Synthesis, biological evaluation, and crystallographic studies of extended guanine-based (lin-benzoguanine) inhibitors for tRNA-guanine transglycosylase (TGT)

Article abstract of DOI:10.1002/hlca.200690062

This paper describes the rational design, synthesis, and biological evaluation of a new generation of inhibitors of the bacterial enzyme tRNA-guanine transglycosylase (TGT), which has been identified as a new target in the fight against bacillary dysenter

Full text of DOI:10.1002/hlca.200690062

Helvetica Chimica Acta – Vol. 89 (2006)
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Synthesis, Biological Evaluation, and Crystallographic Studies of Extended
Guanine-Based (lin-Benzoguanine) Inhibitors for tRNA-Guanine
Transglycosylase (TGT)
by Emmanuel A. Meyer, Nicola Donati, Marine Guillot, W. Bernd Schweizer, and FranÅois Diederich*
Laboratorium für Organische Chemie der Eidgençssischen Technischen Hochschule,
ETH-Hçnggerberg, HCI, CH-8093 Zürich (e-mail: diederich@org.chem.ethz.ch)
and
Bernhard Stengl, Ruth Brenk, Klaus Reuter, and Gerhard Klebe*
Institut für Pharmazeutische Chemie der Philipps-Universität Marburg, Marbacher Weg 6,
D-35032 Marburg
This paper describes the rational design, synthesis, and biological evaluation of a new generation of
inhibitors of the bacterial enzyme tRNA-guanine transglycosylase (TGT), which has been identified as a
new target in the fight against bacillary dysentery (Shigellosis). The enzyme catalyzes the exchange of
guanine in the anticodon wobble position of tRNA by the modified base preQ1, a guanine derivative,
according to a ping-pong mechanism involving a covalent TGT-tRNA intermediate (Fig. 2). Based on
computer modeling (Fig. 3), lin-benzoguanine (6-aminoimidazol[4,5-g]quinazolin-8(7H)-one (2)) was
selected as an extended central scaffold, to form up to seven in-plane intermolecular H-bonds with
the protein while sandwiching between Tyr106 and Met260. Versatile synthetic protocols were developed
for the synthesis of 2, and derivatives with phenyl, benzyl, and 2-phenylethyl side chains (i.e., 16, 17a, and
12a, 12b, 13, 17, resp.) to reach into the lipophilic pocket lined by Val282, Val45, and Leu68 (Schemes
1–3). To account for the limited solubility of the new ligands and in consequence of a recently developed
detailed understanding of the mechanism of TGT catalysis (Fig. 2), the enzyme kinetic assay was com-
pletely redesigned, providing competitive (Kic) and uncompetitive (Kiu) inhibition constants with respect
to tRNA binding by TGT. The modifications of the various parameters in the new assay are described in
detail. Binding affinities of the new inhibitors were found to be in the single-digit micromolar range (Kic
values, Fig. 8). Decoration of the lin-benzoguanine scaffold with lipophilic residues only gave a modest
improvement in biological activity which was explained on structural grounds with the help of four crystal
structures (Fig. 10) obtained by soaking the protein with inhibitors 2 and 12a–12c. Both biochemical and
biostructural analyses reported in this paper provide a fertile basis for the development of more potent
future generations of TGT inhibitors.
1. Introduction. – The bacterial enzyme tRNA-guanine transglycosylase (TGT) is
involved in the infection pathway of Shigella species causing Shigellosis, a bacillary dys-
entery [1]. In the course of our investigations on structure-based drug design of TGT
inhibitors, we recently described a new class of inhibitors based on the 2-aminoquina-
zolin-4(3H)-one scaffold 1a (Fig. 1) [2–4].
In eubacteria, TGT is involved in the posttranslational modification of four specific
tRNAs [5]. It catalyzes the exchange of guanine in the anticodon wobble position by
the modified base preQ1, a 7-deaza-7-aminomethyl derivative of guanine. Kinetic stud-
ꢀ 2006 Verlag Helvetica Chimica Acta AG, Zürich

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Fig. 1. PreQ1 (natural substrate of eubacterial TGT) and queuine (end-product of biosynthetic path-
way) and synthetic TGT inhibitors 1a–1c and 2
Fig. 2. Schematic drawing of the base-exchange mechanism performed by TGT. The exchange of gua-
nine towards preQ1 is performed in sequential steps via a covalent intermediate formed by Asp280.
This intermediate state can be trapped by substrate analogs such as 9-deazaguanine.
ies clearly showed that the reaction pathway follows a ping-pong mechanism (Fig. 2)
[6].
In a first step, unmodified tRNA binds with guanine in position 34 to the active site
of TGT. In a second step, Asp280undergoes nucleophilic attack at C(1 ’) of ribose 34,
cleaving guanine from the tRNA strand. In the resulting intermediate state, tRNA is
covalently bound to Asp280. Guanine is subsequently released from the binding pocket
and replaced by the final substrate preQ1. To activate preQ1 for the nucleophilic attack
on Asp280-C(1’), the base most presumably is deprotonated at position N(9) assisted by

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Asp102 shuffling the proton from the active site via an adjacent H2O molecule. PreQ1 is
then incorporated into tRNA resulting in product formation [7]. The structure of the
covalent intermediate has recently been evidenced by crystal-structure analysis per-
formed on a covalent TGTꢀtRNA intermediate [8]. A complex of TGT, covalently
bound to tRNA, and the unreactive preQ1-substrate analog 9-deazaguanine was trap-
ped in the crystal after guanine had been released at the wobble position. The high rele-
vance of this complex for the proposed enzyme mechanism is confirmed by the fact
that, in the crystal, the non-reactive 9-deazaguanine can be replaced by preQ1, resulting
in the product, which exhibits preQ1 covalently attached to tRNA. In subsequent steps,
performed by other enzymes, the incorporated preQ1 is further modified to queuine,
one of the structurally most dramatically modified nucleobases found in nature [9].
Nucleoside modification is a common feature of tRNA [10], and up to 25% of the
nucleosides show structural changes of the canonical bases [11]. However, the func-
tional role of tRNA modification has yet to be elucidated. Most likely, it is related to
structural stabilization or translational regulation of tRNA via its influence on
codon–anticodon interactions. Variations in tRNA modification have been related to
many human diseases, including cancer or infectious diseases [12]. In this context,
Durand et al. showed that deletion of the gene encoding the tRNA-modifying enzyme
TGT dramatically reduced the virulence of Shigella, the causative agent of bacillary
dysentery (Shigellosis) [1]. Detailed crystallographic studies and biochemical charac-
terization established TGT as a suitable enzymatic target for structure-based drug
design. For these studies TGT from Zymomonas mobilis was used, as TGTs form Shi-
gella are not suited for crystal structure analysis. Both TGTs are identical with respect
to the active site besides one conservative exchange from phenylalanine 106 to tyrosine
[13]. The selective inhibition of TGT could potentially lead to a new class of antibiotics
based upon a unique mode of action. In the following, we present the design, synthesis,
and in vitro evaluation of novel tricyclic TGT inhibitors based on the lin-benzoguanine
scaffold 2.
2. Results and Discussion. – 2.1. Design of the Lead Structure. Available space in the
enzyme active site, as visualized through molecular modeling using the MOLOC soft-
ware package [14], suggested us to use lin-benzoguanine 2 (trivial name for 6-amino-
A
3). Modified nucleobases such as 2 have been introduced nearly three decades ago
by Leonard and co-workers [15][16] but have so far found only limited application
as enzyme inhibitors [17]. In recent work by Kool and co-workers, extended adenine
and thymidine derivatives were employed to construct a stable DNA double helix
with expanded size [18].
Our new lead structure is characterized by the formal insertion of a benzene ring
into the guanine heterocycle, which extends the lateral dimension of the scaffold by
2.4 Š. Compared to the natural substrate preQ1 or 2-aminoquinazolin-4(3H)-one, the
larger aromatic surface is expected to increase the stacking and Van der Waals interac-
tions with Tyr106 and Met260, respectively, two residues forming the top and the bot-
tom of the binding pocket. The scaffold should also retain the same H-bonding pattern
as 1a to Gly230, Gln203, and Asp156, residues involved in the specific recognition of
guanine and preQ1. The H-bond with the C=O of Leu231, seen in the recognition of

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the aminomethyl group in preQ1, is preserved by the presence of the imidazole NH in
lin-benzoguanine.
Attachment of a para-substituted 2-phenylethyl side-chain would be envisaged at
position 4 of the tricyclic scaffold, providing analogous spatial orientation of the lipo-
philic substituent in the apolar enzyme binding pocket as in the previous inhibitor gen-
eration [4].
2.2. Synthesis of the Inhibitors. Commercially available 1H-benzimidazole-6-car-
boxylic acid (3) was esterified to 4 (MeOH, SOCl2) [19] and nitrated (fuming HNO3,
conc. H2SO4) in good yield (Scheme 1). To increase the solubility of benzimidazole 5
in organic solvents, and thus facilitate its purification, the imidazole moiety was pro-
tected by a benzyl group. The two regioisomers 6a and 6b, formed in nearly 1:1
ratio, were readily separated by column chromatography, and their identity was sup-
ported by NOE measurements, whereas correct assignment of the HꢀC(2,4,7) signals
was achieved through 1H,13C-correlation spectroscopy. Further structural evidence was
provided by the crystal structures of two following intermediates (see Sect. 2.3). Reduc-
tion of the NO2 functionality with Zn in AcOH gave anthranilate 7a, and subsequent
iodination (I2, NaHCO3) provided the desired intermediate 8 in high yield.
Scheme 1. Synthesis of Intermediate 8 and Lead Scaffold 2
a) MeOH, SOCl2, 508; 96%. b) HNO3, H2SO4, 508; 68%. c) BnCl, NaH, Bu4NI, THF, D; 93%. d)
G
Zn, AcOH, H2O, 208; 85%. e) I2, NaHCO3, CH2Cl2, H2O, 208; 95%. f) H2, Pd/C (10%), AcOH, D;
98%. g) Chloroformamidinium chloride, Me2SO2, 1508; 32%.
The unsubstituted lead structure 2 was obtained through reduction of 5 (H2, Pd/C,
AcOH), followed by cyclization of the resulting anthranilate 9 with chloroformamidi-
nium chloride.
Sonogashira cross-coupling of 8 with three different alkynes, with [PdCl2(PPh3)2] as
catalyst and CuI as co-catalyst in (i-Pr)2NH provided the desired products 10a–10c in
N
good-to-excellent yield (Scheme 2) [20]. Hydrogenation (H2, Pd/C) of the CꢁC bond
and concomitant deprotection of the benzimidazole moiety yielded 11a–11c.
Interestingly, up to stoichiometric amounts of the Pd catalyst were required for
completion of the two transformations. Finally, cyclization with chloroformamidinium

Helvetica Chimica Acta – Vol. 89 (2006)
Scheme 2. Synthesis of Inhibitors 12a–12c
577
a) [PdCl2(PPh3)2], CuI, (i-Pr)2
ACHTREUNG
chloride provided the desired inhibitors 12a–12c. The tricyclic lin-benzoguanines
showed tautomeric behavior, with respect to the NH position in the imidazole ring,
1
in (CD3)2
G
be eliminated by enhancing the rate of tautomerism through addition of few drops of
D2O and CF3COOD (deuterated TFA). Full protonation of the compounds by TFA
A
was required for the acquisition of highly resolved 13C-NMR spectra. Tautomerism is
influenced by a number of factors including concentration, temperature, solvent
type, and pH (for a minireview, see [21]). Numerous theoretical and experimental stud-
ies have been devoted to the tautomerism of nucleobases, and guanine in particular
[22–24], owing to the possible implication of tautomeric forms in the mispairing and
subsequent replication errors of DNA [25]. Tautomeric equilibria in enzymatic pockets,
although not well understood, can be shifted to higher-energy states through stabiliza-
tion by H2O molecules, binding residues with distinct local pH values, or metal cations
[26]. In the case of TGT–benzoguanine complexation, the interaction of N(3)ꢀH with
the C=O group of Leu231 should provide significant stabilization of one tautomeric
structure as shown in Fig. 3.
Two structural features are responsible for the observed poor water solubility of the
new inhibitors 12a–12c, which could be a major drawback for the development of in
vivo tests: The aminopyrimidone moiety can undergo extensive H-bonding networks,
and the extended flat p–surface of the lin-benzoguanines favors p–p stacking. Solubil-
ity is also very low in apolar solvents as a result of intermolecular H-bonding interac-
tions, which can, however, be disrupted upon addition of dipolar aprotic solvents such
as DMF or Me2SO.
N
To increase the solubility, inhibitors were prepared lacking either the exocyclic NH2
group in position 6, to reduce the H-bonding propensity, or featuring a Ph group
orthogonal to the planar heterocycle, to prevent p–p interactions. Thus, the cyclization
of 11a with formamidinium acetate in EtOH afforded the desired inhibitor 13 without
exocyclic NH2 group (Scheme 3). Moreover, Suzuki cross-coupling of the iodinated

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Fig. 3. Schematic drawing of the binding mode of a lin-benzoguanine-based inhibitor into the active
site of TGT. The H-bonds and selected short apolar contacts are indicated as dotted lines.
Scheme 3. Synthesis of Inhibitors 13, 16, 17a, and 17b
a) Formamidinium acetate, EtOH, 808; 81%. b) Phenylboronic acid, [Pd(PPh3)4], K2CO3, EtOH/
H2O/toluene, 808; 87%. c) H2, Pd/C (10%), EtOH, AcOH, 208; 78%. d) Chloroformamidinium chlo-
ride, Me2SO2, 1508; 38–85%. e) PhCH2CH2Cl, NaH, THF, 808, 12 h. f) H2, Pd/C, AcOH, 208, 12 h;
81%.

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intermediate 8 with phenylboronic acid in a solvent mixture of toluene, EtOH, and H2O
gave ꢁbiaryl-typeꢂ 14 in high yield (87%) [27].
Removal of the Bn group of 14 by hydrogenation and cyclization of the resulting
anthranilate 15 furnished the desired inhibitor 16. Gratifyingly, inhibitor 13 was soluble
in hot MeOH, thus allowing purification by column chromatography (CH2Cl2/MeOH
95 :5), whereas 16 showed no improvement in solubility in protic solvents and could
only be dissolved in Me2SO or DMF.
G
Two additional inhibitors, 17a and 17b with Bn or 2-phenylethyl side chains at the
benzimidazole N-atom were prepared (Scheme 3). Thus, 17b was obtained from 5 by
alkylation with 2-phenylethyl chloride (! 18), reduction (! 19), and cyclization.
2.3. Crystal Structures of Intermediates 7b, 8, and 10b. The constitution of 7b, with
the Bn group adjacent to C(7), was supported by X-ray crystal structure analysis
(Fig. 4,a). Two conformers with different orientations of the Bn group are present in
the unit cell forming two nested coils of H-bonded molecules (N(21)···O(18) 3.02/
3.01 Š) around a 31 axis. The tautomeric structure of 8 was also confirmed by X-ray
crystallography (Fig. 4,b). Also the position of the NH2 group suggests intermolecular
NꢀH/p interactions [28] between a pair of molecules 8 (N-centroid distance: 3.25 Š).
One of the H-atoms forms an intramolecular H-bond with O(19) and the second H-
atom points to C(15) of the Ph ring. Furthermore, the crystal packing features an
edge-to-face aromatic interaction with a centroid-to-centroid distance of 4.96 Š. The
crystal structure of compound 10b revealed a nearly perfect planar alignment of the
central core and the phenylethynyl moiety (Fig. 4,c). It is noticeable that the lowest-
energy conformation of tolane in the gas phase features an orthogonal alignment of
the two aromatic rings [29]. In the crystal lattice of 10b, dimers are formed with a
O(20)···N(21) interaction (shared with an intramolecular N(21)···O(20) H-bond)
through a centre of symmetry that arrange with the next layer to show an antiparallel
dipolar alignment of two C=O groups (distance between the two antiparallel C=O
bonds: 3.56 Š).
2.4. Biological Activity and Crystallographic Studies. All newly synthesized inhibi-
tors were tested for their inhibitory potency against TGT (Z. mobilis). Furthermore,
we succeeded to determine the crystal structures of four inhibitors presented in this
contribution in complex with Z. mobilis TGT.
2.4.1. Adapted Kinetic Assay. Faced by the problem of low solubility of some of our
ligands and in consequence of a more-detailed understanding of the base exchange
mechanism involving competitive and non-competitive inhibition, we had to redesign
our enzyme kinetic assay. To appreciate these findings, several parameters of the pre-
viously applied assay were modified [13]. In the following, we present the motivation
for each modification and its implications on the assay design.
2.4.1.1. Ligand Solubility. Some of our compounds exhibit low solubility under the
applied assay conditions. Effects of low solubility can be manifold. As a consequence of
possible precipitation, the resulting inhibition constants could be underestimated.
However, also the opposite could be observed. As recently reported, the risk of unspe-
cific inhibition exists for a large variety of compounds in particular if low solubility is
given. Such behavior has been referred to as ꢁpromiscuous inhibitionꢂ [30]. These com-
pounds show up repeatedly as inhibitors of various enzymes in biological assays. How-
ever, they do not follow a target-specific mode of action. Instead, such behavior is

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Fig. 4. a) X-Ray crystal structure of 7b (left) with unit cell (right). b) X-Ray crystal structure of 8
(left) with the N–H/p interactions (middle), and the unit cell (right). c) X-Ray crystal structure of 10b
(left) with the unit cell (right). Arbitrary numbering. Atomic displacement parameters in the ORTEP
plots obtained at 293 K are drawn at the 50% probability level.

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581
thought to result from some sort of compound agglomeration that adsorbs the enzyme
onto its aggregated surface. In consequence, the enzyme is inactivated. Adding deter-
gents such as Tween 20 or Triton X-100 to the respective assay solutions obviously
reduces aggregate formation and thus allows to distinguish ꢁpromiscuousꢂ from specific
inhibitors [31]. To avoid possible effects of ꢁpromiscuous inhibitionꢂ or other undesired
precipitation effects in the aqueous TGTassay, Tween 20 was added in 5% of its critical
micellar concentration (CMC) of 59 mM. The amount of Tween 20 used is based on the
results reported by Ryan et al. [31].
2.4.1.2. TGT Solubility and Kinetic Parameters. As a supplementary effect, the use
of detergents influences not only ligand but also protein solubility [31]. In particular
Tween 20 was described to reduce protein absorption onto plastic surfaces of cups usu-
ally used for kinetic measurements. Such artificial immobilization results in a reduced
effective enzyme concentration in the assay solution [31]. Therefore, we reexamined
the kinetic parameters for TGT to quantify the possible impact of Tween 20. Further-
more, accurately determined kinetic parameters are required to derive inhibition con-
stants. Km Values for TGT with respect to both substrates tRNATyr and [8-3H]guanine
(Table) remain within the range already published for Z. mobilis TGT (Km
ACHTREUNG
3
0.2–1.0 mM; Km
ACHTREUNG
as alternative substrate instead of natural preQ1, for which no labeled material was
available.
Table. Kinetic Characterization of Wild Type TGT with tRNATyr and [8-3H]Guanine as Substrate in
Presence of Tween 20( c=5% CMC)
Parameter
[3H]guanine
New values
tRNATyr
Previous valuesa)
New values
Previous valuesa)
Km [mM]
1.2Æ0.2
0.38
0.9Æ0.2
1.0
kcat [sꢀ1
]
2.8·10ꢀ2 Æ0.1·10ꢀ2
1.1·10ꢀ2
2.9·10ꢀ2
2.7·10ꢀ2 Æ0.1·10ꢀ2
3.0·10ꢀ2 Æ0.7·10ꢀ2
1.4·10ꢀ2
1.4·10ꢀ2
kcat/Km [mM sꢀ1
]
2.3·10ꢀ2 Æ0.6·10ꢀ2
ꢀ1
a) Previous values as reported in [33].
In contrast, kcat values significantly higher than those reported in [33] are obtained.
Most likely, this observation results from a higher effective TGT concentration in the
Tween 20-containing assay solution. Accordingly, we assume that the principal catalytic
properties are not affected by added Tween 20.
2.4.1.3. Preincubation of TGTwith Inhibitor. Pre-incubation of Z. mobilis TGTwith
a putative inhibitor was reported to affect the initial velocity of the enzyme-catalyzed
reaction [33]. A 10-min pre-incubation of TGT with the inhibitor to be tested prior to
the addition of substrate showed reduced initial velocity by ca. 50%. In contrast to this
finding, the addition of Tween 20 (c=5% CMC) rather resulted in a slight increase of
the initial velocity after a 10-min pre-incubation. Compared to tests in the absence of
Tween 20, the initial velocity after 10-min preincubation increased by a factor of
three. We assume that the previously observed pre-incubation effect resulted from a
superimposed slow adsorption/desorption process of the enzyme from the plastic
cups, which is unmasked by the use of Tween 20.

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2.4.1.4. Competitive and Uncompetitive Inhibition. Recent kinetic and crystallo-
graphic studies provide a better insight into the molecular details of the TGT base-
exchange reaction [6][8]. Competitive and uncompetitive inhibition of the binding of
tRNA as substrate have to be considered with respect to the stepwise enzyme reaction,
as already assumed in [4]. Due to these considerations, the originally applied assay pro-
tocol to determine inhibition constants (Ki) had to be altered.
The TGT-catalyzed reaction involves two substrates, tRNA and [8-3H]guanine. For
each, TGT provides a distinct binding pocket partially formed upon substrate binding.
While tRNA interacts with apo TGT, [8-3H]guanine binds in a subsequent step into a
modified binding pocket partially formed by the covalently bound tRNA. The crystal
structure of the ꢁternaryꢂ TGT-tRNA·9-deazaguanine complex suggests that com-
pounds of similar size such as the competitive substrate mimic 9-deazaguanine can
be accommodated in this binding pocket via non-covalent interactions (Fig. 2). Accord-
ingly, this site can either accommodate active substrates (e.g., guanine or preQ1) or
other small ligands such as 9-deazaguanine. In the series of our studied inhibitors,
some compounds exhibit molecular dimensions similar to the natural substrates, in par-
ticular our lead structures 1a and 1b, and 2. Once decorated by additional aliphatic sub-
stituents, as in 12a–12c or 16, steric clashes with the covalently bound tRNA ribose
moiety at position 34 can be expected. Accordingly, it can be assumed that these
extended compounds compete only with tRNA binding to uncomplexed TGT (Fig. 5).
To verify this assumption, a trapping experiment, based on the covalent TGT–RNA
intermediate was performed, similar to the study of Xie et al., using 9-deazaguanine [8].
In different reaction mixtures, TGT was first incubated with an excess of tRNA
Fig. 5. Superimposition of TGT crystal structures in complex with tRNA and inhibitor 12a. Shown in
orange/yellow colors is the TGT active site in complex with 9-deazaguanine and tRNA covalently
bound to Asp280. Shown in grey/blue colors is TGT in complex with 12a in two alternative confor-
mations. The 2-phenylethyl substituent of 12a clearly interferes with the tRNA binding site. Color
coding: N: blue, O: red, P: purple.

Helvetica Chimica Acta – Vol. 89 (2006)
583
together with various inhibitors, i.e., 1a, 1b, 2, 12a–12c. Then, an SDS-PAGE analysis of
these mixtures was performed (Fig. 6). Bands are observed for uncomplexed TGT at
43.5 kDa (Fig. 6, Lane 2). Incubation of TGT with tRNA results in two additional,
faint, retarded bands at about 70kDa ( Fig. 6, Lane 3). These bands correspond to cova-
lent TGT–tRNA complexes [34]. The detection of two bands for the covalent complex
results from different tRNA conformers present under the applied PAGE conditions as
already reported in [35]. The occurrence of well-defined bands for TGT–tRNA in the
presence of small inhibitors such as 1a, 1b, and 2 (Fig. 6, Lanes 7–9) clearly indicates
the power of these ligands to stabilize the covalent intermediate similarly to 9-deaza-
guanine [8]. Once exposed to inhibitors exhibiting a large substituent at their basic scaf-
folds such as 12a–12c (Fig. 6, Lanes 4–6), the respective bands are only very faint, sim-
ilar to the situation when pure TGT is mixed with tRNA in absence of a small molecule
inhibitor (Fig. 6, Lane 3). This finding indicates that inhibitors of the size of 12a–12c
are not capable to stabilize the TGT–tRNA complex.
Fig. 6. SDS-PAGE Analysis of trapping experiments performed with TGT/tRNA mixtures together
with inhibitors of different size. While small inhibitors, 1a, 1b, and 2 cause retarded TGT bands by
stabilizing the covalent TGT-tRNA intermediate, functionalized, more extended inhibitors, 12a–12c,
lack this ability. The covalent band is as faint as in pure TGT/tRNA mixtures. SDS-7 Marker (mix-
ture of proteins with different sizes) was used to estimate molecular weight.
In consequence, we assume that size-dependent inhibition models with respect to
tRNA and [3H]guanine as limiting substrates have to be considered in the Ki determi-
nation. Inhibitors with large side chains are expected to be competitive only with
tRNA, binding to TGT as substrate. Small inhibitors of comparable size to the natural
nucleobase substrates exhibit a more complex inhibition potency. They either occupy
the base-exchange site prior to tRNA binding to TGT, a step which is, to some extent,
competitive with tRNA binding. That such kind of binding is possible can be concluded
from the formation of binary TGT·inhibitor complexes seen in many crystal structures
[3][4]. Alternatively, they compete with preQ1 or guanine after tRNA is covalently
attached to TGT. Accordingly, to compare small guanine-sized and larger, extended
inhibitors, their binding constants determined with respect to tRNA binding are
required to provide directly comparable inhibitory values. In our previously applied

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Fig. 7. Inhibition of TGT with respect to tRNA binding, resulting in competitive and uncompetitive
inhibition constants
assay protocol, inhibition constants were determined with respect to [8-3H]guanine as
limiting substrate. In the new protocol [8-3H]guanine is used in 20 mM excess, while the
tRNATyr concentration is now limiting.
Nevertheless, for inhibitors of the guanine-type size two inhibition constants have to
be defined that fully characterize their TGT inhibition with respect to tRNA binding
(Fig. 7).
Small inhibitors such as 1a, 1b, and 2 might act as competitive inhibitors with
respect to tRNA binding (Kic), occupying the base-recognition site of TGT prior to
tRNA binding. If their binding occurs subsequently to tRNA binding, and after cleav-
age and release of the natural substrate guanine from the base-recognition site, stabi-
lization of the covalent TGT–tRNA adduct will be the result. In this case they act as
uncompetitive inhibitors with respect to tRNA binding (Kiu). Such a competitive/
uncompetitive inhibition process can be described by a double reciprocal Linewea-
ver–Burk plot according to Eqn. 1. This relationship exhibits two inhibition constants,
Kic and Kiu. While competitive inhibition results in a raise of Km, uncompetitive inhib-
ition decelerates Vmax. Both inhibition constants contribute to the total inhibition of
TGT with respect to tRNA binding, and both have to be considered to determine
the amount of compound needed to suppress the readout of the assay, namely the incor-
poration of [8-3H]guanine in tRNA replacing unlabeled guanine. In the literature, such
a composite type of inhibition is referred to as mixed or non-competitive inhibition
[36][37].
ꢂ
ꢀ
ꢁꢃ
ꢀ
ꢁ
1
v0
Km
Vmax
½Iꢂ
1
½Sꢂ
1
Vmax
½Iꢂ
¼
Á
1 þ
Á
þ
Á
1 þ
Kic
Kiu
(1)
For inhibitors with a large side chain such as 12a–12c and 16, the competitive inhib-
ition constant Kic is supposedly sufficient to fully describe their inhibition properties, as
these compounds are too large to be significantly accommodated by the binding pocket

Helvetica Chimica Acta – Vol. 89 (2006)
585
as long as the covalent TGT–tRNA adduct is formed. Kiu is hardly relevant in this case,
and the respective inhibition type can be described as predominantly competitive. Eqn.
2 describing the latter situation results from a simplification of Eqn. 1. The competitive
inhibition constant Kic either for guanine-sized or substituted inhibitors describes in
both cases the potential to impede tRNA binding to TGT. Thus, the Kic values for
both types of inhibitors are directly comparable.
ꢂ
ꢀ
ꢁꢃ
1
v0
Km
Vmax
½Iꢂ
1
½Sꢂ
1
Vmax
¼
Á
1 þ
Á
þ
Kic
(2)
The modified assay conditions take impact on the resulting inhibition constants.
Therefore, it is difficult to directly link our previously reported inhibition constants
to the re-evaluated ones presented in this study. In particular the replacement of
[8-3H]guanine by tRNA as limiting substrate, the appropriate consideration of a super-
imposed uncompetitive inhibition mode, and the addition of a detergent to enhance
solubility modulates the inhibition constants to some, however, across the entire data
set not linear fashion. Accordingly, re-evaluation of the most relevant compounds,
reported earlier, appeared essential.
2.4.2. Inhibition Constants. Inhibition constants for lin-benzoguanine 2 and some N-
and 4-substituted derivatives, 12a–12c, 13, 16, 17a and 17b, are given in Fig. 8. As ligand
2 was identified as competitive/uncompetitive inhibitor, we also decided to re-evaluate
several of the small, guanine-size inhibitors, 1a–1d.
Fig. 8. Biological activities (Kic and Kiu values) of quinazolinone-, pteridine-, and lin-benzoguanine-
based inhibitors

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Helvetica Chimica Acta – Vol. 89 (2006)
Ligands 1a and 1b were identified by the trapping experiment as uncompetitive
inhibitors (Sect. 2.4.1.4). Their Kiu constants indicate decreasing power to stabilize
the covalent adduct from 1a (0.6 mM), over 1b (1.7 mM), to 2 (7.9 mM). This descending
order is also reflected by the intensities of the bands at 70kDa ( Fig. 6, Lanes 7–9). Fac-
ing the competitive inhibition constants (Kic) also shows a slightly reduced potency of 2
(4.1 mM) compared to 1a (1.5 mM) and 1b (2.1 mM). Our previously reported, too-low
inhibition constant of 1b (20–50n M) results from the neglect of the uncompetitive
inhibition in the old assay [4]. Therefore, in particular the small guanine-type inhibitors
have been overestimated with respect to their inhibition power. To verify these obser-
vations, the natural compound pteridine 1d, previously described as potent inhibitor in
the higher nanomolar range [38], was also tested for mixed inhibition. With a Kic value
of 2.2 mM, it is a competitive inhibitor in a similar activity range to 1b, while its uncom-
petitive inhibition of 0.7 mM is in the range of that of 1a.
Ligand 1c, a Me-substituted derivative of 1b also shows competitive/uncompetitive
inhibition, although the competitive contribution dominates (Kic =3.7 mM vs. Kiu =19.1
mM). Consistently, the 70-kDa covalent bands in the respective trapping experiment are
less pronounced than for 2 (data not shown). In structural terms, this observation can be
explained by spatial conflicts, with the attached Me group in 1c most likely interfering
with the ribose ring 34 of the covalently bound tRNA. This effect is even more pro-
nounced for inhibitors bearing larger side chains at this position.
Compounds 12a–12c, 13, 16, 17a, and 17b almost purely act as competitive inhibi-
tors. In the trapping experiment, only faint covalent bands remained detectable (Fig. 6;
data shown only for 12a–12c). Ligand 12a exhibits the lowest Kic value (1.0 mM) in the
studied series (Fig. 8), resulting in a modest enhancement over 2 by a factor of 4. How-
ever, a weak uncompetitive contribution of 12a (Kiu =40–70 mM) could be determined
as well. The value of Kiu is separated from Kic by almost two orders of magnitude. Thus,
it does not contribute significantly to the total inhibition. Nevertheless, obviously bind-
ing of 12a is still possible once tRNA is covalently attached to TGT (cf. Fig. 5). Suppos-
edly, the 12a side chain adopts a conformation that allows for its non-repulsive place-
ment next to the bound tRNA. In consequence, a marginal uncompetitive contribution
to inhibition, undetectable by trapping experiments, cannot be excluded also for other
substituted inhibitors. As it was not feasible to determine Kiu for all substituted inhib-
itors, this presumably marginal contribution, indicated by the trapping experiments, has
been neglected in the kinetic measurements. Only Kic values have been determined for
these compounds.
Compound 13 lacks the C(6)ꢀNH2 group compared to ligand 12a. Its loss in affinity
demonstrates the importance of this functionality for binding (13 vs. 12a: Kic 2.8 mM vs.
1.0 mM) mediating polar interactions to Asp156 and Asp102 (see Fig. 3). Additional
substitution of the Ph ring in compound 12a with a p-Me (12b; Kic 6.9 mM) or a p-
MeO group (12c; Kic 3.7 mM) appears detrimental to binding. Immediate attachment
of the Ph group in 16 results in a substantial loss in activity (Kic 29 mM). Similarly, the
N(3)-substituted benzoguanines experience a loss in affinity (17a, 15.4 mM; 17b, 7.3 mM).
Fig. 9 shows the inhibition constants for previously described inhibitors measured
under our new assay conditions [2][4]. All compounds were identified to be primarily
competitive inhibitors via trapping experiments. Competitive inhibition constants (Kic)
for most phenyl-substituted quinazolinones were determined to be in the single-digit

Helvetica Chimica Acta – Vol. 89 (2006)
587
mM range. The large difference in binding affinity between S-, O-, and C-atoms in the
linker is no longer observed. Compounds with substituents other than unsubstituted
Ph rings show reduced affinity.
Fig. 9. Biological activities (Kic values) of quinazolinone-based inhibitors (from [4][28])
2.4.3. Crystal-Structure Analysis. Some of the newly synthesized inhibitors were
crystallized as binary complexes with TGT. Although quite low (see Sect. 2.4.1.1), the
solubility of the lin-benzoguanine-based inhibitors is superior to that of the quinazoli-
none-based ligands presented in [3][4], which allowed the application of soaking meth-
ods. Thus, crystals with unsubstituted 2 and 12a–12c could be obtained, whereas
reduced solubility of 16, 17a, and 17b impeded similar attempts. Details on the crystal
structure analyses will be given in [39]. Fig. 10,a shows how the core scaffold 2 is
involved in an H-bonding pattern, as expected from modeling (Fig. 3). With respect
to structures of uncomplexed TGT, Asn70adopts a shifted conformation, thus inducing
a slightly altered binding pocket. Ligand 12a adopts in the crystal two different, almost
equally populated conformations. The 2-phenylethyl substituent is oriented either
above (up-conformation) or below (down-conformation) a best plane through the tri-
cyclic ring system, respectively. In the up-conformation, the adjacent Asn70is oriented
similarly as in the complex with 2. In contrast, in the down-conformation, Asn70resides
in a geometry similar to uncomplexed TGT (conformational change indicated by arrow
in Fig. 10,b). In ligand 12b, the side chain is nearly exclusively found with limited accu-
racy in the up-conformation (Fig. 10,c), while Asn70occurs equally populated in both
already mentioned conformations. Possibly the down-conformation for the ligand side
chain is hardly adopted due to repulsive contacts of the p-Me group with Asn70in
either of its two orientations. Finally, 12c with a p-MeO group occurs in only one con-
formation with respect to both side chain and Asn70( Fig. 10,d). Surprisingly, in this
case, the side chain adopts the down-conformation; however, with limited accuracy
for the side chain as well as for the scaffold electron density. Asn70is found in a geom-
etry similar to uncomplexed TGT. The latter three structures point to substantial con-
formational adaptations of the TGT binding pocket next to Asn70upon ligand binding.
Depending on the conformational properties of the side chain and its steric demand,
conformational disorder is observed to varying extent.
The crystal structures provide an explanation for the only marginal gain in inhibi-
tion power upon decoration of the central scaffold with a substituted 2-phenylethyl
side chain (e.g., 12a–12c compared to parent scaffold 2). Despite significant gain in lip-
ophilic surface, Kic values do not improve tremendously. The particular positioning of
the 1,2-ethanediyl linkers in 12a–12c, connecting the parent scaffold with the phenyl
moiety, provides a possible explanation for this observation. They protrude into the
highly polar solvation spheres of Asp280, the catalytic nucleophile, and Asp102,

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Helvetica Chimica Acta – Vol. 89 (2006)
Fig. 10. Four different crystal structures of TGT–inhibitor complexes. All complexes were obtained by
soaking TGT crystals with a solution of the inhibitor at pH 5.5; dotted lines: H-bonds. TGT is com-
plexed to: a) 2, red spheres are isolated bound H2O molecules; 1.7 Š resolution. b) 12a, 1.58-Š Reso-
lution; light blue residue: alternative up-conformation of 12a with induced conformational change at
Asn70. c) 12b, 1.58-Š Resolution; arrow: unfavorable intermolecular contact. d) 12c, 2.1-Š Resolu-
tion. Color coding: N: blue, O: red, S: yellow, C: grey.
involved in deprotonation of the substrate base. Thus, unfavorable proximities between
a CH2 moiety of the linker and a side chain O-atom of Asp102 are clearly visible in the
complex of 12b (d(C···O) 3.2 Š) and 12c (d(C···O) 3.1 Š.) Presumably, this penetra-
tion is detrimental to binding affinity. If this area remains unoccupied, such as with
bound 2, these polar residues are involved in a subtle crystal H2O network (Fig.
10,a). Supposedly, any rupture of this solvation network reduces affinity. This loss is

Helvetica Chimica Acta – Vol. 89 (2006)
589
also reflected by the higher Kic value of 1c (3.7 mM) compared to 1b (2.1 mM). Crystal-
structure analyses of both compounds complexed with TGT also indicated repulsive
interactions of the Me group in 1c with Asp102 and the neighboring H2O molecules
[4]. Due to these unfavorable interactions, we assume that 12a–12c suffer significant
affinity loss, which is only partially compensated by the additional hydrophobic inter-
actions experienced in the newly created lipophilic subpocket next to Asn70, lined
by the side chains of Val282, Val45, and Leu68. Across the series, 12a seems to better
match the lipophilic pocket than 12b and 12c, resulting in a better defined electron den-
sity for the side chain. This provides also a structural explanation for the lower Kic value
of 12a.
3. Conclusions. – In this study on the rational development of tRNA-guanine trans-
glycosylase (TGT) inhibitors, we reported the introduction of a new scaffold based on
lin-benzoguanine 2. Versatile synthetic protocols were developed (Schemes 1–3), and
binding affinity (competitive inhibition constants Kic with respect to tRNA binding)
in the single-digit mM range could be achieved (Fig. 8). The crystallographic studies
demonstrated a ligand-induced widening at Asn70of the apolar distal binding pocket
lined by the side chains of Val282, Val45, and Leu68 (Fig. 10). The crystal structures
show unfavorable contacts between the 2-phenylethyl side chains of inhibitors
12a–12c and the carboxylate of Asp102, presumably responsible for the only moderate
binding affinity enhancement of these ligands with respect to the lead scaffold 2. In pre-
vious medicinal chemistry-related projects from this laboratory, modeling was per-
formed based upon different levels of flexibility of the binding pocket, ranging from
rigid in the case of thrombin to highly flexible in plasmepsin II, in which the flap pocket
undergoes major conformational changes (for a short review, see [40]). Our studies on
TGT emphasize its moderate plasticity, with a flip of the peptidic backbone at Leu231/
Ala232 and a rotation of Asp102 as described in [4][33], and ligand-induced conforma-
tional changes at Asn70, as observed in this study. During the design cycle, our under-
standing of TGT evolved, therefore, from a rigid to a fairly flexible enzymatic pocket,
wherein subtle variations in inhibitor structure were responsible for critical changes in
activity. From a biosynthetic point of view, moderate conformational flexibility of TGT
is needed to perform the complex transglycosylation reaction on specific tRNAs with-
out external energy sources (co-factors) and to achieve the functionally required sub-
strate promiscuity.
The presence of tRNA in the binding assay complicates, however, the interpretation
of biological data. Thus, band-shift experiments suggest different mode of action for
our designed inhibitors (Fig. 6). Whereas all inhibitors, including side-chain-bearing
ligands such as 12a–12c, act as competitive inhibitors with respect to tRNA, only
small inhibitors such as 1a, 1b, or 2 additionally stabilize the covalent tRNA–TGT inter-
mediate in a ꢁternaryꢂ complex. The structural information gained in this work provides
fertile ground for further development of TGT inhibitors.
We are grateful to the ETH Research Council and the Fonds der Chemischen Industrie for support of
the work done at the ETH. The work at the Philipps University of Marburg was supported by the Deut-
sche Forschungsgemeinschaft (Grants KL1204/1-3 and KL1204/9-1) and the Graduiertenkolleg ꢁProtein

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Helvetica Chimica Acta – Vol. 89 (2006)
Function at the Atomic Levelꢂ. We also thank Dr. A. Heine for his help in protein X-ray analysis, and P.
Hoffmann and T. Jansen for their help in tRNATyr production (all Philipps University of Marburg).
Experimental Part
General. Solvents and reagents were reagent-grade, purchased from commercial suppliers, and used
without further purification unless otherwise stated. Chloroformamidinium chloride was prepared
according to the procedure described in [41]. THF was freshly distilled from sodium benzophenone
ketyl. Evaporation in vacuo was conducted at H2O aspirator pressure. All products were dried under
high vacuum (h.v., 10ꢀ2 Torr) before anal. characterization. Column chromatography (CC): SiO2 60
(40–63 mm) from Fluka, 0–0.3 bar pressure. TLC: SiO 2 60 F245, Merck, visualization by UV light at
254/356 nm. M.p.: Büchi B540 melting-point apparatus; uncorrected. IR Spectra [cmꢀ1]: Perkin-Elmer
1600-FT spectrometer. NMR Spectra (1H,13C): Varian Gemini-200, Varian Gemini-300, or Bruker
AMX-500; spectra were recorded at 298 K with solvent peaks as reference. MS (m/z (%)): EI-MS:
VG-TRIBRID spectrometer at 70eV; ESI-MS: Perkin-Elmer Sciex API III spectrometer; HR-
MALDI-MS: IonSpec Ultima (2,5-dihydroxybenzoic acid (DHB) matrix). Elemental analyses were per-
formed by the Mikrolabor at the Laboratorium für Organische Chemie, ETH-Zürich. The nomenclature
was generated with the computer program ACD-Name (ACD/Labs) [42].
Crystallization and Soaking. TGT was crystallized and soaked with inhibitors at pH 5.5 as described
in [38].
Determination of Kinetic Parameters and Inhibition Constants. The method has been modified
according to the initial procedure described by Reuter et al. [43] and Graedler et al. [13].
Assay Conditions and Workflow. The determination of TGTactivity was carried out in 75-ml mixtures
containing 150n M Z. mobilis TGT and variable concentrations of E. coli tRNATyr, guanine (7.5% radio-
actively labeled [8-3H]guanine (Hartmann Analytik)), and inhibitor. The assay was performed in 200 mM
HEPES buffer, pH 7.3, 20 mM MgCl2, and 2.95 mM =5% CMC Tween 20 (Roth). As some inhibitors were
added to the assay soln. dissolved in Me
A
contained up to 5% Me
AHCTREUNG
the protein soln. Prior to substrate addition, protein and protein/inhibitor mixtures were pre-incubated
at 378 for 10min to allow TGT to adjust to the assay temp. to reach maximum velocity. The mixture was
kept at 378, and 15-ml aliquots were taken at intervals of 1 to 4 min. Aliquots were immediately transfer-
red to glass fiber (GC-F) filters (Whatman) and quenched with 10% (w/v) Cl3CCOOH at 08 for 15 min.
Unbound guanine was washed from the filters in 7-min intervals twice with 5% (w/v) Cl3CCOOH and
twice with EtOH. Filters were dried at 608 for 45 min. 3H incorporated into tRNA was quantified
using liquid scintillation counting. Results from the aliquots were used to calculate initial velocity
using GraFit [44].
Kinetic Paramters. Michaelis–Menten parameters for tRNA and guanine were determined separately
in triplicate. Average values are given in the Table. Kinetic parameters for guanine were measured using
75 nM TGT, 15 mM tRNA, and variable concentrations of guanine/[8-3H]guanine (0.5–20 mM). Kinetic
parameters for tRNA were measured using 75 nM TGT, 20 mM guanine/[8-3H]guanine, and variable con-
centrations of tRNA (0.25–15 mM). Initial velocities in counts per minute were transferred to [mM/min]
using a calibration constant derived from liquid scintillation counting of guanine/[8-3H]guanine solns.
with variable concentrations. Kinetic parameters were determined via double-reciprocal linearization
and linear regression using GraFit.
Inhibition Constants for Pure Competitive Inhibition. The inhibition assay was performed using 150
nM TGT, 20 mM guanine/[8-3H]guanine, and tRNA at two concentrations (1 and 1.5 mM). Six reaction mix-
tures for each tRNA concentration were prepared. To five of them, inhibitor dissolved in Me2
final volume) at variable concentrations was added. Initial velocities for the reaction mixtures were deter-
mined, and Kic determination was performed using Dixon plots. As Vmax determination is only possible
SO (5%
AHCTREUNG
with limited accuracy for independent measurements, a modified equation published by Graedler et al.
was used to calculate Kic [13]:

Helvetica Chimica Acta – Vol. 89 (2006)
591
(3)
ꢀ
ꢁ
v0 Km þ ½Sꢂ
1
Kic
½Sꢂ
Á
¼
Á ½Iꢂ þ
þ 1
vi
Km
Km
Linear regression of data points derived from Eqn. 3 with GraFit results in a straight line with the
slope 1/Kic.
Inhibition Constants for Mixed Inhibition. The inhibition assay was performed using 75 nM TGT, 20
mM guanine/[8-3H]guanine, and variable tRNA concentrations (0.25–15 mM). Kinetic parameters were
determined once in the absence of inhibitor (Km, Vmax) and twice in the presence of specific inhibitor con-
centrations [I] to calculate (Kampp, V ) via double-reciprocal linearization and linear regression using
app
max
GraFit. Contributions of Kic and Kiu towards pure non-competitive inhibition can be calculated from
the following Eqns. 4 and 5:
½Iꢂ
app
max
ꢄ
ꢅ
Kiu
¼
(4)
(5)
Vmax
V
ꢀ 1
½Iꢂ
ꢄ
ꢅ
Kic
¼
KmappÁVmax
ꢀ 1
KmÁVapp
max
The following nomenclature was applied: Vmax: maximum velocity of uninhibited reaction, v0: initial
velocity at given [S] concentration in the absence of inhibitor, vi: initial velocity at given [S] concentration
in the presence of inhibitor, [S]: tRNA concentration, [I]: inhibitor concentration, Km: Michaelis–Menten
constant. Due to the elaborate Ki determination procedure, the average error for the single values is esti-
mated to be in the range of Æ25%.
SDS-PAGE Trapping Experiment. 5 mM Z. mobilis TGT, 100 mM E. coli tRNATyr, and 1 mM of the
respective inhibitor (dissolved in Me2SO) in 10 ml of 10 0 Mm HEPES buffer, pH 7.3, 20m M MgCl2,
G
and 5 mM dithiothreitol (DTT) were incubated for 1 h at 258. A total of 10 ml of SDS loading buffer
was added and incubated for another 1 h at 258. A total of 10 ml of each sample was loaded onto a
15% SDS gel.
Cyclization with Chloroformamidinium Chloride: General Procedure 1 (GP 1). A mixture of the
anthranilate (1.0equiv.), chloroformamidinium chloride (1.5 equiv.), and dimethyl sulfone (5.00
equiv.) was heated to 1508 for 1.5–3 h. After addition of 25% aq. NH3 soln. (1–2 ml), the mixture was
diluted with H
N
N
crude was precipitated from hot DMF and H2O and dried under h.v. at 808.
Sonogashira Cross Coupling: General Procedure 2 (GP 2). To a suspension of the iodinated 1H-benz-
A
degassed acetylene (1.5 equiv.) was added. The mixture was heated to 508 for 3 h, and the solvent was
removed in vacuo. The residue was taken up in sat. aq. NaCl soln. and extracted with CH2Cl2 (3”).
The combined org. phases were dried (MgSO4) and concentrated in vacuo.
Debenzylation and Hydrogenation: General Procedure 3 (GP 3). To a soln. of benzylated 1H-benz-
A
under H2 (4 bar) for 12 h. The mixture was filtered through Celite, concentrated in vacuo, and the
crude was purified by CC.
Methyl 1H-Benzimidazole-6-carboxylate (4). To a suspension of 1H-benzimidazole-6-carboxylic acid
(2.0g, 12.3 mmol) in MeOH (20ml), SOCl 2 (1.4 ml, 19.0mmol) was added cautiously at 0 8, and the mix-
ture was heated to 508 for 12 h. After slow addition of H2O (35 ml) and removal of MeOH in vacuo, the
U
residue was adjusted to pH 6 with sat. aq. NaHCO3 soln. and extracted with AcOEt (3”). The combined
org. phases were dried (MgSO4) and concentrated in vacuo to give 4 (2.1 g, 96%). Yellow solid. M.p.
133–1358. IR (KBr): 3447m, 3110m, 2803m, 1713s, 1623m, 1428m, 1400m, 1312s, 1226m, 1200m,
1
1081m, 886s, 824s, 785s, 768m, 745m. H-NMR (300 MHz, (CD3)2SO): 12.80(br. s, 1 H); 8.41 (s, 1 H);
8.23 (s, 1 H); 7.83 (d, J=8.4, 1 H); 7.68 (d, J=8.4, 1 H); 3.87 (s, 3 H). 13C-NMR (75 MHz, (CD3)2
ACHTREUNG
A
G
A
ACHTREUNG

592
Helvetica Chimica Acta – Vol. 89 (2006)
Methyl 5-Nitro-1H-benzimidazole-6-carboxylate (5). To fuming HNO3 (10ml), conc. H 2SO4 (10ml)
and subsequently 4 (6.9 g, 39.1 mmol) were added at 08 under stirring. The mixture was heated to 508 for
12 h, cooled to 208, poured on ice, and filtered. The residue was washed with H2O, taken up in H2O/
AHCTREUNG
AcOEt, and the pH adjusted to 7–8 with conc. aq. NaOH soln. The aq. phase was extracted with
AcOEt (3”), and the combined org. phases were dried (MgSO4) and evaporated in vacuo to provide 5
(5.9 g, 68%). Yellow solid. M.p. 100–1018 (THF/hexane). IR (KBr): 3106m, 2950m, 2635m, 1733s,
1628m, 1524s, 1468s, 1435s, 1342s, 1311m, 1253s, 1193m, 1107m, 896m, 860s, 813s, 793s, 781m, 761m.
1H-NMR (300 MHz, (CD3)2SO): 8.66 (s, 1 H); 8.36 (s, 1 H); 8.03 (s, 1 H); 3.86 (s, 3 H). 13C-NMR (75
MHz, (CD3)2
ACHTREUNG
(DHB): 222.0505 ([M+H]+, C9
A
A
ACHTREUNG
3.19, N 19.00; found: C 48.93, H 3.33, N 18.87.
Methyl 1-Benzyl-5-nitro-1H-benzimidazole-6-carboxylate (6a) and Methyl 1-Benzyl-6-nitro-1H-
benzimidazole-5-carboxylate (6b). To a soln. of 5 (100 mg, 0.45 mmol) in abs. THF (4 ml), NaH (60%
A
in oil, 27 mg, 0.68 mmol) was added at 08. After stirring for 30min, BnCl (171 mg, 1.36 mmol) and a
cat. amount of Bu4NI were added, and the mixture was heated to reflux for 12 h. After removal of the
solvent in vacuo, the residue was taken up in H2O and extracted with CH2Cl2 (3”). The combined org.
A
phases were dried (MgSO4) and concentrated in vacuo to give the crude product as brown solid (130
mg, 93%). The regioisomers (6a/6b 45 :55) were separated by CC (AcOEt/hexane 4 :3). Yellowish solids.
Data of 6a. M.p. 144–1468. IR (CHCl3): 2955w, 2253m, 1794w, 1731s, 1628w, 1584w, 1534s, 1462m,
1347s, 1214m, 1120m, 651s. 1H-NMR (300 MHz, CDCl3): 8.42 (s, 1 H); 8.34 (s, 1 H); 7.68 (s, 1 H);
7.38–7.41 (m, 3 H); 7.18–7.22 (m, 2 H); 5.45 (s, 2 H); 3.90( s, 3 H). 13C-NMR (125 MHz, CDCl3):
166.7; 148.1; 144.6; 136.0; 135.2; 134.2; 129.6; 129.2; 128.9; 127.4; 117.7; 112.1; 53.5; 49.7. HR-
MALDI-MS (DHB): 312.0973 ([M+H]+, C16H14N3O4þ, calc. 312.0984).
Data of 6b. M.p. 135–1378. IR (CHCl3): 3020w, 1730s, 1628w, 1533s, 1456m, 1347s, 1120m, 897w. 1H-
NMR (500 MHz, CDCl3): 8.32 (br. s, 1 H); 8.19 (s, 1 H); 8.02 (s, 1 H); 7.36–7.41 (m, 3 H); 7.18–7.20( m, 2
H); 5.44 (s, 2 H); 3.93 (s, 3 H). 13C-NMR (125 MHz, CDCl3): 166.0; 148.4; 144.5; 135.1; 134.1; 129.6;
129.2; 128.7; 127.4; 123.4; 122.4; 107.9; 53.5; 49.6.
Methyl 5-Amino-1-benzyl-1H-benzimidazole-6-carboxylate (7a) and Methyl 6-Amino-1-benzyl-1H-
benzimidazole-5-carboxylate (7b). To a soln. of 6a (50mg, 0.16 mmol) in AcOH (10ml) and H2O (3
ml), Zn (105 mg, 1.6 mmol) was added portionwise at 208. After stirring for 3 h at 208, the mixture
was filtered through Celite, and the filtrate was concentrated in vacuo. The residue was taken up in
H2O and adjusted to pH 7–8 with conc. aq. NaOH soln. The precipitated Zn salts were removed by fil-
tration through Celite, and the aq. phase was extracted with CH2Cl2 (3”). The org. phases were dried
(MgSO4) and evaporated in vacuo. CC (CH2Cl2/MeOH 96 :4) provided 7a (38 mg, 85%). Yellow solids.
Data of 7a. M.p. 172–1748 (CHCl3/hexane). IR (CHCl3): 3382w, 2956m, 1692s, 1640m, 1591s, 1293m,
1102m. 1H-NMR (300 MHz, CDCl3): 8.05 (s, 1 H); 7.89 (s, 1 H); 7.35–7.37 (m, 3 H); 7.19–7.22 (m, 2 H);
7.06 (s, 1 H); 5.31 (s, 2 H); 3.87 (s, 3 H). 13C-NMR (75 MHz, CDCl3): 168.7; 149.1; 147.0; 146.9; 135.5;
129.3; 128.8; 128.5; 127.3; 112.9; 109.4; 105.3; 51.9; 48.9. HR-MALDI-MS (DHB): 282.1270
([M+H]+, C16H16N3Oþ2 , calc. 282.1243). Anal. calc. for C16H15N3O2 (281.31): C 68.31, H 5.37, N 14.94;
found: C 68.18, H 5.52, N 15.09.
Through reduction of the regioisomeric mixture of 6a/6b, the other regioisomer 7b was obtained.
Data of 7b. M.p. 158–1608. IR (CHCl3): 3500w, 3378w, 2253m, 1688m, 1637m, 1591m, 1301m, 1208s,
905s, 665s. 1H-NMR (300 MHz, CDCl3): 8.38 (s, 1 H); 7.94 (s, 1 H); 7.26–7.32 (m, 3 H); 7.14–7.17 (m, 2
H); 6.43 (s, 1 H); 5.23 (s, 2 H); 4.63 (br. s, 2 H); 3.89 (s, 3 H). 13C-NMR (75 MHz, CDCl3): 168.7; 147.3;
143.8; 139.0; 136.1; 135.1; 129.0; 128.2; 126.9; 123.8; 108.7; 94.7; 51.8; 48.6.
Methyl 5-Amino-1-benzyl-4-iodo-1H-benzimidazole-6-carboxylate (8). To a degassed soln. of 7a (2.7
g, 9.6 mmol) in CH2Cl2 (150ml), an aq. NaHCO 3 soln. (0.22M, 75 ml) and I2 (2.9 g, 11.5 mmol) were
added, and the mixture was stirred for 3 h at 208. After addition of sat. aq. Na2SO3 soln. (20ml), the
org. layer was separated, and the aq. phase was extracted with CH2Cl2 (3”). The combined org. phases
were dried (MgSO4) and evaporated in vacuo to give a dark red powder (3.7 g, 95%). For anal. purposes,
a small amount of 8 was purified by CC (AcOEt/hexane 1:1). Yellow solid. M.p. 167–1698 (THF/hex-
ane). IR (CHCl3): 3476w, 3360w, 2954w, 1692s, 1628m, 1585s, 1492m, 1293m, 1120m. 1H-NMR (300
MHz, CDCl3): 8.08 (s, 1 H); 7.89 (s, 1 H); 7.34–7.38 (m, 3 H); 7.17–7.20( m, 2 H); 5.32 (s, 2 H); 3.88

Helvetica Chimica Acta – Vol. 89 (2006)
593
(s, 3 H). 13C-NMR (75 MHz, CDCl3): 167.6; 150.2; 146.1; 146.0; 134.8; 129.0; 128.3; 126.9; 125.0; 113.4;
108.9; 75.6; 52.1; 49.2. HR-MALDI-MS (DHB): 408.0196 ([M+H]+, C16H15IN3O2þ, calc. 408.0209). Anal.
calc. for C16H14IN3O2 (407.21): C 47.19, H 3.47, N 10.32; found: C 47.34, H 3.59, N 10.13.
Methyl 5-Amino-1H-benzimidazole-6-carboxylate (9). To a soln. of 5 (13.0g, 59 mmol) in AcOH
(500 ml), Pd/C (10%, 1.2 g) was added, and the mixture was heated to reflux for 1 h under H2 and sub-
sequently for 12 h at 208. After filtration through Celite, the solvent was evaporated in vacuo. CC
(CH2Cl2/MeOH 96 :4) provided 9 (11.01 g, 98%). White solid. M.p. 169–1708 (CHCl3/hexane). IR
(KBr): 3490m, 3379m, 2951w, 2763w, 1696s, 1647m, 1585s, 1435m, 1304s, 1242m, 1195m, 1141w, 888w,
1
853w, 815w, 793s. H-NMR (300 MHz, (CD3)2
G
H). 13C-NMR (75 MHz, (CD3)2
SO): 169.5; 148.6; 143.6; 141.3; 133.1; 120.4; 109.5; 98.8; 51.7. ESI-MS:
G
191.1 (100, M+), 159.0(96), 131.0(65), 105.0(21).
N 21.98; found: C 56.27, H 4.79, N 21.78.
6-Amino-1,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one (lin-Benzoguanine; 2). GP 1 with 9 (250
mg, 1.31 mmol), chloroformamidinium chloride (225 mg, 1.96 mmol), and dimethylsulfone (2.5 g) pro-
vided 2 (84 mg, 32%). White solid. M.p. >3008 (DMF/H2O). Spectroscopic data were in agreement
with those reported in [45].
Methyl 5-Amino-1-benzyl-4-(2-phenylethynyl)-1H-benzimidazole-6-carboxylate (10a). GP 2 with 8
(100 mg, 0.24 mmol), [PdCl2(PPh3)2] (17 mg, 0.02 mmol), CuI (4.7 mg, 0.02 mmol), and ethynylbenzene
(41 ml, 0.37 mmol). CC (CH2Cl2/MeOH 99 :1) provided 10a (74 mg, 81%). Yellow powder. M.p.
188–1908. IR (CHCl3): 3491w, 3370w, 2954w, 1692s, 1592m, 1269m, 1124m. 1H-NMR (300 MHz,
CDCl3): 8.06 (s, 1 H); 7.88 (s, 1 H); 7.63–7.66 (m, 2 H); 7.32–7.37 (m, 6 H); 7.16–7.19 (m, 2 H); 5.33
(s, 2 H); 3.88 (s, 3 H). 13C-NMR (75 MHz, CDCl3): 168.0; 148.4; 146.3; 134.8; 131.7; 129.0; 128.4;
128.2; 128.1 (2”); 126.9; 125.5; 123.1; 113.6; 108.3; 100.5; 98.0; 81.9; 51.9; 49.0. HR-MALDI-MS
(DHB): 382.1547 ([M+H]+, C24H20N3O2þ, calc. 382.1556).
Methyl 5-Amino-1-benzyl-4-[2-(4-methylphenyl)ethynyl]-1H-benzimidazole-6-carboxylate (10b).
GP 2 with 8 (500 mg, 1.23 mmol), [PdCl2(PPh3)2] (86 mg, 0.12 mmol), CuI (23 mg, 0.12 mmol), and 1-eth-
ynyl-4-methylbenzene (234 ml, 1.84 mmol). CC (CH2Cl2/MeOH 99.25 :0.75) provided 10b (456 mg, 94%).
Dark-yellow powder. M.p. 212–2148 (CHCl3/hexane). IR (CHCl3): 3479m, 3375m, 2805m, 1688s, 1636s,
1588s, 1420s, 1310s, 1277m, 1209s, 1102m, 1068m, 949w, 788m, 627m. 1H-NMR (300 MHz, CDCl3): 8.00 (s,
1 H); 7.86 (s, 1 H); 7.53 (d, J=8.1, 2 H); 7.33–7.38 (m, 3 H); 7.14–7.18 (m, 4 H); 6.38 (br. s, 2 H); 5.32 (s,
2 H); 3.88 (s, 3 H); 2.37 (s, 3 H). 13C-NMR (75 MHz, CDCl3): 168.1; 148.4; 148.3; 146.8; 138.3; 135.1;
131.5; 129.0; 128.9; 128.3; 126.9; 125.7; 120.1; 113.3; 108.1; 100.7; 98.5; 81.5; 51.9; 48.9; 21.7. HR-
MALDI-MS (DHB): 396.1712 ([M+H]+, C25H22N3O2þ, calc. 396.1712). Anal. calc. for C25H21N3O2
(395.45): C 75.93, H 5.35, N 10.63; found: C 75.97, H 5.54, N 10.55.
Methyl 5-Amino-1-benzyl-4-[2-(4-methoxyphenyl)ethynyl]-1H-benzimidazole-6-carboxylate (10c).
GP 2 with 8 (500 mg, 1.23 mmol), [PdCl2(PPh3)2] (86 mg, 0.12 mmol), CuI (23 mg, 0.12 mmol), and 1-eth-
ynyl-4-methoxylbenzene (234 ml, 1.84 mmol). CC (CH2Cl2/MeOH 95.5 :0.5) provided 10c (380mg, 75%).
Dark-yellow powder. M.p. 186–1888. IR (CHCl3): 3484w, 3372w, 3008m, 2358m, 1692s, 1607s, 1564m,
1
1492s, 1438m, 1290s, 1125m, 968w, 833m. H-NMR (300 MHz, CDCl3): 8.00 (s, 1 H); 7.72 (s, 1 H); 7.46
(d, J=8.6, 2 H); 7.25–7.28 (m, 3 H); 7.08–7.11 (m, 2 H); 6.77 (d, J=8.6, 2 H); 6.33 (br. s, 2 H); 5.21
(s, 2 H); 3.80( s, 3 H); 3.74 (s, 3 H). 13C-NMR (75 MHz, CDCl3): 168.0; 159.4; 148.1; 146.8; 146.1;
135.0; 133.0; 128.9; 128.2; 126.8; 125.6; 115.2; 113.7; 113.2; 108.0; 100.5; 98.4; 80.9; 55.3; 51.8; 48.8.
HR-MALDI-MS (DHB): 412.1661 ([M+H]+, C25H22N3O3þ, calc. 412.1661).
Methyl 5-Amino-4-(2-phenylethyl)-1H-benzimidazole-6-carboxylate (11a). GP 3 with 10a (600 mg,
1.57 mmol) and Pd/C (600 mg). CC (CH2Cl2/MeOH 97:3) provided 11a (280mg, 60%). White powder.
M.p. 200–2028. IR (KBr): 3506w, 3392m, 3138s, 2951s, 2588m, 1730m, 1700s, 1583s, 1425s, 1307s, 1204s,
1
1001m, 60 2m. H-NMR (300 MHz, CD3OD): 8.12 (s, 1 H); 8.00 (s, 1 H); 7.12–7.23 (m, 5 H); 3.89 (s, 3
H); 3.12 (t, J=7.8, 2 H); 2.90( t, J=7.8, 2 H). 13C-NMR (125 MHz, (CD3)2SO+2 drops of CF3
N
N
168.1; 147.5; 140.7; 134.9; 128.6; 128.1; 126.1; 121.3; 115.2; 117.8; 113.6; 109.7; 51.5; 32.6; 27.4. HR-
MALDI-MS (DHB): 296.1397 ([M+H]+, C17H18N3O2þ, calc. 296.1399).
Methyl 5-Amino-4-[2-(4-methylphenyl)ethyl]-1H-benzimidazole-6-carboxylate (11b). GP 3 with 10b
(300 mg, 0.76 mmol) and Pd/C (200 mg). CC (CH2Cl2/MeOH 97:3) provided 11b (131 mg, 56%). White
powder. M.p. 238–2408. IR (KBr): 3473w, 3364w, 2949w, 1688s, 1620s, 1555s, 1496s, 1425m, 1342s, 1267s,

594
Helvetica Chimica Acta – Vol. 89 (2006)
1229s, 1065s, 812s. 1H-NMR (300 MHz, CD3OD): 8.11 (s, 1 H); 8.00 (br. s, 1 H); 7.10( d, J=7.8, 2 H); 7.02
(d, J=7.8, 2 H); 3.88 (s, 3 H); 3.08 (t, J=6.6, 2 H); 2.27 (t, J=6.6, 2 H); 2.26 (s, 3 H). 13C-NMR (125 MHz,
(CD3)2SO): 168.4; 147.3; 142.7; 138.5; 135.5; 135.3; 129.3; 129.2; 122.0; 115.7; 110.9; 110.7; 52.9; 32.9;
27.6; 21.3. HR-MALDI-MS (DHB): 310.1547 ([M+H]+, C18H20N3O2þ, calc. 310.1556).
Methyl 5-Amino-4-[2-(4-methoxyphenyl)ethyl]-1H-benzimidazole-6-carboxylate (11c). GP 3 with
10c (350 mg, 0.85 mmol) and Pd/C (10%, 550 mg), reaction time: 7 d. CC (CH2Cl2/MeOH 97:3) provided
11c (122 mg, 44%). White powder. M.p. 208–2098. IR (CHCl3): 3463w, 3374w, 3002w, 2953w, 1685s,
1
1635m, 1610w, 1581w, 1512s, 1437m, 1313m, 1304m, 1248s, 1220s, 1200s. H-NMR (300 MHz, CD3
A
8.10( s, 1 H); 7.99 (s, 1 H); 7.12 (d, J=8.4, 2 H); 6.76 (d, J=8.4, 2 H); 3.88 (s, 3 H); 3.73 (s, 3 H); 3.08
(t, J=7.8, 2 H); 2.84 (t, J=7.8, 2 H). 13C-NMR (75 MHz, CD3
OD): 169.4; 158.3; 144.6; 142.8; 133.7;
129.4; 129.2; 118.1; 116.8; 113.5; 110.0; 109.1; 54.4; 50.9; 32.8; 28.3. HR-MALDI-MS (DHB): 326.1499
([M+H]+, C18 Oþ3 , calc.326.1505).
H20N3
AHCTREUNG
A
N
ACHTREUNG
6-Amino-4-(2-phenylethyl)-1,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one (12a). GP 1 with 11a
(130 mg, 0.44 mmol) and chloroformamidinium chloride (76 mg, 0.66 mmol). Greenish solid (47 mg,
35%). M.p. >3008 (dec.). IR (KBr): 3468w, 3374m, 3109s, 2927m, 1649s, 1600s, 1494s, 1280s, 1121m,
1
889m, 70 2m. H-NMR (300 MHz, (CD3)2SO): 12.46 (br. s, 1 H); 10.86 (br. s, 1 H); 8.30( s, 1 H); 8.03
(s, 1 H); 7.18–7.38 (m, 5 H); 6.15 (br. s, 2 H); 3.35 (t, J=9.0, 2 H); 2.89 (t, J=9.0, 2 H). 13C-NMR (125
MHz, (CD3)2SO+2 drops of CF3
N
N
126.2; 115.8; 113.9; 111.8; 34.4; 26.5. HR-MALDI-MS (DHB): 306.1346 ([M+H]+, C17H16N5O+, calc.
306.1355).
6-Amino-4-[2-(4-methylphenyl)ethyl]-1,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one (12b). GP 1
with 11b (80 mg, 0.26 mmol) and chloroformamidinium chloride (45 mg, 0.39 mmol). Yellow solid (50
mg, 60%). M.p. >3008 (dec.). IR (KBr): 3467w, 3380m, 3108m, 2921m, 1649s, 1610s, 1513s, 1415s,
1
1369s, 1281m, 1123w, 80 8w, 795w. H-NMR (300 MHz, (CD3)2SO): 12.53 (br. s, 1 H); 10.78 (br. s, 1 H);
8.26 (s, 1 H); 8.05 (s, 1 H); 7.25 (d, J=7.8, 2 H); 7.09 (d, J=7.8, 2 H); 6.09 (br. s, 2 H); 3.26 (t, J=8.1,
2 H); 2.81 (t, J=8.1, 2 H); 2.25 (s, 3 H). 13C-NMR (125 MHz, (CD3)2SO): 160.2; 151.4; 145.2; 138.0;
137.2; 135.1; 133.4; 130.3; 128.6; 128.5; 116.0; 113.8; 111.8; 34.1; 26.6; 20.6. HR-MALDI-MS (DHB):
320.1504 ([M+H]+, C18H18N5O+, calc. 320.1511).
6-Amino-4-[2-(methoxyphenyl)ethyl]-1,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one (12c). GP 1
with 11c (92 mg, 0.28 mmol) and chloroformamidinium chloride (49 mg, 0.42 mmol). Yellow solid (62
mg, 66%). M.p. >3008. IR (KBr): 3382m, 3111(sh), 2918m, 1651s, 1615s, 1575s, 1511s, 1413m, 1393m,
1
1372m, 1243s. H-NMR (300 MHz, (CD3)2
A
(s, 1 H); 7.27 (d, J=8.4, 2 H); 6.85 (d, J=8.4, 2 H); 6.15 (br. s, 2 H); 3.72 (s, 3 H); 3.33 (t, J=8.0, 2 H);
2.82 (t, J=8.0, 2 H). 13C-NMR (75 MHz, (CD3)2
SO): 160.0; 157.4; 151.0; 145.1; 138.3; 133.2; 132.1;
130.6; 129.4; 113.3; 113.2; 111.6; 109.6; 54.9; 33.7; 26.8. HR-MALDI-MS (DHB): 336.1461 ([M+H]+,
C18H18N5
Oþ2 , calc. 336.1461).
4-(2-Phenylethyl)-1,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one (13). A mixture of 11a (100 mg,
AHCTREUNG
A
N
ACHTREUNG
0.34 mmol) and formamidinium acetate (46 mg, 0.44 mmol) in EtOH (6 ml) was heated to reflux for
2 d. Another portion of formamidinium acetate was added (46 mg, 0.44 mmol), and the mixture was
heated for further 2 d. After removal of the solvent in vacuo, the residue was washed with H2O and
CH2Cl2. CC (CH2Cl2/MeOH 95 :5) provided 13 (80mg, 81%). White solid. M.p. >3008. IR (KBr):
3425 (sh), 3159m, 3061m, 3025m, 2918m, 1661s, 1615s, 1583m, 1492m, 1454m, 1411s, 1281m, 1185m.
1H-NMR (300 MHz, CD3
3.00 (t, J=8.0, 2 H). 13C-NMR (125 MHz, (CD3)2
142.8; 141.1; 135.1; 130.1; 128.3; 128.1; 125.9; 124.7; 120.7; 109.6; 35.4; 27.7. HR-MALDI-MS (DHB):
291.1238 ([M+H]+, C17 O+, calc. 291.1246).
H15N4
ACHTREUNG
A
N
ACHTREUNG
A
N
ACHTREUNG
Methyl 5-Amino-1-benzyl-4-phenyl-1H-benzimidazole-6-carboxylate (14). A suspension of 8 (500
mg, 1.22 mmol), phenylboronic acid (157 mg, 1.22 mmol), K2CO3 (279 mg, 2.02 mmol), and
[Pd(PPh3)4] (94 mg, 0.08 mmol) in EtOH (10 ml), toluene (5 ml), and H2O (5 ml) was heated to 808
for 2 d. After cooling to 208, the mixture was treated with sat. aq. NH4Cl soln. and extracted with
AcOEt (3”). The combined org. phases were dried (MgSO4) and evaporated in vacuo. CC (CH2Cl2/
MeOH 95.25 :0.75) provided 14 (377 mg, 87%). Yellow solid. M.p. 70–728. IR (CHCl3): 3491w, 3382w,
3027m, 3031m, 1690s, 1627m, 1569m, 1497s, 1437m, 1419m, 1332m, 1123w. 1H-NMR (300 MHz,

Helvetica Chimica Acta – Vol. 89 (2006)
595
(CD3)2SO): 8.32 (s, 1 H); 7.93 (s, 1 H); 7.24–7.51 (m, 10H); 5.78 (br. s, 2 H); 5.46 (s, 2 H); 3.79 (s, 3 H).
13C-NMR (75 MHz, CDCl3): 169.0; 147.7; 147.0; 143.8; 135.7; 135.0; 130.7; 129.4; 129.3; 128.5; 128.0;
127.2; 126.7; 117.6; 112.3; 109.1; 52.0; 48.9. HR-MALDI-MS (DHB): 358.1553 ([M+H]+, C22H20N3O2þ,
calc. 358.1556).
Methyl 5-Amino-4-phenyl-1H-benzimidazole-6-carboxylate (15). A suspension of 14 (0.35 g, 0.98
mmol) and Pd/C (10%, 0.35 g) in EtOH (18 ml) and AcOH (2 ml) was hydrogenated (1 bar) for 24 h
at 208. The mixture was filtered through Celite, and the solvent was removed in vacuo. The residue
was taken up in CH2Cl2/sat. aq. NaHCO3 soln. and extracted with CH2Cl2 (3”). The combined org. phases
were dried (MgSO4) and concentrated in vacuo. CC (CH2Cl2/MeOH 98.6 :1.4) provided 15 (205 mg,
78%). Yellow solid. M.p. 198–2018. IR (CHCl3): 3452w, 3374w, 2996w, 1688s, 1634m, 1603w, 1576m,
1
1501w, 1437m, 1417w, 1372w, 1329s. H-NMR (300 MHz, CDCl3): 11.80(br. s, 1 H) 8.27 (s, 1 H); 7.49
(s, 1 H); 7.32–7.41 (m, 4 H); 7.22–7.26 (m, 1 H); 5.67 (br. s, 2 H); 3.85 (s, 3 H). 13C-NMR (75 MHz,
CDCl3): 169.0; 143.8; 142.1; 138.6; 134.3; 132.7; 129.9; 129.4; 127.9; 120.6; 110.5; 108.5; 51.6. HR-
MALDI-MS (DHB): 268.1084 ([M+H]+, C15
A
G
N
6-Amino-4-phenyl-1,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one (16). GP 1 with 15 (195 mg, 0.73
mmol) and chloroformamidinium chloride (126 mg, 1.09 mmol) provided 16 (76 mg, 38%). Yellow solid.
1
M.p. >3008. IR (KBr): 3310m, 3131s, 1651s, 1605s, 1574s, 1498m, 1387m, 1283w. H-NMR (300 MHz,
(CD3)2
13C-NMR (125 MHz, (CD3)2
130.6; 129.4; 129.1; 116.4; 113.3; 112.3. HR-MALDI-MS (DHB): 278.1033 ([M+H]+, C15
calc. 278.1042).
A
G
R
ACHTREUNG
N
R
ACHTREUNG
N
E
ACHTREUNG
6-Amino-3-benzyl-3,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one (17a). GP 1 with 7b (200 mg,
0.71 mmol) and chloroformamidinium chloride (123 mg, 1.07 mmol) provided 17a (176 mg, 85%).
White solid. M.p. >3008. IR (KBr): 3107m, 2745m, 1699s, 1394s, 1559m, 1497m, 1455m, 1331m, 1211w,
1
704m. H-NMR (300 MHz, (CD3)2
ACHTREUNG
H); 7.16 (s, 1 H); 6.13 (br. s, 2 H); 5.47 (s, 2 H). 13C-NMR (125 MHz, (CD3)2
A
N
ACHTREUNG
A
N
ACHTREUNG
Methyl 6-Nitro-1-(2-phenylethyl)-1H-benzimidazole-5-carboxylate (18). To a soln. of 6 (1.0g, 4.52
mmol) in abs. THF (4 ml), NaH (60% in oil, 217 mg, 5.42 mmol) was added at 08. After 20min stirring,
2-phenylethyl chloride (1.83 ml, 13.5 mmol) was added, and the mixture was heated to reflux for 12 h.
After removal of the solvent in vacuo, the residue was taken up in H2O and extracted with CH2Cl2
G
(3”). The combined org. phases were dried (MgSO4) and concentrated in vacuo. The regioisomers
were separated by CC (AcOEt/hexane 7:3). Yellow oil. IR (CHCl3): 3563 (sh), 3361 (sh), 3034w,
2993w, 2954m, 1737s, 1731s, 1625m, 1589m, 1535s, 1492m, 1438s, 1380s, 1346s, 1285s. 1H-NMR (300
MHz, CDCl3): 8.07 (s, 1 H); 7.82 (s, 1 H); 7.81 (s, 1 H); 7.22–7.27 (m, 3 H); 6.93–6.97 (m, 2 H); 4.49
(t, J=6.6, 2 H); 3.94 (s, 3 H); 3.15 (t, J=6.6, 2 H). 13C-NMR (75 MHz, CDCl3): 166.3; 147.9; 145.4;
144.0; 136.6; 133.8; 128.9; 128.4; 127.3; 122.2; 121.9; 107.2; 53.2; 47.4; 36.5. HR-MALDI-MS (DHB):
326.1138 ([M+H]+, C17
A
G
N
Methyl 6-Amino-1-(2-phenylethyl)-1H-benzimidazole-5-carboxylate (19). A suspension of 18 (292
mg, 0.90 mmol) and Pd/C (10%, 30 mg) in AcOH (10 ml) was hydrogenated (1 bar) for 12 h at 208.
The mixture was filtrated through Celite, and the solvent was removed in vacuo. The residue was
taken up in CH2Cl2/sat. aq. NaHCO3 soln. and extracted with CH2Cl2 (4”). The combined org. phases
were dried (MgSO4) and concentrated in vacuo. CC (AcOEt) provided 19 (216 mg, 81%). Yellow
solid. M.p. 153–1558. IR (CHCl3): 3498m, 3378m, 2993m, 2952m, 1692s, 1637s, 1591s, 1504m, 1454s,
1
1437s, 1360m, 1302s, 1244m. H-NMR (300 MHz, CDCl3): 8.34 (s, 1 H); 7.40( s, 1 H); 7.20–7.42 (m, 3
H); 6.96–7.00 (m, 2 H); 6.47 (s, 1 H); 5.60(br. s, 2 H); 4.20( t, J=6.9, 2 H); 3.87 (s, 3 H); 3.04 (t,
J=6.9, 2 H). 13C-NMR (75 MHz, CDCl3): 169.2; 147.5; 144.0; 138.9; 137.7; 136.4; 129.1; 128.9; 127.3;
124.1; 108.8; 94.6; 51.9; 46.7; 36.0. HR-MALDI-MS (DHB): 296.1396 ([M+H]+, C17
A
G
N
296.1399).
6-Amino-3-(2-phenylethyl)-3,7-dihydro-8H-imidazo[4,5-g]quinazolin-8-one (17b). GP 1 with 19
(0.20 g, 0.68 mmol) and chloroformamidinium chloride (0.12 g, 1.02 mmol) provided 17b (177 mg,
85%). White solid. M.p. >3008. IR (KBr): 3463m, 3096m, 2922w, 1651s, 1557s, 1506s, 1448m, 1358m.

596
1H-NMR (300 MHz, (CD3)2
Helvetica Chimica Acta – Vol. 89 (2006)
AHCTREUNG
5 H); 6.28 (br. s, 2 H); 4.46 (t, J=6.9, 2 H); 3.09 (t, J=6.9, 2 H). 13C-NMR (75 MHz, (CD3)2
150.2; 150.1; 146.1; 139.4; 138.9; 137.9; 128.6; 128.2; 126.3; 116.2; 113.0; 102.5; 45.4; 35.0. HR-MALDI-
MS (DHB): 306.1353 ([M+H]+, C17 O+, calc. 306.1355).
H16N5
SO): 162.8;
A
N
ACHTREUNG
X-Ray Crystal Structures. Cell determination and data collection for all compounds were performed
on a Bruker Kappa CCD instrument, and cell refinement and data reduction with the programs Scale-
pack and Denzo [46].
Compound 7b. Crystals were grown in a soln. of THF with an upper layer of hexane. X-Ray crystal
data for C16H15N3O2 (Mr 281.315): trigonal space group R3 (hexagonal setting), Dx =1.299 g/cmꢀ3, Z=18,
a=28.3050(4), b=28.3050(9), c=9.3331(3) Š, V=6475.6 (3) Š3, MoKa radiation, l=0.71073 Š,
0.9988ꢃqꢃ29.5758, 11415 measured reflections, 6403 independent reflections (Rint =0.039), T=298 K.
The structure was solved by direct methods (SIR97, [47]) and refined by full-matrix least-squares analysis
[48]. All non-H-atoms were refined anisotropically, H-atoms were included in the structure factor calcu-
lation, with positions based on stereochemical considerations. Final agreement factors: R(gt)=0.0513,
wR(gt)=0.1370 for 379 parameters and 4776 observed reflections with I>2s(I). Cambridge Crystallo-
graphic Data Centre deposition No. CCDC-256502.
Compound 8. Crystals were grown by slow diffusion of hexane into a soln. of 8 in THF. X-Ray crystal
data for C16H14IN3O2 (Mr 407.207): Monoclinic space group P21/c, Dx =1.754 g/cmꢀ3
, Z=4,
a=10.2926(2), b=6.87470(10), c=22.0059(4) Š, V=1542.27(5) Š3, MoKa radiation, l=0.71073 Š,
0.9988ꢃqꢃ30.5088, 9089 measured reflections, 4683 independent reflections (Rint =0.028), T=298 K.
The structure was solved by direct methods (SIR97, [47]) and refined by full-matrix least-squares analysis
(SHELXL-97). All non-H-atoms were refined anisotropically, H-atoms were included in the structure
factor calculation, with positions based on stereochemical considerations. Final agreement factors:
R(gt)=0.0445, wR(gt)= 0.1438 for 199 parameters and 3008 observed reflections with I>2s(I). Cam-
bridge Crystallographic Data Centre deposition No. CCDC-256501.
Compound 10b. Crystals were grown by slow diffusion of hexane into a soln. of 8 in CHCl3. X-Ray
crystal data for C25H21N3O2 (Mr 395.462): Monoclinic space group P21/n, Dx =1.275 g/cmꢀ3, Z=4,
a=12.7484(1), b=6.2552(2), c=26.2696(5) Š, V=2060.49(8) Š3, MoKa radiation, l=0.71073 Š,
0.9988ꢃq ꢃ25.3508, 14161 measured reflections, 3750independent reflections ( Rint =0.036), T=172
K. The structure was solved by direct methods (SIR97, [47]) and refined by full-matrix least-squares anal-
ysis (SHELXL-97). All non-H-atoms were refined anisotropically and H-atoms with isotropic temp. fac-
tors. Final agreement factors: R(gt)=0.0582, wR(gt)=0.1706 for 355 parameters and 2727 observed
reflections with I>2s(I). Cambridge Crystallographic Data Centre deposition No. CCDC-256500.
Crystallographic data (excluding structure factors) for the structures reported in this paper have
been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained,
free of charge, on application to the CCDC, 12 Union Road, Cambridge CB21EZ UK (fax:
+44(1223)336033; e-mail: deposit@ccdc.cam.ac.uk).
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Received November 10, 2005