Structure-activity relationships of benzimidazole-based glutaminyl cyclase inhibitors featuring a heteroaryl scaffold

Article abstract of DOI:10.1021/jm4001709

Glutaminyl cyclase (hQC) has emerged as a new potential target for the treatment of Alzheimer's disease (AD). The inhibition of hQC prevents of the formation of the Aβ_3(pE)-40,42 species which were shown to be of elevated neurotoxicity and are likely to act as a seeding core, leading to an accelerated formation of Aβ-oligomers and fibrils. This work presents a new class of inhibitors of hQC, resulting from a pharmacophore-based screen. Hit molecules were identified, containing benzimidazole as the metal binding group connected to 1,3,4-oxadiazole as the central scaffold. The subsequent optimization resulted in benzimidazolyl-1,3,4-thiadiazoles and -1,2,3-triazoles with an inhibitory potency in the nanomolar range. Further investigation into the potential binding mode of the new compound classes combined molecular docking and site directed mutagenesis studies.

Full text of DOI:10.1021/jm4001709

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Article
Structure-Activity-Relationships of Benzimidazole-based
Glutaminyl Cyclase Inhibitors Featuring a Heteroaryl-scaffold
Daniel Ramsbeck, Mirko Buchholz, Birgit Koch, Livia Böhme,
Torsten Hoffmann, Hans-Ulrich Demuth, and Ulrich Heiser
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4001709 • Publication Date (Web): 25 Jul 2013
Downloaded from http://pubs.acs.org on August 1, 2013
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StructureꢀActivityꢀRelationships of Benzimidazoleꢀ
based Glutaminyl Cyclase Inhibitors Featuring a
Heteroarylꢀscaffold
Daniel Ramsbeck†§, Mirko Buchholz†, Birgit Koch‡, Livia Böhme∞, Torsten Hoffmann∞, HansꢀUlrich
Demuth†,‡,∞ and Ulrich Heiser*,†.
†Department of Medicinal Chemistry,
‡Department of Enzymology
∞Department of Preclinical Pharmacology,
Probiodrug AG, Weinbergweg 22, 06120 Halle, Germany.
§present address: Fraunhofer Institute for Cell Therapy and Immunology, Perlickstraße 1, 04103
Leipzig, Germany
RECEIVED DATE
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ABSTRACT. Glutaminyl cyclase (hQC) has emerged as a new potential target for the treatment of
Alzheimer’s Disease (AD). The inhibition of hQC prevents of the formation of the Aβ3(pE)ꢀ40,42ꢀspecies
which were shown to be of elevated neurotoxicity and are likely to act as a seeding core leading to an
accelerated formation of Aβꢀoligomers and fibrils. This work presents a new class of inhibitors of hQC,
resulting from a pharmacophoreꢀbased screen. Hit molecules were identified, containing benzimidazole
as the metal binding group connected to 1,3,4ꢀoxadiazole as the central scaffold. The subsequent
optimization resulted in benzimidazolylꢀ1,3,4ꢀthiadiazoles and ꢀ1,2,3ꢀtriazoles with an inhibitory
potency in the nanoꢀmolar range. Further investigation into the potential binding mode of the new
compound classes combined molecular docking and site directed mutagenesis studies.
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KEYWORDS. Human glutaminyl cyclase, metalꢀbindingꢀgroup, benzimidazole, ligandꢀbased
screening, pharmacophore model, siteꢀdirected mutagenesis, Alzheimer’s Disease, pGluꢀAβ peptide,
docking, 1,2,3ꢀtriazoles, 1,2,4ꢀoxadiazoles, 1,3,4ꢀoxadiazoles, 1,3,4ꢀthiadiazoles, positional isomers.
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Introduction. Postꢀtranslational protein modifications are playing a key role in the maturation of the
majority of bioactive peptides. Among those, the formation of Nꢀterminal pyroglutamate (pGlu)
residues is commonly found in nature.1ꢀ3 This modification on one hand increases the proteolytic
stability or, on the other hand, contributes to the formation and stabilization of the bioactive
conformation of the respective peptides and proteins. The formation of the Nꢀterminal pGlu residue is
catalyzed by human glutaminyl cyclase (hQC)4 and its isoꢀenzyme (hisoQC).5 QCs are highly abundant
in secretory tissue such as secretory glands or brain tissue.6,7 Both isoꢀenzymes are zincꢀdependent and
exhibit a high structural identity and similar substrate specificity. hQC is highly abundant in secretory
vesicles and is coꢀsecreted with its substrates, whereas hisoQC is a Golgi resident enzyme.5
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Targeting the Nꢀterminal modification of Glu or Gln into the respective pGlu has come into the focus of
drug development for the treatment of Alzheimer’s Disease (AD). pGluꢀmodified Aβꢀpeptides are
discussed to be a crucial species involved in the early onset of AD.8,9 These pGluAβ peptides exhibit an
increased neurotoxicity and accelerated aggregation kinetics as compared to native Aβ−peptides, e.g.
Aβ(1ꢀ40) or Aβ(1ꢀ42)
.
10,11 Furthermore, it was shown that Aβ(pE3ꢀ42) coꢀoligomerizes with an excess of Aβ(1ꢀ
to form metastable lowꢀn oligomers that then exhibit a different morphology and an elevated
42)
neurotoxicity as compared to oligomers formed by Aβ(1ꢀ42) alone. This neurotoxic effect requires the
presence of the Tauꢀprotein, showing a link between the Aβꢀ and Tauꢀpathology.12 In vivo evidence
regarding the involvement of hQC in the formation of pGluAβ and its effects on learning and memory
was gained by hQCꢀtransgenic and ꢀknockꢀout mice models of AD.13,14 Furthermore, the application of
QC inhibitors successfully reduced the amount of pGluAβ in the brain of other transgenic mouse
models of AD resulting in positive effects on learning and memory.15,16
Recently, we presented the first generation of QC inhibitors, based on imidazoꢀpropylthiourea, which
were later subsequently modified by bioꢀisosteric replacements and structure based approaches.17,18
Based on these results, a pharmacophoreꢀbased filter was designed, applying a flexible alignment of
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known QCꢀinhibitors and a QCꢀsubstrate. The subsequent screen of a virtual library led to the medium
potent benzimidazolylꢀ1,3,4ꢀoxadiazole hitꢀcompounds as a suitable chemoꢀtype for further
optimization. The optimization and SAR as well as a discussion of the potential binding mode in the
active site are presented here.
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Results and Discussion
3D Similarity Filtering. A flexible alignment of potent inhibitors identified earlier17 and the substrate
HꢀGlnꢀPheꢀAlaꢀNH2 resulted in a match of pharmacophoric features (Fig 1).17 The resulting
pharmacophore was then applied in a 3Dꢀsimilarity filtering of a library containing 653218 preꢀ
processed leadꢀlike compounds19ꢀ21 setting a smartꢀkey constraint in the metalꢀbinding part, considering
only imidazoleꢀrelated substructures. This was done in order to limit the number of hits to compounds
containing imidazole or imidazoleꢀrelated heterocycles. The rationale for this approach was the earlier
identification of imidazole out of a screen of different heterocycles as potent metalꢀbinder for hQC.17
The search resulted in a number of 4878 hits corresponding to a hit rate of 0.75%. Among these hits,
besides the expected imidazoleꢀderivatives also benzimidazoleꢀbased compounds were identified in a
remarkable high number (1760 molecules corresponding to 36.1% of all hits). This finding prompted us
to examine the inhibitory potency of benzimidazole as a new type of metalꢀbindingꢀgroup (MBG) in
QCꢀinhibitors, resulting in a Kiꢀvalue of 150 ꢀΜ (Fig.2), which is indeed comparable to the previously
identified imidazole (Ki = 100 ꢀΜ).17 Consequently, with the goal to broaden the chemical space of
possible QCꢀinhibitors by compounds featuring this new type of MBG, only benzimidazoleꢀcontaining
compounds were further considered. The next filtering step was set by the limitation of the allowed
value for the topological polar surface area (TPSA) to be below 70 Å2. This was done with respect to the
potential ability of the compounds to pass the bloodꢀbrainꢀbarrier (BBB). The application of the TPSAꢀ
filter and the commercially availability of the screening compounds reduced the number of the tested
compounds to be 69. Among those, 9 compounds were found to be active (Ki, hQC < 5 ꢀM). The analysis
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of the chemical accessibility and possible ways for derivatization led to three oxadiazoleꢀsubstituted
benzimidazoles (Fig. 2), now serving as starting points for further potency optimization.
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Chemistry. The synthesis of the different 1,3,4ꢀoxadiazoleꢀsubstituted derivatives (5, 6 and 7) was
accomplished starting from benzimidazoleꢀ5(6)ꢀcarboxylic acid hydrazide (3) according to Scheme 1.
The alkylthioꢀ1,3,4ꢀoxadiazole derivatives (5a-h) were yielded from 3 after reaction with
carbondisulfide under basic conditions followed by alkylation of the resulting 3Hꢀ1,3,4ꢀoxadiazoleꢀ2ꢀ
thione (4) with different alkylhalides.22 The respective aminoalkylꢀsubstituted 1,3,4ꢀoxadiazole
derivative (6) resulted from 3 by acylation with benzylisothiocyanate and subsequent cyclization of the
crude thiosemicarbazide by means of dicyclohexylcarbodiimide.23 In case of the alkyl substituted 1,3,4ꢀ
oxadiazole derivates (7a and 7e-g), the benzimidazole carbohydrazide 3 was acylated with different acid
chlorides followed by subsequent POCl3 mediated cyclization of the bisacylhydrazide intermediates.
The derivatives with inverse etherꢀ, thioꢀetherꢀ or sulfoneꢀspacer (7b-d), as well as the alkyl substituted
1,3,4ꢀoxadiazoles 7h-k were accessible by direct treatment of 3 with different carboxylic acids in neat
POCl3.24
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The 1,3,4ꢀthiadiazoles (8a-d) were synthesized according to Scheme 1 by acylation of 3 with different
acid chlorides followed by cyclization of the intermediates by means of Lawesson´s reagent.25 The
compounds 8e and 8f-i were synthesized in a oneꢀpot manner. For that purpose, 3 was either condensed
with carboxylic acid by means of dicyclohexylcarbodiimide and then treated with Lawesson´s reagent
as done for 8e, or reacted with the corresponding carboxylic acids and a mixture of Lawesson´s reagent
and POCl3 leading to compounds 8f-i.
The thiazole 11 was prepared starting from 2 (Scheme 2). After conversion into the carboxylic acid
chloride, the oxazole 9 was yielded by the reaction with isocyanoethylacetate and DBU. The
hydrolyzation and decarboxylation led to the aminomethylketone 10.26 The thiazole 11 then resulted
from the subsequent treatment of 10 with phenylpropionic acid, Lawesson´s reagent, POCl3 and TEA.
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The isomeric thiazole 13 was accessible through the reaction of benzimidazoleꢀ5(6)ꢀcarboxylic acid (2)
with the aminomethylketone 12, that was readily prepared from the respective carboxylic acid using
carbonyldiimidazole, isocyanoethylacetate, DBU and aqueous HCl.
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The 1,2,4ꢀoxadiazole (17) and 1,2,4ꢀtriazole (19) were synthesized from 5(6)ꢀCyanobenzimidazole (15).
15 was treated either with hydroxylamine hydrochloride yielding Nꢀhydroxybenzimidazoleꢀ5(6)ꢀ
carboxamidine (16) or hydrazine hydrate, yielding the amidrazone 18 (Scheme 3). The latter was
synthesized by a Pinnerꢀreaction involving the imidatester as intermediate which was then converted
into 18 by aminolysis with hydrazine hydrate. Both building blocks were acylated and cyclized in the
same manner,27 applying phenylpropionic acid and carbonyldiimidazole leading to the respective 1,2,4ꢀ
oxadiazoleꢀ (17) or 1,2,4ꢀtriazoleꢀsubstituted benzimidazole derivative (19).
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The preparation of the 1,2,3ꢀtriazoleꢀsubstituted benzimidazoles (22a-d, Scheme 4) started from 4ꢀ
ethynylꢀ2ꢀnitroaniline (20),28,29 which was subjected to a click reaction that enabled a oneꢀpot synthesis
of the 1,2,3ꢀtriazoleꢀsubstituted nitroꢀanilines (21a-d) applying different phenethylꢀbromides.30 21a-d
were then converted into the corresponding benzimidazoles (22a-d) by reduction and cyclization using
sodium formiate, palladium, formic acid and triethylꢀorthoformiate.
Structure-Activity Relationship. Using 1a (Fig. 2) as a starting point, the structureꢀactivity
relationship was investigated by the systematic modification of key scaffold elements: the central 5ꢀ
membered heteroaromatic ring (Fig. 2: green), the spacer between the fiveꢀmembered heteroaromatic
ring and the terminal hydrophobic substituent (Fig. 2 yellow) as well as the substitution pattern at the
hydrophobic substituent itself (Fig. 2: blue).
Initially, the pyridineꢀmethyleneꢀmoiety was replaced by hydrophobic residues of different size and
character (5a-h, Table1). This led to a minor improvement of the inhibitory potency as compared to 1a
only in case of the benzylꢀ and naphtylꢀmethylꢀderivatives 5e and 5f. In contrast, the exchange of the
pyridineꢀmethylene substituent for plain aliphatic residues (5aꢀ5d) resulted in a 5ꢀ to 20ꢀfold decreased
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activity of the corresponding derivatives as compared to the starting point 1a. Furthermore, keeping a
terminal phenylꢀmoiety, the extension of the spacer length as in 5g-h led to a decrease in the inhibitory
potency in general. These findings led to the conclusion that a terminal phenyl moiety connected to the
central fiveꢀmembered heterocycle via a spacer of two heavy atoms length resembled the general shape
and size of the inhibitory structure best.
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Thus, the influence of this spacer on the inhibitory activity of the compounds was further explored. The
exchange of the thioꢀether present in 5e by an aminoꢀfunctionality (6, Table 2) led to a drop of potency,
whereas the introduction of a methylene unit (7a) led to a slightly increased activity. Hence, this
simplified analog was utilized as basis for further structural modifications. Furthermore, the exchange of
the methylene unit proximate to the phenylꢀmoiety into an ether (7b) led to a decreased activity while
the introduction of a sulfide (7c), or a sulfone (7d) was well tolerated and resulted in a doubled potency
as compared to compound 7a.
The impact of the central heterocycle on the activity of the compounds was also explored starting from
the simplified analog 7a (Table 3). Thereby the change of the central oxadiazole in 7a into a thiadiazole
(8a) resulted in a 2ꢀtimes improvement of the inhibitory potency. The introduction of a thiazole resulted
in a slight drop of potency in case of the 2ꢀ(benzimidazoleꢀ5(6)ꢀyl)ꢀthiazole (13) and a total loss of
potency in case of the isomeric 5ꢀ(benzimidazoleꢀ5(6)ꢀyl)ꢀthiazole 11. Also, the introduction of the
1,2,4ꢀoxadiazole (17) or the 1,2,4ꢀtriazole (19) resulted in a moderate drop of potency. The potency of
the 1,2,3ꢀtriazole 22a was found to be 3ꢀtimes higher as seen for 7a.
Keeping the ethyleneꢀspacer constant, the influence of the substitution pattern at the terminal phenyl
moiety was investigated for the 1,3,4ꢀoxadiazole as well as for the 1,3,4ꢀthiadiazole derivatives (Table
4). Here, the introduction of electronꢀenriching methoxyꢀsubstituents in position 3 and 4 (7e, 8b) or 2
and 3 (7g, 8d) of the terminal phenyl, or their incorporation into a dioxolaneꢀmoiety (7h, 8e) led to an
improvement or perpetuation of the inhibitory potency, as compared to the unꢀsubstituted equivalents. A
slight drop of potency resulted from the introduction of methoxyꢀsubstituents in the positions 2 and 4 of
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the phenyl ring (7f, 8c). In all cases, the 1,3,4ꢀthiadiazoles were found to be more potent as the
corresponding 1,3,4ꢀoxadiazoles, paralleling the findings with the unꢀsubstituted derivatives (Table 3).
The introduction of electronꢀwithdrawing fluoroꢀ or trifluoroꢀmethyl substituents as in 7i, 7j, 7k, 8f and
8g led to a decrease (7i, 7j, 8f) or a total loss of inhibitory potency (7k, 8g). Since we observed an
increased activity caused by electronꢀdonating substituents, methoxyꢀsubstituents were also introduced
to the 1,2,3ꢀtriazole derivative 22a. This modification also led to an improved potency in case of the 4ꢀ
methoxyꢀsubstituted derivative 22b and the 3,4ꢀdimethoxy derivative 22d. However, the introduction of
a methoxyꢀsubstituent in position 3 of the phenylꢀmoiety (22c) led to a slightly decreased activity
compared to 22a.
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The combination of the above findings resulted in the most potent compounds of the study: 8h and 8i
(Table 5). These compounds contained a central 1,3,4ꢀthiadiazole and a terminal 3,4ꢀdimethoxyphenyl
moiety. Furthermore, the exchange of the methylene unit of the spacer proximate to the 3,4ꢀ
dimethoxyphenyl substituent into a sulfide (8h) or sulfone (8i) led to an additional gain of potency,
more pronounced for the sulfideꢀmodification. Obviously, here the combination of a central 1,3,4ꢀ
thiadiazole, the spacer modification and the introduction of the terminal 3,4ꢀmethoxyphenyl moiety
summed up in a cooperative manner, resulting in a 40ꢀfold and 12ꢀfold improvement as compared to the
starting compound 1a for 8h and 8i, respectively.
Docking and mutagenesis studies. First information regarding the possible binding mode of this novel
class of QCꢀinhibitors was gained by an inꢀsilico docking analysis. This analysis was paralleled by an
inꢀvitro mutagenesis study, in which the influence of the character of amino acid side chains in the
active site on the inhibitory potency of selected compounds was evaluated. The two potent inhibitors, 8h
of the 1,3,4ꢀthiadiazole series and 22b of the 1,2,3ꢀtriazole series were selected for these studies.
The docking study was performed in consideration of the two different tautomeric forms of the metalꢀ
binding benzimidazole moiety, resulting in either 5ꢀ or 6ꢀsubstituted derivatives of the inhibitors.
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The results of the docking analysis, here exemplary shown for the results of the GoldScore® scoring
function (for solutions of the other scoring functions see supporting information) and application of the
PostDOCK script31 are shown in Figure 3. The results of the fitness value for all 30 solutions are found
in a range of 74.6 – 83.0 for the 6ꢀyl tautomer and 79.1 – 87.0 in case of the 5ꢀyl tautomer of 8h. For
compound 22b this range was found within 70.4 – 78.0 for the 6ꢀyl tautomer and within 78.2 – 84.0 for
the respective 5ꢀyl tautomer. In general, these findings could suggest a preferred binding of the 5ꢀyl over
the 6ꢀyl tautomers. However, with the range of the scoring values being relatively narrow and
differences between the single solutions being small, a certain pose of the inhibitors with a distinct
minimum of binding energy cannot be concluded. Taking the uncertainty of scoringꢀfunctions in
predicting bindingꢀposes into account, all poses found have to be regarded as equally possible.
However, taking a closer look on the results of the docking study, a pattern for the binding of the
inhibitors is visible. In case of compound 8h, when enabling the metalꢀcontact of the benzimidazoleꢀ
nitrogen leading to a 6ꢀyl substituted inhibitor, all 30 dockingꢀsolutions are placed with the spacerꢀpart
and the terminal phenylꢀmoiety pointing towards a hydrophilic binding region formed by K144 and
H206 (Fig 3A). Thereby the central 1,3,4ꢀthiadiazole forms a πꢀπ stacking interaction with the side
chain of W207, located at the backside of the activeꢀsite opening. The solutions for the 5ꢀyl isomer are
almost equally distributed between two binding modes: one, occupying the hydrophilic binding region
mentioned above, the other with the terminal phenyl residue part of the inhibitor adopting a hydrophobic
binding area (Fig 3B) that is formed by the three amino acids I303, F325 and W329. A similar binding
mode of an inhibitor was former identified by crystal structures of drosophila QC containing the first
potent hQC inhibitor PBD150.32 For compound 22b the placement of the solutions is stricter, depending
on which isomer of the metalꢀbinding benzimidazole is considered. In all solutions for the 6ꢀyl isomer,
the phenylꢀmoiety of the compound is directed towards the hydrophilic binding region (Fig 3C), while
almost all solutions of the 5ꢀyl isomer are placed at the hydrophobic pocket (Fig 3D). Summing up, the
docking experiments suggest two major ways of binding for each compound, which are differently
populated, depending on which isomeric form of the inhibitor is considered.
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For gaining a deeper insight in the likely “true” binding mode of the inhibitors, amino acids located at
the active site and supposed to be involved in binding of the inhibitor molecules, were altered by inꢀ
vitro mutagenesis experiments. The effect of these changes on the potency of 8h and 22b was then
elucidated by measurement of the inhibition constants for the mutant protein. A similar approach was
recently published for the first potent QCꢀinhibitor PBD150.33 According to the results of the above
docking study, the aminoꢀacids altered were either located in the hydrophobic region, as I303, F325 and
W329 or in the hydrophilic region, as W207, H206 and K144 (Fig 3). The results of this study are
summarized in Table 6. Clear changes in the inhibitory potency for both compounds 8h and 22b were
visible for the mutagenesis of F325 and W329, as well as of W207. All other mutations did not
remarkably affect the inhibitory potency. In the case of F325, the change of the aromatic side chain into
the aliphatic, more polar asparagine or the aromatic and polar tyrosine led to a loss of the inhibitory
potency for both tested inhibitors. In case of the aromatic W329, the inhibitory potency was maintained
when a phenylalanine was placed in this position, but abolished, when a polar tyrosine was introduced.
Also, the binding site at W207 was affected when its aromatic character was changed into the aliphatic
leucine or glutamine. These findings can be interpreted in a way, that the binding of 8h and 22b in the
hQC active site, besides the metal contact of the benzimidazole part, is enabled by interactions,
maintained through F325, W329 and W207 (Fig 4).
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The finding of the mutagenesis study would support the general binding mode for the 5ꢀylꢀ
benzimidazole isomers (Fig 3B and D) as suggested by docking. In these cases the aromatic moiety at
the spacer end is bent towards the hydrophobic region. However, taking a closer look on the results, it
turns out that none of the suggested solutions of the 5ꢀyl tautomers of 8h and 22b does exactly match
the results of the mutagenesis study. The majority of the docking solutions suggests an interaction with
all three key aminoꢀacids of the hydrophobic region, the mutagenesis study gives rise only for a contact
with F325 and W329, leaving out I303. Also, the interaction of the inhibitor with W207, identified with
the mutagenesis experiment, was not seen for the majority of docking solutions with the bent
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conformation. A π−π interaction of the central heterocycle was suggested only in those cases in which
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the spacer part with the terminal phenyl moiety pointed towards the pocket formed by H206 and K144.
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The only exception showing a good match of the docking and the mutagenesis results was found in one
single high ranking docking solution, but for a 6ꢀyl tautomer and only in case of compound 22b. In this
single case a π−π interaction of the central heterocycle with W207 can be considered as well as an
interaction of the terminal methoxyꢀphenyl moiety of 22b with F325 and W329.
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For a conclusive discussion of the possible binding mode of the new type of inhibitors in the active site
of hQC the results of the analysis gained by mutagenesis study should be preferred over the ones
suggested by docking. That is why the detailed analysis of the ligandꢀreceptor interactions also in
comparison to PBD150 is made based on the single docking result of 22b mentioned above (see
supporting information for details).
Conclusion. A new chemical class of inhibitors for human Glutaminyl Cyclase based on compounds
identified by a ligandꢀbased pharmacophore approach is presented. Thereby benzimidazole as a new
metalꢀbinding group is introduced. The optimization process, starting with inhibitors of a potency in the
micromolar range, finally led to compounds with activities in the nanomolar range, with the most active
compound 8h exhibiting an inhibition constant of 23 nM. This result is in line with the most potent
inhibitors of human Glutaminyl Cyclase known so far, that exhibit inhibitory constants between 60 and
6 nM.
Insight into the possible binding mode into the active site of hQC was gained by docking and
mutagenesis studies. As the chemical structure of the new inhibitors can exist in two tautomeric forms,
both, the 5ꢀylꢀ as well as 6ꢀyl substituted derivatives of the two potent representatives 8h and 22b were
subjected to docking studies, resulting in the suggestion of two general binding modes. A siteꢀdirected
mutagenesis approach identified amino acids in the active site that are involved in the inhibitor binding.
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The comparison with the docking results indicated, that the inhibitors are likely to bind in the 5ꢀyl
substituted isomeric form, adopting a bent conformation. The most conclusive interpretation and best
match of the mutagenesis and the docking experiments, however, was found for only one single docking
solution of 22b in its 6ꢀyl tautomeric form. This means, a final conclusion on which tautomeric form of
22b binds in the receptor cannot be drawn.
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We consider the approach of combining docking studies with a site directed mutagenesis being a
meaningful tool when, as seen here, docking studies are not conclusive and crystallographic data are not
available for the molecules of interest. Further efforts towards the latter are now in focus of further
investigations
Experimental Section.
Chemistry. Starting materials and solvents were purchased from Aldrich, Acros Organics, Alfa Aesar
and Maybridge Co. The purity of the compounds was assessed by HPLC and confirmed to be ≥ 95%.
The analytical HPLCꢀsystem consisted of a MerckꢀHitachi device (model LaChrom®) utilizing a
Phenomenex Luna 5 ꢁM C18(2) column (125x4,0 mm) with λ = 214 nm as the reporting wavelength.
The compounds were analyzed using a gradient at a flow rate of 1 mL/min; whereby eluent (A) was
acetonitrile, eluent (B) was water, both containing 0.04 % (v/v) trifluoro acetic acid applying the
following gradient: Method [A] 0 min – 25 min, 20ꢀ80% MeCN; 25 min – 30 min, 80ꢀ95% MeCN; 30
min ꢀ31 min, 95ꢀ20% MeCN; 31 min – 40 min, 20% MeCN; Method [B] 0 min – 15 min, 5ꢀ50%
MeCN; 15 min – 20 min, 50ꢀ95% MeCN; 20 min ꢀ23 min, 95% MeCN; 23 min ꢀ24 min, 95ꢀ5% MeCN;
24 min – 30 min, 5% MeCN. The purities of all reported compounds were determined by the percentage
of the peak area at 214 nm.
ESIꢀMass spectra were obtained with a SCIEX API 150 and a SCIEX API 365 spectrometer (Perkin
Elmer) utilizing the positive ionization mode. The high resolution positive ion ESI mass spectra were
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obtained from a Bruker Apex III 70e Fourier transform ion cyclotron resonance mass spectrometer
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(Bruker Daltonics, Billerica, USA) equipped with an Infinity cell. The melting points were detected
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utilizing a Kofler melting point device and are uncorrected. The H NMRꢀSpectra were recorded at a
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VARIAN GEMINI 2000 (400 MHz) or at a BRUKER AC 500 (500 MHz). DMSOꢀD6 was used as
solvent unless otherwise specified. Chemical shifts are expressed as parts per million (ppm). The solvent
was used as internal standard. Splitting patterns have been designated as follows: s (singulet), d
(doublet), dd (doublet of doublet), t (triplet), m (multiplet) and br (broad signal). Semi preparative
HPLC was performed on a Prepstar device (Varian) equipped with a Phenomenex Luna 10ꢂM C18(2)
column (250x21 mm). The compounds were eluted using the same solvent system as described above,
applying a flow rate of 21 mL/min.
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Benzimidazole-5(6)-carboxylic acid hydrazide 3
Step 1: Benzimidazole-5(6)-carboxylic acid ethyl ester: 2 (12.9 g; 80 mmol; 1 eq.) was dissolved in
EtOH (200 ml), treated with conc. H2SO4 (5 ml; 88 mmol; 1.1 eq.) and heated to reflux for 24 h. After
cooling to room temperature the mixture was poured into ice, basified by means of 5 N aqueous NaOH
and extracted with EtOAc (3x100 ml). The combined organic layers were dried over Na2SO4 and
evaporated. The remains were used without further purification. Yield: 12.6 g (82.9%); MS m/z: 191.3
[M+H]+
Step 2: Benzimidazole-5(6)-carboxylic acid hydrazide 3: Benzimidazolꢀ5(6)ꢀcarboxylic acid ethyl
ester (12.6 g; 66 mmol; 1 eq.) was dissolved in EtOH (75 ml) and treated with N2H4*H2O (8.2 g; 264
mmol; 4 eq.). The mixture was heated to reflux for 48 h. The precipitate was collected by filtration after
cooling, washed by means of heptane and used without further purification. Yield: 10.2 g (87.8%); MS
m/z: 177.2 [M+H]+
Benzimidazole-5(6)-(3H-1,3,4-oxadiazol-5-yl-2-thione) 4
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Benzimidazoleꢀ5(6)ꢀcarboxylic acid hydrazide (9.0 g; 51 mmol; 1 eq.) was suspended in EtOH (85 ml).
KOH (1.7 g; 30 mmol; 0.6 eq.) dissolved in water (17 ml) and CS2 (3 ml; 50 mmol; 0.98 eq.) were
added and the mixture was heated to reflux for 10 h. After cooling to room temperature the mixture was
concentrated in vacuo and poured into ice. Concentrated HCl(aq) was added dropwise until a precipitate
was formed. The solid was collected by filtration and used without further purification. Yield: 6.3 g
(56.9%); MS m/z: 219.4 [M+H]+
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General procedure for the synthesis of the thioalkyl-substituted 1,3,4-oxadiazoles 5a-h
Benzimidazoleꢀ5(6)ꢀ(3Hꢀ1,3,4ꢀoxadiazolꢀ5ꢀylꢀ2ꢀthione) (330 mg; 1.5 mmol; 1 eq.) and triethylamine
(0.209 ml; 1.5 mmol; 1 eq.) were dissolved in EtOH (10 ml). The respective alkylhalide (1.5 mmol; 1
eq.) was added and the mixture was heated to reflux over night. The solvent was evaporated and the
residue was purified by flash chromatography on silica using a CHCl3/MeOH gradient.
5(6)-(5-(4-Benzylamino)-1,3,4-oxadiazol-2-yl)benzimidazole 6
3 (0.354 g; 2 mmol; 1 eq.) was suspended in EtOH (20 ml). After addition of benzylisothiocyanate
(298mg; 0.2 mmol; 1 eq.) the mixture was refluxed for 24 h. The solvent was evaporated and the residue
was taken up in THF (30 ml). Dicyclohexylcarbodiimide (0.62 g; 3 mmol; 1.5 eq.) was added and the
mixture was heated to reflux for 1 h. The solvent was evaporated and the remains were purified by flash
chromatography on Al2O3 using a CHCl3/MeOH gradient. Yield: 0.161 g (28.4%); mp: 109ꢀ111 °C; MS
m/z: 292.4 [M+H]+; 1HꢀNMR, 500 MHz, DMSO d6: δ 4.45 (d, 2H, 3J=6.2 Hz); 7.25ꢀ7.28 (m, 1H); 7.33ꢀ
3
7.36 (m, 2H); 7.39ꢀ7.40 (m, 2H); 7.67ꢀ7.71 (m, 2H); 7.97 (br s, 1H); 8.26 (t, 1H, J=6.3 Hz); 8.33 (s,
1H); 12.69 (br s, 1H); ESIꢀFTICRꢀMS m/z: 292.11893 [M+H]+; calc. for C16H14N5O+: 292.11929;
HPLC (method B): rt 8.99 min (99.7%)
General procedure for the synthesis of the alkyl-substituted 1,3,4-oxadiazoles 7a, e-g
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3 (176 mg; 1 mmol; 1 eq.) was suspended in THF (10 ml). Triethylamine (0.153 ml; 1.1 mmol; 1.1 eq.)
and the respective acid chloride (1.1 mmol; 1.1 eq.) were added sequentially. After stirring at room
temperature for 3 h the mixture was diluted by means water and extracted with EtOAc (3x25 ml). The
combined organic layers were dried over Na2SO4 and evaporated. The residue was taken up in MeCN
(10 ml), treated with POCl3 (0.5 ml; 5.5 mmol; 5.5 eq.) and heated to reflux over night. After cooling
the mixture was carefully basified by means of saturated aqueous NaHCO3ꢀsolution and extracted with
EtOAc (3x25 ml). The combined organic layers were dried over Na2SO4 and evaporated. The residue
was purified by semiꢀpreparative HPLC.
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General procedure for the synthesis of the alkyl-substituted 1,3,4-oxadiazoles 7b-d, h-k
3 (176 mg; 1 mmol; 1 eq.) and the respective carboxylic acid were dissolved in POCl3 (3 ml) and heated
to reflux over night. After cooling the mixture was poured into ice water, basified by means of 2N
aqueous NaOH and extracted with EtOAc (3x25 ml). The combined organic layers were dried over
Na2SO4 and evaporated. The residue was purified by semiꢀpreparative HPLC.
General procedure for the synthesis of the 1,3,4-thiadiazoles 8a-d
3 (176 mg; 1 mmol; 1 eq.) was suspended in THF (10 ml). Triethylamine (0.153 ml; 1.1 mmol; 1.1 eq.)
and the respective acid chloride (1.1 mmol; 1.1 eq.) were added sequentially. After stirring at room
temperature for 3 h the mixture was diluted by means of water and extracted with EtOAc (3x25 ml).
The combined organic layers were dried over Na2SO4 and evaporated. The residue was taken up in THF
(10 ml), treated with Lawesson´s reagent (606 mg; 1.5 mmol; 1.5 eq.) and heated to reflux for 1h. After
cooling the mixture was diluted with water, basified by means of aqueous 2 N NaOH and extracted with
EtOAc (3x25 ml). The combined organic layers were dried over Na2SO4 and evaporated. The remains
were purified by flash chromatography on silica using a CHCl3/MeOH gradient.
5(6)-(5-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-1,3,4-thiadiazol-2-yl)benzimidazole 8e
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3ꢀ(1,3ꢀBenzodioxolꢀ5ꢀyl)propionic acid (195mg; 1 mmol; 1 eq.) was dissolved in THF (10 ml), treated
with DCC (206 mg; 1 mmol; 1 eq.) and stirred at room temperature for 1h. Benzimidazoleꢀ5(6)ꢀ
carboxylic acid hydrazide (176 mg; 1 mmol; 1 eq.) was added and the mixture was heated to 50°C over
night. After cooling to room temperature, Lawesson´s reagent (606 mg; 1.5 mmol; 1.5 eq.) was added
and the mixture was heated to reflux for 5h. After cooling to room temperature, the mixture was diluted
by means of saturated aqueous NaHCO3 solution and extracted with EtOAc (3x25 ml). The combined
organic layers were dried over Na2SO4 and evaporated. The residue was purified by semiꢀpreparative
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HPLC. Yield: 0.019 g (5.4%); mp: 125ꢀ128 °C; MS m/z: 351.3 [M+H]+; HꢀNMR, 400 MHz, DMSO
d6: δ 3.02 (t, 2H, 3J=7.5 Hz); 3.42 (t, 2H, 3J=7.5 Hz); 5.96 (s, 2H); 6.73 (dd, 1H, 3J=7.9 Hz, 4J=1.7 Hz);
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6.81 (d, 1H, J=7.9 Hz); 6.92 (d, 1H, J=1.7 Hz); 7.83 (d, 1H, J=8.3 Hz); 7.92 (dd, 1H, J=8.3 Hz,
4
4J=1.7 Hz); 8.22 (d, 1H, J=1.7 Hz); 8.93 (s, 1H); ESIꢀFTICRꢀMS m/z: 351.09063 [M+H]+; calc. for
C18H15N4O2S+: 351.09102; HPLC (method 2): rt 12.40 min (99.4%)
General procedure for the synthesis of the 1,3,4-thiadiazoles 8f-i
3 (176 mg; 1 mmol; 1 eq.) and the respective carboxylic acid (1 mmol; 1eq.) were suspended in MeCN
(10 ml). Lawesson´s reagent (606 mg; 1.5 mmol; 1.5 eq.) and POCl3 (0.137 ml; 1.5 mmol; 1.5 eq.) were
added and the mixture was heated to reflux over night. After cooling to room temperature, the mixture
was carefully basified by means of saturated aqueous NaHCO3 solution and extracted with EtOAc (3x25
ml). The combined organic layers were dried over Na2SO4 and evaporated. The remains were purified
by semiꢀpreparative HPLC.
5-(Benzimidazole-5(6)-yl)oxazole-4-carboxylic acid ethylester 9
Benzimidazoleꢀ5(6)ꢀcarboxylic acid (1.62 g; 10 mmol; 1 eq.) was suspended in toluene (50 ml).
Thionylchloride (3.63 ml; 50 mmol; 5 eq.) was added and the mixture was heated to reflux over night.
After cooling the mixture was concentrated to dryness. The remains were resuspended in THF (50 ml),
cooled to 0°C and treated with DBU (2.23 ml; 15 mmol; 1.5 eq.). A mixture of isocyanoethylacetate
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(1.32 ml; 12 mmol; 1.2 eq.) and DBU (4.46 ml; 30 mmol; 3 eq.) in THF (50 ml) was added dropwise.
After complete addition the reaction was allowed to warm to room temperature and stirring was
continued for 24 h. The mixture was diluted by means of water and extracted with EtOAc (3x100 ml).
The combined organic layers were dried over Na2SO4 and evaporated. The remains were purified by
flashchromatography on silica using a CHCl3/MeOH gradient. Yield: 2.0 g (78.0%); MS m/z: 258.3
[M+H]+
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Benzimidazole-5(6)-aminomethylketone-dihydrochloride 10
9 (2.0 g; 7.8 mmol) was dissolved in MeOH (20 ml). Concentrated aqueous HCl (40 ml) was added and
the solution was heated to reflux over night. The solvent was evaporated to dryness and the residue was
used without further purification. MS m/z: 176.3 [M+H]+
5(6)-(2-Phenethylthiazol-5-yl)benzimidazole 11
10 (248 mg; 1 mmol; 1 eq.) and 3ꢀPhenylpropionic acid (150 mg; 1 mmol; 1 eq.) were suspended in
MeCN (10 ml). After addition of Lawesson´s reagent (606 mg; 1.5 mmol; 1.5 eq.), POCl3 (0.137 ml; 1.5
mmol; 1.5 eq.) and triethylamine (0.22 ml; 3 mmol; 3 eq.) the mixture was heated to reflux over night.
After cooling to room temperature the mixture was carefully diluted by means of saturated aqueous
NaHCO3 solution and extracted with EtOAc (3x25 ml). The combined organic layers were dried over
Na2SO4 and evaporated. The remains were purified by semiꢀpreparative HPLC. Yield: 0.025 g (8.2%);
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mp: 113ꢀ116 °C; MS m/z: 306.2 [M+H]+; HꢀNMR, 400 MHz, DMSO d6: δ 3.09 (t, 2H, J=7.5 Hz);
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3.33 (t, 2H, J=7.5 Hz); 7.18ꢀ7.21 (m, 1H); 7.28ꢀ7.29 (m, 4H); 7.71ꢀ7.73 (m, 1H); 7.80ꢀ7.82 (m, 1H);
7.93 (br s, 1H); 8.15 (s, 1H); 9.22 (br s, 1H); ESIꢀFTICRꢀMS m/z: 306.10634 [M+H]+; calc. for
C18H16N3S+: 306.10594; HPLC (method B): rt 13.00 min (100%).
Amino-4-phenylbutan-2-on-hydrochloride 12
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Phenylpropionic acid (451 mg; 3 mmol; 1 eq.) was dissolved in THF (10 ml), treated with CDI (486 mg;
3 mmol; 1 eq.) and stirred at room temperature for 1 h. The mixture was cooled to 0°C and a mixture of
isocyanoethylacetate (0.393 ml; 3.6 mmol; 1.2 eq.) and DBU (1.34 ml; 9 mmol; 3 eq.) in THF (10 ml)
was added dropwise. After complete addition, the mixture was stirred at room temperature for 24 h.
After dilution by means of water, the mixture was extracted with EtOAc (3x25 ml). The combined
organic layers were dried over Na2SO4 and evaporated. The remains were purified by
flashchromatography on silica using a CHCl3/MeOH gradient. Yield: 319 mg (43.4%); MS m/z: 246.3
[M+H]+.
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The residue was dissolved in MeOH (5 ml), treated with aqueous concentrated HCl (15 ml) and heated
to reflux over night. The solvent was evaporated to dryness. The compound was used without further
purification. MS m/z: 164.4 [M+H]+;
5(6)-(5-Phenethylthiazol-2-yl)benzimidazole 13
The compound was synthesized from Aminoꢀ4ꢀphenylbutanꢀ2ꢀonꢀhydrochloride (12; 163 mg) and 2
(162 mg) according to the method described for 11. Yield: 0.042 g (13.8%); mp: 109ꢀ111 °C; MS m/z:
306.1 [M+H]+; 1HꢀNMR, 400 MHz, DMSO d6: δ 2.98 (t, 2H, 3J=7.5 Hz); 3.20 (t, 2H, 3J=7.5 Hz); 7.17ꢀ
7.22 (m, 1H); 7.25ꢀ7.31 (m, 4H); 7.62 (s, 1H); 7.80 (d, 1H, 3J=8.7 Hz); 7.90 (dd, 1H, 3J=8.3 Hz, 4J=1.7
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Hz); 8.16 (d, 1H, J=1.7 Hz); 9.02 (s, 1H); ESIꢀFTICRꢀMS m/z: 306.10603 [M+H]+; calc. for
C18H16N3S+: 306.10594; HPLC (method B): rt 13.73 min (97.4%)
5(6)-Cyanobenzimidazole 15
14 (5.0 g; 37 mmol; 1 eq.) was dissolved in 5 N aqueous HCl (200 ml). After addition of formic acid (20
ml) the mixture was heated to reflux for 3 h. After cooling the mixture was basified by means of
aqueous ammonia and stored in a fridge over night. The formed precipitate was collected by filtration,
washed with water and used without further purification. Yield: 3.6 g (67.6%); MS m/z: 144.2 [M+H]+
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N-Hydroxy-benzimidazole-5(6)-carboxamidine 16
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A solution of 5(6)ꢀcyanobenzimidazole (1.43 g; 10 mmol; 1 eq.) in EtOH (30 ml) was treated with
K2CO3 (2.76 g; 20 mmol; 2 eq.) and H2NOH*HCl (0.77 g; 11 mmol; 1.1 eq.) and heated to reflux over
night. After cooling to room temperature the mixture was diluted by means of diethylether. The
precipitate was collected by filtration, washed with water and dried in vacuo. The compound was used
without further purification. Yield: 1.28 g (72.7%); MS: 177.3 [M+H]+
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5(6)-(5-Phenethyl-1,2,4-oxadiazol-3-yl)benzimidazole 17
3ꢀPhenylpropionic acid (150 mg; 1 mmol; 1 eq.) was dissolved in DMF (5 ml), treated with
carbonyldiimidazole (162 mg; 1 mmol; 1 eq.) and stirred at room temperature for 1h. After addition of
16 (176 mg; 1 mmol; 1eq.) the temperature was increased to 110°C and stirring was continued over
night. After cooling the mixture was diluted by means of water and saturated aqueous NaHCO3 solution
and extracted with EtOAc (3x25 ml). The combined organic layers were dried over Na2SO4 and
evaporated. The remains were purified by flash chromatography on silica using a CHCl3/MeOH
gradient. Yield: 0.053 g (18.3%); mp: 109ꢀ110 °C; MS m/z: 291.3 [M+H]+; 1HꢀNMR, 400 MHz, DMSO
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3
d6: δ 3.15 (t, 2H, J=7.9 Hz); 3.33 (t, 2H, J=7.9 Hz); 7.18ꢀ7.22 (m, 1H); 7.27ꢀ7.30 (m, 4H); 7.66ꢀ7.87
(br m, 2H), 8.15ꢀ8.24 (br m, 1H); 8.34ꢀ8.36 (m, 1H); 12.69ꢀ12.72 (m, 1H); ESIꢀFTICRꢀMS m/z:
291.12362 [M+H]+; calc. for C17H15N4O+: 291.12404; HPLC (method B): rt 13.21 min (99.1%)
Benzimidazole-5(6)-carboxamidrazone 18
Gaseous HCl was bubbled through an ice cooled solution of 15 (1.43 g; 10 mmol; 1 eq.) in dry EtOH
(20 ml). The tube was sealed and stirred at room temperature for 48 h. The mixture was diluted by
means of diethylether. The precipitate was collected by filtration, washed with a small amount of EtOH
and dietyhlether and was dried in vacuo. The residue was resuspended in EtOH (20 ml), treated with
N2H4*H2O (1.5 g; 30 mmol; 3 eq.) and stirred at room temperature for 3 h. The precipitate was collected
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by filtration and washed by means of water. The residue was used without further purification. Yield:
0.75 g (42.9%); MS m/z: 176.4 [M+H]+
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5(6)-(5-Phenethyl-4H-1,2,4-triazol-3-yl)benzimidazole 19
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A solution of 3ꢀPhenylpropionic acid (150 mg; 1 mmol; 1 eq.) in DMF (5 ml) was treated with
carbonyldiimidazole (162 mg; 1 mmol; 1 eq.) and stirred at room temperature for 1 h. After addition of
18 (175 mg; 1 mmol; 1 eq.) the temperature was increased to 110°C and stirring was continued for 24h.
The mixture was cooled to room temperature, diltuted by means of water and saturated aqueous
NaHCO3 solution and extracted with EtOAc (3x25 ml). The combined organic layers were dried over
Na2SO4 and evaporated. The remains were purified by flash chromatography on silica using a
CHCl3/MeOH gradient. Yield: 0.062 g (21.5%); mp: 115ꢀ119 °C; MS m/z: 290.1 [M+H]+; 145.8
[M+2H]2+; 1HꢀNMR, 400 MHz, DMSO d6: δ 2.94 (br s, 2H); 3.30 (br s, 2H); 7.00 (s, 1H); 7.25ꢀ7.28 (m,
4H); 7.59ꢀ7.65 (m, 1H); 7.85ꢀ7.87 (m, 1H); 8.16ꢀ8.30 (m, 1H); ESIꢀFTICRꢀMS m/z: 290.14006
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[M+H]+; calc. for C17H16N5 : 290.14002; HPLC (method B): rt 10.06 min (100%)
+
4-Ethinyl-2-nitroaniline 20
The compound was synthesized as described in:28,29
Step 1: 2-Nitro-4-(trimethylsilylethinyl)aniline: A solution of 4ꢀiodoꢀ2ꢀnitroaniline (5.32 g; 20 mmol;
1 eq.) in THF (20 ml) was treated with PdCl2(PPh3)2 (154 mg; 0.22 mmol; 0.011 eq.), CuI (84 mg; 0.44
mmol; 0.022 eq.) and DIPEA (10.5 ml; 60 mmol; 3 eq.) under argon atmosphere.
Trimethylsilylacetylene (3.1 ml; 22 mmol; 1.1 eq.) was added dropꢀwise at room temperature. After
complete addition, stirring was continued for 3h. The mixture was diluted by means of water and
extracted with EtOAc (3x100 ml). The combined organic layers were dried over Na2SO4 and
evaporated. The remains were purified by flash chromatography on silica using a CHCl3/MeOH
gradient. Yield: 3.96 g (84.6%); MS m/z: 235.3 [M+H]+; 469.4 [2M+H]+
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Step 2: 4-Ethinyl-2-nitroaniline 20: A solution of 2ꢀnitroꢀ4ꢀ(trimethylsilylethinyl)aniline (3.96 g; 16.9
mmol; 1 eq.) in MeOH/CH2Cl2 (100 ml; 1:1 v/v) was treated with K2CO3 (9.33 g; 67.6 mmol; 4 eq.).
The mixture was stirred at room temperature for 5 h. After dilution by means of water, the organic layer
was separated. The aqueous layer was extracted with CH2Cl2 (2x100 ml). The combined organic layers
were dried over Na2SO4 and evaporated. The remains were purified by flash chromatography on silica
using a CHCl3/MeOH gradient. Yield: 2.06 g (75.1%); MS m/z: 163.4 [M+H]+
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General procedure for the synthesis of the 4-(1-Alkyl-1,2,3-triazol-4-yl)-2-nitroanilines 21a-d
Sodium azide (65 mg; 1 mmol; 1 eq.) was dissolved in DMSO (2 ml), treated with the respective
alkylbromide (1 mmol; 1 eq.) and stirred at room temperature for 24h. The mixture was diluted with
water (2 ml), treated with sodium ascorbate (20 mg; 0.1 mmol; 0.1 eq.), 20 (162 mg; 1 mmol; 1 eq.) and
an aqueous 1 M solution of CuSO4*5 H2O (0.2 ml; 2 mmol; 0.2 eq.) and stirred at room temperature for
3h. The formed precipitate was collected by filtration, washed by means of water and dried in vacuo.
The residue was used without further purification.
General procedure for the synthesis of the 1,2,3-triazoles 22a-d
A solution of the respective 4ꢀ(1ꢀAlkylꢀ1,2,3ꢀtriazolꢀ4ꢀyl)ꢀ2ꢀnitroaniline in formic acid and
triethylorthoformiate (10 ml; 1:1 v/v) was treated with sodium formiate (2.04 g; 30 mmol) and
palladium on charcoal. The sealed tube was heated to 110°C for 24h. After cooling to room temperature
the mixture was filtered through Celite and concentrated in vacuo. The residue was redissolved in water,
basified by means of aqueous saturated NaHCO3 solution an extracted with EtOAc (3x25 ml). The
combined organic layers were dried over Na2SO4 and evaporated. The remains were purified by flash
chromatography on silica using a CHCl3/MeOH gradient.
Computational Chemistry
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For the flexible alignment of PBD150 and the substrate HꢀGlnꢀPheꢀAlaꢀNH2 the Molecular Operating
Environment (MOE) in the version 2003.02 was used (Chemical Computing Group, Montreal, Canada).
For the pharmacophore search itself and for the visualization of the docking results MOE in the version
2011.10 was used. Molecular Docking was done by using GOLD vers. 5.0 (CCDC Software Ltd.,
Cambridge, U.K.).
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Flexible Alignment and Pharmacophore search. The flexible alignment of HꢀGlnꢀPheꢀAlaꢀNH2, 1ꢀ(3ꢀ
(1HꢀImidazolꢀ1ylꢀ)propyl)ꢀ3ꢀ(3,4ꢀdimethoxyphenyl)thiourea (PBD150) and Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀ
yl)propyl)ꢀ1ꢀ(3,4ꢀdimethoxyphenyl)cyclopropanecarbothioamide was accomplished as described
elsewhere17. After sorting the list concerning the average energies, the alignment on position 37 (U = ꢀ
4.32 kcal/mol, S = 97.91) was used for the pharmacophore creation. Thereby the pharmacophore editor
was opened and the pharmacophore was built as follows by using the PCH_All scheme: F1: Metal
ligator & “[n]1ccnc1”, R=1.2, essential: yes, N3 of the imidazole moiety of PBD150; F2: Acceptor,
R=1.2, essential: no, sulfur of the thiourea moiety of PBD150; F3: Donor, R=1.2, essential: no, N of the
thioamide moiety of the corresponding derivative, F4: Aromatic|Hydrophobic, R=1.4, essential: no,
center of the phenyl side chain of HꢀGlnꢀPheꢀAlaꢀNH2; F5: Acceptor, R=1.1, essential: no, oxygen of
the 4ꢀmethoxygroup of PBD150. Compounds had to fulfill at least 4 of the 5 features. The search was
done by using the preꢀprocessed databases “leadlike_conf_001.mdb” – “leadlike_conf_008.mdb” that
are provided with the MOEꢀpackage. They contain more than 650.000 chemical structures that are preꢀ
20,21
filtered concerning the criteria of Oprea et al.
including their conformational space. After this the
compounds were successively filtered for compounds containing a benzimidazole as substructure and
the TPSAꢀvalue below or equal to 70 Å2. The remaining compounds were analyzed for there
commercially availability and their synthetic access, resulting in 69 compounds that were tested using
the assay described below. The compounds sharing in this paper as the starting point for further
chemical exploration were purchased from Asinex (1a: ASN6346968; 1b: ASN6346970; 1c:
ASN06346980)..
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Journal of Medicinal Chemistry
Docking. For the molecular docking of the compounds 8h and 22b the pdb file 3si0 (www.rcsb.org)
was used. Thereby the protonate 3D functionality of MOE® (vers. 2011.10) was applied in order to
protonate the system. The coꢀcrystallized imidazole was removed from the system. After the protein file
was converted into the mol2ꢀformat the subsequent steps were performed in GOLD®. Any water outside
the active site was removed. All remaining water molecules were set to toggle. The active site was
defined using the zinc as the center with a radius of 15 Å. A substructure constraint for the
benzimidazole moiety as the metal binding group was used, with a force constant of 10. Both tautomeric
forms of the inhibitors were created in MOE, energy minimized and saved as mol2 file. The resulting 4
compounds were then docked 30 times applying each of the available scoring functions. The solutions
for each tautomer and scoring function with the corresponding score values were converted into MOE
databases, applying a svlꢀscript provided by CCG. The postdockꢀ1.2.svl script, available via the svl
exchange server (svl.chemcomp.com), was used to create the corresponding figures.
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Biochemistry
Inhibitor Testing. QC activity was assayed essentially as described in34. For a fluorometric detection
the assay consisted of varying concentrations of HꢀGlnꢀAMC (7ꢀaminoꢀ4ꢀmethylcoumarin, Bachem,
Bubendorf, Switzerland) in a concentration range between 0.25 and 4 Km in 50 m TrisꢀHCl, pH 8.0;
M
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0.4 /ml recombinant pyroglutamyl aminopeptidase (pGAP) from Bacillus amyloliqefaciens (Qiagen,
Hilden, Germany) as auxiliary enzyme and the inhibitory compound. For excitation/emission a
wavelength of 380/460 nm (HꢀGlnꢀAMC) was used. Reactions were started by addition of QC. The
activity was determined from a standard curve of the fluorophore under assay conditions. All
determinations were carried out at 30°C by using a BMG Fluostar reader for microplates (BMG
Labtechnologies, Offenburg, Germany). Evaluation of all kinetic data was performed with GraFit as the
analyzing software (version 5.0.4. for windows, ERITHACUS SOFTWARE Ltd., Horley, UK).
Mutagenesis. Cloning, expression and purification of hQC and the diverse variants were performed as
previously described.33 For all cloning procedures the Escherichia coli strain DH5α was applied. The
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hQC cDNA was inserted into the yeast expression vector pPICZαB (Invitrogen, Karlsruhe, Germany)
with additionally introduction of an Nꢀterminal His6ꢀtag (primer pair Cs/Cas, Table S1). The mutations
in hQC cDNA were introduced according to the quikꢀchange II siteꢀdirected mutagenesis protocol
(Stratagene, Santa Clara, CA, USA) using appropriate primer pairs (Table S1). The cDNA was verified
by sequencing (primer Ss, Sas for K144 mutation, Table S1). Plasmid DNA was amplified, purified
(Plasmid Miniprep Kit, Qiagen, Hilden, Germany) and linearized (Pme1 endonuclease).
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For heterologous target protein expression strain X33 (AOX1, AOX2) of the methylotrophic yeast
Pichia pastoris was used. Yeast was grown, transformed and analyzed according to the manufacturer’s
instructions (Invitrogen, Karlsruhe, Germany). Transformation of competent yeast cells was performed
by electroporation according to the manufacturer’s instructions (BioꢀRad, Munich, Germany).
Transgenics were screened for target protein expression. Clones displaying highest QC activity were
chosen for large expression. To confirm the insertion of the correct hQC variant, genomic DNA was
prepared according to standard molecular biological techniques. The target DNA was amplified by PCR
using gene specific primer pairs flanking the mutation site and the sequence was analyzed.
For large scale expression, high cell density fermentation in a 5 l reactor (Biostad B; Braun Biotech,
Melsungen, Germany) or expression in shake flasks were applied, essentially as described elsewhere.
35,36
The purification of hQC variants followed three liquid chromatographic steps: Ni2+ꢀIMAC
(immobilized metal affinity chromatography), hydrophobic interaction and anion exchange
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chromatography.
QCꢀcontaining fractions were pooled and purity was analyzed by SDSꢀPAGE
(sodium dodecyl sulfate polyacrylamide gel electrophoresis, Servagel TG 4ꢀ20, Serva, Heidelberg,
Germany) and Coomassie Blue staining. The purified enzyme was stored at ꢀ20°C after addition of
glycerol [50% (V/V)] or without glycerol at ꢀ80°C.
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Journal of Medicinal Chemistry
Acknowledgment. The authors are grateful for helpful discussions with Stephan Schilling. We thank
Antje Hamann and UlfꢀTorsten Gärtner for technical synthesis support as well as Guido Kirsten and
Markus Kossner (Chemical Computing Group) for providing useful svlꢀscripts.
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Supporting Information Available. Compound data, detailed experimental data of the mutagenesis
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study and the docking experiments are available free of charge via the Internet at http://pubs.acs.org.
ABBREVIATIONS. hQC, human Glutaminyl Cyclase; pGlu, pyroꢀglutamyl; MBG, metalꢀbindingꢀ
group.
*To whom correspondence should be addressed. Phone: +49 (0) 345 5559908. Fax: +49 (0) 345
5559901. Eꢀmail: ulrich.heiser@probiodrug.de
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13
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17
18
19
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Table 1.
1
2
3
4
5
6
7
8
9
10
11
12
13
entry
R
IC50 [ꢂM]
24.80
Ki [ꢂM]
entry
R
IC50 [ꢂM]
4.24
Ki [ꢂM]
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17.8 ± 0.35
0.78 ± 0.07
5a
5b
5c
5d
5e
14.70
15.00
7.23
9.67 ± 0.02
8.67
7.26
7.56
0.60 ± 0.03
6.07 ± 0.46
4.56 ± 0.17
5f
5g
5h
n.d.
5.26 ± 0.19
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