MetS is therefore intimately linked to important biochemical Keywords benzimidazole; benzothiadiazole; inhibition; methionine synthase; molecular modelling Correspondence A.. Kinetic ana
Trang 1by substituted benzo-fused heterocycles
Elizabeth C Banks1, Stephen W Doughty2,*, Steven M Toms1, Richard T Wheelhouse1
and Anna Nicolaou1
1 School of Pharmacy, University of Bradford, UK
2 School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, UK
Methionine synthase (MetS)
(5-methyltetrahydrofolate-homocysteine transmethylase)
two established mammalian enzymes that utilize a
bio-logically active cobalamin derivative [methylcobalamin
(CH3-Cbl)] as a cofactor [1] MetS catalyses the transfer
of the methyl group from 5-methyltetrahydrofolate to
homocysteine via the CH3-Cbl cofactor, with cycling
of cobalamin between the +1 [Cbl(I)] and +3 [Cbl(III)]
valency states (Fig 1) Studies on the Escherichia coli
and Homo sapiens cobalamin-dependent MetS have
revealed that it is a large, conformationally flexible
pro-tein, consisting of four functional domains arranged
in a linear manner Each one of these domains binds a
different substrate or cofactor In detail, the N-terminal module is the homocysteine (Hcy)-binding domain; the second domain binds 5-methyltetrahydrofolate, the third domain binds CH3-Cbl; and the fourth domain (C-term-inal module) binds S-adenosyl-methionine (S-AdoMet),
an allosteric cofactor required for reductive reactivation [2] X-ray crystal structures of the cobalamin-, S-AdoMet-and 5-methyltetrahydrofolate-binding sites have only been reported for the bacterial enzyme [3–5]
The reaction products methionine and tetrahydro-folate are further metabolized through the one-carbon methionine transmethylation and folate cycles MetS
is therefore intimately linked to important biochemical
Keywords
benzimidazole; benzothiadiazole; inhibition;
methionine synthase; molecular modelling
Correspondence
A Nicolaou, School of Pharmacy, University
of Bradford, Richmond Road, Bradford BD7
1DP, UK
Fax: +44 1274 235600
Tel: +44 1274 234717
E-mail: a.nicolaou@bradford.ac.uk
*Present address
Faculty of Health and Biological Sciences,
School of Pharmacy, University of
Notting-ham Malaysia Campus, Jalan Broga, 43500
Semenyih, Selangor Darul Ehsan, Malaysia
(Received 14 July 2006, revised 7 November
2006, accepted 9 November 2006)
doi:10.1111/j.1742-4658.2006.05583.x
The cobalamin–dependent cytosolic enzyme, methionine synthase (EC.2.1.1.13), catalyzes the remethylation of homocysteine to methionine using 5-methyltetrahydrofolate as the methyl donor The products of this remethylation – methionine and tetrahydrofolate – participate in the active methionine and folate pathways Impaired methionine synthase activity has been implicated in the pathogenesis of anaemias, cancer and neurological disorders Although the need for potent and specific inhibitors of methion-ine synthase has been recognized, there is a lack of such agents In this study, we designed, synthesized and evaluated the inhibitory activity of a series of substituted benzimidazoles and small benzothiadiazoles Kinetic analysis revealed that the benzimidazoles act as competitive inhibitors of the rat liver methionine synthase, whilst the most active benzothiadiazole (IC50¼ 80 lm) exhibited characteristics of uncompetitive inhibition A model of the methyltetrahydrofolate-binding site of the rat liver methionine synthase was constructed; docking experiments were designed to elucidate,
in greater detail, the binding mode and reveal structural requirements for the design of inhibitors of methionine synthase Our results indicate that the potency of the tested compounds is related to a planar region of the inhibitor that can be positioned in the centre of the active site, the presence
of a nitro functional group and two or three probable hydrogen-bonding interactions
Abbreviations
CH 3 -Cbl, methylcobalamin; DHPS, dihydropteroate synthase; Hcy, homocysteine; IC 50 , half-inhibitory concentration; MeTr, methyltransferase protein; MetS, methionine synthase; S-AdoMet, S-adenosylmethionine.
Trang 2pathways These include the reactions of
trans-sulfura-tion through the productrans-sulfura-tion of homocysteine,
biologi-cal methylations of DNA, lipids and proteins, and
polyamine biosynthesis through the production of
methionine and S-AdoMet [6,7] Furthermore, MetS is
the only human enzyme that metabolizes
methyltetra-hydrofolate to tetramethyltetra-hydrofolate, thereby facilitating the
recycling of this major form of folates to other
bio-active folates that provide one-carbon units for purine
and pyrimidine synthesis Impaired function of MetS
has been linked to megaloblastic anaemias and
neuro-logical disorders [8], atherosclerosis [7] and
carcinogen-esis [9,10]
Although the need to develop inhibitors of MetS as
drug candidates has long been recognized [11], there
are a limited number of reports on agents inhibiting
this enzyme The anaesthetic gas N2O is possibly the
only selective inhibitor of MetS reported to date, its
action mediated through the oxidation of the
cobala-min cofactor [12] Other compounds that have been
shown to inhibit this enzyme are the cell-signalling
molecule nitric oxide [13,14], chloroform and carbon
tetrachloride [15], methylmercury [16], ethanol and
acetaldehyde [17], hydrazine [18], S-AdoMet
deriva-tives [19] and a series of cobalamin analogues [20]
Polyamines have been shown to stimulate MetS
activity [21], whilst methotrexate has been shown to
indirectly inhibit the enzyme in vivo through depletion
of its substrate, 5-methyltetrahydrofolate [22]
In a new strategy for discovering specific inhibitors
of MetS, drug-like, benzo-fused heterocycles that
mimic substructures of 5-methyltetrahydrofolate have
been evaluated in a cell-free system The inhibitory
activity and mechanism of action have been probed by
kinetic studies using purified rat liver enzyme, whilst a
diamines were prepared by selective reduction of nitroanilines [23] and cyclized with formic acid [24] to give the desired substituted benzimidazoles The benz-imidazoles 1a–b and the benzothiadiazoles 2a–c were commercially available
All the compounds were tested against highly puri-fied rat liver MetS [14] and the half-inhibitory con-centrations (IC50) are presented in Table 1 The benzimidazoles 1c, 1h and 1k, and the nitrobenzothiad-iazole 2b, gave IC50 values close to or below 100 lm, with 2b being the most potent inhibitor (IC50¼
80 lm) From these results it was apparent that the presence of a nitro group at the 5-position was associ-ated with stronger inhibition (1c compared with 1d; 1h with 1i) and this was positively associated with the presence of the 3-methoxy group (1c compared with 1h) However, the aminobenzimidazole 1k showed a marginally stronger inhibition than the corresponding nitrobenzimidazole 1j This result may indicate that there is more than one mode of interaction with the active site that affects the activity of more-highly sub-stituted molecules such as 1j and 1k Furthermore, the presence of an N-methyl group on the benzimidazole ring (position similar to the one in the substrate 5-methyltetrahydrofolate; Fig 1) was detrimental to the IC50 (comparing 1c with 1f and 1h with 1j) Finally, the inhibitory activity of the benzothiadiazoles was improved by the nitro substitution (2b compared with 2a or 2c)
To explore further the molecular mechanism of action of those two classes of substituted benzohetero-cycles, the kinetic parameters of inhibition were meas-ured Compounds 1c and 2b were chosen as being representative of each class as a result of their good inhibitory activity and availability Figure 2 shows the Lineweaver–Burk, Dixon and Cornish–Bowden plots for the uninhibited and inhibited reactions The Km
values for the uninhibited reaction were calculated to
be 25 lm for 5-methyltetrahydrofolate and 0.6 lm for homocysteine, both results being in fair agreement
Fig 1 The cobalamin-dependent methionine synthase catalysed
reaction Cbl(I), cob(I)alamin; CH 3 -Cbl, methylcobalamin; R,
ptero-glutamate.
Trang 3with previously published data for the pig liver enzyme
(16.8 and 2.16 lm, respectively) [25] The Lineweaver–
Burk plots for the inhibited reactions showed that the
nitrobenzimidazole 1c exhibits the characteristics of
mixed inhibition (Fig 2A) (Ki¼ 26 lm), whilst the
nitrobenzothiadiazole 2b is an uncompetitive inhibitor
of MetS with respect to 5-methyltetrahydrofolate
(Ki¼ 17 lm) (Fig 2B) These findings were confirmed
by the Dixon (Fig 2C,D) [26] and Cornish–Bowden
(Fig 2E,F) [27] plots for the two compounds When
1c and 2b were assessed with homocysteine as the
vari-able substrate, the Lineweaver–Burk, Dixon and
Corn-ish–Bowden plots indicated that both compounds
exhibited characteristics of mixed inhibition (Fig 3),
with the nitrobenzothiadiazole 2b presenting a strong
component of uncompetitive inhibition (Fig 3D) It
must also be noted that both compounds were very
weak inhibitors when assessed with respect to
homo-cysteine, with detectable inhibition noted mainly at
high concentrations of the inhibitors (0.5 and 1 mm;
Fig 3A)
The results of these studies suggest that the two
clas-ses of substituted benzo-fused heterocycles may act by
two distinct mechanisms The mixed inhibition
exhib-ited by the nitrobenzimidazole 1c is a pattern usually
observed in multisubstrate enzyme-catalysed reactions
such as MetS However, the uncompetitive inhibition,
shown by the nitrobenzothiadiazole 2b, indicates that
there is no reversible link between the inhibitor and
the variable substrates Plausible rationalization
includes the possibility that the relatively small nitro-benzothiadiazole may displace the dimethylbenzimidaz-ole side chain of the cobalamin-cofactor or that it may act on the binding site of the MetS allosteric cofactor, S-AdoMet, both effects which could explain the observed uncompetitive inhibition Furthermore, clo-sely related 1,2,3-benzothiadiazoles and 1,2,4-thiazoles act as potent electron acceptors in biological systems Thus, 1,2,4-thiadiazoles can be used to trap cysteine residues by mixed disulfide formulation [28] Alternat-ively, 1,2,3-benzothiazoles have been shown to inhibit cytochrome P450 metabolites by interference with elec-tron transport within the catalytic cycle of cytochrome P450 [29] Details of the potential binding and electron transfer events that may account for the uncompetitive inhibition of MetS by nitrobenzothiadiazole 2b are the subject of continuing investigation in this laboratory
To elucidate further the mechanism of action of the substituted benzo-fused heterocycles, to explore the interactions occurring at the binding site, and to develop a tool that could assist further optimization
of inhibitors, a molecular model of the methyltetra-hydrofolate-binding domain of the rat liver MetS was constructed In the absence of a high-resolution struc-ture of the methyltetrahydrofolate-binding site for the mammalian enzyme, a model based on the X-ray crys-tal structure of the methyltetrahydrofolate corrinoid iron-sulfur methyltransferase protein (MeTr) from Clostridium thermoaceticum, as determined by Doukov
et al [30], was constructed It has been suggested that
Table 1 Structures and half inhibitory concentrations (IC 50 ) of the series 1 and 2 substituted benzo-fused heterocycles IC 50 values were determined using highly purified rat liver methionine synthase.
N N
R X
Y
1 5
7
N S N
Z
Trang 4the methyltetrahydrofolate-binding domain of MetS,
and indeed of other methyltransferases, share
architec-tural similarities [30] We therefore constructed the rat
liver MetS methyltetrahydrofolate-binding site
(resi-dues 359–639) model based on the homology of this
protein with that of MeTr (residues 1–262) Using
information from the Brookhaven protein data bank, sequence homologues of the two proteins were obtained (Fig 4) This led to the deduction of a back-bone structure that was modelled to the conserved TIM barrel fold (triose phosphate isomerase type structure), a common feature for globular proteins that
D C
F E
Fig 2 Lineweaver–Burk plots for nitrobenzimidazole 1c (A) and nitrothiadiazole 2b (B), with respect to methyltetrahydrofolate (MTHF), at inhibitor concentrations of 1000 l M (·), 500 l M (m), 100 l M (d) and 0 l M (j) Dixon plots for nitrobenzimidazole 1c (C) and nitrothiadiazole 2b (D), and Cornish–Bowden plots for nitrobenzimidazole 1c (E) and nitrothiadiazole 2b (F), with respect to the inhibitor, at methyltetrahydro-folate (MTHF) concentrations of 11 l M (·), 22 l M (m), 67 l M (d) and 224 l M (j).
Trang 5has been predicted to occur in the pterin-binding site
of related methyltransferases [30] The nonconserved
sequences were altered and any insertions or deletions
were applied using the molecular modelling program,
sybyl
3 , to construct and refine the model A gradual refinement of the resulting structure was performed using minimization through application of the charmm
4 program and force field [31]
A
C
E
B
D
F
Fig 3 Lineweaver–Burk plots for nitrobenzimidazole 1c (A) and nitrothiadiazole 2b (B), with respect to homocysteine (Hcy), at inhibitor con-centrations of 1000 l M (·), 500 l M (m), 100 l M (d) and 0 l M (j) Dixon plots for nitrobenzimidazole 1c (C) and nitrothiadiazole 2b (D), and Cornish–Bowden plots for nitrobenzimidazole 1c (E) and nitrothiadiazole 2b (F), with respect to the inhibitor, at homocysteine (Hcy) concen-trations of 1.1 l M (·), 2.2 l M (m), 6.5 l M (d) and 11 l M (j).
Trang 6Docking of the substrate 5-methyltetrahydrofolate in
the model of the active site was based on the approach
followed by Doukov et al [30] to identify the
inter-actions between the pterin cofactor and MeTr This
approach was based on the assumption that the strong
structural homology of MeTr and the dihydropteroate
synthases (DHPS) allows the prediction of interactions
between the pterin ring and MeTr based on the
binding of hydroxymethylpterin pyrophosphate to
DHPS Following the same approach, and in order to
identify the orientation of the substrate when bound
to the active site, we superimposed the rat liver MetS
model on the experimentally determined structure of
hydroxymethlypterin pyrophosphate bound to DHPS
[32] The result of this approach indicated the
orienta-tion of 5-methyltetrahydrofolate in the MetS active
site Figure 5 shows the superimposed structures of
DHPS and 5-methyltetrahydrofolate The model of the
methyltetrahydrofolate-binding site of MetS with one
molecule of the substrate included was further
opti-mized using charmm, and the ligand was
parameter-ized using partial atomic charges and other parameters
obtained from quantum mechanic modelling
(Hartree-Fock 6–31G* within the Spartan PCPro package)
the ligand structure Electrostatic surfaces of the
meth-yltetrahydrofolate-binding site domain were generated
to show the size of the active site (Fig 6) The
negat-ively charged areas may indicate the need of the
inhib-itor to have positively charged regions for favourable
interactions to take place Figure 7 highlights the amino acyl residues that are proposed to interact with 5-methyltetrahydrofolate, according to this model Calculations using interaction potentials produced predicted values for the percentage inhibition of each of the tested compounds These data were then compared with the experimentally determined data (percentage inhibition at 100 lm), and the results are presented in Fig 8 The predicted activities of seven heterocycles (1j, 1k, 1f, 1g, 1a, 2a, 2c) were found to have good cor-relation with the experimentally determined inhibition
Fig 4 Sequence homology of the template sequence of MeTr and the methyltetrahydrofolate-binding domain of rat liver MetS The residues shown in bold indicate conserved homology between the MeTr and MetS proteins Marked with a cross (+) are the residues that have a high degree of similarity so that although the sequence is not identical, the function of the residues is expected to remain the same.
Fig 5 The orientation of hydroxymethylpterin pyrophosphate (HMPP) when bound to dihydropteroate synthase, and the super-imposed structure of methyltetrahydrofolate showing the proposed orientation in the methionine synthase active site HMPP is repre-sented as a stick structure; methyltetrahydrofolate is reprerepre-sented
as a wire structure.
Trang 7(percentage inhibition ± 10, Fig 8), including two of
the five most active compounds (i.e the benzimidazoles
1j and 1k) Interestingly, 2b, the most active compound
of this series, was not predicted to have strong inhibitory
activity This finding is consistent with the kinetic
evalu-ation for this inhibitor that suggests a different mode of
action (i.e through noncompetitive inhibition, as shown
in Fig 2) unrelated to direct binding to the methyltetra-hydrofolate site
Overall, the biological evaluation and molecular modelling studies indicate two routes for the develop-ment of the next generation of inhibitors Specifically, the molecules need to be relatively small or not carry bulky substituents in order to enter the active site They require a planar region that can be positioned in the centre of the active site, a nitro functional group and two or three possible hydrogen-bonding groups Further refinement of this model could assist the dis-covery of the next generation of inhibitors for MetS Moreover, the observed noncompetitive inhibition pat-tern, with respect to the methyltetrahydrofolate-bind-ing site of MetS, implies the existence of other bindmethyltetrahydrofolate-bind-ing sites that may also be investigated for the development
of inhibitors, whilst the potential reactivity of the benzothiadiazole ring opens the possibility for design-ing mechanism-based inhibitors Overall, this approach may lead to the identification of compounds with potential therapeutic value, in particular as chemo-therapeutic agents for methionine-dependent cancers in combination with methionine-depleted treatments [33]
As the enzyme and its related metabolites have been involved in many disorders, including cardiovascular disease, neurodegenerative diseases and cancer, potent and specific inhibitors will also be valuable tools for defining the exact role of MetS in the pathophysiology
of these diseases
Experimental procedures
hydroxycobalamin, dimethylsulfoxide, ascorbic acid, phenyl-methanesulfonyl fluoride, Na-p-tosyl-l-lysylchloromethyl ketone, trypsin inhibitor, aprotinin, DEAE-cellulose, and phosphate buffers were purchased from Sigma (Poole, UK) 5-[14C]-methyl]methyltetrahydrofolic acid (barium salt) (56 mCiÆmmol)1) was purchased from Amersham (Little Chalfont, UK) AG1-X8 resin (200–400 mesh chloride form)
6
and the Protein Assay kit were from Bio-Rad (Hemel Hemp-stead, UK) Q-Sepharose Fast Flow and Hydroxyapatite were from Pharmacia
HiSafe 3 scintillation cocktail was from Fisher Scientific (Leicester, UK) Amicon ultrafiltration membranes, of
30 kDa, were purchased from
Benzimidazole (1a), 1-methylbenzimidazole (1b), 2,1,3-ben-zothiadiazole (2a), 4-nitro-2,1,3-benzothiadiazole (2b), 4-amino-2,1,3-benzothiadiazole (2c) and 4-methoxyaniline were obtained from Aldrich (Poole, UK); and 1,3-dinitroben-zene was from Avocado (Hewsham, UK) Solvents were
of the highest purity commercially available and were
A
B
C
Fig 6 Snapshot pictures showing the electrostatic surfaces of the
methyltetrahydrofolate-binding domain of rat liver MetS (A) with
5-methyltetrahydrofolate bound showing the open cleft binding side
from a side angle, (B) from a reverse angle, and (C) from above.
Red indicates negatively charged surfaces, and blue indicates
posi-tively charged surfaces.
Trang 8purchased from Sigma or BDH (Poole, UK) TLC plates
(sil-ica gel 60 F254) and silica gel (particle size 40–63 lm) for
chromatography were from Merck (Beeston, UK)
Deuterat-ed solvents were from Goss (Glossop, UK) Melting points
were determined using an Electrothermal IA9200 digital melt-ing point apparatus IR spectra were recorded on a Perkin Elmer
Elmer, Seer Green, UK) 1H and 13C NMR spectra were acquired at 270.05 and 67.80 MHz, respectively, on a JEOL GX270 spectrometer (JEOL UK, Welwyn, UK)
assign-ments were made using the DEPT135 experiment Mass spectra were obtained from the EPSRC National Mass Spectrometry Service Centre, University of Wales (Swansea, UK)
Synthesis of the substituted benzimidazoles 1c–k 1,3,5-Trinitrobenzene [34]
1,3-Dinitrobenzene 50 g (0.297 mol) was dissolved in fum-ing nitric acid (130.5 mL) and fumfum-ing sulphuric acid (243.5 mL), then heated under reflux at 150C for 7 days The reaction was cooled slowly to room temperature On addition to ice-cold distilled water, a solid precipitated which was collected by filtration and recrystallized from glacial acetic acid to give 1,3,5-trinitrobenzene (61.01 g, 97%), melting point (m.p.) 118–119C, literature
[34] 1H NMR (CDCl3) d: 9.41 (s
NMR (CDCl3) d: 149.6 (C-NO2), 124.4 (CH-Ar) MS (EI):
213 (M+) IR vmaxÆcm)13104s
13;14 (C-H aromatic), 1624s (C¼C aromatic), 1475m
13;14 (C¼C aromatic), 1544s (N¼O, asym-metric), 1345s (N¼O, symasym-metric), 900s (C-H bend)
Fig 7 Detailed view into the 5-methyltetrahydrofolate-binding pocket of rat liver MetS Atoms within 7 A ˚ of the docked ligand are shown: hydrophobic amino acid residues are coloured bronze, and hydrophilic acid residues are coloured blue Blue lines indicate the putative hydro-gen bonding interactions with ASN100 H-bond lengths are shown on the drawing, deviations from linearity are < 15 for both.
Fig 8 Correlation of experimentally determined inhibition
(percent-age inhibition at 100 l M ) with computer-predicted inhibition, based
on the calculation of interaction potentials.
Trang 93,5-Dinitroanisole [35]
1,3,5-Trinitrobenzene (5 g, 0.023 mol) was dissolved in
methanol (75 mL) with gentle heating To this hot solution,
a hot solution of potassium bicarbonate (0.5 mol, 7.5 g) in
water (30 mL) and methanol (20 mL) was added The
mix-ture was heated at reflux for 2.5 h, cooled to room
tem-perature and the methanol evaporated under reduced
pressure The aqueous residue was extracted with
chloro-form (3· 40 mL), the chloroform extracts combined, dried
over MgSO4and the solvent evaporated The product was
recrystallized from ethanol to give 3,5-dinitroanisole
(3.36 g, 74%), m.p
NMR (CDCl3) d: 8.65 (d
J¼ 2 Hz, 2H, 2-H, 6-H), 4.01 (s, 3H, OCH3) 13C NMR
(dimethylsulfoxide) d: 164.3 (C-1), 152.7 (C-3,5), 118.9
(C-2,6), 114.3 (C-4), 61.07 (CH3) MS (EI): 198 (M+) IR,
vmaxÆcm)1: 3098s (C-H aromatic), 2862w
(C¼C aromatic), 1544s (N¼O, asymmetric), 1345s (N¼O,
symmetric), 1080s (C-O)
2,3,5-Trinitroanisole [36]
3,5-Dinitroanisole (1.5 g, 0.007 mol) was dissolved in
con-centrated sulfuric acid (20 mL) with gentle heating The
solution was placed in an ice bath and fuming nitric acid
(4.2 mL) was added dropwise over a period of 10 min
The mixture was kept on ice for 20 min, with constant
stopped by the addition of distilled water (50–100 mL)
and the product was extracted into ether (3· 40 mL)
The ether extracts were combined, dried over MgSO4and
the solvent evaporated The product was recrystallized
from ethanol to give 2,3,5-trinitroanisole (1.6 g, 96%),
m.p 100–104C, lit 104 C [36] 1
6-H), 4.06 (s, 3H, OCH3) 13C NMR
(dimethylsulfoxide-d6) d: 155.1 (C-1), 152.5 (C-5), 143.9 (C-3), 140.0 (C-2),
116.5 (C-6), 107.4 (C-4), 60.2 (CH3) MS (EI): 243 (M+)
IR vmaxÆcm)1: 3117m (C-H aromatic), 2992w (C-H sp3),
1600m (C¼C aromatic), 1469m (C¼C aromatic), 1046s
(C-O symmetric)
2-Amino-3,5-dinitroanisole [37]
2,3,5-Trinitroanisole (1 g, 0.004 mol) was dissolved in
abso-lute ethanol (54 mL), cooled to 2C, and concentrated
reflux for 3 h, cooled to room temperature and the solvent
evaporated under reduced pressure After isolation by flash
chromatography [chloroform⁄ petroleum ether (70 : 30;
v⁄ v)], the product was recrystallised from ethanol to give
2-amino-3,5-dinitroanisole (0.86 g, 80%), m.p 182–184C,
lit 180C [37] 1H NMR (CDCl3) d: 8.82 (d, J¼ 2 Hz,
1H, 4-H), 9.0–5.0 (br
vmax⁄ cm)1: 3465s (NH asymmetric), 3322s (NH symmetric), 3098w (C-H aromatic), 2992w (C-H sp3), 1600m (C¼C aro-matic), 1456m (C¼C aroaro-matic), 1550s (N¼O asymmetric), 145s (N¼O symmetric), 1059s (C-O)
2-N-Methylamino-3,5-dinitroanisole [37]
2,3,5-Trinitroanisole (1 g, 0.004 mol) was dissolved in tetrahydrofuran (THF) (5 mL) and methylamine in THF
19
(10 mL, 2 m), then the solution was heated in a Young’s tube for 4 h, cooled and the solvent evaporated under reduced pressure The product was isolated by flash chro-matography [diethyl ether⁄ hexane (60 : 40, v ⁄ v)] to give 2-aminomethyl-3,5-dinitroanisole (0.8 g, 89%), 220–222C, lit 230C [37] 1
H NMR (CDCl3) d: 8.76 (d, J¼ 2 Hz, 1H, 4-H), 8.49 (br, 1H, NH), 7.65 (d, J¼ 2 Hz, 1H, 6-H), 3.94 (s, 3H, OCH3), 3.37 (d, J¼ 6 Hz, 3H, NCH3) 13C NMR (CDCl3) d: 149.6 (C-1), 148.4 (C-5), 138.8 (C-3), 137.2 (C-2), 124.3 (C-6), 118.9 (C-4), 57.2 (OCH3), 33.6 (NCH3) MS (EI): 198 (M+)
2,3-Diamino-5-nitroanisole, 3-amino-2-methylamino-5-nitroanisole [37]
The same method was
2-amino-3,5-dinitro-anisole and 2-N-methylamino-3,5-dinitro2-amino-3,5-dinitro-anisole The appro-priate compound (1.0 mmol) was dissolved in methanol (30 mL) and water (2 mL) Ammonium chloride (573 mg,
10 mmol) and ammonium carbonate (350 mg, 3.64 mmol) were added to the solution A hot solution of sodium sulfide (110 mg, 1.41 mmol in 1.5 mL water) was added dropwise over a time-period of 5 min and the solution heated at reflux for 2 h The reaction mixture was allowed
to cool slowly to room temperature, the volatile solvent evaporated under reduced pressure and the product was isolated by flash chromatography, eluted with methanol:
c NH3: chloroform (1 : 1 : 98) 2,3-Diamino-5-nitroanisole: (0.150 mg, 77%), m.p 173–175C, lit.165–167 C [37] 1H NMR (CDCl3) d: 7.41 (d, J¼ 2 Hz, 1H, 4-H), 7.39 (d,
J¼ 2 Hz, 1H, 6-H), 4.05 (s, 2H, NH2), 3.93 (s, 3H,
3-Amino-2-methylamino-5-nitroanisole: (0.181 mg, 80%), m.p 158–160C 1
H NMR (CDCl3) d: 7.32 (d, J¼ 2 Hz, 1H, 4-H), 7.24 (d,
J¼ 2 Hz, 1H, 6-H), 4.1 (br, s, 3H, NH), 3.87 (s, 3H, OCH3), 2.82 (s, 3H, NCH3)
5-Nitro-7-methoxybenzimidazole (1h) and 1-methyl-5-nitro-7-methoxybenzimidazole (1j) [37]
The same method was
2,3-diamino-5-nit-roanisole and 3-amino-2-methylamino-5-nit2,3-diamino-5-nit-roanisole The compound (1.0 mmol) was dissolved in formic acid (5 mL) and heated at reflux for 2 h The reaction was removed
Trang 10(s, 3H, OCH3), 3.29 (s, 3H, NCH3) HRMS (ES) (M + H)
208.0717, C9H10N3O3requires 208.0717
5-Amino-7-methoxybenzimidazole (1i) [37] and
5-amino-7-methoxy-N1-methylbenzimidazole (1k) [38]
The same method was
5-nitro-7-methoxy-benzimidazole (1h) and
1-methyl-5-nitro-7-methoxybenzimi-dazole (1j) The compound (0.6 mmol) was dissolved in
ethanol (30 mL) with 2 drops of concentrated HCl, and
10% weight of palladium on a carbon catalyst was added
The system was evacuated and the mixture stirred
vigo-rously under a hydrogen atmosphere until the reaction was
complete (approximately 2–3 h by TLC) The catalyst was
removed by filtration through celite and washed with
copi-ous amounts of ethanol The solvent was evaporated under
reduced pressure and the product was recrystallized from
ethanol and ethyl acetate An alternative method involved
using eight equivalents of ammonium formate as the
hydro-gen source and reacting the mixture, as above, for 2 h in an
evacuated system 1i: (0.08 mg, 82%), m.p 220–223C, lit
216–218C [37].1H NMR (dimethylsulfoxide-d6) d: 8.72 (s,
1H, 2-H), 8.20 (d, J¼ 2 Hz, 1H, 4-H), 7.65 (d, J ¼ 2 Hz,
1H, 6-H), 5.81–6.12 (br, 3H, NH, NH2) 4.07 (s, 3H,
OCH3) 1 k: (0.081 mg, 76%), m.p 180–182, lit 178C
[38].1H NMR (dimethylsulfoxide-d6) d: 8.64 (s, 1H, 2-H),
8.26 (d, J¼ 2 Hz, 1H, 4-H), 7.12 (d, J ¼ 2 Hz, 1H, 6-H),
5.23–5.65 (br, 2H, NH2), 4.01 (s, 3H, OCH3), 3.26 (s, 3H,
NCH3)
5-Nitrobenzimidazole (1c) and 5-aminobenzimidazole
(1d) [39]
These compounds were synthesized, according to the
meth-ods described above, from 2,4 dinitroaniline 1c: (2.5 g,
88%), m.p 203–204C, lit 204–205 C [39] 1
H NMR (dimethylsulfoxide-d6) d: 8.54 (s, 1H, 2-H), 8.51 (d, J¼ 2 Hz,
1H, 4-H), 8.44 (s, 1H, NH), 8.13 (dd
1H, 6-H) 7.09 (d, J¼ 8 Hz, 1H, 7-H) MS (EI): 164(M+
)
1dÆ2HClÆ0.2H2O: (3.1 g, 90%), m.p decomposition
(2· 30 mL) The combined organic extracts were dried over MgSO4 and evaporated under reduced pressure The residue was dissolved in isopropanol, treated with 5 mL of concentrated HCl, evaporated twice from isopropanol then recrystallized from isopropanol-ether to yield a grey solid, 1eÆHCl: (0.50 g, 59%), m.p 202–206C, lit 199–202 C [40] 1H NMR (dimethylsulfoxide-d6) d: 15.01 (br, 2H,
2· NH), 9.43 (d, J ¼ 5 Hz, 1H, 2-H), 7.71 (d, J ¼ 9 Hz, 7-H), 7.23 (d, J¼ 2 Hz, 1H, 4-H), 7.14 (dd, J ¼ 9Hz, J ¼
2 Hz, 1H, 6-H), 3.83 (s, 3H, OCH3) MS (EI) (free base):
148 (M+)
1-Methyl-5-nitro-benzimidazole (1f)
Chlorodinitrobenzene (0.81 g, 4.0 mmol), dissolved in THF
for 12 h at 90C in a Young’s tube The reaction was monitored using TLC with diethyl ether as the eluant The mixture was cooled to room temperature and the solvent evaporated under reduced pressure The resulting diamino compound was then cyclized in formic acid (5 mL) heated
at reflux for 2 h, after which the reaction was cooled to room temperature and toluene (20 mL) and water (1 mL) were added The volatile solvent was evaporated under reduced pressure and the residue was poured into water (30 mL) and extracted with ethyl acetate (3· 30 mL) The
(20 mL), dried over MgSO4and evaporated under reduced pressure The product was recrystallized from ethyl acetate (0.23 g, 32%), m.p 213–215C, lit 209–211 C [41] 1
H NMR (dimethylsulfoxide-d6) d: 8.73 (d, J¼ 2 Hz, 1H, 4-H), 8.25 (d, J¼ 9 Hz, 2H, 6-H), 8.04 (s, 1H, 2-H), 7.44 (d, J¼ 9 Hz, 1H, 7-H), 3.92 (s, 3H, NCH3) MS (EI):
178 (M+)
1-Methyl-5-amino-benzimidazoleÆ2HClÆ0.2H2O (1g)
1-Methyl-5-nitro-benzimidazole (1f) (0.097 g, 0.548 mmol) was dissolved in ethanol (20 mL), 10% palladium on carbon