Research ArticleReceived: 20 July 2016 Revised: 8 September 2016 Accepted article published: 17 November 2016 Published online in Wiley Online Library: 2 January 2017
(wileyonlinelibrary.com) DOI 10.1002/ps.4488
Design, synthesis and biological evaluation ofnovel nicotinamide derivatives bearing asubstituted pyrazole moiety as potential SDHinhibitorsXian-Hai Lv,a,b,† Zi-Li Ren,a,† Peng Liu,a Bing-Xin Li,a Qing-Shan Li,c Ming-JieChub and Hai-Qun Caoa*
Abstract
BACKGROUND: Succinate dehydrogenase (SDH) plays an important role in the Krebs cycle, which is considered as an attractivetarget for development of succinate dehydrogenase inhibitors (SDHIs) based on antifungal agents. Thus, in order to discovernovel molecules with high antifungal activities, SDH as the target for a series of novel nicotinamide derivatives bearingsubstituted pyrazole moieties were designed and synthesised via a one-pot reaction.
RESULTS: The biological assay data showed that compound 3 l displayed the most potent antifungal activity with EC50 valuesof 33.5 and 21.4𝛍M against Helminthosporium maydis and Rhizoctonia cerealis, respectively. Moreover, 3 l exhibited the bestinhibitory ability against SDH enzymes. The results of docking simulation showed that 3 l was deeply embedded into the SDHbinding pocket, and the binding model was stabilised by a cation–𝝅 interaction with Arg 43, Tyr 58 and an H-bond with Trp 173.
1 INTRODUCTIONThe extensive use of existing antifungal agents has resulted insevere resistance to these drugs.1,2 Therefore, the discovery ofnovel antifungal chemical scaffolds with new mechanisms ofaction is urgently needed.3 Succinic dehydrogenase (SDH),4,5 asthe target of SDH inhibitors (SDHIs), has been considered a promis-ing target for antifungal development because it plays an impor-tant role in the mitochondrial respiratory system.6,7 SDH catalyseselectrons to move to ubiquinone (QH2) from succinate while pro-ducing energy in the Krebs cycle8 – 10 and SDHIs react directly withSDH to inhibit the transfer of electrons,11,12 blocking the energymetabolism and resulting in the death of the target organisms.13,14
In recent years, many SDHIs have been applied15,16 (Fig. 1).They have a novel structure, high- and broad-spectrum fungicidalactivity,17,18 such as carboxin, boscalid, isopyrazam, penthiopyrad,fluopyram etc.19,20 Scalliet et al.21 compared the structure–activityof Mycosphaerella graminicola SDH (PDB entry: 2fbw) and severalSDHIs via molecular docking, and found the following results: O,N and SDH can form stable hydrogen bonds in amide groups;22
the pyrazole ring binds more closely to protein compared withpyridine ring;23 the aromatic ring attached to amino can forma hydrophobic bond and 𝜋–𝜋 interaction with correspondingamino acids in the binding pocket of target proteins.24 The
common characteristics of these SDHI compounds can be seen:all compounds contain an amide bond, an acyl group connectedto aromatic ring or hydrogen bond acceptor and an amino groupconnected to an aromatic ring.
Therefore, we chose SDH as target, and boscalid and penthiopy-rad as primer molecules to design and synthesise target com-pounds (Fig. 2). On the basis of the molecular structures of SDHIs,we introduced the pyrazole ring and pyridine ring in an attemptto bind more closely with the target protein. In order to ver-ify this assumption, molecular docking was performed by tar-get compounds into the binding site of SDH (PDB entry: 2fbw).Compared with the positive drug boscalid, it was clearly seen thatcompound 3 l showed obviously lower interaction energy than
∗ Correspondence to: H Cao, School of Plant Protection, Anhui Agricultural Uni-versity, Hefei 230036, P.R. China. E-mail: [email protected]
† Xian-Hai Lv and Zi-Li Ren contributed equally to this work.
a School of Plant Protection, Anhui Agricultural University, Hefei, P.R. China
b School of Science, Anhui Agricultural University, Hefei, P.R. China
c School of Medical Engineering, Hefei University of Technology, Hefei, P.R. China
Figure 3. The CDOCKER_INTERACTION_ENERGY (kcal mol−1) obtainedfrom the docking study of all synthesised compounds by the CDOCKERprotocol.
boscalid, which reached −38.2725 kcal mol−1, demonstrating thatcompound 3 l is likely to exhibit more potent inhibitory activ-ity against SDH (Fig. 3). The results of these preliminary analysesserved as a modest stimulant to induce us to synthesise thesecompounds.
Herein, a series of novel nicotinamide-bearing substituted pyra-zole derivatives was designed and synthesised as SDHIs and their
antifungal activities are also reported in this study. In addition, aninnovative method has been proposed for a one-pot conversionto amides using phosphorus oxychloride in pyridine under mildconditions. The reactions could be carried out at low tempera-tures without the utilisation of catalysts, which have better yields,shorter reaction time and easier preparation.
2 EXPERIMENTAL2.1 Materials and measurementsAll chemicals (reagent grade) used were purchased from SigmaAldrich (St Louis, MO, USA) and Sinopharm Chemical Reagent Co.,Ltd (Beijing, China). All the 1H NMR spectra were measured on anAgilent DD2 600 Hz spectrometer (Agilent Technologies Corpora-tion, Santa Clara, CA, USA). Chemical shifts were reported in ppm(𝛿). ESI-MS spectra were recorded on a Mariner System 5304 massspectrometer (Applied Biosystems, Foster City, CA, USA). Elemen-tal analyses were performed on a CHN-O-Rapid instrument (Leco,Tres Cantos, Madrid, Spain) and were within 0.4% of the theoreticalvalues. Melting points were measured without correction.
2.2 General procedure for the synthesisof 5-amino-1-aryl-1H-pyrazole-4-carbonitriles 2a–2dIntermediates 2a–2d were synthesised according to the reportby Harden et al.25 A stirred mixture of para-substituted phenylhy-drazine hydrochloride (0.025 mol) was dissolved in H2O (30 mL),then the pH of the mixture was adjusted to 7–8 by the dropwise
Nicotinamide derivatives as SDH inhibitors www.soci.org
R1 COOH
1a-1e
N NN
NC
H
O
R1
R2
N
N
NH2
N
R2
2a-2d 3a-3q
R2 NHNH2 O
N
N
N
N
NH2
N
R2
2a:R=H
2b:R=F
2c:R=Cl
2d:R=CH3
i
(2a-2d)
ii
1a:
N
COOH
1b:
N
COOH
H3CO
1c:
N
COOH
Cl
1d:
N
COOH
Cl
1e:
N
COOH
Scheme 1. General synthesis of compounds 3a–3q. Reagents and conditions: (i) H2O, NaOH, ethanol, 3 h, reflux, (ii) POCl3, Py, 40 ∘C.
addition of 10% NaOH solution to form the free para-substitutedphenyl hydrazines, which were then refluxed for 3 h with ethoxymethylene malononitrile in an ethanol medium. After completionof the reaction, the reaction mixture was allowed to cool at roomtemperature, and the solid, 2a–2d, was filtered under vacuum. Thecrude products obtained were recrystallised from ethanol to givethe pure products.
2.3 General procedure for the synthesis of nicotinamidebearing substituted pyrazole derivatives 3a–3qNiacin (1 mmol) and compound 2 (1 mmol) were dissolved inpyridine (3 mL), POCl3 (0.25 mL) was added under an ice-bathand stirred for 1 h under ice, then at 40 ∘C overnight beforepouring into 30 mL saturated Na2CO3 solution and sufficientlystirred. Mostly, a precipitate formed which was then collectedby suction filtration and recrystallised in ethanol. For data oncompounds 3a–3q, see the supporting information.
2.4 Biological testing2.4.1 Antifungal bioassayThe test fungi, H. maydis and R. cerealis, were provided by theLaboratory of Plant Disease Control, Anhui Agricultural University.After retrieval from the storage tube, the strains were incubated inpotato dextrose agar (PDA) at 25 ∘C for a week to allow the growthof new mycelia for the antifungal assay.
The fungicidal activity of the synthetic compounds was testedin vitro against two plant pathogenic fungi using a mycelia growthinhibition method.26 The synthesised compounds were dissolvedin dimethyl sulfoxide (DMSO) to prepare the 10 mg mL−1 stocksolution before mixing with molten agar below 60 ∘C. The mediacontaining compounds at a concentration of 25 mg mL−1 for theinitial screening were then poured into sterilised Petri dishes. Afteran appropriate time at 25 ∘C, the colony diameter of each strain
was measured with the original mycelial disc diameter (5 mm)subtracted from this measurement. Percentage inhibition wascalculated as (1− a/b)× 100%, where a is the colony diameter inPetri dishes with compounds and b is the mean colony diameter inPetri dishes without tested compounds. DMSO served as negativecontrol, whereas the commercially available agricultural fungicideboscalid was used as a positive control. Each measurement con-sisted of at least three replicates. The concentration-dependentcurve was the values of inhibition rates for the Y axis against thetest sample concentrations (mg mL−1) for the X axis. The EC50 valuewas defined as the concentration required for 50% inhibition ofmycelial growth.
2.4.2 Enzyme assay in vitroIsolation of H. maydis and R. cerealis. Fungus mitochondria wereisolated according to a previously reported method.27 Cultureswere inoculated at 0.05 OD600nm and grown on a reciprocal shaker(180 rpm, 25 ∘C) for 5 days in Sabouraud maltose broth (SMB)medium. Cells were harvested by vacuum filtration and disruptedin liquid nitrogen using a mortar and pestle. The resultant powderwas resuspended to 10% w/v in mitochondrial extraction buffer[10 mM KH2PO4, pH 7.2, 10 mM KCl, 10 mM MgCl2, 0.5 M sucrose,0.2 mM EDTA, 2 mM phenyl methyl sulfonyl fluoride (PMSF)]. Theextract was clarified by centrifugation (5000× g, 4 ∘C for 10 min,twice), and intact mitochondria were then pelleted at 10 000× gfor 20 min at 4 ∘C and resuspended in the same buffer. Mitochon-drial suspensions were brought to a concentration of 10 mg mL−1
and stored at −80 ∘C until use.Mitochondrial suspensions were diluted 1/20 in extraction buffer
and preactivated at 30 ∘C for 30 min in the presence of 10 mM suc-cinate. Succinate:ubiquinone/2,6-dichlorophenol indophenol(DCPIP)activity inhibition measurements were performed byadding 10𝜇L of preactivated mitochondria to 200𝜇L of assaybuffer (50 mM sodium phosphate, pH 7.2, 250 mM sucrose, 3 mM
All the synthetic compounds were analysed by elemental and spectroscopic methods, which showed that all compounds were in full accordance withthe structures depicted in the table. The crystal data and refinement parameters for some of the title compounds are listed in Table 2. The crystalstructure is shown in Fig. 4. The structure was solved by direct methods and refined on F2 by full-matrix least-squares methods for using SHELXTL.25
NaN3, 10 mM succinate) supplemented with 140 μM dichlorophe-nolindophenol (DCIP) and 1 mM 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0). Inhibitor concentrations ranged between4.4 and 150 μM, with uniform 2× dilution factor steps (six inhibitorconcentrations+DMSO control). A total of 96-well plates were
pre-equilibrated at reaction temperature (30 ∘C) for 10 min before
the reactions were started by the addition of 10𝜇L of preac-
tivated H. maydis mitochondrial suspension. DCPIP reduction
was conducted at 30∘Cand monitored at 595 nm. Calculated
absorbance slopes (OD h−1) were used for half-inhibitory con-centration (IC50) calculations using GraphPad Prism 5.0 software(GraphPad Software, USA).
2.5 Determination of crystal structureCrystal structure determination of compounds 3a, 3f and 3 k wascarried out on a Bruker D8 VENTURE PHOTON (Bruker Corporation,Rheinstetten, Germany) equipped with graphite monochromatedMo-Ka (l 1∕4 0.71073 Å) radiation. The structure was solved bydirect methods and refined on F2 by full-matrix least squaresmethods using SHELXTL.28 All non-hydrogen atoms were refinedwith anisotropic thermal parameters. All hydrogen atoms with theexception of those on nitrogen atoms were geometrically fixedand refined using a riding model.
2.6 Molecular dockingThe crystal structures of succinate dehydrogenase (PDB entry:2wqy) were retrieved from the Protein Data Bank. The moleculardocking procedure was performed by using CDOCKER protocol forreceptor–ligand interactions section of DS 3.1 (Discovery Studio3.1; Accelrys, Inc., San Diego, CA, USA).29
3 RESULTS AND DISCUSSION3.1 ChemistryA series of novel nicotinamide bearing substituted pyrazole deriva-tives was synthesised and the general pathway is outlined inScheme 1. The intermediate structure 2a–2d was obtained via
refluxing the mixture of substituted phenyl hydrazine hydrochlo-rides and ethoxymethylenemalononitrile in the ethanol mediumfor 3 h. The target compound was obtained by a convenient, effi-cient method for one pot under mild conditions by convertingamines and acids to amides using POCl3 in pyridine.
All the synthetic compounds were analysed by elemental andspectroscopic methods, which showed that all compounds were infull accordance with the structures depicted in Table 1. The crystaldata and refinement parameter for some of the title compoundsare listed in Table 2, and the crystal structures are shown in Fig. 4.The structure was solved by direct methods and refined on F2 byfull-matrix least-squares methods by using SHELXTL.28
3.2 Biological activity3.2.1 Antifungal activityAll the compounds 3a–3q were evaluated for their antifungalactivities against Helminthosporium maydis and Rhizoctonia cere-alis using a mycelia growth inhibition method. The EC50 of thosecompounds against two plant pathogenic fungi are presented inTable 3. The standard antifungal agent boscalid was also screenedunder identical conditions for comparison. The results revealedthat some of the synthetic compounds exhibited significant anti-fungal activities. Among these compounds, compound 3 l dis-played the best activity with EC50 values of 33.5 and 21.4 μM againstHelminthosporium maydis and Rhizoctonia cerealis, respectively.
Compound 3a, containing a para-substituted pyridyl ring andwithout any substituent group on the aromatic ring, showed
Values are the mean± standard deviation (SD) of three replicates.
general antifungal activity against Helminthosporium maydis andRhizoctonia cerealis (EC50 > 250, >250 μM). Then, we changedthe position of substitution on the pyridyl ring and introduceddifferent substituent groups on the benzene ring in 3a, and
Table 4. IC50 values of fungal SDH inhibition activity (in vitro)
IC50 (𝜇M)
Compound H. maydis R. cerealis
3 g 35.8± 0.3 36.4± 0.13 h 38.5± 0.4 40.2± 0.23 l 20.2± 0.3 25.4± 0.13 m 31.7± 0.4 34.4± 0.33q 24.5± 0.2 26.3± 0.3
Table 5. -CDOCKER_INTERACTION_ENERGY of the amide tautomersin 3 l
Tautomer-CDOCKER_INTERACTION_ENERGY
(kcal mol−1)
O
NH
38.1609
O
N
36.9073
O
N
38.2725
found that, when compounds contain a meta-substituted pyridylring, the activity of the compounds 3e, 3j, 3o (3e, EC50 = 186.4,197.7 μM; 3j, EC50 = 162.3, 154.2 μM; 3o, EC50 = 125.3, 134.4 μM)is slightly superior to that of para-substituted compounds 3a,3f, 3 k (3a, EC50 > 250, >250 μM; 3f, EC50 = 173.5, 165.3 μM; 3 k,
Nicotinamide derivatives as SDH inhibitors www.soci.org
Figure 5. (a) Binding model of 3 l in the active site of Mycosphaerella graminicola SDH (PDB entry: 2fbw). (b) Binding model of boscalid in the active siteof Mycosphaerella graminicola SDH (PDB entry: 2fbw). The H-bond is displayed as a green dashed line and 𝜋 interaction is displayed as a solid yellow line.
EC50 = 134.7, 151.8 μM). We can conclude that the substituted posi-tion on the pyridyl ring can affect the antifungal activity and, morespecifically, compounds whose pyridyl ring is meta-substitutedshow better activity than those that are para-substituted.
Subsequently, we introduced different substituent groups onthe pyridyl ring which had been meta-substituted to evaluate anti-fungal activity. When substituent groups were introduced on thepyridyl ring, the activity of compounds 3b, 3c, 3d (3b, EC50 = 114.4,107.6 μM; 3c, EC50 = 124.1, 92.2 μM; 3d, EC50 = 132.3, 144.5 μM) wasgreatly increased compared with that of compound 3e, espe-cially compound 3b, with methoxy on the 6-position, displayingthe greatest antifungal activity. When introducing different sub-stituent group(s) on the benzene ring, we found that compounds3 k, 3 l, 3 m, 3n, 3o (3 k, EC50 = 134.7, 151.8 μM; 3 l, EC50 = 33.5,21.4 μM; 3 m, EC50 = 64.2, 82.1 μM; 3n, EC50 = 113.6, 129.4 μM; 3o,EC50 = 125.3, 134.4 μM) with a chlorine atom on the 4-positiondisplayed greater antifungal activity than compounds 3f, 3 g,3 h, 3i, 3j (3f, EC50 = 173.5, 165.3 μM; 3 g, EC50 = 71.4, 85.4 μM;3 h, EC50 = 96.5, 106.4 μM; 3i, EC50 = 132.5, 8.3 μM; 3j, EC50 = 162.3,154.2 μM) with a fluorine atom on the 4-position. In short, thestructure–activity relationships of these compounds can be sum-marised: compounds whose pyridyl ring is meta-substituted showbetter activity; methoxy has more advantages for antifungal activ-ity at the 6-position on the pyridyl ring; and a chlorine atom at the4-position on the benzene ring may enhance antifungal activity.
In summary, we changed the position of substitution on thepyridyl ring and introduced different substituent group(s) on thebenzene ring in 3a and found compound 3 l displayed the bestactivity compared with other compounds, indicating that com-pound 3 l can be used as a new antifungal agent.
3.2.2 Fungal SDH inhibition activitiesIn order to determine the relationship between compounds andantifungal effect, five compounds tested against SDH enzymes invitro had EC50 values of <100 μM. As shown in Table 4, 3 l showed
the best inhibitory ability against SDH enzymes with IC50 valuesof 20.2, 25.4 μM. Furthermore, these compounds show differentinhibitory abilities with IC50 values from 20 to 40 μM and exhibitedthe same tendency as the data acquired by using the myceliagrowth inhibition assay in vitro. SDH inhibitory activity assays fullyproved that the target compounds displayed as good inhibitoryeffects as other successfully developed nicotinamide fungicides.Therefore, we assumed that compound 3 l could affect the func-tion of SDH and lead to metabolic dysfunction and antifungaldeath.
3.3 Molecular dockingTo gain more understanding of the interaction betweencompound 3 l and SDH, we explored their binding modelgenerated by molecular docking based on the Mycosphaerellagraminicola SDH (PDB entry: 2fbw) and preprocessed by theDS 3.1 (Discovery Studio 3.1, Accelrys, Inc.). We calculatedthe−CDOCKER_INTERACTION_ENERGY (internal ligand strainenergy and receptor–ligand interaction energy) of three tau-tomers of 3 l listed in Table 5. As shown in Table 5, the imine formby annular tautomerisation has the best structural characteristics,because it is more suitable for binding with the SDH binding site.Furthermore, the binding mode of 3 l with the best structuralcharacteristics and the antifungal agent boscalid within the SDHstructure are shown in Fig. 5.
As shown in Fig. 5a, the substituted amide skeleton was deeplyembedded into the binding pocket, suggesting the pose of 3 linto the SDH-binding site which revealed that it has suitable shapecomplementarity with the binding pocket. On the other hand,the binding mode was stabilised by different interactions formedbetween 3 l and amino acid residues in the binding site.
The results of docking simulation suggested there was thesame binding mode of 3 l compared with boscalid; the phenylmoiety is located at the surface of the protein, they have acation–𝜋 interaction established between Arg 43, Tyr 58 and the
electron-rich 𝜋 system of benzene and the pyridine ring, and astable H-bond was also detected in the binding model betweenO in the amide and Trp 173 (3 l, angle= 136.9∘, distance= 2.1 Å;boscalid, angle= 144.7∘, distance= 2.1 Å ). It is worth noting that,for compound 3 l, a greater H-bond interaction was formed by Nin the amide and Tyr 58 in the SDH-binding site (angle= 86.0∘, dis-tance= 2.3 Å), which provides valuable information for the furtherdesign of SDH inhibitors.
4 CONCLUSIONA series of novel nicotinamide derivatives bearing substitutedpyrazole moieties were synthesised and evaluated for their anti-fungal activity against H. maydis and R. cerealis. Compound 3 lshowed the most potent antifungal activity with EC50 values of33.5 and 21.4 μM against H. maydis and R. cerealis, respectively, andexhibited the best inhibitory ability against SDH enzymes. Dock-ing simulation showed that the substituted amide skeleton wasdeeply embedded into the binding pocket, and the conformationwas stabilised by cation–𝜋 interaction with Arg 43, Tyr 58 andan H-bond with Trp 173. Above all, the results obtained from thisstudy suggest that compound 3 l may serve as a potential SDHinhibitor which can be used as a novel antifungal agent and pro-vide valuable information for the design SDH inhibitors.
ACKNOWLEDGEMENTThis work was supported by National Natural Science Foundationof China (No.21302002), and by Anhui Provincial Natural ScienceFoundation (1408085QB33).
SUPPORTING INFORMATIONSupporting information may be found in the online version of thisarticle.
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