Imidazole- and benzimidazole-based inhibitors of the kinase IspE: Targeting the substrate-binding site and the triphosphate-binding loop of the ATP site

Article abstract of DOI:10.1002/ejoc.201201467

The enzymes of the mevalonate-independent biosynthetic pathway to isoprenoids are attractive targets for the development of new drug candidates, in particular against malaria and tuberculosis, because they are present in major human pathogens but not in humans. Herein, the structure-based design, synthesis, and biological evaluation of a series of inhibitors featuring a central imidazole or benzimidazole scaffold for the kinase IspE from E. coli, a model for the corresponding malarial enzyme, are described. Optimization of the binding preferences of the hydrophobic sub-pocket at the substrate-binding site allowed IC₅₀ values in the lower micromolar range to be reached. Structure-activity relationship studies using a 1,2-disubstituted imidazole central core revealed that alicyclic moieties fit the sub-pocket better than acyclic aliphatic and aromatic residues. The phosphate-binding region in the ATP-binding site of IspE, a neutral glycine-rich loop, was addressed for the first time by an additional vector attached to the central core. Polar functional groups, such as trifluoromethyl or nitriles, were introduced to undergo orthogonal dipolar interactions with the amide groups in the loop. Alternatively, small hydrogen-bond-accepting heterocyclic residues, capable of binding to the convergent NH groups in the loop, were explored. The biological data showed slightly improved inhibitory potency in some cases and confirmed the challenges in addressing, with gain in binding affinity, the highly water-exposed sections of enzyme active sites, such as the glycine-rich loop of IspE. Inhibitors of the kinase E. coli IspE, an enzyme involved in the synthesis of isoprenoids and a model for IspE from P. falciparum, were developed. Decorated imidazole- and benzimidazole-based scaffolds address the cytidine-binding pocket, the hydrophobic sub-pocket, and the phosphate-binding region in the active site. In vitro activities in the micromolar IC ₅₀-range were measured. Copyright

Full text of DOI:10.1002/ejoc.201201467

FULL PAPER
DOI: 10.1002/ejoc.201201467
Imidazole- and Benzimidazole-Based Inhibitors of the Kinase IspE: Targeting
the Substrate-Binding Site and the Triphosphate-Binding Loop of the ATP Site
Paolo Mombelli,[a] Camille Le Chapelain,[a] Noah Munzinger,[a] Evelyne Joliat,[a]
Boris Illarionov,[b] W. Bernd Schweizer,[a] Anna K. H. Hirsch,[c] Markus Fischer,*[b]
Adelbert Bacher,*[b] and François Diederich*[a]
Keywords: Drug design / Inhibitors / Ligand design / Molecular recognition
The enzymes of the mevalonate-independent biosynthetic
pathway to isoprenoids are attractive targets for the develop-
ment of new drug candidates, in particular against malaria
and tuberculosis, because they are present in major human
pathogens but not in humans. Herein, the structure-based
design, synthesis, and biological evaluation of a series of in-
hibitors featuring a central imidazole or benzimidazole scaf-
fold for the kinase IspE from E. coli, a model for the corre-
sponding malarial enzyme, are described. Optimization of
the binding preferences of the hydrophobic sub-pocket at the
substrate-binding site allowed IC50 values in the lower
micromolar range to be reached. Structure–activity relation-
ship studies using a 1,2-disubstituted imidazole central core
revealed that alicyclic moieties fit the sub-pocket better than
acyclic aliphatic and aromatic residues. The phosphate-bind-
ing region in the ATP-binding site of IspE, a neutral glycine-
rich loop, was addressed for the first time by an additional
vector attached to the central core. Polar functional groups,
such as trifluoromethyl or nitriles, were introduced to un-
dergo orthogonal dipolar interactions with the amide groups
in the loop. Alternatively, small hydrogen-bond-accepting
heterocyclic residues, capable of binding to the convergent
NH groups in the loop, were explored. The biological data
showed slightly improved inhibitory potency in some cases
and confirmed the challenges in addressing, with gain in
binding affinity, the highly water-exposed sections of enzyme
active sites, such as the glycine-rich loop of IspE.
biosynthetic route, with acetyl coenzyme A serving as the
only carbon source. Thus, the enzymes of the non-mevalon-
ate pathway are widely recognized as attractive targets for
the generation of lead compounds with potential as selec-
tive antibacterial, antimalarial, or agrochemical agents
featuring novel modes of action, which is a fundamental
issue that needs to be overcome to fight fast-developing re-
sistance.[5]
The fourth enzyme and only kinase involved in the non-
mevalonate pathway, IspE [4-diphosphocytidyl-2C-methyl-
d-erythritol (CDP-ME) kinase, EC 2.7.1.148], catalyzes the
ATP- and Mg2+-dependent phosphorylation of the tertiary
alcohol of CDP-ME (5) to give 4-diphosphocytidyl-
2C-methyl-d-erythritol 2-phosphate (CDP-ME2P, 6;
Scheme 1).[6] Due to the lack of structural information on
IspE orthologues from pathogens until the recent report of
X-ray crystal structures of the M. tuberculosis enzyme,[6h]
our initial efforts in mapping the molecular recognition
properties of IspE used the enzyme from Escherichia (E.)
coli as a model system. In our previous studies, we exten-
sively explored the binding preferences of the small hydro-
phobic and the ribose sub-pockets of the substrate-binding
site.[6g,7]
Introduction
Discovered in the early 1990s, the non-mevalonate path-
way is a biosynthetic route to assemble the universal iso-
prenoid precursors, isopentenyl diphosphate (IPP; 1) and
dimethylallyl diphosphate (DMAPP; 2), in seven enzymatic
steps starting from pyruvate (3) and d-glyceraldehyde 3-
phosphate (4) (Scheme 1).[1] The enzymes of this pathway
are essential in plants[2] and many human pathogens, in-
cluding the tuberculosis-causing Mycobacterium (M.) tuber-
culosis[3] and the causative agent of malaria, Plasmodium
(P.) falciparum.[4] In humans, in contrast, isoprenoids are
exclusively assembled through the mevalonate-dependent
[a] Laboratorium für Organische Chemie, ETH Zürich,
Hönggerberg, HCI, 8093 Zürich, Switzerland
Fax: +41-44-632-1109
E-mail: diederich@org.chem.ethz.ch
Homepage: http://www.diederich.chem.ethz.ch
[b] Hamburg School of Food Science, Institut für
Lebensmittelchemie, Universität Hamburg,
Grindelallee 117, 20146 Hamburg, Germany
Fax: +49-40-42838-4342
E-mail: markus.fischer@hsfs.uni-hamburg.de
Homepage: http://www.chemie.uni-hamburg.de/lc/fischer.html
[c] Stratingh Institute for Chemistry, University of Groningen,
Nijenborgh 7, 9747 AG Groningen, The Netherlands
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201467.
Herein, we report the structure-based design, synthesis,
and biological evaluation of imidazole- and benzimidazole-
based inhibitors targeting the phosphate recognition site –
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Imidazole- and Benzimidazole-Based Inhibitors of the Kinase IspE
Scheme 1. The non-mevalonate biosynthetic pathway to isoprenoid precursors, i.e., isopentenyl diphosphate (IPP; 1) and dimethylallyl
diphosphate (DMAPP; 2). DXS: 1-deoxy-d-xylulose 5-phosphate synthase (EC 2.2.1.7); IspC: 1-deoxy-d-xylulose 5-phosphate reducto-
isomerase (EC 1.1.1.267); IspD: 4-diphosphocytidyl-2C-methyl-d-erythritol 4-phosphate synthase (EC 2.7.7.60); IspE: 4-diphosphocyti-
dyl-2C-methyl-d-erythritol kinase (EC 2.7.1.148); IspF: 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12); IspG: 2C-
methyl-d-erythritol 2,4-cyclodiphosphate reductase (EC 1.17.7.1); IspH: (2E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase
(EC 1.17.1.2).
a “neutral” glycine-rich loop – in the ATP-binding pocket, fectly oriented into the cavity by a propargylic sulfonamide
in addition to the cytidine-binding pocket and the hydro- moiety in its preferred conformation (staggered with the N-
phobic sub-pocket of the substrate-binding site of E. coli lone pair bisecting the O=S=O moiety), allowed inhibitory
IspE. In addition to expanding our knowledge of the mo- constants (Ki) down to the triple-digit nanomolar range to
lecular recognition properties of IspE, this approach is be obtained.[7a]
interesting in view of the large number of such neutral
phosphate recognition sites in biological systems and the
lack of systematic studies addressing them.[8]
The introduction of an additional third vector towards
the glycine-rich loop required the installation of a new cen-
tral core in the ligands. Modeling suggested a 1,2,4-trisub-
stituted imidazole ring as an appropriate scaffold to direct
the three exit vectors, with the heterocycle stacking on the
polarized side chain of Asp141 (Figure 2).
Results and Discussion
We first investigated the appropriate filling of the hydro-
phobic sub-pocket by introducing different lipophilic sub-
Ligand Design
Molecular modeling was performed with the software stituents at imidazole-C-2. Subsequently, the triphosphate-
MOLOC[9] using the X-ray crystal structure of E. coli IspE binding region of E. coli IspE was addressed by the intro-
in complex with CDP-ME (5) and the ATP-analogue aden- duction of pharmacophores linked to position C-4 of the
osine-5Ј-(γ-imino)triphosphate (AMP-PNP, PDB accession imidazole moiety by a methylene spacer. In IspE, a glycine-
code 1OJ4, 2.0 Å resolution).[6c] The active site of E. coli rich loop (Gly101–Ser108; Figure 1) is responsible for bind-
IspE can be divided into three main pockets, corresponding ing the ATP-triphosphate through multiple hydrogen bonds
to the cytidine- and methylerythritol-binding regions for the with the converging backbone NH groups, without the in-
recognition of substrate 5, and the adenosine-binding volvement of basic amino acids or metal cations.[6c,6f,8] This
pocket at the other end of the active site (Figure 1).
confers a certain lipophilic character to the loop, which,
In our previous studies, we have addressed the cytidine- together with a balance between preorganization and the
binding pocket with a 1,5-disubstituted cytosine scaffold ability to wrap around a guest, makes it appealing for in-
stacking between the aromatic side chains of Tyr25 and creasing binding affinity, also taking advantage of a poten-
Phe185, similar to the natural substrate. Saturated five- tial bisubstrate inhibition mode.[10] In addition to hydrogen-
membered heterocycles such as 2-tetrahydrothienyl or 2- bond-accepting heterocycles such as tetrazole or triazole,
tetrahydrofuranyl moieties were shown to address the ribose we considered small and negatively polarized functional
sub-pocket best.[6g,7b] Modeling revealed the presence of a groups, such as nitriles and trifluoromethyl groups, as phos-
small, hydrophobic sub-pocket lined by Leu15, Leu28, and phate surrogates suitable for this neutral glycine-rich loop,
Phe185 adjacent to the cytidine-binding pocket. Appropri- due to their ability to undergo favorable orthogonal dipolar
ate filling, achieved with small lipophilic residues per- interactions.[11]
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Figure 1. Schematic view of the active site of E. coli IspE depicting the different pockets, of which the inhibitors described here address
the cytidine-binding pocket, the hydrophobic sub-pocket, and the phosphate-binding loop.
Figure 2. Design strategy of new ligands bearing a 1,2,4-trisubstituted imidazole central core and proposed binding mode of the imidazole
fragment at the active site of E. coli IspE (PDB code: 1OJ4)[6c] as modeled with MOLOC. Distances are given in Å units. Color code:
gray Cenzyme, green Cligand, blue N, red O, yellow S. If not otherwise stated, this color code is used throughout.
Optimization of the Binding towards the Sub-Pocket
and ammonium hydrogen carbonate in water, following a
literature procedure, to afford the 2-substituted imidazole
Ligands (Ϯ)-7–(Ϯ)-12 (see Table 1 for the structures) were 14.[12] Alkylation of imidazole 14 with propargyl bromide
synthesized in two or three steps, as illustrated in Scheme 2 gave alkyne 15, which was subsequently cross-coupled with
for compound (Ϯ)-10 [for the syntheses of the other ligands, the
previously
described
1-thiolanylated
5-iodo-
see the Supporting Information, Scheme S1]. In the first cytosine (Ϯ)-16[7a] under Sonogashira conditions to provide
step, cyclohexanecarbaldehyde (13) was treated with glyoxal the desired ligand (Ϯ)-10 in moderate yield.
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Imidazole- and Benzimidazole-Based Inhibitors of the Kinase IspE
Scheme 2. Synthesis of ligands (Ϯ)-10 and (Ϯ)-17. Reagents and conditions: (a) Glyoxal, NH4HCO3, H2O, 25 °C, 18 h, 25%; (b) propargyl
bromide, Cs2CO3, DMF, 0 to 25 °C, 9 h, 28%; (c) [PdCl2(PPh3)2], CuI, Et3N, DMF, 25 °C, 3.5 h, 25%; (d) Me2NSO2Cl, Et3N, CH2Cl2,
25 °C, 14 h, 62%; (e) i. nBuLi, THF, –78 °C, 45 min; ii. DMF, –78 to 25 °C, 2 h, 83%; (f) Me3SiCF3, nBu4NF, THF, 0 to 25 °C, 2 h,
73%; (g) NaH, MeI, DMF, 0 to 25 °C, 16 h, 85%; (h) 5% aq. HCl, 25 °C, 7 h, 90%; (i) propargyl bromide, Cs2CO3, DMF, 0 to 25 °C,
8 h, 48%; (j) (Ϯ)-16, [PdCl2(PPh3)2], CuI, Et3N, DMF, 25 °C, 7 h, 47%.
Table 1. Structures and inhibitory activities of ligands (Ϯ)-7–(Ϯ)- ated by pyruvate kinase dephosphorylating phosphoenol
12 against E. coli IspE.
pyruvate. The resulting pyruvate is reduced to l-lactate by
the lactate dehydrogenase using NADH as reducing agent.
The consumption of NADH is monitored photometrically
at 340 nm. This photometric assay has previously been
proven to work effectively for structurally related com-
pounds by comparison with a direct NMR spectroscopic
assay.[13]
The inhibitory potency towards E. coli IspE was found
to be strongly dependent on the substituent filling the
hydrophobic sub-pocket (Table 1). Compound (Ϯ)-7, un-
substituted at imidazole-C-2, did not display any significant
inhibition at 500 μM, whereas an additional substituent at
C-2 resulted in a substantial gain in binding affinity. Linear
or branched alkyl chains, such as n-propyl and isobutyl,
led to IC50 values in the double-digit micromolar range for
compounds (Ϯ)-8 and (Ϯ)-9.
[a] The clogD values were calculated with the Advanced Chemistry
Development (ACD/Labs) Software v. 12.5 (1994–2012, ACD/
Labs). [b] Ki values were calculated from IC50 values and Km
=
150 μM using the Cheng–Prusoff equation.[20]
The half-maximal (50%) inhibitory concentration (IC50)
Replacement of the alkyl chains with saturated alicyclic
values were determined in an enzyme-coupled assay using moieties in inhibitors (Ϯ)-10 and (Ϯ)-11 resulted in an ad-
pyruvate kinase and lactate dehydrogenase as auxiliary ditional five- to sixfold enhancement in activity, with IC50
enzymes to enable photometric monitoring (see values of 12 and 15 μM, respectively. Interestingly, the
Scheme S2).[13] For the phosphorylation of the substrate phenyl-substituted analogue (Ϯ)-12 was found to be sub-
CDP-ME (5), IspE consumes ATP, which is then regener- stantially less potent (IC50 value = 144 μM), indicating that
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the inhibitory activity increase of compounds (Ϯ)-10 and The nitrile functional group of ligands (Ϯ)-18 and (Ϯ)-19
(Ϯ)-11 is not solely a result of increasingly favorable par- were introduced by Horner–Wadsworth–Emmons reaction;
titioning (increasing clogD values), but also of a better fit the common precursor of dinitriles (Ϯ)-20 and (Ϯ)-21 was
of the respective substituents into the sub-pocket (Figure 3). obtained by Knoevenagel condensation (see Scheme S3).
Reduction of the aldehyde functional group to give the cor-
responding alcohol and subsequent activation by treatment
with thionyl chloride allowed the introduction by nucleo-
philic substitution of the 1H-tetrazole and 1,2,3-1H-triazole
moieties in ligands (Ϯ)-22 and (Ϯ)-23, respectively (see
Scheme S4).
Table 2. Structures and inhibitory activities of ligands (Ϯ)-17–(Ϯ)-
23 against E. coli IspE.
Figure 3. Close-up of the binding mode of (Ϯ)-10 in the hydro-
phobic sub-pocket of E. coli IspE as modeled with MOLOC (PDB
code: 1OJ4). Distances in Å.
Addressing the Phosphate-Binding Loop
The cyclohexyl substituent was maintained as the moiety
filling the hydrophobic sub-pocket, due to the superior
binding affinity of (Ϯ)-10 compared with the other com-
pounds included in Table 1. Therefore, the new series of
compounds was based on the structure of inhibitor (Ϯ)-10
bearing an additional vector at imidazole-C-4 for targeting
the glycine-rich P-loop.
The introduction of the additional substituent in ligands
(Ϯ)-17–(Ϯ)-23 (see Table 2 for the structures) was achieved
through the organolithium-mediated formylation of the
imidazole moiety and subsequent chain extension by in-
tergroup conversion of the resulting aldehyde. As an exam-
ple, the synthesis of compound (Ϯ)-17 is shown in
Scheme 2. The 2-substituted imidazole 14 was protected
with the ortho-directing N,N-dimethylsulfamoyl group
using the corresponding sulfamoyl chloride and triethyl-
amine as base to give 24 in moderate yield.[14] Lithiation of
[a] The clogD values were calculated with the Advanced Chemistry
Development (ACD/Labs) Software v. 12.5 (1994–2012, ACD/
Labs). [b] Ki values were calculated from IC50 values and Km
=
150 μM using the Cheng–Prusoff equation.[20]
The inhibitory activity of ligands (Ϯ)-17–(Ϯ)-23 against
imidazole 24 with nBuLi, followed by reaction with N,N- E. coli IspE is summarized in Table 2. Trifluoromethyl de-
dimethylformamide (DMF), afforded 5-formylimidazole 25 rivative (Ϯ)-17 was the most active compound of the series,
as a single regioisomer in very good yield.[15] Aldehyde 25 with an IC50 value of 9.9 μM. The more polar nitriles
was then treated with the Ruppert–Prakash reagent[16] and (Ϯ)-(E)-18 and (Ϯ)-19 and dinitriles (Ϯ)-20 and (Ϯ)-21
tetrabutylammonium fluoride to afford alcohol (Ϯ)-26 as a were less active, with IC50 values between 22 and 38 μM.
racemate in good yield. Methylation of the secondary Tetrazole (Ϯ)-22 and triazole (Ϯ)-23 displayed a substan-
alcohol and subsequent cleavage of the protecting group of tially lower affinity, also compared to reference compound
(Ϯ)-27 under acidic conditions gave key intermediate (Ϯ)-10.
(Ϯ)-28. The imidazole moiety was then N-alkylated with
According to molecular modeling studies, all the com-
propargyl bromide using cesium carbonate as base, afford- pounds in this series should establish favorable orthogonal
ing a mixture of regioisomers, which was separated by col- dipolar interactions with the peptide bonds of the glycine-
umn chromatography to provide the desired 1,2,4-trisubsti- rich loop (see Figure S1). Only the trifluoromethyl deriva-
tuted imidazole (Ϯ)-29 in moderate yield. Finally, Sonoga- tive (Ϯ)-17, however, was found to be slightly more active
shira cross-coupling with 5-iodocytosine (Ϯ)-16 yielded the than the parent compound (Ϯ)-10 unsubstituted at imid-
target compound (Ϯ)-17 in 47% yield. All other 1,2,4-tri- azole-C-4. This might indicate that the methylene linker
substituted imidazoles were prepared starting from alde- carrying the phosphate surrogate is too flexible to efficiently
hyde 25 as a common precursor following similar strategies. position the pharmacophore in the phosphate-binding re-
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Imidazole- and Benzimidazole-Based Inhibitors of the Kinase IspE
gion. This flexibility allows phosphate surrogates, in par- derived ligands, see Scheme S5). Reductive cyclization of 2-
ticular the more polar versions, to orient towards bulk amino-3-nitrophenol (35) with aldehyde 13 in the presence
water rather than binding to the loop.
of sodium dithionite according to a literature procedure
gave 2,4-disubstituted benzimidazole 36 in 80% yield.[17]
Treatment of alcohol 36 with 2-propanol under Mitsunobu
conditions provided ether 37 in moderate yield after
Benzimidazole-Based Ligands
The next series of compounds was designed with the spe- recrystallization from ethyl acetate. Whereas the alkylation
cific aim of establishing a more direct and preorganized ac- of 2,5-disubstituted benzimidazoles afforded a ca. 1:1 mix-
cess to the glycine-rich loop. For this purpose, the imidazole ture of regioisomers, usually separable with column
core was extended to include a benzimidazole moiety. In chromatography with the structural assignment based on
this way, the methylene linker could be omitted and the ten- 2D NMR spectroscopy, the outcome of the alkylation of 4-
dency of the phosphate mimic to orient toward the bulk alkoxybenzimidazole 37 with sodium hydride and propargyl
solvent should thereby be attenuated. Molecular modeling bromide proved to be strongly dependent on the solvent
studies suggested that positions C-4 and C-5 of the benz- (see Figure S2). In DMF, the undesired and sterically more
imidazole core were well-suited for targeting the glycine- hindered 1,2,7-trisubstituted benzimidazole 38 was the
rich loop, depending on the phosphate surrogate chosen.
major isomer formed (ratio 38/39 = 4:1), indicating elec-
Benzimidazoles (Ϯ)-30–(Ϯ)-34 (see Figure 4 for the tronic rather than steric control of the reaction. The re-
structures) were synthesized by applying the strategy de- gioselectivity of the alkylation was inverted in tetra-
scribed previously for the imidazole-based ligands. This in- hydrofuran (THF), in which a 1:3 ratio of 38/39 was ob-
volved construction of the heterocyclic core and its eventual served. The ratio in favor of the desired isomer was signifi-
modification by intergroup conversion, followed by N-alk- cantly improved (38/39 = 1:5) in a less polar, noncoordina-
ylation and final Sonogashira cross-coupling. As an exam- ting solvent, such as dichloromethane. This observation
ple, the synthesis of 4-alkoxybenzimidazole (Ϯ)-34 is shown supports a mechanism involving coordination of the Na+
in Scheme 3 (for the syntheses of the other benzimidazole- cation to the deprotonated nitrogen atom of the benzimid-
Figure 4. Structures and inhibitory activities of ligands (Ϯ)-30–(Ϯ)-34 against E. coli IspE. [a] clogD values were calculated with Advanced
Chemistry Development (ACD/Labs) Software V12.5 (1994–2012 ACD/Labs). [b] Ki values were calculated from IC50 values and Km
=
150 μM using the Cheng–Prusoff equation.[20]
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Scheme 3. Synthesis of ligand (Ϯ)-34 and ORTEP plot at 100 K (atomic displacement parameters are shown at the 50% probability level)
of the X-ray crystal structure of (Ϯ)-34. For the sake of clarity, solvent molecules are omitted and only one position of the disordered
tetrahydrothienyl ring and isopropyl group are shown. Reagents and conditions: (a) 1 m aq. Na2S2O4, EtOH, 70 °C, 5 h, 80%; (b) PPh3,
diisopropyl azodicarboxylate, iPrOH, THF, 25 °C, 2 h, 49%; (c) propargyl bromide, NaH, CH2Cl2, 0 to 25 °C, 24 h, 44%; (d) (Ϯ)-16,
[PdCl2(PPh3)2], CuI, iPr2NEt, DMF, 25 °C, 24 h, 36%.
azole moiety assisted by the proximal alkoxy group. As a a CF3 group at position C-5 of the benzimidazole core re-
consequence, the N-3 position is blocked, and alkylation is sulted in a slight loss of potency for (Ϯ)-31, with an IC50
directed to position N-1. In polar solvents such as DMF, value of 25 μM. The more polar nitrile (Ϯ)-32 (IC50
=
the solvent competes for Na+ complexation, and electronic 13 μM) was found to be slightly more potent. Interestingly,
control of the regioselectivity is established. Sonogashira the trend is reversed as compared to the imidazole-based
cross-coupling reaction of 1,2,4-isomer 39 with 5-iodocyto- inhibitors with a flexible linker, in which activity was lost
sine (Ϯ)-16 finally afforded ligand (Ϯ)-34 in moderate yield. with the introduction of more polar residues and was high-
Crystals of benzimidazole (Ϯ)-34 suitable for X-ray crys- est for the CF3 derivative (see Table 1). With an IC50 value
tal structure analysis were grown by slow evaporation of a of 85 μM, phosphate (Ϯ)-33 is a weaker inhibitor than con-
CH2Cl2/MeOH solution (Scheme 3), subsequent measure- trol compound (Ϯ)-30. The dramatic difference in clogD
ments confirmed the correct assignment of the regioisomers values [(Ϯ)-30: 5.97; (Ϯ)-33: –0.37], however, suggests that
formed in the alkylation reaction. In the crystal structure, the expected energetic gains from phosphate binding to the
the isopropoxy substituent is disordered and adopts two glycine-rich loop, in analogy to the β-phosphate of AMP-
orientations. In both conformations, however, the O(10)– PNP in the cocrystal structure (see Figure S4), might well
C(11) bond is almost antiperiplanar to the C(4)–C(9) bond, be overcompensated for by the highly unfavorable partition-
with torsion angles |τ|(C8–C9–O10–C11) of 7° and 2°, ing. The lipophilic isopropoxy counterpart (Ϯ)-34 is in-
respectively, allowing conjugation of the oxygen atom with active at inhibitor concentrations below 500 μM, despite
the aromatic system. The propargylic unit of (Ϯ)-34 is per- strain-free binding suggested by the modeling. This finding
pendicular to the aromatic ring, as expected, with a torsion also supports attractive polar interactions of the substitu-
angle τ(C2–N1–C20–C21) = 100°. These conformational ents in (Ϯ)-31–(Ϯ)-33 with the P-loop.
preferences were corroborated by searches in the Cam-
bridge Structural Database (CSD)[18] performed on similar
molecular fragments (see Figure S3).
Conclusions
The inhibitory activities of ligands (Ϯ)-30–(Ϯ)-34 against
E. coli IspE were determined by using the photometric as-
We have used the structure-based approach to design li-
say described above and are summarized in Figure 4. The gands featuring trisubstituted imidazole or benzimidazole
unsubstituted control compound (Ϯ)-30 displayed an IC50 central cores to address the cytidine-binding pocket, the
value of 17 μM, which is in a similar range to that of the hydrophobic sub-pocket, and the glycine-rich loop at the
corresponding imidazole (Ϯ)-10 (Table 1). Introduction of active site of the kinase IspE from E. coli. The syntheses of
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Imidazole- and Benzimidazole-Based Inhibitors of the Kinase IspE
twice with CH2Cl2. The combined organic phases were washed
with water (3ϫ) and brine, dried with MgSO4, filtered, and concen-
trated. The residue was purified by column chromatography (CC).
the inhibitors relied on the construction of the heterocyclic
moieties and their subsequent regioselective functionaliza-
tion, such as by formylation of imidazoles and alkylation
of 4-alkoxybenzimidazoles. Biological evaluation of the li-
gands demonstrated that appropriate filling of the hydro-
phobic sub-pocket is essential to gain binding affinity
towards IspE, as this cavity represents one of the few lipo-
philic environments of the otherwise highly polar active site.
Addressing the water-exposed phosphate-binding region re-
quires careful choice of the linker. Whereas flexible linkers
are not appropriate to orient polar phosphate surrogates
into the loop, they seem to be better tolerated for apolar
phosphate mimics. We are currently focused on further en-
hancing the degree of preorganization of the ligands, which
should allow additional binding affinity in the glycine-rich
loop to be gained. The study has provided valuable new
insight into the molecular recognition properties at the
active site of IspE. The fact that the best biological activities
Sonogashira Cross-Coupling Reaction. General Procedure B: A solu-
tion of 5-iodocytosine (Ϯ)-16 (1.0 equiv.), the acetylene derivative
(1.0–3.0 equiv.), and the base (2.0–3.0 equiv.) in anhydrous DMF
was deoxygenated thoroughly by bubbling with argon for 45 min.
[PdCl2(PPh3)2] (0.01–0.10 equiv.) and CuI (0.02–0.20 equiv.) were
added and the suspension was stirred at 25 °C for 1–48 h. The re-
sulting suspension was filtered through a plug of SiO2 (washing
with CH2Cl2/MeOH, 92:8) and concentrated. The residue was puri-
fied by CC. If Et3N was used in the eluting solvent, the fractions
containing the desired product were concentrated, dissolved in
CH2Cl2, washed with water (2ϫ) and brine (2ϫ), dried with
MgSO4, filtered, and the solvents evaporated.
N-Alkylation of Benzimidazoles. General Procedure C: A solution
of the benzimidazole (1.0 equiv.) in DMF, THF, or CH2Cl2
(7.7 mL/mmol) was treated with NaH (60% dispersion in oil,
1.1 equiv.) and stirred at 25 °C for 1 h. The alkyl halide (1.0 equiv.)
only reach into the lower micromolar IC50-range, despite was added at 0 °C. The suspension was stirred at 0 °C for 0.5 h and
at 25 °C for 6–20 h and the solvents evaporated. The residue was
treated with water and extracted twice with CH2Cl2. The combined
organic phases were washed with water (3ϫ) and brine, dried with
MgSO4, filtered, and concentrated. The residue was purified by
CC.
convincing molecular modeling predictions, underlines once
more the challenges of efficiently addressing highly polar
binding sites with synthetic ligands.
2-Cyclohexyl-1H-imidazole (14):
A suspension of NH4HCO3
Experimental Section
(2.82 g, 35.6 mmol) and 40% aq. glyoxal (2.0 mL, 17.8 mmol) in
water (3.5 mL) was treated dropwise with cyclohexanecarbaldehyde
(13; 2.2 mL, 17.8 mmol). The mixture was stirred at 25 °C for 18 h,
diluted with water (80 mL), and extracted with CH2Cl2/iPrOH (3:1,
3ϫ 100 mL). The combined organic phases were washed with brine
(150 mL), dried with MgSO4, filtered, and concentrated. CC (SiO2;
CH2Cl2/MeOH, 95:5Ǟ90:10) gave 14 (0.66 g, 25%) as a yellow-
In vitro Assays, Materials: [1,3,4-13C3]4-Diphosphocytidyl-2C-
methyl-d-erythritol (CDP-ME, 5) was prepared as described
earlier.[13] E. coli IspE was prepared according to a published pro-
cedure.[6a] NADH and phosphoenolpyruvate potassium salt (PEP)
were purchased from Biomol; ATP, the pyruvate kinase, and the
lactate dehydrogenase from Sigma–Aldrich.
1
beige solid, m.p. 136 °C (decomp.). H NMR (300 MHz, CDCl3):
Enzyme-Coupled Photometric Assay for IC50 Determination: Assays
were conducted in 96-well plates (Nunc, Cat. No 781602) with
transparent flat bottoms. Assay mixtures were prepared as de-
scribed previously[13] with some minor modifications: A solution
containing 100 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 2 mm dithio-
threitol, 2.5 mm potassium phosphoenolpyruvate, 2 mm ATP,
0.46 mm NADH, 1 U of lactate dehydrogenase, 1 U of pyruvate ki-
nase, and 1 U of IspE protein (total volume 60 μL) was added to
the inhibitor solution (60 μL). Dilution series (1:2) of inhibitors
covered the concentration range of 200 to 0.8 μM. The reaction
was started by addition of CDP-ME (60 μL; final concentration
250 μM) and monitored at a wavelength of 340 nm at 27 °C. IC50
values were evaluated with a nonlinear regression method using the
program Dynafit.[19]
δ = 1.22–1.44 (m, 3 H, 4Ј-Hax and 3Ј,5Ј-Hax), 1.46–1.58 (m, 2 H,
2Ј,6Ј-Hax), 1.69–1.75 (m, 1 H, 4Ј-Heq), 1.80–1.86 (m, 2 H, 3Ј,5Ј-
Heq), 2.03–2.10 (m, 2 H, 2Ј,6Ј-Heq), 2.75 (tt, J = 11.5, 3.6 Hz, 1 H,
1Ј-H), 6.95 (s, 2 H, 4-H and 5-H) ppm, NH not visible. 13C NMR
(100 MHz; CDCl3/CD3OD, 97:3): δ = 25.78, 25.97 (2 C), 31.77
(2 C), 37.55, 120.76 (2 C), 152.62 ppm. IR (ATR): ν = 3049 (w),
˜
2928 (s), 2852 (m), 1674 (w), 1571 (m), 1480 (w), 1435 (m), 1384
(m), 1284 (w), 1230 (w), 1182 (w), 1152 (w), 1134 (w), 1098 (s),
1074 (m), 1012 (w), 976 (m), 913 (m), 882 (s), 818 (m), 767 (s), 756
(m), 739 (s), 708 (s), 689 (m) cm–1. HRMS (EI): calcd. for
C9H14N2 [M]+ 150.1151, found 150.1151 (25%); calcd. for
+
C5H7N2 [M – C4H7]+ 95.0604, found 95.0604 (100%); calcd. for
+
C4H6N2 [M – C5H8]+ 82.0525, found 82.0520 (34%).
+
2-Cyclohexyl-1-(prop-2-yn-1-yl)-1H-imidazole (15): Imidazole 14
(220 mg, 1.47 mmol) was treated with Cs2CO3 (525 mg, 1.61 mmol)
and propargyl bromide (130 μL, 1.47 mmol) in DMF (11 mL) ac-
cording to General Procedure A. The suspension was stirred at
25 °C for 9 h. CC (SiO2; pentane/EtOH, 9:1Ǟ7:1) gave 15 (77 mg,
28%) as a yellow solid. Rf = 0.31 (SiO2; pentane/EtOH, 9:1), m.p.
48–52 °C. 1H NMR (300 MHz, CDCl3): δ = 1.31–1.43 (m, 3 H, 4Ј-
Hax and 3Ј,5Ј-Hax), 1.59–1.74 (m, 3 H, 2Ј,6Ј-Hax and 4Ј-Heq), 1.82–
1.93 (m, 4 H, 3Ј,5Ј-Heq and 2Ј,6Ј-Heq), 2.42 (t, J = 2.6 Hz, 1 H,
HCϵC), 2.64 (tt, J = 11.6, 3.4 Hz, 1 H, 1Ј-H), 4.64 (d, J = 2.6 Hz,
2 H, NCH2), 6.93 and 6.96 (2d, J = 1.4 Hz, 2 H, 4-H and 5-
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 25.99, 26.53 (2 C), 31.94
(2 C), 35.14, 35.94, 73.86, 77.30, 118.41, 127.19, 151.63 ppm. IR
Chemistry. General: For a detailed description of the chemicals, an-
alytical equipment, and experimental methods used, see the Sup-
porting Information. In the following, the general synthetic pro-
cedures and experimental details for the syntheses of all com-
pounds shown in Scheme 1 and Scheme 3 are described. All other
synthetic and analytical protocols are included in the Supporting
Information. For the atom numbering used for the assignment of
1
the H NMR spectra, see the spectra appended in the Supporting
Information.
N-Alkylation of Imidazoles. General Procedure A: A solution of the
imidazole (1.0 equiv.) in DMF (7.7 mL/mmol) was treated with
Cs2CO3 (1.1 equiv.) and the alkyl halide (1.0 equiv.) at 0 °C. The
suspension was stirred at 0 °C for 0.5 h and at 25 °C for 6–20 h and
concentrated. The residue was treated with water and extracted
(ATR): ν = 3231 (w), 3200 (w), 3104 (w), 2928 (m), 2853 (m), 2116
˜
(w), 1674 (w), 1523 (w), 1484 (m), 1452 (m), 1376 (w), 1337 (w),
Eur. J. Org. Chem. 2013, 1068–1079
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1075

M. Fischer, A. Bacher, F. Diederich et al.
FULL PAPER
1274 (s), 1245 (w), 1159 (m), 1144 (w), 1107 (w), 1097 (m), 1066
was added dropwise over 40 min to a solution of 24 (9.01 g,
(w), 1008 (w), 942 (w), 921 (m), 891 (w), 859 (w), 839 (w), 821 (w), 35.0 mmol) in THF (160 mL) at –78 °C. The resulting solution was
779 (m), 748 (m), 720 (s) cm–1. HRMS (EI): calcd. for C12H16N2
[M]+ 188.1308, found 188.1298 (20%); calcd. for C12H15N2
[M – H]+ 187.1230, found 187.1228 (21%); calcd. for C10H11N2
[M – C2H5]+ 159.0917, found 159.0920 (28%); calcd. for C8H9N2
[M – C4H7]+ 133.0760, found 133.0763 (100%); calcd. for C7H8N2
[M – C5H8]+ 120.0682, found 120.0681 (40%).
stirred at –78 °C for 45 min, treated dropwise with DMF (13.0 mL,
168.0 mmol), and stirred at –78 °C for 1 h. The solution was slowly
warmed to 25 °C over 1 h, stirred at 25 °C for 1 h, cooled to –78 °C,
and treated with sat. aq. NaHCO3 (30 mL). The residue was diluted
with water (20 mL) at 25 °C and extracted with CH2Cl2 (2 ϫ
50 mL). The combined organic phases were washed with water
(50 mL) and brine (50 mL), dried with MgSO4, filtered, and con-
centrated. CC (SiO2; pentane/EtOAc, 3:1 Ǟ1:2) gave 25 (8.31 g,
83%) as a yellow solid. Rf = 0.53 (SiO2; pentane/EtOAc, 1:1), m.p.
94–96 °C. 1H NMR (300 MHz, CDCl3): δ = 1.23–1.43 (m, 3 H, 4Ј-
Hax and 3Ј,5Ј-Hax), 1.63–1.77 (m, 3 H, 2Ј,6Ј-Hax and 4Ј-Heq), 1.85–
1.97 (m, 4 H, 3Ј,5Ј-Heq and 2Ј,6Ј-Heq), 2.92 (s, 6 H, Me), 3.27 (tt,
J = 11.6, 3.3 Hz, 1 H, 1Ј-H), 7.78 (s, 1 H, 4-H), 10.05 (s, 1 H,
CHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 25.63, 26.19 (2 C),
32.54 (2 C), 37.96, 38.14 (2 C), 132.92, 138.04, 160.63, 180.47 ppm.
+
+
+
+
+
(؎)-5-[3-(2-Cyclohexyl-1H-imidazol-1-yl)prop-1-yn-1-yl]-1-(tetra-
hydrothien-2-yl)cytosine [(؎)-10]: Alkyne 15 (54 mg, 0.29 mmol)
was treated with 5-iodocytosine (Ϯ)-16 (93 mg, 0.29 mmol), Et3N
(120 μL, 0.86 mmol), [PdCl2(PPh3)2] (20 mg, 0.03 mmol), and CuI
(11 mg, 0.06 mmol) in DMF (7 mL) according to General Pro-
cedure B. The suspension was stirred at 25 °C for 3.5 h. CC (SiO2;
CH2Cl2/MeOH, 95:5Ǟ92:8) gave (Ϯ)-10 (28 mg, 25%) as an off-
white solid. Rf = 0.31 (SiO2; CH2Cl2/MeOH, 93:7), m.p. 99 °C (de-
1
comp.). H NMR (400 MHz, CDCl3, assignment based on DFQ-
IR (ATR): ν = 3102 (w), 2933 (m), 2854 (m), 1671 (m), 1538 (m),
˜
COSY experiment): δ = 1.24–1.42 (m, 3 H, 4ЈЈЈ-Hax and 3ЈЈЈ,5ЈЈЈ-
Hax), 1.66–1.94 (m, 8 H, 4ЈЈЈ-Heq, 3ЈЈЈ,5ЈЈЈ-Heq, 2ЈЈЈ,6ЈЈЈ-H2, and 4Ј-
Ha), 2.04–2.17 (m, 2 H, 3Ј-Ha and 4Ј-Hb), 2.29–2.37 (m, 1 H, 3Ј-
Hb), 2.69 (tt, J = 11.7, 3.3 Hz, 1 H, 1ЈЈЈ-H), 2.92–2.98 (m, 1 H, 5Ј-
Ha), 3.16–3.21 (m, 1 H, 5Ј-Hb), 4.89 (s, 2 H, NCH2), 5.59–5.68 (br.
s, 1 H, NH), 6.30 (dd, J = 6.1, 3.5 Hz, 1 H, 2Ј-H), 6.96 (d, J =
1.3 Hz, 1 H, 5ЈЈ-H), 6.99 (d, J = 1.3 Hz, 1 H, 4ЈЈ-H), 7.67–7.76 (br.
s, 1 H, NH), 8.19 (s, 1 H, 6-H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 25.77, 26.36 (2 C), 28.19, 31.89 (2 C), 33.42, 35.82, 35.91, 38.88,
66.11, 76.88, 88.99, 89.23, 118.57, 127.64, 146.27, 151.96, 154.56,
1458 (s), 1414 (s), 1389 (s), 1249 (m), 1164 (s), 1094 (s), 1047 (s),
962 (s), 888 (m), 781 (m), 763 (m), 720 (s), 630 (w) cm–1. HRMS
(ESI): 318.1483 (100%); calcd. for C12H19N3NaO3S+ [M + Na]+
308.1039, found 308.1038 (13 %); calcd. for C12H20N3O3S+
[M + H]+ 286.1220, found 286.1219 (49 %); 206.1646 (30 %).
C12H19N3O3S (285.4): calcd. C 50.51, H 6.71, N 14.72; found
C 50.52, H 6.67, N 14.61.
(؎)-2-Cyclohexyl-N,N-dimethyl-5-(2,2,2-trifluoro-1-hydroxyethyl)-
1H-imidazole-1-sulfonamide [(؎)-26]: A solution of aldehyde 25
(1.20 g, 4.21 mmol) in THF (20 mL) was treated dropwise with
Me3SiCF3 (0.69 mL, 4.63 mmol) and a solution of nBu4NF (1 m in
THF, 0.42 mL, 0.42 mmol) at 0 °C. The solution was warmed to
25 °C over 0.5 h, stirred at 25 °C for 1 h, treated with water
(20 mL), and extracted with CH2Cl2 (3ϫ 50 mL). The combined
organic phases were washed with water (70 mL) and brine (70 mL),
dried with MgSO4, filtered, and concentrated. CC (SiO2; pentane/
EtOAc, 2:1Ǟ1:2) gave (Ϯ)-26 (310 mg, 21%) as a white solid. The
early fractions were evaporated, dissolved in THF (15 mL), and
treated with a solution of nBu4NF (1 m in THF, 2.0 mL, 2.0 mmol).
The mixture was stirred at 25 °C for 1 h and concentrated. CC
(SiO2; pentane/EtOAc, 2:1Ǟ1:2) gave additional (Ϯ)-26 (779 mg,
52%). Total yield: 1.09 g (73%). Rf = 0.44 (SiO2; pentane/EtOAc,
1:1), m.p. 115–117 °C. 1H NMR (400 MHz, CDCl3): δ = 1.25–1.38
(m, 3 H, 4Ј-Hax and 3Ј,5Ј-Hax), 1.60–1.73 (m, 3 H, 2Ј,6Ј-Hax and
4Ј-Heq), 1.84–1.90 (m, 4 H, 3Ј,5Ј-Heq and 2Ј,6Ј-Heq), 2.92 (s, 6 H,
Me), 3.12 (tt, J = 11.7, 3.1 Hz, 1 H, 1Ј-H), 3.98–4.12 (br. s, 1 H,
OH), 5.58 [q, 3J(H,F) = 6.7 Hz, 1 H, HC–CF3], 7.13 (d, J = 0.7 Hz,
1 H, 4-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 25.61, 26.38,
26.39, 32.50, 32.58, 37.81 (2 C), 38.03, 64.75 [q, 2J(C,F) = 33.6 Hz],
124.02 [q, 1J(C,F) = 281.7 Hz], 127.11 (br.), 129.01 [q, 4J(C,F) =
3.0 Hz], 157.22 ppm. 19F NMR (376 MHz, CDCl3): δ = –75.84 [d,
164.18 ppm. IR (ATR): ν = 3104 (sh. w), 2926 (m), 2851 (w), 2231
˜
(w), 1637 (s), 1482 (s), 1447 (m), 1401 (m), 1326 (m), 1297 (m),
1270 (m), 1226 (m), 1179 (w), 1160 (w), 1100 (m), 970 (w), 923 (w),
890 (w), 778 (s), 721 (m) cm–1. HRMS (MALDI; 3-HPA): calcd.
for C20H25KN5OS+ [M + K]+ 422. 1411, found 422.1402 (2%);
calcd. for C20H25N5NaOS+ [M + Na]+ 406.1672, found 406.1670
(3%); calcd. for C20H26N5OS+ [M + H]+ 384.1853, found 384.1852
(100%); calcd. for C16H20N5O+ [M – C4H6S + H]+ 298.1662, found
298.1661 (14%).
2-Cyclohexyl-N,N-dimethyl-1H-imidazole-1-sulfonamide (24): N,N-
Dimethylsulfamoyl chloride (6.3 mL, 58.6 mmol) was added to a
solution of 14 (8.00 g, 53.3 mmol) and Et3N (8.5 mL, 61.2 mmol)
in CH2Cl2 (80 mL). The suspension was stirred at 25 °C for 14 h,
filtered (washing with CH2Cl2), and the filtrate was concentrated.
CC (SiO2; pentane/EtOAc, 2:1Ǟ1:2) gave 24 (8.48 g, 62%) as a
white solid. Rf = 0.30 (SiO2; pentane/EtOAc, 1:1), m.p. 95–97 °C.
1H NMR (300 MHz, CDCl3): δ = 1.25–1.43 (m, 3 H, 4Ј-Hax and
3Ј,5Ј-Hax), 1.59–1.74 (m, 3 H, 2Ј,6Ј-Hax and 4Ј-Heq), 1.83–1.97 (m,
4 H, 3Ј,5Ј-Heq and 2Ј,6Ј-Heq), 2.88 (s, 6 H, Me), 3.14 (tt, J = 11.7,
3.4 Hz, 1 H, 1Ј-H), 6.95 (d, J = 1.7 Hz, 1 H, 4-H), 7.17 (d, J =
1.7 Hz, 1 H, 5-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 25.84,
26.43 (2 C), 32.65 (2 C), 37.23, 38.17 (2 C), 118.95, 127.22,
3J(H,F) = 6.9 Hz] ppm. IR (ATR): ν = 3130 (w), 2932 (w), 2858
˜
153.88 ppm. IR (ATR): ν = 3102 (w), 2949 (w), 2927 (m), 2853
˜
(w), 1580 (w), 1497 (w), 1450 (w), 1385 (m), 1367 (m), 1276 (m),
1157 (s), 1128 (s), 1114 (m), 1104 (m), 1080 (s), 1018 (w), 967 (m),
859 (m), 806 (m), 766 (m), 723 (s), 705 (s), 655 (w) cm–1. HRMS
(EI): calcd. for C13H20F3N3O3S+ [M]+ 355.1172, found 355.1170
(11%); calcd. for C11H14F3N2O+ [M – Me2NSO2]+ 247.1053, found
247.1060 (100%); 180.0495 (35%); 108.0114 (51%).
(m), 1673 (w), 1533 (w), 1482 (m), 1466 (m), 1458 (m), 1446 (m),
1415 (m), 1388 (s), 1345 (w), 1321 (w), 1298 (w), 1261 (m), 1226
(w), 1186 (m), 1174 (s), 1163 (s), 1152 (s), 1133 (s), 1093 (s), 1072
(m), 1047 (s), 999 (m), 961 (s), 909 (m), 887 (m), 856 (w), 817
(w), 765 (m), 737 (m), 721 (s), 708 (s) cm–1. HRMS (EI): calcd.
for C11H19N3O2S+ [M]+ 257.1192, found 257.1199 (7%); calcd. for
(؎)-2-Cyclohexyl-N,N-dimethyl-5-(2,2,2-trifluoro-1-methoxyethyl)-
1H-imidazole-1-sulfonamide [(؎)-27]: NaH (60% dispersion in oil,
62 mg, 1.55 mmol) was added in two portions to a solution of
(Ϯ)-26 (500 mg, 1.41 mmol) in DMF (5 mL) at 0 °C. The mixture
was stirred at 0 °C for 0.5 h, at 25 °C for 0.5 h, and treated with
MeI (96 μL, 1.55 mmol) at 0 °C. The mixture was stirred at 25 °C
for 16 h, poured onto ice/water, and extracted with CH2Cl2 (3ϫ
C9H13N2 [M – Me2NSO2]+ 149.1073, found 149.1064 (100 %);
+
calcd. for C5H7N2 [M – Me2NSO2 – C3H6]+ 95.0604, found
+
95.0594 (33 %). C11H19N3O2S (257.4): calcd. C 51.34, H 7.44,
N 16.33; found C 51.64, H 7.39, N 16.05.
2-Cyclohexyl-5-formyl-N,N-dimethyl-1H-imidazole-1-sulfonamide
(25): A solution of nBuLi (1.6 m in hexane, 26.3 mL, 42.1 mmol)
1076
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2013, 1068–1079

Imidazole- and Benzimidazole-Based Inhibitors of the Kinase IspE
40 mL). The combined organic phases were washed with water (3ϫ
100 mL) and brine (100 mL), dried with MgSO4, filtered, and con-
centrated. CC (SiO2; pentane/EtOAc, 5:1 Ǟ 2:1) gave (Ϯ)-27
IR (ATR): ν = 3307 (w), 2932 (m), 2856 (w), 2123 (w), 1504 (w),
˜
1450 (w), 1385 (m), 1378 (w), 1271 (m), 1165 (s), 1120 (s), 1100 (s),
1018 (w), 971 (w), 891 (w), 869 (w), 838 (w), 806 (w), 698 (m) cm–1.
(440 mg, 85 %) as an off-white solid. Rf = 0.44 (SiO2; pentane/ HRMS (ESI): calcd. for C15H20F3N2O+ [M + H]+ 301.1522, found
EtOAc, 7:3), m.p. 76–78 °C. 1H NMR (300 MHz, CDCl3): δ =
1.26–1.43 (m, 3 H, 4Ј-Hax and 3Ј,5Ј-Hax), 1.59–1.76 (m, 3 H, 2Ј,6Ј-
Hax and 4Ј-Heq), 1.85–1.93 (m, 4 H, 3Ј,5Ј-Heq and 2Ј,6Ј-Heq), 2.92
(s, 6 H, NMe2), 3.14 (tt, J = 11.5, 3.3 Hz, 1 H, 1Ј-H), 3.51 (d, J =
301.1528 (100%).
(؎)-5-{3-[2-Cyclohexyl-4-(2,2,2-trifluoro-1-methoxyethyl)-1H-imid-
azol-1-yl]prop-1-yn-1-yl}-1-(tetrahydrothien-2-yl)cytosine [(؎)-17]:
Alkyne (Ϯ)-29 (56 mg, 0.19 mmol) was treated with 5-iodocytosine
(Ϯ)-16 (60 mg, 0.19 mmol), Et3N (78 μL, 0.56 mmol),
[PdCl2(PPh3)2] (13 mg, 0.02 mmol), and CuI (7 mg, 0.04 mmol) in
DMF (4.6 mL) according to General Procedure B. The suspension
was stirred at 25 °C for 7 h. CC (SiO2–NH2; CH2Cl2 ǞCH2Cl2/
EtOH, 95:5) gave (Ϯ)-17 (43 mg, 47%) as a light-yellow solid. Rf
= 0.34 (SiO2; CH2Cl2/MeOH, 94:6), m.p. 117–120 °C. 1H NMR
(300 MHz, CDCl3): δ = 1.25–1.44 (m, 3 H, 4ЈЈЈ-Hax and 3ЈЈЈ,5ЈЈЈ-
Hax), 1.63–1.94 (m, 8 H, 4ЈЈЈ-Heq, 3ЈЈЈ,5ЈЈЈ-Heq, 2ЈЈЈ,6ЈЈЈ-H2, and 4Ј-
Ha), 2.05–2.20 (m, 2 H, 3Ј-Ha and 4Ј-Hb), 2.29–2.41 (m, 1 H, 3Ј-
Hb), 2.68 (tt, J = 11.5, 3.3 Hz, 1 H, 1ЈЈЈ-H), 2.92–3.01 (m, 1 H, 5Ј-
Ha), 3.17–3.26 (m, 1 H, 5Ј-Hb), 3.50 (s, 3 H, OMe), 4.66 [q, 3J(H,F)
= 6.8 Hz, 1 H, HC–CF3], 4.91 (s, 2 H, NCH2), 5.74–5.85 (br. s, 1 H,
NH), 6.30 (dd, J = 6.1, 3.5 Hz, 1 H, 2Ј-H), 7.10 (s, 1 H, 5ЈЈ-H),
7.71–7.88 (br. s, 1 H, NH), 8.23 (s, 1 H, 6-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 25.62, 26.27 (2 C), 28.23, 31.74, 31.78,
33.42, 35.94, 36.20, 38.86, 58.51, 66.16, 77.05 [q, 2J(C,F) =
31.9 Hz], 77.25, 88.64, 89.31, 117.68 [br. q, 4J(C,F) = 1.2 Hz],
124.05 [q, 1J(C,F) = 282.0 Hz], 133.11 [br. q, 3J(C,F) = 1.4 Hz],
146.39, 151.96, 154.39, 164.04 ppm. 19F NMR (376 MHz, CDCl3):
3
0.6 Hz, 3 H, OMe), 5.31 [qd, J(H,F) = 6.2, J = 0.6 Hz, 1 H, HC–
CF3], 7.18 (br. s, 1 H, 4-H) ppm. 13C NMR (100 MHz, CDCl3): δ
= 25.68, 26.39 (2 C), 32.58, 32.64, 37.62 (2 C), 38.02, 58.58, 72.84
[q, 2J(C,F) = 32.1 Hz], 123.84 [q, 1J(C,F) = 283.2 Hz], 125.55 (br.),
130.34 [q, 4J(C,F) = 2.3 Hz], 157.35 ppm. 19F NMR (376 MHz,
CDCl ): δ = –74.31 [d, 3J(H,F) = 6.4 Hz] ppm. IR (ATR): ν = 3152
˜
3
(w), 2931 (w), 2859 (w), 1570 (w), 1497 (w), 1475 (w), 1461 (w),
1417 (w), 1389 (m), 1364 (m), 1347 (m), 1262 (m), 1239 (w), 1208
(w), 1159 (s), 1128 (s), 1113 (m), 1099 (m), 1084 (s), 1015 (w), 969
(m), 856 (m), 844 (m), 767 (m), 726 (s), 705 (s), 640 (w) cm–1
.
HRMS (EI): calcd. for C14H22F3N3O3S+ [M]+ 369.1328, found
369.1330 (16 %); calcd. for C12H16F3N2O+ [M – Me2NSO2]+
+
261.1209, found 261.1215 (100 %); calcd. for C11H12F3N2
[M – Me2NSO2 – MeOH]+ 229.0947, found 229.0947 (73 %);
194.0669 (45%); 108.0111 (60%). C14H22N3O3F3S (369.4) calcd.
C 45.52, H 6.00, N 11.38; found C 45.81, H 6.00, N 11.26.
(؎)-2-Cyclohexyl-4-(2,2,2-trifluoro-1-methoxyethyl)-1H-imidazole
[(؎)-28]: A suspension of (Ϯ)-27 (300 mg, 0.81 mmol) in 5% aq.
HCl (10 mL) was stirred at 25 °C for 7 h. The mixture was cooled
to 0 °C, neutralized with sat. aq. NaHCO3, and extracted with
CH2Cl2 (3ϫ 20 mL). The combined organic phases were washed
with brine (30 mL), dried with MgSO4, filtered, and concentrated.
CC (SiO2; pentane/EtOAc, 2:1Ǟ1:1) gave (Ϯ)-28 (191 mg, 90%)
δ = –76.12 [t, 3J(H,F) = 7.4 Hz] ppm. IR (ATR): ν = 3326 (w),
˜
3178 (w), 2931 (m), 2854 (w), 2235 (w), 1639 (s), 1496 (s), 1448
(m), 1402 (m), 1328 (w), 1297 (w), 1271 (m), 1228 (w), 1162 (s),
1120 (s), 1097 (s), 1017 (w), 969 (w), 884 (w), 838 (w), 806 (w), 779
(m), 697 (w) cm–1. HRMS (MALDI; 3-HPA): calcd. for
C23H28F3KN5O2S+ [M + K]+ 534.1547, found 534.1555 (8%);
calcd. for C23H28F3N5NaO2S+ [M + Na]+ 518.1808, found
518.1816 (12%), calcd. for C23H29F3N5O2S+ [M + H]+ 496.1989,
1
as a white crystalline solid, m.p. 154–155 °C. H NMR (400 MHz,
CDCl3): δ = 1.23 (qt, J = 12.2, 3.3 Hz, 1 H, 4Ј-Hax), 1.36 (qt, J =
12.2, 3.2 Hz, 2 H, 3Ј,5Ј-Hax), 1.48 (qd, J = 12.2, 2.8 Hz, 2 H, 2Ј,6Ј-
H
ax), 1.69–1.74 (m, 1 H, 4Ј-Heq), 1.79–1.84 (m, 2 H, 3Ј,5Ј-Heq),
+
2.02–2.06 (m, 2 H, 2Ј,6Ј-Heq), 2.74 (tt, J = 11.7, 3.5 Hz, 1 H, 1Ј-
found 496.1993 (100%); calcd. for C19H23F3N5O2 [M – C4H6S +
3
H]+ 410.1798, found 410.1799 (48%).
H), 3.44 (s, 3 H, OMe), 4.63 [q, J(H,F) = 6.7 Hz, 1 H, HC–CF3],
7.03 (s, 1 H, 5-H), 9.40–9.86 (br. s, 1 H, NH) ppm. 13C NMR
(100 MHz, CDCl3): δ = 25.76, 25.94 (2 C), 31.94 (2 C), 37.80,
58.15, 114.26 (br.), 153.32 (br.) ppm, the signals of C(4) and CF3
are not visible. 19F NMR (376 MHz, CDCl3): δ = –76.27 [d,
2-Cyclohexyl-1H-benzimidazol-4-ol (36): A solution of 2-amino-3-
nitrophenol (35; 1.10 g, 7.13 mmol) and cyclohexanecarbaldehyde
(13; 0.86 mL, 7.13 mmol) in EtOH (28 mL) was treated with aq.
Na2S2O4 (1 m, 21.4 mL, 21.4 mmol) at 25 °C and heated to 70 °C
for 5 h. The mixture was cooled to 25 °C, treated dropwise with
aq. NH3 (1 m, 14.3 mL), and cooled to 0 °C. The resulting precipi-
tate was filtered off, washed with H2O, and dried in a desiccator
over CaCl2 to give 36 (1.24 g, 80%) as a white solid, m.p. 225–
227 °C. 1H NMR [300 MHz, (CD3)2SO]: δ = 1.22–1.43 (m, 3 H, 4Ј-
Hax and 3Ј,5Ј-Hax), 1.55–1.72 (m, 3 H, 2Ј,6Ј-Hax and 4Ј-Heq), 1.77–
1.81 (m, 2 H, 3Ј,5Ј-Hax), 1.96–2.00 (m, 2 H, 2Ј,6Ј-Heq), 2.80 (tt, J
= 11.5, 3.6 Hz, 1 H, 1Ј-H), 6.45–6.52 (m, 1 H, 5-H), 6.84–6.94 (m,
2 H, 6-H and 7-H), 9.52 (br. s, 1 H, NH), 11.93 (br. s, 1 H,
OH) ppm. 13C NMR [100 MHz, (CD3)2SO]: δ = 25.59, 25.63 (2 C),
31.36 (2 C), 37.78, 106.13, 121.74, 157.38 ppm, four aromatic sig-
3J(H,F) = 4.8 Hz] ppm. IR (ATR): ν = 2930 (m), 2856 (w), 1577
˜
(w), 1531 (w), 1451 (w), 1371 (w), 1270 (m), 1240 (m), 1167 (s),
1120 (s), 1104 (s), 1015 (w), 971 (m), 879 (w), 806 (w), 707 (m) cm–1.
HRMS (ESI): calcd. for C12H18F3N2O+ [M + H]+ 263.1366, found
263.1376 (100%).
(؎)-2-Cyclohexyl-1-(prop-2-yn-1-yl)-4-(2,2,2-trifluoro-1-methoxy-
ethyl)-1H-imidazole [(؎)-29]: Imidazole (Ϯ)-28 (170 mg,
0.65 mmol) was treated with Cs2CO3 (232 mg, 0.71 mmol) and pro-
pargyl bromide (56 μL, 0.65 mmol) in DMF (5 mL) according to
General Procedure A. The suspension was stirred at 25 °C for 8 h.
CC (SiO2; pentane/EtOAc, 4:1Ǟ1:1) gave (Ϯ)-29 (94 mg, 48%) as
a colorless oil. Rf = 0.34 (SiO2; pentane/EtOAc, 4:1). 1H NMR
(300 MHz, CDCl3): δ = 1.31–1.44 (m, 3 H, 4Ј-Hax and 3Ј,5Ј-Hax),
1.60–1.75 (m, 3 H, 2Ј,6Ј-Hax and 4Ј-Heq), 1.84–1.93 (m, 4 H, 3Ј,5Ј-
Heq and 2Ј,6Ј-Heq), 2.46 (t, J = 2.6 Hz, 1 H, HCϵC), 2.64 (tt, J =
11.7, 3.4 Hz, 1 H, 1Ј-H), 3.47 (d, J = 0.5 Hz, 3 H, OMe), 4.65 [br.
q, 3J(H,F) = 6.8 Hz, 1 H, HC–CF3], 4.65 (d, J = 2.6 Hz, 2 H,
NCH2), 7.08 (s, 1 H, 5-H) ppm. 13C NMR (75 MHz, CDCl3): δ =
25.81, 26.44 (2 C), 31.74, 31.76, 35.55 and 35.98 (1 C), 58.34, 74.50,
74.56, 76.17, 76.65 [q, 2J(C,F) ≈ 34.6 Hz], 117.66, 123.79 [q, 1J(C,F)
nals are not visible. IR (ATR): ν = 3211 (w), 3062 (w), 3030 (w),
˜
2925 (m), 2851 (w), 2373 (w), 1896 (w), 1731 (w), 1625 (w), 1597
(m), 1519 (w), 1430 (m), 1381 (m), 1300 (m), 1239 (m), 1194 (m),
1154 (m), 1128 (m), 1064 (s), 1044 (s), 887 (m), 850 (w), 788 (s),
732 (s), 713 (m), 665 (w), 606 (w) cm–1. HRMS (ESI): calcd. for
C13H17N2O+ [M + H]+ 217.1335, found 217.1330 (100%).
2-Cyclohexyl-4-isopropoxy-1H-benzimidazole (37): A solution of
alcohol 36 (700 mg, 3.24 mmol), PPh3 (1.02 g, 3.89 mmol), and
= 280.7 Hz], 132.48 [br. q, 4J(C,F) ≈ 1.7 Hz], 151.66 ppm. 19F iPrOH (0.30 mL, 3.89 mmol) in THF (30.8 mL) was treated with
3
NMR (376 MHz, CDCl3): δ = –76.25 [d, J(H,F) = 6.5 Hz] ppm. DIAD (94% in toluene, 0.82 mL, 3.89 mmol) at 25 °C. The mixture
Eur. J. Org. Chem. 2013, 1068–1079
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1077

M. Fischer, A. Bacher, F. Diederich et al.
FULL PAPER
was stirred at 25 °C for 2 h and concentrated. CC (SiO2; pentane/
EtOAc, 2:1) and recrystallization in EtOAc gave 37 (410 mg, 49%)
as a white solid. Rf = 0.57 (SiO2; pentane/EtOAc, 1:1), m.p. 216 °C.
1H NMR (300 MHz, CDCl3): δ = 1.18–1.48 (m, 3 H, 4Ј-Hax and
OCH), 5.08 (s, 2 H, NCH2), 5.50 (br. s, 1 H, NH), 6.27–6.30 (m,
1 H, 2Ј-H), 6.72 (d, J = 7.9 Hz, 1 H, 5ЈЈ-H), 6.92 (br. s, 1 H, NH),
6.99 (d, J = 8.0 Hz, 1 H, 7ЈЈ-H), 7.18 (t, J = 8.0 Hz, 1 H, 6ЈЈ-H),
8.13 (s, 1 H, 6-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 22.20
3Ј,5Ј-Hax), 1.40 (d, J = 6.0 Hz, 6 H, Me), 1.60–1.89 (m, 5 H, 2Ј,6Ј- (2 C), 25.63, 26.33 (2 C), 28.17, 31.47 (2 C), 33.40, 33.77, 36.74,
Hax, 4Ј-Heq, and 3Ј,5Ј-Heq), 2.13–2.19 (m, 2 H, 2Ј,6Ј-Heq), 2.91 (br.
s, 1 H, 1Ј-H), 4.72 (br. s, 1 H, OCH), 6.66 (d, J = 8.1 Hz, 1 H, 5-
38.88, 66.11, 70.88, 76.32, 88.93, 89.15, 101.45, 106.20, 123.24,
133.16, 136.38, 146.18, 149.82, 154.53, 156.58, 163.95 ppm. IR
H), 7.10 (t, J = 8.0 Hz, 1 H, 6-H), 7.29 (br. s, 1 H, 7-H), 9.07 (br. (ATR): ν = 3602 (w), 3427 (w), 3366 (w), 3059 (w), 2931 (m), 2852
˜
s, 1 H, NH) ppm. 13C NMR (100 MHz, CDCl3): δ = 22.16 (2 C),
(w), 2224 (w), 1670 (s), 1637 (s), 1611 (m), 1598 (m), 1495 (s), 1449
(m), 1403 (s), 1385 (m), 1372 (w), 1343 (m), 1330 (m), 1296 (m),
1271 (s), 1247 (m), 1222 (s), 1197 (m), 1178 (m), 1160 (w), 1138
(w), 1116 (s), 1066 (m), 1010 (w), 976 (m), 921 (w), 892 (w), 838
(w), 817 (w), 777 (s), 740 (s), 719 (w), 676 (w), 661 (w) cm–1. HRMS
(MALDI): calcd. for C27H34N5O2S+ [M + H]+ 492.2428, found
25.83, 26.04 (2 C), 31.93 (2 C), 38.57, 70.30, 104.86, 122.21,
157.85 ppm, four aromatic signals are not visible. IR (ATR): ν =
˜
3675 (w), 2975 (m), 2926 (m), 2852 (m), 1618 (w), 1601 (m), 1539
(m), 1495 (w), 1444 (m), 1416 (m), 1380 (m), 1370 (m), 1357 (m),
1322 (m), 1292 (w), 1265 (m), 1242 (s), 1167 (w), 1140 (w), 1116
(s), 1088 (m), 1061 (m), 1041 (w), 985 (m), 903 (m), 891 (w), 856
492.2450 (100%); calcd. for C23H28N5O2 [M – C4H6S + H]+
+
(m), 782 (m), 767 (w), 735 (s), 669 (w), 632 (w), 609 (w) cm–1. 406.2238, found 406.2231 (66%).
HRMS (EI): calcd. for C16H22N2O+ [M]+ 258.1727, found
Supporting Information (see footnote on the first page of this arti-
258.1729 (39%); calcd. for C13H16N2O+ [M – C3H6]+ 216.1257,
found 216.1251 (25%); calcd. for C9H9N2O+ [M – C3H6 – C4H7]+
161.0709, found 161.0706 (100%).
cle): Figures (modeling, regioselective alkylation, CSD searches)
and schemes (biological assay, syntheses not shown in the main
manuscript) referred to in this article, materials and general meth-
ods, synthetic and analytical protocols, X-ray diffraction data,
NMR spectra of new compounds.
2-Cyclohexyl-4-isopropoxy-1-(prop-2-yn-1-yl)-1H-benzimidazole
(39): Benzimidazole 37 (320 mg, 1.24 mmol) was treated with NaH
(60% dispersion in oil, 50 mg, 1.24 mmol) and propargyl bromide
(106 μL, 1.24 mmol) in CH2Cl2 (24 mL) according to General Pro-
cedure C. The suspension was stirred at 25 °C for 24 h. CC (SiO2;
pentane/EtOAc, 95:5Ǟ90:10) gave 39 (160 mg, 44%) as a white
crystalline solid. Rf = 0.85 (SiO2; pentane/EtOAc, 4:1), m.p. 99–
101 °C. 1H NMR (400 MHz, CDCl3): δ = 1.32–1.41 (m, 3 H, 4Ј-
Hax and 3Ј,5Ј-Hax), 1.46 (d, J = 6.1 Hz, 6 H, Me), 1.74–2.05 (m,
7 H, 2Ј,6Ј-H2, 4Ј-Heq, and 3Ј,5Ј-Heq), 2.33 (t, J = 2.5 Hz, 1 H,
HCϵC), 2.87 (tt, J = 11.6, 3.7 Hz, 1 H, 1Ј-H), 4.84 (d, J = 2.5 Hz,
2 H, NCH2), 4.96 (sept., J = 6.1 Hz, 1 H, OCH), 6.70 (br. d, J =
7.9 Hz, 1 H, 5-H), 6.98 (dd, J = 8.1, 0.8 Hz, 1 H, 7-H), 7.15 (t, J
= 8.0 Hz, 1 H, 6-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 22.20
(2 C), 25.67, 26.31 (2 C), 31.29 (2 C), 32.85, 36.63, 70.82, 73.12,
77.12, 101.69, 106.23, 122.87, 133.23, 136.53, 149.70, 156.55 ppm.
Acknowledgments
This research was supported by the Swiss National Science Foun-
dation and the Hans-Fischer Gesellschaft, Germany. The authors
thank Dr. Daniel Zimmerli (Hoffmann-La Roche AG, Basel) for
providing the amino-modified silica and for advice on purification
of compounds.
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˜
2119 (w), 1863 (w), 1608 (m), 1595 (m), 1566 (w), 1508 (m), 1497
(m), 1467 (w), 1448 (m), 1435 (m), 1407 (m), 1381 (w), 1365 (m),
1339 (m), 1300 (w), 1293 (w), 1270 (m), 1250 (s), 1222 (m), 1197
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found 296.1889 (48 %); calcd. for C18H21N2O+ [M – CH3]+
281.1648, found 281.1650 (75 %); calcd. for C16H18N2O+
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C 76.98, H 8.15, N 9.29.
(؎)-5-{3-[2-Cyclohexyl-4-(propan-2-yloxy)-1H-benzimidazol-1-yl]-
prop-1-yn-1-yl}-1-(tetrahydrothien-2-yl)cytosine [(؎)-34]: Alkyne 39
(64 mg, 0.22 mmol) was treated with 5-iodocytosine (Ϯ)-16 (35 mg,
11 mmol), iPr2NEt (116 μL, 0.65 mmol), [PdCl2(PPh3)2] (8 mg,
0.01 mmol), and CuI (4 mg, 0.02 mmol) in DMF (2.5 mL) accord-
ing to General Procedure B. The suspension was stirred at 25 °C for
24 h. CC (SiO2; CH2Cl2/MeOH, 96:4) gave (Ϯ)-34 (19 mg, 36%) as
a yellow-brown solid. Rf = 0.17 (SiO2; CH2Cl2/MeOH, 96:4), m.p.
165–170 °C (decomp.). 1H NMR (400 MHz, CDCl3): δ = 1.35–1.41
(m, 3 H, 4ЈЈЈ-Hax and 3ЈЈЈ,5ЈЈЈ-Hax), 1.46 (d, J = 6.1 Hz, 6 H, Me),
1.76–2.13 (m, 10 H, 2ЈЈЈ,6ЈЈЈ-H2, 4ЈЈЈ-Heq, 3ЈЈЈ,5ЈЈЈ-Heq, 3Ј-Ha, and
4Ј-H2), 2.27–2.34 (m, 1 H, 3Ј-Hb), 2.87–2.96 (m, 2 H, 1ЈЈЈ-H and
5Ј-Ha), 3.13–3.19 (m, 1 H, 5Ј-Hb), 4.95 (sept., J = 6.1 Hz, 1 H,
1078
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: November 2, 2012
Published Online: January 4, 2013
Eur. J. Org. Chem. 2013, 1068–1079
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