Structure-based design and synthesis of the first weak non-phosphate inhibitors for IspF, an enzyme in the non-mevalonate pathway of isoprenoid biosynthesis

Article abstract of DOI:10.1002/hlca.200790105

In this paper, we describe the structure-based design, synthesis, and biological evaluation of cytosine derivatives and analogues that inhibit IspF, an enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. This pathway is responsible for the biosynthesis of the C₅ precursors to isoprenoids, isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2; Scheme 1). The non-mevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non-mevalonate pathway have been identified as attractive new drug targets in the fight against malaria. Based on computer modeling (cf. Figs. 2 and 3), new cytosine derivatives and analogues (Fig. 1) were selected as potential drug-like inhibitors of IspF protein, and synthesized (Schemes 2-5). Determination of the enzyme activity by ¹³C-NMR spectroscopy in the presence of the new ligands showed inhibitory activities for some of the prepared cytosine and pyridine-2,5-diamine derivatives in the upper micromolar range (IC₅₀ values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the polar diphosphate binding site and the side chain of Asp56′, which interacts with the ribose moiety of the substrate and substrate analogues. Furthermore, a new spacious sub-pocket was discovered which accommodates aromatic spacers between cytosine derivatives or analogues (binding to 'Pocket III') and rings that occupy the flexible hydrophobic region of 'Pocket II'. The proposed binding mode remains to be further validated by X-ray crystallography.

Full text of DOI:10.1002/hlca.200790105

Helvetica Chimica Acta – Vol. 90 (2007)
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Structure-Based Design and Synthesis of the First Weak Non-Phosphate
Inhibitors for IspF, an Enzyme in the Non-Mevalonate Pathway of Isoprenoid
Biosynthesis
by Corinne Baumgartner, Christian Eberle, and FranÅois Diederich*
Laboratorium für Organische Chemie, Department of Chemistry and Applied Biosciences, ETH Zürich,
Hçnggerberg, HCI, CH-8093 Zürich (e-mail: diederich@org.chem.ethz.ch)
and
Susan Lauw, Felix Rohdich*, Wolfgang Eisenreich, and Adelbert Bacher
Laboratorium für Organische Chemie und Biochemie, TU München, Lichtenbergstr. 4,
D-85748 Garching (e-mail: felix.rohdich@ch.tum.de)
In this paper, we describe the structure-based design, synthesis, and biological evaluation of cytosine
derivatives and analogues that inhibit IspF, an enzyme in the non-mevalonate pathway of isoprenoid
biosynthesis. This pathway is responsible for the biosynthesis of the C5 precursors to isoprenoids,
isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2; Scheme 1). The non-
mevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since
mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non-mevalonate
pathway have been identified as attractive new drug targets in the fight against malaria. Based on
computer modeling (cf. Figs. 2 and 3), new cytosine derivatives and analogues (Fig. 1) were selected as
potential drug-like inhibitors of IspF protein, and synthesized (Schemes 2 – 5). Determination of the
enzyme activity by 13C-NMR spectroscopy in the presence of the new ligands showed inhibitory activities
for some of the prepared cytosine and pyridine-2,5-diamine derivatives in the upper micromolar range
(IC50 values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the
polar diphosphate binding site and the side chain of Asp56’, which interacts with the ribose moiety of the
substrate and substrate analogues. Furthermore, a new spacious sub-pocket was discovered which
accommodates aromatic spacers between cytosine derivatives or analogues (binding to ꢀPocket IIIꢁ) and
rings that occupy the flexible hydrophobic region of ꢀPocket IIꢁ. The proposed binding mode remains to
be further validated by X-ray crystallography.
1. Introduction. – Caused by the protozoan Plasmodium parasites, malaria is one of
the most important tropical diseases, infecting 300 – 500 million and killing 1 – 3 million
people annually [1]. To combat the rapidly growing resistance of the Plasmodium
parasites against currently available antimalarial agents, there is ongoing search for
new drugs with a novel mode of action.
In the early 1990s, the non-mevalonate pathway for the biosynthesis of the C5
precursors to isoprenoids, isopentenyl diphosphate (IPP; 1) and dimethylallyl
diphosphate (DMAPP; 2), was discovered [2]. The non-mevalonate pathway starts
with the condensation of pyruvate (3) and glyceraldehyde 3-phosphate (4). The
ꢂ 2007 Verlag Helvetica Chimica Acta AG, Zürich

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cyclization of 4-(diphosphocytidyl)-2C-methyl-d-erythritol 2-phosphate (5) to the
cyclic diphosphate intermediate 2C-methyl-d-erythritol 2,4-cyclodiphosphate
(MECDP; 6) is catalyzed by the fifth enzyme in the pathway, IspF (¼2C-methyl-d-
erythritol 2,4-cyclodiphosphate synthase; EC 4.6.1.12; Scheme 1) [3].
Scheme 1. Biosynthesis of the C5 Precursors to Terpenes IPP (1) and DMAPP (2), via the Non-
Mevalonate Pathwaya)
a) DXS ¼ 1-Deoxy-d-xylulose-5-phosphate synthase; CMP ¼ cytosine 5’-monophosphate; MECDP ¼
2C-methyl-d-erythritol 2,4-cyclodiphosphate; IPP ¼ isopentenyl diphosphate; DMAPP ¼ dimethylallyl
diphosphate.
While mammals use the mevalonate pathway for the biosynthesis of IPP (1) and
DMAPP (2), many pathogenic bacteria, such as Myobacterium tuberculosis, as well as
the protozoan Plasmodium parasites, exclusively use the non-mevalonate pathway. The
antibiotic Fosmidomycin has recently been shown to be an efficient inhibitor of IspC
(¼1-deoxy-d-xylulose 5-phosphate reductoisomerase ¼ 2C-methylerythritol-4-phos-
phate synthase; EC 1.1.1.267), the second enzyme in the non-mevalonate pathway,
and has been submitted to clinical tests [4a – c]. Because of the discovery of the potent
antimalarial activity of Fosmidomycin, the absence of the enzymes in humans, and the
general fact that isoprenoids are essential for protozoa, the enzymes of the non-
mevalonate pathway have been validated as attractive new drug targets [4].
Since these enzymes process phosphate- and diphosphate-based substrates, their
active sites are highly polar. They feature only limited hydrophobic surfaces, so that the
development of low-molecular-weight inhibitors appears quite challenging. Most of the

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few inhibitors known to date, such as Fosmidomycin, indeed are phosphates or
phosphonates [5][6].
Here, we describe the structure-based design, synthesis, and in vitro evaluation of
cytosine derivatives and analogues 7 – 21 (Fig. 1), targeting the inhibition of IspF
protein (for an illustration of our structure-based design approach for the development
of new antimalarials, see [7]). We show how modeling-based variation of the spacer
between cytosine or its replacements, and terminal aromatic rings led from inactive to
the first weakly active non-phosphate inhibitors of IspF, exhibiting IC50 values
(concentration of inhibitor at which 50% maximal initial velocity is observed) in the
upper micromolar range, i.e., featuring higher affinities than previously described
diphosphate-based ligands [6].
Fig. 1. Cytosine derivatives and analogues prepared as potential inhibitors of IspF
2. Results and Discussion. – 2.1. Ligand Design. Several X-ray crystal structures of
IspF have been solved [6][8]. They show oligomeric IspF in most cases as a C3-
symmetric homotrimer, with active sites located at the interface of adjacent subunits.
Each of the topologically equivalent active sites possesses two pockets. The rigid, well-
conserved ꢀPocket IIIꢁ of one monomer binds the cytidine moiety of substrate 5. The
larger, more flexible ꢀPocket IIꢁ in an adjacent monomer hosts the phosphate and
erythritol moieties (for the protein residues lining these pockets, see Figs. 2 and 3,a).
Additionally, ꢀPocket IIꢁ also contains one or two divalent metal ions (Mg2þ or Mn2þ,
and Zn2þ) which participate in the binding of the diphosphate moiety of 5 as well as in
catalysis.
Recently, we described the synthesis and biological activity of the first inhibitors for
IspF [6]. These compounds closely resembled substrate 5, featuring a cytosine
diphosphate moiety attached to a fluorescent anthranilate (¼2-aminobenzoate) or

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dansylamide (¼ 5-(dimethylamino)naphthalene-1-sulfamoyl) residue to reach into the
hydrophobic region of ꢀPocket IIꢁ. These compounds were developed as fluorescent
probes for assay development, and their binding mode was elucidated by X-ray crystal-
structure analysis. To obtain more drug-like molecules, the new cytosine analogues and
derivatives shown in Fig. 1 were designed using the molecular modeling software
MOLOC [9]. This new approach was based on two published X-ray crystal structures,
one of IspF in complex with cytidine 5’-diphosphate (CDP), and Zn2þ and Mn2þ ions
(Protein Data Bank (PDB) code: 1GX1 [8a]) and the other in complex with cytidine
5’-monophosphate (CMP), MECDP (6), and a Zn2þ ion (PDB code: 1JY8 [8b]).
Compared to the first-generation inhibitors, major structural changes were planned: i)
modification of the cytosine nucleobase, ii) by-passing the ribose pocket lined with
Asp56’ (see below), and iii) by-passing the highly polar phosphate binding site with the
metal ion centers. In the modeling, the coordinates of the enzyme, as seen in the X-ray
crystal structures, were left unchanged, while geometry and energy of the docked-in
ligand were minimized.
Whereas a rich variety of adenine substitutes has been developed as part of the
intense ongoing search for selective kinase inhibitors [10], cytosine analogues are much
less known [11]. In our case, we chose 2,5-diaminopyridine and 5-amino-1H-
imidazo[4,5-b]pyridine scaffolds as substitutes for cytosine.
The nucleobase in CMP bound to ꢀPocket IIIꢁ forms four H-bonds with the protein
backbone, specifically with Leu106 (O ··· N 2.9 Š), Met105 (N ··· N 2.9 Š), Pro103
(N ··· O 3.1 Š), and Ala100 (N ··· O 3.0 Š) ( (PDB code: 1 H48 [8g]; Fig. 2,a). In
addition, the ribose moiety interacts with the side chain of Asp56’ (from the adjacent
monomer). Replacing cytosine by the synthetically readily accessible 2,5-diaminopyr-
idine moiety in 7 – 12 maintains three of the four H-bonds to the protein backbone
(Fig. 2,b) while providing an exit vector to reach into the hydrophobic portion of
ꢀPocket IIꢁ, by-passing both Asp56’ and the polar diphosphate binding site. A more
complete, but synthetically more challenging cytosine substitute is the 5-amino-1H-
imidazo[4,5-b]pyridine scaffold introduced into the potential ligands 13 and 14
(Fig. 2,c). While maintaining all four H-bonds to the protein backbone as seen for
cytosine, this heterocycle also features an exit vector for reaching the hydrophobic
portion of ꢀPocket IIꢁ, while avoiding the polar regions. Cytosine derivatives 15 – 21
should bind to ꢀPocket IIIꢁ in a similar fashion to CMP.
Examination of the active site of IspF protein by molecular modeling suggested that
both cytosine and its substitutes bound to ꢀPocket IIIꢁ could be connected by relatively
rigid linkers to the aromatic ring filling the hydrophobic portion of ꢀPocket IIꢁ, lined by
Ala71’, Phe68’, Phe61’, and Leu76’, while by-passing both the polar ribose (Asp56’) and
diphosphate binding sites. This is shown for ligand 11 in Fig. 3,a. In fact, all compounds
depicted in Fig. 1 could be docked in a similar fashion into the active site and energy-
minimized, while avoiding any repulsive contacts with the enzyme. Fig. 3,b shows a
superimposition of the proposed preferential binding mode for compounds 11 and 19,
which later were shown to be active inhibitors. It suggests that the site hosting the
aromatic linker is quite large, allowing different, nearly orthogonal orientations of this
ring.
2.2. Ligand Synthesis. – 2.2.1. 2,5-Diaminopyridine Derivatives. The synthesis of 7
and 8 started from 4-nitrobenzyl alcohol (22) and 3-methyl-4-nitrobenzyl alcohol (23),

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Fig. 2. Schematic representation of CMP bound to ꢀPocket IIIꢁ of IspF (a) (PDB code: 1 H48 [8g]) and
proposed complexation of cytosine analogues 7 (b) and 13 (c). Potential H-bonds are depicted in red.
respectively, which were converted into the corresponding phthalimides 24 and 25 via a
Mitsunobu reaction (Scheme 2) [12]. Deprotection using NH2Me afforded the amines
26 and 27, respectively. Amide coupling with PhCOOH, activated as the N-
hydroxysuccinimide ester, provided the amides 28 and 29, and Pd-catalyzed hydro-
genation yielded amines 30 and 31, respectively. Buchwald – Hartwig cross-coupling
[13] with 5-bromo-2-nitropyridine, using [Pd2(dba)3] as catalyst and (Æ)-BINAP as
ligand, afforded 32 and 33, which were converted into the desired target compounds 7
and 8, respectively, by Pd-catalyzed hydrogenation. Methoxy derivative 10 was
prepared in a similar fashion starting from 4-methoxybenzoic acid via 34 ! 35 ! 36 !
10.
An alternative protocol to prepare the 2,5-diaminopyridine-based ligands is shown
in Scheme 3. Alcohol 23 was silyl-protected (! 37) and then reduced to the aniline
derivative 38. Buchwald – Hartwig cross-coupling reaction with 5-bromo-2-nitropyr-

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Fig. 3. a) Computer model (MOLOC) of the proposed binding mode of 11 in the active site of IspF (PDB
code: 1GX1 [8a]). Potential H-bonds are depicted as thin green lines, and distances are given in Š.
b) Superimposition of the proposed binding modes for 11 and 19. Color code: C-skeleton of ligand 11:
green, C-skeleton of ligand 19: dark green, C-skeleton of the protein: grey, N-atoms: blue, O-atoms: red,
S-atoms: yellow.

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Scheme 2. Synthesis of the Pyridine-diamines 7, 8, and 10
a) Phthalimide, PPh3, DIAD, THF, 258, 17 h; 88% (24); 99% (25). b) NH2Me (33% in EtOH), 258, 20 h;
quant. (26); 60% (27). c) 1. Benzoic acid or 4-methoxybenzoic acid, HO-Su, EDC· HCl, CH2Cl2, 258, 2 h;
2. 26 or 27, Et3N, CH2Cl2, 258, 20 h; 76% (28); 97% (29); 23% (34). d) Pd/C, H2, MeOH/CH2Cl2, 258, 3 h;
91% (30); 93% (31); 74% (35). e) 5-Bromo-2-nitropyridine, (Æ)-BINAP, [Pd2(dba)3], Cs2CO3, DME,
1108, 20 h; 59% (32); 47% (33); 70% (36). f) Pd/C, H2, MeOH/CH2Cl2, 258, 3 h; 90% (7); 89% (8); 89%
(10). DIAD ¼ Diisopropyl azodicarboxylate; HO-Su ¼ N-hydroxysuccinimide; NPht ¼ N-phthalimido;
EDC · HCl ¼ (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride; BINAP ¼ 2,2’-bis(di-
phenylphosphino)-1,1’-binaphthyl; dba ¼ dibenzylideneacetone.
idine yielded 39, which was deprotected to the alcohol 40. Conversion of 40 into amine
41 was achieved via the azido derivative 42. Amide couplings with the corresponding
benzoic acids, activated as the N-hydroxysuccinimide esters, gave 43 and 44, which were
transformed by catalytic hydrogenation into the desired target molecules 11 and 12,
respectively. The potential ligand 9 was prepared by reducing both the N3 and the NO2
group in 42 to give 45, followed by amide coupling. Performing the reduction of the
NO2 group by catalytic hydrogenation in the last step, as described for 11 and 12, led to
partial dechlorination under formation of an inseparable mixture.
2.2.2. Imidazopyridine Derivatives. The imidazopyridines 13 and 14 were synthe-
sized starting from 2-amino-6-chloro-3-nitropyridine (46) (Scheme 4). Reduction of
the NO2 group afforded the corresponding diamine 47. Cyclization with triethyl
orthoformate yielded the chlorinated imidazopyridine 48. A modified Ullmann
coupling [14] with 4-iodobenzonitrile provided the desired compound 49 and by-
product 50. Similarly, 51 and 52 were obtained starting from 4-iodo-3-methylbenzoni-

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Scheme 3. Synthesis of the Pyridine-diamines 9, 11, and 12
a) Pd/C, H2, MeOH, 258, 3 h; 55%. b) 5-Bromo-2-nitropyridine, (Æ)-BINAP, [Pd2(dba)3], Cs2CO3,
DME, 1108, 20 h; 39%. c) Bu4NF, THF, 258, 2 h; 88%. d) DPPA, DBU, THF, 08, 2 h, 258, 16 h; 98%. e)
PPh3, THF, H2O, 258, 20 h; 95%. f) 1. Benzoic acid derivative, HO-Su, EDC · HCl, CH2Cl2, 258, 2 h; 2. 41
or 45, Et3N, CH2Cl2, 258, 20 h; 61% (43); 34% (44); 47% (9). g) Pd/C, H2, MeOH/CH2Cl2, 258, 3 h; 82%
(45); 78% (11); 72% (12). DME ¼ 1,2-dimethoxyethane; DPPA ¼ diphenylphosphoryl azide; DBU ¼
1,3-diazabicyclo[5.4.0]undecane.
trile. Reduction of the nitriles 49 and 51 to amines 53 and 54, amide coupling to 55 and
56, and Buchwald – Hartwig cross-coupling afforded imines 57 and 58, respectively.
Finally, cleavage of the imines under acidic conditions afforded the potential inhibitors
13 and 14, respectively.
2.2.3. Cytosine Derivatives. The cytosine derivatives 16 – 21 were synthesized
starting from naphthalene-1,4-dicarboxylic acid 59 (Scheme 5; the synthesis of cytosine
derivative 15 (cf Fig. 1) is not described in this paper). Reduction afforded diol 60,
which, after mono-protection (!61), was transformed into bromide 62, and reaction
with Cbz-protected cytosine 63 provided 64. Deprotection of the alcohol (!65),
transformation via azido derivative 66 into amine 67, and amide couplings yielded 68 –
73. Finally, removal of the cytosine protecting group by catalytic hydrogenation
afforded the desired inhibitors 16 – 21.
2.3. Biological Results. The inhibitory affinity (IC50 values) of the new potential
ligands towards IspF protein was determined using a 13C-NMR assay [15]. This assay

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Scheme 4. Synthesis of the Imidazopyridines 13 and 14
a) 1. SnCl2 · 2 H2O, AcOEt, t-BuOH, 608, 1 h; 2. NaBH4, 608, 3 h; 66%. b) HC(OEt)3, TsOH · H2O,
toluene, 1408, 4 h; 95%. c) 4-Iodobenzonitrile or 4-iodo-3-methylbenzonitrile, 1,10-phenanthroline, CuI,
Cs2CO3, DMF, 1108, 24 h; 33% (49) and 27% (50); 11% (51) and 20% (52). d) CoCl2 · H2O, NaBH4,
MeOH, 508, 24 h; 82% (53); 79% (54). e) 1. Benzoic acid, HO-Su, EDC · HCl, CH2Cl2, 258, 2 h; 2. 53 or
54, Et3N, CH2Cl2, 258, 20 h; 68% (55); 53% (56). f) Benzophenone imine, [Pd2(dba)3], (Æ)-BINAP,
Cs2CO3, DME, 1108, 20 h; 34% (57); 57% (58). g) HCl, THF, 258, 2 h; 75% (13); 52% (14).
directly shows the conversion of 13C-labeled substrate to the product catalyzed by IspF.
Activity was first checked in a single-point measurement at an inhibitor concentration
of 1 mm. For ligands that displayed substantial activity at this concentration, the IC50
value was determined by variation of the ligand concentration. In addition, the
logarithmic partitioning coefficients clogP were calculated with the program ACD/
LogP (ACD/Labs) to estimate the influence of lipophilicity (and partitioning) on
binding.
Unexpectedly, the imidazopyridines 13 and 14 showed no detectable binding
affinity, despite offering an array of three H-bond donor/acceptor sites similar to that of
cytosine to bind to ꢀPocket IIIꢁ and the favorable accommodation into the active site
suggested by the molecular modeling. Gratifyingly, several of the 2,5-diaminopyridine-
and cytosine-based ligands showed activities (Table) that exceed, by one order of
magnitude or more, the IC50 values of the previously reported CDP-derived inhibitors,
CDP, and CMP, determined under identical conditions [6]. All compounds shown in
Fig. 1 but excluded from the Table do not show measurable binding affinity.
It can be assumed that the new active ligands bind to ꢀPocket IIIꢁ in the way
proposed in Fig. 2. This binding mode had been validated by X-ray crystallography for

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Scheme 5. Synthesis of the Cytosine Derivatives 16 – 21
a) LiAlH4, THF, 708, 20 h; 70%. b) (i-Pr)3SiCl, 1H-imidazole, THF, 08, 2 h, 258, 19 h; 46%. c) CBr4,
PPh3, CH2Cl2, 08, 3 h; 63%. d) 1. 63, NaH, DMF, 258, 1.5 h; 2. 62, DMF 258, 17 h; 79%. e) Bu4NF, THF,
258, 2 h; 90%. f) DPPA, DBU, THF, 08, 2 h, 258, 16 h; 93%. g) PPh3, THF, H2O, 258, 20 h; 98%. h) 1.
Benzoic acid derivative, HO-Su, EDC · HCl, CH2Cl2, 258, 2 h; 2. 67, Et3N, CH2Cl2, 258, 20 h; 81% (68);
74% (69); 22% (70); 42% (71); 78% (72); 31% (73). i) Pd/C, H2, MeOH/CH2Cl2, 258, 3 h; 76% (16);
65% (17); 54% (18); 59% (19); 40% (20); 72% (21).
Table. Biological Activities (IC50 [mm]a)) and clogP Values of the Inhibitors of IspF Protein
Inhibitor
Inhibition at
c(inhibitor) ¼ 1 mma)
IC50 [mm]
clogP
8
9
10
11
12
16
17
18
30%
14%
19%
60%
n.d.b)
n.d.
n.d.
0.49
n.d.
0.45
0.54
n.d.
0.65
n.d.
n.d.
15.0
7.3
2.7 Æ 0.6
3.5 Æ 0.6
2.9 Æ 0.6
3.7 Æ 0.6
2.6 Æ 0.6
2.3 Æ 0.5
3.0 Æ 0.5
2.4 Æ 0.5
3.2 Æ 0.5
2.2 Æ 0.5
2.7 Æ 0.5
n.d.
5%
59%c)
52%c)
0%c)
35%c)
0%c)
0%c)
19
20
21
CMPd)
CDPd)
n.d.
a) Uncertainties in IC50 values: Æ 2 – 10%. Values are determined from at least two experiments. b) Not
determined. c) c(inhibitor) ¼ 0.5 mm. d) Adopted from [6].

Helvetica Chimica Acta – Vol. 90 (2007)
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the weaker CDP-based ligands [6]. In agreement with the earlier results, filling the
hydrophobic portion of ꢀPocket IIꢁ does not seem to provide a large gain in binding free
energy due to the very high flexibility of the surrounding protein. Although clear
structure – activity relationships are not yet visible and currently also lack support by X-
ray structural analysis, it seems that filling this pocket with a phenyl residue bearing
electron-accepting substituents (e.g., 11 (CF3): IC50 ¼ 490 mm; 17 (Cl): IC50 ¼ 540 mm; 19
(CF3): IC50 ¼ 650 mm) is more favorable than filling it with a phenyl residue with an
electron-donating group (e.g., 10 and 18 (MeO), or 21 (Me2N) with no or very weak
measurable activity) or a larger residue (12 and 20 with a quinolinyl residue).
Partitioning, as estimated by the clogP values, also does not seem to greatly affect
binding affinity, although the clogP values of the most active compounds are more on
the positive side.
On the other hand, the investigation shows that it is possible to design ꢀdrug-likeꢁ
inhibitors, by-passing the ribose and diphosphate binding sites occupied by the natural
substrate 5 and the previously reported CDP-based ligands [6]. In particular, the
discovery of the sub-pocket that is filled by the 2-methyl-1,4-phenylene spacer in 11 and
the naphthalene-1,4-diyl moiety in 16, 17, and 19 (Fig. 3) opens new perspectives for
future lead developments.
Examination of the known crystal structures of IspF [8] revealed a preorganized,
open sub-pocket in all co-crystals, except in the crystal of pure enzyme (PBD code:
1JN1, [8d]). It is lined by Pro62’, Asp63’, Gly58’, Asp56’, Ala131, Thr132, Thr133,
Lys104, and Leu106. While most of the residues display highly conserved geometries in
the various crystal structures, Pro62’, Asp63’, and Lys104 seem to be rather flexible.
Overall, the sub-pocket is quite large, as suggested by the two different orientations of
the aromatic spacers in the modeling experiments (Fig. 3,b); at present, it is clearly not
optimally filled. We performed additional modeling studies with the truncated putative
ligands 74 and 75. We deliberately removed in these model compounds the vector
reaching into the highly flexible hydrophobic part of ꢀPocket IIꢁ, since, in some
structures, this region of the protein indeed arranges to give an open sub-pocket while
some others show no sub-pocket at all. Compounds 74 and 75 could be docked in a
strain-free way into all crystal structures featuring a preorganized open sub-pocket
adjacent to the cytosine binding site. Further optimization of the occupancy of this
pocket is currently under investigation.
3. Conclusions. – With its highly polar active site and the flexibility of the
hydrophobic part of ꢀPocket IIꢁ lined by Ala71’, Phe68’, Phe61’, and Leu76’, which
limits the binding free enthalpy that can be gained through its occupancy, IspF presents
itself as a rather ꢀtough targetꢁ. Nevertheless, the change from the first-generation
inhibitors, highly polar, CDP-derived derivatives, to the new, much more ꢀdrug-likeꢁ

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ligands described in this paper has yielded an increase in efficacy of about one order of
magnitude, as determined by IC50 values. From the new data, the following conclusions
can be drawn: i) It is possible to inhibit IspF without addressing the highly polar
diphosphate binding site of the protein and the side chain of Asp56’. The latter was
found to undergo ionic H-bonding with the OH residues of the ribose moieties in CMP
[8b], CDP [8a], and analogues [6]. This contrasts our findings in the development of
bisubstrate inhibitors of the enzyme catechol O-methyltransferase, which revealed a
crucial role of the interaction between a ribose ring in the substrate and a glutamate
side chain of the enzyme [16]. ii) Appropriate binding to the cytosine site in ꢀPocket IIIꢁ
by 2,5-diaminopyridines and derivatives of the nucleobase presumably yields the
largest part of the measured driving force for complexation. It still remains to be
clarified, however, why the 5-amino-1H-imidazo[4,5-b]pyridine scaffold, which should
undergo H-bonding interactions with the protein similar to cytosine, is not a suitable
substitute of the nucleobase. iii) An appropriate residue for occupancy of the
hydrophobic region of ꢀPocket IIꢁ, which makes a substantial contribution to the overall
binding free enthalpy, has not yet been identified. This desirable objective remains
difficult in view of the demonstrated large conformational flexibility in this region of
the protein [6][8]. iv) We consider the identification of a new spacious sub-pocket,
which presumably accommodates the aromatic spacers in our ligands between cytosine
(and analogues) and the terminal ring for occupancy of ꢀPocket IIIꢁ as the most
important finding of the present study. The occupation of this site (for the modeling, see
Fig. 3) provides room for much optimization, which is the subject of ongoing research.
Of course, we are well aware that the proposed binding mode of our inhibitors needs to
be further validated by X-ray crystallography, and efforts in this direction are also
pursued.
This research was supported by the ETH Research Council, F. Hoffmann-La Roche, Ltd., Basel, and
Chugai Pharmaceuticals.
Experimental Part
General. Solvents and reagents were reagent-grade, purchased from commercial suppliers, and used
without further purification unless otherwise stated. The following compounds were prepared according
to literature procedures: 6-chloropyridine-2,3-diamine (47) [17], (naphthalene-1,4-diyl)dimethanol (60)
[18], and benzyl (1,2-dihydro-2-oxopyrimidin-4-yl)carbamate (63) [19]. THF was freshly distilled from
sodium benzophenone ketyl, CH2Cl2 from CaH2. All products were dried under high vacuum (10ꢀ2 Torr)
before anal. characterization. Column chromatography (CC): SiO2 60 (40 – 63 mm) from Fluka, 0 – 0.3 bar
pressure. TLC: SiO2 60 F245 (on glass), Merck, visualization by UV light at 245 nm or staining with a soln.
of KMnO4 (3 g) and K2CO3 (20 g) in 5% aq. NaOH soln. (5 ml) and H2O (300 ml). M.p.: Büchi B540
melting point apparatus; uncorrected. IR Spectra: Perkin Elmer Spectrum BX FTIR System
spectrometer (ATR unit, Attenuated Total Reflection, Golden Gate); in cmꢀ1. NMR spectra (1H, 13C,
and 19F): Varian Gemini-300 and Bruker AMX-500; spectra were recorded at 258, with solvent peak as
reference. High-resolution mass spectra (HR-MS): MALDI: IonSpec Ultima spectrometer, 2,5-
dihydroxybenzoic acid (DHB) as matrix; EI: VG TRIBRID spectrometer at 70 eV. Elemental analyses
were performed by the Mikrolabor at the Laboratorium für Organische Chemie, ETH Zürich. The
nomenclature was generated with the computer program ACD-Name (ACD/Labs).

Helvetica Chimica Acta – Vol. 90 (2007)
1055
General Procedure for the Synthesis of a Phthalimide by a Mitsunobu Reaction (GP A). To a soln. of
an alcohol (1.0 equiv.), phthalimide (1.2 equiv.), and PPh3 (1.2 equiv.) in THF (4 ml per 1 mmol), DIAD
(1.2 equiv.) was added. The mixture was stirred at 258 for 17 h, and the solvent was concentrated in vacuo.
General Procedure for the Deprotection of a Phthalimide (GP B). A soln. of a phthalimide (1.0
equiv.) in 33% NH2Me in EtOH (10 ml per 1 mmol) was stirred at 258 for 20 h. The solvent was
concentrated in vacuo, and the resulting residue was taken up in 10% aq. AcOH soln. (15 ml per 1 mmol)
and washed with CH2Cl2/i-PrOH 3 :1 (6 ꢁ 10 ml per 1 mmol). The aq. phase was treated with 1m aq.
NaOH soln. until pH > 12 was reached and extracted with CH2Cl2/i-PrOH 3 :1 (6 ꢁ 10 ml per 1 mmol).
The combined org. phases were dried (MgSO4) and filtered. The filtrate was concentrated in vacuo and
dried.
General Procedure for the Conversion of a Primary Amine to an Amide (GP C). A soln. of a benzoic
acid, N-hydroxysuccinimide, and EDC · HCl in CH2Cl2 was stirred at 258 for 2 h. The mixture was added
to an amine. After the addition of Et3N and CH2Cl2, the soln. was stirred at 258 for 20 h. CH2Cl2 was
added, and the org. phase was washed with sat. aq. NaCl soln., dried (MgSO4), and filtered. The filtrate
was concentrated in vacuo.
General Procedure for the Reduction of an Aromatic NO2 Group to an Amino Group or for the
Deprotection of a Cbz-Protected Amine (GP D). To a soln. of a nitrobenzene derivative or a Cbz-
protected cytosine derivative (1.0 equiv.) in MeOH or CH2Cl2/MeOH, 10% Pd/C (w/w) was added under
Ar. The flask was evacuated and refilled with H2 (3ꢁ). The black suspension was stirred at 258 for 3 h
under H2. The mixture was filtered over Celite, and the Celite was washed with MeOH and CH2Cl2. The
filtrate was concentrated in vacuo.
General Procedure for a Buchwald – Hartwig Cross-Coupling Reaction (GP E). [Pd2(dba)3] (5 mol-
%), (Æ)-BINAP (10 mol-%), an aromatic halide (1.0 equiv.), and Cs2CO3 (2.5 equiv.) were added to an
oven-dried sealed tube. The flask was evacuated and refilled with Ar. An aromatic amine or
benzophenone imine (1.2 equiv.) and DME (2 ml per 1 mmol) were added, and the mixture was stirred at
1108 for 20 h. The suspension was allowed to cool to 258 and taken up in CH2Cl2 (30 ml per 1 mmol). The
org. phase was washed with sat. aq. NaCl soln. (30 ml per 1 mmol), dried (MgSO4), and filtered. The
filtrate was concentrated in vacuo.
General Procedure for the Deprotection of a (i-Pr)3Si-Protected Alcohol (GP F). To a soln. of a silyl
ether (1.0 equiv.) in THF (15 ml per 1 mmol), Bu4NF (1m in THF, 1.2 equiv.) was added, and the mixture
was stirred at 258 for 2 h. After addition of H2O (60 ml per 1 mmol), the aq. layer was extracted with
CH2Cl2 (100 ml per 1 mmol). The org. phase was dried (MgSO4) and filtered. The filtrate was
concentrated in vacuo.
General Procedure for the Conversion of an Alcohol to an Azido Derivative (GP G). A soln. of an
alcohol (1.0 equiv.) and DPPA (1.4 equiv.) in THF (15 ml per 1 mmol) was stirred at 258 for 10 min, then
cooled to 08. DBU (1.4 equiv.) was added dropwise, and the mixture was stirred at 08 for 2 h and at 258 for
16 h. CH2Cl2 (60 ml per mmol) and H2O (50 ml per mmol) were added, and the phases were separated.
The aq. layer was extracted with CH2Cl2 (30 ml per 1 mmol). The combined org. phases were washed
with sat. aq. NaCl soln. (50 ml per 1 mmol), dried (MgSO4), and filtered. The filtrate was concentrated in
vacuo.
General Procedure for the Staudinger Reduction of an Azido Derivative to an Amine (GP H). To a
soln. of an azido derivative (1.0 equiv.) in THF (10 ml per 1 mmol) and H2O (0.4 ml per 1 mmol), PPh3
(2.0 equiv.) was added, and the mixture was stirred at 258 for 20 h. AcOEt (40 ml per 1 mmol) was added,
and the org. phase was extracted with 1m aq. HCl soln. (3 ꢁ 30 ml per 1 mmol). The aq. phase was treated
with 4m aq. NaOH soln. until pH > 12 was reached and extracted with CH2Cl2 (2 ꢁ 30 ml per 1 mmol) and
CH2Cl2/i-PrOH 3 :1 (2 ꢁ 40 ml per 1 mmol). The combined org. phases were dried (MgSO4) and filtered.
The filtrate was concentrated in vacuo and dried.
General Procedure for a modified Ullmann Coupling Reaction with an Imidazopyridine and an
Aromatic Iodide (GP J). CuI, an imidazopyridine, and Cs2CO3 or K2CO3 were added to an oven-dried
sealed tube. The flask was evacuated and refilled with Ar. An aromatic iodide, 1,10-phenanthroline, and
DMF were added, and the mixture was stirred at 1108 for 24 h. The suspension was allowed to cool to 258
and taken up in AcOEt. The solvent was concentrated in vacuo.

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Helvetica Chimica Acta – Vol. 90 (2007)
General Procedure for the Reduction of an Aromatic Nitrile to an Aromatic Amine (GP K). To a
suspension of a nitrile (1.0 equiv.) and CoCl2 · 6 H2O (4.0 equiv.) in MeOH or CH2Cl2/MeOH 1:1, NaBH4
(4.0 equiv.) was added, and the mixture was stirred at 508 for 24 h. The suspension was allowed to cool to
258, taken up in 4m aq. HCl soln. (10 ml per 1 mmol) and H2O (40 ml per 1 mmol), and washed with
CH2Cl2 (3 ꢁ 40 ml per 1 mmol). The aq. phase was treated with 1m aq. NaOH soln. until pH > 12 was
reached and extracted with CH2Cl2 (2 ꢁ 60 ml per 1 mmol) and CH2Cl2/i-PrOH 3 :1 (3 ꢁ 60 ml per
1 mmol). The combined org. phases were washed with sat. aq. NaCl soln. (150 ml per 1 mmol), dried
(MgSO4), and filtered. The filtrate was concentrated in vacuo and dried.
General Procedure for the Deprotection of an Aromatic Benzophenone Imine to an Aromatic Amine
(GP L). To a soln. of a benzophenone imine (1.0 equiv.) in THF (10 ml per mmol), 2m aq. HCl soln. (1 ml
per 1 mmol) was added. The suspension was stirred at 258 for 2 h, 1m aq. HCl soln. (100 ml per 1 mmol)
was added, and the aq. phase was washed with hexane/AcOEt 2 :1 (150 ml per 1 mmol). The aq. phase
was treated with 1m aq. NaOH soln. until pH > 12 was reached and extracted with CH2Cl2 (3 ꢁ 300 ml per
1 mmol). The combined org. phases were dried (MgSO4) and filtered. The filtrate was concentrated in
vacuo.
2-(4-Nitrobenzyl)-1H-isoindole-1,3(2H)-dione (24) [20]. GP A started from 4-nitrobenzyl alcohol
(22) (5.01 g, 32.7 mmol), phthalimide (5.77 g, 39.2 mmol), PPh3 (10.3 g, 39.2 mmol), THF (100 ml), and
DIAD (7.8 ml, 39.2 mmol) to yield, after purification by CC (SiO2; CH2Cl2), 24 (8.11 g, 88%). White
solid. M.p. 173 – 1748 ([20]: 171 – 1748). IR (neat): 3074w, 2931w, 2843w, 1767m, 1698s, 1598m, 1509s,
1466m, 1422m, 1392s, 1343s, 1327s, 1101m, 942s. 1H-NMR (300 MHz, CDCl3): 4.93 (s, 2 H); 7.58 (d, J ¼
8.4, 2 H); 7.73 – 7.76 (m, 2 H); 7.86 – 7.89 (m, 2 H); 8.18 (d, J ¼ 8.7, 2 H). 13C-NMR (75 MHz, CDCl3):
41.0; 123.6; 123.9; 129.3; 131.8; 134.3; 143.2; 147.4; 167.6. HR-EI-MS: 282.0637 (Mþ, C15H10N2O4þ ; calc.
282.0641). Anal. calc. for C15H10N2O4 (282.25): C 63.83, H 3.57, N 9.92; found C 63.80, H 3.60, N 9.81.
1-(4-Nitrophenyl)methanamine (26) [21]. GP B started from 24 (4.0 g, 14.2 mmol) and 33% NH2Me
in EtOH (150 ml) to yield 26 (2.17 g, quant.). Yellow oil. The crude product was used in the next step
without further purification and was not fully characterized. 1H-NMR (300 MHz, CDCl3): 4.01 (s, 2 H);
7.50 (d, J ¼ 8.4, 2 H); 8.19 (d, J ¼ 8.7, 2 H).
N-(4-Nitrobenzyl)benzamide (28) [22]. GP C started from PhCOOH (200 mg, 1.63 mmol), N-
hydroxysuccinimide (244 mg, 2.12 mmol), EDC · HCl (469 mg, 2.45 mmol), CH2Cl2 (3 ml), 26 (372 mg,
2.45 mmol), Et3N (1.1 ml, 8.15 mmol), and CH2Cl2 (2 ml) to yield, after purification by CC (SiO2;
CH2Cl2/MeOH 199 :1 ! 99 :1), 28 (290 mg, 76%). Yellow solid. M.p. 155 – 1568 ([22]: 155 – 1568). IR
(neat): 3310w, 3076w, 2941w, 2850w, 1633s, 1603m, 1529m, 1512s, 1489m, 1346s, 1311s, 1290s, 1234m,
1106m, 854s. 1H-NMR (300 MHz, CDCl3): 4.76 (d, J ¼ 6.2, 2 H); 6.61 (s, 1 H); 7.44 – 7.57 (m, 5 H); 7.81 (d,
J ¼ 8.1, 2 H); 8.20 (d, J ¼ 8.7, 2 H). 13C-NMR (75 MHz, CDCl3): 43.4; 123.9; 126.9; 128.2; 128.7; 131.9;
133.6; 145.8; 147.2; 167.4. HR-EI-MS: 256.0845 (Mþ, C14H12N2O3þ ; calc. 256.0848). Anal. calc. for
C14H12N2O3 (256.26): C 65.62, H 4.72, N 10.93; found C 65.34, H 4.80, N 10.80.
N-(4-Aminobenzyl)benzamide (30) [23]. GP D started from 28 (264 mg, 1.03 mmol), 10% Pd/C
(27 mg, 10% (w/w)), and CH2Cl2/MeOH 2 :1 (9 ml) to yield, after purification by CC (SiO2; CH2Cl2/
MeOH 98 :2), 30 (211 mg, 91%). White solid. M.p. 142 – 1438 ([23]: 142 – 1438). IR (neat): 3439m,
3338m, 3000w, 2926w, 1623s, 1610s, 1576m, 1530s, 1516s, 1486s, 1282s, 1077m, 841m. 1H-NMR (300 MHz,
(CD3)2SO): 4.30 (d, J ¼ 5.9, 2 H); 4.94 (s, 2 H); 6.51 (d, J ¼ 8.4, 2 H); 6.84 (d, J ¼ 8.4, 2 H); 7.42 – 7.54 (m,
3 H); 7.87 (d, J ¼ 8.4, 2 H); 8.84 (t, J ¼ 5.6, 1 H). 13C-NMR (75 MHz, 608, (CD3)2SO): 42.4; 113.7; 126.6;
127.0; 127.9; 128.0; 130.7; 134.6; 147.2; 165.7. HR-EI-MS: 226.1100 (Mþ, C14H14N2Oþ; calc. 226.1106).
Anal. calc. for C14H14N2O (226.28): C 74.31, H 6.24, N 12.38; found C 74.02, H 6.32, N 12.26.
N-{4-[(6-Nitropyridin-3-yl)amino]benzyl}benzamide (32). GP E started from [Pd2(dba)3] (25 mg,
0.03 mmol, 5 mol-%), BINAP (34 mg, 0.06 mmol, 10 mol-%), 5-bromo-2-nitropyridine (114 mg,
0.56 mmol), Cs2CO3 (455 mg, 1.40 mmol), amine 30 (151 mg, 0.67 mmol), and DME (1.5 ml) to yield,
after purification by CC (SiO2; CH2Cl2/MeOH 98 :2), 32 (124 mg, 59%). Orange solid. M.p. 198 – 2008.
IR (neat): 3238m, 3066w, 2853w, 1633s, 1574s, 1500s, 1472m, 1444m, 1358m, 1332s, 1305s, 1290s, 1256s,
1105s, 1013m. 1H-NMR (300 MHz, (CD3)2SO): 4.47 (d, J ¼ 6.2, 2 H); 7.24 (d, J ¼ 8.4, 2 H); 7.35 (d, J ¼ 8.4,
2 H); 7.45 – 7.56 (m, 4 H); 7.90 (d, J ¼ 8.4, 2 H); 8.15 – 8.18 (m, 2 H); 9.05 (t, J ¼ 5.6, 1 H); 9.46 (s, 1 H).
13C-NMR (75 MHz, (CD3)2SO): 42.2; 119.8; 120.4; 127.1; 128.1; 128.4; 131.1; 134.1; 134.6; 135.1; 137.9;

Helvetica Chimica Acta – Vol. 90 (2007)
1057
146.0; 147.2; 165.9 (one arom. signal missing due to overlap). HR-EI-MS: 348.1215 (Mþ, C19H16N4O3þ ;
calc. 348.1222).
N-{4-[(6-Aminopyridin-3-yl)amino]benzyl}benzamide (7). GP D started from 32 (103 mg,
0.30 mmol), 10% Pd/C (10 mg, 10% (w/w)), and CH2Cl2/MeOH 2 :1 (12 ml) to yield, after purification
by CC (SiO2; CH2Cl2/MeOH 95 :5), 7 (83 mg, 90%). Grey solid. M.p. 79 – 818. IR (neat): 3297m, 3030w,
2922w, 1614m, 1575m, 1514s, 1494s, 1290s, 1178m. 1H-NMR (300 MHz, (CD3)2SO): 4.33 (d, J ¼ 5.9, 2 H);
5.62 (s, 2 H); 6.44 (d, J ¼ 8.7, 1 H); 6.69 (d, J ¼ 8.4, 2 H); 7.08 (d, J ¼ 8.4, 2 H); 7.21 (dd, J ¼ 8.7, 2.8, 1 H);
7.42 – 7.54 (m, 4 H); 7.72 (d, J ¼ 2.8, 1 H); 7.87 (d, J ¼ 8.4, 2 H); 8.89 (t, J ¼ 5.3, 1 H). 13C-NMR (75 MHz,
(CD3)2SO): 42.2; 108.1; 113.4; 127.0; 128.1; 128.2; 128.3; 128.6; 130.9; 132.0; 134.3; 141.0; 145.2; 155.4;
165.6. HR-EI-MS: 318.1474 (Mþ, C19H18N4Oþ; calc. 318.1481). Anal. calc. for C19H18N4O (318.37): C
71.68, H 5.70, N 17.60; found C 71.39, H 5.92, N 17.51.
2-(3-Methyl-4-nitrobenzyl)-1H-isoindole-1,3(2H)-dione (25). GP A started from 3-methyl-4-nitro-
benzyl alcohol (23) (3.00 g, 17.9 mmol), phthalimide (3.17 g, 21.5 mmol), PPh3 (5.65 g, 21.5 mmol), THF
(60 ml), and DIAD (4.2 ml, 21.5 mmol) to yield, after purification by CC (SiO2; CH2Cl2), 25 (5.27 g,
99%). Yellow solid. M.p. 154 – 1568. IR (neat): 3074w, 2988w, 1765m, 1710s, 1610m, 1588m, 1515s, 1464m,
1435m, 1395s, 1333s, 1319s, 1103m, 935m. 1H-NMR (300 MHz, CDCl3): 2.57 (s, 3 H); 4.86 (s, 2 H); 7.37 –
7.39 (m, 2 H); 7.72 – 7.77 (m, 2 H); 7.84 – 7.88 (m, 2 H); 7.93 (d, J ¼ 8.7, 1 H). 13C-NMR (75 MHz, CDCl3):
20.7; 40.8; 123.5; 125.2; 126.8; 131.8; 132.6; 134.1; 134.2; 141.4; 148.4; 167.7. HR-EI-MS: 296.0790 (Mþ,
C16H12N2Oþ4 ; calc. 296.0797). Anal. calc. for C16H12N2O4 (296.28): C 64.86, H 4.08, N 9.45; found C 64.82,
H 4.13, N 9.71.
1-(3-Methyl-4-nitrophenyl)methanamine (27). GP B started from 25 (1.83 g, 6.19 mmol) and 33%
NH2Me in EtOH (60 ml) to yield 27 (616 mg, 60%). Yellow oil. IR (neat): 3376w, 2930w, 2855w, 1610m,
1587m, 1509s, 1449m, 1337s, 1158w, 833s. 1H-NMR (300 MHz, CDCl3): 2.92 (s, 3 H); 3.94 (s, 2 H); 7.29 (d,
J ¼ 8.4, 1 H); 7.31 (s, 1 H); 7.97 (d, J ¼ 8.1, 1 H). 13C-NMR (75 MHz, CDCl3): 20.6; 45.4; 125.0; 125.2;
131.1; 134.0; 147.7; 148.6. HR-EI-MS: 165.0659 ([M ꢀ H]þ, C8H9N2O2þ ; calc. 165.0659).
N-(3-Methyl-4-nitrobenzyl)benzamide (29). GP C started from PhCOOH (801 mg, 6.56 mmol), N-
hydroxysuccinimide (981 mg, 8.52 mmol), EDC · HCl (1.88 g, 9.81 mmol), CH2Cl2 (13 ml), 27 (1.64 g,
9.89 mmol), Et3N (4.5 ml, 32.5 mmol), and CH2Cl2 (9 ml) to yield, after purification by CC (SiO2;
CH2Cl2/MeOH 199 :1 ! 99 :1), 29 (1.72 g, 97%). Yellow solid. M.p. 127 – 1288. IR (neat): 3326m, 3058w,
1639m, 1578m, 1538m, 1511s, 1488s, 1335s, 1291m, 1237m, 986m, 835s. 1H-NMR (300 MHz, CDCl3): 2.59
(s, 3 H); 4.68 (d, J ¼ 5.9, 2 H); 6.61 (s, 1 H); 7.29 – 7.31 (m, 2 H); 7.43 – 7.56 (m, 3 H); 7.80 – 7.82 (m, 2 H);
7.96 (d, J ¼ 9.0, 1 H). 13C-NMR (75 MHz, CDCl3): 20.7; 43.2; 125.1; 125.7; 126.9; 128.6; 131.6; 131.8;
133.7; 134.1; 143.9; 148.1; 167.4. HR-EI-MS: 270.1000 (Mþ, C15H14N2O3þ ; calc. 270.1004). Anal. calc. for
C15H14N2O3 (270.28): C 66.66, H 5.22, N 10.36; found C 66.47, H 5.05, N 10.31.
N-(4-Amino-3-methylbenzyl)benzamide (31). GP D started from 29 (1.50 g, 5.55 mmol), 10% Pd/C
(150 mg, 10% (w/w)), and CH2Cl2/MeOH 2 :1 (39 ml) to yield, after purification by CC (SiO2; CH2Cl2/
MeOH 98 :2), 31 (1.24 g, 93%). Light pink solid. M.p. 113 – 1148. IR (neat): 3431w, 3308m, 3018w, 2920w,
2857w, 1629s, 1577m, 1532s, 1505s, 1487m, 1304m, 1277s, 1228m, 1148m, 832m. 1H-NMR (300 MHz,
(CD3)2SO): 2.02 (s, 3 H); 4.28 (d, J ¼ 5.9, 2 H); 4.72 (s, 2 H); 6.54 (d, J ¼ 7.8, 1 H); 6.83 – 6.88 (m, 2 H);
7.41 – 7.54 (m, 3 H); 7.85 – 7.89 (m, 2 H); 8.82 (t, J ¼ 5.8, 1 H). 13C-NMR (75 MHz, (CD3)2SO): 17.5; 42.4;
113.6; 120.6; 125.7; 126.7; 127.0; 128.1; 129.2; 130.9; 134.4; 145.2; 165.6. HR-EI-MS: 240.1260 (Mþ,
C15H16N2Oþ; calc. 240.1263). Anal. calc. for C15H16N2O (240.30): C 74.97, H 6.71, N 11.66; found C 74.85,
H 6.74, N 11.57.
N-{3-Methyl-4-[(6-nitropyridin-3-yl)amino]benzyl}benzamide (33). GP E started from [Pd2(dba)3]
(58 mg, 0.06 mmol, 5 mol-%), BINAP (77 mg, 0.12 mmol, 10 mol-%), 5-bromo-2-nitropyridine (249 mg,
1.21 mmol), Cs2CO3 (987 mg, 3.03 mmol), 31 (350 mg, 1.46 mmol), and DME (3.5 ml) to yield, after
purification by CC (SiO2; CH2Cl2/MeOH 98 :2), 33 (207 mg, 47%). Orange solid. M.p. 206 – 2088. IR
1
(neat): 3300m, 2872w, 1633s, 1574s, 1512s, 1469s, 1325s, 1280s, 1260s, 1218s, 1104s. H-NMR (300 MHz,
(CD3)2SO): 2.19 (s, 3 H); 4.47 (d, J ¼ 5.9, 2 H); 7.04 (dd, J ¼ 9.0, 2.8, 1 H); 7.20 – 7.29 (m, 3 H); 7.46 – 7.57
(m, 3 H); 7.90 – 7.92 (m, 2 H); 8.03 (d, J ¼ 2.8, 1 H); 8.13 (d, J ¼ 9.0, 1 H); 9.04 (s, 1 H); 9.05 (t, J ¼ 5.8,
1 H). 13C-NMR (75 MHz, (CD3)2SO): 17.7; 42.2; 119.0; 120.5; 124.2; 125.8; 127.1; 128.1; 130.0; 131.1;
132.7; 133.8; 134.1; 135.7; 137.2; 146.8; 147.6; 165.9. HR-MALDI-MS: 385.1268 ([M þ Na]þ
C20H18N4NaOþ3 ; calc. 385.1271).

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Helvetica Chimica Acta – Vol. 90 (2007)
N-{4-[(6-Aminopyridin-3-yl)amino]-3-methylbenzyl}benzamide (8). GP D started from 33 (109 mg,
0.30 mmol), 10% Pd/C (11 mg, 10% (w/w)), and CH2Cl2/MeOH 1:2 (18 ml) to yield, after purification
by CC (SiO2; CH2Cl2/MeOH 95 :5), 8 (89 mg, 89%). Grey solid. M.p. > 2208 (dec.). IR (neat): 3464w,
3370m, 3288m, 3032w, 2929w, 1624m, 1542m, 1497s, 1424m, 1316s, 1293s. 1H-NMR (300 MHz,
(CD3)2SO): 2.17 (s, 3 H); 4.33 (d, J ¼ 5.9, 2 H); 5.59 (s, 2 H); 6.43 (d, J ¼ 8.4, 1 H); 6.60 (d, J ¼ 8.1,
1 H); 6.65 (s, 1 H); 6.93 (d, J ¼ 8.4, 1 H); 7.03 (s, 1 H); 7.15 (dd, J ¼ 8.7, 2.8, 1 H); 7.42 – 7.54 (m, 3 H); 7.70
(d, J ¼ 2.5, 1 H); 7.86 – 7.88 (m, 2 H); 8.88 (t, J ¼ 5.9, 1 H). 13C-NMR (75 MHz, (CD3)2SO): 18.0; 42.2;
108.0; 112.7; 123.9; 125.6; 127.0; 128.1; 129.0; 129.2; 129.7; 130.9; 132.5; 134.3; 141.6; 143.5; 155.5; 165.6.
HR-EI-MS: 332.1634 (Mþ, C20H20N4Oþ; calc. 332.1632).
4-Methoxy-N-(3-methyl-4-nitrobenzyl)benzamide (34). GP C started from 4-methoxybenzoic acid
(367 mg, 2.41 mmol), N-hydroxysuccinimide (360 mg, 3.13 mmol), EDC · HCl (692 mg, 3.61 mmol),
CH2Cl2 (5 ml), 27 (592 mg, 3.56 mmol), Et3N (1.7 ml, 12.2 mmol), and CH2Cl2 (5 ml) to yield, after
purification by CC (SiO2; CH2Cl2/MeOH 199 :1), 34 (1.67 g, 23%). Grey solid. M.p. 145 – 1478. IR
(neat): 3375w, 2941m, 2864m, 1626m, 1509s, 1462m, 1367w, 1280m, 1158m, 1145m, 1092m, 1063s, 1012m,
882s. 1H-NMR (300 MHz, CDCl3): 2.60 (s, 3 H); 3.86 (s, 3 H); 4.67 (d, J ¼ 6.1, 2 H); 6.42 (br. s, 1 H); 6.95
(d, J ¼ 9.0, 2 H); 7.29 – 7.33 (m, 2 H); 7.78 (d, J ¼ 8.7, 2 H); 7.97 (d, J ¼ 8.7, 1 H). 13C-NMR (75 MHz,
CDCl3): 20.8; 43.3; 55.6; 114.1; 125.4; 126.0; 126.2; 129.0; 132.0; 134.4; 144.5; 148.4; 162.7; 167.2. HR-
MALDI-MS: 301.1186 ([M þ H]þ, C16H17N2O4þ ; calc. 301.1183). Anal. calc. for C16H16N2O4 (300.31): C
63.99, H 5.37, N 9.33; found C 63.91, H 5.53, N 9.28.
N-(4-Amino-3-methylbenzyl)-4-methoxybenzamide (35). GP D started from 34 (497 mg,
1.65 mmol), 10% Pd/C (50 mg, 10% (w/w)), and CH2Cl2/MeOH 2 :1 (12 ml) to yield, after purification
by CC (SiO2; CH2Cl2/MeOH 99 :1 ! 98 :2), 35 (332 mg, 74%). White solid. M.p. 134 – 1368. IR (neat):
3301w, 2920w, 1625s, 1574m, 1540m, 1503s, 1445m, 1360m, 1300m, 1283s, 1257s, 1226s, 1177s, 1107m,
1026s, 970m, 846s, 825s. 1H-NMR (300 MHz, CDCl3): 2.16 (s, 3 H); 3.61 (br. s, 2 H); 3.84 (s, 3 H); 4.49 (d,
J ¼ 5.3, 2 H); 6.16 (br. s, 1 H); 6.65 (d, J ¼ 8.1, 1 H); 6.91 (d, J ¼ 8.7, 2 H); 7.02 – 7.05 (m, 2 H); 7.74 (d, J ¼
9.0, 2 H). 13C-NMR (75 MHz, CDCl3): 17.2; 43.8; 55.3; 113.6; 114.9; 122.5; 126.8; 128.0; 128.6; 130.4;
144.0; 162.0; 166.5 (one arom. signal missing due to overlap). HR-EI-MS: 270.1361 (Mþ, C16H18N2O2þ ;
calc. 270.1363). Anal. calc. for C16H18N2O2 (270.33): C 71.09, H 6.71, N 10.36; found C 70.93, H 6.72, N
10.26.
4-Methoxy-N-{3-methyl-4-[(6-nitropyridin-3-yl)amino]benzyl}benzamide (36). GP E started from
[Pd2(dba)3] (35 mg, 0.04 mmol, 5 mol-%), BINAP (48 mg, 0.08 mmol, 10 mol-%), 5-bromo-2-nitro-
pyridine (155 mg, 0.77 mmol), Cs2CO3 (631 mg, 1.93 mmol), 35 (250 mg, 0.92 mmol), and DME (3 ml) to
yield, after purification by CC (SiO2; CH2Cl2/MeOH 99 :1 ! 98 :2), 36 (212 mg, 70%). Orange solid.
M.p. 186 – 1888. IR (neat): 3277w, 2924w, 1634w, 1605m, 1573m, 1503s, 1385w, 1286s, 1251s, 1177m,
1104m, 1027m, 1006m, 841s. 1H-NMR (300 MHz, (CD3)2SO): 2.18 (s, 3 H); 3.81 (s, 3 H); 4.45 (d, J ¼ 5.9,
2 H); 7.01 (d, J ¼ 8.7, 2 H); 7.08 (dd, J ¼ 9.0, 2.8, 1 H); 7.18 – 7.27 (m, 3 H); 7.89 (d, J ¼ 9.0, 2 H); 8.02 (d,
J ¼ 2.8, 1 H); 8.13 (d, J ¼ 9.0, 1 H); 8.90 (t, J ¼ 6.1, 1 H); 8.99 (s, 1 H). 13C-NMR (125 MHz, (CD3)2SO):
18.4; 42.8; 56.0; 114.2; 119.9; 121.3; 125.1; 126.6; 127.2; 129.8; 130.9; 133.5; 134.6; 136.6; 138.3; 147.7;
148.5; 162.3; 166.3. HR-MALDI-MS: 415.1384 ([M þ Na]þ, C21H20N4NaOþ4; calc. 415.1377).
N-{4-[(6-Aminopyridin-3-yl)amino]-3-methylbenzyl}-4-methoxybenzamide (10). GP D started from
36 (160 mg, 0.41 mmol), 10% Pd/C (16 mg, 10% (w/w)), and CH2Cl2/MeOH 2 :1 (21 ml) to yield, after
purification by CC (SiO2; AcOEt/pentane/MeOH 8 :1:0.1), 10 (132 mg, 89%). Light grey solid.
M.p. 206 – 2088. IR (neat): 3318w, 2926w, 1605m, 1573w, 1542w, 1493s, 1381w, 1291m, 1250s, 1177m,
1118w, 1027m, 843m, 814m. 1H-NMR (300 MHz, (CD3)2SO): 2.16 (s, 3 H); 3.80 (s, 3 H); 4.30 (d, J ¼ 5.9,
2 H); 5.59 (br. s, 2 H); 6.43 (d, J ¼ 8.4, 1 H); 6.59 (d, J ¼ 8.1, 1 H); 6.65 (s, 1 H); 6.91 (d, J ¼ 8.1, 1 H); 6.98
(d, J ¼ 9.0, 2 H); 7.00 (br. s, 1 H); 7.15 (dd, J ¼ 8.7, 2.8, 1 H); 7.69 (d, J ¼ 2.5, 1 H); 7.85 (d, J ¼ 9.0, 2 H);
8.72 (t, J ¼ 5.9, 1 H). 13C-NMR (75 MHz, (CD3)2SO): 18.0; 42.2; 55.3; 108.2; 113.0; 113.5; 124.2; 125.8;
126.8; 129.1; 129.5; 129.9; 132.7; 141.8; 143.7; 155.7; 161.5; 165.4 (one arom. signal missing due to
overlap). HR-MALDI-MS: 362.1722 ([M þ Na]þ, C21H22N4NaOþ2; calc. 362.1737).
Triisopropyl[(3-methyl-4-nitrobenzyl)oxy]silane (37). To a soln. of 3-methyl-4-nitrobenzyl alcohol
(23) (10.0 g, 59.8 mmol) in THF (100 ml), (i-Pr)3SiCl (15.4 ml, 71.8 mmol) and 1H-imidazole (4.89 g,
71.8 mmol) were added at 08. The mixture was stirred at 08 for 2 h and at 258 for 21 h. After addition of
CH2Cl2 (200 ml), the mixture was washed with sat. aq. NaHCO3 soln. (3 ꢁ 200 ml) and sat. aq. NaCl soln.

Helvetica Chimica Acta – Vol. 90 (2007)
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(200 ml). The org. phase was dried (MgSO4) and filtered. The filtrate was concentrated in vacuo, and the
crude product was purified by CC (SiO2, CH2Cl2) to yield 37 (17.5 g, 90%). Yellow oil. IR (neat): 2943m,
2866m, 1614w, 1591w, 1519s, 1462m, 1340s, 1164m, 1107s, 1070m, 881s, 836m. 1H-NMR (300 MHz,
CDCl3): 1.07 – 1.25 (m, 21 H); 2.62 (s, 3 H); 4.87 (s, 2 H); 7.31 (s, 1 H); 7.32 (d, J ¼ 8.1, 1 H); 7.99 (d, J ¼
8.4, 1 H). 13C-NMR (75 MHz, CDCl3): 12.1; 18.1; 21.0; 64.1; 123.7; 124.8; 129.5; 133.7; 147.3; 147.6. HR-
EI-MS: 280.1364 ([M ꢀ C3H7]þ, C14H22NO3Siþ; calc. 280.1364). Anal. calc. for C17H29NO3Si (323.51): C
63.12, H 9.04, N 4.33; found C 63.11, H 8.79, N 4.41.
2-Methyl-4-{[(triisopropylsilyl)oxy]methyl}aniline (38). GP D started from 37 (4.21 g, 13.0 mmol),
10% Pd/C (420 mg, 10% (w/w)), and MeOH (40 ml) to yield, after purification by CC (SiO2; AcOEt/
pentane/Et2O 9 :1 ! 5 :1), 38 (2.08 g, 55%). Red oil. IR (neat): 3319m, 2928w, 1633s, 1605s, 1549s, 1523s,
1505s, 1439m, 1354s, 1343s, 1296s, 1247s, 1186s, 1110m, 1024s, 834s. 1H-NMR (300 MHz, CDCl3): 1.08 –
1.17 (m, 21 H); 2.17 (s, 3 H); 3.57 (br. s, 2 H); 4.70 (s, 2 H); 6.65 (d, J ¼ 8.7, 1 H); 7.02 – 7.04 (m, 2 H).
13C-NMR (75 MHz, CDCl3): 12.0; 17.4; 18.0; 64.9; 114.7; 122.0; 124.8; 128.4; 131.8; 143.3. HR-EI-MS:
293.2171 (Mþ, C17H31NOSiþ; calc. 293.2170). Anal. calc. for C17H31NOSi (293.52): C 69.56, H 10.64, N
4.77; found C 69.38, H 10.60, N 4.85.
N-(2-Methyl-4-{[(triisopropylsilyl)oxy]methyl}phenyl)-6-nitropyridin-3-amine (39). GP E started
from [Pd2(dba)3] (196 mg, 0.21 mmol, 5 mol-%), BINAP (270 mg, 0.43 mmol, 10 mol-%), 5-bromo-2-
nitropyridine (864 mg, 4.26 mmol), Cs2CO3 (3.47 g, 10.7 mmol), 38 (1.49 g, 5.08 mmol), and DME (9 ml)
to yield, after purification by CC (SiO2; pentane/AcOEt 5 :1), 39 (691 mg, 39%). Orange solid.
M.p. 106 – 1088. IR (neat): 3246m, 3120w, 3060w, 2880w, 1570m, 1544m, 1505s, 1474m, 1387m, 1326s,
1288s, 1269s, 1156m, 1110s, 1045m, 1000s, 827s. 1H-NMR (300 MHz, CDCl3): 1.09 – 1.23 (m, 21 H); 2.25
(s, 3 H); 4.83 (s, 2 H); 6.05 (br. s, 1 H); 7.05 (dd, J ¼ 9.0, 3.1, 1 H); 7.20 – 7.30 (m, 3 H); 8.04 (d, J ¼ 2.8,
1 H); 8.12 (d, J ¼ 8.7, 1 H). 13C-NMR (75 MHz, CDCl3): 12.2; 18.2; 64.6; 119.8; 120.2; 124.7; 124.7; 128.9;
133.3; 134.4; 134.9; 140.4; 146.9; 148.3 (one aliph. signal missing due to overlap). HR-MALDI-MS:
416.2362 ([M þ H]þ, C22H34N3O3Siþ; calc. 416.2364).
{3-Methyl-4-[(6-nitropyridin-3-yl)amino]phenyl}methanol (40). GP F started from 39 (600 mg,
1.44 mmol), THF (20 ml), and Bu4NF (1m in THF, 1.7 ml, 1.73 mmol) to yield, after purification by CC
(SiO2; CH2Cl2/MeOH 97:3), 40 (331 mg, 88%). Orange solid. M.p. 176 – 1788. IR (neat): 3252w, 2942m,
1
2864m, 1574s, 1506s, 1471m, 1354m, 1329s, 1262s, 1214m, 1155m, 1105s, 1009m, 882m, 811s. H-NMR
(300 MHz, CDCl3/CD3OD 6 :1): 2.11 (s, 3 H); 4.49 (s, 2 H); 6.91 (dd, J ¼ 9.1, 3.0, 1 H); 7.05 – 7.11 (m,
2 H); 7.17 (s, 1 H); 7.85 (d, J ¼ 3.0, 1 H); 7.96 (d, J ¼ 9.1, 1 H). 13C-NMR (75 MHz, CDCl3/CD3OD 6 :1):
17.3; 63.6; 119.0; 120.2; 124.6; 125.5; 129.9; 133.5; 134.0; 135.8; 139.3; 147.0; 147.9. HR-EI-MS: 259.0955
(Mþ, C13H13N3O3þ ; calc. 259.0952). Anal. calc. for C13H13N3O3 (259.26): C 60.23, H 5.05, N 16.21; found C
60.02, H 5.14, N 15.93.
N-[4-(Azidomethyl)-2-methylphenyl]-6-nitropyridin-3-amine (42). GP G started from 40 (310 mg,
1.20 mmol), DPPA (0.36 ml, 1.68 mmol), THF (30 ml), and DBU (0.25 ml, 1.68 mmol) to yield, after
purification by CC (SiO2; CH2Cl2/MeOH 99 :1), 42 (332 mg, 98%). Orange oil. IR (neat): 3294w, 3054w,
1
2921w, 2092s, 1570s, 1502s, 1470m, 1385m, 1328s, 1285s, 1260s, 1158m, 1104s, 1007m, 832m. H-NMR
(300 MHz, CDCl3): 2.28 (s, 3 H); 4.35 (s, 2 H); 6.17 (br. s, 1 H); 7.13 (dd, J ¼ 9.1, 3.1, 1 H); 7.20 – 7.42 (m,
3 H); 8.08 (d, J ¼ 3.1, 1 H); 8.14 (d, J ¼ 9.1, 1 H). 13C-NMR (125 MHz, CDCl3): 18.1; 54.5; 120.4; 120.7;
124.6; 127.4; 130.3; 131.8; 133.7; 135.1; 137.1; 146.7; 149.0. HR-MALDI-MS: 285.1100 ([M þ H]þ,
C13H13N6Oþ2 ; calc. 285.1095).
N-[4-(Aminomethyl)-2-methylphenyl]-6-nitropyridin-3-amine (41). GP H started from 42 (325 mg,
1.14 mmol), PPh3 (0.60 g, 2.28 mmol), THF (14 ml), and H2O (0.6 ml) to yield 41 (280 mg, 95%). Yellow
solid. M.p. 96 – 988. IR (neat): 2840w, 1569s, 1503s, 1340m, 1381m, 1327s, 1265s, 1218m, 1159m, 1105s,
1000m, 897m, 835s. 1H-NMR (300 MHz, CDCl3): 2.25 (s, 3 H); 3.88 (s, 2 H); 6.06 (br. s, 1 H); 7.05 (dd,
J ¼ 9.0, 2.5, 1 H); 7.21 – 7.23 (m, 2 H); 7.29 (s, 1 H); 8.05 (d, J ¼ 2.5, 1 H); 8.12 (d, J ¼ 9.0, 1 H). 13C-NMR
(125 MHz, CDCl3): 18.1; 46.1; 120.2; 120.4; 125.1; 126.4; 130.7; 132.3; 133.8; 134.8; 135.4; 142.2; 147.1.
HR-EI-MS: 258.1105 (Mþ, C13H14N4O2þ ; calc. 258.1117).
N-{3-Methyl-4-[(6-nitropyridin-3-yl)amino]benzyl}-4-(trifluoromethyl)benzamide (43). GP C start-
ed from a,a,a-trifluoro-4-toluic acid (148 mg, 0.78 mmol), N-hydroxysuccinimide (115 mg, 0.99 mmol),
EDC · HCl (218 mg, 1.14 mmol), CH2Cl2 (5 ml), 41 (135 mg, 0.52 mmol), Et3N (0.5 ml, 2.60 mmol), and
CH2Cl2 (5 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 98 :2), 43 (137 mg, 61%). Yellow

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Helvetica Chimica Acta – Vol. 90 (2007)
solid. M.p. 158 – 1608. IR (neat): 3232w, 2925w, 1638m, 1572m, 1535m, 1499s, 1470m, 1322s, 1264s, 1222m,
1158m, 1125s, 1106s, 1067m, 1004m, 837m. 1H-NMR (300 MHz, (CD3)2SO): 2.19 (s, 3 H); 4.49 (d, J ¼ 5.6,
2 H); 7.09 (dd, J ¼ 9.0, 3.0, 1 H); 7.21 – 7.30 (m, 3 H); 7.87 (d, J ¼ 8.1, 2 H); 8.03 (d, J ¼ 2.5, 1 H); 8.11 (d,
J ¼ 8.1, 2 H); 8.13 (d, J ¼ 8.7, 1 H); 9.00 (s, 1 H); 9.29 (t, J ¼ 5.9, 1 H). 13C-NMR (75 MHz, (CD3)2SO):
17.7; 42.4; 119.0; 120.5; 124.2; 125.2; 125.8; 128.0; 130.1; 132.7; 133.8; 135.8; 136.7; 137.8; 146.8; 147.6;
164.8 (CF3 signal missing, one arom. signal missing due to overlap). 19F-NMR (282 MHz, (CD3)2SO):
ꢀ 60.3. HR-EI-MS: 430.1249 (Mþ, C21H17F3N4O3þ ; calc. 430.1248).
N-{4-[(6-Aminopyridin-3-yl)amino]-3-methylbenzyl}-4-(trifluoromethyl)benzamide (11). GP D
started from 43 (110 mg, 0.26 mmol), 10% Pd/C (11 mg, 10% (w/w)), and CH2Cl2/MeOH 1:2 (15 ml)
to yield, after purification by CC (SiO2; CH2Cl2/MeOH 98 :2 ! 95 :5), 11 (80 mg, 78%). Light grey solid.
M.p. 208 – 2108. IR (neat): 3467w, 3370w, 3267w, 1627m, 1549m, 1499s, 1425m, 1326s, 1314s, 1293s, 1176m,
1129s, 1068s, 1019s, 861s, 808m. 1H-NMR (300 MHz, (CD3)2SO): 2.17 (s, 3 H); 4.34 (d, J ¼ 5.9, 2 H); 5.60
(br. s, 2 H); 6.43 (d, J ¼ 8.4, 1 H); 6.59 (d, J ¼ 8.4, 1 H); 6.66 (s, 1 H); 6.93 (dd, J ¼ 8.4, 1.8, 1 H); 7.03 (d,
J ¼ 1.8, 1 H); 7.15 (dd, J ¼ 8.7, 2.8, 1 H); 7.70 (d, J ¼ 2.5, 1 H); 7.84 (d, J ¼ 8.1, 2 H); 8.06 (d, J ¼ 8.4, 2 H);
9.12 (t, J ¼ 5.9, 1 H). 13C-NMR (75 MHz, (CD3)2SO): 18.0; 42.4; 108.0; 112.7; 123.9; 125.1 (d, 3J(C,F) ¼
2
3.7); 125.6; 128.0; 128.5; 129.1; 129.8; 130.8 (q, J(C,F) ¼ 31.0); 132.6; 138.1; 141.7; 143.7; 155.5; 164.5
(CF3 signal missing). 19F-NMR (282 MHz, (CD3)2SO): ꢀ 60.9. HR-MALDI-MS: 401.1577 ([M þ H]þ,
C21H20F3N4Oþ; calc. 401.1584). Anal. calc. for C21H19F3N4O (400.40): C 62.99, H 4.78, N 13.99; found C
62.89, H 4.70, N 13.76.
N-{3-Methyl-4-[(6-nitropyridin-3-yl)amino]benzyl}quinoline-6-carboxamide (44). GP C started
from quinoline-6-carboxylic acid (140 mg, 0.81 mmol), N-hydroxysuccinimide (115 mg, 0.99 mmol),
EDC · HCl (218 mg, 1.14 mmol), CH2Cl2 (5 ml), 41 (135 mg, 0.52 mmol), Et3N (0.5 ml, 2.60 mmol), and
CH2Cl2 (5 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 98 :2), 44 (73 mg, 34%). Orange
solid. M.p. 127 – 1298. IR (neat): 3266w, 2921w, 1643m, 1573s, 1496s, 1383w, 1326s, 1285s, 1262s, 1158m,
1104s, 1006m, 842m. 1H-NMR (300 MHz, (CD3)2SO): 2.20 (s, 3 H); 4.54 (d, J ¼ 5.9, 2 H); 7.09 (dd, J ¼ 9.0,
2.9, 1 H); 7.27 – 7.34 (m, 3 H); 7.62 (dd, J ¼ 8.3, 4.2, 1 H); 8.03 (d, J ¼ 2.7, 1 H); 8.10 (d, J ¼ 9.0, 1 H); 8.13
(d, J ¼ 9.2, 1 H); 8.24 (dd, J ¼ 8.8, 2.0, 1 H); 8.49 (dd, J ¼ 8.5, 1.1, 1 H); 8.58 (d, J ¼ 1.9, 1 H); 8.96 – 9.07
(m, 2 H); 9.29 (t, J ¼ 5.9, 1 H). 13C-NMR (75 MHz, (CD3)2SO): 17.7; 42.4; 119.2; 120.6; 122.1; 124.4;
126.0; 127.1; 127.7; 128.0; 129.0; 130.3; 132.1; 132.9; 133.9; 136.0; 137.1; 137.1; 147.0; 147.8; 148.6; 152.0;
165.7. HR-MALDI-MS: 414.1555 ([M þ H]þ, C23H20N5O3þ ; calc. 414.1561).
N-{4-[(6-Aminopyridin-3-yl)amino]-3-methylbenzyl}quinoline-6-carboxamide (12). GP D started
from 44 (60 mg, 0.15 mmol), 10% Pd/C (6 mg, 10% (w/w)), and CH2Cl2/MeOH 1:2 (9 ml) to yield, after
purification by CC (SiO2; CH2Cl2/MeOH 92 :8), 12 (40 mg, 72%). Light grey solid. M.p. > 1988 (dec.).
IR (neat): 3466w, 3371m, 3283w, 2925w, 1622s, 1538m, 1496s, 1424m, 1379m, 1332m, 1293s, 1137w, 1118w,
841m. 1H-NMR (300 MHz, (CD3)2SO): 2.18 (s, 3 H); 4.39 (d, J ¼ 5.6, 2 H); 5.60 (s, 2 H); 6.43 (d, J ¼ 8.4,
1 H); 6.61 (d, J ¼ 8.4, 1 H); 6.67 (s, 1 H); 6.98 (dd, J ¼ 9.0, 2.1, 1 H); 7.07 (d, J ¼ 1.8, 1 H); 7.16 (dd, J ¼ 8.4,
2.5, 1 H); 7.60 (dd, J ¼ 8.3, 4.2, 1 H); 7.70 (d, J ¼ 2.5, 1 H); 8.07 (d, J ¼ 8.7, 1 H); 8.20 (dd, J ¼ 8.7, 2.2,
1 H); 8.47 (dd, J ¼ 8.1, 1.6, 1 H); 8.53 (d, J ¼ 1.6, 1 H); 8.98 (dd, J ¼ 4.2, 1.8, 1 H); 9.12 (t, J ¼ 5.9, 1 H).
13C-NMR (125 MHz, (CD3)2SO): 18.6; 43.2; 108.9; 113.7; 122.8; 124.9; 126.6; 127.8; 128.5; 128.6; 129.6;
129.7; 130.1; 130.7; 133.1; 133.4; 137.7; 142.5; 144.5; 149.3; 152.7; 156.4; 166.2. HR-MALDI-MS: 384.1813
([M þ H]þ, C23H22N5Oþ; calc. 384.1819).
5-{[4-(Aminomethyl)-2-methylphenyl]amino}pyridin-2-amine (45). GP D started from 42 (106 mg,
0.37 mmol), 10% Pd/C (11 mg, 10% (w/w)), and CH2Cl2/MeOH 1:2 (6 ml) to yield 45 (70 mg, 82%).
Yellow oil. The crude product was used in the next step without further purification and was not fully
characterized. 1H-NMR (300 MHz, CDCl3): 2.16 (s, 3 H); 3.62 (s, 2 H); 6.45 (d, J ¼ 8.7, 1 H); 6.65 (d, J ¼
8.1, 1 H); 6.87 (br. d, J ¼ 7.5, 1 H); 6.97 (br. s, 1 H); 7.19 (dd, J ¼ 8.7, 2.4, 1 H); 7.67 (d, J ¼ 2.7, 1 H). HR-
EI-MS: 228.1363 (Mþ, C13H16N4þ ; calc. 228.1369).
N-{4-[(6-Aminopyridin-3-yl)amino]-3-methylbenzyl}-4-chlorobenzamide (9). GP C started from 4-
chlorobenzoic acid (67 mg, 0.43 mmol), N-hydroxysuccinimide (62 mg, 0.53 mmol), EDC · HCl (119 mg,
0.62 mmol), CH2Cl2 (2 ml), 45 (65 mg, 0.28 mmol), Et3N (0.2 ml, 1.35 mmol), and CH2Cl2 (2 ml) to yield,
after purification by CC (SiO2; CH2Cl2/MeOH 95 :5), 9 (48 mg, 47%). Light grey solid. M.p. > 1758
(dec.). IR (neat): 3468w, 3310w, 2927w, 1624s, 1598m, 1543m, 1497s, 1417m, 1381m, 1278s, 1118w, 1090m,
1016m, 807m. 1H-NMR (300 MHz, (CD3)2SO): 2.17 (s, 3 H); 4.31 (d, J ¼ 5.6, 2 H); 5.60 (br. s, 2 H); 6.43

Helvetica Chimica Acta – Vol. 90 (2007)
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(d, J ¼ 8.7, 1 H); 6.59 (d, J ¼ 8.1, 1 H); 6.66 (s, 1 H); 6.92 (br. d, J ¼ 8.1, 1 H); 7.02 (br. s, 1 H); 7.15 (dd, J ¼
9.0, 2.5, 1 H); 7.53 (d, J ¼ 8.4, 2 H); 7.69 (d, J ¼ 2.5, 1 H); 7.89 (d, J ¼ 8.7, 2 H); 8.96 (t, J ¼ 5.9, 1 H).
13C-NMR (75 MHz, (CD3)2SO): 18.0; 42.4; 108.2; 112.9; 124.2; 125.8; 128.4; 129.0; 129.2; 129.4; 130.0;
132.8; 133.3; 136.0; 141.9; 143.9; 155.8; 164.9. HR-MALDI-MS: 367.1325 ([M þ H]þ, C20H20ClN4Oþ;
calc. 367.1320).
5-Chloro-1H-imidazo[4,5-b]pyridine (48) [24]. A soln. of 47 (2.86 g, 19.9 mmol), TsOH · H2O
(379 mg, 2.00 mmol), and HC(OEt)3 (6.6 ml, 39.8 mmol) in toluene (60 ml) was stirred under reflux for
4 h. The solvent was concentrated in vacuo, and the crude product was purified by CC (SiO2, 98 :2 !
95 :5) to yield 48 (2.89 g, 95%). Light brown solid. M.p: 230 – 2328. IR (neat): 3068w, 3039w, 2959w,
1
2896w, 2824m, 1620w, 1568m, 1488w, 1450m, 1385s, 1342s, 1266m, 1205m, 1110s, 920s, 804s. H-NMR
(300 MHz, (CD3)2SO): 7.29 (d, J ¼ 8.4, 1 H); 8.06 (d, J ¼ 8.1, 1 H); 8.49 (s, 1 H). 13C-NMR (125 MHz,
(CD3)2SO): 117.6; 126.1; 128.5; 143.7; 145.1; 151.4. HR-EI-MS: 153.0090 (Mþ, C6H4ClN3þ ; calc. 153.0089).
Anal. calc. for C6H4ClN3 (153.57): C 46.93, H 2.63, N 27.36; found C 46.89, H 2.66, N 27.33.
4-(5-Chloro-1H-imidazo[4,5-b]pyridin-1-yl)benzonitrile (49) and 4-(5-Chloro-3H-imidazo[4,5-
b]pyridin-3-yl)benzonitrile (50). GP J started from CuI (114 mg, 0.60 mmol, 10 mol-%), 48 (1.10 g,
7.16 mmol), Cs2CO3 (4.08 g, 12.5 mmol), 4-iodobenzonitrile (1.37 g, 5.97 mmol), 1,10-phenanthroline
(215 mg, 1.19 mmol, 20 mol-%), and DMF (5 ml) to yield, after purification by CC (SiO2; CH2Cl2/Et2O
9 :1 ! 5 :1 ! 1:1), 49 (509 mg, 33%) and 50 (407 mg, 27%).
Data of 49: Violet solid. M.p. 313 – 3158. IR (neat): 3358w, 3099w, 2227m, 1600m, 1507s, 1480m,
1413s, 1364m, 1317m, 1244m, 1203m, 1178m, 1157m, 1128m, 1102m, 976m, 922m, 805s. 1H-NMR
(300 MHz, (CD3)2SO): 7.49 (d, J ¼ 8.7, 1 H); 7.99 (d, J ¼ 9.0, 2 H); 8.15 (d, J ¼ 9.0, 2 H); 8.27 (d, J ¼ 8.4,
1 H); 9.05 (s, 1 H). 13C-NMR (125 MHz, (CD3)2SO): 111.3; 118.9; 119.8; 123.5; 124.8; 134.1; 135.0; 139.6;
145.5; 147.2; 156.4. HR-EI-MS: 254.0356 (Mþ, C13H7ClN4þ ; calc. 254.0354).
Data of 50: White solid. M.p. 247 – 2498. IR (neat): 3080w, 2228m, 1595m, 1518s, 1488m, 1450m,
1
1408m, 1361m, 1299m, 1288m, 1242m, 1167s, 1105m, 921m, 838s. H-NMR (300 MHz, (CD3)2SO): 7.52
(d, J ¼ 8.4, 1 H); 8.15 (d, J ¼ 9.0, 2 H); 8.22 (d, J ¼ 9.0, 2 H); 8.32 (d, J ¼ 8.4, 1 H); 9.08 (s, 1 H). 13C-NMR
(75 MHz, (CD3)2SO): 109.9; 118.3; 119.5; 123.1; 131.3; 133.9; 135.0; 138.4; 144.6; 144.8; 145.2. HR-EI-
MS: 254.0354 (Mþ, C13H7ClN4þ ; calc. 254.0354). Anal. calc. for C13H7ClN4 (254.68): C 61.31, H 2.77, N
22.00; found C 61.09, H 2.78, N 21.84.
1-[4-(5-Chloro-1H-imidazo[4,5-b]pyridin-1-yl)phenyl]methanamine (53). GP K started from 49
(300 mg, 1.18 mmol), CoCl2 · 6 H2O (1.12 g, 4.71 mmol), NaBH4 (178 mg, 4.71 mmol), and MeOH
(100 ml) to yield 53 (250 mg, 82%). Brown solid. The crude product was used in the next step without
further purification and was not fully characterized. 1H-NMR (300 MHz, (CD3)2SO): 3.82 (s, 2 H); 7.43
(d, J ¼ 8.4, 1 H); 7.59 (d, J ¼ 8.1, 2 H); 7.65 (d, J ¼ 8.7, 2 H); 8.12 (d, J ¼ 8.7, 1 H); 8.89 (s, 1 H).
N-[4-(5-Chloro-1H-imidazo[4,5-b]pyridin-1-yl)benzyl]benzamide (55). GP C started from
PhCOOH (61 mg, 0.50 mmol), N-hydroxysuccinimide (75 mg, 0.65 mmol), EDC · HCl (144 mg,
0.75 mmol), CH2Cl2 (2 ml), 53 (155 mg, 0.60 mmol), Et3N (0.35 ml, 2.50 mmol), and CH2Cl2 (2 ml) to
yield, after purification by CC (SiO2; CH2Cl2/MeOH 98 :2), 55 (123 mg, 68%). Yellow solid. M.p. 165 –
1678. IR (neat): 3436w, 3348w, 3045w, 2923w, 1651s, 1602m, 1515s, 1481s, 1410s, 1368m, 1328m, 1290m,
1237s, 1209m, 1159m, 1097s, 1076m, 976m, 922s, 798s. 1H-NMR (300 MHz, CDCl3): 4.77 (d, J ¼ 5.9, 2 H);
6.74 (br. s, 1 H); 7.30 (d, J ¼ 8.4, 1 H); 7.43 – 7.62 (m, 7 H); 7.78 (d, J ¼ 8.4, 1 H); 7.83 – 7.86 (m, 2 H); 8.28
(s, 1 H). 13C-NMR (75 MHz, CDCl3): 43.6; 119.6; 121.3; 124.3; 125.3; 127.2; 128.9; 129.9; 132.1; 134.1;
134.7; 139.9; 145.1; 146.8; 156.0; 167.8. HR-EI-MS: 362.0932 (Mþ, C20H15ClN4Oþ; calc. 362.0929).
N-(4-{5-[(Diphenylmethylidene)amino]-1H-imidazo[4,5-b]pyridin-1-yl}benzyl)benzamide (57).
GP E started from [Pd2(dba)3] (14 mg, 0.02 mmol, 5 mol-%), BINAP (20 mg, 0.03 mmol, 10 mol-%),
55 (107 mg, 0.29 mmol), Cs2CO3 (245 mg, 0.78 mmol), benzophenone imine (0.06 ml, 0.36 mmol), and
DME (2 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 98 :2 ! 97:3 ! 95 :5), 57 (50 mg,
34%). Yellow solid. M.p. > 1158 (dec.). IR (neat): 3284w, 3055w, 1733w, 1639m, 1599m, 1575s, 1515s,
1
1481m, 1404m, 1292m, 1236m, 977m, 693s. H-NMR (300 MHz, CDCl3): 4.73 (d, J ¼ 6.2, 2 H); 6.58 (d,
J ¼ 8.7, 1 H); 6.78 (br. s, 1 H); 7.22 (s, 4 H); 7.37 – 7.54 (m, 11 H); 7.56 (d, J ¼ 8.4, 1 H); 7.81 – 7.86 (m, 4 H);
8.13 (s, 1 H). 13C-NMR (75 MHz, CDCl3): 43.5; 112.2; 120.2; 122.5; 123.8; 127.3; 128.1; 128.3; 128.8;
128.9; 129.6; 129.7; 129.9; 130.3; 131.4; 131.9; 134.3; 135.2; 136.5; 139.2; 143.5; 155.6; 160.4; 167.7; 170.7.
HR-MALDI-MS: 508.2145 ([M þ H]þ, C33H26N5Oþ; calc. 508.2132).

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Helvetica Chimica Acta – Vol. 90 (2007)
N-[4-(5-Amino-1H-imidazo[4,5-b]pyridin-1-yl)benzyl]benzamide (13). GP L started from 57
(43 mg, 0.09 mmol), THF (1.0 ml), and 2m aq. HCl soln. (0.1 ml) to yield, after purification by CC
(SiO2; CH2Cl2/MeOH 95 :5 ! 9 :1), 13 (22 mg, 75%). Yellow solid. M.p. 112 – 1148. IR (neat): 3393w,
3326m, 3198m, 2927w, 1636m, 1600m, 1576s, 1517s, 1490s, 1410s, 1283m, 1248s, 1114m, 979m, 803s.
1H-NMR (300 MHz, (CD3)2SO): 4.55 (d, J ¼ 6.2, 2 H); 5.83 (s, 2 H); 6.48 (d, J ¼ 8.7, 1 H); 7.47 – 7.61 (m,
7 H); 7.71 (d, J ¼ 8.7, 1 H); 7.92 (d, J ¼ 6.9, 2 H); 8.40 (s, 1 H); 9.15 (t, J ¼ 5.6, 1 H). 13C-NMR (75 MHz,
(CD3)2SO): 42.2; 105.5; 117.7; 120.9; 122.5; 127.1; 128.2; 128.6; 131.2; 134.0; 134.5; 138.8; 141.7; 155.0;
156.9; 166.0. HR-MALDI-MS: 344.1512 ([M þ H]þ, C20H18N5Oþ; calc. 344.1506).
4-(5-Chloro-1H-imidazo[4,5-b]pyridin-1-yl)-3-methylbenzonitrile (51) and 4-(5-Chloro-3H-imida-
zo[4,5-b]pyridin-3-yl)-3-methylbenzonitrile (52). GP J started from CuI (124 mg, 0.65 mmol, 20 mol-%),
48 (501 mg, 3.26 mmol), K2CO3 (1.35 g, 9.77 mmol), 4-iodo-3-methylbenzonitrile (1.19 g, 4.89 mmol),
1,10-phenanthroline (235 mg, 1.30 mmol, 40 mol-%), and DMF (8 ml) to yield, after purification by CC
(SiO2; CH2Cl2/Et2O 9 :1 ! 5 :1 ! 1:1), 51 (99 mg, 11%) and 52 (171 mg, 20%).
Data of 51: Yellow solid. M.p. 198 – 2008. IR (neat): 3072w, 3040w, 2923w, 2236m, 1727w, 1667w,
1603m, 1504s, 1484s, 1435m, 1410s, 1313m, 1285m, 1246m, 1206m, 1174m, 1118m, 1100m, 978m, 917m,
895m, 829s. 1H-NMR (300 MHz, CDCl3): 2.20 (s, 3 H); 7.32 (d, J ¼ 8.4, 1 H); 7.46 (d, J ¼ 8.4, 1 H); 7.46 (d,
J ¼ 8.4, 1 H); 7.73 (dd, J ¼ 8.3, 1.8, 1 H); 7.78 (br. s, 1 H); 8.19 (s, 1 H). 13C-NMR (75 MHz, CDCl3): 17.9;
114.4; 117.6; 120.0; 121.0; 128.5; 131.2; 131.6; 135.8; 136.8; 137.9; 145.3; 147.2; 155.5. HR-EI-MS: 268.0510
(Mþ, C14H9ClNþ4 ; calc. 268.0511). Anal. calc. for C14H9ClN4 (268.70): C 62.58, H 3.38, N 20.85; found C
62.37, H 3.20, N 20.79.
Data of 52: White solid. M.p. 238 – 2408. IR (neat): 3100w, 3036w, 2923w, 2228m, 1596m, 1581m,
1506s, 1486m, 1449m, 1409s, 1355m, 1312m, 1292m, 1225s, 1167s, 1123m, 1102m, 1040m, 985m, 891m,
845s, 816s. 1H-NMR (300 MHz, CDCl3): 2.25 (s, 3 H); 7.36 (d, J ¼ 8.4, 1 H); 7.47 (d, J ¼ 8.1, 1 H); 7.70 (d,
J ¼ 8.4, 1 H); 7.75 (br. s, 1 H); 8.09 (s, 1 H); 8.13 (d, J ¼ 8.4, 1 H). 13C-NMR (75 MHz, CDCl3): 18.5;
113.9; 118.0; 120.0; 128.8; 131.1; 131.2; 134.2; 135.5; 137.2; 137.3; 143.8; 145.3; 147.1. HR-EI-MS: 268.0512
(Mþ, C14H9ClNþ4 ; calc. 268.0511). Anal. calc. for C14H9ClN4 (268.70): C 62.58, H 3.38, N 20.85; found C
62.32, H 3.21, N 20.56.
1-[4-(5-Chloro-1H-imidazo[4,5-b]pyridin-1-yl)-3-methylphenyl]methanamine (54). GP K started
from 51 (129 mg, 0.48 mmol), CoCl2 · 6 H2O (460 mg, 1.94 mmol), NaBH4 (37 mg, 1.94 mmol), and
CH2Cl2/MeOH 1:1 (50 ml) to yield 54 (103 mg, 79%). Yellow oil. The crude product was used in the next
1
step without further purification and was not fully characterized. H-NMR (300 MHz, CDCl3): 2.08 (s,
3 H); 3.98 (s, 2 H); 7.25 – 7.46 (m, 5 H); 8.16 (s, 1 H). HR-EI-MS: 271.0745 ([M ꢀ H]þ, C14H12ClN4þ ; calc.
271.0745).
N-[4-(5-Chloro-1H-imidazo[4,5-b]pyridin-1-yl)-3-methylbenzyl]benzamide (56). GP C started
from PhCOOH (59 mg, 0.39 mmol), N-hydroxysuccinimide (57 mg, 0.49 mmol), EDC · HCl (110 mg,
0.57 mmol), CH2Cl2 (2 ml), 54 (71 mg, 0.26 mmol), Et3N (0.2 ml, 1.30 mmol), and CH2Cl2 (2 ml) to yield,
after purification by CC (SiO2; CH2Cl2/MeOH 98 :2), 56 (52 mg, 53%). Yellow solid. M.p. 78 – 808. IR
(neat): 3328w, 3065w, 2924w, 1640m, 1602m, 1531m, 1505s, 1476s, 1401s, 1358m, 1306m, 1244m, 1226s,
1170s, 1113m, 1098m, 978m, 920s, 806m. 1H-NMR (300 MHz, CDCl3): 2.09 (s, 3 H); 4.74 (d, J ¼ 5.9, 2 H);
6.58 (br. s, 1 H); 7.26 – 7.29 (m, 2 H); 7.38 – 7.55 (m, 6 H); 7.84 (d, J ¼ 8.1, 2 H); 8.15 (s, 1 H). 13C-NMR
(75 MHz, CDCl3): 17.8; 43.6; 119.5; 121.3; 126.2; 127.0; 127.2; 127.8; 128.9; 131.3; 132.0; 133.1; 134.2;
135.6; 141.0; 146.0; 146.6; 155.4; 167.7. HR-MALDI-MS: 377.1156 ([M þ H]þ, C21H18ClN4Oþ; calc.
377.1164).
N-(4-{5-[(Diphenylmethylidene)amino]-1H-imidazo[4,5-b]pyridin-1-yl}-3-methylbenzyl)benzamide
(58). GP E started from [Pd2(dba)3] (9 mg, 0.01 mmol, 10 mol-%), BINAP (12 mg, 0.02 mmol, 20 mol-
%), 56 (37 mg, 0.10 mmol), Cs2CO3 (160 mg, 0.49 mmol), benzophenone imine (0.02 ml, 0.12 mmol),
and DME (1 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 98 :2 ! 97:3 ! 95 :5), 58
(29 mg, 57%). Yellow solid. M.p. 118 – 1208. IR (neat): 3276w, 3057w, 2922w, 2852w, 1634m, 1599m,
1575m, 1532m, 1505s, 1479s, 1446m, 1401s, 1361m, 1292s, 1249m, 1194m, 1142m, 1101m, 1075w, 1028w,
978s, 821m, 693s. 1H-NMR (300 MHz, CDCl3): 2.05 (s, 3 H); 4.71 (d, J ¼ 6.0, 2 H); 6.53 (d, J ¼ 8.6, 1 H);
6.57 (t, J ¼ 6.0, 1 H); 7.18 – 7.56 (m, 15 H); 7.81 – 7.84 (m, 4 H); 8.01 (s, 1 H). 13C-NMR (125 MHz,
CDCl3): 17.8; 43.7; 112.1; 120.1; 123.6; 126.7; 127.2; 128.0; 128.0; 128.3; 128.9; 129.7; 130.0; 131.2; 131.3;

Helvetica Chimica Acta – Vol. 90 (2007)
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132.0; 133.9; 134.3; 135.7; 136.6; 139.3; 140.3; 144.6; 155.1; 160.3; 167.7; 170.6 (one arom. signal missing
due to overlap). HR-MALDI-MS: 520.2130 ([M ꢀ H]þ, C34H26N5Oþ; calc. 520.2132).
N-[4-(5-Amino-1H-imidazo[4,5-b]pyridin-1-yl)-3-methylbenzyl]benzamide (14). GP L started from
58 (28 mg, 0.05 mmol), THF (1.0 ml), and 2m aq. HCl soln. (0.1 ml) to yield, after purification by CC
(SiO2; CH2Cl2/MeOH 95 :5 ! 9 :1), 14 (10 mg, 52%). Grey solid. M.p. 176 – 1788. IR (neat): 3202w,
2924w, 1601s, 1575m, 1538m, 1505s, 1489s, 1411s, 1349w, 1306s, 1248m, 1229m, 1168w, 1105w, 1027w, 983w,
806m. 1H-NMR (300 MHz, (CD3)2SO): 2.05 (s, 3 H); 4.54 (d, J ¼ 5.9, 2 H); 5.78 (s, 2 H); 6.42 (d, J ¼ 8.4,
1 H); 7.25 (d, J ¼ 8.7, 1 H); 7.33 (br. s, 2 H); 7.41 (s, 1 H); 7.46 – 7.55 (m, 3 H); 7.92 (d, J ¼ 8.4, 2 H); 8.17 (s,
1 H); 9.15 (t, J ¼ 5.9, 1 H). 13C-NMR (75 MHz, (CD3)2SO): 17.3; 42.2; 105.5; 119.2; 120.4; 125.8; 127.0;
127.1; 128.2; 129.9; 131.2; 133.0; 133.9; 134.0; 140.4; 142.8; 154.2; 156.8; 166.0. HR-EI-MS: 357.1586 (Mþ,
C21H19N5Oþ; calc. 357.1585).
{4-[(Triisopropylsilyl)oxy)methyl]naphthalen-1-yl}methanol (61). To a soln. of 60 (500 mg,
2.66 mmol) in THF (10 ml), (i-Pr)3SiCl (0.6 ml, 2.66 mmol) and 1H-imidazole (181 mg, 2.66 mmol)
were added at 08. The mixture was stirred at 08 for 2 h and at 258 for 19 h. After addition of CH2Cl2
(50 ml), the mixture was washed with sat. aq. NaHCO3 soln. (3 ꢁ 50 ml) and sat. aq. NaCl soln. (50 ml).
The org. phase was dried (MgSO4) and filtered. The filtrate was concentrated in vacuo, and the crude
product was purified by CC (SiO2, CH2Cl2) to yield 61 (418 mg, 46%). White solid. M.p. 88 – 908. IR
(neat): 3256w, 2937m, 2862m, 1718w, 1516w, 1461m, 1391m, 1270m, 1243m, 1117s, 1006s, 880s. 1H-NMR
(300 MHz, CDCl3): 1.11 – 1.30 (m, 21 H); 1.69 (t, J ¼ 5.9, 1 H); 5.15 (d, J ¼ 5.9, 2 H); 5.30 (s, 2 H); 7.50 –
7.59 (m, 3 H); 7.65 (d, J ¼ 7.5, 1 H); 8.00 (dd, J ¼ 7.2, 2.5, 1 H); 8.19 (dd, J ¼ 7.2, 2.5, 1 H). 13C-NMR
(75 MHz, CDCl3): 12.3; 18.3; 63.5; 64.1; 123.0; 123.9; 124.6; 125.5; 126.0; 126.2; 131.1; 131.4; 135.6; 137.6.
HR-EI-MS: 301.1619 ([M ꢀ C3H7]þ, C18H25O2Siþ; calc. 301.1619).
{[4-(Bromomethyl)naphthalen-1-yl]methoxy}triisopropylsilane (62). To a soln. of 61 (1.48 g,
4.30 mmol) in CH2Cl2 (30 ml), PPh3 (1.24 g, 4.73 mmol) was added at 08, and the mixture was stirred
for 10 min. CBr4 (1.57 g, 4.73 mmol) was added portionwise, and the mixture was stirred for 3 h at 08. The
solvent was concentrated in vacuo, and the crude product was purified by CC (SiO2, pentane ! pentane/
CH2Cl2 10 :1) to yield 62 (1.11 g, 63%). Yellow oil. IR (neat): 2940m, 2863m, 1517w, 1461m, 1388w,
1245m, 1206m, 1164m, 1112s, 880s. 1H-NMR (300 MHz, CDCl3): 1.18 – 1.27 (m, 21 H); 4.98 (s, 2 H); 5.29
(s, 2 H); 7.49 – 7.64 (m, 4 H); 7.98 (d, J ¼ 7.2, 1 H); 8.19 (d, J ¼ 8.4, 1 H). 13C-NMR (75 MHz, CDCl3):
12.3; 18.3; 63.4; 63.7; 122.9; 123.9; 124.6; 126.3; 126.4; 127.8; 131.2; 132.5; 136.1; 139.0. HR-EI-MS:
363.0777 ([M ꢀ C3H7]þ, C18H24BrOSiþ; calc. 363.0775).
Benzyl {1,2-Dihydro-2-oxo-1-[(4-{[(triisopropylsilyl)oxy]methyl}naphthalen-1-yl)methyl]pyrimidin-
4-yl}carbamate (64). To a suspension of 63 (708 mg, 2.89 mmol) and NaH (115 mg, 2.89 mmol, as a 60%
dispersion in mineral oil) in anh. DMF stirred at 258 for 1.5 h, 62 (1.07 g, 2.62 mmol) in anh. DMF was
added slowly. The mixture was stirred at 258 for 17 h. MeOH was added, the solvent was concentrated in
vacuo, and the crude product was purified by CC (SiO2, CH2Cl2/MeOH 99 :1 ! 98 :2) to yield 64 (1.18 g,
79%). White solid. M.p. 67 – 698. IR (neat): 2940m, 2863m, 1743m, 1661s, 1627s, 1553m, 1495s, 1455m,
1366s, 1207s, 1050m. 1H-NMR (300 MHz, CDCl3): 1.12 – 1.31 (m, 21 H); 5.17 (s, 2 H); 5.31 (s, 2 H); 5.50
(s, 2 H); 7.13 (d, J ¼ 7.2, 1 H); 7.30 – 7.35 (m, 6 H); 7.45 (d, J ¼ 7.2, 1 H); 7.50 – 7.57 (m, 2 H); 7.71 (d, J ¼
7.2, 1 H); 7.76 (br. s, 1 H); 7.90 – 8.02 (m, 2 H). 13C-NMR (75 MHz, CDCl3): 12.2; 18.3; 50.5; 63.1; 67.9;
95.0; 122.6; 123.7; 124.0; 126.4; 127.0; 128.3; 128.6; 129.1; 130.0; 131.0; 134.9; 138.9; 146.8; 152.1; 155.8;
161.8 (two arom. signals missing due to overlap). HR-MALDI-MS: 572.2930 ([M þ H]þ,
C33H42N3O4Siþ; calc. 572.2939). Anal. calc. for C33H41N3O4Si (571.78): C 69.32, H 7.23, N 7.35; found C
69.04, H 7.29, N 7.45.
Benzyl (1,2-Dihydro-1-{[4-(hydroxymethyl)naphthalen-1-yl]methyl}-2-oxopyrimidin-4-yl)carbamate
(65). GP F started from 64 (182 mg, 0.32 mmol), THF (5 ml), and Bu4NF (1m in THF, 0.38 ml,
0.38 mmol) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 98 :2), 65 (120 mg, 90%). White
solid. M.p. > 908 (dec.). IR (neat): 3264w, 2930w, 1742m, 1646s, 1626s, 1553m, 1494s, 1455m, 1366s, 1191s,
1
1058m, 998m, 744s. H-NMR (300 MHz, CDCl3): 1.84 (br. s, 1 H); 5.18 (s, 2 H); 5.20 (s, 2 H); 5.52 (s,
2 H); 7.04 (d, J ¼ 7.2, 1 H); 7.30 – 7.43 (m, 7 H); 7.55 – 7.60 (m, 3 H); 7.92 – 7.95 (m, 1 H); 8.15 – 8.18 (m,
1 H). 13C-NMR (75 MHz, CDCl3): 50.2; 63.0; 67.8; 95.0; 123.7; 124.2; 124.5; 126.7; 127.1; 127.9; 128.2;
128.6; 130.1; 131.2; 131.6; 134.8; 138.3; 146.9; 152.1; 155.9; 161.9 (one arom. signal missing due to
overlap). HR-MALDI-MS: 416.1602 ([M þ H]þ, C24H22N3O4þ ; calc. 416.1605).

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Helvetica Chimica Acta – Vol. 90 (2007)
Benzyl (1-{[4-(Azidomethyl)naphthalen-1-yl]methyl}-1,2-dihydro-2-oxopyrimidin-4-yl)carbamate
(66). GP G started from 65 (1.13 g, 2.73 mmol), DPPA (0.82 ml, 3.82 mmol), THF (15 ml), and DBU
(0.57 ml, 3.82 mmol) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 99 :1 ! 98 :2), 66 (1.12 g,
93%). White solid. M.p. > 658 (dec.). IR (neat): 3030w, 2094m, 1738m, 1657m, 1626m, 1548m, 1494s,
1454m, 1365s, 1190s, 1055m, 995m, 743s. 1H-NMR (300 MHz, CDCl3): 5.18 (s, 2 H); 5.30 (s, 2 H); 5.54 (s,
2 H); 7.08 (d, J ¼ 7.5, 1 H); 7.33 – 7.41 (m, 7 H); 7.48 (s, 1 H); 7.49 (d, J ¼ 7.2, 1 H); 7.57 – 7.66 (m, 2 H);
7.97 – 8.00 (m, 1 H); 8.07 – 8.10 (m, 1 H). 13C-NMR (75 MHz, CDCl3): 50.3; 52.9; 68.0; 95.1; 124.0; 124.3;
126.4; 127.2; 127.6; 128.3; 128.6; 131.4; 131.5; 131.7; 132.8; 134.7; 146.8; 151.9; 155.6; 161.7 (two arom.
signals missing due to overlap). HR-MALDI-MS: 441.1664 ([M þ H]þ, C24H21N6O3þ ; calc. 441.1670).
Anal. calc. for C24H20N6O3 (440.46): C 65.45, H 4.58, N 19.08; found C 65.49, H 4.59, N 18.79.
Benzyl (1-{[4-(Aminomethyl)naphthalen-1-yl]methyl}-1,2-dihydro-2-oxopyrimidin-4-yl)carbamate
(67). GP H started from 66 (1.12 g, 2.54 mmol), PPh3 (1.33 g, 5.08 mmol), THF (20 ml), and H2O
(0.8 ml) to yield 67 (1.03 g, 98%). White solid. M.p. 156 – 1578. IR (neat): 3352w, 3287w, 2917w, 1732m,
1
1652s, 1584s, 1507s, 1454m, 1422m, 1364s, 1222s, 1189s, 1066s, 934s, 745s. H-NMR (300 MHz, CDCl3):
3.65 (s, 2 H); 4.38 (s, 2 H); 5.17 (s, 2 H); 5.51 (s, 2 H); 7.03 (d, J ¼ 7.2, 1 H); 7.29 – 7.43 (m, 7 H); 7.51 – 7.62
(m, 4 H); 7.91 – 7.94 (m, 1 H); 8.13 – 8.16 (m, 1 H). 13C-NMR (125 MHz, CDCl3): 44.1; 50.6; 68.2; 95.2;
124.1; 124.4; 127.0; 127.4; 128.6; 128.8; 128.9; 131.8; 132.1; 132.3; 132.4; 133.9; 134.0; 141.1; 147.0; 152.4;
156.2; 162.1. HR-MALDI-MS: 415.1758 ([M þ H]þ, C24H23N4O3þ ; calc. 415.1765).
Benzyl [1-({4-[(Benzoylamino)methyl]naphthalen-1-yl}methyl)-1,2-dihydro-2-oxopyrimidin-4-yl]-
carbamate (68). GP C started from PhCOOH (49 mg, 0.40 mmol), N-hydroxysuccinimide (60 mg,
0.52 mmol), EDC · HCl (116 mg, 0.60 mmol), CH2Cl2 (3 ml), 67 (200 mg, 0.48 mmol), Et3N (0.3 ml,
2.01 mmol), and CH2Cl2 (3 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 98 :2 ! 97:3), 68
(168 mg, 81%). White solid. M.p. 208 – 2108. IR (neat): 3157w, 3066w, 2961w, 1720m, 1641s, 1613s, 1536s,
1487s, 1456m, 1424s, 1384s, 1361s, 1287s, 1249s, 1200s, 1029s, 998s, 751s, 695s. 1H-NMR (300 MHz, CDCl3/
CD3OD 6 :1): 4.95 (d, J ¼ 5.6, 2 H); 5.04 (s, 2 H); 5.37 (s, 2 H); 7.01 (d, J ¼ 7.0, 1 H); 7.18 – 7.23 (m, 5 H);
7.28 – 7.32 (m, 3 H); 7.34 – 7.50 (m, 4 H); 7.66 – 7.72 (m, 3 H); 7.77 – 7.81 (m, 1 H); 8.03 – 8.06 (m, 1 H).
13C-NMR (75 MHz, CDCl3/CD3OD 6 :1): 41.9; 50.5; 67.7; 96.3; 123.8; 124.4; 125.5; 127.1; 127.2; 127.3;
127.6; 128.2; 128.6; 128.6; 130.3; 131.4; 131.8; 132.0; 134.0; 135.2; 135.5; 147.1; 153.1; 157.0; 163.1; 168.4
(one arom. signal missing due to overlap). HR-MALDI-MS: 519.2019 ([M þ H]þ, C31H27N4O4þ ; calc.
519.2027). Anal. calc. for C31H26N4O4 (518.57): C 71.80, H 5.05, N 10.80; found C 71.79, H 5.06, N 10.73.
N-({4-[(4-Amino-2-oxopyrimidin-1(2H)-yl)methyl]naphthalen-1-yl}methyl)benzamide (16). GP D
started from 68 (150 mg, 0.29 mmol), 10% Pd/C (15 mg, 10% (w/w)), and CH2Cl2/MeOH 1:1 (15 ml) to
yield, after purification by CC (SiO2; CH2Cl2/MeOH 9 :1), 16 (85 mg, 76%). White solid. M.p. 305 – 3068.
IR (neat): 3352m, 3072m, 1641s, 1615s, 1519s, 1484s, 1423s, 1381s, 1341m, 1296s, 1278s, 1207m, 786s.
1H-NMR (300 MHz, (CD3)2SO): 4.93 (d, J ¼ 5.6, 2 H); 5.34 (s, 2 H); 5.66 (d, J ¼ 7.2, 1 H); 7.04 (br. s,
1 H); 7.11 (br. s, 1 H); 7.21 (d, J ¼ 7.2, 1 H); 7.43 – 7.55 (m, 5 H); 7.57 – 7.64 (m, 2 H); 7.89 (d, J ¼ 8.4, 2 H);
8.14 – 8.17 (m, 1 H); 8.22 – 8.25 (m, 1 H); 9.06 (t, J ¼ 5.6, 1 H). 13C-NMR (75 MHz, (CD3)2SO): 40.8;
48.5; 94.0; 124.1; 124.3; 124.9; 125.2; 126.4; 127.4; 128.3; 130.9; 131.2; 131.3; 132.8; 134.3; 134.8; 145.3;
155.9; 165.9; 166.3 (one arom. signal missing due to overlap). HR-MALDI-MS: 407.1473 ([M þ Na]þ,
C23H20N4NaOþ2 ; calc. 407.1479). Anal. calc. for C23H20N4O2 (384.44): C 71.86, H 5.24, N 14.57; found C
71.58, H 5.24, N 14.41.
Benzyl {1-[(4-{[(4-Chlorobenzoyl)amino]methyl}naphthalen-1-yl)methyl]-1,2-dihydro-2-oxopyri-
midin-4-yl}carbamate (69). GP C started from 4-chlorobenzoic acid (63 mg, 0.41 mmol), N-hydroxy-
succinimide (59 mg, 0.51 mmol), EDC · HCl (114 mg, 0.59 mmol), CH2Cl2 (4 ml), 67 (112 mg,
0.27 mmol), Et3N (0.2 ml, 1.35 mmol), and CH2Cl2 (4 ml) to yield, after purification by CC (SiO2;
CH2Cl2/MeOH 98 :2), 69 (110 mg, 74%). White solid. M.p. 184 – 1868. IR (neat): 3200w, 3060w, 2924w,
1749m, 1647s, 1542m, 1488s, 1437m, 1420m, 1365s, 1320m, 1191s, 1119s, 1095s, 1060m, 997s, 805s, 786s,
752s, 720s, 694s. 1H-NMR (300 MHz, CDCl3/CD3OD 6 :1): 4.92 (br. s, 2 H); 5.04 (s, 2 H); 5.36 (s, 2 H);
7.01 (d, J ¼ 7.2, 1 H); 7.19 – 7.49 (m, 12 H); 7.64 (d, J ¼ 8.4, 2 H); 7.77 – 7.80 (m, 1 H); 8.01 – 8.05 (m, 1 H).
13C-NMR (75 MHz, CDCl3/CD3OD 6 :1): 41.7; 50.4; 67.6; 96.1; 123.5; 124.1; 125.2; 126.8; 127.0; 127.2;
127.9; 128.3; 128.5; 128.6; 130.0; 130.4; 131.1; 131.6; 134.9; 135.1; 137.6; 146.9; 152.8; 156.7; 162.8; 167.0
(one arom. signal missing due to overlap). HR-MALDI-MS: 575.1451 ([M þ Na]þ, C31H25ClN4NaO4þ ;
calc. 575.1457).

Helvetica Chimica Acta – Vol. 90 (2007)
1065
N-({4-[(4-Amino-2-oxopyrimidin-1(2H)-yl)methyl]naphthalen-1-yl}methyl)-4-chlorobenzamide
(17). GP D started from 69 (120 mg, 0.22 mmol), 10% Pd/C (12 mg, 10% (w/w)), and CH2Cl2/MeOH 1:1
(20 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 9 :1), 17 (60 mg, 65%). White solid. M.p.
301 – 3038. IR (neat): 3349m, 3074m, 1660s, 1616s, 1519s, 1377s, 1309m, 1280s, 1206m, 1130m, 1094m,
1016m, 848m, 786s. 1H-NMR (300 MHz, (CD3)2SO): 4.92 (d, J ¼ 5.9, 2 H); 5.34 (s, 2 H); 5.66 (d, J ¼ 7.2,
1 H); 7.05 (br. s, 1 H); 7.10 (br. s, 1 H); 7.21 (d, J ¼ 7.5, 1 H); 7.45 (d, J ¼ 7.2, 1 H); 7.49 (d, J ¼ 7.2, 1 H);
7.54 (d, J ¼ 8.4, 2 H); 7.59 – 7.64 (m, 2 H); 7.92 (d, J ¼ 8.4, 2 H); 8.15 – 8.18 (m, 1 H); 8.21 – 8.24 (m, 1 H);
9.14 (t, J ¼ 5.9, 1 H). 13C-NMR (75 MHz, (CD3)2SO): 40.9; 48.5; 94.0; 124.1; 124.3; 125.1; 126.4; 127.4;
128.4; 129.3; 130.9; 132.2; 132.9; 133.1; 134.5; 136.1; 145.4; 155.6; 165.2; 165.9 (one arom. signal missing
due to overlap). HR-MALDI-MS: 419.1260 ([M þ H]þ, C23H20ClN4O2þ ; calc. 419.1269).
Benzyl {1,2-Dihydro-1-[(4-{[(4-methoxybenzoyl)amino]methyl}naphthalen-1-yl)methyl]-2-oxopyri-
midin-4-yl}carbamate (70). GP C started from 4-methoxybenzoic acid (61 mg, 0.40 mmol), N-
hydroxysuccinimide (60 mg, 0.52 mmol), EDC · HCl (116 mg, 0.60 mmol), CH2Cl2 (2 ml), 67 (250 mg,
0.60 mmol), Et3N (0.3 ml, 2.01 mmol), and CH2Cl2 (2 ml) to yield, after purification by CC (SiO2;
CH2Cl2/MeOH 98 :2), 70 (48 mg, 22%). White solid. M.p. 192 – 1948. IR (neat): 3140w, 3076w, 2967w,
1749m, 1652s, 1604s, 1549s, 1494s, 1418m, 1364s, 1323m, 1256s, 1204s, 1180s, 1057s, 1028m, 997s, 786s, 738s.
1H-NMR (300 MHz, CDCl3/CD3OD 6 :1): 3.70 (s, 3 H); 4.92 (d, J ¼ 5.7, 2 H); 5.04 (s, 2 H); 5.36 (s, 2 H);
6.78 (d, J ¼ 9.0, 2 H); 7.00 (d, J ¼ 6.9, 1 H); 7.19 – 7.30 (m, 6 H); 7.36 – 7.49 (m, 4 H); 7.65 (d, J ¼ 9.0, 2 H);
7.76 – 7.80 (m, 1 H); 8.02 – 8.04 (m, 1 H). 13C-NMR (75 MHz, CDCl3/CD3OD 6 :1): 41.8; 50.4; 55.3; 67.7;
96.3; 113.7; 123.7; 124.4; 125.4; 126.2; 127.1; 127.3; 127.6; 128.1; 128.6; 128.8; 129.0; 130.1; 131.9; 132.0;
132.5; 135.7; 147.1; 155.0; 157.0; 162.4; 163.1; 168.0. HR-MALDI-MS: 571.1943 ([M þ Na]þ,
C32H28N4NaOþ5 ; calc. 571.1952).
N-({4-[(4-Amino-2-oxopyrimidin-1(2H)-yl)methyl]naphthalen-1-yl}methyl)-4-methoxybenzamide
(18). GP D started from 70 (48 mg, 0.09 mmol), 10% Pd/C (5 mg, 10% (w/w)), and CH2Cl2/MeOH 1:1
(10 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 9 :1), 18 (20 mg, 54%). White solid. M.p.
302 – 3048. IR (neat): 3349m, 3076w, 1660m, 1619s, 1575m, 1522m, 1503s, 1485s, 1418m, 1398m, 1381s,
1298s, 1279m, 1252s, 1180s, 1129m, 1108m, 1022m, 846m, 786s. 1H-NMR (300 MHz, (CD3)2SO): 3.80 (s,
3 H); 4.91 (d, J ¼ 5.3, 2 H); 5.34 (s, 2 H); 5.66 (d, J ¼ 7.2, 1 H); 6.99 (d, J ¼ 9.0, 2 H); 7.04 (br. s, 1 H);
7.10 (br. s, 1 H); 7.21 (d, J ¼ 7.5, 1 H); 7.44 (d, J ¼ 7.2, 1 H); 7.48 (d, J ¼ 7.5, 1 H); 7.58 – 7.62 (m, 2 H); 7.88
(d, J ¼ 8.7, 2 H); 8.14 – 8.17 (m, 1 H); 8.22 – 8.25 (m, 1 H); 8.90 (t, J ¼ 5.6, 1 H). HR-MALDI-MS:
437.1583 ([M þ Na]þ, C24H22N4NaOþ3 ; calc. 437.1584). Anal. calc. for C24H22N4O3 (414.46): C 69.55, H
5.35, N 13.52; found C 69.62, H 5.54, N 13.30.
Benzyl (1,2-Dihydro-2-oxo-1-{[4-({[4-(trifluoromethyl)benzoyl]amino}methyl)naphthalen-1-yl]me-
thyl}pyrimidin-4-yl)carbamate (71). GP C started from a,a,a-trifluoro-p-toluic acid (44 mg, 0.23 mmol),
N-hydroxysuccinimide (35 mg, 0.30 mmol), EDC · HCl (67 mg, 0.35 mmol), CH2Cl2 (2 ml), 67 (144 mg,
0.35 mmol), Et3N (0.2 ml, 1.16 mmol), and CH2Cl2 (2 ml) to yield, after purification by CC (SiO2;
CH2Cl2/MeOH 98 :2), 71 (56 mg, 42%). White solid. M.p. 247 – 2498. IR (neat): 3240w, 3150w, 2973m,
1752m, 1651s, 1629m, 1616m, 1552m, 1493s, 1454m, 1419m, 1362s, 1319s, 1260m, 1165s, 1123s, 1061s, 997s,
855s, 803s, 745s, 693s. 1H-NMR (300 MHz, (CD3)2SO): 4.96 (d, J ¼ 5.6, 2 H); 5.18 (s, 2 H); 5.50 (s, 2 H);
7.02 (d, J ¼ 7.2, 1 H); 7.21 (d, J ¼ 7.2, 1 H); 7.33 – 7.40 (m, 5 H); 7.48 (d, J ¼ 7.2, 1 H); 7.62 – 7.65 (m, 2 H);
7.85 (d, J ¼ 8.4, 2 H); 8.03 (d, J ¼ 7.5, 1 H); 8.09 (d, J ¼ 8.1, 2 H); 8.15 – 8.17 (m, 1 H); 8.23 – 8.26 (m,
1 H); 9.30 (t, J ¼ 5.6, 1 H); 10.82 (s, 1 H). 13C-NMR (125 MHz, (CD3)2SO): 40.9; 49.3; 66.4; 94.6; 123.7;
1
3
123.8 (q, J(C, F) ¼ 270.8, CF3); 124.2; 125.0; 125.2 (d, J(C, F) ¼ 3.8); 126.3; 126.4; 127.8; 128.0; 128.2;
128.4; 130.6; 131.1 (q, 2J(C, F) ¼ 31.2); 131.1; 131.7; 134.5; 135.8; 137.9; 149.4; 153.0; 155.0; 162.8; 165.0
(one arom. signal missing due to overlap). 19F-NMR (282 MHz, (CD3)2SO): ꢀ 60.9. HR-MALDI-MS:
587.1892 ([M þ H]þ, C32H26F3N4Oþ4 ; calc. 587.1901).
N-({4-[(4-Amino-2-oxopyrimidin-1(2H)-yl)methyl]naphthalen-1-yl}methyl)-4-(trifluoromethyl)benz-
amide (19). GP D started from 71 (37 mg, 0.06 mmol), 10% Pd/C (4 mg, 10% (w/w)), and CH2Cl2/
MeOH 1:1 (6 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 9 :1), 19 (16 mg, 59%). White
solid. M.p. > 3088 (dec.) IR (neat): 3355m, 3076w, 1660m, 1643s, 1617s, 1539m, 1521m, 1486s, 1425m,
1
1381m, 1325s, 1279m, 1180s, 1134s, 1109s, 1071s, 1020m, 860m, 784s. H-NMR (300 MHz, (CD3)2SO):
4.95 (d, J ¼ 5.6, 2 H); 5.35 (s, 2 H); 5.66 (d, J ¼ 7.5, 1 H); 7.04 (br. s, 1 H); 7.10 (br. s, 1 H); 7.20 (d, J ¼ 7.2,
1 H); 7.47 (d, J ¼ 6.9, 1 H); 7.49 (d, J ¼ 7.2, 1 H); 7.58 – 7.63 (m, 2 H); 7.85 (d, J ¼ 8.1, 2 H); 8.08 (d, J ¼

1066
Helvetica Chimica Acta – Vol. 90 (2007)
8.1, 2 H); 8.15 – 8.18 (m, 1 H); 8.21 – 8.24 (m, 1 H); 9.30 (t, J ¼ 5.9, 1 H). 13C-NMR (75 MHz, (CD3)2SO):
41.0; 48.5; 93.8; 123.9; 124.0; 124.8; 124.9; 125.2 (d, 3J(C, F) ¼ 3.8); 126.2; 126.2; 128.1; 130.7; 131.0; 132.8;
134.1; 137.8; 145.1; 155.6; 164.8; 165.0 (CF3 signal missing, one arom. signal missing due to overlap). 19F-
NMR (282 MHz, (CD3)2SO): ꢀ 60.9. HR-MALDI-MS: 453.1524 ([M þ H]þ, C24H20F3N4O2þ ; calc.
453.1533). Anal. calc. for C24H19F3N4O2 (452.43): C 63.71, H 4.23, N 12.38; found C 63.43, H 4.42, N 12.08.
Benzyl (1,2-Dihydro-2-oxo-1-{[4-({[(4-quinolin-6-yl)carbonyl]amino}methyl)naphthalen-1-yl]meth-
yl}pyrimidin-4-yl)carbamate (72). GP C started from quinoline-6-carboxylic acid (125 mg, 0.72 mmol),
N-hydroxysuccinimide (106 mg, 0.92 mmol), EDC · HCl (204 mg, 1.06 mmol), CH2Cl2 (6 ml), 67
(200 mg, 0.48 mmol), Et3N (0.3 ml, 2.41 mmol), and CH2Cl2 (6 ml) to yield, after purification by CC
(SiO2; CH2Cl2/MeOH 98 :2 ! 95 :5), 72 (212 mg, 78%). White solid. M.p. > 1538 (dec.). IR (neat):
3200w, 3062w, 1746m, 1646s, 1616s, 1548s, 1494s, 1418m, 1366s, 1300m, 1255m, 1202s, 1056m, 1000m,
846m, 785s, 747s, 696s. 1H-NMR (300 MHz, CDCl3/CD3OD 6 :1): 5.01 (d, J ¼ 5.6, 2 H); 5.05 (s, 2 H); 5.38
(s, 2 H); 7.03 (d, J ¼ 7.2, 1 H); 7.21 – 7.23 (m, 5 H); 7.31 (d, J ¼ 7.5, 1 H); 7.39 (dd, J ¼ 8.4, 4.4, 1 H); 7.43
(d, J ¼ 7.4, 1 H); 7.47 – 7.52 (m, 3 H); 7.79 – 7.82 (m, 1 H); 7.96 (d, J ¼ 9.0, 1 H); 8.03 (dd, J ¼ 8.7, 1.9,
1 H); 8.08 – 8.12 (m, 1 H); 8.18 – 8.21 (m, 2 H); 8.29 (d, J ¼ 1.6, 1 H); 8.78 (dd, J ¼ 4.4, 1.6, 1 H).
13C-NMR (75 MHz, CDCl3/CD3OD 6 :1): 42.0; 50.5; 67.7; 96.3; 122.0; 123.8; 124.4; 125.6; 127.1; 127.3;
127.4; 127.8; 127.9; 128.2; 128.6; 128.6; 128.9; 130.4; 131.4; 132.0; 132.3; 135.2; 135.4; 138.0; 147.2; 148.7;
151.6; 153.1; 157.0; 163.1; 167.5 (one arom. signal missing due to overlap). HR-MALDI-MS: 570.2127
([M þ H]þ, C34H28N5Oþ4 ; calc. 570.2136).
N-({4-[(4-Amino-2-oxopyrimidin-1(2H)-yl)methyl]naphthalen-1-yl}methyl)quinoline-6-carbox-
amide (20). GP D started from 72 (177 mg, 0.31 mmol), 10% Pd/C (18 mg, 10% (w/w)), and CH2Cl2/
MeOH 1:1 (40 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 9 :1), 20 (54 mg, 40%). White
solid. M.p. > 2608 (dec.). IR (neat): 3351w, 3105w, 1659m, 1616s, 1519m, 1484s, 1420m, 1377m, 1326m,
1280m, 1204m, 1130m, 1032w, 785s, 649s. 1H-NMR (300 MHz, (CD3)2SO): 5.00 (d, J ¼ 5.9, 2 H); 5.35 (s,
2 H); 5.66 (d, J ¼ 7.2, 1 H); 7.04 (br. s, 1 H); 7.11 (br. s, 1 H); 7.22 (d, J ¼ 7.8, 1 H); 7.50 (d, J ¼ 7.2, 1 H);
7.52 (d, J ¼ 7.2, 1 H); 7.58 – 7.65 (m, 3 H); 8.08 (d, J ¼ 8.7, 1 H); 8.16 – 8.29 (m, 3 H); 8.45 – 8.74 (m, 1 H);
8.55 (d, J ¼ 1.6, 1 H); 8.98 (dd, J ¼ 4.4, 1.6, 1 H); 9.30 (t, J ¼ 5.9, 1 H). 13C-NMR (75 MHz, (CD3)2SO):
41.0; 48.5; 94.0; 122.2; 124.1; 124.3; 125.1; 126.4; 127.2; 127.9; 128.1; 129.0; 130.9; 131.3; 132.2; 133.0;
134.6; 137.2; 145.4; 148.7; 152.1; 155.9; 165.9; 165.9 (two arom. signals missing due to overlap). HR-
MALDI-MS: 458.1588 ([M þ Na]þ, C26H21N5NaO2þ ; calc. 458.1588).
Benzyl (1-{[4-({[4-(Dimethylamino)benzoyl]amino}methyl)naphthalen-1-yl]methyl}-1,2-dihydro-2-
oxopyrimidin-4-yl)carbamate (73). GP C started from 4-(dimethylamino)benzoic acid (66 mg,
0.40 mmol), N-hydroxysuccinimide (60 mg, 0.52 mmol), EDC · HCl (116 mg, 0.60 mmol), CH2Cl2
(3 ml), 67 (200 mg, 0.48 mmol), Et3N (0.3 ml, 2.01 mmol), and CH2Cl2 (3 ml) to yield, after purification
by CC (SiO2; CH2Cl2/MeOH 98 :2), 73 (70 mg, 31%). White solid. M.p. 176 – 1788. IR (neat): 3187w,
3070w, 2925w, 1751m, 1734m, 1653s, 1604s, 1558s, 1488s, 1443m, 1418m, 1368s, 1278s, 1253s, 1205s, 1065s,
1042s, 1008s, 804s, 787s, 753s. 1H-NMR (300 MHz, CDCl3/CD3OD 6 :1): 3.88 (s, 6 H); 4.94 (s, 2 H); 5.06
(s, 2 H); 5.38 (s, 2 H); 6.55 (d, J ¼ 9.0, 2 H); 7.02 (d, J ¼ 6.5, 1 H); 7.22 – 7.30 (m, 5 H); 7.38 – 7.50 (m,
5 H); 7.58 (d, J ¼ 9.0, 2 H); 7.78 – 7.81 (m, 1 H); 8.03 – 8.06 (m, 1 H). 13C-NMR (75 MHz, CDCl3/CD3OD
6 :1): 40.0; 41.8; 50.5; 67.8; 96.3; 111.2; 123.8; 124.5; 125.5; 127.1; 127.3; 127.8; 128.2; 128.6; 128.6; 128.7;
130.1; 131.4; 132.0; 135.2; 136.0; 147.1; 152.8; 163.1; 168.3 (one arom. signal missing due to overlap, no
resonance for two C¼O groups). HR-MALDI-MS: 584.2259 ([M þ Na]þ, C33H31N5NaO4þ ; calc.
584.2268).
N-({4-[(4-Amino-2-oxopyrimidin-1(2H)-yl)methyl]naphthalen-1-yl}methyl)-4-(dimethylamino)-
benzamide (21). GP D started from 73 (60 mg, 0.11 mmol), 10% Pd/C (6 mg, 10% (w/w)), and CH2Cl2/
MeOH 1:1 (15 ml) to yield, after purification by CC (SiO2; CH2Cl2/MeOH 9 :1), 21 (34 mg, 72%).
White solid. M.p. 294 – 2968. IR (neat): 3355m, 3104s, 1659m, 1610s, 1512s, 1484s, 1420m, 1380m, 1299s,
1277m, 1205m, 785s. 1H-NMR (300 MHz, (CD3)2SO): 2.96 (s, 6 H); 4.89 (d, J ¼ 5.6, 2 H); 5.33 (s, 2 H);
5.65 (d, J ¼ 7.5, 1 H); 6.69 (d, J ¼ 8.7, 2 H); 7.03 (br. s, 1 H); 7.09 (br. s, 1 H); 7.21 (d, J ¼ 7.5, 1 H); 7.41 (d,
J ¼ 7.5, 1 H); 7.46 (d, J ¼ 7.2, 1 H); 7.57 – 7.60 (m, 2 H); 7.77 (d, J ¼ 8.7, 2 H); 8.13 – 8.16 (m, 1 H); 8.22 –
8.25 (m, 1 H); 8.67 (t, J ¼ 5.6, 1 H). HR-MALDI-MS: 450.1895 ([M þ Na]þ, C25H25N5NaO2þ ; calc.
450.1901).

Helvetica Chimica Acta – Vol. 90 (2007)
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Biological Assay. Materials. [1,3,4-13C3]-5 was prepared as described in [15]. IspF E. coli was
prepared according to the procedure reported in [6].
Inhibition of the Reaction Catalyzed by IspF Monitored via 13C-NMR Spectroscopy. Assay mixtures
contained 100 mm Tris · HCl, pH 8.0, 10 mm MgCl2, 2 mm dithiothreitol, 1 mm [1,3,4-13C3]-5, and 12 mg
IspF protein. Inhibitory substances to final concentrations of 0, 0.5, or 1 mm and additional Me2SO (final
concentration of 5% (v/v)) were added to a total volume of 515 ml. The mixtures were incubated at 378
for 40 min, and the reaction was terminated by the addition of EDTA to a final concentration of 25 mm.
D2O was added to a final concentration of 10% (v/v). The soln. was analyzed by 13C-NMR spectroscopy
on a Bruker DRX-500 (125 MHz) NMR spectrometer. The dependence of the intensity of the 13C-signals
of the substrate and the product (measured by integration) on the concentration of a given inhibitor was
used to determine a % inhibition. Substrate/product ratios were obtained from the signals of C(1) of
[1,3,4-13C3]-5 and C-1 of [1,3,4-13C3]-6 [15].
Determination of IC50 Values via 13C-NMR Spectroscopy. The same assay mixture as described above
was used. Inhibitory substances were added at various concentrations. IC50 Values were calculated by
non-linear least-squares regression fit (GraphPad Prism 4 Software, San Diego, CA, 2003).
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Received February 14, 2007