Activity of 6-aryl-pyrrolo[2,3-d]pyrimidine-4-amines to Tetrahymena

Article abstract of DOI:10.1016/j.bioorg.2012.06.003

A series 6-aryl-pyrrolo[2,3-d]pyrimidine-4-amines (43 compounds), some of which are epidermal growth factor tyrosine kinase inhibitors, were tested for their protozoal toxicity using an environmental Tetrahymena strain as model organism. The protozoacidal activity of the analogues was found to be highly dependent on a 4-hydroxyl group at the 6-aryl ring, and a chiral 1-phenylethanamine substituent in position 4. Further, the potency was affected by the aromatic substitution pattern of the phenylethanamine: the unsubstituted, the meta-fluoro and the para-bromo substituted derivatives had the lowest minimum protozoacidal concentrations (8-16 μg/mL). Surprisingly, both enantiomers were found to have high potency suggesting that this compound class could have several modes of action. No correlation was found between the compounds protozoacidal activity and the in vitro epidermal growth factor receptor tyrosine kinase inhibitory potency. This suggests that the observed antimicrobial effects are related to other targets. Testing towards a panel of kinases indicated several alternative modes of action.

Full text of DOI:10.1016/j.bioorg.2012.06.003

Bioorganic Chemistry 44 (2012) 35–41
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Activity of 6-aryl-pyrrolo[2,3-d]pyrimidine-4-amines to Tetrahymena
Svein Jacob Kaspersen a, Eirik Sundby b, Colin Charnock c, Bård Helge Hoff a,
⇑
a Norwegian University of Science and Technology, Høgskoleringen 5, NO-7491 Trondheim, Norway
b Sør-Trøndelag University College, E.C. Dahls gate 2, 7004 Trondheim, Norway
c Oslo and Akershus University College of Applied Sciences, P.O. Box 4, St. Olavs plass, 0130 Oslo, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 April 2012
Available online 6 July 2012
A series 6-aryl-pyrrolo[2,3-d]pyrimidine-4-amines (43 compounds), some of which are epidermal growth
factor tyrosine kinase inhibitors, were tested for their protozoal toxicity using an environmental Tetrahy-
mena strain as model organism. The protozoacidal activity of the analogues was found to be highly
dependent on a 4-hydroxyl group at the 6-aryl ring, and a chiral 1-phenylethanamine substituent in posi-
tion 4. Further, the potency was affected by the aromatic substitution pattern of the phenylethanamine:
the unsubstituted, the meta-fluoro and the para-bromo substituted derivatives had the lowest minimum
Keywords:
Pyrrolopyrimidine
Antiprotozoal agent
Tetrahymena
Kinase
protozoacidal concentrations (8–16 lg/mL). Surprisingly, both enantiomers were found to have high
potency suggesting that this compound class could have several modes of action. No correlation was
found between the compounds protozoacidal activity and the in vitro epidermal growth factor receptor
tyrosine kinase inhibitory potency. This suggests that the observed antimicrobial effects are related to
other targets. Testing towards a panel of kinases indicated several alternative modes of action.
Ó 2012 Elsevier Inc. All rights reserved.
Benzylamine
1. Introduction
infections attributed to members of this genus are a problem in
closed fish farming. Low molecular weight compounds such as
Diseases caused by parasitic protozoa, as for instance malaria,
dysentery, leishmaniasis, and human African trypanosomiasis are
major causes of mortality throughout the world, thus, the study
of effects of organic compounds on protozoa is important. Thera-
peutic agents are available [1,2] however, many of the drugs have
critical side effects [3,4] and also resistance is emerging [5,6].
Therefore, identification of new lead compounds is required, and
inhibition of cellular kinase activity has been recognised as a useful
strategy [7–11]. Among others, tyrosine kinase inhibitors such as
Erlotinib, Canertinib and Sunitinib designed for cancer chemother-
apy have been identified as efficient antiprotozoal agents [11].
Tetrahymena is a genus of ciliated protozoa. Its members are
easily grown and relatively safe to handle making them useful
model systems for biochemical mechanistic studies in eukaryotes
[12]. The motility behaviour of Tetrahymena is conveniently used
to monitor bioactivity and cell toxicity of chemicals [13–15].
Compounds such as diphenols, aminophenols, diaminoaromatics,
halogenated aromatic nitro compounds, aromatic aldehydes and
Menadione (I) [22], and anti-infective agents such as Niclosamide
(II) have been proposed as treatment alternatives [23]. Other com-
pounds with in vitro activity towards Tetrahymena include among
others Climacostol (III) [24], known antimicrobial agents as Chlo-
roquine [25] and Chloroamphenicol [26,27] and antineoplastics
such as Necodazole [28], Fig. 1.
Tetrahymena are known to have epidermal growth factor
(EGF)-like receptors which are involved in cell division [29], and
cyst formation [30]. Also other processes such as chemotaxis
[31], hormonal imprinting [32], cell division [33,34], stress
response [34,35], and GTP signalling [36], are triggered and
controlled by kinase activity. Using an environmental Tetrahymena
isolate as model, we have evaluated the potency of a series of
6-aryl-7H-pyrrolo[2,3-d]pyrimidine-4-amines as antiprotozoal agents.
One goal has been to identify new lead compounds for combating
protozoa. Secondly, the study could shed light on the toxicity
profile of this compound class since some of the derivatives are
efficient inhibitors of the epidermal growth factor receptor tyro-
sine kinase (EGFR-TK) in vitro [37].
a-haloketones are generally toxic to Tetrahymena. This is due to
their ability to undergo various reactions with biomacromolecules
[16–19]. Tetrahymena do not pose a serious threat to human
health. However, Legionella in symbiosis with Tetrahymena tropi-
calis appears more resistant and aggressive [20,21]. In addition,
2. Materials and methods
2.1. General
⇑
1H and 13C NMR spectra were recorded with a Bruker Avance
400 spectrometer operating at 400 MHz and 100 MHz,
Corresponding author. Fax: +47 73544256.
E-mail address: bard.helge.hoff@chem.ntnu.no (B.H. Hoff).
0045-2068/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2012.06.003

36
S.J. Kaspersen et al. / Bioorganic Chemistry 44 (2012) 35–41
O
O
NO2
OH
O
CH3
Cl
N
H
Cl
HO
OH
C6H13
Menadione (I)
Niclosamide (II)
Climacostol (III)
F
N
O
H3C
N
H
H
O
Cl
N
N
N
O
CH3
N
N
H
F
H
O
N
N
O
H
Canertinib (IV)
Sunitinib (V)
Fig. 1. Structure of compounds I–III with potency towards Tetrahymena and the kinase inhibitors Canertinib (IV) and Sunitinib (V).
respectively. 19F NMR was performed on a Bruker Avance 600
operating at 564 MHz. The 19F NMR shift values are relative to
hexafluorobenzene. Coupling constants are in Hertz. HPLC (Agilent
110-Series) with a G1379A degasser, G1311A Quatpump, G1313A
ALS autosampler and a G1315D Agilent detector (230 nm) was
used to determine the purity of the synthesised compounds. Con-
ditions: a Omrisphere 5 C18 (100 ꢀ 3.0 mm) column, flow rate
1.0 mL/min, elution starting with H2O + 1% TFA/acetonitrile (98/
2), linear gradient elution for 15 min. ending at acetonitrile/
water+1% TFA (90/10), then 15 min isocratic elution. The software
used with the HPLC was Agilent ChemStation. Accurate mass
determination was performed with EI (70 eV) using a Finnigan
MAT 95 XL. FTIR spectra were recorded on a Thermo Nicolet Avatar
330 infrared spectrophotometer. All melting points are uncor-
rected and measured by a Büchi melting point instrument. Optical
rotation was measured with a PerkinElmer Instruments Model 341
Polarimeter.
E. coli for 48 h under a humidified atmosphere in the dark at
22 2 °C. After incubation, protozoa were harvested and washed
as previously described [38], and resuspended in pasteurised
E. coli (corresponding to
a MacFarland 0.5 standard) at
1 ꢀ 104 cells/mL. After incubation for 48 h at 22 2 °C, wells were
examined for motile cells using an inverted microscope. This ap-
proach enabled the whole content of the well to be visualised.
The estimated minimum protozoacidal concentration (MPC; 48 h)
was the lowest concentration at which no motile cells were seen.
After examination in the microscope, the whole content of wells
was transferred to culture dishes containing NNA/pasteurised
E. coli. Cultures were examined over a 7-day period with an in-
verted microscope to see if a cell population developed. The MPC
value measured (MPC; 7 days) was the lowest concentration that
prevented the development of even a single viable cell in the 7-
day period. Each test was performed in triplicate and the results
were averaged to give the MPC value.
2.4. Kinase profiling
2.2. Isolation and characterisation of Tetrahymena
Compound (R)-25e was profiled utilising a panel of 124 protein
kinases in the MRC National Centre for Protein Kinase Profiling Ser-
vice at the University of Dundee (http://www.kinase-screen.mrc.a-
c.uk). The compound was tested in vitro, in duplicate, at a final
concentration of 50 nM. For further details of the methodology
see Bain et al. [39].
The Tetrahymena strain used was originally isolated from pond
water in Norway and was identified to the genus level based on its
phenotype and on partial sequencing of the 18S rDNA-gene. The
sequence had 100% identity with reported sequences for Tetrahy-
mena iwoffi, Tetrahymena tropicalis and Tetrahymena furgosoni.
The strain was maintained on non-nutrient agar (CCAP, Scotland)
seeded with a thick suspension of pasteurised Escherichia coli prior
to testing. The strain and further information on the sequencing
studies can be made available on request.
2.5. Synthesis
Detailed description of the synthesis and characterisation of
most of the intermediates and tested compounds can be found
elsewhere [37,40]. The synthesis and characterisation of the new
chemical entities are given below.
2.3. Determination of minimum protozoacidal (MPC) concentrations
Stock solutions of the agents were made in DMSO at a concen-
tration of 5120
included as control. Water was used as dilutant producing dou-
bling concentrations of the agents at 128–4 g/mL. These interme-
diate dilutions (50 L) were pipetted in triplicate into a 96-well,
NuncÒ round-bottomed microtiter plate system (Thermo Fischer
Scientific, USA). Addition of 50 L of the inoculum gave the final
tested concentration range (2–64 g/mL) and maximally 1.25%
DMSO. A positive control (no agent), and a negative control (with-
out Tetrahymena) tests were also included. Tetrahymena was
grown on NNA seeded with a thick pasteurised suspension of
lg/mL. Benzalkonium chloride (stock in water) was
2.5.1. General procedure thermal amination to 20–24
l
The following is representative: 4-chloro-6-(4-methoxy-
phenyl)-7H-pyrrolo[2,3-d]pyrimidine (14) (275 mg, 1.06 mmol)
and (S)-1-phenylethanamine ((S)-19i) (0.44 mL, ꢁ3.5 mmol) were
added to a dry round bottle flask containing 1-butanol (3.5 mL) un-
der argon atmosphere. The mixture was heated at 145 °C for 24 h.
The precipitate formed upon cooling to rt. was isolated by filtra-
tion, washed with diethyl ether (25 mL) and dried resulting in a
solid.
l
l
l

S.J. Kaspersen et al. / Bioorganic Chemistry 44 (2012) 35–41
37
2.5.1.1. (S)-6-(4-Methoxyphenyl)-N-(1-phenylethyl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine ((S)-20e). The compound was prepared as
described in Section 2.5.1 starting with 4-chloro-6-(4-methoxy-
phenyl)-7H-pyrrolo[2,3-d]pyrimidine (14) (159 mg, 0.61 mmol)
and (S)-1-phenylethanamine (19e) (222 mg, 1.84 mmol). This gave
179 mg (0.52 mmol, 85%) of a white solid, mp. 226–228 °C,
(285 mg, 1.66 mmol). This gave 162 mg (0.41 mmol, 74%) of a
white solid, mp. 274–276 °C, ½a D20
= ꢃ432 (c 0.37, DMSO). Spectro-
ꢂ
scopic properties were in correspondence with that reported previ-
ously for the racemate [37]. 1H NMR (400 MHz, DMSO-d6) d: 11.93
(s, 1H, NH, H-7), 8.24 (d, J = 8.2, 1H), 8.04 (s, 1H, H-2), 7.94 (m, 1H),
7.87 (m, 1H), 7.81 (m, 1H), 7.71–7.64 (m, 3H), 7.59–7.45 (m, 3H),
7.01 (m, 2H), 6.97 (s, 1H, H-5), 6.27 (m, 1H), 3.79 (s, 3H), 1.67 (d,
J = 6.8, 3H). IR (neat, cmꢃ1): 3131, 2962 1624, 1251, 828, 775.
HRMS (EI): 394.1783 (calcd C25H22N4O, 394.1794, M+).
½
a 2D0
= + 289 (c 0.17, DMSO). Spectroscopic properties were in cor-
ꢂ
respondence with that reported previously for the (R)-enantiomer
[40]. 1H NMR (400 MHz, DMSO-d6) d: 11.92 (s, 1H, NH, H-7), 8.04
(s, 1H, H-2), 7.73 (m, 3H), 7.43 (m, 2H), 7.30 (m, 2), 7.19 (m, 1H),
7.02 (d, J = 8.8, 2H), 6.96 (bs, 1H, H-5), 5.50 (m, 1H), 3.80 (s, 3H),
1.53 (d, J = 7.0, 3H). HRMS (EI): 344.1634 (calcd C21H20N4O,
344.1632, M+).
2.5.1.6. (R)-N-(1-(Naphthalen-1-yl)ethyl)-6-phenyl-7H-pyrrolo[2,3-
d]pyrimidin-4-amine ((R)-21p). The compound was prepared as
described in Section 2.5.1 starting with 4-chloro-6-(4-phenyl)-
7H-pyrrolo[2,3-d]pyrimidine (15) (87 mg, 0.38 mmol) and (R)-1-
(naphthalen-1-yl)ethanamine (19p) (195 mg, 1.14 mmol). This
gave 107 mg (0.29 mmol, 76%) of a white solid. The solid melted
at 155–157 °C, but partly re-solidified to a solid melting at
2.5.1.2. (R)-6-(4-Methoxyphenyl)-N-(1-(4-methoxyphenyl)ethyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine ((R)-20h). The compound was
prepared as described in Section 2.5.1 starting with 4-chloro-6-
(4-metoxyphenyl)-7H-pyrrolo-[2,3-d]-pyrimidine (14) (238 mg,
200 °C, ½a 2D0
ꢂ
= ꢃ481 (c 1.00, DMSO), purity: 98% (by HPLC). 1H
0.92 mmol)
and
(R)-(4-methoxyphenyl)ethanamine
(19h)
NMR (400 MHz, DMSO-d6) d: 12.07 (s, 1H, NH, H-7), 8.26–8.24
(m, 1H), 8.08 (s, 1H, H-2), 8.01–7.93 (m, 2H), 7.82–7.77 (m, 3H),
7.67–7.66 (m, 1H), 7.59–7.41 (m, 5H), 7.30–7.27 (m, 1H), 7.14 (s,
1H, H-5), 6.32–6.25 (m, 1H), 1.68 (d, J = 6.8, 3H). 13C NMR
(100 MHz, DMSO-d6) d: 155.3, 152.3, 152.0 141.3, 133.92, 133.87,
132.3, 131.1, 129.42 (2C), 129.1, 127.7, 127.6, 126.6, 126.0, 125.9,
124.9 (2C), 123.8, 122.7, 104.4, 60.2, 96.7, 22.3. IR (neat, cmꢃ1):
2976, 1586, 1471, 1311, 774, 749. HRMS (EI): 364.1683 (calcd
(288 mg, 1.90 mmol). This gave 220 mg (0.59 mmol, 64%) of an
off-white solid, mp. 249–251 °C, ½a D20
= ꢃ330.1 (c 0.14, DMSO),
ꢂ
purity > 99% (by HPLC). 1H NMR (400 MHz, DMSO-d6) d: 11.89 (s,
1H, NH, H-7), 8.04 (s, 1H), 7.71 (d, J = 8.9, 2H), 7.64 (d, J = 8.4, 1H,
NH), 7.34 (d, J = 8.7, 2H), 7.02 (d, J = 8.9, 2H), 6.94 (s, 1H, H-5),
6.86 (d, J = 8.7, 2H), 5.45 (m, 1H), 3.80 (s, 3H), 3.71 (s, 3H), 1.50
(d, J = 7.0, 3H). 13C NMR (100 MHz, DMSO-d6) d: 158.6, 157.9,
154.8, 151.3 (2C), 137.5, 133.4, 127.2 (2C), 125.9 (2C), 124.5,
114.4 (2C), 113.5 (2C), 103.9, 94.6, 55.2, 55.0, 48.0, 22.9. HRMS
(ESI): 375.1814 (calcd C22H22N4O2, 375.1816, M + H+). IR (neat,
cmꢃ1): 3099, 2973, 1588, 1244, 830.
C
24H20N4, 364.1682, M+).
2.5.1.7. (S)-N-(1-(Naphthalen-1-yl)ethyl)-6-phenyl-7H-pyrrolo[2,3-
d]pyrimidin-4-amine ((S)-21p). The compound was prepared as
described in Section 2.5.1 starting with 4-chloro-6-(4-phenyl)-
7H-pyrrolo[2,3-d]pyrimidine (15) (70 mg, 0.30 mmol) and (S)-1-
phenylethanamine (19e) (156 mg, 0.91 mmol). This gave 84 mg
2.5.1.3. (S)-N-(1-(4-Bromophenyl)ethyl)-6-(4-methoxyphenyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine ((S)-20i). The compound was pre-
pared as described in Section 2.5.1 starting with 4-chloro-6-(4-
methoxyphenyl)-7H-pyrrolo-[2,3-d]-pyrimidine (14) (190 mg,
0.73 mmol) and (S)-1-(4-bromophenyl)ethanamine (19i) (439 mg,
2.19 mmol). This gave 245 mg (0.58 mmol, 79%) of a white solid,
(23 mmol, 77%) of a white solid, mp. 162–166 °C, ½a D20
ꢂ
= +422 (c
1.00, DMSO), purity: 98% (by HPLC). The spectroscopic properties
corresponded with that reported for (S)-21p in Section 2.5.1.6.
HRMS (EI): 364.1682 (calcd C24H20N4, 364.1682, M+).
mp. 274–275 °C, ½a D20
ꢂ
= +309 (c 0.21, DMSO). Spectroscopic proper-
ties were in correspondence with that reported previously for the
(R)-enantiomer [37]. 1H NMR (400 MHz, DMSO-d6) d: 11.93 (s,
1H, NH, H-7), 8.03 (s, 1H, H-2), 7.76 (s, 1H, NH), 7.73 (m, 2H),
7.49 (m, 2H), 7.38 (m, 2H), 7.02 (m, 2H), 6.94 (d, J = 1.8, 1H, H-5),
5.44 (m, 1H), 3.80 (s, 3H), 1.51 (d, J = 7.0, 3H). HRMS (EI):
422.0739 (calcd C21H19BrN4O, 422.0737, M+)
2.5.1.8.
(R)-6-(4-Fluorophenyl)-N-(1-(naphthalen-1-yl)ethyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine ((R)-22p). The compound was
prepared as described in Section 2.5.1 starting with 4-chloro-6-
(4-fluorophenyl)-7H-pyrrolo[2,3-d]pyrimidine
(16)
(64 mg,
0.26 mmol) and (R)-1-(naphthalen-1-yl)ethanamine
(19p)
(133 mg, 0.78 mmol). This gave 70 mg (0.18 mmol, 70%) of a white
solid, mp. 147–150 °C, ½a D20
= ꢃ444 (c 0.40, DMSO), purity: 99% (by
ꢂ
2.5.1.4. 6-(4-Methoxyphenyl)-N-(1-naphthalen-1-ylmethyl)-7H-pyr-
rolo[2,3-d]pyrimidin-4-amine (20o). The compound was prepared
as described in Section 2.5.1 starting with 4-chloro-6-(4-methoxy-
phenyl)-7H-pyrrolo[2,3-d]pyrimidine (14) (154 mg, 0.59 mmol)
and naphthalen-1-ylmethanamine (19o) (280 mg, 1.78 mmol).
This gave 173 mg (0.45 mmol, 77%) of a white solid, mp 278–
281 °C. 1H NMR (400 MHz, DMSO-d6) d: 11.98 (s, 1H, NH, H-7),
8.21–8.19 (m, 1H), 8.14 (s, 1H, H-2), 7.97–7.94 (m, 2H), 7.86–
7.84 (m, 1H), 7.71–7.69 (m, 2H), 7.57–7.52 (m, 3 H), 7.49–7.46
(m, 1H), 7.01–7.00 (m, 2H), 6.89 (s, 1H, H-5), 5.20 (d, J = 5.6, 2H),
3.79 (s, 3H). 13C NMR (100 MHz, DMSO-d6), d: 159.1, 155.8, 151.8
(2C, overlap), 135.7, 134.2, 133.8, 131.5, 128.9, 127.8, 126.6,
126.4 (2C), 126.2, 125.9, 125.7, 124.9, 124.0, 114.9 (2C), 104.4,
95.0, 55.6, 41.9. IR (neat, cmꢃ1): 3152, 1597,1254,769. HRMS (EI):
380.1632 (calcd C24H20N4O, 380.1632, M+).
HPLC). 1H NMR (400 MHz, DMSO-d6) d: 12.07 (s, 1H, NH, H-7),
8.26–8.24 (m, 1H), 8.08 (s, 1H, H-2), 7.99–7.93 (m, 2H), 7.82–
7.80 (m, 3H), 7.67–7.65 (m, 1H), 7.58–7.45 (m, 3H), 7.31–7.27
(m, 2H), 7.09 (s, 1H, H-5), 6.31–6.25 (m, 1H), 1.68 (d, J = 6.8, 3H).
13C NMR (100 MHz, DMSO-d6), d: 161.9 (d, J = 244.5), 155.3,
152.3 (2C), 152.0, 141.3, 139.9, 133.0, 131.1, 129.1, 128.9 (d,
J = 2.9), 127.6, 126.9 (d, J = 7.9), 126.6, 126.0 (d, J = 3.5, 2C), 123.8,
122.7, 116.4 (d, J = 21.7, 2C), 104.4, 96.6, 60.2, 22.2. 19F NMR
(564 MHz, DMSO-d6, C6F6) d: ꢃ117.1 (m). IR (neat, cmꢃ1): 2985,
1585, 1496, 1312, 1233, 834, 774. HRMS (EI): 382.1585 (calcd
C
24H19FN4, 382.1586, M+).
2.5.1.9. (S)-6-(4-Fluorophenyl)-N-(1-(naphthalen-1-yl)ethyl)-7H-pyr-
rolo[2,3-d]pyrimidin-4-amine ((S)-22p). The compound was
prepared as described in Section 2.5.1 starting with 4-chloro-6-
(4-fluorophenyl)-7H-pyrrolo[2,3-d]pyrimidine
(16)
(66 mg,
2.5.1.5. (R)-6-(4-Methoxyphenyl)-N-(1-(naphthalen-1-yl)ethyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine ((R)-20p). The compound was
prepared as described in Section 2.5.1 starting with 4-chloro-6-
(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (14) (144 mg,
0.27 mmol) and (S)-1-phenylethanamine (19p) (137 mg,
0.80 mmol). This gave 76 mg (0.20 mmol, 74%) of a white solid,
mp. 149–152 °C, ½a D20
ꢂ
= +380 (c 0.13, DMSO), purity: 98% (by HPLC).
The spectroscopic properties corresponded with that reported for
0.55 mmol)
and
(R)-1-(naphthalen-1-yl)ethanamine
(19p)
(R)-22p in Section 2.5.1.8.

38
S.J. Kaspersen et al. / Bioorganic Chemistry 44 (2012) 35–41
2.5.2. General procedure demethylation of 20–25
dence with that reported previously for the racemate [37]. 1H
NMR (400 MHz, DMSO-d6) d: 12.98 (s, 1H, NH, H-7), 9.82 (br s,
1H, OH), 9.55 (br s, 1H, NH), 8.30 (s, 1H), 8.14 (s, 1H, H-2), 8.00
(m, 1H), 7.91 (m, 1H), 7.64–7.57 (m, 5H), 7.54–7.50 (m, 1H), 7.26
(s, 1H, H-5), 6.89–6.87 (m, 2H), 6.09 (m, 1H), 1.78 (d, J = 6.5, 3H).
HRMS (EI): 380.1635 (calcd C24H20N4O, 380,1637, M+).
The following is representative: (S)-6-(4-methoxyphenyl)-N-(1-
phenylethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (20i) was dis-
solved in dry CH2Cl2 (2 mL) under argon atmosphere. BBr3
(0.17 ml, ꢁ1.8 mmol) in dry CH2Cl2 (1.5 mL) was added drop wise
over 1 h. at 0 °C using a syringe pump. Then the mixture was al-
lowed to react at 20 °C for 24 h. The reaction was quenched by
addition of water (10 mL), and the mixture was extracted with
EtOAc (3 ꢀ 25 mL). The combined organic phase was washed with
brine (15 mL), dried over MgSO4 and concentrated. The resulting
residue was purified by precipitation from acetone (0.5 mL). The
solid formed was isolated by filtration, washed with diethyl ether
(10 mL) and dried.
2.5.2.5.
(S)-4-(4-(1-(Naphthalen-1-yl)ethylamino)-7H-pyrrolo[2,3-
d]pyrimidin-6-yl)phenol hydrobromide ((S)-25p). The compound
was synthesised as described in Section 2.5.2 starting (S)-6-(4-
methoxyphenyl)-N-(1-(naphthalen-1-yl)ethyl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine ((S)-20p) (73 mg, 0.19 mmol). This gave
48 mg (0.13 mmol, 68%) of a white solid, mp > 300 °C. ½a D20
ꢂ
= +389
(c 0.16, DMSO) purity > 99% (by HPLC). Spectroscopic properties
were in correspondence with that reported previously for the race-
mate [37], and that described for (R)-25p in Section 2.5.2.4.
2.5.2.1. (S)-4-(4-(1-Phenylethylamino)-7H-pyrrolo[2,3-d]pyrimidin-
6-yl)phenol hydrobromide ((S)-25e). The compound was
synthesised as described in Section 2.5.2 starting from (S)-
6-(4-methoxyphenyl)-N-(1-phenylethyl)-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine (20e) (99 mg, 0.29 mmol). This gave
3. Result and discussion
56 mg, (0.14 mmol, 47%) of
a white solid, mp > 300 °C,
a 2D0
= +289 (c 0.17, DMSO). purity > 98% (by HPLC) Spectro-
3.1. Synthesis
½ ꢂ
scopic properties were in correspondence with that reported
previously for the (R)-enantiomer [37]. 1H NMR (400 MHz,
DMSO-d6) d: 12.98 (s, 1H, NH, H-7), 9.83 (br s, 1H, OH), 9.55
(br s, 1H, NH), 8.30 (s, 1H, H-2), 7.66 (m, 2H), 7.50–7.48 (m,
2H), 7.39 (m, 2H), 7.32–7.29 (m, 1H), 7.24 (s, 1H, H-5), 6.89
(m, 2H), 5.37 (m, 1H), 1.66 (d, J = 6.5, 3H). HRMS (EI):
330.1475 (calcd C20H18N4O, 330.1475, M+).
The pyrrolopyrimidines were synthesised as described previ-
ously, Scheme 1 [37,41,42].
The first step forming the pyrroles 1–5 gave mediocre yields.
We found that formation of 6–8 was the main reason for the loss
in yield. The alcohol 6 may be caused by water generated in the
pyrrole cyclisation, whereas the esters 7 and 8 most likely originate
from fragmentation of the unstable aminoimidate. An increase in
yield of 1–5 was seen when using >2 equivalents of the aminoim-
idate and 3 equivalents of sodium ethoxide. It has previously been
suggested that such pyrroles are UV labile [42]. Discolouration of
the products was seen on storage in DMSO for 1 day at room tem-
perature. Cyclisation of 1–5 using formamide gave the 4-hydroxy-
pyrrolopyrimidines 9–13 which all were crystalline and easily
isolated. Standard chlorination gave 14–18, which also were con-
veniently isolated and purified if full conversion was obtained in
the reaction. In the next step, thermal nucleophilic aromatic
substitution on 14–18 was performed using various amines.
Compound 14 was reacted with 19a–q giving 20a–q, while the
4-chloropyrrolopyrimidines 15–18 were mainly substituted with
19e–f and 19p, giving the corresponding 4-amino derivatives
21–24. Deprotection of the methoxy derivatives 20 with boron
tribromide gave the phenolic compounds 25. Twelve of the
compounds reported in this study are new chemical entities.
2.5.2.2.
(S)-4-(4-(1-(4-bromophenyl)ethylamino)-7H-pyrrolo[2,3-
d]pyrimidin-6-yl)phenol hydrobromide ((S)-25i). The compound
was synthesised as described in Section 2.5.2 starting from (S)-N-
(1-(4-bromophenyl)ethyl)-6-(4-methoxyphenyl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine (20i) (134 mg, 0.32 mol). This gave 83 mg
(0.17 mmol, 52%) of a white solid, mp. 274–276 °C, ½a D20
ꢂ
= +272 (c
0.28, DMSO), purity > 97% (by HPLC). Spectroscopic properties
were in correspondence with that reported previously for the
(R)-enantiomer [37]. 1H NMR (400 MHz, DMSO-d6) d: 12.94 (s,
1H, NH, H-7), 9.82 (br s, 1H, OH), 9.47 (br s, 1H, NH), 8.29 (s, 1H,
H-2), 7.65 (m, 2H), 7.58 (m, 2H), 7.44 (m, 2H), 7.16 (s, 1H, H-5),
6.88 (m, 2H), 5.37 (m, 1H), 1.63 (d, J = 6.6, 3H. HRMS (EI):
408.0581 (calcd C20H17Br79N4O, 408.0580, M+).
2.5.2.3. 4-(4-(Naphthalen-1-ylmethylamino)-7H-pyrrolo[2,3-d]pyr-
imidin-6-yl)phenol hydrobromide (25o). The compound was
synthesised as described in Section 2.5.2 starting from 6-(4-
methoxyphenyl)-N-(naphthalen-1-ylmethyl)-7H-pyrrolo[2,3-d]-
pyrimidin-4-amine (20o) (117 mg, 0.31 mmol) and BBr3 (0.29 mL,
3.1 mmol). This gave 74 mg (0.20 mmol, 66%) of a white solid,
mp > 300 °C. purity > 98% (by HPLC). 1H NMR (400 MHz, DMSO-
d6) d: 13.01 (s, 1H, H-7), 9.82 (s, 1H, OH), 8.36 (s, 1H, H-2), 8.15–
8.13 (m, 1H), 8.04–8.02 (m, 1H), 7.97–7.95 (m, 1H), 7.66–7.60
(m, 4H), 7.54–7.7.50 (m, 2H), 7.17 (s, 1H, H-5), 6.87–6.89 (m,
2H), 5.26 (bs, 2H). 13C NMR (100 MHz, DMSO-d6) d: 158.5, 149.9,
148.6, 142.8, 138.1, 133.9, 132.1, 131.3, 129.1, 128.9, 127.2 (2C),
127.1, 126.7, 126.1, 125.4, 124.1, 121.7, 116.4 (2C), 103.5, 96.8,
43.9. IR (neat, cmꢃ1): 3123, 1643, 1612, 1493, 1178, 757. HRMS
(EI): 366.1471 (calcd C23H18N4O, 366.1475, M+).
3.2. Toxicity towards Tetrahymena
The Tetrahymena strain used in this study was originally iso-
lated from pond water in Norway.
It was observed that this isolate grew faster and was more vig-
orous than several of our culture collection Tetrahymena strains.
These observations were considered important when choosing it
as a test strain for the present study. Benzylalkonium chloride
was used as a control in the testing showing a MPC value of
8 lg/mL. We first investigated the effect of compounds having ben-
zylamines, chiral 1-phenylethanamines and 1-naphthylethanam-
ines as substituents in Fragment B (see Table 1), and methoxy,
hydrogen, fluoro, bromo and cyano as R in Fragment A.
2.5.2.4.
(R)-4-(4-(1-(naphthalen-1-yl)ethylamino)-7H-pyrrolo[2,3-
Most of the compounds were synthesised as their (R)-enantio-
mers, but the (S)-enantiomers of the methoxy substituted com-
pound 20e, and 20p, 21p and 22p having a 1-naphthylethanamine
substituent at C-4 were included to investigate the importance of
stereochemistry. Testing revealed that the (R)-naphthyl derivative
20p had an MPC-value of 32 (Table 1, entry 7), while other deriva-
tives with R = OMe, H, F, Br and CN did not possess protozoacidal
d]pyrimidin-6-yl)phenol ((R)-25p). The compound was synthesised
as described in Section 2.5.2 starting (R)-6-(4-methoxyphenyl)-N-
(1-(naphthalen-1-yl)ethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
((R)-20p) (113 mg, 0.29 mmol). This gave 58 mg (0.15 mmol, 53%)
of a white solid, mp > 300 °C. ½a D20
= ꢃ379 (c 0.26, DMSO) pur-
ꢂ
ity > 98% (by HPLC). Spectroscopic properties were in correspon-

S.J. Kaspersen et al. / Bioorganic Chemistry 44 (2012) 35–41
39
Scheme 1. Synthesis of 20–25 using the amines 19a-q.
Table 1
Activity of the pyrrolopyrimidines 20–24 and 25o-p towards Tetrahymena.
Entry
Substance
R
R1
Ar/R2
MPC l
g/mL (48 h)a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
20a
20b
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
F
F
F
F
F
Br
Br
CN
CN
OH
OH
OH
OH
H
H
H
p-F
H
>64
>64
>64
>64
>64
>64
32b
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
(R)-20e
(S)-20e
(R)-20h
20o
(R)-20p
(S)-20p
(R)-20r
21c
CH3
CH3
CH3
H
CH3
CH3
Et
H
p-MeO
C10H7
C10H7
C10H7
H
m-F
H
H
21e
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
(R)-21f
(R)-21p
(S)-21p
22c
(R)-22e
(R)-22f
(R)-22p
(S)-22p
(R)-23e
(R)-23f
(R)-24e
(R)-24f
25o
p-F
C10H7
C10H7
m-F
H
p-F
C10H7
C10H7
H
p-F
H
p-F
C
10H7
(rac)-25p
(R)-25p
(S)-25p
CH3
CH3
CH3
C10H7
C10H7
C10H7
64
32a
64
a
The MPC values were determined by averaging three parallel measurements.
MPC = 32 also after 7 days.
b
activity with respect to Tetrahymena in the lower
tion range.
Solubility challenges were encountered when some of these
compounds were tested, the investigation was continued with
l
g/mL concentra-
the naphthylic compounds 25o-p which had a hydrophilic pheno-
lic group in fragment A, (Table 1, entries 24–27). However, no dras-
tic improvement in potency was observed. The (R)-enantiomer of
25p (Table 1, entry 26) was the most potent and gave a similar

40
S.J. Kaspersen et al. / Bioorganic Chemistry 44 (2012) 35–41
Table 2
the concentration range tested (Table 2, entries 1–4). However,
to our satisfaction (R)-25e, PKI-166 [43], having a para-hydroxy-
Activity of compounds 25 towards Tetrahymena.
phenyl at C-6 and a 1-phenylethanamine substituent as C-4,
proved to be potent (MPC: 8–16 lg/mL Entry 5), indicating the
importance of a chiral centre. Keeping the chiral R1 group as
methyl, and introducing rather conservative variations in the para
position in terms of size and electronic properties, gave an
MPC = 32
analogue 25g was inactive. By changing the para substituent to
bromo, (R)-25i, a MPC value of 8 g/mL was obtained. Compared
lg/mL for the fluoro derivative 25f, while the methyl
l
to the activity of the fluoro containing compound 25f this indicates
that a combination of both increased size and polarisability might
be beneficial for achieving good potency. Also, the racemic trifluo-
romethyl derivative (rac)-25j showed activity (MPC = 32 lg/mL).
Ortho and meta substitution by fluoro or a methyl substituent low-
ered the toxicity, but the meta-fluoro derivative 25m showed
Entry
Substance
R
R2
MPC
l
g/ml
MPC lg/ml
(48 h)a
(7 days)a
appreciable protozoacidal activity (MPC: 16 lg/mL, entry 13). It
was further investigated how the potency was affected by extend-
ing the chain length of R1, but the result for 25q containing a 1-
phenylpropanamine substituent at C-4 (MPC = 32 lg/mL, entry
14) did not encourage further evaluation. To verify the importance
of stereochemistry for the toxicity profile of these compounds we
also synthesised and analysed for the effect of the (S)-enantiomers
of the 1-phenylethanamine containing 25e and its para-bromo
substituted derivative 25i. Both were found to be highly potent
(Table 2, entries 15–16).
Some of the compounds evaluated in this study are efficient
inhibitors of EGFR-TK in vitro [37]. These kinases depends on acti-
vation from the epidermal growth factor (EGF), which is a known
signalling polypeptide in Tetrahymena [44]. However, we did not
find any correlation between the in vitro activity towards EGFR-
TK and the MPC values. This might indicate that receptors found
in Tetrahymena are structurally different to the human version.
To investigate if other kinases might be the target, the para-
hydroxyphenyl derivative 25e was evaluated against a panel of
124 kinases. Low inhibitory potency was observed in most cases
(data not shown). Kinases which were inhibited to a degree of
25% or more at 50 nM are compiled in Fig. 2. Of these kinases, pro-
tein kinase B [45–47], calmodulin dependant enzymes [48,49], pro-
tein kinase C [9,50], and ERK1 type proteins [51] are found in
protozoa and represent possible sites of action for the
pyrrolopyrimidines.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
25a
25b
25c
25d
H
H
H
H
H
p-F
m-F
o-F
H
>64
>64
>64
>64
8/16b
32
>64
8/8b
32
64
64
16
32
>64
>64
>64
>64
16/16b
32
>64
8/8b
32
(R)-25e
(R)-25f
(R)-25g
(R)-25i
(rac)-25j
(R)-25k
(Rac)-25l
(R)-25m
(Rac)-25n
(R)-25q
(S)-25e
(S)-25i
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Et
p-F
p-CH3
p-Br
p-CF3
o-F
o-CH3
m-F
m-CH3
H
>64
>64
16
32
32
32
CH3
CH3
H
Br
8/16b
8
8/16b
16
a
The MPC values were determined by averaging three parallel measurements.
Values given represent a second triplicate ground of testing.
b
MPC value to that of the methoxy derivative (R)-20p (Table 1, entry
7).
Then we decided to evaluate the effect of the substitution pat-
tern in fragment B by varying R1 (hydrogen, methyl and ethyl)
and including mono ortho, meta or para R2-groups, while keeping
the phenolic unit in fragment A. The compounds tested and their
potencies towards Tetrahymena are compiled in Table 2.
The unsubstituted benzylamine derivative 25a and three fluoro
substituted benzylamine derivatives, 25b-d, were all inactive in
80
70
60
50
40
30
20
10
0
TrkA
IKKe
JNK2
YES1
PKBb
PKCa
ERK1
MINK1
CAMK1
VEGFR
MAPKAP-K3
Fig. 2. Effect of 25e (50 nM) on the activity of a selection of kinases. PKC
Neurotrophic tyrosine kinase receptor type 1, ERK1: extracellular-signal-regulated kinase; IKKe: inhibitory
a
: Protein kinase C alpha; JNK2: c-Jun N-terminal kinase/mitogen-activated protein kinase, TrkA:
B kinase; MAPKAP-K3: MAPK-activated protein kinase 3; CAMK
j
1: calmodulin-dependent kinase: MINK1: misshapen-like kinase 1 VEGFR: vascular endothelial growth factor receptor, Yes 1: Yamaguchi sarcoma viral oncogene homologue.
PKBb: protein kinase B.

S.J. Kaspersen et al. / Bioorganic Chemistry 44 (2012) 35–41
41
[8] M. Parsons, J.A. Ledbetter, G.L. Schieven, Inhibitors of tyrosine phosphorylation
in protozoa as drugs, EP 507256, 1992.
[9] F.J. Gamo, L.M. Sanz, J. Vidal, C. de Cozar, E. Alvarez, J.L. Lavandera, D.E.
Vanderwall, D.V.S. Green, V. Kumar, S. Hasan, J.R. Brown, C.E. Peishoff, L.R.
Cardon, J.F. Garcia-Bustos, Nature 465 (2010) 305–310.
[10] R. Diaz-Gonzalez, F.M. Kuhlmann, C. Galan-Rodriguez, L. Madeira da Silva, M.
Saldivia, C.E. Karver, A. Rodriguez, S.M. Beverley, M. Navarro, M.P. Pollastri,
PLoS Neglected Trop. Dis. 5 (2011) e1297.
[11] K. Mensa-Wilmot, Tyrosine kinase inhibitors as anti-kinetolastid and anti-
apicomplexan protozoal agents, WO 2008066755, 2007.
[12] M.P. Sauvant, D. Pepin, E. Piccinni, Chemosphere 38 (1999) 1631–1669.
[13] M. Hewitt, M.T.D. Cronin, P.H. Rowe, T.W. Schultz, SAR QSAR Environ. Res. 22
(2011) 621–637.
[14] T.W. Schultz, C.L. Sparfkin, A.O. Aptula, SAR QSAR Environ. Res. 21 (2010) 681–
691.
[15] S.J. Enoch, M.T.D. Cronin, T.W. Schultz, J.C. Madden, Chemosphere 71 (2008)
1225–1232.
[16] F. Bajot, M.T.D. Cronin, D.W. Roberts, T.W. Schultz, SAR QSAR Environ. Res. 22
(2011) 51–65.
[17] T.I. Netzeva, T.W. Schultz, Chemosphere 61 (2005) 1632–1643.
[18] A.O. Aptula, D.W. Roberts, M.T.D. Cronin, T.W. Schultz, Chem. Res. Toxicol. 18
(2005) 844–854.
[19] T.W. Schultz, G.D. Sinks, M.T.D. Cronin, Aquat. Toxicol. 39 (1997) 267–278.
[20] M. Koubar, M.H. Rodier, R.A. Garduno, J. Frere, FEMS Microbiol. Lett. 325
(2011) 10–15.
[21] W.K. Whitekettle, Control of protozoa and protozoan cysts that harbor
Legionella using quaternary ammonium salts, US 20050027010, 2005.
[22] P.J. Jakobsen, O. Enger, Treatment of parasite diseases using vitamin K3, WO
2009063044, 2009.
[23] M.P. Leibowitz, J.K. Chettri, R. Ofir, D. Zilberg, J. Fish Dis. 33 (2010) 473–480.
[24] Y. Muto, Y. Tanabe, K. Kawai, Y. Okano, H. Iio, Cent. Eur. J. Biol. 6 (2011) 99–
104.
[25] H. Hegyesi, P. Kovacs, G. Csaba, Acta Microbiol. Hung. 39 (1992) 289–293.
[26] J.R. Nilsson, Protoplasma 135 (1986) 1–11.
The toxicity/potency of the compounds presented in this study
against Tetrahymena crucially depended on the presence of a para
phenolic group in fragment A. This might be due to better solubil-
ity and bioavailability than for 20–24, or that the hydroxyl function
engages in critical bonding interaction. In Tetrahymena pyriformis
there is evidence for a transmembrane efflux pump as a detoxifica-
tion mechanism, preferably excluding lipophilic compounds [52].
This might be an alternative explanation for the observed toxicity
differences seen on introduction of the 4-hydroxyl group in frag-
ment A. As Tetrahymena has only limited CYP-450 dependent
metabolism activity [53], it is less likely that the toxicity is due
to oxidation of the para-hydroxyphenyl unit of 25 leading to aro-
matic 1,2-dienones, which are typical Michael acceptors in reac-
tion with bio macromolecules [16,18]. The low level of CYP-450
enzymes should also exclude the possible formation of active com-
pounds from putative precursors such as the C-6 phenyl substi-
tuted derivative 21. Furthermore, the MPC values were affected
by the substitution pattern and the presence of a chiral centre in
fragment B. All the above indicates that there is a specific mode
of action involving a defined 3-dimentional receptor target. How-
ever, the fact that both enantiomers showed toxicity is suggesting
that the pyrrolopyrimidines could have multiple modes of action,
or that the target is rather flexible.
As a model for unspecific toxicity the study shows that intro-
duction of a 4-hydroxyl group in fragment A could be problematic
for EGFR-TK inhibitors, but also that the toxicity profile could be
modulated by the substitution pattern both in fragment A and B.
Human toxicity issues have in fact been seen for 25e [54].
[27] C. Wu, P. Clift, C.H. Fry, J.A. Henry, Arch. Toxicol. 70 (1996) 850–853.
[28] A. Kaczanowski, M. Ramel, J. Kaczanowska, D. Wheatley, Exp. Cell Res. 195
(1991) 330–337.
[29] G. Csaba, P. Kovacs, E. Pallinger, Cell Biol. Int. 28 (2004) 491–496.
[30] L. Chiu, Pharmaceutical compositions containing protein kinase B inhibitors
and epidermal growth factor receptor tyrosine kinase inhibitors for treating
cancer, CN 101653606, 2008.
4. Conclusion
[31] F. Chen, V. Leick, J. Microbiol. Methods 59 (2004) 233–241.
[32] P. Kovacs, G. Csaba, Cell Biochem. Funct. 10 (1992) 267–271.
[33] F. Hans, S. Dimitrov, Oncogene 20 (2001) 3021–3027.
[34] W. Li, S. Zhang, O. Numata, Y. Nozawa, S. Wang, Cell Biochem. Funct. 27 (2009)
364–369.
[35] S. Nakashima, S. Wang, N. Hisamoto, H. Sakai, M. Andoh, K. Matsumoto, Y.
Nozawa, J. Biol. Chem. 274 (1999) 9976–9983.
[36] J. Bartholomew, J. Reichart, R. Mundy, J. Recktenwald, S. Keyser, M. Riddle, H.
Kuruvilla, Purinergic Signalling 4 (2008) 171–181.
[37] S.J. Kaspersen, C. Sørum, V. Willassen, E. Fuglseth, E. Kjøbli, G. Bjørkøy, E.
Sundby, B.H. Hoff, Eur. J. Med. Chem. 46 (2011) 6002–6014.
[38] E. Otterholt, C. Charnock, Water Res. 45 (2011) 2527–2538.
[39] J. Bain, L. Plater, M. Elliott, N. Shpiro, C.J. Hastie, H. McLauchlan, I. Klevernic, J.S.
Arthur, D.R. Alessi, P. Cohen, Biochem. J. 408 (2007) 297–315.
[40] C. Sørum, N. Simic, E. Sundby, B.H. Hoff, Magn. Reson. Chem. 48 (2010) 244–
248.
[41] X. Cai, C. Qian, S. Gould, Fused bicyclic pyrimidines as PTK inhibitors
containing a zinc binding moiety, WO 2008033745, 2007.
[42] E. Toja, A. DePaoli, G. Tuan, J. Kettenring, Synthesis (1987) 272–274.
[43] G. Caravatti, J. Bruggen, E. Buchdunger, R. Cozens, P. Furet, N. Lydon, T. O’Reilly,
P. Traxler, ACS Symposium Series 796 (2001) 231–244.
A series of pyrrolopyrimidines have been tested for their proto-
zoacidal activity against Tetrahymena. Five compounds were found
to be highly active (MPC 8–16 lg/mL). The identified compounds
do not contain the typical groups which trigger non-specific toxic-
ity effects. The presence of a para phenolic group in position 6
(fragment A), and a chiral centre in the 4-benzylamine (fragment
B) enhanced the potency considerably. Ortho-substitution in frag-
ment B, and an electron donating methyl group in the para position
reduced the toxicity. There is no evidence that EGFR-TK kinases are
targets for these compounds in Tetrahymena, however, kinase pro-
filing identified other potential sites of action. The detailed mech-
anism will be investigated in continuing work. The presented
structure–activity relationships could be used as guidelines for tar-
geting other, medically more important protozoa.
Acknowledgments
[44] I.V. Schemarova, G.V. Selivanova, T.D. Vlasova, Tsitologiya 49 (2007) 156–160.
[45] A. Vaid, P. Sharma, J. Biol. Chem. 281 (2006) 27126–27133.
[46] J. Guergnon, F. Dessauge, F. Traincard, X. Cayla, A. Rebollo, P.E. Bost, G.
Langsley, A. Garcia, Apoptosis 11 (2006) 1263–1273.
[47] V. Pascuccelli, C. Labriola, M.T. Tellez-Ino, A.J. Parodi, Mol. Biochem. Parasitol.
102 (1999) 21–33.
The group is thankful to the Anders Jahres foundation for finan-
cial support. Roger Aarvik is thanked for technical support. Susana
Villa Gonzalez is acknowledged for HRMS experiments.
[48] K. Gonda, M. Katoh, K. Hanyu, Y. Watanabe, O. Numata, J. Cell Sci. 112 (1999)
3619–3626.
References
[49] J. Hirano-Ohnishi, Y. Watanabe, J. Biochem. 105 (1989) 858–860.
[50] D.L. Hassenzahl, N.K. Yorgey, M.D. Keedy, A.R. Price, J.A. Hall, C.C. Myzcka, H.G.
Kuruvilla, J. Comp. Physiol. A. 187 (2001) 171–176.
[1] M.J.G. Farthing, Nat. Clin. Pract. Gastroenterol. Hepatol. 3 (2006) 436–445.
[2] H.B. Fung, T.L. Doan, Clin. Ther. 27 (2005) 1859–1884.
[3] J.A. Castro, M. Montalto de Mecca, L.C. Bartel, Hum. Exp. Toxicol. 25 (2006)
471–479.
[4] H. Cerecetto, M. Gonzalez, Pharmaceuticals 3 (2010) 810–838.
[5] A. Klokouzas, S. Shahi, S.B. Hladky, M.A. Barrand, H.W. van Veen, Int. J.
Antimicrob. Agents 22 (2003) 301–317.
[51] M. Arslanyolu, J. Appl. Biol. Sci. 1 (2007) 1–11.
[52] M. Bamdad, P. Brousseau, F. Denizeau, FEBS Lett. 456 (1999) 389–393.
[53] H. Iida, J. Kimura, J.J. Johnson, L.J. Marnett, Comp. Biochem. Physiol. C 63C
(1979) 381–387.
[54] T. Takada, H.M. Weiss, O. Kretz, G. Gross, Y. Sugiyama, Drug Metab. Dispos. 32
(2004) 1272–1278.
[6] S.M. Townson, P.F.L. Boreham, P. Upcroft, J.A. Upcroft, Acta Trop. 56 (1994)
173–194.
[7] M. Matsushita, K.D. Janda, Bioorg. Med. Chem. 10 (2002) 855–867.