Selective synthesis of fluorinated benzyl- or phenylpyrimidines from the heterocyclisation of α-trifluoroacetylarylpropanenitriles

Article abstract of DOI:10.1016/j.jfluchem.2007.05.014

Some 5-benzyl-6-trifluoromethyl-2,4-diaminopyrimidines analogous to trimethoprim and 6-aryl-5-trifluoroethyl-2,4-diaminopyrimidines analogous to pyrimethamine were prepared from the same synthons, trifluoromethylated β-ketonitriles. The heterocyclisation

Full text of DOI:10.1016/j.jfluchem.2007.05.014

Journal of Fluorine Chemistry 128 (2007) 1039–1045
www.elsevier.com/locate/fluor
Selective synthesis of fluorinated benzyl- or phenylpyrimidines from the
heterocyclisation of a-trifluoroacetylarylpropanenitriles
a
b
Hatice Berber a, , Catherine Mirand , Francis Derouin
*
a
´ ´
FRE 2715/CNRS, IFR 53, Universite de Reims Champagne-Ardenne, Faculte de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France
b
´ ˆ ´
Laboratoire de Parasitologie-Mycologie (EA 3520), Universite Denis Diderot, Hopital Saint-Louis, AP-HP, Faculte de Medecine,
´
´
15 rue de l’Ecole de Medecine, 75006 Paris, France
Received 25 January 2007; received in revised form 12 May 2007; accepted 15 May 2007
Available online 18 May 2007
Abstract
Some 5-benzyl-6-trifluoromethyl-2,4-diaminopyrimidines analogous to trimethoprim and 6-aryl-5-trifluoroethyl-2,4-diaminopyrimidines
analogous to pyrimethamine were prepared from the same synthons, trifluoromethylated b-ketonitriles. The heterocyclisation between enol
ether of b-ketonitrile and guanidine leading to these compounds was studied.
These fluorinated novel compounds were tested for their in vitro activity against Toxoplasma gondii, a widespread apicomplexan protozoa
responsible for congenital toxoplasmosis and cerebral toxoplasmosis in immunocompromised patients.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Diaminopyrimidine; Solvent and fluorine effects on heterocyclisation; Benzylpyrimidine; Phenylpyrimidine; Toxoplasmosis
1. Introduction (Fig. 1)
Furthermore, the introduction of fluorine significantly alters
the physico-chemical properties of a molecule (due in part to its
large electronegativity) and thus can have surprising effects on
chemical reactivity [5,6].
A surprisingly vast number of fluorine-containing molecules
are marketed as drugs or are in clinical trials [1,2] considering
organo-fluorine compounds are very rare in Nature, the
traditional source of bioactives (only 13 secondary metabolites
identified) [3]. The special properties of the fluorine atom can
have considerable impacts on the behaviour of a molecule in a
biological environment [4–6]. The various uses of fluorine to
increase metabolic stability and lipophilicity are examples of
directed strategies of fluorine substitution [1–6].
In a previous paper concerning the preparation of fluori-
nated aminopyrimidines analogous to trimethoprim, we have
described an unexpected synthesis of 5-trifluoroethyl-2,4-
diaminopyrimidines from the heterocyclisation of a-trifluoro-
acetylpropanenitriles [8d]. These compounds can be considered
as inverted (5,6-substituents) pyrimethamine derivatives.
Diaminopyrimidines trimethoprim (tmp) and pyrimetha-
mine (pyr) are the reference drugs used for the treatment of
opportunistic infections due to Toxoplasma gondii.
These compounds result from the impressive development
of synthetic methodologies in organic fluorine chemistry and
efforts in this way are in constant progress [6,7]. Two general
strategies are employed: the direct fluorination method [7a] and
the building-block method [7a,b].
T. gondii is an apicomplexan protozoa, which is character-
ized by a bifunctional enzyme, called dihydrofolate reductase-
thymidylate synthase (DHFR-TS) which plays a crucial role
in pyrimidine biosynthesis [9]. Recently, more attention has
been given to T. gondii as a frequent cause of toxoplasmic
encephalitis in immunocompromized patients with AIDS or in
immunosuppressed organ transplant recipients [9].
Along the second route, we have developed syntheses
of trifluoromethylated b-enaminoketones and demonstrated
their usefulness in the preparation of various CF3 nitrogen
heterocycles [8].
These DHFR inhibitors (tmp and pyr) are always used in
combination for synergistic effect with sulfonamides such as
sulfadiazine (with pyr) that is known to induce severe allergic
reactions in many patients [9a]. Despite the continuing success
* Corresponding author. Tel.: +33 326 913733; fax: +33 326 918029.
E-mail address: hatice.berber@univ-reims.fr (H. Berber).
0022-1139/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2007.05.014

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H. Berber et al. / Journal of Fluorine Chemistry 128 (2007) 1039–1045
1a,b were converted into enol ethers 2a,b (78 and 88%,
respectively) as Z,E isomeric mixtures by reaction with triethyl
orthoformate. The heterocyclisation with guanidine as a free
base in acetonitrile at reflux gave 3a,b (91 and 66%,
respectively).
2.2. Synthesis of 6-aryl-5-trifluoroethylpyrimidines
analogous to pyr (Scheme 1)
Fig. 1. Structure of trimethoprim (tmp) and pyrimethamine (pyr).
When methanol was used instead of acetonitrile as the
solvent of the reaction, phenylpyrimidines 5c,d were isolated
with good yields (51 and 63%, respectively) by procedure
described previously for 5a,b [8d].
In our previous paper, a mechanism was also proposed for
this unexpected heterocyclisation after observing the tautomer-
ization of enol ether 2a to enenitrile 4a when 2a was treated
with guanidine hydrochloride in the presence of potassium
carbonate at reflux in acetonitrile [8d].
of these compounds, there is currently a need for more effective
derivatives for the treatment of toxoplasmosis as this drug
combination is frequently responsible for adverse side effects.
To complete this work, we now wish to report: (i) the
synthesis of novel fluorinated pyrimidines, 6-trifluoromethyl-
2,4-diaminopyrimidines analogous to tmp and 5-trifluoroethyl-
2,4-diaminopyrimidines analogous to pyr obtained from
heterocyclisation between the same trifluoromethylated syn-
thons and guanidine; (ii) the study of the effect of the
trifluoromethyl group on the heterocyclisation and the role
played by the solvent; (iii) the evaluation of anti-toxoplasma
activity of these compounds.
Indeed, tautomerization occurs in strong basic conditions
since guanidine acts as a base. This reaction is also very slow:
after 20 h at reflux 4a was obtained (42%) and 2a was recovered
(25%). Furthermore, in order to study the equilibrium
displacement of the tautomerization, isolated enenitrile 4a
was treated in the same conditions as above mentioned;
surprisingly, after 20 h at reflux, isomerization of 4a–2a did not
occur (4a was recovered). So the equilibrium is favourably but
slowly displaced from 2 toward 4 with these base-catalysed
conditions.
2. Results and discussion
For the synthesis of these 2,4-diaminopyrimidines we have
employed a general method that consists of condensing enol
ethers of b-ketonitriles with guanidine.
Thus, phenylpyrimidines no doubt originate from a 1,4
¨
2.1. Synthesis of 5-benzyl-6-trifluoromethylpyrimidines
analogous to tmp (Scheme 1)
Michael addition of guanidine on the tautomer 4, followed by
ethanol elimination, then cyclisation on the nitrile carbon [8d].
To confirm this, the isolated enenitrile 4a was treated with
guanidine ‘‘base’’ in methanol at reflux and phenylpyrimidine
5a was obtained (30%).
Benzylpyrimidines 3a,b were prepared from b-ketonitriles
1a,b isolated in their more stable hydrated forms. Compounds
Scheme 1.

H. Berber et al. / Journal of Fluorine Chemistry 128 (2007) 1039–1045
1041
Scheme 2.
2.3. Solvent effect on heterocyclisation and role of the CF3
group (Schemes 1–3)
nucleophilic substitution on a CF3-substituted carbon atom is
not an easy process. The difficulty is attributed to destabiliza-
tion of the transition state by fluorine and also to electrostatic
repulsion between a nucleophile and the lone pairs of electrons
of the fluorine atoms [6,7].
Depending on the solvent used, and starting from the same
trifluoromethylated synthon, benzylpyrimidine or phenylpyr-
imidine was obtained by condensation with guanidine.
In order to elucidate the mechanism and especially the role
played by the trifluoromethyl group, we achieved the reaction
in the nonfluorinated series by reacting enol ether 6 with
guanidine in methanol at reflux (Scheme 2). In this case, only
benzylpyrimidine 7 [10] was obtained with good yields (73%)
instead of phenylpyrimidine as observed in the fluorinated
series. It is also interesting to note that enol ether 6 was
only obtained by reaction between diazomethane (a strong
methylating agent) and the corresponding b-ketonitrile (the
attempt with triethyl orthoformate as in the fluorinated series
was unsuccessful). And so probably, tautomerization of enol
ether 6 to the corresponding enenitrile does not occur with these
basic conditions if we compare enolization in these two series.
Evidently, trifluoromethyl group has a great influence on
tautomerization and heterocyclisation when methanol, a polar
and protic solvent, was used.
Since no hydrogen bonding is possible between acetoni-
trile, a polar and aprotic solvent, and enenitrile 4a, the
heterocyclisation can only occur with enol ether 2a in a
¨
Michael addition/elimination mechanism. To check this, 4a
was treated with guanidine ‘‘base’’ in acetonitrile at reflux
(20 h) and neither benzylpyrimidine 3a nor phenylpyrimidine
5a was obtained, only 4a was recovered with 11% yields
(Scheme 1).
We have also noted that the reaction of guanidine with 2a in
dimethylformamide at room temperature gave no product and
we observed total destruction of the reagent 2a.
According to these results, we can conclude that the
tautomerization equilibrium is very slow and displaced from 2
toward 4 in guanidine base-catalysed conditions. And when a
large excess of guanidine as a free base (4 equiv.) is used in
acetonitrile, isomerization occurs but slowly and, as 4 is not
activated in this solvent, guanidine acts also as a nucleophile
and prefers reacting with 2. Even though in methanol, 4 is
activated (by hydrogen bonding) and becomes more reactive
than 2 and so reacts with guanidine after base-catalysed
tautomerization.
This solvent is able to form hydrogen bonds with the oxygen
of the ethoxy group of 4a which induces ethanol elimination.
Thus two types of mechanism can be considered (Scheme 3):
formation of stable allyl/benzyl carbocations (the positive
charge is also delocalized around the benzene ring) or a
concerted allylic SN20 process. In the first case, a SN1
mechanism is possible because such cations (with a CF3 group
placed on the a-position) can be generated, studied, and used in
synthetic applications [11].
2.4. Synthesis of the non fluorinated inverted pyr derivative
(Scheme 4)
Compound 11 was prepared in order to compare the
bioactivity of 5d with the non fluorinated derivative and thus to
evaluate the influence of fluorine on the bioactivity.
And in both cases, the cyclocondensation with guanidine
starts preferentially on the benzyl carbon (Scheme 3) because
Scheme 3.

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H. Berber et al. / Journal of Fluorine Chemistry 128 (2007) 1039–1045
Scheme 4.
The synthetic approach used to prepare 11 was adapted from
So the introduction of a trifluoromethyl group at the 6-
position of the pyrimidine ring, seems to decrease the
inhibitory activity while slightly enhancing the cytotoxicity.
The weak activity of the benzyl derivative 3a can also be
explained by the absence of substituents on the aromatic
group. Indeed, structure–activity relationships studies gen-
erally showed that the presence of one or more substituent(s)
on the benzyl group can play an important role in the activity
(by binding process) [14].
the method developed by Hitchings [12]. It was synthesized
from the corresponding 4-hydroxy derivative 9 which was
obtained by refluxing an ethanolic solution of guanidine
hydrochloride with keto ester 8 in the presence of potassium
carbonate (70%). The hydroxy aminopyrimidine 9 was then
converted into the chloro derivative 10 by treatment with
excess phosphoryl chloride under reflux conditions with low
yields (30%). The 4-chloroaminopyrimidine 10 was finally
reacted with saturated solution of ethanolic ammonia to give
diaminopyrimidine 11 (56%).
For compounds related to pyr (5c,d and 11), a marked
decrease of the inhibitory effect was noted, about 100-fold
lower compared to pyr; however estimated IC50 were close to
that of tmp. Moreover, these results show that there is no
influence of fluorine on the activity, when comparing IC50 of 5d
and 11. Compound 5c is the less active because of the ortho-
position of Cl on the aromatic ring. In this series, activities were
described only for molecules with substituent(s) in the meta or
para position [15].
2.5. Biological results: in vitro activity of tmp/pyr
derivatives on T. gondii (Table 1)
Anti-toxoplasma activities of derivatives 3a,b and 5c,d and
11 were assessed in vitro.
All these compounds were tested individually at 10
concentrations ranging from 0.01 to 40 mg Lꢀ1 (final
concentration in the culture). Each concentration was tested
in eight replicate wells and in two replicate culture plates. A
reference drug (i.e. pyrimethamine or trimethoprim) was tested
in each set of experiments.
3. Conclusion
Herein we have described a novel method which allowed the
preparation of fluorinated benzyl- or phenylpyrimidines from
the same reagents, only by varying the nature of the solvent.
The presence of the CF3 group induces this unusual reactivity,
which may be ascribed to the strong electron withdrawing
effect of this substituent.
In vitro, all the compounds started to show inhibitory effects
on Toxoplasma growth, at concentrations >1 mg Lꢀ1 for
compounds 5c, >2 mg Lꢀ1 for 3b and 5d and >5 mg Lꢀ1 for
3a and 11. A cytotoxic effect on the monolayers was recorded
for compounds 3b, 5d and 11 at concentrations ꢁ20 mg Lꢀ1
.
Concerning their bioactivity on T. gondii, they were found of
the same range of activity than the reference drug tmp, even if
less active than pyr. Moreover the most active pyr derivative 5d
(IC50 = 3.5 mg Lꢀ1) revealed to be two to threefold more
cytotoxic than pyr. Comparing 5d with 11, no influence of the
CF3 group on bioactivity could be highlighted.
Estimated IC50 by linear regression analysis ranged between
3.5 mg Lꢀ1 (5d) and 16 mg Lꢀ1 (5c) (Table 1). In comparison,
the IC50 of trimethoprim and pyrimethamine were estimated at
2.8 and 0.06 mg Lꢀ1, respectively, i.e. concentrations in
agreement with our previously reported results using the same
assay [13].
Surprisingly, the tmp analogue 3b revealed itself to be less
active than tmp and cytotoxic at concentrations >40 mg Lꢀ1
4. Experimental
.
4.1. General experimental techniques
Table 1
Melting points were taken on a Stuart Scientific melting
point apparatus (smp3) and were uncorrected. IR spectra were
Activities of diaminopyrimidines against T. gondii on MRC5 fibroblast tissue
cultures
1
Compound
T. gondii IC50 (mg Lꢀ1
)
Cytotoxicity (mg Lꢀ1
)
recorded on a BOMEM MB Series apparatus. H (300 MHz)
and 13C (75 MHz) NMR spectra were recorded on a Bruker AC
300 spectrometer. Chemical shifts are expressed in ppm from
TMS as internal reference. Mass spectra (EIMS and HRMS)
were recorded on a Micromass GCT spectrometer. Thin layer
chromatography (TLC) was carried out on aluminium-baked
Merck silica gel 60 F254. Column chromatography was
performed on silica gel.
3a
3b
5c
5d
11
pyr
tmp
15.4
9.7
16
100
40
100
40
3.5
5
20
0.06
2.8
>50
>100

H. Berber et al. / Journal of Fluorine Chemistry 128 (2007) 1039–1045
1043
4.2. General procedure for the preparation of 2a–d
120.0 (q, 1JC,F = 278.3 Hz, CF3), 129.1 (CH-ar), 129.2 (CH-ar),
129.9 (CH-ar), 130.0 (CH-ar), 133.6 (Cq-ar), 134.1 (Cq-ar),
153.0 (q, 2JC,F = 33.7 Hz, Cq-CF3). EIMS, m/z: 291 [M]+ (16),
289 [M]+ (100), 260 (16), 254 (16). HRMS, calcd for
C13H11F3NO35Cl: 289.0481. Found: 289.0482; and for
C13H11F3NO37Cl: 291.0452. Found: 291.0455.
A solution of 1a–d in HC(OEt)3 (3 mL/1.5 mmol of 1) was
refluxed (140 8C) and the reaction was monitored by TLC. The
excess of HC(OEt)3 was removed by distillation in vacuo and
2a–d as Z,E isomeric mixtures were obtained by purification on
column chromatography.
4.3. General procedure for the synthesis of 3a,b
4.2.1. 2-Benzyl-3-ethoxy-4,4,4-trifluorobut-2-enenitrile
(2a)
A solution of guanidine hydrochloride (4 equiv.) in anhydrous
methanol (0.3 mL mmolꢀ1 of guanidine hydrochloride) was
added to a stirred solution of sodium methoxide (4 equiv.)
prepared in situ in anhydrous methanol (0.3 mL mmolꢀ1 of
sodium methoxide) under an atmosphere of nitrogen at rt.
The mixture was stirred for 5 min, filtered, and MeOH was
evaporated. Guanidine (4 equiv.) as a free base was dissolved in
CH3CN (4 mL mmolꢀ1 of enol ether 2) and 2a,b was added. The
reactionmixturewasheatedtorefluxand wasmonitoredbyTLC.
Itwas then cooledat rt, concentrated invacuo and the residuewas
purified by column chromatography to yield compounds 3a,b.
Oil; 78%; IR (KBr); n 2992, 2223, 1638, 1331, 1186, 1140,
1008, 700 cmꢀ1; 1H NMR (CDCl3): d 1.39 (3H, m, CH3), 3.66
(2H, s, CH2), 4.10 and 4.25 (2H, q, J = 7.0 Hz, CH2O), 7.15–
7.39 (5H, m, H-ar); 13C NMR (CDCl3): d 15.1 and 15.2 (CH3),
33.7 and 34.5 (CH2), 70.5 and 71.8 (CH2O), 104.7 (Cq), 114.8
1
and 115.7 (CN), 119.2 (q, JC,F = 279.9 Hz, CF3), 127.5 and
127.6 (CH-ar), 128.5 and 128.6 (2ꢂ CH-ar), 128.9 (2ꢂ CH-ar),
135.2 and 135.5 (Cq-ar), 152.8 (q, 2JC,F = 33.3 Hz, Cq). EIMS,
m/z: 255 [M]+ (92), 226 (26), 158 (100), 130 (87). HRMS, calcd
for C13H12F3NO: 255.0871. Found: 255.0871.
4.2.2. 3-Ethoxy-4,4,4-trifluoro-2-(30,40,50-
trimethoxybenzyl)-but-2-enenitrile (2b)
Oil; 88%; IR (KBr); n 3000, 2946, 2215, 1638, 1593, 1510,
4.3.1. 5-Benzyl-2,4-diamino-6-(trifluoromethyl)pyrimidine
(3a)
Solid; 91%; mp 169–170 8C; IR (KBr); n 3500, 3328, 3192,
1645, 1565, 1466, 1451, 1420, 1177, 1130, 723 cmꢀ1; 1H NMR
(CD3OD): d 3.90 (2H, s, CH2), 7.00–7.30 (5H, m, H-ar); 13C
NMR (CD3OD): d 30.9 (CH2), 104.4 (C-5), 123.3 (q,
1JC,F = 276.5 Hz, CF3), 127.5 (CH-ar), 128.7 (2ꢂ CH-ar),
129.6 (2ꢂ CH-ar), 139.4 (Cq-ar), 153.1 (q, 2JC,F = 31.1 Hz, C-
6), 163.1 and 166.7 (C-2, C-4). EIMS, m/z: 268 [M]+ (100), 267
(46), 218 (29), 191 (25). HRMS, calcd for C12H11F3N4:
268.0936. Found: 268.0921.
1462, 1424, 1333, 1248, 1184, 1130, 1011, 847, 824, 716 cmꢀ1
;
1H NMR (CDCl3): d 1.42 (3H, t, J = 7.0 Hz, CH3), 3.61 (2H, s,
CH2), 3.86 (3H, s, CH3O), 3.87 (6H, s, 2ꢂ CH3O), 4.15 and
4.28 (2H, q, J = 7.0 Hz, CH2O), 6.46 (2H, s, H-ar); 13C NMR
(CDCl3): d 15.1 and 15.3 (CH3), 34.8 (CH2), 56.0 (2ꢂ CH3O),
60.8 (CH3O), 70.5 and 71.8 (CH2O), 104.6 (Cq), 105.5 and
105.6 (2ꢂ CH-ar), 114.9 and 115.8 (CN), 119.2 (q,
1JC,F = 279.7 Hz, CF3), 130.7 and 131.1 (Cq-ar), 137.4 (Cq-
2
ar), 152.7 (q, JC,F = 33.0 Hz, Cq-CF3), 153.4 (2 ꢂ Cq-ar).
EIMS, m/z: 346 [M + H]+ (18), 345 (100), 330 (22), 181 (36).
4.3.2. 2,4-Diamino-5-(30,40,50-trimethoxybenzyl)-6-
(trifluoromethyl)pyrimidine (3b)
4.2.3. 2-(20-Chlorobenzyl)-3-ethoxy-4,4,4-trifluorobut-2-
enenitrile (2c)
Solid; 66%; mp 235–237 8C; IR (KBr); n 3459, 3424, 3343,
3221, 1667, 1640, 1595, 1472, 1457, 1422, 1230, 1178, 1127,
1
Oil, 69%, IR (KBr); n 2989, 2225, 1636, 1475, 1446, 1332,
1184, 1141, 1010, 748 cmꢀ1; 1H NMR (CDCl3): d 1.40 (3H, m,
CH3), 3.83 (2H, s, CH2), 4.12 and 4.30 (2H, q, J = 7.1 Hz,
CH2O), 7.18–7.49 (4H, m, H-ar); 13C NMR (CDCl3): d 15.2 and
15.3 (CH3), 31.3 and 32.2 (CH2), 70.4 and 72.0 (CH2O), 102.7
725 cmꢀ1; H NMR (DMSO-d6): d 3.63 (3H, s, CH3O), 3.71
(6H, s, 2ꢂ CH3O), 3.79 (2H, s, CH2), 6.36 (2H, s, NH2), 6.44
(2H, s, H-ar), 6.58 (2H, br s, NH2); 13C NMR (DMSO-d6): d
29.7 (CH2), 56.0 (2ꢂ CH3O), 60.2 (CH3O), 102.0 (C-5), 105.2
1
(2ꢂ CH-ar), 122.5 (q, JC,F = 277.4 Hz, CF3), 134.9 (Cq-ar),
1
(Cq), 114.4 and 115.2 (CN), 119.1 (q, JC,F = 280.0 Hz, CF3),
127.3 (CH-ar), 129.1 (CH-ar), 129.8 (CH-ar), 130.6 (CH-ar),
136.0 (Cq-ar), 151.2 (q, 2JC,F = 30.3 Hz, C-6), 152.9 (2 ꢂ Cq-
ar), 161.8 and 165.0 (C-2, C-4). EIMS, m/z: 358 [M]+ (100), 343
(29), 327 (26), 311 (24), 219 (48), 69 (23). HRMS, calcd for
C15H17F3N4O3: 358.1253. Found: 358.1266.
2
133.2 (Cq-ar), 134.2 (Cq-ar), 153.5 (q, JC,F = 33.2 Hz, Cq-
CF3). EIMS, m/z: 291 [M]+ (4), 289 [M]+ (22), 226 (100), 218
(53), 131 (25), 125 (23). HRMS, calcd for C13H11F3NO35Cl:
289.0481. Found: 289.0488; and for C13H11F3NO37Cl:
291.0452. Found: 291.0466.
4.4. 3-Ethoxy-4,4,4-trifluoro-2-[1-phenyl-methylidene]-
butyronitrile (4a)
4.2.4. 2-(40-Chlorobenzyl)-3-ethoxy-4,4,4-trifluorobut-2-
enenitrile (2d)
Oil; 73%; 1H NMR (CDCl3): d 1.42 (3H, m, CH3), 3.66 (2H,
s, CH2), 4.13 and 4.28 (2H, q, J = 7.0 Hz, CH2O), 7.18 (2H, d,
J = 8.4 Hz, H-ar), 7.32 (2H d, J = 8.4 Hz, H-ar); 13C NMR
(CDCl3): d 15.2 and 15.3 (CH3), 33.2 and 34.0 (CH2), 70.5 and
71.9 (CH2O), 104.0 (Cq), 114.7 and 115.7 (CN), 119.2 and
Enol ether 2a (165 mg, 0.65 mmol) was added to a solution
of guanidine hydrochloride (143 mg, 1.5 mmol) and K2CO3
(270 mg, 1.95 mmol) in CH3CN (10 mL). The reaction mixture
was heated to reflux for 20 h, the solvent was then evaporated.
The residue was purified by column chromatography (CH2Cl2/
cyclohexane; 50/50) to give 30 mg of 4a and 80 mg of a mixture
of 4a and 2a (49/51).

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H. Berber et al. / Journal of Fluorine Chemistry 128 (2007) 1039–1045
Oil; 42%; IR (KBr); n 2917, 2218, 1620, 1454, 1358, 1266,
white solid (70%). 1H NMR (DMSO-d6): d 0.99 (3H, t,
1184, 1143, 1121, 1101, 756, 688 cmꢀ1; H NMR (CDCl3): d
1.32 (3H, t, J = 7.0 Hz, CH3), 3.76 (2H, m, CH2O), 4.34 (1H, q,
3JH,F = 5.9 Hz, CHCF3), 7.39 (1H, s, CH), 7.46 (3H, m, H-ar),
7.85 (2H, m, H-ar); 13C NMR (CDCl3): d 14.9 (CH3), 67.3
J = 7.1 Hz, CH3), 2.20 (2H, q, J = 7.1 Hz, CH2), 6.41 (2H, br s,
NH2), 7.41 (2H, d, J = 8.3 Hz, H-ar), 7.50 (2H, d, J = 8.3 Hz, H-
ar), 10.99 (1H, br s, OH); 13C NMR (DMSO-d6): d 14.2 (CH3),
19.2 (CH2), 113.6 (C-5), 128.2 (2ꢂ CH-ar), 130.0 (2ꢂ CH-ar),
133.0 (Cq-ar), 138.8 (Cq-ar), 153.4 (C-6), 160.6 and 163.4 (C-2,
C-4).
1
2
(CH2O), 78.6 (q, JC,F = 32.1 Hz, CHCF3), 103.7 (Cq), 116.1
(Cq), 123.0 (q, 1JC,F = 283.0 Hz, CF3), 129.0 (2ꢂ CH-ar), 129.6
(2ꢂ CH-ar), 131.7 (CH-ar), 132.1 (Cq-ar), 148.6 (CH). EIMS,
m/z: 255 [M]+ (26), 186 [M ꢀ CF3]+ (54), 158 (100), 140 (49).
HRMS, calcd for C13H12F3NO: 255.0871. Found: 255.0873.
4.6.1. 2-Amino-4-chloro-6-(40-chlorophenyl)-5-
ethylpyrimidine (10)
The above compound 9 (111 mg, 0.445 mmol) was refluxed
with POCl3 (5 mL) for 2 h. The excess POCl3 was removed in
vacuo and the residue was poured on to ice and ammonia, then
filtered off. The solid was purified by column chromatography
(CH2Cl2/MeOH, 98/2) to yield 36 mg of 10 as a pale yellow
solid (30%). 1H NMR (CDCl3): d 1.10 (3H, t, J = 7.4 Hz, CH3),
2.59 (2H, q, J = 7.4 Hz, CH2), 5.72 (2H sl, NH2), 7.39 (2H, d,
J = 8.4 Hz, H-ar), 7.45 (2H, d, J = 8.4 Hz, H-ar); 13C NMR
(CDCl3): d 14.0 (CH3), 21.8 (CH2), 121.9 (C-5), 128.7 (2ꢂ CH-
ar), 129.4 (2ꢂ CH-ar), 135.5 (Cq-ar), 135.9 (Cq-ar), 159.7 (C-
6), 163.1 and 166.6 (C-2, C-4).
4.5. General procedure for the synthesis of 5c,d and 7 was
previously described [8d]
4.5.1. 6-(20-Chlorophenyl)-2,4-diamino-5-(20,20,20-
trifluoroethyl)pyrimidine (5c)
Solid; 51%; mp 182 8C; IR (KBr); n 3356, 3183, 1620, 1557,
1447, 1261, 1141, 1100, 760 cmꢀ1; 1H NMR (DMSO-d6): d 3.3
(2H, m, CH2), 6.13 (2H, br s, NH2), 6.60 (2H, br s, NH2), 7.23–
7.59 (4H, m, H-ar); 13C NMR (DMSO-d6): d 30.1 (q,
1
2JC,F = 29.5 Hz, CH2), 93.3 (C-5), 126.7 (q, JC,F = 277.2 Hz,
CF3), 127.1 (CH-ar), 129.3 (CH-ar), 129.9 (CH-ar), 130.9 (CH-
ar), 131.0 (Cq-ar), 138.0 (Cq-ar), 162.2, 164.0 and 165.0 (C-2,
C-4, C-6). EIMS, m/z: 304 [M]+ (39), 302 [M]+ (100), 267 (35),
235 (28), 233 (84), 198 (56), 69 (33). HRMS, calcd for
C12H10F3N4Cl: 302.0546. Found: 302.0562.
4.6.2. 2,4-Diamino-6-(40-chlorophenyl)-5-ethylpyrimidine
(11)
The chloro compound 10 (35 mg, 0.13 mmol) was heated in
a closed system at 135 8C with 10 mL saturated solution of
ethanolic ammonia for 20 h. The solvent was then evaporated
and the residue was washed with aqueous EtOH to give 18 mg
of 11 as a white solid (56%). IR (KBr); n 3301, 3156, 1680,
4.5.2. 6-(40-Chlorophenyl)-2,4-diamino-5-(20,20,20-
trifluoroethyl)pyrimidine (5d)
1
1
Solid; 63%; mp 208–209 8C; H NMR (DMSO-d6): d 3.41
1634, 1514, 1113, 1091 cmꢀ1; H NMR (DMSO-d6): d 0.94
(2H, q, 3JH,F = 11.0 Hz, CH2), 6.10 (2H, br s, NH2), 6.58 (2H, br
s, NH2), 7.37 (2H, d, J = 8.5 Hz, H-ar), 7.48 (2H, d, J = 8.5 Hz,
H-ar); 13C NMR (DMSO-d6): d 29.6 (q, 2JC,F = 29.2 Hz, CH2),
92.5 (C-5), 126.8 (q, 1JC,F = 277.5 Hz, CF3), 128.3 (2ꢂ CH-ar),
130.2 (2ꢂ CH-ar), 132.7 (Cq-ar), 138.8 (Cq-ar), 162.1, 164.2
and 166.3 (C-2, C-4, C-6). EIMS, m/z: 304 [M]+ (21), 303 (31),
302 [M]+ (72), 301 (100), 235 (9), 233 (28), 198 (19). HRMS,
calcd for C12H10F3N4Cl: 302.0546. Found: 302.0548.
(3H, t, J = 7.3 Hz, CH3), 2.28 (2H, q, J = 7.3 Hz, CH2), 7.27
(2H, sl, NH2), 7.55 (2H, d, J = 8.5 Hz, H-ar), 7.65 (2H, d,
J = 8.5 Hz, H-ar), 7.94 (2H, sl, NH2); 13C NMR (DMSO-d6): d
13.4 (CH3), 18.2 (CH2), 108.4 (C-5), 129.0 (2ꢂ CH-ar), 130.6
(2ꢂ CH-ar), 131.6 (Cq-ar), 135.1 (Cq-ar), 155.2 (C-6), 164.4
(C-2, C-4). EIMS, m/z: 250 [M]+ (23), 248 [M]+ (66), 235 (33),
233 (100), 198 (37), 130 (31). HRMS, calcd for C12H13N4Cl:
248.0829. Found: 248.0825.
4.5.3. 5-Benzyl-2,4-diamino-6-methylpyrimidine (7)
4.7. Biological studies: anti-T. gondii activity
Solid; 73%; IR (KBr); n 3375, 3150, 1665, 1575, 1408, 881,
618, 557 cmꢀ1; 1H NMR (DMSO-d6): d 2.03 (3H, s, CH3), 3.76
(2H, s, CH2), 5.72 (2H, br s, NH2), 6.08 (2H, br s, NH2), 7.06–
7.36 (5H, m, H-ar); 13C NMR (DMSO-d6): d 21.5 (CH3), 30.5
(CH2), 102.6 (C-5), 125.9 (CH-ar), 128.0 (2ꢂ CH-ar), 128.4
(2ꢂ CH-ar), 140.7 (Cq-ar), 161.5, 161.3, 163.5 (C-2, C-4, C-6).
EIMS, m/z: 214 [M]+ (100), 213 (36). HRMS, calcd for
C12H14N4: 214.1218. Found: 214.1223.
Briefly, following a previously described method [13] the
virulent RH strain of T. gondii was maintained in mice by
intraperitoneal passage every 2 days. For each experiment,
tachyzoites were collected from the peritoneal cavity of
infected mice then resuspended in physiological saline. Tissue
cultures and drug tests were carried out using MRC5 fibroblast
tissue cultures. Confluent monolayers prepared in 96-well tissue
culture plates were inoculated with 2000 fresh tachyzoites. After
4 h, 10 serial dilutions of each drug, ranging form 0.01 to
40 mg Lꢀ1 for test compounds and 0.001–5 for pyrimethamine
were added into the culture medium. Each culture plate
comprised eight negative control (without T. gondii) and eight
positive control wells (without drug). After 72 h of incubation,
the plates were examined microscopically for cytopathic effects
then fixed with cold methanol for 5 min. Toxoplasma growth was
assessed by enzyme linked immunoassay (ELISA) performed
4.6. 2-Amino-6-(40-chlorophenyl)-4-hydroxy-5-
ethylpyrimidine (9)
8 (1 g, 4.16 mmol) was refluxed with guanidine hydro-
chloride (400 mg, 4.19 mmol) and K2CO3 (400 mg, 2.90 mmol)
in EtOH for 24 h. The mass was diluted with water (15 mL) and
then made acid with acetic acid (5 mL). The crystalline mass
was filtered off and washed with EtOH to yield 729 mg of 9 as a

H. Berber et al. / Journal of Fluorine Chemistry 128 (2007) 1039–1045
1045
´
[8] (a) M. Soufyane, C. Mirand, J. Levy, Tetrahedron Lett. 34 (1993) 7737–
directly on the fixed cultures using a peroxydase-labeled
monoclonal antibody directed against the SAG-1 surface protein
of T. gondii. After addition of the substrate, spectrophotometric
readings were performed at 405 nm with blanking on the
negative control well. For each wells, the results were expressed
as optical density (OD) values. The effect of each drug at various
concentrations was described by plotting the OD values as a
function of the logarithm of the concentration and a linear
regression model was used to summarize the concentration–dose
effect relationship and to determine the IC50.
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(b) M. Soufyane, S. Van den Broek, L. Khamliche, C. Mirand, Hetero-
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[10] Benzylpyrimidine (7) was previously reported:
E.A. Falco, S. Dubreuil, G.H. Hitchings, J. Am. Chem. Soc. 73 (1951)
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Acknowledgements
We thank C. Petermann (NMR) and P. Sigaut (MS) for
spectroscopic recordings. This study was supported by the
French National Reference Centre on toxoplasmosis.
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