Synthesis of a series of N6-substituted adenosines with activity against trypanosomatid parasites

Article abstract of DOI:10.1016/j.ejmech.2009.02.010

The involvement of purine salvage in the accumulation of current trypanocidal drugs is important for the treatment of African sleeping sickness. The substrate specificity of essential nucleoside transporters is therefore of physiological and pharmacological interest. With the intention to contribute to the knowledge in the field, a series of 16 adenosine derivatives with substituents in N⁶-position were prepared in order to evaluate their potential to inhibit Trypanosoma brucei spp. in vitro. An unmodified ribose moiety was selected to conserve key molecular recognition motifs of the arsenal of integral membrane proteins expressed in large numbers on the protozoan plasma membrane. Two of the new compounds prepared using a polymer-assisted acylation protocol showed antitrypanosomal activities in the single digit micromolar concentration range.

Full text of DOI:10.1016/j.ejmech.2009.02.010

European Journal of Medicinal Chemistry 44 (2009) 3665–3671
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of a series of N6-substituted adenosines with activity against
trypanosomatid parasites
,
b
c
c
Andreas Link a , Philipp Heidler , Marcel Kaiser , Reto Brun
*
a Institute of Pharmacy, Ernst-Moritz-Arndt-University, Friedrich-Ludwig-Jahn-Straße 17, 17487 Greifswald, Germany
b Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35032 Marburg, Germany
c Swiss Tropical Institute, Socinstraße 57, 4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The involvement of purine salvage in the accumulation of current trypanocidal drugs is important for the
treatment of African sleeping sickness. The substrate specificity of essential nucleoside transporters is
therefore of physiological and pharmacological interest. With the intention to contribute to the
knowledge in the field, a series of 16 adenosine derivatives with substituents in N6-position were
prepared in order to evaluate their potential to inhibit Trypanosoma brucei spp. in vitro. An unmodified
ribose moiety was selected to conserve key molecular recognition motifs of the arsenal of integral
membrane proteins expressed in large numbers on the protozoan plasma membrane. Two of the new
compounds prepared using a polymer-assisted acylation protocol showed antitrypanosomal activities in
the single digit micromolar concentration range.
Received 11 September 2008
Received in revised form
11 February 2009
Accepted 12 February 2009
Available online 20 February 2009
Dedicated to Prof. Dr. Detlef Geffken on the
occasion of his 65th birthday.
Keywords:
Adenosine
Ó 2009 Elsevier Masson SAS. All rights reserved.
Nucleoside transporter
Transport proteins
Polymer-bound reagents
1. Introduction
disclosed 11 nucleoside/nucleobase transporters, and 15 ion pumps
and channels [3]. Thus, the activity and selective toxicity of aden-
One prominent peculiarity widespread among protozoan para-
site species is that these pathogens have lost the capability to
synthesize purines de novo. This energy saving biochemical
handicap is the cause for fundamental differences in the nucleoside
utilization machinery of trypanosomes and mammalian organisms.
Each parasite has developed a distinct and unique complement of
purine transporters and salvage enzymes that are necessary to use
purines from the surrounding environment [1]. As a consequence
of this efficient parasitic scavenging system, Trypanosoma brucei,
the parasite that causes sleeping sickness, is solely dependent on its
host to supply purines and therefore susceptible to pharmaceutical
intervention with nucleoside antimetabolites and nucleoside
derived ligands of nucleoside binding pockets such as cordycepin
(30-deoxyadenosine) [2].
osine derived antitrypanosomal agents will be strongly influenced
by these individual cell surface transporters that mediate access to
the cell. This is because substrate recognition by nucleobase and
nucleoside transporters is strikingly different in humans and
protozoa, and purine salvage by the parasites is far more efficient at
low substrate concentrations [1].
Some current antiprotozoal agents accumulate in the parasites
thus obtaining a reasonable therapeutic selectivity by a concentra-
tion effect rather than by interaction with specific intracellular, or
even so-called ‘‘clean’’ targets [4,5]. Following this rationale, the use
of purine derivatives for the treatment of various protozoan
infections, including malaria [6–8] and leishmaniasis [9–13], has
been investigated by us as well as others. This report is a continu-
ation of this project.
Recently, a thorough characterization of the plasma membrane
subproteome of bloodstream forms of T. brucei revealed that it
contains a large number of integral membrane proteins. Besides the
abundance of numerous adenylate cyclases, the investigation
2. Chemistry
Recently, the endogenous occurrence of various N6-benzylade-
nosine derivatives such as the derivative 1, prepared by our group
(Fig. 1) [6], and their cytokinin activity has been unraveled by
Dolezal et al. [14]. Thus, the nucleophilic displacement of the
chlorine atom in 2 with various amines is a straightforward and
* Corresponding author. Tel.: þ49 3834 864893; fax: þ49 3834 864895.
E-mail address: link@uni-greifswald.de (A. Link).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.02.010

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A. Link et al. / European Journal of Medicinal Chemistry 44 (2009) 3665–3671
1
phthalimide followed by treatment with methylamine (not shown).
N
H
N
6
The resulting amine (3) is best purified and stored as its dihydro-
chloride salt. Prior to use, 3 dihydrochloride was suspended in
methanol and eluted through a column of strongly basic ion
exchange resin with methanol. Aminolysis of 2 with an excess of 3
as free base furnished 4 in good yield.
3N
HO
OMe
O
N
N7
MeO
HO
OH
Meanwhile, polymer-bound acylation reagents were prepared.
For the connection of carboxylic acids with primary amines,
diverse types of functional anchor groups have been described
such as pyrimidinone-linkers [17], N-hydroxysuccinimide-linkers
[18], N-acylindol-linkers [19,20], and 2,4,6-trichloro[1,3,5]triazene-
1
Fig. 1. N6-(2,4-Dimethoxybenzyl)-adenosine (1) – a natural product (prepared by us
unknowingly).
linkers [21], to name only
a few. Here, 2,3,5,6-tetrafluoro-
highly promising approach for the generation of bioactive
compounds. In this project, we aimed at the introduction of amines
with a flexible spacer long enough to reach putative lipophilic
binding pockets surrounding the targeted adenosine binding
motifs. More specific aspects of the rationale for the synthesis of the
N6-substituted adenosines presented here are conferred in the
Discussion section. In order to gain access to the target compounds,
it would have been one option to synthesize various amines with
such a spacer and a lipophilic binding motif attached to it. These
amines could have been reacted with 2 to yield novel adenosine
derivatives. However, nucleosides formed by the nucleophilic
displacement reaction of 2 with amines are often obtained in less
than 80% yield and have to be purified by demanding column
chromatography.
Therefore, the sequential introduction of the amine-based linker
3 and parallel decoration of the resulting nucleoside 4 with various
substituents were envisioned. Thus, one common nucleoside
derivative 4 with a spacer of suitable length and polarity was
selected and synthesized by the route depicted in Scheme 1 [15].
Only this common scaffold 4 had to be purified by challenging
column chromatography. The subsequent introduction of diversity
elements through polymer-assisted acylation led to the target
compounds that could be isolated by mere filtration over short
chromatography columns to remove particulates and invisible
impurities.
4-hydroxybenzoic acid (5) was chosen as a commercially available
couple & release linker [22]. The labile carboxylic acid-linker bond
results from strong electron withdrawing properties of fluoro
substituents of the benzene ring. However, the relative high reac-
tivity of this bond is the reason for the limited applicability in
solid-phase synthesis protocols. Nevertheless this construct is an
attractive choice for the transfer of simple carboxylic acid residues
onto nucleophiles such as 4. During the attachment of 2,3,5,6-
tetrafluoro-4-hydroxybenzoic acid (5) to an aminomethylated
polystyrene through in situ activation by using N,N0-diisopropyl-
carbodiimide (DIC) and benzotriazol-1-ol (N-hydroxy benzo-
triazole, HOBt) the unintended formation of phenolic esters occurs.
This side reaction can easily be detected by monitoring the diag-
nostic infrared absorption at 1765 cmꢀ1. Thus, treatment of the
resin with a weak base like piperidine and subsequent washing
with hydrochloric acid in N,N-dimethyl formamide (DMF) are
advisable. With this simple procedure side-products are destroyed
quantitatively and resulting unbound 5 is removed. The appro-
priate 16 carboxylic acids were then coupled to polymer-bound
linker 6 separately. In situ activation by means of DIC treatment in
the presence of catalytic amounts of 4-dimethylamino-pyridine
(DMAP) yielded 16 polymer reagents of the general structure 7
(Scheme 2).
The quantitative acylation of template 4 with an excess of the
individual polymer-bound reagents was performed in parallel by
reaction in CH2Cl2 or THF. After completion of the reactions, the
resulting amides 8–23 formed 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-chromatography columns by using a medium
pressure LC system without individual optimization of chromato-
graphic parameters [23].
Initially, a 2-(2-amino-ethoxy)-ethyl substituent was intro-
duced into commercially available 6-deamino-6-chloro adenosine
2 [16] by using an excess of 2-(2-amino-ethoxy)-ethylamine (3).
The required 2-(2-amino-ethoxy)-ethylamine (3), although
commercially available, was prepared starting from inexpensive
but toxic 1-chloro-2-(2-chloroethoxy)ethane and potassium
N
NH2
O
Cl
N
HO
O
3. Results
+
N
N
H2N
After an operationally simple, parallel chromatographic purifi-
cation step, 16 adenosine derivatives 8–23 were obtained in high
yields and excellent purity. The products were subjected to both
spectroscopic characterization and biological evaluation. The
results of these investigations are reported in Table 1.
OH
HO
2
3
i
(87.7 %)
4. Discussion
N
In general, the introduction of structural diversity into the sugar
and/or the base moiety of nucleosides represents a validated
approach for the generation of active compounds in bioorganic and
medicinal chemistry. Specifically, nucleoside transporters of
kinetoplastids such as T. brucei display high affinity for their
substrates and thus can be exploited for the selective accumulation
of toxic nucleoside derivatives in pathogenic protozoa [5].
Nonetheless, a critical analysis of the various adenosine trans-
porters that are functionally expressed during the various life-cycle
H
N
N
HO
NH2
O
N
N
O
HO
OH
4
Scheme 1. Preparation of N6-spacer-modified amino scaffold 4. (i) 1-Propanol, Hu¨nig’s
base, 12 h 60 ꢁC, 24 h 5 ꢁC, chromatographic purification.

A. Link et al. / European Journal of Medicinal Chemistry 44 (2009) 3665–3671
3667
N
O
H
N
F
F
F
F
F
F
F
F
F
F
F
N
R
O
O
O
iii
i
ii
HO
N
H
OH
O
NH2
OH
R
+
O
O
N
N
R
R
R
N
H
HO
N
H
F O
OH
HO
8-23
5
6
7
R
Cl
H
N
OMe
OMe
F
O
NO2
F
F
O
Cl
8
9
10
11
12
13
14
15
OMe
OMe
OMe
O
N
H
16
17
18
19
20
21
22
23
Scheme 2. Polymer-assisted synthesis of target compounds 8–23. (i) Coupling conditions (as described [22]): 2,3,5,6-tetrafluoro-4-hydroxybenzoic acid (5, 1.7 equiv), DIC
(1.5 equiv), DMF, HOBt (1.5 equiv), 25 ꢁC, 16 h, piperidine in DMF, HCl in DMF; (ii) RCOOH (2 equiv) (R s. above), DMAP (0.2 equiv), DIC (2 equiv), DMF, 20 ꢁC, 16 h. (iii) 16 reactions
separately: 4, CH2Cl2 or THF, 20 ꢁC, 12 h.
stages of T. brucei, has been hampered by the fact that in
trypanosomes, which express most genes constitutively in large
polycistrons, the presence of mRNA does not guarantee the
expression of the corresponding protein product [24].
the H-bond-accepting ring nitrogen atoms, N3 and N7, are replaced
by carbon atoms [28]. Whereas the ribose sugar of adenosine is
known not to be involved in the binding of the P2 transporter, the
ribose is important for binding to another P1-like transporter,
NT9/AT-Dl [28]. For this reason we chose to synthesize adenosine
derivatives with an intact sugar moiety in this project. Concerning
the aminopurine moiety, the relatively large difference between
the adenosine and inosine binding energies can be explained by the
existence of a strong H-bond between the 6-amino group and the
NT9/AT-D binding pocket, and a possible weak H-bond with N1.
The aminopurine nucleoside cordycepin was shown to be
a potent trypanocide and its uptake is mediated by the purine
transporters P1 and P2. While cordycepin is highly active in vitro, it
shows poor antitrypanocidal activity in vivo unless coadministered
with an adenosine deaminase inhibitor. Furthermore, it interacts
with multiple mammalian proteins. For instance, it could be shown
that cordycepin inhibits the growth of B16-BL6 mouse melanoma
cells through the stimulation of the adenosine A3 receptor followed
These membrane proteins have been divided into P1- and
P2-type transporters [25]. Both P1 and P2 transporters efficiently
transport numerous purine nucleoside analogs, whereas P2 is
additionally involved in the accumulation of the front-line trypa-
nocidal drugs pentamidine and melarsoprol by the parasite.
Based on the existing knowledge of P2 substrate recognition
motifs, melamine-based nitroheterocycles specifically targeted to
the trypanosome were designed and synthesized as novel trypa-
nocides by Baliani et al. [26]. However, it was shown that resistance
to the current front-line drugs against African trypanosomiasis
might correlate with loss of P2 transporter activity [5]. In this
context, studying the P1 adenosine transporters is an important
field of research given that the T. brucei genome contains multiple
equilibrative nucleoside transporter (ENT) family genes encoding
P1-type transporters [27].
by glycogen synthase kinase-3
b (GSK) activation and cyclin D1
Al-Salabi et al. reported that the high affinity for purine nucle-
osides of the NT10/AT-B P1-type transporter is much reduced when
suppression. This is of interest for antineoplastic chemotherapy
because both inhibitors of cyclin dependant kinases (CDKs) and
GSK are promising therapeutic targets [29,30]. Obviously, removal
of the 30OH group is compatible with high trypanocidal activity
(cordycepin lacks the 30-OH function), even if the potentially
important influence on the favored sugar pucker in solution at
37 ꢁC remains elusive. However, published models for binding of
adenosine to T. b. brucei nucleoside transporters suggest that the
optimal affinity of adenosine analogs for the T. b. brucei P1 nucle-
oside transporter in some stages of the parasite life-cycle may
require intact 30 and 50 OH groups, in addition to the N-3 and N-7
H-bond acceptors [28,4,31]. Affinity for the P2 adenine/adenosine
transporter is reported to involve the presence of an N6 H-bond
donor in conjunction with an N-1 H-bond acceptor, an aromatic
Table 1
Purity and biological activity of target compounds 8–23.
b
Entry
Puritya (%)
IC50
[
m
M]
8
9
100
99.8
100
99.6
100
100
100
93.5
97.4
99.3
100
99.8
100
99.5
99.2
100
49.7
>170
n.d.c
13.6
167.3
88.7
3.4
131.7
>170
131.9
125.3
37.6
85.9
4.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
ring contributing to
p–p interactions, and a group at the position
corresponding to N-9, which takes on electrostatic interactions [5].
On the basis of these findings, various di- or trisubstituted adeno-
sine derivatives are also of interest as potential antitrypanosomal
agents. However, di- or trisubstituted adenosine derivatives tend to
have a higher log PO/W and are thus prone to passive diffusion
through membranes, are not ideal substrates for transport proteins
and have poor pharmacokinetic properties. In order to identify
active compounds that might effectively use P1 and P2 binding
motifs we decided to design N6-monoalkylated adenosines with
10.0
9.2
a
HPLC, 100% method, detection at 254 nm.
T. b. rhodesiense. IC50 for standard drug (melarsoprol): 10 nM
Compound 10 was not included in the biological investigation (n.d. ¼ not
b
c
determined).

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A. Link et al. / European Journal of Medicinal Chemistry 44 (2009) 3665–3671
a balanced polar surface area, lipophilicity and molmass as well as
stability to adenosine deaminases.
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
The molecular target of the compounds synthesized is not
known. Our assumption is that the compounds are accumulated
inside the trypanosome and bind to a large number of enzymes,
some of which comprise adenosine binding motifs with varying
affinities. In this respect, promiscuity in ligand–target interaction
with many different enzymes of the adenosine salvage pathway or
enzymes utilizing adenosine containing substrates such as ATP or
NADþ might be regarded as an advantage in terms of the parasites
ability to acquire resistance. Additionally, the fact that purine
analogs enter the parasites through multiple distinct transport
proteins as well as passive diffusion might be suited to prevent the
onset of resistance to this class of compounds [32].
The highly analogous compounds 14 and 21 show the same
desired activity in the selected model. While single digit micro-
molar activity is only moderate it seems warranted to make addi-
tional modifications to the adenosine molecule in order to generate
enough data for a complete SAR-study. The synthesis of new
adenosine derived amino-functionalized templates can be achieved
either by established procedures or by using novel glycosylation
reactions [33,34]. The parallel derivatization, however, can be
achieved fast and efficiently with the polymer-bound acylating
species reported here.
at 10
6.1. 2-(2-Amino-ethoxy)-ethylamine (3)
To solution of 30 mmol (3.51 ml) 1-chloro-2-(2-chlor-
ml/min with polyethylene glycol as reference).
a
ethoxy)ethane in 300 ml DMF was added 120 mmol (22.2 g) of
potassium phthalimide. The resulting suspension was kept at reflux
and stirred for 24 h. The suspension was poured onto crushed ice
and the resulting precipitate was filtrated and washed with 70 ml
aqueous sodium hydroxide solution (0.1 mol/l), subsequently with
70 ml water. The residue was dried under reduced pressure.
Intermediate product: 1-phthaloyl-2-(2-phthaloyl-ethoxy)-
ethane 1H NMR (500 MHz, [D6]-DMSO and 1 drop D2O) ¼
d (ppm)
7.80–7.77 (m, 4H, benzene), 7.69–7.66 (m, 4H, benzene), 3.71–3.66
(m, 4H, ethylene), 3.65–3.60 (m, 4H, ethylene). An amount of
13.7 mmol (5 g) of this material was dissolved in 200 ml of a solu-
tion of 33% methylamine in methanol. The mixture was stirred for
5 h. Afterwards the solvent was removed under reduced pressure
and the remaining residue was suspended in 8 ml of water. Column
chromatography using strongly basic ion exchange resin (Dowex
1 ꢂ 2, 200–400 mesh, OHꢀ-form) yielded an aqueous solution of
the desired amine, which was further concentrated in vacuo. Yield
5. Conclusion
49% (699 mg). 1H NMR (500 MHz, [D6]-DMSO þ 1 drop D2O) ¼
d
(ppm) 3.34 (t, 4H, J ¼ 5.8 Hz, ethylene), 2.65 (t, 4H, J ¼ 5.6 Hz,
Through introduction of a spacer fragment decorated with
a nucleophilic aliphatic primary amine, adenosine was transformed
into a scaffold for the introduction of diversity at a predefined
position. By using polymer-bound acylating species, clean and high
yielding transformation of the unprotected, enantiomerically pure
amino alcohol was easily achieved. The complex scaffold did not
have to be attached to a linker or polymer but nevertheless the
resulting mixtures could easily be separated by filtration. After
consumption of the limiting nucleoside derivative, the suspensions
obtained contain only the target molecules in solution along with
polymer-bound reagents. The already highly pure test compounds
could be further purified rapidly and efficiently by flash or medium
pressure chromatography, yielding substances of sufficient purity
for biological evaluation. This approach enables the rapid genera-
tion of complex molecules that are not found in presently known
libraries. Such compounds will be useful in providing new insights
into the complexities of nucleoside transport in trypanosomes.
ethylene).
Storage as hydrochloric acid salt: amine 3 is dissolved in ice
cooled diethylether. Subsequent addition of hydrochloric acid leads
to the formation of the corresponding crystalline dihydrochloride.
Yield 93% 1H NMR (500 MHz, [D6]-DMSO þ 1 drop D2O) ¼
d (ppm)
3.95 (t, 4H, J ¼ 4.6 Hz, ethylene), 3.29 (t, 4H, J ¼ 4.6 Hz, ethylene).
6.2. N6-[2-(2-Amino-ethoxy)-ethyl]adenosine (4)
To a solution of 500 mg (1.74 mmol) 1-(6-Chloro-purin-9-yl)-
-1-deoxyribofuranose (2) in 22.5 ml 1-propanol were added 1.40 g
(13.44 mmol) of 2-(2-Amino-ethoxy)-ethylamine (3) and 300
b-
D
ml
Hu¨nig’s base. The mixture was held at 60 ꢁC for 12 h. Reaction
control via TLC indicated quantitative conversion. The solvent was
removed under reduced pressure and the remaining oily residue
was dissolved in 20 ml methanol/water (1:4). Chromatographic
purification was achieved using a short column filled with strongly
basic ion exchange resin (Dowex 1 ꢂ2, 200–400 mesh, OHꢀ-form).
The product containing fractions was collected and the solvent was
removed under in vacuo. Yield 88% (543 mg). 1H NMR (500 MHz,
6. Experimental section
1H NMR spectra were recorded on a Jeol Eclipseþ 500 spec-
trometer, using tetramethylsilane as internal standard. The purity
of the target compounds was deduced from 1H NMR data as well as
evaluated by HPLC, using a Dionex Summit HPLC-system with
a diode array detector and CC 125/4 Nucleodur 100-5 C18 ec
columns, supplied by Macherey-Nagel and water/methanol gradi-
ents. 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 coefficients at the standard wavelength 254 nm, which
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 reac-
tion control was performed on Macherey-Nagel Polygram Sil
G/UV254 precoated microplates, spots were visualized under
UV-illumination at 254 nm. IR-spectra for the detection of errone-
ously formed 4-hydroxy-2,3,5,6-tetrafluorobenzoic acid esters on
[D6]-DMSO þ 1 drop D2O) ¼
d (ppm) 8.35 (s, 1H, C8–H), 8.22 (bs, 1H,
C2–H), 7.82 (bs, 1H very broad, N6–H), 5.89 (d, 1H, J ¼ 6,1 Hz, 10H),
4.61 (m, 1H, 20H), 4.15 (m, 1H, 30H), 3.97 (m, 1H, 40H), 3.69–3.54 (m,
6H, 50H overlapping ethylene), 3.39 (t, 2H, J ¼ 5.7 Hz, ethylene), 2.65
(m, 2H, ethylene). 13C NMR: (126 MHz, [D6]-DMSO þ 1 drop
D2O) ¼
d (ppm) 154.6, 152.2, 148.4, 139.7, 119.7, 87.9, 85.8, 73.4, 72.5,
70.5, 68.5, 61.5, 41.2 (one signal is missing due to peak overlapping)
HR-ESI-MS [M þ H]þ calcd 355.1730 found 355.1741; mp 147 ꢁC.
6.3. General procedure for the preparation of simple polymer
supported acid, using 4-hydroxy-2,3,5,6-tetrafluorobenzoic
acid (5)
To a flask containing 1 g of 4-hydroxy-2,3,5,6-tetrafluorobenzoic
acid loaded onto aminomethylated polystyrene (6) (prepared from
very high load aminomethylated polystyrene, purchased from
Novabiochem, Switzerland, batch number A20540) with an initial
loading level of 1.1 mmol/g was added 20 ml DMF and the resin was

A. Link et al. / European Journal of Medicinal Chemistry 44 (2009) 3665–3671
3669
allowed to swell for 10 min [22]. Subsequently, 2 equiv (2.2 mmol)
6.8. N6-{2-[2-(3-Fluorobenzamido)ethoxy]ethyl}adenosine (11)
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
Conversion rate 96%. Purity (HPLC after MPLC) ¼ 99.6%
(195
ml) of DIC, the mixture was agitated at room temperature for
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.57 (t, 1H,
16 h. The resin beads were filtered off and washed exhaustively
with DMF (three times 15 ml), dichloromethane (three times 15 ml)
and THF (three times 15 ml) and afterwards dried under in vacuo.
After careful drying the increase in weight was determined.
J ¼ 5.6 Hz, amido), 8.34 (s, 1H, C8–H), 8.20 (bs, 1H, C2–H), 7.74–7.67
(m, 2H, N6–H overlapping benzene), 7.63 (m, 1H, benzene), 7.50 (m,
1H, benzene), 7.36 (m, 1H, benzene), 5.88 (d, 1H, J ¼ 6,1 Hz, 10H),
5.39 (m, 1H, 30OH), 5.33 (m, 1H, 50OH), 5.13 (m, 1H, 20OH), 4.60 (m,
1H, 20H), 4.15 (m, 1H, 30H), 3.96 (m, 1H, 40H), 3.71–3.53 (m, 8H, 50H
overlapping ethylene and H2O), 3.43 (m, 2H, ethylene). HR-ESI-MS
[M þ Na]þ calcd 499.1717 found 499.1743.
6.4. General procedure for the preparation of compounds 8–23
using chemset 7
6.9. N6-{2-[2-(3,4-Difluorobenzamido)ethoxy]ethyl}adenosine (12)
Conversion rate 100%. Purity (HPLC after MPLC) ¼ 100%
200 mg of resin loaded with the appropriate carboxylic acid
(loading level approximately 1 mmol carboxylic acid per g) was
swollen in 2 ml dichloromethane (or alternatively THF). A solution
of 4 in a few drops THF (as little solvent as possible) was added to
the suspension. After 12 h agitation at ambient temperature
(20 ꢁC) in most cases complete conversion of the amine 4 to the
target carboxylic acid amide 8–23 could be observed via TLC.
Subsequently the resin was washed with dichloromethane and
THF and the collected fractions were evaporated under reduced
pressure. To ensure product purity, all samples were further
purified via MPLC.
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.59 (t, 1H,
J ¼ 5.4 Hz, amido), 8.34 (s, 1H, C8–H), 8.20 (bs, 1H, C2–H), 8.10 (bs,
1H, N6–H), 7.88 (m, 1H, benzene), 7.72 (m, 1H, benzene), 7.53 (m,
1H, benzene), 5.89 (d, 1H, J ¼ 6.1 Hz, 10H), 5.38 (m, 1H, 30OH), 5.33
(m, 1H, 50OH), 5.13 (m, 1H, 20OH), 4.60 (m, 1H, 20H), 4.15 (m, 1H,
30H), 3.96 (m,1H, 40H), 3.69–3.61 (m, 4H, 50H overlapping ethylene),
3.55 (m, 2H, ethylene), 3.42 (m, 2H, ethylene), two protons are
missing due to signal overlapping with water. HR-ESI-MS [M þ H]þ
calcd 495.1804 found 495.1778.
6.5. N6-(2-{2-[2-(2,6-Dichlorophenyl)acetamido]-
ethoxy}ethyl)adenosine (8)
6.10. N6-(2-{2-[4-(4-Nitrophenyl)butanamido]ethoxy}
ethyl)adenosine (13)
Conversion rate 98%. Purity (HPLC after MPLC) ¼ 100% (254 nm).
Conversion rate 95%. Purity (HPLC after MPLC) ¼ 100% (254 nm).
1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.35 (s, 1H, C8–H), 8.22
1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.34 (s, 1H, C8–H), 8.20
(bs, 1H, C2–H), 8.10 (m, 1H, amido), 7.72 (bs, 1H, N6–H), 7.42 (m, 2H,
benzene), 7.29 (m, 1H, benzene), 5.88 (d, 1H, J ¼ 5.6 Hz, 10H), 5.38
(m, 1H, 30OH), 5.33 (m, 1H, 50OH), 5.12 (m, 1H, 20OH), 4.60 (m, 1H,
20H), 4.15 (m, 1H, 30H), 3.96 (m, 1H, 40H), 3.80 (s, 2H, benzyl–CH2),
3.70–3.58 (m, 6H, 50H overlapping ethylene), 3.47 (m, 2H, ethylene),
3.36 (m, 2H, ethylene overlapping H2O). HR-ESI-MS [M þ H]þ calcd
541.1369 found 541.1387.
(bs, 1H, C2–H), 8.14 (d, 2H, J ¼ 8.7 Hz, benzene), 7.81 (t, 1H,
J ¼ 5.4 Hz, amido), 7.70 (bs, 1H, N6–H), 7.46 (d, 2H, J ¼ 8.7 Hz,
benzene), 5.88 (d, 1H, J ¼ 6.0 Hz, 10H), 5.38 (d, 1H, J ¼ 6.1 Hz, 30OH),
5.33 (m, 1H, 50OH), 5.13 (d, 1H, J ¼ 4.7 Hz, 20OH), 4.60 (m, 1H, 20H),
4.15 (m, 1H, 30H), 3.97 (m, 1H, 40H), 3.70–3.53 (m, 6H, 50H ethylene),
3.44 (t, 2H, J ¼ 5.8 Hz, ethylene), 3.20 (m, 2H, ethylene overlapping
H2O), 2.69 (t, 2H, J ¼ 7.6 Hz, butanamido), 2.09 (t, 2H, J ¼ 7.4 Hz,
butanamido), 1.82 (m, 2H, butanamido). HR-ESI-MS [M þ H]þ calcd
546.2312 [M þ H]þ found 546.2338.
6.6. N6-{2-[2-(3,4-Dimethoxybenzamido)ethoxy]ethyl}adenosine
(9)
6.11. N6-(2-{2-[4-(Phenoxy)butanamido]ethoxy}ethyl)adenosine
(14)
Conversion rate 83%. Purity (HPLC after MPLC) ¼ 99.8%
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.34 (m, 2H,
C8–H overlapping amido), 8.20 (bs, 1H, C2–H), 7.71 (bs, 1H, N6H),
7.44 (m, 2H, benzene), 6.98 (d, 1H, J ¼ 8.5 Hz, benzene), 5.89 (d, 1H,
J ¼ 6.1 Hz,10H), 5.37 (d,1H, J ¼ 6.2 Hz, 30OH), 5.32 (m,1H, 50OH), 5.11
(d, 1H, J ¼ 4.7 Hz, 20OH), 4.60 (m,1H, 20H), 4.14 (m, 1H, 30H), 3.96 (m,
1H, 40H), 3.79 (s, 3H, methoxy), 3.78 (s, 3H, methoxy), 3.70–3.60 (m,
4H, 50H overlapping ethylene), 3.55 (m, 2H, ethylene), 3.40 (m, 2H,
ethylene), 3.36 (m, 2H, ethylene overlapping H2O). HR-ESI-MS
[M þ H]þ calcd 519.2203 found 519.2226.
Conversion rate 98%. Purity (HPLC after MPLC) ¼ 100% (254 nm).
1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.34 (s, 1H, C8–H), 8.21
(bs, 1H, C2–H), 7.86 (t, 1H, J ¼ 5.4 Hz, amido), 7.70 (bs, 1H, N6–H),
7.26 (m, 2H, phenoxy), 6.90 (m, 3H, phenoxy), 5.88 (d,1H, J ¼ 6.1 Hz,
10H), 5.39 (d, 1H, J ¼ 6.2 Hz, 30OH), 5.33 (m, 1H, 50OH), 5.13 (d, 1H,
J ¼ 4.7 Hz, 20OH), 4.59 (m, 1H, 20H), 4.15 (m, 1H, 30H), 3.94 (m, 3H,
40H overlapping butanamido), 3.70–3.52 (m, 6H, 50H overlapping
ethylene), 3.43 (t, 2H, J ¼ 5.9 Hz, ethylene), 3.21 (m, 2H, ethylene
overlapping H2O), 2.24 (t, 2H, J ¼ 7.4 Hz, butanamido), 1.91 (m, 2H,
butanamido). HR-ESI-MS [M þ H]þ calcd 517.2411 found 517.2442.
6.7. N6-(2-{2-[2-(4-
Methylphenyl)acetamido]ethoxy}ethyl)adenosine (10)
6.12. N6-{2-[2-(4-Acetamido-benzamido)ethoxy]ethyl}
Conversion rate 100%. Purity (HPLC after MPLC) ¼ 100%
adenosine (15)
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.33 (s, 1H,
C8–H), 8.20 (bs, 1H, C2–H), 7.98 (bs, 1H, N6–H), 7.69 (m, 1H, amido),
7.10 (m, 2H, Benzene), 7.05 (m, 2H, Benzene), 5.88 (m, 1H, 10H), 5.37
(m, 1H, 30OH), 5.32 (m, 1H, 50OH), 5.11 (m, 1H, 20OH), 4.59 (m, 1H,
20H), 4.14 (m, 1H, 30H), 3.95 (m, 1H, 40H), 3.71–3.51 (m, 6H, 50H
overlapping ethylene and benzyl–CH2), 3.43 (m, 2H, ethylene), 3.33
(m, 2H, ethylene), 3.18 (m, 2H, ethylene), 2.23 (s, 3H, methyl). HR-
ESI-MS [M þ H]þ calcd 487.2305 found 487.2295.
Conversion rate 93%. Purity (HPLC after MPLC) ¼ 93.5%
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 10.09 (s, 1H,
acetamido), 8.34 (s, 1H, C8–H), 8.32 (t, 1H, J ¼ 4.6 Hz, amido), 8.21
(bs, 1H, C2–H), 7.78 (d, 2H, J ¼ 8.7 Hz, benzene), 7.73 (bs, 1H, N6–H),
7.62 (d, 2H, J ¼ 8.6 Hz, benzene), 5.88 (d, 1H, J ¼ 6.1 Hz, 10H), 5.39
(m, 1H, 30OH), 5.33 (m, 1H, 50OH), 5.13 (m, 1H, 20OH), 4.60 (m, 1H,
20H), 4.15 (m, 1H, 30H), 3.96 (m, 1H, 40H), 3.83–3.52 (m, 8H, 50H

3670
A. Link et al. / European Journal of Medicinal Chemistry 44 (2009) 3665–3671
overlapping ethylene and water), 3.41 (m, 2H, ethylene), 2.06 (s, 3H,
6.17. N6-(2-{2-[2-(1,10-Diphenyl)acetamido]ethoxy}-
ethyl)adenosine (20)
acetyl–CH3). HR-ESI-MS [M þ H]þ calcd 538.2026 found 538.2052.
Conversion rate 90%. Purity (HPLC after MPLC) ¼ 100% (254 nm).
1H NMR (500 MHz, [D6]-DMSO) ¼
d
(ppm) 8.42 (bs, 1H, N6H), 8.38
6.13. N6-(2-{2-[2-(1-Naphthyl)acetamido]ethoxy}ethyl)
adenosine (16)
(m, 2H, C8H overlapping amido), 8.25 (s, 1H, C2H), 7.36 (m, 4H,
4-phenoxybenzyl), 7.20 (m, 3H, phenyl), 7.18 (m,1H, phenyl), 7.10 (t,
1H, 4-phenoxybenzyl, J ¼ 7.4 Hz), 6.96 (m, 4H, 4-phenoxybenzyl),
5.87 (d, 1H, 10H, J ¼ 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 overlapped by water). HR-ESI-MS [M þ H]þ calcd 549.2462
found 549.2486.
Conversion rate 98%. Purity (HPLC after MPLC) ¼ 97.4%
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.35 (s, 1H,
C8–H), 8.22 (bs, 1H, C2–H), 8.17 (m, 1H, amido), 8.07 (d, 1H,
J ¼ 7.4 Hz, naphthyl), 7.89 (m, 1H, naphthyl), 7.79 (m, 1H, naphthyl),
7.71 (bs, 1H, N6–H), 7.49 (m, 2H, naphthyl), 7.41 (m, 2H, naphthyl),
5.89 (d, 1H, J ¼ 6.0 Hz, 10H), 5.40 (m, 1H, 30OH), 5.33 (m, 1H, 50OH),
5.14 (m, 1H, 20OH), 4.60 (m, 1H, 20H), 4.15 (m, 1H, 30H), 3.96 (m, 1H,
40H), 3.90 (s, 2H, methylene), 3.71–3.51 (m, 6H, 50H overlapping
ethylene and water), 3.46 (m, 2H, ethylene), 3.24 (m, 2H, ethylene
overlapping H2O). HR-ESI-MS [M þ H]þ calcd 523.2305 found
523.2288.
6.18. N6-{2-[2-(4-Phenylbutanamido)ethoxy]ethyl}adenosine (21)
Conversion rate 89%. Purity (HPLC after MPLC) ¼ 99.5%
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.34 (s, 1H,
C8–H), 8.20 (bs, 1H, C2–H), 7.79 (t, 1H, J ¼ 5.4 Hz, amido), 7.70 (bs,
1H, N6–H), 7.26 (m, 2H, benzene), 7.16 (m, 3H, benzene), 5.89 (d, 1H,
J ¼ 6.1 Hz, 10H), 5.38 (m, 2H, 30OH overlapping 20OH), 5.33 (m, 1H,
50OH), 4.61 (m, 1H, 20H), 4.15 (m, 1H, 30H), 3.97 (m, 1H, 40H), 3.83–
3.75 (m, 2H, butyryl), 3.70–3.53 (m, 6H, 50H overlapping ethylene),
3.45 (t, 2H, J ¼ 6.0 Hz, ethylene), 3.19 (m, 2H, ethylene), 2.54 (t, 2H,
J ¼ 7.7 Hz, butyryl), 2.08 (t, 2H, J ¼ 7.4 Hz, butyryl). HR-ESI-MS
[M þ H]þ calcd 501.2462 found 501.2437.
6.14. N6-(2-{2-[2-(4-Methoxyphenyl)acetamido]ethoxy}
ethyl)adenosine (17)
Conversion rate 89%. Purity (HPLC after MPLC) ¼ 99.3%
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.35 (s, 1H,
C8–H), 8.21 (bs, 1H, C2–H), 8.03 (t, 1H, J ¼ 5.4 Hz, amido), 7.70 (bs,
1H, N6–H), 7.17 (t, 1H, J ¼ 7.8 Hz, benzene), 6.83–6.75 (m, 3H,
benzene), 5.89 (d, 1H, J ¼ 6.1 Hz, 10H), 5.37 (d, 1H, J ¼ 6.2 Hz, 30OH),
5.33 (m, 1H, 50OH), 5.13 (d, 1H, J ¼ 4.6 Hz, 20OH), 4.60 (m, 1H, 20H),
4.15 (m, 1H, 30H), 3.96 (m, 1H, 40H), 3.72 (s, 3H, methoxy), 3.69–3.52
(m, 6H, 50H overlapping ethylene), 3.45 (t, 2H, J ¼ 5.8 Hz, ethylene),
3.37 (s, 2H, methylene), 3.12 (m, 2H, ethylene). HR-ESI-MS [M þ H]þ
calcd 503.2254 found 503.2273.
6.19. N6-(2-[2-(4-Phenoxybenzamido)ethoxy]ethyl)adenosine (22)
Conversion rate 89%. Purity (HPLC after MPLC) ¼ 99.2%
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.41 (t, 1H,
J ¼ 5.6 Hz, amido), 8.33 (s, 1H, C8–H), 8.21 (bs, 1H, C2–H), 7.86 (d,
2H, J ¼ 8.8 Hz, benzene), 7.73 (bs, 1H, N6–H), 7.43 (m, 2H, benzene),
7.20 (t, 1H, J ¼ 7.0 Hz, benzene), 7.08 (d, 2H, J ¼ 7.6 Hz, benzene),
7.02 (d, 2H, J ¼ 8.8 Hz, benzene), 5.89 (d, 1H, J ¼ 6.1 Hz, 10H), 5.38 (d,
2H, J ¼ 6.1 Hz, 30OH), 5.33 (m, 1H, 50OH), 5.13 (d, 1H, J ¼ 4.7 Hz,
20OH), 4.61 (m, 1H, 20H), 4.15 (m, 1H, 30H), 3.96 (m, 1H, 40H), 3.83–
3.53 (m, 8H, 50H overlapping ethylene and H2O), 3.42 (t, 2H,
J ¼ 6.0 Hz, ethylene). HR-ESI-MS [M þ H]þ calcd 551.2254 found
551.2246.
6.15. N6-{2-[2-(3,3-Dimethylacrylamido)ethoxy]ethyl}
adenosine (18)
Conversion rate 92%. Purity (HPLC after MPLC) ¼ 100% (254 nm).
1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.35 (s, 1H, C8–H), 8.22
6.20. N6-(2-[2-(3,4-Dimethoxyphenylacetamido)ethoxy]
ethyl)adenosine (23)
(bs, 1H, C2–H), 7.72 (m, 2H, amido overlapping N6–H), 5.89 (d, 1H,
J ¼ 6.0 Hz, 10H), 5.65 (s, 1H; acryl–CH), 5.38 (d, 1H, J ¼ 6.2 Hz, 30OH),
5.34 (m, 1H, 50OH), 5.12 (d, 1H, J ¼ 4.7 Hz, 20OH), 4.60 (m, 1H, 20H),
4.15 (m, 1H, 30H), 3.97 (m, 1H, 40H), 3.71–3.52 (m, 6H, 50H over-
lapping ethylene), 3.45 (t, 2H, J ¼ 5.8 Hz, ethylene), 3.22 (m, 2H,
ethylene), 2.06 (s, 3H, CH3), 1.77 (m, 3H, CH3). HR-ESI-MS [M þ H]þ
calcd 437.2149 found 437.2164.
Conversion rate 100%. Purity (HPLC after MPLC) ¼ 100%
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d (ppm) 8.37 (s, 1H,
C8–H), 8.20 (bs, 1H, C2–H), 8.05 (t, 1H, J ¼ 5.5 Hz, amido), 7.71 (bs,
1H, N6–H), 6.83 (m, 2H, benzene), 6.78 (m, 1H, benzene), 5.93 (d,
1H, J ¼ 6.1 Hz, 10H), 5.37 (d, 1H, J ¼ 6.2 Hz, 30OH), 5.33 (m, 1H, 50OH),
5.12 (d, 1H, J ¼ 4.6 Hz, 20OH), 4.60 (m, 1H, 20H), 4.15 (m, 1H, 30H),
3.96 (m, 1H, 40H), 3.74 (s, 6H, methoxy), 3.69–3.51 (m, 6H, 50H
overlapping ethylene), 3.45 (t, 2H, J ¼ 5.8 Hz, ethylene), 3.36 (s, 2H,
benzyl–CH2), 3.12 (m, 2H, ethylene). HR-ESI-MS [M þ H]þ calcd
533.2360 found 533.2377.
6.16. N6-(2-{2-[4-(3-Indolyl)-butanamido]ethoxy}ethyl)
adenosine (19)
Conversion rate 92%. Purity (HPLC after MPLC) ¼ 99.8%
(254 nm). 1H NMR (500 MHz, [D6]-DMSO) ¼
d
(ppm) 10.70 (s, 1H,
6.21. In vitro microplate assay against T. brucei rhodesiense
indole–NH), 8.35 (s, 1H, C8–H), 8.21 (bs, 1H, C2–H), 7.79 (m, 1H,
amido), 7.70 (bs, 1H, N6–H), 7.49 (d, 1H, J ¼ 8.2 Hz, indole), 7.32 (d,
1H, J ¼ 8.1 Hz, indole), 7.08 (m,1H, indole), 7.04 (m,1H, indole), 6.95
(m, 1H, indole), 5.89 (d, 1H, J ¼ 6.1 Hz, 10H), 5.38 (d, 1H, J ¼ 6.1 Hz,
30OH), 5.34 (m, 1H, 50OH), 5.13 (m, 1H, 20OH), 4.61 (m, 1H, 20H), 4.15
(m, 1H, 30H), 3.97 (m, 1H, 40H), 3.70–3.52 (m, 6H, 50H overlapping
ethylene and H2O), 3.44 (t, 2H, J ¼ 5.9 Hz, ethylene), 3.20 (m, 2H,
ethylene), 2.66 (t, 2H, J ¼ 7.3 Hz, butyryl), 2.14 (m, 2H, butyryl), 1.85
(m, 2H, butyryl). HR-ESI-MS [M þ H]þ calcd 540.2571 found
540.2595.
Minimum essential medium (50 ml) supplemented according to
a known procedure with 2-mercaptoethanol and 15% heat-inacti-
vated horse serum was added to each well of a 96-well microtiter
plate [35]. Serial drug dilutions were prepared covering a range
from 90 to 0.123
desiense STIB 900 in 50
incubated at 37 ꢁC under a 5% CO2 atmosphere for 72 h. Alamar Blue
(10 l containing 12.5 mg resazurin dissolved in 1000 ml distilled
water) was then added to each well and incubation continued for
m
g/ml. Then 104 bloodstream forms of T. b. rho-
ml were added to each well and the plate
m

A. Link et al. / European Journal of Medicinal Chemistry 44 (2009) 3665–3671
3671
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a further 2–4 h. The Alamar blue dye is an indicator of cellular
growth and/or viability. The blue, non-fluorescent, oxidized form
becomes pink and fluorescent upon reduction by living cells. The
plate was then read in a Spectramax Gemini XS microplate fluo-
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536 nm and emission wavelength of 588 nm [36]. Fluorescence
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control. Data were transferred into the graphic programme Softmax
Pro (Molecular Devices) which calculated IC50 values. Melarsoprol
served as standard.
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Acknowledgment
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