Inhibitors of adenosine consuming parasites through polymer-assisted solution phase synthesis of lipophilic 5′-amido-5′-deoxyadenosine derivatives

Article abstract of DOI:10.1016/j.bmc.2009.01.026

Given the more or less global spread of multidrug-resistant plasmodia, structurally diverse starting points for the development of chemotherapeutic agents for the treatment of malaria are urgently needed. Thus, a series of 20 adenosine derivatives with a

Full text of DOI:10.1016/j.bmc.2009.01.026

Bioorganic & Medicinal Chemistry 17 (2009) 1428–1436
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitors of adenosine consuming parasites through polymer-assisted
solution phase synthesis of lipophilic 50-amido-50-deoxyadenosine derivatives
Philipp Heidler a, Vida Zohrabi-Kalantari a, Marcel Kaiser b, Reto Brun b, Thomas Emmrich c, Andreas Link c,
*
a Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35032 Marburg, Germany
b Swiss Tropical Institute, Socinstraße 57, 4002 Basel, Switzerland
c Institute of Pharmacy, Ernst-Moritz-Arndt-University, Friedrich-Ludwig-Jahn-Straße 17, 17487 Greifswald, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Given the more or less global spread of multidrug-resistant plasmodia, structurally diverse starting
points for the development of chemotherapeutic agents for the treatment of malaria are urgently needed.
Thus, a series of 20 adenosine derivatives with a large lipophilic substituent in N6-position were prepared
in order to evaluate their potential to inhibit the chloroquine resistant Plasmodium falciparum strain K1
in vitro. The rationale for synthesis of these structures was the high probability of interactions with mul-
tiple adenosine associated targets and the assumption that a large hydrophobic N6-(4-phenoxy)benzyl
substitution should allow the molecules to diffuse across parasite membranes. Starting from readily
available inosine, the new compounds were prepared as single isomers using a polymer-assisted acyla-
tion protocol enabling the straightforward isolation of the target compounds in pure form. Heterocyclic
ring systems were synthesized on-bead on Kenner’s safety-catch linker prior to acylation of the scaffold
in solution. Most of the highly pure compounds displayed anti-plasmodial activity in the low micromolar
or even submicromolar concentration range.
Received 27 October 2008
Revised 12 January 2009
Accepted 13 January 2009
Available online 20 January 2009
Keywords:
Anti-plasmodial activity
Modified nucleosides
Polymer-bound reagents
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
before5 as exemplified by our lead structure (Fig. 1) 1. Herein we
report the polymer-assisted parallel synthesis of a series of 20 car-
The year 2008 marked the centennial anniversary of the award
of the Nobel Prize to Paul Ehrlich. His impact, however, extended
far beyond his prize-winning achievements in immunology.1 Dur-
ing his early research with lipophilic dyes, he had investigated the
effects of methylene blue on malaria causing parasites, and so Ehr-
lich as well pioneered the quest for drugs against plasmodia. Given
the more or less global spread of multidrug-resistant malaria these
are once again in urgent demand, today. Several new anti-malarial
agents are currently undergoing clinical trials, mainly those resur-
rected from earlier anti-malarial drug discovery programs.2 In
addition truly novel compounds are being advanced through the
drug development process. At present there are four such com-
pounds in Phase III resulting from research funded by the Medi-
cines for Malaria Venture.3 But looking at the notoriously high
failure rate typical of drug discovery, there is still a need for novel
starting-points for the development of new low-cost effective ther-
apies for drug-resistant malaria. In continuation of our efforts to
identify new adenosine derived nucleosides with anti-plasmodial
activity, we focused on the synthesis of adenosine derivatives with
dual substitution in 50- and N6-position4 because we already were
able to identify active compounds with this substitution pattern
boxylic acid amides of 50-amino-50-deoxy-N6-(4-phenoxy)benzyl
adenosine (6) (see Scheme 1).
2. Chemistry
The 4-phenoxybenzyl-substituent in template 6 is introduced
into 6-deamino-6-chloro adenosine6 (2) using an excess of 4-phen-
oxybenzylamine (3). Compound 3 is accessible starting from cheap
4-phenoxybenzoic acid (not shown). The corresponding carboxylic
acid chloride is generated by treatment with thionyl chloride, re-
acted with ammonia and reduced using lithium alanate following
established procedures. The resulting 4-phenoxybenzylamine (3)
is best purified and stored as its hydrochloride salt. The displace-
O
O
CH3
NH
HO
N
O
H
N
N
N
H
NO2
N
N
OH
1
* Corresponding author. Tel.: +49 3834 864893; fax: +49 3834 864895.
E-mail address: link@uni-greifswald.de (A. Link).
Figure 1. Lead Structure: 50,N6-disubstituted adenosine derivative 1.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.026

P. Heidler et al. / Bioorg. Med. Chem. 17 (2009) 1428–1436
1429
ment of the 50-hydroxyl group of N6-substituted adenosine 4 by an
amino group is performed using a standard Mitsunobu protocol.7
In this way, a phthalimide residue is introduced chemoselectively
at the most acidic, primary alcohol function and consequently
regioselectively in 50-position. The mandatory removal of the
phthalic acid group is usually initiated by the addition of hydra-
zine. However, in this case this reaction was sluggish and incom-
plete in our hands, even under forcing conditions. Resort to the
alternative methyl hydrazine did not improve the situation, while
the use of methylamine lead to the desired removal of the phthal-
oyl group under mild conditions at room temperature. Necessary
purification of 6 was performed using column chromatography
over strongly basic ion exchange resin (see Scheme 1).
Meanwhile, polymer-bound acylation reagents were prepared.
For the connection of carboxylic acids with primary amines, di-
verse types of functional anchor groups have been described such
as pyrimidinone-linkers,8 N-hydroxysuccinimide-linker,9 N-acylin-
dol-linkers,10 2,4,6-trichloro[1,3,5]triazene-linkers,11 and pyrazo-
lone active esters12 to name only a few of the most popular. For
the first subset of simple carboxylic acids 2,3,5,6-tetrafluoro-4-
hydroxy benzoic acid as commercially available couple&release
linker was chosen.13 The labile carboxylic acid-linker bond of acids
attached to this linker results from strong electron withdrawing
properties of fluoro substituents of the benzene ring. However,
the comparably high reactivity of this bond is the reason for the
limited applicability in solid-phase synthesis protocols, but ren-
ders this construct an attractive choice for the transfer of simple
carboxylic acid residues onto nucleophiles. During the attachment
of 2,3,5,6-tetrafluoro-4-hydroxy benzoic acid on aminomethylated
polystyrene through in situ activation using N,N0-diisopropyl-car-
bodiimide (DIC) and benzotriazol-1-ol (N-hydroxy benzotriazole,
HOBt) the unintended formation of phenolic esters 8 had to be ta-
ken into account.
This side reaction can easily be monitored by investigation of
the diagnostic infrared absorption at 1765 cmꢀ1. Thus, treatment
of 8 with a weak base like piperidine and subsequent washing with
hydrochloric acid in N,N-dimethyl formamide (DMF) is mandatory
in order to destroy the side-product quantitatively, remove un-
bound 2,3,5,6-tetrafluoro-4-hydroxy benzoic acid and thus gain ac-
cess to linker-construct 7 with free phenolic OH-groups. The
appropriate carboxylic acids were coupled to this linker using
in situ activation by means of DIC via the corresponding O-acyli-
soureas in the presence of catalytic amounts of 4-dimethyl-
amino-pyridine (DMAP) (see Scheme 2).
Because Caddick et al. reported the possibility to transform ac-
rylic acid derivatives bound to this linker (9) into branched ali-
phatic carboxylic acids like 10{6} via radicalic addition of
alkyliodides in the presence of tributyl tin hydride and azobisi-
sobutyronitrile (AIBN), we tried to adapt this reaction using 10 dif-
ferent alkyl iodides.14 Due to the fact that this reaction requires
harsh conditions (100 °C, 90 min), that possibly lead to the disinte-
gration of the polymer backbone, in most of the cases no suitable
polymer-bound reagents could be obtained. Only in the case of
tert-butyliodide and 2-iodopropane lead the reaction to the forma-
tion of appropriate polymer-bound carboxylic acid equivalents in
sufficient purity that could be used in the derivatization of tem-
plate 6. In order to avoid ambiguous results, test samples for bio-
logical evaluation were not prepared following this route.
Identical specimen of 50-deoxy-50-[(4-methyl)pentanamido]-N6-
(4-phenoxybenzyl)adenosine (21) were synthesized via coupling
of 4-methylpentanoic acid to linker construct 7, and used for test-
ing instead (see Scheme 3).
For a second subset of more demanding carboxylic acids we
envisioned to prepare benzimidazoles and benzimidazolinones
via nuclophilic aromatic substitution. Thus, Kenner’s safety-catch
linker was attached to aminomethylated polystyrene and loaded
with 4-fluoro-3-nitrobenzoic acid to give 11.15 This aromatic car-
boxylic acid has been shown by us16 and others17 to be well suited
for the quantitative transformation into anilines by treatment with
various amines and subsequent reduction of the nitro group using
tin-II-chloride-solution in DMF. The resulting substituted ortho-
O
H2N
+
Cl
HO
N
O
3
N
N
N
HO
OH
2
i
(87 %)
HO
O
N
O
H
N
N
F
F
F
F
O
N
N
HO
OH
4
OH
NH
ii (73 %)
7
i
O
N
O
O
F
F
F
N
O
H
N
N
O
F
F
F
O
5
N
N
NH
OH
HO
OH
F
O
iii (94 %)
F
8
H2N
O
ii
N
O
H
N
N
7
N
N
HO
OH
6
Scheme 2. Aminolysis of active ester 8 resulting from overacylation of linker
construct 7. Reagents and conditions: (i) Unintended overacylation during coupling
(conditions as described13): 2,3,5,6,-tetrafluoro-4-hydroxybenzoic acid (1.7 equiv),
DIC (1.5 equiv), DMF, HOBt (1.5 equiv), 25 °C, 16 h; (ii) piperidine in DMF, HCl in
DMF.
Scheme 1. Synthesis of 50-amino-50-deoxy-N6-(4-phenoxybenzyl)adenosine (6).
Reagents and conditions: (i) 1-Propanol, Hünig’s base, 60 °C, 24 h; 5 °C, 24 h; (ii) dry
THF, PPh3, phthalimide, di-tert-butyl-azodicarboxylate 0.5 h, ꢀ20 °C; (iii) EtOH,
CH3NH2, 20 °C, 2 h.

1430
P. Heidler et al. / Bioorg. Med. Chem. 17 (2009) 1428–1436
F
F
F
O
S
N
O
O
O
O
ii
CH2
O
O
7
NH
NH
i
N
13{
}
1-5
F
9
N
14
{
}
1-8
N
i
iii
R
F
F
F
F
F
F
F
F
O
S
N
O
O
O
CH3
CH3
O
O
O
O
O
O
NH
NH
R
NH
ii
N
13{
}
2,5,6,7,8
NH
10{
1-10
}
10{ }
6
15{
2,5,6,7,8
}
N
R
O
Scheme 3. Loading of and C-bond formation on couple&release linker 7 yielding
Chemset 10{1–10}. Reagents and conditions: (i) DMF, carboxylic acid RCOOH (for R
see Table 1, entries 16–25) (2 equiv), DMAP (0.2 equiv) DIC (2 equiv), 16 h (ii) as (i),
(conditions as described14): DMF, acrylic acid (5 equiv) DMAP (0.2 equiv) DIC
(5 equiv), 16 h iii) (conditions as described14): 2-iodopropane (5 equiv), Bu3SnH
(5 equiv), AIBN (1 equiv), toluene, 100 °C, 1.5 h.
Scheme 5. Formation of heterocyclic carboxylic acid equivalents 14{1–5} and
15{2,5,6,7,8} as polymer-supported acylation reagents. Reagents and conditions: (i)
Triethyl orthoformate (2.5 equiv) in CH2Cl2; activation with bromo acetonitril in 1-
methyl-pyrrolidin-2-one (NMP), Hünig’s base (ii) CDI (4 equiv) in DMF, THF or
CH2Cl2, repeat; activation with bromo acetonitril in NMP, Hünig’s base.
O
S
O
O
O
O
NH
HN
11
NH
HO
O
R
i
NO2
N
6
O
H
N
N
F
i
N
N
OH
16-35
O
O
O
S
HN
O
Scheme 6. Polymer-assisted synthesis of target compounds 16–35. Reagents and
conditions: (i) 10{1–10}, 14{1–5}, 15{2,5,6,7,8}, separately.
NH
12{
}
1-8
NO2
NHR
were filtered over glass filter funnels and the solvent was removed
under reduced pressure. In order to remove notorious invisible
impurities such as inorganic salts or tiny polymeric fragments of
polystyrene beads all compounds were purified over flash-chroma-
tography columns using a medium pressure LC system (see
Scheme 6).19
ii
O
S
O
O
O
NH
N
3. Results
N
NH2
NHR
13
{
}
1-8
After an operationally simple to perform parallel chromato-
graphic purification protocol, 20 adenosine derivatives were ob-
tained in high yield and purity and subjected to spectroscopic
characterization and biological evaluation. The results of these
investigations are listed in Table 1.
Scheme 4. Nucleophilic aromatic substitution and subsequent reduction yielding
ortho-dianiline Chemset 13{1–8}. Reagents and conditions: (i) Allylamine, naph-
thalen-1-ylmethylamine, pentylamine, pyridin-2-methylamine, 3-methoxy-propyl-
amine, 2-cyclohex-1-enyl-ethylamine, 4-methoxy-benzylamine, or butylamine,
separately; R groups in Chemsets 12–15{1–8} can be deduced from the residues
of these amines; (ii) SnCl2 (excess) in DMF, 25 °C, 10 h, twice.
4. Discussion
The common key step in the on-bead syntheses of the two types
of heterocyclic carboxylic acids are the nucleophilic substitution
reactions using various nucleophilic amines. This nucleophilic aro-
matic substitution is especially well-suited for solid-phase trans-
formations because it is generally possible to achieve complete
conversion of the starting material. This is a prerequisite for prod-
uct purity at the end of multi-step syntheses. Because the treat-
ment with nucleophilic amines will cleave the reactive phenol
ester bond, this versatile reaction cannot be applied using a simple
couple&release linker intended to react with amines. Quite to the
contrary, Kenner’s safety-catch linker especially the variants in-
vented by Ellman et al. is stable towards the amines used in the
nucleophilic substitution step und thus the appropriate choice
for the construction of the heterocyclic carboxylic acid equiva-
dianilines 13{1–8} enabled the formation of heterocyclic rings typ-
ical for drug-like small molecules (see Scheme 4).18
By treatment of the first five members of chemset 13{1–8} with
carbonic acid ortho-esters and subsequent activation of the safety-
catch, the benzimidazole chemset 14{1–5} was obtained. Reaction
of 13{2,5,6,7,8} with carbonyl diimidazole (CDI) yielded substituted
benzimidazolinones that could be activated to chemset
15{2,5,6,7,8}. The polymer bound heterocyclic carboxylic acid
equivalents could be used as polymer-bound reagents to generate
target compounds 26–35 (see Scheme 5).
The quantitative acylation of template 6 with an excess of the
individual polymer-bound reagents was performed in parallel.
After completion of the reactions, the resulting amides formed

P. Heidler et al. / Bioorg. Med. Chem. 17 (2009) 1428–1436
1431
Table 1
Residues in target compounds 16–35 as depicted in scheme 6, purity and biological activity
Yielda (%)
Purityb (%)
IC50 (lM)
c
Entry
R
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
2-Methyl-prop-1-en-1-yl
3-Phenoxy-prop-1-yl
3-Phenyl-prop-1-yl
(Indol-3-yl)prop-1-yl
(Naphthalen-1-yl)methyl
3-Methyl-but-1-yl
(3-Chlorophenyl)methyl
(3-Methoxyphenyl)methyl
99
98
100
98
98
97
95
97
83
98
100
100
99
96
98
84
98
68
98
80
97.2
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
92.3
100.0
97.4
100.0
67.4
100.0
77.1
85.4
4.3
2.8
2.6
>8.5
0.3
4.6
3.1
3.4
3.5
3.3
2.7
2.2
1.5
4.0
2.6
2.9
3.3
2.1
3.1
0.9
3,5-Dichlorophenyl
(4-Phenoxy)phenyl
1-Allyl-1H-benzimidazol-5-yl
1-(Naphthalen-1-yl)methyl-1H-benzimidazol-5-yl
1-(Pent-1-yl)-1H-benzimidazol-5-yl
1-(Pyridin-2-yl)methyl-1H-benzimidazol-5-yl
1-(Methoxyprop-1-yl)-1H-benzimidazol-5-yl
1-[2-(Cyclohex-1-en-1-yl)ethyl]2-oxo-2,3-dihydro-1H-benzimidazol-5-yl
1-(4-Phenoxymethyl)-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl
1-[(Pyrid-2-yl)-methyl]-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl
1-(But-1-yl)-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl
1-(3-Methoxyprop-1-yl)-2-oxo-2,3-dihydro-1H-benzimidazol-5-yl
100.0
89.4
96.5
a
Yields as conversion rates (LC ratio of product/starting material prior to work-up).
HPLC, 100% method, detection at 254 nm.
P. falciparum chloroquine resistant K1 strain. IC50 for standard drug (chloroquine): 0.17 lM.
b
c
lents.20 A disadvantage of this linker is the fact, that an additional
activation step at the end of the on-bead transformation is neces-
sary. Therefore, for simple acid residues, that are transferred with-
out chemical manipulation of their structure or subjected to
reactions with neutral reagents, phenol based couple&release link-
ers are advantageous. The second special advantage of the Kenner
linker lies in its remarkable selectivity for nitrogen nucleophiles. It
has been demonstrated by us21 and others,22 that weakly basic
amino groups can be acylated with carboxylic acids attached to
the activated form of Kenner’s linker in the presence of reactive
primary alcoholic functions chemoselectively. However, due to
the fact that the selected amines in this study contain only reactive
primary amino groups attached to primary carbon atoms and less
reactive secondary alcoholic functions, this chemoselectivity is
easy to achieve with less N-selective linkers, as well.
From a biological viewpoint, the lipophilic character of the com-
pounds prepared is necessary in order to allow the molecules to
diffuse across parasite membranes and subsequently interact with
a variety of unknown targets, randomly. The molecular target of
the compounds synthesized is not known. Our assumption is that
the compounds bind to a higher number of different enzymes,
some of which comprise adenosine binding motifs with varying
affinity. Lately, 50-amido-50-deoxyadenosine derivatives have been
reported to interact with novel and highly relevant drug targets
such as poly(ADP-ribose) polymerase-123 and MbtA,24 an adenyla-
tion enzyme required for siderophore biosynthesis of the mycobac-
tins. Ribose-modified N6-substituted nucleosides are under
investigation as ribonucleotide reductase inhibitors.25 Very re-
cently, the endogenous occurrence of various N6-benzyladenosine
derivatives and their cytokinin activity has been unraveled by
Dolezal et al.26
The compounds show the desired activity in the selected model.
Therefore it seems rewarding to perform modifications of the ade-
nine ring or seek to reduce the high molecular mass of the com-
pounds and introduce functional groups that improve aqueous
solubility. In order to do so, it will be necessary to synthesize
new adenosine derived amino-functionalized templates in multi-
step syntheses with the associated laborious chromatographic
purification or preferably following novel glycosylation ap-
proaches.27 The parallel derivatization, however, can be achieved
fast and efficiently using the polymer-bound acylating species re-
ported here.
5. Conclusion
Polymer-bound acylating species prepared by simple coupling
to a suitable linker or by multi-step solid-phase synthesis are
known to be well-suited for the parallel synthesis of carboxylic
acid derivatives such as amides. They enable clean and high yield-
ing transformation of unprotected enantiomerically pure amino
alcohols like amino deoxynucleosides in solution. The complex
scaffolds do not have to be attached to a linker or polymer but nev-
ertheless the resulting mixture can easily be separated by filtra-
tion. After consumption of the amine added in limiting amounts,
the suspensions obtained contain only the target molecules in
solution along with polymer-bound reagents. The already highly
pure test compounds can thus be purified rapidly and efficiently
for biological evaluation by flash or medium pressure chromato-
graphy. As could be demonstrated here, the degree of diversity of
a set of carboxylic acids can be broadened by this approach. Espe-
cially the convergent connection of heterocycles formed on poly-
mer-support with delicate multifunctional scaffolds enables the
rapid generation of complex molecules that are not contained in
present portfolios.
In this respect, promiscuity in ligand-target interaction with
many different enzymes of the adenosine salvage pathway or en-
zymes utilizing adenosine containing substrates such as ATP or
NAD+ might be regarded as an advantage in terms of the parasites
ability to acquire resistance. From a medicinal chemistry viewpoint
however, the high lipophilicity is associated with potentially low
bioavailability and problematic solubility but as well might offer
the chance for high plasma albumin binding and therefore a cheap
galenic depot form causing elevated plasma half-life.
6. Experimental
1H NMR spectra were recorded on a Bruker AMX 400 spectrom-
eter, using tetramethylsilane as internal standard. The purity of the
target compounds was deduced from 1H NMR data as well as eval-
uated by HPLC, using a Dionex Summit HPLC-system with a diode

1432
P. Heidler et al. / Bioorg. Med. Chem. 17 (2009) 1428–1436
array detector and CC 125/4 Nucleodur 100-5 C18 ec columns, sup-
plied by Macherey-Nagel and water/methanol gradients. Purity
was calculated using the UV data at 254 nm using Chromeleon
6.50 software. A second, lower frequency (220 nm) was monitored
in order to be able to identify impurities with low absorption coef-
ficients at the standard wavelength 254 nm, that are notoriously
present in small molecule libraries. Due to the fact, that all samples
were purified over reversed-phase columns prior to analysis, these
impurities—if present—were effectively removed prior to analysis
and thus absent in detectable amounts. TLC reaction control was
performed on Macherey-Nagel Polygram Sil G/UV254 precoated
microplates, spots were visualized under UV-illumination at
254 nm. LC reaction control was performed on the HPLC system
described above. IR-spectra for the detection of erroneously
formed 2,3,5,6-tetrafluoro4-hydroxy-benzoic acid esters on the
solid support were recorded as KBr tablets on a Nicolet 510P FT-
IR spectrometer. High resolution MS data were obtained on a
Micromass Autospec instrument (ESI, methanol (1/1, v/v) infusion
155.1, 152.3, 134.3, 131.4, 129.8, 128.8, 123.1, 123.0, 123.8,
118.6, 118.1, 87.7, 81.2, 72.5, 71.4. HR-ESI-MS [M+H]+ calcd
579.1992, found 579.2004, mp 202 °C.
6.3. 50-Amino-50-deoxy-N6-(4-phenoxybenzyl)adenosine (6)
To a solution of 1.9 mmol of 50-deoxy-N6-(4-phenoxybenzyl)-
50-phthalimido-1-yl-adenosine (5) in 130 mL ethanol were added
6 mL of a solution of 33% methylamine in ethanol. After 2 h TLC
indicated completion of the reaction and the residue was concen-
trated in vacuo. After purification with a column packed with
strongly basic ion exchange resin, the desired amine could be
obtained applying a methanol water gradient starting with 5%
methanol. The product containing fractions were collected and
the solvent was removed in vacuo. Yield 94% (801 mg). 1H NMR
(400 MHz, DMSO-d6) + one drop D2O = d (ppm) 8.40 (s, 1H,
C8–H), 8.36 (br s, 1H, N6H), 8.22 (s, 1H, C2–H), 7.36 (m, 4H, 4-
phenoxybenzyl), 7.10 (t, 1H, J = 7.4 Hz, 4-phenoxybenzyl), 6.95
(m, 4H, 4-phenoxybenzyl), 5.87 (d, 1H, J = 6.0 Hz, 10H), 4.72–4.66
(m, 3H, 20H overlapping 4-phenoxybenzyl-CH2), 4.16 (m, 1H,
30H), 3.88 (m, 1H, 40H), 2.86–2.72 (m, 2H, 50CH2). 13C NMR
(126 MHz, DMSO-d6) = d (ppm) 156.9, 155.2, 154.3, 152.4, 140.2,
140.0, 135.2, 129.9, 128.8, 123.1, 118.6, 118.2, 87.4, 86.0, 73.0,
70.7, 43.7. HR-ESI-MS [M+H]+ calcd 449.1937, found 449.1956,
mp 147 °C.
at 10 lL/min with polyethylene glycol as reference). Microanalyses
were obtained using a Hewlett–Packard CHN-analyzer type 185.
6.1. N6-(4-Phenoxybenzyl)adenosine (4)
To
a solution of 5.7 mmol) 1-(6-Chloro-purin-9-yl)-b-d-1-
deoxyribofuranose (2) in 1-propanol were added 1.1 equiv of 4-
phenoxybenzyl amine (3) and 1 equiv of Hünig’s base. The solution
was stirred for 24 h at 60 °C. The reaction was monitored by TLC.
After completion of the reaction the solution was stored at 5 °C
for 24 h. The product precipitated as a white amorphous powder.
After filtration the resulting solid was crystallized from methanol.
Yield 87% (2.223 g). 1H NMR: (400 MHz, DMSO-d6) = d (ppm) 8.43
(br s, 1H, N6H), 8.37 (s, 1H, C8–H), 8.22 (s, 1H, C2–H), 7.36 (m, 4H,
4-phenoxybenzyl), 7.10 (t, 1H, 4-phenoxybenzyl, J = 7.4 Hz), 6.95
(m, 4H, 4-phenoxybenzyl), 5.90 (d, 1H, 10H, J = 6.72 Hz), 5.44 (d,
1H, 30OH, J = 7.3 Hz), 5.37 (m, 1H, 50OH), 5.18 (d, 1H, 20OH,
J = 5.5 Hz), 4.70 (br s, 2H, 4-phenoxybenzyl), 4.61 (q, 1H, 20H,
J = 5.5 Hz), 4.01 (m, 1H, 30H), 3.96 (m, 1H, 40H), 3.63–3.72 (m, 1H,
50CH2), 3.52–3.58 (m, 1H, 50CH2). 13C NMR: (126 MHz, DMSO-
d6) = d (ppm) 156.9, 155.2, 154.4, 152.3, 148.5, 139.8, 135.2,
129.9, 128.9, 123.1, 120.8, 118.6, 118.2, 87.9, 85.9, 73.5, 70.6,
61.6, 42.5. Combustion analysis: %C, 61.44 (calcd 61.46), %H, 5.18
(calcd 5.16), %N, 15.23 (calcd 15.58), mp 141 °C.
6.4. General procedure for the preparation of simple polymer
supported acid, using the 2,3,5,6-tetrafluorobenzoic-4-hydroxy-
acid linker
To a flask containing 1 g of 2,3,5,6-tetrafluoro-4-hydroxy-ben-
zoic acid loaded aminomethylated polystyrene (7) (prepared from
very high load aminomethylated polystyrene, purchased from
Novabiochem, Switzerland, batch number A20540) with an initial
loading level of 1.1 mmol/g were added 20 mL DMF and the resin
13
was allowed to swell for 10 min.
Subsequently, 2 equiv
(2.2 mmol) of the appropriate carboxylic acid and 0.22 mmol
(27 mg) of DMAP were added to the suspended resin. After the
addition of 2.2 mmol (195 lL) of DIC, the mixture was agitated at
room temperature for 16 h. The resin beads were filtered off and
washed exhaustively with DMF (three times 15 mL), dichlorometh-
ane (three times 15 mL) and THF (three times 15 mL) and after-
wards dried under vacuo. After careful drying the increase in
weight was monitored.
6.2. 50-Deoxy-N6-(4-phenoxybenzyl)-50-phthalimido-1-yl-
adenosine (5)
6.5. General procedure for the preparation of compounds 16–
25 using the 2,3,5,6-tetrafluoro-4-hydroxy benzoic acid linker
construct 7
To a solution of 4.45 mmol of N6-(4-phenoxybenzyl)adenosine
(4) in 31 mL of freshly dried THF (sodium/benzophenone) were
added 2.92 g (11.13 mmol) triphenylphosphine and 1.64 g (11.13
mmol) phthalimide. The suspension was cooled down to ꢀ20 °C.
After the addition of 1.94 g (11.13 mmol) di-tert-butyl-azodicar-
boxylate the suspension was stirred for 30 min at ꢀ20 °C and then
slowly allowed to warm to room temperature. The reaction was
monitored by TLC. After 90 min quantitative conversion could be
observed and the solvent was removed under vacuo. Subsequent
addition of 10 mL of methanol and storage at 5 °C for 24 h yielded
a white solid that was isolated by filtration. The resulting solid was
crystallized in methanol. Yield 73% (1.880 g). 1H NMR (500 MHz,
DMSO-d6) = d (ppm) 8.38 (s, 1H, C8–H), 8.35 (br s, 1H, N6H), 8.01
(s, 1H, C2–H), 7.84 (m, 5H, phthalimido), 7.36 (m, 4H, 4-phenoxyb-
enzyl), 7.11 (t, 1H, 4-phenoxybenzyl, J = 7.4 Hz), 6.96 (m, 4H, 4-
phenoxybenzyl), 5.86 (d, 1H, 10H, J = 5.5 Hz), 5.49 (d, 1H, 30OH,
J = 6.3 Hz), 5.30 (d, 1H, 20OH, J = 6.3 Hz), 4.77 (q, 1H, 20H,
J = 5.9 Hz), 4,68 (br s, 2H, 4-phenoxybenzyl), 4.27 (m, 1H, 30H),
4.14 (m, 1H, 40H), 3.94–4.01 (m, 1H, 50CH2), 3.83–3.90 (m, 1H,
50CH2). 13C NMR (126 MHz, DMSO-d6) = d (ppm) 167.7, 156.9,
Resin (200 mg) loaded with the appropriate carboxylic acid
(loading level approximately 1 mmol carboxylic acid per gram)
were swollen in 2 mL dichloromethane (or alternatively THF). A
solution of 9 mg (0.02 mmol) 50-amino-50-deoxy-N6-(4-phenoxyb-
enzyl)adenosine (6) in a few drops THF (as little solvent as possi-
ble) were added to the suspension. After 12 h in most cases
complete conversion of the amine 6 to the target carboxylic acid
amide 16–25 could be observed via TLC. Subsequently the resin
was washed with dichloromethane and THF and the collected frac-
tions were evaporated under reduced pressure. To ensure product
purity, all samples were further purified via MPLC.
6.6. 50-Deoxy-50-[(3,3-dimethyl)acrylamido]-N6-(4-phenoxyb-
enzyl)adenosine (16)
Conversion rate 99%. Purity HPLC after MPLC = 97.2% (254 nm).
1H NMR (400 MHz, hexadeutero [D6] dimethylsulfoxide DMSO) = d

P. Heidler et al. / Bioorg. Med. Chem. 17 (2009) 1428–1436
1433
(ppm) 8.43 (br s, 1H, N6H), 8.36 (s, 1H, C8–H), 8.25 (s, 1H, C2–H),
8.09 (t, 1H, amido, J = 5.8 Hz), 7.36 (m, 4H, 4-phenoxybenzyl),
7.10 (t, 1H, 4-phenoxybenzyl, J = 7.4 Hz), 6.96 (m, 4H, 4-phenoxyb-
enzyl), 5.85 (d, 1H, 10H, J = 6.6 Hz), 5.70 (br s, 1H, acryl-CH), 5.32
(br s, 2H, 30OH and 20OH), 4.69 (m, 3H, 20H overlapping 4-phen-
oxybenzyl), 4.05 (m, 1H, 30H), 3.97 (m, 1H, 40H), 3.35–3.46(m, 2H,
50CH2 overlapping H2O), 2.08 (s, 3H, methyl), 1.78 (s, 3H, methyl).
HR-ESI-MS [M+H]+ calcd 530.2278, found 530.2281.
20H overlapping benzyl), 4.08 (m, 1H, 30H), 3.98 (m, 1H, 40H),
3.94 (s, 2H, CH2) 3.40–3.50 (m, 2H, 50CH2). HR-ESI-MS [M+H]+ calcd
617.2512, found 617.2495.
6.11. 50-Deoxy-50-[(4-methyl)pentanamido]-N6-(4-phenoxy-
benzyl)adenosine (21)
The biological test sample was not synthesized via 9 but as the
other amides 16–25 by coupling of the appropriate carboxylic acid
(in this case 4-methylpentanoic acid) to the linker construct 7. Con-
version rate 97%. Purity HPLC after MPLC = 100.0% (254 nm). 1H NMR
(400 MHz, DMSO-d6) = d (ppm) 8.44 (br s, 1H, N6H), 8.36 (s, 1H, C8–
H), 8.25 (s, 1H, C2–H), 8.13 (t, 1H, amido, J = 5.6 Hz), 7.36 (m, 4H, 4-
phenoxybenzyl), 7.10 (t, 1H, 4-phenoxybenzyl, J = 7.4 Hz), 6.96 (m,
4H, 4-phenoxybenzyl), 5.85 (d, 1H, 10H, J = 6.4 Hz), 4.68 (m, 3H, 20H
overlapping benzyl), 4.04 (m, 1H, 30H), 3.95 (m, 1H, 40H), 3.38–3.45
(m, 2H, 50H overlapping H2O), 2.10 (t, 2H, CH2, J = 8.0 Hz), 1.48 (m,
1H, CH), 1.37 (m, 2H, CH2), 0.83 (d, 6H, CH3, J = 6.6 Hz). HR-ESI-MS
[M+H]+ calcd 547.2669, found 547.2692.
6.7. 50-Deoxy-50-[(4-phenoxy)butanamido]-N6-(4-phenoxy-
benzyl)adenosine (17)
Conversion rate 98%. Puritiy HPLC after MPLC = 100.0% (254
nm). 1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.44 (br s, 1H, N6H),
8.36 (s, 1H, C8–H), 8.28 (m, 2H, C2–H overlapping amido), 7.36
(m, 4H, 4-phenoxybenzyl), 7.24 (m, 2H, phenoxy), 7.10 (t, 1H, 4-
phenoxybenzyl, J = 7.4 Hz), 6.96 (m, 4H, 4-phenoxybenzyl), 6.89
(m, 3H, phenoxy), 5.85 (d, 1H, 10H, J = 6.4 Hz), 5.43 (d, 1H, 30OH,
J = 5.6 Hz), 5.25 (d, 1H, 20OH, J = 4.3 Hz), 4.70 (m, 3H, 20H overlap-
ping 4-phenoxybenzyl), 4.05 (m, 1H, 30H), 3.95 (m, 3H, 40H overlap-
ping CH2), 3.39–3.59 (m, 2H, 50CH2 overlapping H2O), 2.31 (t, 2H,
CH2, J = 7.3 Hz), 1.94 (m, 2H, CH2). HR-ESI-MS [M+H]+ calcd
610.2540, found 610.2544.
6.12. 50-Deoxy-50-[(3-chlorophenyl)acetamido]-N6-(4-phenoxy-
benzyl)adenosine (22)
Conversion rate 95%. Purity HPLC after MPLC = 100.0% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.42 (br s, 1H, N6H), 8.38
(m, 2H, C8–H overlapping amido), 8.25 (s, 1H, C2–H), 7.36 (m,
4H, 4-phenoxybenzyl), 7.20 (m, 3H, benzyl), 7.18 (m, 1H, benzyl),
7.10 (t, 1H, 4-phenoxybenzyl, J = 7.4 Hz), 6.96 (m, 4H, 4-phenoxyb-
enzyl), 5.87 (d, 1H, 10H, J = 5.9 Hz), 4.68 (m, 3H, 20H overlapping
benzyl), 4.07 (m, 1H, 30H), 3.94 (m, 1H, 40H), 3.48 (s, 2H, CH2),
3.36–3.46 (m, 2H, 50H overlapping water). HR-ESI-MS [M+H]+ calcd
601.1966, found 601.1957.
6.8. 50-Deoxy-50-[(4-phenyl)butanamido]-N6-(4-phenoxy-
benzyl)adenosine (18)
Conversion rate 100%. Purity HPLC after MPLC = 100.0% (254
nm). 1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.43 (br s, 1H, N6H),
8.36 (s, 1H, C8–H), 8.19 (m, 2H, C2–H C2–H overlapping amido),
7.36 (m, 4H, 4-phenoxybenzyl), 7.24 (m, 2H, phenyl), 7.08–7.16
(m, 4H, phenyl overlapping 4-phenoxybenzyl), 6.95 (m, 4H, 4-
phenoxybenzyl), 6.89 (m, 3H, phenoxy), 5.85 (d, 1H, 10H,
J = 6.4 Hz), 5.42 (d, 1H, 30OH, J = 6.1 Hz), 5.26 (d, 1H, 20OH,
J = 4.6 Hz), 4.69 (m, 3H, 20H overlapping 4-phenoxybenzyl), 4.05
(m, 1H, 30H), 3.97 (m, 1H, 40H), 3.39–3.59 (m, 2H, 50CH2 overlap-
ping H2O), 2.54 (t, 2H, CH2, J = 7.7 Hz), 2.14 (t, 2H, CH2,
J = 7.4 Hz), 1.79 (m, 2H, CH2). HR-ESI-MS [M+H]+ calcd 595.2669,
found 595.2363.
6.13. 50-Deoxy-50-[(3-methoxyphenyl)acetamido]-N6-(4-phen-
oxybenzyl)adenosine (23)
Conversion rate 97%. Purity HPLC after MPLC = 100.0% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.42 (br s, 1H, N6H), 8.34
(m, 2H, C8–H overlapping amido), 8.24 (s, 1H, C2–H), 7.36 (m,
4H, 4-phenoxybenzyl), 7.17 (t, 1H, methoxyphenyl, J = 7.9 Hz),
7.10 (t, 1H, 4-phenoxybenzyl, J = 7.4 Hz), 6.96 (m, 4H, 4-phenoxyb-
enzyl), 6.89 (m, 3H, phenoxy), 6.80 (m, 3H, methoxyphenyl), 5.86
(d, 1H, 10H, J = 6.1 Hz), 5.43 (br s, 2H, 30OH overlapping 20OH),
4.70 (m, 3H, 20H overlapping 4-phenoxybenzyl), 4.07 (m, 1H,
30H), 3.95 (m, 1H, 40H), 3.69 (s, 3H, methoxy), 3.42 (s, 2H, CH2),
3.31 (m, 2H, 50H overlapping water). HR-ESI-MS [M+H]+ calcd
597.2462, found 597.2439.
6.9. 50-Deoxy-50-[(3-indolyl)butanamido]-N6-(4-phenoxy-
benzyl)adenosine (19)
Conversion rate 98%. Purity HPLC after MPLC = 100.0% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 10.71 (s, 1H, indol-NH), 8.42
(br s, 1H, N6H), 8.36 (s, 1H, C8–H), 8.21 (s, 1H, C2–H), 8.18 (t, 1H, ami-
do, J = 5.6 Hz), 7.47 (d, 1H, J = 7.9 Hz, indol), 7.36 (m, 5H, 4-phen-
oxybenzyl and indol), 7.08 (m, 1H, indol), 7.02 (t, 1H, 4-
phenoxybenzyl, J = 6.9 Hz), 6.96 (m, 5H, 4-phenoxybenzyl and in-
dol), 5.86 (d, 1H, 10H, J = 6.4 Hz), 5.44 (br s, 1H, 30OH), 5.26 (br s,
1H, 20OH), 4.70 (m, 3H, 20H overlapping benzyl-CH2), 4.06 (m, 1H,
30H), 3.98 (m, 1H, 40H), 3.34–3.48(m, 2H, 50CH2 overlapping H2O),
2.66 (t, 2H, 500CH2, J = 7.4 Hz), 2.20 (t, 2H, 500CH2, J = 7.4 Hz), 1.87
(m, 2H, 500CH2). HR-ESI-MS [M+H]+ calcd 634.2814, found 634.2778.
6.14. 50-Deoxy-50-(3,5-dichlorobenzamido)-N6-(4-phenoxy-
benzyl)adenosine (24)
Conversion rate 83%. Purity HPLC after MPLC = 100.0% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.88 (m, 1H, amido), 8.39
(br s, 1H, N6H), 8.37 (s, 1H, C8–H), 8.15 (s, 1H, C2–H), 7.86 (s, 1H,
phenyl), 7.85 (s, 1H, phenyl), 7.80 (t, 1H, phenyl, J = 1.9 Hz), 7.35
(m, 4H, 4-phenoxybenzyl), 7.10 (m, 1H, 4-phenoxybenzyl), 6.96
(m, 4H, 4-phenoxybenzyl), 5.87 (d, 1H, 10H, J = 5.9 Hz), 5.46 (br s,
1H, 30OH), 5.28 (br s, 1H, 20OH), 4.76 (m, 1H, 20H), 4.69 (br s, 2H,
CH2), 4.20 (m, 1H, 30H), 4.07 (m, 1H, 40H), 3.55–3.65 (m, 2H,
50CH2). HR-ESI-MS [M+H]+ calcd 621.1447, found 621.1420.
6.10. 50-Deoxy-50-[(1-naphthyl)acetamido]-N6-(4-phenoxy-
benzyl)adenosine (20)
Conversion rate 98%. Purity HPLC after MPLC = 100.0% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.47 (t, 1H, amido,
J = 5.9 Hz), 8.42 (br s, 1H, N6H), 8.38 (s, 1H, C8–H), 8.25 (s, 1H,
C2–H), 8.03 (m, 1H, naphthyl), 7.90 (m, 1H, naphthyl), 7.80 (m,
1H, naphthyl), 7.49 (m, 2H, naphthyl), 7.32–7.43 (m, 6H, 4-phen-
oxybenzyl overlapping naphthyl), 7.10 (m, 1H, 4-phenoxybenzyl),
6.95 (m, 4H, 4-phenoxybenzyl), 5.88 (d, 1H, 10H, J = 6.1 Hz), 5.44
(d, 1H, 30OH, J = 5.9), 5.24 (d, 1H, 20OH, J = 4.6 Hz), 4.72 (m, 3H,
6.15. 50-Deoxy-50-(4-phenoxybenzamido)-N6-(4-phenoxyben-
zyl)adenosine (25)
Conversion rate 98%. Purity HPLC after MPLC = 92.3% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.62 (m, 1H, amido), 8.39

1434
P. Heidler et al. / Bioorg. Med. Chem. 17 (2009) 1428–1436
(br s, 1H, N6H), 8.38 (s, 1H, C8–H), 8.15 (s, 1H, C2–H), 7.87 (d, 2H, 4-
phenoxyphenyl, J = 8.9 Hz), 7.43 (m, 2H, 4-phenoxyphenyl), 7.35
(m, 4H, 4-phenoxybenzyl), 7.20 (t, 1H, phenoxyphenyl,
J = 7.4 Hz), 7.09 (m, 3H, 4-phenoxybenzyl overlapping 4-phenoxy-
phenyl), 7.01 (d, 2H, 4-phenoxyphenyl, J = 8.7 Hz), 6.96 (m, 4H, 4-
phenoxybenzyl), 5.87 (d, 1H, 10H, J = 6.4 Hz), 5.44 (br s, 2H, 30OH
overlapping 20OH), 4.76 (t, 1H, 20H, J = 5.6 Hz), 4.69 (br s, 2H,
CH2), 4.19 (m, 1H, 30H), 4.09 (m, 1H, 40H), 3.60 (m, 2H, 50CH2).
HR-ESI-MS [M+H]+ calcd 645.2462, found 645.2467.
1H, J = 1.4/17.4 Hz, allyl), 4.95 (d, 2H, J = 5.6 Hz, allyl), 4.79 (m,
1H, 20H), 4.69 (br s, 2H, benzyl-CH2), 4.20 (m, 1H, 30H), 4.11 (m,
1H, 40H), 3.62 (m, 2H, 50H). HR-ESI-MS [M+H]+ calcd 633.2574,
found 633.2575.
6.19. 50-Deoxy-50-{5-[1-(1-naphthalen-1-yl)methyl]-1H-benz-
imidazolyl}carboxamido-N6-(4-phenoxybenzyl)adenosine (27)
Conversion rate 100%. Purity HPLC after MPLC = 97.4% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.65 (t, 1H, J = 5.6 Hz, ami-
do-NH), 8.47 (s, 1H, amidino-CH), 8.38 (m, 2H, C8–H overlapping
amino), 8.28 (s, 1H, C2–H), 8.19 (m, 2H, naphthyl overlapping ben-
zene), 7.98 (m, 1H, naphthyl), 7.88 (m, 1H, benzene), 7.76 (dd, 1H,
J = 1.5/8.4 Hz, benzene), 7.60 (m, 3H, naphthyl), 7.44 (m, 1H, naph-
thyl), 7.35 (m, 4H, 4-phenoxybenzyl), 7.10 (t, 1H, J = 6.4 Hz, 4-
phenoxybenzyl), 7.05 (d, 1H, J = 6.6 Hz, naphthyl), 6.96 (m, 4H, 4-
phenoxybenzyl), 6.06 (s, 2H, naphthylmethyl), 5.87 (d, 1H,
J = 6.4 Hz, 10H), 5.62 (m, 1H, 30OH), 5.25 (d, 1H, J = 4.6 Hz, 20OH),
4.79 (m, 1H, 20H), 4.69 (br s, 2H, benzyl-CH2), 4.19 (m, 1H, 30H),
4.10 (m, 1H, 40H), 3.61 (m, 2H, 50H). HR-ESI-MS [M+H]+ calcd
733.2887, found 733.2859.
6.16. General procedure for the solid phase synthesis of
benzimidazole carboxylic acids
(A) Nucleophilc substitution. To a suspension of 400 mg (ap-
prox. 0.4 mmol) resin 11 in 6 mL DMF a tenfold excess of the
appropriate amine (4 mmol) in neat form or a solution thereof in
DMF is added. The suspension is agitated for 10 h and a color
change of the yellowish slurry to bright orange is visible The resin
is washed with DMF (five times) and THF (five times) and dried un-
der reduced pressure. (B) Reduction of the nitro group. 400 mg (ap-
prox. 0.4 mmol) of the nitrobenzene-containing resin 12 are
suspended in a freshly prepared solution of SnCl2 in DMF (c = 2
mol/l) and agitated for 10 h. The suspension is filtered, resus-
pended in another aliquot of freshly prepared SnCl2-solution
(c = 2 mol/l) in DMF and agitated for 10 h once again. The orange
color of the resin changes to pale yellow. The resin is washed with
DMF (five times) and immediately used for following transforma-
tions or washed THF (five times) as well and dried under reduced
pressure for storage. (C) Ring closing reaction. An amount of 1 g
(approx. 1 mmol) of the appropriate resin 13{1–5} is suspended
in 20 mL freshly distilled CH2Cl2 (distillation over CaCl2). To this
6.20. 50-Deoxy-50-{5-[1-(1-pentyl)-1H-benzimidazolyl]-
carboxamido}-N6-(4-phenoxybenzyl)adenosine (28)
Conversion rate 99%. Purity HPLC after MPLC = 100% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.65 (t, 1H, J = 5.6 Hz, ami-
do-NH), 8.39 (m, 2H, C8–H overlapping amino), 8.31 (s, 1H, C2–H),
8.25 (m, 1H, amidino-CH), 8.19 (br s, 1H, benzene), 7.82 (m, 1H,
benzene), 7.69 (d, 1H, J = 8.4 Hz, benzene), 7.35 (m, 4H, 4-phen-
oxybenzyl), 7.10 (t, 1H, J = 8.4 Hz, 4-phenoxybenzyl), 6.95 (m, 4H,
4-phenoxybenzyl), 5.87 (d, 1H, J = 6.4 Hz, 10H), 5.62 (m, 2H, 30OH
overlapping 20OH), 4.80 (m, 1H, 20H), 4.69 (br s, 2H, benzyl-CH2),
4.20 (m, 1H, 30H), 4.10 (m, 3H, 40H overlapping pentyl), 3.62 (m,
2H, 50H), 0.90–0.82 (m, 7H, pentyl); one signal of 2H is missing
due to overlapping with DMSO signal. HR-ESI-MS [M+H]+ calcd
649.2887, found 649.2848.
suspension 420 lL (2.5 mmol) triethyl orthoformate are added
and the mixture is shaken for 48 h. The resin is filtered over a glass
filter funnel, washed with CH2Cl2 (five times) and THF (three
times) and dried under reduced pressure. (D) Activation of the Ken-
ner linker. The sulfonamide linker of 400 mg (approx. 0.4 mmol)
resin was activated for cleavage by alkylation with 520
lL
(6.4 mmol) bromoacetonitrile, and 580 L (3.4 mmol) Hünig’s base
in 6 mL NMP overnight and washed with dry DMSO (five times
6 mL) and THF (five times 10 mL).
l
6.21. 50-Deoxy-N6-(4-phenoxybenzyl)-50-{5-[1-(1-pyrid-2-
ylmethyl)-1H-benzimidazolyl]carboxamido}adenosine (29)
6.17. Acylation of adenosine template 6
Conversion rate 96%. Purity HPLC after MPLC = 67.4% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.66 (t, 1H, J = 5.7 Hz, ami-
do-NH), 8.50 (m, 1H, pyridyl), 8.46 (s, 1H, C8–H), 8.37 (m, 2H, C2–H
overlapping amino), 8.26 (m, 1H, amidino-CH), 8.19 (br s, 1H, ben-
zene), 7.88–7.74 (m, 2H, benzene overlapping pyridyl), 7.56 (d, 1H,
J = 8.7 Hz, benzene), 7.35 (m, 4H, 4-phenoxybenzyl), 7.29 (m, 2H,
pyridyl), 7.10 (t, 1H, J = 8.4 Hz, 4-phenoxybenzyl), 6.96 (m, 4H, 4-
phenoxybenzyl), 5.88 (d, 1H, J = 6.4 Hz, 10H), 5.63 (s, 2H, methy-
lene), 5.21 (m, 2H, 30OH overlapping 20OH), 4.77–4.65 (m, 2H,
20H overlapping benzyl-CH2), 4.18 (m, 1H, 30H), 4.10 (m, 1H, 40H),
3.62 (m, 2H, 50H). HR-ESI-MS [M+H]+ calcd 684.2683, found
684.2706.
Resin (300 mg) loaded with the appropriate carboxylic acid
(loading level approximately 1 mmol carboxylic acid per gram)
were swollen in 3 mL THF. A solution of 13 mg (0.029 mmol) 50-
Amino-50-deoxy-N6-(4-phenoxybenzyl)adenosine (6) in
a few
drops THF (as little solvent as possible) were added to the suspen-
sion. After 12 h in most cases complete conversion of the amine (6)
to the target carboxylic acid amides 26–30 could be observed via
TLC. Subsequently the resin was washed with dichloromethane
and THF and the collected fractions were evaporated under re-
duced pressure. To ensure product purity, all samples were further
purified via MPLC.
6.22. 50-Deoxy-50-{5-[1-(3-methoxypropyl)-1H-benzimidazolyl]
carboxamido}-N6-(4-phenoxybenzyl)adenosine (30)
6.18. 50-Deoxy-50-[5-(1-prop-2-enyl)-1H-benzimidazolyl]car-
boxamido-N6-(4-phenoxybenzyl)adenosine (26)
Conversion rate 98%. Purity HPLC after MPLC = 100% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.66 (t, 1H, J = 5.4 Hz, ami-
do-NH), 8.38 (m, 2H, C8–H overlapping amino), 8.30 (s, 1H, C2–H),
8.25 (m, 1H, amidino-CH), 8.19 (br s, 1H, benzene), 7.82 (m, 1H,
benzene), 7.65 (d, 1H, J = 8.7 Hz, benzene), 7.36 (m, 4H, 4-phen-
oxybenzyl), 7.10 (t, 1H, J = 7.4 Hz, 4-phenoxybenzyl), 6.95 (m, 4H,
4-phenoxybenzyl), 5.87 (d, 1H, J = 6.6 Hz, 10H), 4.79 (m, 1H, 20H),
4.69 (br s, 2H, benzyl-CH2), 4.32 (t, 2H, J = 7.1 Hz, propylene),
4.20 (m, 1H, 30H), 4.11 (m, 1H, 40H), 3.61 (m, 2H, 50H), 3.21 (s,
Conversion rate 100%. Purity HPLC after MPLC = 100% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.66 (t, 1H, J = 6.1 Hz, ami-
do-NH), 8.38 (m, 2H, C8–H overlapping amino), 8.32 (s, 1H, ami-
dino-CH), 8.26 (s, 1H, C2–H), 8.20 (s, 1H, benzene), 7.81 (dd, 1H,
J = 1.5/8.7 Hz, benzene), 7.60 (d, 1H, J = 8.7 Hz, benzene), 7.36 (m,
4H, 4-phenoxybenzyl), 7.10 (t, 1H, J = 7.4 Hz, 4-phenoxybenzyl),
6.96 (m, 4H, 4-phenoxybenzyl), 6.11–5.99 (m, 1H, allyl), 5.87 (d,
1H, J = 6.4 Hz, 10H), 5.21 (dd, 1H, J = 1.4/10.2 Hz, allyl), 5.10 (dd,

P. Heidler et al. / Bioorg. Med. Chem. 17 (2009) 1428–1436
1435
3H, methoxy), 2.03 (m, 2H, propylene); one signal of 2H is missing
due to overlapping with DMSO signal. HR-ESI-MS [M+H]+ calcd
665.2836, found 665.2861.
zyl), 7.23 (m, 3H, pyridyl), 7.10 (m, 1H, benzene), 7.01 (m, 1H,
4-phenoxybenzyl), 6.96 (m, 4H, 4-phenoxybenzyl), 5.86 (d, 1H,
J = 6.1 Hz, 10H), 5.41 (d, 1H, J = 6.1 Hz, 30OH), 5.24 (d, 1H,
J = 4.6 Hz, 20OH), 5.13 (s, 2H, pyridylmethyl), 4.75 (m, 1H, 20H),
4.70 (br s, 2H, 4-phenoxyphenyl-CH2), 4.16 (m, 1H, 30H), 4.08
(m, 1H, 40H), 3.60 (m, 2H, 50H). HR-ESI-MS [M+H]+ calcd
700.2632, found 700.2666.
6.23. General procedure for the solid-phase synthesis of
benzimidazolinone carboxylic acids
(A) Nucleophilc substitution (see Section 6.16) (B) reduction of
the nitro group (see Section 6.16) (C) ring closing reaction. An
6.27. 50-Deoxy-50-{5-[1-(1-butyl)-2-oxo-2,3-dihydro-1H-
benzimidazolyl]carboxamido}-N6-(4-phenoxybenzyl)adenosine
(34)
amount of
1 g (approx. 1 mmol) of the appropriate resin
13{2,5,6,7,8} is suspended in 20 mL dry DMF (stored over molsi-
eve), THF or CH2Cl2. To this suspension 660 mg (4 mmol) CDI are
added and the mixture is shaken for 12 h. The color of the resin of-
ten changes from pale yellow to red. The resin is filtered over a
glass filter funnel and subjected to the same procedure once again.
The resin is filtered over a glass filter funnel, washed extensively
with DMF (five times), CH2Cl2 (five times) and THF (five times)
and dried under reduced pressure, (D) activation of the Kenner lin-
ker (see Section 6.16).
Conversion rate 98%. Purity HPLC after MPLC = 89.4% (254
nm). 1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.55 (br s, 1H,
urea-NH), 8.38 (m, 2H, amido-NH overlapping C8–H), 8.18 (s,
1H, C2–H), 7.57 (m, 1H, benzene), 7.49 (m, 1H, benzene), 7.35
(m, 4H, phenoxybenzyl), 7.15 (d, 1H, J = 8.1 Hz, benzene), 7.10
(m, 1H, 4-phenoxybenzyl), 6.96 (m, 4H, 4-phenoxybenzyl), 5.87
(d, 1H, J = 6.4 Hz, 10H), 5.37 (m, 2H, 30OH overlapping 20OH),
4.77 (m, 1H, 20H), 4.69 (br s, 2H, 4-phenoxyphenyl-CH2), 4.18
(m, 1H, 30H), 4.09 (m, 1H, 40H), 3.80 (t, 2H, J = 7.0 Hz, butyl),
3.60 (m, 2H, 50H), 1.61 (m, 2H, butyl), 1.27 (m, 2H, butyl), 0.88
(t, 2H, J = 7.3 Hz, butyl). HR-ESI-MS [M+H]+ calcd 665.2836,
found 665.2802.
Acylation of adenosine template 6 to yield the target carbox-
ylic acid amides 31–35 was performed as described above for
26–30.
6.24. 50-Deoxy-50-{5-[1-(2-cyclohex-1-enylethyl)-2-oxo-2,3-
dihydro-1H-benzimidazolyl]carboxamido}-N6-(4-
phenoxybenzyl)adenosine (31)
6.28. 50-Deoxy-50-{5-[1-(3-methoxypropyl)-2-oxo-2,3-dihydro-
1H-benzimidazolyl]carboxamido}-N6-(4-phenoxybenzyl)adenosine
(35)
Conversion rate 84%. Purity HPLC after MPLC = 77.1% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 8.49 (m, 1H, amido-NH),
8.35 (m, 2H, C8–H overlapping amino), 8.18 (s, 1H, C2–H), 7.50
(m, 1H, benzene), 7.44 (s, 1H, benzene), 7.35 (m, 4H, 4-phenoxyb-
enzyl), 7.10 (t, 1H, J = 7.4 Hz, 4-phenoxybenzyl), 7.05 (m, 1H, ben-
zene), 6.96 (m, 4H, 4-phenoxybenzyl), 5.87 (d, 1H, J = 6.4 Hz, 10H),
5.25 (m, 1H, cyclohexenyl), 4.77 (m, 1H, 20H), 4.69 (br s, 2H, ben-
zyl-CH2), 4.18 (m, 1H, 30H), 4.09 (m, 1H, 40H), 3.88 (t, 2H,
J = 6.9 Hz, ethylene), 3.59 (m, 2H, 50H), 2.23 (t, 2H, J = 7.7 Hz, ethyl-
ene), 1.98 (m, 2H, cyclohexenyl), 1.80 (m, 2H, cyclohexenyl), 1.53
(m, 2H, cyclohexenyl), 1.42 (m, 2H, cyclohexenyl). HR-ESI-MS
[M+H]+ calcd 717.3149, found 717.3137.
Conversion rate 80%. Purity HPLC after MPLC = 96.5% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 11.1 (br s, 1H, urea-NH),
8.55 (m, 1H, amido-NH), 8.37 (m, 2H, C8–H overlapping amino),
8.18 (s, 1H, C2–H), 7.57 (m, 1H, benzene), 7.49 (m, 1H, benzene),
7.35 (m, 4H, phenoxyphenyl), 7.10 (m, 2H, benzene overlapping
4-phenoxyphenyl), 6.96 (m, 4H, 4-phenoxyphenyl), 5.88 (d, 1H,
J = 6.4 Hz, 10H), 5.46 (m, 2H, 30OH overlapping 20OH), 4.76 (m, 1H,
20H), 4.69 (br s, 2H, 4-phenoxyphenyl-CH2), 4.18 (m, 1H, 30H),
4.09 (m, 1H, 40H), 3.84 (t, 2H, J = 6.7 Hz, propylene), 3.60 (m, 2H,
50H), 3.31 (m, 2H, propylene overlapping H2O), 3.20 (s, 3H, meth-
oxy), 1.85 (m, 2H, propylene). HR-ESI-MS [M+H]+ calcd 681.2785,
found 681.2748.
6.25. 50-Deoxy-50-{5-[1-(4-methoxybenzyl)-2-oxo-2,3-dihydro-
1H-benzimidazolyl]carboxamido}-N6-(4-phenoxybenzyl)-
adenosine (32)
6.29. Growth of Plasmodium falciparum parasites
Conversion rate 98%. Purity HPLC after MPLC = 85.4% (254 nm).
1H NMR (400 MHz, DMSO-d6) = d (ppm) 11.47 (br s, 1H, urea-NH),
8.83 (t, 1H, J = 6.0 Hz, amido-NH), 8.66 (m, 2H, C8–H overlapping
benzene), 8.48 (s, 1H, C2–H), 7.84 (dd, 1H, J = 1.6/8.2 Hz, benzene),
7.81 (d, 1H, J = 1.6 Hz, benzene), 7.66 (m, 4H, 4-phenoxybenzyl),
7.57 (d, 2H, J = 8.7 Hz, benzyl), 7.41 (m, 2H, benzene überlappt 4-
phenoxybenzyl), 7.26 (m, 4H, phenoxybenzyl), 7.17 (d, 2H,
J = 8.7 Hz, benzyl), 6.17 (d, 1H, J = 6.3 Hz, 10H), 5.78 (m, 1H, 30OH),
5.60 (m, 1H, 20OH), 5.26 (br s, 2H, benzyl-CH2), 5.03 (m, 3H, benzyl
overlapping 20H), 4.48 (m, 1H, 30H), 4.38 (m, 1H, 40H), 4.00 (s, 3H,
methoxy), 3.89 (m, 2H, 50H). HR-ESI-MS [M+H]+ calcd 751.2605,
found 751.2594.
In vitro activity against erythrocytic stages of P. falciparum
was determined using a [3H]hypoxanthine incorporation assay,
using the chloroquine and pyrimethamine resistant K1 strain.28
Compounds, including chloroquine (Sigma C6628) as standard,
were dissolved in DMSO at 10 mg/mL and added to parasite cul-
tures incubated in RPMI 1640 medium without hypoxanthine,
supplemented with HEPES (5.94 g/L), NaHCO3 (2.1 g/L), neomycin
(100 U/mL), AlbuMAX (5 g/L), and washed human red cells A+ at
2.5% hematocrit (0.3% parasitemia). Serial doubling dilutions of
each drug were prepared in 96-well microtiter plates and incu-
bated in a humidified atmosphere at 37 °C; 4% CO2, 3% O2, 93%
N2. After 48 h, 50 l lCi) in medium
L of [3H]hypoxanthine (=0.5
6.26. 50-Deoxy-50-{5-[1-(1-pyrid-2yl-methyl)-2-oxo-2,3-
dihydro-1H-benzimidazolyl]carboxamido}-N6-(4-
phenoxybenzyl)adenosine (33)
was added to each well of the plate. The plates were incubated
for a further 24 h under the same conditions. The plates were
then harvested with a Betaplate cell harvester (Wallace, Zurich,
Switzerland), and the red blood cells were transferred onto a
glass fiber filter and then washed with distilled water. The dried
filters were inserted into a plastic foil with 10 mL of scintillation
fluid and counted in a Betaplate liquid scintillation counter
(Wallace, Zürich, Switzerland). IC50 values were calculated from
sigmoidal inhibition curves using Microsoft Excel.29
Conversion rate 68%. Purity HPLC after MPLC = 100% (254
nm). 1H NMR (400 MHz, DMSO-d6) = d (ppm) 11.2 (br s, 1H,
urea-NH), 8.55 (m, 1H, amido-NH), 8.47 (m, 1H, pyridyl), 8.37
(m, 2H, C8–H überlappt amino), 8.17 (s, 1H, C2–H), 7.74 (m,
1H, benzene), 7.52 (m, 1H, benzene), 7.35 (m, 4H, phenoxyben-

1436
P. Heidler et al. / Bioorg. Med. Chem. 17 (2009) 1428–1436
14. Caddick, S.; Hamza, D.; Wadman, S. N.; Wilden, J. D. Org. Lett. 2002, 4,
Acknowledgments
1775.
15. Heidler, P.; Link, A. Bioorg. Med. Chem. 2005, 13, 585.
16. Golisade, A.; Herforth, C.; Quirijnen, L.; Maes, L.; Link, A. Bioorg. Med. Chem.
2002, 10, 159.
17. Yeh, C. M.; Tung, C. L.; Sun, C. M. J. Comb. Chem. 2000, 2, 341.
18. Larsen, T.; Link, A. Angew. Chem., Int. Ed. 2005, 44, 4432.
19. Isbell, J. J. Comb. Chem. 2008, 10, 150.
The graphical abstract was generated using the program PyMOL
(Version 0.98). This investigation received financial support from
the UNICEF/UNDP/World Bank/WHO Special Programme for Re-
search and Training in Tropical Diseases (TDR).
20. (a) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem. Soc., Chem.
Commun. 1971, 636; (b) Backes, B. J.; Virgilio, A. A.; Ellman, J. A. J. Am. Chem.
Soc. 1996, 118, 3055; (c) Thompson, L. A.; Moore, F. L.; Moon, Y.-C.; Ellman, J. A.
J. Org. Chem. 1998, 63, 2066; (d) Backes, B. J.; Ellman, J. A. J. Org. Chem. 1999, 64,
2322; (e) Tang, T. P.; Ellman, J. A. J. Org. Chem. 2002, 67, 7819.
21. Golisade, A.; Wiesner, J.; Herforth, C.; Jomaa, H.; Link, A. Bioorg. Med. Chem.
2002, 10, 769.
22. Bressi, J. C.; Verlinde, C.; Aronov, A. M.; Le Shaw, M.; Shin, S. S.; Nguyen, L. N.;
Suresh, S.; Buckner, F. S.; Van Voorhis, W. C.; Kuntz, I. D.; Hol, W. G. J.; Gelb, M.
H. J. Med. Chem. 2001, 44, 2080.
23. Jagtap, P. G.; Southan, G. J.; Baloglu, E.; Ram, S.; Mabley, J. G.; Marton, A.;
Salzman, A.; Szabo, C. Bioorg. Med. Chem. Lett. 2004, 14, 81.
24. Neres, J.; Labello, N. P.; Somu, R. V.; Boshoff, H. I.; Wilson, D. J.; Vannada, J.;
Chen, L.; Barry, C. E., 3rd; Bennett, E. M.; Aldrich, C. C. J. Med. Chem. 2008, 51,
5349.
25. Cappellacci, L.; Franchetti, P.; Vita, P.; Petrelli, R.; Lavecchia, A.; Jayaram,
H. N.; Saiko, P.; Graser, G.; Szekeres, T.; Grifantini, M. J. Med. Chem. 2008,
51, 4260.
References and notes
1. Gensini, G. F.; Conti, A. A.; Lippi, D. J. Infect. 2007, 54, 221.
2. Schlitzer, M. Arch. Pharm. 2008, 341, 149.
3. Bathurst, I.; Hentschel, C. Trends Parasitol. 2006, 22, 301.
4. Herforth, C.; Wiesner, J.; Franke, S.; Golisade, A.; Jomaa, H.; Link, A. J. Comb.
Chem. 2002, 4, 302.
5. Herforth, C.; Wiesner, J.; Heidler, P.; Sanderbrand, S.; Van Calenbergh, S.; Jomaa,
H.; Link, A. Bioorg. Med. Chem. 2004, 12, 755.
6. Zhao, H.; Pagano, A. R.; Wang, W. M.; Shallop, A.; Gaffney, B. L.; Jones, R. A. J.
Org. Chem. 1997, 62, 7832.
7. Mitsunobu, O. Synthesis 1981, 1.
8. Petricci, E.; Botta, M.; Corelli, F.; Mugnaini, C. Tetrahedron Lett. 2002, 43,
6507.
9. Shao, H.; Zhang, Q.; Goodnow, R.; Chen, L.; Tam, S. Tetrahedron Lett. 2000, 41,
4257.
10. (a) Todd, M. H.; Oliver, S. F.; Abell, C. Org. Lett. 1999, 1, 1149; (b) Todd, M. H.;
Oliver, S. F.; Abell, C. Org. Lett. 1999, 1, 1687.
26. Dolezal, K.; Popa, I.; Hauserova, E.; Spichal, L.; Chakrabarty, K.; Novak, O.;
Krystof, V.; Voller, J.; Holub, J.; Strnad, M. Bioorg. Med. Chem. 2007, 15,
3737.
27. Bookser, B. C.; Raffaele, N. B. J. Org. Chem. 2007, 72, 173.
28. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents
Chemother. 1979, 16, 710.
11. Masala, S.; Taddei, M. Org. Lett. 1999, 1, 1355.
12. Byun, J.-W.; Lee, D.-H.; Lee, Y.-S. Tetrahedron Lett. 2003, 44, 8063.
13. Salvino, J. M.; Kumar, N. V.; Orton, E.; Airey, J.; Kiesow, T.; Crawford, K.;
Mathew, R.; Krolikowski, P.; Drew, M.; Engers, D.; Krolikowski, D.; Herpin, T.;
Gardyan, M.; McGeehan, G.; Labaudiniere, R. J. Comb. Chem. 2000, 2, 691.
29. Matile, H.; Pink, J. R. L. In Immunological Methods; Lefkovits, I., Pernis, B., Eds.;
Academic Press: San Diego, 1990; p 221.