3-

carboxanilide (3’-methylcarboxin) or 2-n-heptyl-4-hydroxyquiloline-N-oxide (HQNO), 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 41

known inhibitors of other members of the SQOR superfamily, are ineffective for W. succinogenes QFR.13 Therefore, from a medicinal point of view the identification and characterization of potent inhibitors targeting QFRs in epsilon-proteobacteria like H. pylori and C. jejuni or the non-pathogen W. succinogenes is of great interest. Such molecules would also be useful as mechanistic probes to investigate the quinol oxidation site in these membrane protein complexes. Given, that precise structural information of the quinone binding-site is currently unknown, the use of a structure-based approach to inhibitor design is not feasible. Therefore, we used an alternative method to discover such molecules, by creation of analogues of the natural substrate, following a substrate-based inhibitor design strategy.

Figure 1. Constitution of the natural substrate MK-6, modified substrates (1-2) and substratebased inhibitors (3-5). 1,4-Naphthoquinones including menaquinone-6 (MK-6) are substrates for diheme-containing SQORs.14 Starting from the natural substrate MK-6, we designed different substituted naphthoquinones with the purpose of discovering potent substrate-based inhibitors (Figure 1). When the isoprenoid side chain R2 in MK-6 at the 3-position was replaced by a decyl-group, no changes in enzymatic activity were detected compared to the natural substrate MK-6. Both compounds (1) and (2) were accepted and converted by the enzyme as substrates. In contrast, the substitution of the 2-position by a hydroxyl-group (3), bromine (4) or methoxy-group (5) resulted in compounds that inhibited the enzymatic activity of QFR.


Page 5 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


This observation suggests that the variation of the substituent at the 2-position in the quinone-scaffold is crucial for determining the substrate or inhibitor behavior of MK-6 designed derivatives. Alkyl-2-hydroxy-1,4-naphthoquinones such as compound 3 are known for their wide range of biological properties. They act as antimalarial,15-17 anticancer,18 antileishmanial19-21 and insecticidal agents22. They are also able to inhibit quinoloxidizing/quinone-reducing membrane protein complexes such as cytochrome bc1 complex,2325

photosystem II26 and diheme-containing succinate:quinone oxidoreductase (SQOR)

membrane protein complexes with IC50 values in the µM range.9-10 Recently, Trumpower et al. investigated the influence of the length of the alkyl side chain in related alkyl-3-hydroxy1,4-naphthoquinones on bc1 complex inhibition, for which a chain of eight carbon atoms were required to achieve significant enzymatic inhibition with IC50 values below 100 nM.23-25 These results suggest the possibility to design and synthesize analogues of a lead structure in an analogue-based strategy, through monoalkylation and variation of the length and bulkiness of the alkyl-residue.4 To investigate the influence of size and length of the hydrocarbon tail structure of the substrate-based 3-alkyl-2-hydroxy-1,4-naphthoquinones series on inhibition, the hydroxy-naphthoquinone scaffold (lawsone 3a) was kept unchanged, while the 3-position was systematically altered with different hydrocarbon tail structures (3b-k) (Figure 2). In the first step, we changed the length of the hydrocarbon tail (3a-e). In the second variation step, we changed the bulkiness of the hydrophobic tail structure; here, representative tertiary butyl(3g) and cyclohexyl- (3i and 3j) substituents were introduced. Taking the possibility of hydrogen-bonding into account, bromine and hydroxyl-groups were attached (3f and 3h) with increasing inhibitor hydrophilicity. Finally, in order to estimate the strength of the cation-π and π-π interactions for binding, compound (3k), a rigid analogue of (3i), was prepared containing an aromatic ring system. The same strategy was applied for the 3-alkyl-2-bromo1,4-naphthoquinones (4a-c) and 3-alkyl-2-methoxy-1,4-naphthoquinones series (5a-b). Here, the SAR study was focused only on the influence of the side chain length alteration (figure 2). 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Constitution of the substrate-based inhibitors (3-5) used in this SAR-study. We report the design and synthesis of 23 naphthoquinones as substrate-based analogues, along with their testing in enzyme activity assay against QFR from W. succinogenes. A good correlation was found for the hydrophobicity and the inhibition properties of the hydroxyl based derivatives. Furthermore, we also report on the investigation of the predominant tautomer and structural features important for the binding. Results and discussion Synthesis The synthetic routes to the desired naphthoquinones series are shown in schemes 1-5. The alkyl side chain in (1-5) was introduced by a radical alkylation of the corresponding naphthoquinones. Different radical alkylation methods are described in the literature, namely by using alkyl iodides in the presence of tributyltin hydride and 2,2’-azobisisobutyronitrile as radical initiator, via a photosensitive thiohydroxamic ester,23-25, 27-28 or using alkyl iodides in the presence of benzoyl peroxide in acetic acid. These methods are multistep syntheses and suffer from low yield or the need of using protecting groups. In this study, the alkyl side-chain was introduced in one step via Hunsdiecker oxidative decarboxylation followed by an oxa6 ACS Paragon Plus Environment

Page 6 of 41

Page 7 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Michael addition of the corresponding acids.29 The alkylation is promoted by silver nitrate as a catalyst in the presence of ammonium peroxydisulfate in an acetonitrile/water mixture. We have successfully used this reaction for the alkylation of a wide range of different quinone scaffolds.9-10, 30 Similar to other known radical alkylation reactions, the yield of the resulting alkylated products by this method was low as well. However, for this reaction, we were able to increase the yield up to 40-50% by reducing the solvent volume and by addition of the corresponding acid in two separate portions (see experimental section). Under these conditions, the desired product precipitated directly from the solvent and no further purification was required. The monoalkylated naphthoquinones (1a, 2, 7 and 8) could be synthesized using these optimized conditions to give pure products in high yield. After the first alkylation step, compound 1a was further alkylated using the same conditions to obtain the desired dialkylated product 1. Surprisingly, when trifluoroacetic acid was used for the alkylation of (1a) under Hunsdiecker conditions, the epoxidated product of (1a), (±)-1amethyl-1a,7a-dihydro-naphth[2,3-b]oxirene-2,7-dione

(9),

was

formed.

The

bromo-

substituted series (4a-h) was synthesized in a similar manner to the monoalkylated naphthoquinones (1a, 2, 7 and 8), starting from 2-bromo-1,4-naphthoquinone 1031 by using the same protocol. For the 3-alkyl-2-hydroxy-1,4-naphthoquinone series (3a-k), four different synthetic methods were applied. The hydroxy group was introduced by a sequence of in situ ∆2-Weitz-Scheffer-type epoxidation/epoxide cleavage reaction32 (scheme 1) or via nucleophilic substitution of bromine (scheme 2).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 41

Scheme 1. Synthesis of substituted hydroxyquinones, method 1.

(a) Radical Hundsdiecker oxidative decarboxylation/oxa-Michael addition; i) corresponding acid 1.5 equiv.; ii) 1.8 equiv. (NH4)2S2O8; iii) 0.5 equiv. AgNO3, CH3CN, H2O, 80 °C, 7h; (b) Weitz-Scheffer-type epoxidation/epoxide cleavage i) H2O2, Na2CO3; ii) H2SO4; (c) trifluoroacetic acid 1.5 equiv.; ii) 1.8 equiv. (NH4)2S2O8; iii) 0.5 equiv. AgNO3, CH3CN/H2O, 60-70 °C, 7 h. Scheme 2. Synthesis of substituted hydroxyquinones, method 2.

(a) Radical Hunsdiecker oxidative decarboxylation/oxa-michael addition; i) corresponding acid 1.5 equiv.; ii) 1.8 equiv. (NH4)2S2O8 iii) 0.5 equiv. AgNO3, CH3CN/H2O, 60-70 °C, 7h; (b) nucleophilic substitution: KOH/MeOH, reflux, 1 h. The epoxidation/epoxide cleavage turned out to be an effective approach to hydroxylsubstituted naphthoquinones. The ∆2-Weitz-Scheffer-type epoxidation of the endocyclic double bond was carried out in ethanol as solvent by adding a solution of alkaline hydrogen peroxide at room temperature. The pure epoxide was formed after a short reaction time and in 8 ACS Paragon Plus Environment

Page 9 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


high yield. The conversion was monitored during the reaction by thin layer chromatography (TLC), and by high field shift of the diagnostic signal for the α-hydrogen in the 1H-NMR spectrum. Without an isolation step, the epoxides were directly cleaved under acidic conditions to generate the desired hydroxyl quinones. Compound (3i) was also synthesized simultaneously by Hooker oxidation33 (scheme 3). Scheme 3. Synthesis of substituted hydroxyquinones, method 3. O

O OH

OH a

3j

O

3i

O

(a) Hooker oxidation. KMnO4/ NaOH, 0 °C. For compound (3h), a new synthetic route was applied. Interestingly the alkaline ether hydrolysis of either α- or β-lapachones34 as precursor resulted in the formation of the product (3h) (scheme 4). Scheme 4. Synthesis of substituted hydroxyquinones, method 4.

(a) Alkaline ether hydrolysis of α-(11) or β-lapachone (12). 1 % aqueous NaOH, reflux, 2 h. The

methoxyalkynaphthoquinones

(5a-b)

were

obtained

by

methylation

of

the

hydroxyalkynaphthoquinones analogues with dimethyl sulfate in acetone at room temperature (scheme 5).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 41

Scheme 5. Synthesis of substituted methoxyalkynaphthoquinone.

(a) Potassium carbonate, dimethyl sulfate, acetone, 2 h room temperature. The compounds were characterized by

1

H-NMR,

13

C-NMR spectroscopy, elementary

analyses and X-ray diffraction. The chemistry shown in schemes 1-5 proved to be a clear and robust route to obtain a diverse set of analogues for our SAR study, and an attractive strategy for a follow-up hit-to-lead phase. As reported for some related 2-alkyl-3-hydroxy-1,4-naphthoquinones with anticancer properties, these molecules exist in two different tautomeric forms,18 namely the 1,2- and a 1,4-quinone form, which are connected by a prototopic tautomerism (Figure 3). The 1,2- and 1,4-naphthoquinone tautomers possess different thermodynamic stability and different biological properties. For example, β-lapachone (12), a 1,2-isomer inhibits topoisomerase II activity tenfold better than its 1,4-quinone isomer α-lapachone (11).18 O

O OH

O R

R O

OH

Figure 3. Tautomerism of 2-alkyl-3-hydroxy-1,4-naphtoquinones. To predict which hydroxy-alkylnaphthoquinones tautomer is thermodynamically more stable, we studied the tautomerism of 2-alkyl-3-hydroxy-1,4-naphthoquinones in solution by 1H- and


Page 11 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13

C-NMR spectroscopy, in solid state by single crystal analysis and by quantum mechanical

methods. For the study in solution, we selectively trapped the 1,4- and 1,2-tautomer by preparing the Oalkylated naphthoquinones (13) and (14) (Scheme 6). Scheme 6. Constitution of trapped 1,4- and 1,2-tautomers, 2-(nonyloxy)-1,4-naphthoquinone (13) and 4-(nonyloxy)-1,2-naphthoquinone (14). O

O OH

3a

O

O OR

a 13

O

14

O

OR

R= -(CH2)8CH3

(a) 1-bromononane, K2CO3, DMF, reflux 5h. This method in combination with

1

H- and

13

C-NMR spectroscopy has already been

successfully applied to study the tautomerism of 4-hydroxy-4(1H) quinolone and assignment of lapachone isomers.34-35 For the constitutional isomers (13) and (14), significant differences in the aromatic region of the proton signals in the 1H-NMR spectrum (Figure 4) as well as chemical shift differences for the carbonyl signals in the

13

C-NMR spectrum (not shown)

were observed.

Figure 4. Aromatic region of the proton signals in the 1H NMR spectra of (3), (13) and (14). Perspective view of the X-ray structure and dihedral angle χ (C12-C11-C10-C9) of (3), 11 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 41

showing the atom numbering with displacement ellipsoids drawn at the 50% probability level. Selected bond distances (Å) and bond angles (deg): O(2)-C(2) 1.228(3), O(3)-C(9) 1.235(2), O(1)-C(1) 1.344(3), C(2)-C(1) 1.484(3), C(1)-C(10) 1.355(3), O(2)-C(2)-C(1) 119.5(2), O(1)C(1)-C(10) 120.2(2), C(10)-C(9)-O(3) 119.9(2). Whereas the 1,4-isomer (11) reveals two sets of multiplet signals for the four aromatic protons in the 1H-NMR spectrum and two signals for the carbonyl groups in the 13C-NMR spectrum at 185.9 and 180.1 ppm, isomer 12 possess four well-resolved signals for each aromatic proton in the 1H-NMR spectrum and two signals for the carbonyl groups in the 13C-NMR spectrum at 179.5 and 168.9 ppm. In case of (3), we observed the similar chemical shift pattern in the aromatic region of the 1H-NMR spectrum as present in 1,4-isomer (13) (Figure 4), furthermore a broad singlet at 7.2 ppm, which was assigned to the free hydroxy proton. In analogy to isomer (14), the carbonyl signals of (3) appear at 184.3 and 181.4 ppm, in accordance with the predominate presence of the 1,4 tautomer. Single crystals of (3, 3g, 3i) suitable for X-ray analysis were obtained from dichloromethane. The molecular structure of (3) (monoclinic, P21/c), is shown in Figure 4. The alkyl side chain has an all-trans conformation and the naphthoquinone scaffold shows a significant deviation from planarity (mean deviation of carbon atoms from plane: 3 pm). The C12-C11 bond is orientated almost perpendicular to the naphthoquinone plane (torsion angle χ: C12-C11-C10-C9: 89.1(2)°). Interestingly for stigmatellin, an inhibitor of bacterial reaction center and the cytochrome bc1 complex,36 this orientation of the side-chain relative to the head group is one of the two preferred geometries (referred to as type 1). The dihedral angle χ has been reported to be essential factor for stigmatellin binding.37 The conformation is significantly influenced by an intramolecular O-H...O hydrogen bond between the hydroxy group and the adjacent carbonyl O atom. A similar geometry was observed for 2-hydroxy-3-(3-oxobutyl)-1,4-naphthoquinone, a related 2-hydroxy-1,412 ACS Paragon Plus Environment

Page 13 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


naphthoquinone.38 The distances O(2)-C(2) 122.8(3) pm, O(3)-C(9) 123.5(2) pm confirm the double bond character while O(1)-C(1) distance of 134.4(3) pm points out the single bond. Furthermore, crystal structures of (3g) and (3i), similar to (3) emphasize the presence of the 1,4-tautomer with the free hydroxy group (see supplementary material). This finding is supported by the results of density functional calculations applying a solvent model, which demonstrate that the 1,4 tautomer is energetically preferred by 17.6 kJ mol-1 compared to the 1,2 tautomer (see supplementary material). In fact, the thermodynamic more stable tautomer is not necessarily the bioactive form and it has been shown that proteins prefer a specific tautomer during the binding and shift the tautomeric equilibrium in favor of one species.39 In order to identify the bioactive tautomer, we tested our trapped isomers (13) and (14) in the enzyme activity assay.

Biological testing The inhibition effect of the substituted 3-alkyl-1,4-naphthoquinones (3a-k, 4a-c and 5a-b) on quinol:fumarate reductase activity and on succinate:quinone reductase activity of QFR from W. succinogenes are given in figures 5-7. The inhibition effect was monitored as the ratio of specific activity in the presence of 20 µM of the respective compound and in its absence (“% of activity: residual activity”). For the quinol:fumarate reductase activity of QFR, the quinones were pre-reduced to the quinol before the measurements were taken. The hydrogen and methyl substituted compounds (1) and (2) were recognized and converted by the enzyme as substrate. As represented in figures 5-7, all of the designed naphthoquinones (3-5) were inhibiting the enzyme activity in both direction, the quinol:fumarate reductase activity as well as the reverse reaction, succinate:quinone reductase activity. Amongst the series, the aliphatic side chains in the 3-position and substituents in the 2-position clearly influence the enzymatic activity and therefore endow these derivatives with a considerable inhibition potential. 13 ACS Paragon Plus Environment


150

-OH red -OH ox

125

% residual activity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 41

100 75 50 25 0

3a

3b

3c

3d

3

3e

3f

3g

3h

3i

3j

3k

Figure 5. 3-alkyl-2-hydroxy-1,4-naphthoquinones (3a-k) and their inhibition performance on succinate:quinone reductase activity (gray bars) and on the quinol:fumarate reductase activity (white bars) of QFR from W. succinogenes, respectively.

150

-OMe red -OMe ox

125 100 75 50 25 0

5a

5b

5

Figure 6. 3-alkyl-2-methoxy-1,4-naphthoquinones (5a-b) and their inhibitory effect on the quinol:fumarate reductase activity of QFR from W. succinogenes. In contrast to the measurements summarized in the previous tables, the residual activity of substrate reduction could not be determined due to the high level of non-enzymatic reaction.


Page 15 of 41

-Br red -Br ox

150

% residual activity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


125 100 75 50 25 0

4a

4b

4c

4

Figure 7. 3-alkyl-2-bromo-1,4-naphthoquinones (4a-c) and their inhibitory effect on the succinate:quinone reductase activity (gray bars) and on the quinol:fumarate reductase activity (white bars) of QFR from W. succinogenes. The systematic elongation of the alkyl side chain in the hydroxyl series (3a to 3e) results in an increase in inhibition potency as indicated in a decrease in residual enzyme activity. Changing the hydrogen group in 3a by a methyl group (3b) results in a substantially increased inhibition. Further elongation from methyl to hexyl group (3b to 3d) leaves the inhibition unchanged. However, when the length of six carbon atom was exceeded (3 and 3e) a second increase in inhibition was observed. Restriction in conformational flexibility of the alkyl side chain was analyzed by comparing the linear decyl side chain in (3) with (10) carbon atoms to compounds (3i, 3j and 3k) with cyclohexane and phenyl substituents with similar number of carbon atoms. As represented in figure 5, these conformationally restricted derivatives were less potent. A similar result was obtained for compound (3g) with a bulky group side chain. No inhibition was observed with compounds containing side chains including polar groups (3h and 3f). These findings clearly visualize the enzyme pocket preference for inhibitors with lipophilic, flexible and less bulky side chains. For the 3-alkyl-2-methoxy-1,4-naphthoquinones (5a-b) series also a clear correlation between the length of the side chain and inhibition potency was observed. Elongation of the side chain length from (5) to (5b) results in an increase in 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

inhibition potency, manifested in the decreased rates of enzyme activity (figure 6). This trend implies that the methoxy and hydroxy substituted naphthoquinones share the same inhibition and binding mode, where the oxygen atom probably acts as a hydrogen bond acceptor. In contrast to the methoxy and hydroxyl series, the inverse effect was observed for bromosubstituted naphthoquinones (4a-c). An increase in length resulted in a decrease in inhibition (figure 7). Compounds 4a and 4b, used at a single top concentration of 20µM, showed complete inhibition of QFR activities (figure 7). In order to quantify the potency of these compounds concentration-response experiments were conducted with W. succinogenes QFR. The IC50-values for compound 4a were determined to be 0.061 µM for the activity of quinol oxidation by fumarate and 1.094 µM for the activity of succinate oxidation by quinone. For compound 4b, the respective IC50-values were determined to be 0.612 µM and 0.604 µM (see supporting information). The selectivity of our synthesized naphthoquinones for QFR was examined by testing the biological effects of 4a and 4b in a DT-diaphorase secondary assay. DT-diaphorase or NAD(P)H dehydrogenase is a related enzyme, that catalyzes the reduction of quinones substrates by oxidation of NAD(P)H. Compounds 4a and 4b, show no effect on the DTdiaphorase enzymatic reaction (see supporting Information).

The biological testing of the trapped compounds (13) and (14) showed a clear differentiation in the enzymatic conversion. The 1,4-tautomer (13) was recognized and converted by the enzyme, but not the 1,2-tautomer (14). Based on the fact, that the 1,2-isomer (14) was left unrecognized by the enzyme, whereas all the 1,4-isomers (1), (2) and (13) were active, we conclude that (3) would bind in its 1,4-tautomeric form. For a better understanding of the hydroxy-naphthoquinone inhibition, we investigated some physico-chemical properties of these molecules. Hydrophobic interactions and steric parameters are important driving forces in stabilizing the protein-inhibitor complex. The 16 ACS Paragon Plus Environment

Page 16 of 41

Page 17 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


overall hydrophobicity of an inhibitor is an indicator for such attractive interactions and can be characterized by the clogP values (partition coefficient in octanol/water).40 The partition coefficient clogP were calculated using CLOGP (v4.62, daylight chemical information system Inc.) and correlated against the enzyme residual activity for both enzymes (figure 8). As represented a correlation was found between clogP representing the hydrophobic nature of the inhibitors and the enzyme inhibition.

Figure 8. Correlation between clogP of hydroxyl naphthoquinones series (3a-k) against enzyme residual activity: hollow circles: quinol:fumarate reductase residual activity reverse reaction (like-QFR), solid circle: succinate:quinone reductase residual activity (like-SQR). Dashed line: fit for like-QFR activity residual activity = (122.2±8.5) + (-8.2±1.6) clogP; R = 0.85. Dotted line: fit for like-SQR activity residual activity = (115.6±10.9) + (-8.1±2.1) clogP, R = -0.78. Homology model In order to highlight the structural similarity of QFR from W. succinogenes, with the QFR from H. pylori and C. jejuni homology models were generated (figure 9).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 41

Figure 9: Comparison of QFR C-subunits structure model. W. succinogenes (PDB 2bs2; on the left) is compared to the homology models of C-subunits from H. pylori (middle) and C. jejuni (right) QFR. The atoms are colored according to sequence conservation. Red: fully conserved; yellow: conserved in at least two structures, similar residue in the third structure; white: not conserved. The inset shows a zoom to the putative quinone binding pocket. Multiple-sequence alignments between the sequence of the template structure (PDBid: 2BS2) and the target sequence H. pylori and C.jejuni C-subunit of QFR were generated using ClustalX1. The primary constraints for the alignments of the C-subunit were derived from the sequence alignment: (1) the requirement for ligation of the two hemes by the fully conserved 4 histidine residues, (2) the predicted (and not consensus) TM (trans-membrane) helix sequence regions and (3) the ability to satisfy the lipid-facing propensities of the individual helices as far as possible. The default modeling schedule of MODELLER was used, with “Thorough Variable Target Function Schedule” and “Slow MD Annealing”.41-42 The actual charges on the redox active metals do not influence the results to any larger degree, because the active site surroundings are identical in the template (PDB entry code 2BS2) and thus 18 ACS Paragon Plus Environment

Page 19 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


copied directly to the model. Also, the nomenclature of atoms was unified between the templates and the topology files. These changes did not affect the modeling procedure. Five models with the lowest value of the MODELLER objective function were selected from the 50 generated each time, based on the objective function value and its restraints violation profile. The stereochemical features of selected models were assessed by COOT41-42 and the structures were studied visually. The alignment modeling procedure was iterated, until the results satisfied the previous data. Conclusion Based on the natural substrate we identified novel small molecule candidates, which inhibited the quinol:fumarate reductase activity as well as the reverse reaction, succinate:quinone reductase activity of QFR from W. succinogenes, closely related to QFRs from the human pathogen H. pylori and C. jejuni.7, 43 In order to visualize the sequence homology between these enzymes, we build homology models of QFRs from H. pylori and C. jejuni. Remarkably well conserved sequence motifs are localized in the vicinity of the putative quinol binding site, suggesting that our results can serve as a good model for the corresponding QFRs from the human pathogen organisms. The structurally unresolved binding pocket of the protein complexes were explored by using the identified hit compounds. A follow up SAR study revealed the importance of hydrophobic tail at 3-position and substituents at 2-position on the ligand site and gave a clear picture of the structurally unexplored binding pocked as being highly lipophilic. This observation is in line with the detected path of conserved residues in the modeled QFRs, leading from the putative quinol binding-pocket (Figure 9, red color), and providing support for the conclusion that the hydrophobic tail at 3-position is involved in specific non-bonding interactions with these residues. However, more results from functional characterization of mutants in the conserved path are needed to fully solidify this conclusion.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 41

For compounds 4a and 4b the IC50-values were determined in the primary assay and the selectivity tested in a counter DT-diaphorase assay. Both compounds showed activity in low

µM range and no effect in the counter assay. For the hit compound (3) we also showed that the 1,4- tautomer is thermodynamically more stable than the 1,2-tautomer and presumably the bioactive form. Taken together the biological activities indicated that not only the tautomerism and hereby the position of the carbonyl groups, but also the free hydroxy group, as a hydrogen bond acceptor, is important for the recognition and formation of inhibitor protein complex. We conclude that tautomerism, structural analogy, hydrophobicity and steric requirements of a ligand are critical for recognition, conversion or inhibition by a given target enzymes. Similar to our findings, the reported crystal structure of 5-undecyl-6-hydroxy-4,7dioxobenzothiazole (UHDBT), a related hydroxyquinone inhibitor bonded to the Qo site of the cytochrome bc1 complex,23-25 represents the ligand in the 1,4 tautomeric form and the hydroxy group as a hydrogen-bound acceptor. The hydroxy group, as its deprotonated negative charged form, is hydrogen-bonded to the protonated histidine (His181). The presence of the hydroxyl group is also crucial for the activity of 3-alkyl-2-hydroxy-1,4-naphtoquinones as antimalarial and anticancer agents as shown by Fieser et al.44 and Kongkathip et al.18 Our results are also consistent with the calculated structure of atovaquone, an antimalaria agent, bound to the ubiquinol oxidation site of cytochrome bc1 complex28 and may provide a powerful tool for design and systematic study of binding mode of substrate based inhibitors for related quinone converting membrane proteins.45

Experimental Section Chemistry General Experimental. NMR spectra were recorded on Bruker DPX250 and AVII300 spectrometers operating at a 1H frequency of 250 MHz or 300 MHz and at a 13C frequency of 20 ACS Paragon Plus Environment

Page 21 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


62.9 MHz. Elementary analyses were measured on a Foss Heraeus CHN-O-RAPID instrument. All reactions were monitored by thin-layer chromatography (TLC), performed on silica gel POLYgram® (Macherey-Nagel). Chromatographic purifications were done with Merck silica gel 60. Compounds (1a), (3a) and (6) were commercially available. Elemental microanalysis was used to determine the purity of purchased starting materials and synthesized compounds with purity ≥95%.

Radical Hundsdiecker decarboxylation To a mixture of 2-bromo-naphthoquinone (10),31 (4 g, 16.8 mmol), 1 eq of corresponding acid (25.3 mmol), silver nitrate (1.2 g, 7 mmol) in 80 mL acetonitrile was slowly added a solution of ammonium persulfate (8 g, 35 mmol) in 25 mL water. After 1h heating a second portion (0.5 eq) of the corresponding acid was added. To the reaction mixture also a second portion of ammonium persulfate (8 g, 35 mmol) in 25 mL water was slowly added. After further 3hrs heating at 80° the products (4a-h) were precipitated and isolated by filtration.

Hydroxyl nucleophilic substitution of bromo substituents. General procedure. 1 mmol of corresponding bromo-naphthoquinones (4a-h) were dissolved in boiling methanol (20 mL) and a 0.7 N potassium hydroxide (8 mL) was added and the mixture was heated to reflux for 1h. After cooling to room temperature and acidification, the organic layer was extracted by chloroform, dried and evaporated in vacuum. The crude products (3a-k) were purified by silica chromatography (hexane/ethyl acetate 6:1).

Alkaline ether hydrolysis

α- or β-lapachone34 (150 mg, 0.62 mmol) were suspended in 1% aqueous sodium hydroxide solution (4 mL) and refluxed for 2h. After cooling to room temperature, the solution was 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 41

acidified, extracted with chloroform and dried. After concentration in vacuo and chromatographic purification (hexane/ethyl acetate 6:1) hydroxy naphtoquinone (3h) was obtained.

O-Methylation with dimethyl sulfate Dimethyl sulfate (0.056 mL, 0.5 mmol) was slowly added to a mixture of 2-alkyl-3-hydroxy1,4-naphthoquinones (3, 3c, 3d) (0.22 mmol), potassium carbonate (276 mg, 2 mmol) in 10 mL acetone at room temperature. After 2h the solvents were removed and the crude product extracted in chloroform and washed with water and brine. After chromatographic purification (hexane/ethyl acetate 4:1) 2-alkyl-3-methoxy-1,4-naphthoquinones (5, 5a, 5b) were obtained.

O-Alkylation of lawsone Compounds (13) and (14) were prepared starting from 2-hydroxy-1,4-naphthoquinone (3a) by alkylation with 1-bromononane in the presence of potassium carbonate under heating to reflux in N,N-dimethylformamide (DMF) for 5h. Under this condition only the O-alkylated products (13) and (14) were obtained.46 To a solution of 2-hydroxy-1,4-naphthoquinone (3a) (1 g, 5.75 mmol) and potassium carbonate (0.8 g, 5.8 mmol), 1-bromononane 2.6 g (2.2 eq, 12.6 mmol, 2.4 mL in 2.5 mL DMF) was added dropwise. The reaction mixture was heated to reflux for 5h. The mixture was diluted with water and extracted twice with chloroform, dried and concentrated under reduced pressure. Purification by chromatography on silica gel (hexane:ethyl acetate 4:1) afford crystals of (13) and (14).

2-Decyl-3-methyl-1,4-naphthoquinone (1)


Page 23 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

H-NMR (250.13 MHz, CDCl3): δ [ppm]= 8.1 – 8.0 (m, 2H, aromatic), 7.8 – 7.6 (m, 2H,

aromatic), 2.7 – 2.6 (t, J = 7,5 Hz, 2H, CH2), 2.2 (s, 3H, CH3), 1.5 – 1.2 (m, 16H, CH2), 0.9 (t, J = 6,5 Hz, 3H, CH3). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]= 185.8 ; 184.7 (C=O), 147.6; 143.0; 132.2; 132.1 (C),

133.3; 133.2; 126.2; 126.1 (CH), 31.8; 30.1; 29.5; 29.5; 29.4; 29.3; 28.7; 27.1; 22.6 (CH2), 14.1; 12.6 (CH3). Anal.Calcd. for C21H28O2: C 80.73, H 9.03; Found: C 80.82, H 9.14. (yield: 53%).

2-Decyl-1,4-naphthoquinone (2) and 2-Decyl-3-hydroxy-1,4-naphthoquinone (3) were prepared as described.9

2- Hydroxy-3-methyl-1,4-naphthoquinone (3b) 1


aromatic), 7.3 (s br, 1H, OH ), 2.0 (s, 3H, CH3). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]=185.0 ; 181.1 (C=O), 153.1; 132.8; 120.5; 131.1 (C),

134.8; 132.8; 126.7; 126.1 (CH), 8.6 (CH3). (yield: 51%). Anal.Calcd. for C11H8O3: C 70.21, H 4.29; Found: C 70.38, H 4.4. Crystal structure of this compound has already been reported. Reference code CCDC640776.47

2-Ethyl-3-hydroxy-1,4-naphthoquinone (3c) 1


aromatic), 7.2 (s, 1H, OH), 2.5 (q, J = 7.5 Hz, 2H, CH2), 1.1 (t, J = 7.5 Hz, 3H, CH3). 13


134.8; 132.8; 126.7; 126.0 (CH), 16.7 (CH2), 12.6 (CH3). 23 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 41

Anal.Calcd. for C12H10O3: C 71.28, H 4.98; Found: C 71.01, H 5.21. (yield: 57%).

2-Hexyl-3-hydroxy -1,4-naphthoquinone (3d) 1


aromatic), 7.2 (s, 1H, OH), 2.5 (t, J = 7.5 Hz, 2H, CH2), 1.5 – 1.4 (m, 2H, CH2), 1.3 – 1.1 (m, 6H, 3x CH2), 0.9 – 0.7 (t, J = 7.5 Hz, 3H, CH3). 13


134.8; 132.8; 126.7; 126.0 (CH), 31.6; 29.4; 28.2; 23.3; 22.5 (CH2), 14.0 (CH3). Anal.Calcd. for C16H18O3: C 74.4, H 7.02; Found: C 74.14, H 7.09. (yield: 41%).

2-Hydroxy-3-tetradecyl-1,4-naphthoquinone (3e) 1


aromatic), 7.2 (s, 1H, OH), 2.5 (t, J = 7.5 Hz, 2H, CH2), 1.5 – 1.1 (m, 24H, CH2), 0.8 (t, J = 7.5 Hz, 3H, CH3). 13


134.8; 132.8; 126.7; 126.0 (CH), 31.9; 29.8; 29.7; 29.6; 29.6; 29.6; 29.5;29.5; 29.4; 29.3; 28.3; 23.4; 22.7 (CH2), 14.1 (CH3). Anal.Calcd. for C24H34O3: C 77.8, H 9.25; Found: C 78.03, H 9.06. (yield: 44%).

2-(10-Bromodecyl)-3-hydroxy-1,4-naphthoquinone (3f) 1


aromatic), 7.2 (s, 1H, OH), 3.3 (t, J = 7.5 Hz, 2H, CH2-Br), 2.5 (t, J = 7.5 Hz, 2H, C=C-CH2), 1.7 (t, J = 7,5 Hz, 2H, CH2), 1.5 – 1.1 (m, 14H, CH2).


Page 25 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13


134.8; 132.8; 126.7; 126.0 (CH), 34.0; 32.8; 29.7; 29.3; 29.3; 29.3; 28.7; 28.2; 28.1; 23.3 (CH2). Anal.Calcd. for C20H25BrO3: C 61.08, H 6.41; Found: C 61.24, H 6.43. (yield: 23%).

2-Hydroxy-3-neopentyl-1,4-naphthoquinone (3g) was prepared as described.38

2-Hydroxy-3-(3-hydroxy-3-methylbutyl)-1,4-naphthoquinone (3h) 1


aromatic), 7.5 (s, 1H, OH), 2.6 (t, J = 7.8 Hz, 2H, CH2), 1.6 (t, J = 7.8 Hz, 2H, CH2), 1.2 (s, 6H, 2x CH3). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]= 184.8 ; 181.3 (C=O), 153.3; 132.8; 129.5; 124.8;

71.0 (C), 134.7; 132.9; 126.7; 126.1 (CH), 41.5; 18.3 (CH2), 29.0 (CH3). Anal.Calcd. for C15H16O4: C 69.22, H 6.2; Found: C 69.04, H 6.39. (yield: 89%).

2-(3-Cyclohexylpropyl)-3-hydroxy-1,4-naphthoquinone (3i) 1


aromatic), 7.23 (s br, 1H, OH), 2.5 (t, J = 7.5 Hz, 2H, CH2), 1.7 – 0.7 (m, 15H, 7x CH2, CH). 13

C-NMR (62.9 MHz, CDCl3) δ [ppm]= 184.7; 181.4 (C=O), 153.0; 132.9; 129.4; 124.9 (C),

134.8; 132.8; 126.7; 126.0; 37.5 (CH), 37.5; 33.3; 26.7; 26.4; 25.6; 23.6 (CH2). Anal.Calcd. for C19H22O3: C 76.48, H 7.43; Found: C 76.60, H 7.39. (yield: 48%).

2-(4-Cyclohexylbutyl)-3-hydroxy-1,4-naphthoquinone (3j)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Page 26 of 41


aromatic), 7.5 (s br, 1H, OH), 2.5 (t, J = 7.5 Hz, 2H, CH2), 1.8 – 0.7 (m, 17H, 8x CH2, CH). 13

C-NMR (62.9 MHz, CDCl3) δ [ppm]=184.6; 181.4 (C=O), 153.0; 132.9; 129.4; 124.9 (C),

134.7; 132.7; 126.7; 126.0; 37.3 (CH), 37.6; 33.4; 28.6; 27.1; 26.7; 26.4; 23.4 (CH2). Anal.Calcd. for C20H24O3: C 76.89, H 7.74; Found: C 76.78, H 7.89. (yield: 46%).

2-Hydroxy-3-(3-phenylpropyl)-1,4-naphthoquinone (3k) 1


aromatic), 7.25 (s br, 1H, OH), 7.2 – 7.0 (m, 5H, aromatic), 2.7 – 2.5 (m, 4H, CH2), 1.9 – 1.7 (m, 2H, CH2). 13

C-NMR (62.9 MHz, CDCl3) δ [ppm]= 184.6; 181.4 (C=O), 153.1; 142.1; 132.9; 129.4;

124.3 (C), 134.9; 132.9; 128.4; 128.2; 126.8; 126.1; 125.7 (CH), 36.0; 29.6; 23.3 (CH2). Anal.Calcd. for C19H16O3: C 78.06, H 5.52; Found: C 77.78, H 5.58. (yield: 58%).

2-Bromo-3-decyl-1,4-naphthoquinone (4) 1


aromatic), 2.9 (t, J = 7.7 Hz, 2H, CH2), 1.8 – 1.7 (m, 2H, CH2), 1.7 – 1.2 (m, 14H, 7x CH2), 0.8 (t, J = 6.9 Hz, 3H, CH3). 13


134.1; 133.8; 127.4; 127.1 (CH), 31.9; 31.7; 29.9; 29.6; 29.6; 29.5; 29.3; 27.8; 22.7 (CH2), 14.1 (CH3). Anal.Calcd. for C20H25BrO2: C 63.66, H 6.68; Found: C 63.62, H 6,67. (yield: 33%).

2-Bromo-3-methyl-1,4-naphthoquinone (4a)47


Page 27 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2-Bromo-3-ethyl-1,4-naphthoquinone (4b) 1


aromatic), 2.7 (q, J = 7.5 Hz, 2H, CH2), 1.3 (t, J = 7.5 Hz, 3H, CH3). 13


134.0; 133.8; 127.3; 127.0 (CH), 25.1 (CH2), 11.9 (CH3). Anal.Calcd. for C12H9BrO2: C 54.37, H 3.42; Found: C 54.13, H 3.57. (yield: 38%).

2-Bromo-3-hexyl-1,4-naphthoquinone (4c) 1


aromatic), 2.7 (t, J = 6.0 Hz, 2H, CH2), 1.5 – 1.4 (m, 2H, CH2), 1.3 – 1.2 (m, 6H, 3x CH2), 0.8 (t, J = 6 Hz, 3H, CH3). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]=181.6 ; 177.6 (C=O), 152.1; 138.9; 131.5; 130.9 (C),

133.8; 133.5; 127.6; 127.0 (CH), 31.5; 31.4; 29.5; 27.7; 22.3 (CH2), 14.0 (CH3). Anal.Calcd. for C16H17BrO2: C 59.83, H 5.33; Found: C 60.00, H 5.39. (yield: 44%).

2-Bromo-3-tetradecyl-1,4-naphthoquinone (4e) 1


aromatic), 2.8 (t, J = 7.5 Hz, 2H, CH2), 1.7 – 1.2 (m, 24H, CH2), 0.8 (t, J = 7.5 Hz, 3H, CH3). 13


134.0; 133.8; 127.4; 127.1 (CH), 31.9; 31.7; 29.9; 29.7; 29.7; 29.6; 29.6; 29.6; 29.5; 29.4; 29.3; 27.8; 22.7 (CH2), 14.1 (CH3). Anal.Calcd. for C24H33BrO2: C 66.51, H 7.67; Found: C 66.76, H 7.68. (yield: 42%).

2-Bromo-3-(10-bromodecyl)-1,4-naphthoquinone (4f)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Page 28 of 41


aromatic), 3.3 (t, J = 7.5 Hz, 2H, CH2), 2.6 (t, J = 7.5 Hz, 2H, CH2), 1.8 (t, J = 7.5 Hz, 2H, CH2), 1.6 – 1.1 (m, 14H, CH2). 13


134.0; 133.8; 127.4; 127.0 (CH), 33.9; 32.8; 31.6; 31.5; 29.7; 29.3; 29.2; 28.7; 28.1; 27.7 (CH2). Anal.Calcd. for C20H24Br2O2: C 52.65, H 5.3; Found: C 52.87, H 5.40. (yield: 49%).

2-Bromo-3-(3-cyclohexylpropyl)-1,4-naphthoquinone (4g) 1


aromatic), 2.7 (t, J = 7.5 Hz, 2H, CH2), 1.8 – 0.7 (m, 15H, 7x CH2, CH). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]= 181.7; 177.7 (C=O), 152.2; 138.0; 131.6; 131.1 (C),

134.0; 133.8; 127.4; 127.0; 37.5 (CH), 37.4; 33.3; 31.9; 26.6; 26.3; 25.2 (CH2). Anal.Calcd. for C19H21BrO2: C 63.17, H 5.86; Found: C 63.11, H 5.88. (yield: 40%).

2-Bromo-3-(3-phenylpropyl)-1,4-naphthoquinone (4h) 1

H-NMR (250.13 MHz, CDCl3): δ[ppm]= 8.1 – 7.9 (m, 2H, aromatic), 7.7 – 7.6 (m, 2H,

aromatic), 7.4 – 7.0 (m, 5H, aromatic protons), 2.8 – 2.6 (m, 4H, CH2), 1.9 – 1.7 (m, 2H, CH2). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]= 181.7; 177.7 (C=O), 151.8; 141.3; 138.8; 131.6;

131.2 (C), 134.1; 133.9; 128.5; 128.3; 127.4; 127.1; 126.0 (CH), 36.0; 31.4; 29.0 (CH2). Anal.Calcd. for C19H15BrO2: C 64.24, H 4.26; Found: C 64.25, H 4.35. (yield: 51%).

2-decyl-3-methoxy-1,4-naphthoquinone (5)


Page 29 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1


aromatic), 4.1 (s, 3H, OCH3), 2.6 (t, J = 7.5 Hz, 2H, CH2), 1.8 – 1.1 (m, 16H, 8x CH2), 0.65 (t, J = 7.5 Hz, 3H, CH3). 13


133.6; 133.1; 126.1; 125.9 (CH), 31.8; 29.8; 29.6; 29.5; 29.4; 29.3; 28.9; 23.7; 22.6 (CH2), 61.1; 14.1 (CH3). Anal.Calcd. for C21H28O3: C 76.79, H 8.59; Found: C 76.73, H 8.52. (yield: 86%).

2-ethyl-3-methoxy-1,4-naphthoquinone (5a) 1


aromatic), 3.8 (s, 3H, OCH3), 2.3 (q, J = 7.4 Hz, 2H, CH2), 0.8 (t, J = 7.5 Hz, 3H, CH3). 13


133.6; 133.1; 126.8; 126.4 (CH), 17.1 (CH2), 61.1; 13.3 (CH3). Anal.Calcd. for C13H12O3: C 72.21, H 5,59; Found: C 72.15, H 5.72. (yield: 96%).

2-hexyl-3-methoxy-1,4-naphthoquinone (5b) 1

H-NMR (250.13 MHz, CDCl3): δ [ppm]= 7.9 – 7.8 (m, 2H, aromatic), 7.6 –7.5 (m, 2H,

aromatic), 4.0 (s, 3H, OCH3), 2.5 (t, J = 7.3 Hz, 2H, CH2), 1.4 – 1.1 (m, 8H, 4x CH2), 0.65 (t, J = 7.2 Hz, 3H, CH3). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]= 188.5 ; 184.6 (C=O), 160.8; 139.2; 135.; 134.7 (C),

136.7; 136.2; 129.2; 129.1 (CH), 34.7; 32.6; 32.0; 26.8; 25.6 (CH2), 64.2; 17.1 (CH3). Anal.Calcd. for C17H20O3: C 74.97, H 7.40; Found: C 74.73, H 7.40. (yield: 92%).

2-neopentyl-1,4-naphthoquinone (7)10



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 41

2(4-cyclohexylbutyl)-1,4-naphthoquinone (8) 1


aromatic), 6.6 (s, 1H, CH), 2.5 (t, J = 7.5 Hz, 2H, CH2), 1.8 – 0.7 (m, 17H, 8x CH2, CH). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]= 185.2; 185.1 (C=O), 151.9; 132.3; 132.0 (C), 134.6;

135.5; 135.5; 126.5; 125.9; 37.1 (CH), 37.5; 37.3; 28.5; 28.3; 26.6; 26.6; 26.3 (CH2). Anal.Calcd. for C20H24O2: C 81.04, H 8.16; Found: C 80.86, H 8.14. (yield: 60%).

2,3-epoxy-2-methyl-1,4-naphthoquinone (9) 1


aromatic), 3.8 (s, 1H, CH), 1.7 (s, 3H, CH3). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]= 191.6; 191.5 (C=O), 132.1; 132.0; 61.4; (C), 134.5;

134.3; 127.4; 126.3; 61.4 (CH), 14.7 (CH3). Anal.Calcd. for C11H8O3: C 70.21, H 4.29; Found: C 70.34, H 4.37.

α- and β-lapachones (11 and 12) were prepared as described.51 Spectroscopic data were published elsewhere.34

2-(Nonyloxy)-1,4-naphthoquinone (13) 1


aromatic), 6.18 (s, 1H, CH), 4.02 (t, J = 6.8 Hz, 2H, OCH2CH2), 1.92 (m, 2H, OCH2CH2), 1.31 (m, 12H, CH2), 0.94 (m, 3H, CH3).


Page 31 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13

C-NMR (62.9 MHz, CDCl3): δ [ppm]=185.9; 180.1 (C=O), 159.8; 132.0; 131.2 (C), 134.1;

133.2; 126.6; 126.1; 110.1 (CH), 69.6; 31.8; 29.4; 29.2; 29.2; 28.2; 25.8; 22.6 (CH2), 14.0 (CH3). (yield: 55%).

4-(Nonyloxy)-1,2-naphthoquinone (14) 1

H-NMR (250.13 MHz, CDCl3): δ [ppm]= 8.05 (dd, J = 7.6 Hz, 4J = 1.5 Hz, 1H, H8), 7.79

(dd, J = 7.6 Hz, J = 1.5 Hz, 1H, H5), 7.62 (dt, J = 7.6 Hz, J = 1.5 Hz, 1H, H7), 7.2 (dt, J = 7.6 Hz, J = 1.5 Hz, 1H, H6), 6.2 (s, 1H, CH), 4.07 (t, J = 6.4 Hz, 2H, OCH2CH2), 1.84 (m, 2H, OCH2CH2), 1.21 (m, 12H, CH2), 0.8 (m, 3H, CH3). 13

C-NMR (62.9 MHz, CDCl3): δ [ppm]=179.5; 168.9 (C=O), 179.6; 132.2; 130.4 (C), 134.9;

131.4; 129.0; 124.7; 103.3 (CH), 70.0; 31.8; 29.4; 29.2; 29.2; 28.4; 26.0; 22.6 (CH2), 14.0 (CH3). (yield: 21%).

Crystal structure determinations CCDC-940665,

CCDC-939744,

CCDC-940666

and

CCDC-940667

contain

the

crystallographic data of compounds (1), (3), (13) and (14). Copies of the data can be obtained free

of

charge

from

Cambridge

Crystallographic

Data

Centre

via

www.ccdc.cam.ac.uk/conts/retrieving.html. The crystal structures of compounds (3g), (3i), (4b), (4c), (9) and (12) have already been reported.48 clogP Hydrophobicity, expressed as clogP values of compounds, were calculated with the programme CLOGP, v4.62, Daylight Chemical Information System Inc. (Aliso Viejo, CA, USA) Biological activity tests In order to determine the rates of redox reaction as catalyzed by quinol:fumarate reductase (QFR) from W. succinogenes, i.e. fumarate reduction by quinol oxidation, into 50 mM 31 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 41

phosphate buffer adjusted to pH 7.4, containing 150 µM DMN, synthesized as described earlier,49 12 µg mL-1 enzyme was added at 37°C in anaerobized cuvettes (path length 0.4 cm). The quinone was pre-reduced to quinol before the measurement by the addition of small aliquots of NaBH4 solution, the individual samples were incubated in the cuvettes for 90 s prior to the measurement and the reaction was started by the addition of 40 µM of fumarate. The progress was monitored by subtracting the change in absorbance at 290 nm from that at 270 nm (ε = 15.2 mM-1cm-1).11 The effect of the compounds on succinate:quinone reductase activity of QFR50 (i.e. the reverse reaction to the canonical QFR activity, succinate oxidation by quinone reduction) was determined accordingly, except that the quinone was not prereduced to quinol before the measurement and the reaction was started by the addition of 400

µM of succinate instead of fumarate. All measurements were performed in triplicate. The effect is expressed as the ratio of specific activity in presence of 20 µM of the respective compound and in its absence (“% residual activity”).

ASSOCIATED CONTENT Supporting Information. Multiple-sequence alignments for the homology modeling together with the density functional calculations. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Corresponding authors: Prof. Dr. Harald Schwalbe and Dr. Hamid R. Nasiri E-mail address: [email protected];


Page 33 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Present Addresses

‖ Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge 18 CB2 1EW, UK.

┴ Department of Physiology, University of California, Los Angeles, CA 90095, U.S.A. # Department of Structural Biology, Center of Human and Molecular Biology, Saarland University, Faculty of Medicine, Building 60, D-66421 Homburg, Germany

Funding Sources This work was supported by the Deutsche Forschungsgemeinschaft through SFB 472 “Molecular Bioenergetics” (to H.S. and to C.R.D.L.), the Fonds der Chemischen Industrie (general support to H.S.) and the state of Hessen (Center for Biomolecular Magnetic Resonance (BMRZ)). ABBREVIATIONS H. pylori, Helicobacter pylori; TLC, thin layer chromatography. C. jejuni, Campylobacter jejuni; MK-6, Menaquinone-6; SQOR, succinate:quinone oxidoreductase; QFR, Quinol:fumarate reductase; SAR, structure-activity relationship; REFERENCES 1.

Ge, Z.; Feng, Y.; Dangler, C. A.; Xu, S.; Taylor, N. S.; Fox, J. G., Fumarate reductase

is essential for Helicobacter pylori colonization of the mouse stomach. Microbial Pathogenesis 2000, 29, 279-287. 2.

Cover, T. L.; Blaser, M. J., Helicobacter pylori factors associated with disease.

Gastroenterology 1999, 117, 257-261. 33 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.

Page 34 of 41

Peek, R. M., Jr.; Blaser, M. J., Helicobacter pylori and gastrointestinal tract

adenocarcinomas. Nature reviews. Cancer 2002, 2, 28-37. 4.

Kim, C. U.; Lew, W.; Williams, M. A.; Wu, H.; Zhang, L.; Chen, X.; Escarpe, P. A.;

Mendel, D. B.; Laver, W. G.; Stevens, R. C., Structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors. J. Med. Chem. 1998, 41, 2451-2460. 5.

Weingarten, R. A.; Taveirne, M. E.; Olson, J. W., The dual-functioning fumarate

reductase is the sole succinate:quinone reductase in Campylobacter jejuni and is required for full host colonization. J. Bacteriol. 2009, 191, 5293-5300. 6.

Lancaster, C. R. D., Respiratory Chain Complex II and Succinate:Quinone

Oxidoreductases. In The Encyclopedia of Biological Chemistry, Lennarz, W. J.; Lane, M. D., Eds. Elsevier: Waltham, MA, 2013; Vol. 4. 7.

Lancaster, C. R. D.; Simon, J., Succinate:quinone oxidoreductases from epsilon-

proteobacteria. Biochim. Biophys. Acta 2002, 1553, 84-101. 8.

Lancaster, C. R. D.; Kröger, A.; Auer, M.; Michel, H., Structure of fumarate reductase

from Wolinella succinogenes at 2.2 A resolution. Nature 1999, 402, 377-385. 9.

Madej, M. G.; Nasiri, H. R.; Hilgendorff, N. S.; Schwalbe, H.; Lancaster, C. R. D.,

Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex. EMBO J. 2006, 25, 4963-4970. 10. Madej, M. G.; Nasiri, H. R.; Hilgendorff, N. S.; Schwalbe, H.; Unden, G.; Lancaster, C. R. D., Experimental evidence for proton motive force-dependent catalysis by the dihemecontaining succinate:menaquinone oxidoreductase from the Gram-positive bacterium Bacillus licheniformis. Biochemistry 2006, 45, 15049-15055.


Page 35 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


11. Lancaster, C. R. D.; Groß, R.; Haas, A.; Ritter, M.; Mäntele, W.; Simon, J.; Kröger, A., Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 13051-13056. 12. Lancaster, C. R. D., The di-heme family of respiratory complex II enzymes. Biochimica et Biophysica Acta (BBA) - Bioenergetics 2013, 1827, 679-687. 13. Lancaster, C. R. D., Structure and function of succinate:quinone oxidoreductases and the role of quinol:fumarate reductases in fumarate respiration. In Respiration in Archaea and Bacteria, Zannoni, D., Ed. Kluwer Academic: Dordrecht, 2004; Vol. 1, pp 57-85. 14. Lemma, E.; Hagerhall, C.; Geisler, V.; Brandt, U.; von Jagow, G.; Kröger, A., Reactivity of the Bacillus subtilis succinate dehydrogenase complex with quinones. Biochim. Biophys. Acta 1991, 1059, 281-285. 15. Gokhale, N. H.; Padhye, S. B.; Croft, S. L.; Kendrick, H. D.; Davies, W.; Anson, C. E.; Powell, A. K., Transition metal complexes of buparvaquone as potent new antimalarial agents. 1. Synthesis, X-ray crystal-structures, electrochemistry and antimalarial activity against Plasmodium falciparum. J. Inorg. Biochem. 2003, 95, 249-258. 16. Wiesner, J.; Ortmann, R.; Jomaa, H.; Schlitzer, M., New antimalarial drugs. Angew. Chem. 2003, 42, 5274-5293. 17. Martin, Y. C.; Bustard, T. M.; Lynn, K. R., Relationship between physical properties and antimalarial activities of 1,4-naphthoquinones. J. Med. Chem. 1973, 16, 1089-1093. 18. Kongkathip, N.; Luangkamin, S.; Kongkathip, B.; Sangma, C.; Grigg, R.; Kongsaeree, P.; Prabpai, S.; Pradidphol, N.; Piyaviriyagul, S.; Siripong, P., Synthesis of novel



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 41

rhinacanthins and related anticancer naphthoquinone esters. J. Med. Chem. 2004, 47, 44274438. 19. Teixeira, M. J.; de Almeida, Y. M.; Viana, J. R.; Holanda Filha, J. G.; Rodrigues, T. P.; Prata, J. R., Jr.; Coelho, I. C.; Rao, V. S.; Pompeu, M. M., In vitro and in vivo Leishmanicidal activity of 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone (lapachol). Phytother. Res. 2001, 15, 44-48. 20. Chan-Bacab, M. J.; Pena-Rodriguez, L. M., Plant natural products with leishmanicidal activity. Nat. Prod. Rep. 2001, 18, 674-688. 21. Mantyla, A.; Garnier, T.; Rautio, J.; Nevalainen, T.; Vepsalainen, J.; Koskinen, A.; Croft, S. L.; Jarvinen, T., Synthesis, in vitro evaluation, and antileishmanial activity of watersoluble prodrugs of buparvaquone. J. Med. Chem. 2004, 47, 188-195. 22. Jewess, P. J.; Chamberlain, K.; Boogaard, A. B.; Devonshire, A. L.; Khambay, B. P., Insecticidal 2-hydroxy-3-alkyl-1,4-naphthoquinones: correlation of inhibition of ubiquinol cytochrome c oxidoreductase (complex III) with insecticidal activity. Pest Manage. Sci. 2002, 58, 243-247. 23. Fry, M.; Pudney, M., Site of action of the antimalarial hydroxynaphthoquinone, 2[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80). Biochem. Pharmacol. 1992, 43, 1545-1553. 24. Palsdottir, H.; Lojero, C. G.; Trumpower, B. L.; Hunte, C., Structure of the Yeast Cytochrome bc1 Complex with a Hydroxyquinone Anion Qo Site Inhibitor Bound. J. Biol. Chem. 2003, 278, 31303-31311.


Page 37 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25. Kessl, J. J.; Moskalev, N. V.; Gribble, G. W.; Nasr, M.; Meshnick, S. R.; Trumpower, B. L., Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex. Biochim. Biophys. Acta 2007, 1767, 319-326. 26. Jewess, P. J.; Higgins, J.; Berry, K. J.; Moss, S. R.; Boogaard, A. B.; Khambay, B. P., Herbicidal action of 2-hydroxy-3-alkyl-1,4-naphthoquinones. Pest Manage. Sci. 2002, 58, 234-242. 27. Derbre, S.; Duval, R.; Roue, G.; Garofano, A.; Poupon, E.; Brandt, U.; Susin, S. A.; Hocquemiller, R., Semisynthesis and screening of a small library of pro-apoptotic squamocin analogues: selection and study of a benzoquinone hybrid with an improved biological profile. ChemMedChem 2006, 1, 118-129. 28. Kessl, J. J.; Lange, B. B.; Merbitz-Zahradnik, T.; Zwicker, K.; Hill, P.; Meunier, B.; Palsdottir, H.; Hunte, C.; Meshnick, S.; Trumpower, B. L., Molecular basis for atovaquone binding to the cytochrome bc1 complex. J. Biol. Chem. 2003, 278, 31312-31318. 29. Jacobsen, N.; Torssell, K., Radikalische Alkylierung von Chinonen: Erzeugung von Radikalen in Redoxreaktionen. Liebigs Ann. Chem. 1972, 763,135–147. 30. Angerer, H.; Nasiri, H. R.; Niedergesass, V.; Kerscher, S.; Schwalbe, H.; Brandt, U., Tracing the tail of ubiquinone in mitochondrial complex I. Biochim. Biophys. Acta 2012, 1817, 1776-1784. 31. Van, T. N.; Kimpe, N. D., Synthesis of 6H-naphtho[2,3-c]chromene-7,12-diones via palladium-catalyzed

intramolecular

cyclization

of

2-bromo-3-aryloxymethyl-1,4-

naphthoquinones. Tetrahedron 2003, 59, 5941-5946. 32. Weitz, E.; Scheffer, A., Über die Einwirkung von alkalischem Wasserstoffsuperoxyd auf ungesättigte Verbindungen. Ber. dtsch. Chem. Ges. A/B 1921, 54, 2327-2344. 37 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 41

33. Lee, K.; Turnbull, P.; Moore, H. W., Concerning the Mechanism of the Hooker Oxidation. J. Org. Chem. 1995, 60, 461-464. 34. Nasiri, H. R.; Bolte, M.; Schwalbe, H., Electrochemical and crystal structural analysis of alpha- and dehydro-alpha-lapachones. Nat. Prod. Res. 2008, 22, 1225-1230. 35. Nasiri, H. R.; Bolte, M.; Schwalbe, H., Tautomerism of 4-Hydroxy-4(1H)Quinolon. Heterocycl. Commun. 2011, 12, 319-322. 36. Lancaster, C. R. D.; Michel, H., The coupling of light-induced electron transfer and proton uptake as derived from crystal structures of reaction centres from Rhodopseudomonas viridis modified at the binding site of the secondary quinone, QB. Structure 1997, 5, 13391359. 37. Hunte, C.; Koepke, J.; Lange, C.; Rossmanith, T.; Michel, H., Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae cocrystallized with an antibody Fv fragment. Structure 2000, 8, 669-684. 38. Nasiri, H. R.; Madej, M. G.; Lancaster, C. R. D.; Schwalbe, H.; Bolte, M., 2-Hydroxy3-(3-oxobutyl)naphthalene-1,4-dione. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 2006, 62, o671-o673. 39. Kierdaszuk, B.; Modrak-Wojcik, A.; Wierzchowski, J.; Shugar, D., Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme-->ligand fluorescence resonance energy transfer. Biochim. Biophys. Acta 2000, 1476, 109-128. 40. Smith, D. A.; van de Waterbeemd, H.; Walker, D. K., Physicochemistry. Wiley-VCH Verlag GmbH: 2001; pp 1-13.


Page 39 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


41. Šali, A.; Blundell, T. L., Comparative Protein Modelling by Satisfaction of Spatial Restraints. J. Mol. Biol. 1993, 234, 779-815. 42. Emsley, P.; Cowtan, K., Coot: model-building tools for molecular graphics. 2004, 60, 2126-2132. 43. Herzog, E.; Gu, W.; Juhnke, H. D.; Haas, A. H.; Mäntele, W.; Simon, J.; Helms, V.; Lancaster, C. R. D., Hydrogen-bonded networks along and bifurcation of the E-pathway in quinol:fumarate reductase. Biophys. J. 2012, 103, 1305-1314. 44. Fieser, L. F.; Richardson, A. P., Naphthoquinone antimalarials; correlation of structure and activity against P. lophurae in ducks. J. Am. Chem. Soc. 1948, 70, 3156-3165. 45. Hunte, C.; Zickermann, V.; Brandt, U., Functional modules and structural basis of conformational coupling in mitochondrial complex I. Science 2010, 329, 448-451. 46. Kongkathip, N.; Kongkathip, B.; Siripong, P.; Sangma, C.; Luangkamin, S.; Niyomdecha, M.; Pattanapa, S.; Piyaviriyagul, S.; Kongsaeree, P., Potent antitumor activity of synthetic 1,2-Naphthoquinones and 1,4-Naphthoquinones. Bioorg. Med. Chem. 2003, 11, 3179-3191. 47. Nasiri, H. R.; Panisch, R.; Madej, M. G.; Bats, J. W.; Lancaster, C. R. D.; Schwalbe, H., The correlation of cathodic peak potentials of vitamin K(3) derivatives and their calculated electron affinities. The role of hydrogen bonding and conformational changes. Biochim. Biophys. Acta 2009, 1787, 601-608. 48. Nasiri, H.; Bolte, M. Private communication to the Cambridge Structural Database, 2009. Refcodes OBEWAV, OBEXOK, OBEWID, OBEWEZ, OBEYEB, KEMBAG01. 49. Lancaster, C. R. D.; Sauer, U. S.; Groß, R.; Haas, A. H.; Graf, J.; Schwalbe, H.; Mäntele, W.; Simon, J.; Madej, M. G., Experimental support for the “E pathway hypothesis” 39 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 41

of coupled transmembrane e– and H+ transfer in dihemic quinol:fumarate reductase. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 18860-18865. 50. Unden, G.; Kröger, A., The function of the subunits of the fumarate reductase complex of Vibrio succinogenes. Eur. J. Biochem. 1981, 120, 577-584. 51. Schaffner-Sabba, K.; Schmidt-Ruppin, K. H.; Wehrli, W.; Schuerch, A. R.; Wasley, J. W. F., beta.-Lapachone: synthesis of derivatives and activities in tumor models. J. Med. Chem. 1984, 27, 990-994.


Page 41 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TABLE OF CONTENTS (TOC) GRAPHIC


Design, Synthesis, and Biological Testing of Novel ... - ACS Publications

Recommend Documents