Synthesis of novel diarylpyrimidine analogues of TMC278 and their antiviral activity against HIV-1 wild-type and mutant strains

Article abstract of DOI:10.1016/j.ejmech.2006.11.014

Novel diarylpyrimidines (DAPY), which represent next generation of non-nucleoside reverse transcriptase inhibitors (NNRTIs), were synthesized and their activities against human immunodeficiency virus type I (HIV-1) assessed. Modulations at positions 2 and

Full text of DOI:10.1016/j.ejmech.2006.11.014

European Journal of Medicinal Chemistry 42 (2007) 567e579
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel diarylpyrimidine analogues of TMC278 and their
antiviral activity against HIV-1 wild-type and mutant strains
a
a
a
a
´
Celine Mordant , Benoit Schmitt , Elisabeth Pasquier , Christophe Demestre ,
Laurence Queguiner a, Chantal Masungi b, Anik Peeters b, Liesbeth Smeulders b,
Eva Bettens b, Kurt Hertogs b, Jan Heeres c, Paul Lewi c, Jerome Guillemont a,
*
a Johnson & Johnson Pharmaceutical Research and Development, Medicinal Chemistry Department,
Campus de Maigremont BP315, F-27106 Val de Reuil Cedex, France
b Tibotec BVBA, Generaal de Wittelaan L 11B 3, B-2800 Mechelen, Belgium
c Center for Molecular Design, Janssen Pharmaceutica, Antwerpsesteenweg 37, B-2350 Vosselaar, Belgium
Received 20 September 2006; accepted 30 November 2006
Available online 15 December 2006
Abstract
Novel diarylpyrimidines (DAPY), which represent next generation of non-nucleoside reverse transcriptase inhibitors (NNRTIs), were synthe-
sized and their activities against human immunodeficiency virus type I (HIV-1) assessed. Modulations at positions 2 and 6 of the left phenyl ring
generated interesting derivatives of TMC278 displaying high potency against wild-type and mutant viruses compared to nevirapine and efavirenz.
The pharmacokinetic profile of the best newly synthesized DAPY was evaluated and compared with TMC278 now in phase II clinical trials.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: NNRTI; HIV-1; Diarylpyrimidine (DAPY)
1. Introduction
which terminates DNA synthesis. This class of inhibitors is ac-
tive in HIV-1 as well as HIV-2 strains. NNRTIs, however, bind
to an allosteric site, in the proximity of the active site of the en-
zyme and are specifically active against HIV-1 [4,5].
The application of the approved NNRTIs is limited to some
extent. This is mainly due to relatively low genetic barrier of
this class of drugs, and cross-resistance between these structur-
ally unrelated drugs. The intensive use of approved NNRTIs
(efavirenz, nevirapine and delavirdine, Fig. 1) [6e8] has led
to the emergence of HIV-resistant strains. Actually, for the first
generation of NNRTIs, only a single mutation is sufficient to
confer resistance, leaving some patients with no further
NNRTI therapeutic solutions [9].
Issues related to the use of NNRTI drugs have stirred our
effort and allowed the discovery of new series of NNRTIs
that belong to the diarylpyrimidine (DAPY) family [10].
DAPY compounds, with TMC125 (Fig. 1) as prototype of
the early generation of DAPY series, display a broad anti-
HIV activity spectrum against wild-type and mutant strains.
TMC125, which is currently in phase III clinical trials [11],
Multi-drug therapy against the human immunodeficiency
virus (HIV), known as highly active antiretroviral therapy
(HAART) [1,2], has drastically reduced the morbidity and
mortality of HIV-infected patients during the last decade and
slowed down the progression of acquired immunodeficiency
syndrome (AIDS). A number of inhibitors targeting reverse
transcriptase (RT) and protease enzymes, and the fusion process
are included in multi-drug therapy. Typically, reverse transcrip-
tase inhibitors serve as a mainstay of most frontline HIV com-
bination therapies. The reverse transcriptase enzyme can be
inhibited by two classes of drugs belonging either to the nucle-
oside reverse transcriptase inhibitors (NRTIs) or to the non-nu-
cleoside reverse transcriptase inhibitors (NNRTIs) [3]. NRTIs
act at the active site of RT. They are incorporated in the growing
DNA strand and prevent further elongation of the DNA-chain,
* Corresponding author. Tel.: þ33 2 32 61 74 58; fax: þ33 2 32 61 72 98.
E-mail address: jguillem@prdfr.jnj.com (J. Guillemont).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.11.014

568
C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
MeSO2NH
H
N
O
N
HN
N
CF3
Cl
O
N
N
N
H
N
N
O
N
H
O
Efavirenz
Nevirapine
Delavirdine
CN
O
CN
NH
CN
CN
N
N
HN
N
NH
Br
NH2
N
TMC125
TMC278, 1
Fig. 1. Structure of reference compounds, TMC125 and TMC278.
is the first NNRTI to demonstrate beneficial effects on heavily
experienced HIV-infected patients failing the traditional
NNRTI therapies [12,13]. Structureeactivity relationship stud-
ies combined with molecular modeling and X-ray analyses
have provided keys on the capability of DAPY compounds
to accommodate in the binding pocket, to amino acid residues
that confer resistance to NNRTIs. This has resulted into the
synthesis of a second-generation of DAPY compounds dis-
playing high potency against a whole panel of HIV-1 relevant
mutant strains. One of the second-generation of DAPY com-
pounds, TMC278 (Fig. 1) [14,15], is now in phase II clinical
trials [16].
To pursue our efforts in improving the anti-HIV activity of
DAPY derivatives against a clinically relevant panel of mutant
strains, the design and synthesis of TMC278 analogues were
undertaken.
Modulations on the left wing A of the diarylpyrimidine
were investigated (Fig. 2) on both sides:
The results show that newly synthesized DAPY derivatives
were endowed with nanomolar activity against a relevant panel
of viruses, including single and double NNRTI mutant strains.
2. Chemistry
The new series of DAPY derivatives (compounds 2e11)
was synthesized following two main pathways based on clas-
sical reactions namely the Heck reaction and the WittigeWad-
swortheEmmons reaction, which had been developed for the
synthesis of 1 (TMC278) (Scheme 1) [14]. These two syn-
thetic routes start from the same building block, chloropyrimi-
dine derivative 12 [10].
On one hand, the Wittig precursor, the aldehyde derivative
C, was synthesized in three steps from 12 with moderate
yields. The ester intermediate A was first obtained by heating
the chloropyrimidine 12 with the desired 3,5-disubstituted
ethyl 4-aminobenzoate at 150 ꢀC or 110 ꢀC in the presence
of 3 N hydrochloric acid. Thereafter, the ester function was
classically reduced with lithium aluminum hydride and the re-
sulting alcohol B underwent manganese-mediated oxidation to
give the expected aldehyde C. The latter was thus involved in
a key Wittig reaction and condensed with diethyl cyanomethyl
phosphonate in the presence of t-BuOK to afford the following
(i) The influence of an X spacer between the substituted
phenyl and the position 4 of the pyrimidine was
evaluated;
(ii) The effect of the nature of the substituents at positions 2
and 6 of the phenyl ring was assessed.
CN
CN
CN
CN
Left wing A
R1
R2
N
HN
NH
X
N
NH
X Linker
N
N
TMC278, 1
2 R1=H, R2=Me, X=NH
3 R1=R2=H, X=NH
7 R1=Me, R2=Et, X=NH
8 R1=Me, R2=iPr, X=NH
9 R1= Me, R2=OMe, X=NH
10 R1=Me, R2=Cl, X=NH
11 R1=OMe, R2=Cl, X=NH
4 R1=H, R2=Me, X=NMe
5 R1=R2=Me, X=O
6 R1=R2=Me, X=S
Fig. 2. Modulations of an X spacer between the left wing A and the pyrimidine and introduction of various 2,6-substitutions on the phenyl ring.

C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
569
CO2Et
O
OEt
OH
O
CN
NH
CN
NH
CN
NH
CN
NH
R2
R1
NH2
(a)
R2
R1
R2
N
R2
N
R1
R1
HN
HN
Cl
HN
N
N
(b)
(c)
N
N
N
N
Br
12
A
B
C
(d)
(e)
R1
R2
CN
NH
R3
CN
R3
CN
NH
CN
NH
Br
N
CN
NH
R2
N
R2
N
R1
R2
N
R1
R1
N
HN
R3
(f)
N
N
N
4, 7, 8, 9
D
1, 2, 3, 10, 11
Scheme 1. Synthetic routes via Wittig or Heck reactions to 2,6-disubstituted rings. Wittig pathway: (a) 2, 3: 150 ꢀC, 2e4 h; 10, 11: HCl 3 N, reflux, 5e16 h; (b)
LiAlH4, THF, 0 ꢀC to RT, 20 h; (c) MnO2, CH2Cl2/DMF, RT, 20 h; (d) (EtO)2P(O)CH2CN, t-BuOK, THF, 0 ꢀC to RT, 4e20 h. Heck pathway: (e) 4, 9: NMP,
140 ꢀC, 20 h; 7: HCl 3 N, reflux, 20 h; 8: heated neat; (f) acrylonitrile, Pd(OAc)2, P(o-Tol)3, NEt3, CH3CN, 140 ꢀC, 20 h.
acrylonitrile derivatives 2, 3, 10 and 11. These products were
isolated and further crystallized with a complete (E )-stereose-
lectivity, except for compound 3, which exhibited a (E )/(Z )
ratio of 80/20.
On the other hand, the Heck precursor was readily prepared
from 12. The bromo-derivative D was synthesized either by
heating (140 ꢀC) the chloropyrimidine 12 and the correspond-
ing bromo-aniline in N-methyl-pyrrolidinone as solvent (com-
pounds 4 and 9) or according to the protocols described above
for carboxylic esters of type A (7 and 8). The coupling
reaction was then conducted on different bromo-derivatives
of D according to classical Heck conditions using palladiu-
m(II) diacetate as a catalyst in the presence of triethylamine
in acetonitrile. The following acrylonitrile derivatives 4, 7, 8
and 9 were thus prepared according to this procedure and ob-
tained with moderate yields. The stereoselectivity ranged from
80/20 to 96/4 ratio in favor of the (E ) diastereoisomer.
The synthesis of the corresponding phenoxy and phenylsul-
fanyl derivatives 5 and 6 was also based on a Wittig reaction in
order to introduce the acrylonitrile function (Scheme 2).
CN
O
CHO
CN
CN
NH
CN
NH
OH
(a)
Cl
NH
O
N
N
N
O
N
N
(b)
N
12
5a
5
CN
Br
O
OEt
CN
NH
CN
CN
NH
CN
NH
Br
S
6e
SH
NH
Cl
S
N
N
S
N
N
(c)
(d), (e), (b)
(a)
N
N
N
N
12
6a
6b
6
Scheme 2. Synthesis of compounds 5 and 6. (a) NaH, NMP, dioxane, 150 ꢀC, 12e48 h; (b) (Et2O)2P(O)CH2CN, t-BuOK, THF, 0 ꢀC to RT, 20 h; (c) P(CO) 10 bar,
Pd(OAc)2, PPh3, K2CO3, EtOH, DMF, 90 ꢀC, 72 h; (d) LiAlH4, THF, 0 ꢀC to RT, 20 h; (e) MnO2, CH2Cl2, RT, 72 h.

570
C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
Compound 5 was synthesized in two steps from derivative
All DAPY derivatives were considerably more potent than
13, 14 or 15 on the whole panel of viruses.
12. Introduction of the phenoxy moiety on the pyrimidine
scaffold was realized by heating (150 ꢀC) the chloropyrimi-
dine 12 and commercially available 3,5-dimethyl-4-hydroxy-
benzaldehyde with sodium hydride in a 1:1 mixture of
NMP:dioxane. A Wittig reaction on 5a allowed us to isolate
5 with a complete (E )-stereoselectivity and an excellent yield.
The same coupling conditions were used, started from 12, to
synthesize bromo-derivative 6a, which was then submitted to
a palladium(II) catalyzed CO-insertion leading to the corre-
sponding ethyl ester 6b with a moderate yield. A standard se-
quence LiAlH4-reduction/MnO2-oxidation/Wittig reaction
was finally performed to achieve the synthesis of 6 with
a (E )/(Z ) ratio of 85/15.
First the influence on activity of the X spacer connecting
the left phenyl ring and the pyrimidine was evaluated (Table 1).
Compounds 5 and 6, in which the NH-linker of TMC278 (1)
was respectively replaced by an O- and S-linker, were thus
compared to TMC278 (1) and to the reference compounds
(13, 14 and 15).
In line with the results obtained for 1, compounds 5 and 6
demonstrated high potency on the whole panel with an en-
hanced resistance profile on the double mutant strains as com-
pared to marketed drugs (13, 14 and 15). The K103N single
mutant strain showed hypersusceptibility towards the DAPY
compounds. However, the sulfanyl derivative 6 displayed
a slight decrease of activity to some extent compared to
TMC278 (1). These results prompted us to consider the
NH-group as the most appropriate spacer to achieve high-level
potency on single and double mutant strains. Therefore, we
pursued the optimization process with this linker.
All these A-ring analogues of 1 were synthesized to allow
further exploration of the influence of a heteroatom linker X
and the effect of a 2,6-disubstitution on the activity.
The effect of the left phenyl A-ring’s 2,6-disubstitution on
activity was then investigated. Modeling data with a 2,6-
dimethyl DAPY derivative had shown that pep main interac-
tions were present between the phenyl ring A of the substrate
and residues Tyr181 and Tyr188 of the binding pocket [12].
The conformation favoring these specific interactions was par-
tially due to the presence of the two methyl groups at positions
2 and 6, which prevented great conformational shifts of this
left wing A, especially by limiting the rotational freedom. Re-
moval of the substituents might therefore increase the confor-
mational degrees of freedom and weaken the pep interaction.
In addition, the two methyl groups were involved in hydropho-
bic interactions with a number of side chains exposed in the
NNRTI pocket. One methyl group interacted with Pro95,
Leu100 and Tyr181 and to a lesser extent with Glu138 and
Trp229, while the other methyl group was located close to
3. Results and discussion
The anti-HIVactivity of compounds 2e11 was measured us-
ing an HIV-1 replication assay and compared to TMC278 (1).
The cells were infected with HIV-1 wild-type virus (LAI) or
with single (L100I, K103N, Y181C, Y188L, F227C) or double
mutant (L100I/K103N, K103N/Y181C, F227C/V106A) strains
derived from wild-type LAI. The results are reported as the con-
centration required to achieve 50% inhibition of cellular activ-
ity (EC50). In addition, the cytotoxicity (CC50) of the
compounds was determined. The selective index (SI ¼ CC50/
EC50), which indicates the specificity of the antiviral effect, is
listed for the wild-type virus.
Tables 1 and 2 list the results for compounds 2e11 in com-
parison with those for compound 1 and three reference com-
pounds efavirenz (13), nevirapine (14) and delavirdine (15).
Table 1
Influence of the X spacer on the inhibition of wt LAI and mutant strains of HIV-1
CN
CN
NH
N
X
N
Compound
X
E/Z
LAI
CC50
SIa
EC50 (nM)
L100I
K103N
Y181C Y188L F227C L100I þ
K103N þ
F227C þ
K103N
Y181C
V106A
1
NH
O
100/0
100/0
85/15
1.0
0.9 >25000
3.7 >25000 >6821
2000
2020
0.5
0.6
0.1
0.2
1.3
3.2
5.8
3.9
1.2
1.6
0.5
0.7
7.5
3.7
3.2
4.3
0.5
0.6
5
28302
6
S
2.6
31.0
209
0.7
46.4
2.8
55.0
1.4
46.9
79.2
18.6
55.0
1.1
4.1
13
14
15
a
Efavirenz
Nevirapine
Delavirdine
1.6
33.3
32314
31631
67188
19850
>949
1460
3532
1980
10220
7078
9035
5910
1022
827
4560
14505
38352
2697
721
122.1
>550 9530
20583
SI ¼ CC50/EC50
.

C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
571
Table 2
Influence of the 2,6-substitution on the inhibition of wt LAI and mutant strains of HIV-1
CN
CN
R1
R2
N
NH
X
N
Compound
X
R1
R2
E/Z
LAI CC50
SIa
EC50 (nM)
L100I K103N Y181C Y188L F227C L100I/
K103N/ F227C/
V106A
K103N Y181C
1
NH
NH
NH
NMe
NH
NH
NH
NH
NH
Me
H
Me
Me
H
100/0
100/0
80/20 33.9 >25000
1.0
2000
2020
0.5
9.2
0.1
0.6
1.3
13.4
1.2
43.6
303.3
16.8
2.6
0.5
1.2
98.4
1.0
1.1
3.9
0.3
0.5
0.8
7.5
478.7
6440
103.8
8.2
3.2
89.6
0.5
1.2
76.1
1.5
0.9
3.2
0.3
0.5
0.3
2
0.9 >25000 >29372
3
H
>737 585.1
57.7
0.7
1523
3952
160.3
5.3
4
H
Me
Et
80/20
85/15
96/04
91/09
100/0
100/0
1.0
3.0
4.0
0.7
0.8
0.1
11625
2553
11791
633
11351
841
2.5
0.9
3.1
0.5
0.4
0.2
12.0
4.0
7
Me
Me
Me
Me
0.5
1.3
8
iPr
OMe
Cl
2947
846
11.1
1.9
3.8
0.9
38.3
21.1
6.6
10.6
4.3
9
<0.1
0.1
10
11
a
2606
398
3316
3981
2.3
1.0
2.3
2.5
4.1
2.0
OMe Cl
0.3
15.8
SI ¼ CC50/EC50
.
Val106 and Val179 and might also interact weakly with the
side chains of K103 and Y188.
double mutants L100I/K103N and K103N/Y181C, which
were fully resistant to efavirenz (13) and nevirapine (14)/dela-
virdine (15), respectively, remained strongly inhibited by any
of the substituents.
The antiviral results for the compounds with variations of
the 2,6-substitution on the left phenyl ring are listed in Table 2.
The mono-substituted derivative 2 exhibited an excellent
potency against wild-type virus but a rather moderate activity
on mutant strains. The decrease in activity on the single mu-
tant strains L100I, Y181C and Y188L was due to the loss of
favorable hydrophobic interactions and the weakening of the
pep interactions. In the double mutant L100I/K103N, com-
pound 2 was more than 100 times less active than in the
wild-type enzyme. The non-2,6-substituted compound 3 had
submicromolar activity against wild-type (EC50 ¼ 0.034 mM)
and was considerably less potent compared to compound 2.
This decrease in activity of 3 might be explained by additional
degrees of freedom for the molecule due to the absence of sub-
stituents on the phenyl ring, causing further weakening of the
pep interactions.
Introduction of small R1 and R2 groups, with electronic
properties different from the previous alkyl substitutions, at
positions 2 and 6 of the phenyl ring was also investigated.
Electron-donating and electron-withdrawing groups were con-
sidered (9e11). The results showed antiviral profiles for these
three compounds similar to the 2,6-dimethyl substituted com-
pound TMC278 (1); the electronic nature of the substituents
did not seem to highly impact the activity. The potency of
compounds 9 and 11 on the double mutant strain L100I/
K103N was slightly decreased compared to 1.
Derivatives 10 and 11 were selected for further in vitro and
in vivo studies and compared to TMC278 (1). The metabolic
stability profile of these compounds was assessed using rat,
dog and human liver microsomes (Table 3).
Interesting results were also observed when considering the
methylation of the NH-linker (compound 4). This compound
showed a nanomolar activity on wild-type and single mutant
strains that was comparable to the other substituted DAPY de-
rivatives. However, a drop in activity was observed when con-
sidering L100I/K103N and K103N/Y181C mutations.
Superposition of 1 and 4 after docking in the NNRTI pocket
showed that the methyl group of the NeCH3 linker in com-
pound 4 is located further away from residues Pro95,
Leu100 and Tyr181.
It is also interesting to notice that introduction of an ethyl
(7) or isopropryl (8) group at position 6 had only minor effects
on the activity. In the series methyleethyleisopropyl, the size
of the substituent seems to be inversely proportional to the ac-
tivity. This could be explained by the presence of some steric
hindrance generated by the larger substituents. However, the
As outlined in Table 3, the metabolic stability in rat, dog
and human liver microsomes of compounds 1, 10 and 11
was similar. In rat and dog liver microsomes, high rates of
recovery were observed (60e70%) whereas in human
microsomes, 10 and 11 seemed to be less metabolically stable
(up to 74% metabolized). However, since TMC278 demon-
strated the same metabolic stability, further evaluation of the
Table 3
Metabolic stability of compounds 1, 10 and 11
Compound
Microsome stability
(% recovered after 15 min)
Rat
Dog
Human
1 (TMC278)
73
72
53
65
60
58
26
39
26
10
11

572
C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
pharmacokinetic profile of the two new compounds 10 and 11
in rat and dog species was sensible.
Proton NMR spectra were recorded on a Bruker Avance 300
(300 MHz) and a Bruker Avance 400 (400 MHz) spectrometer
using internal deuterium lock. Chemicals shifts are reported to
internal DMSO (d 2.54) in parts per million and coupling
constants (J ) in hertz. Exact mass spectra (TOF) were re-
corded with a Micromass LCT instrument. Elemental analyses
were performed with a Thermo Electron Corporation instru-
ment EA 1110 or EA 1108 for C, H, and N, and the results
were within ꢁ0.4% of the theoretical values. Chemicals and
solvents were purchased from either Acros Co. or Aldrich
Chemical Co. Yields refer to purified products and are not op-
timized. The synthesis of TMC278 was described before [14].
The results presented in Table 4 show mean values of phar-
macokinetic parameters. Following a single dose of an oral
formulation, TMC278 was well absorbed in rats and dogs
with mean AUC values of 9650 and 23,790 ng.h/mL and
high mean Cmax levels of 1940 and 1046 ng/mL, respectively.
Absorption of TMC278 was prolonged with a T1/2 of 6 h in
rats and 23 h in dogs. The derivatives 10 and 11 were highly
absorbed in dogs as well. Compound 10 had lower mean
AUC (17,600 ng.h/mL) and Cmax (1040 ng/mL) values com-
pared to 1 whereas the mean AUC (44,500 ng.h/mL) and
Cmax (2184 ng/mL) values of compound 11 were significantly
higher. Nevertheless, the half-life of these derivatives was
somewhat lower (14.5 h) compared to 1 in both animal spe-
cies. The pharmacokinetic profile of TMC278 allows once-
daily dosing in HIV-infected patients.
5.2. General procedure 1 for the synthesis of 4-[2-(4-
cyanophenylamino)-pyrimidin-4-ylamino]-3,5-
disubstitutedbenzoic acid ethyl ester A
Method A: a mixture of 4-amino-3,5-disubstitutedbenzoic
acid ethyl ester (1 eq.) and 4-(4-chloropyrimidin-2-ylamino)-
benzonitrile 12 [10] (1 eq.) was heated at 150 ꢀC in an oil
bath for 2e4 h. The residue was extracted with CH2Cl2/
MeOH and washed with 10% K2CO3. The organic layer was
dried over MgSO4 and concentrated under reduced pressure.
The residue was crystallized from diisopropyl ether. Method
B: a mixture of 4-amino-3,5-disubstituted-benzoic acid ethyl
ester (1 eq.) and 4-(4-chloropyrimidin-2-ylamino)-benzonitrile
12 [10] (1 eq.) in the presence of 3 N hydrochloric acid was
heated at reflux in an oil bath for 16 h. After cooling down,
the precipitate was filtered and rinsed successively with water
and diisopropyl ether.
4. Conclusion
This exploratory study clearly highlights the necessity to
maintain a 2,6-disubstitution pattern on the left wing A within
the DAPY series to achieve a high level of inhibition on
NNRTI-resistant viruses. Furthermore, it points out that the
methyl groups in TMC278 could be efficiently replaced by
other functionalities (9, 10, and 11) without any loss of activ-
ity. The newly synthesized derivatives had an enhanced po-
tency to some extent on specific mutant strains. Compounds
10 and 11 not only demonstrate an excellent overall profile
on a selection of clinically relevant mutant strains compared
to the approved drugs, but also interesting pharmacokinetic
characteristics in dogs. This promising broad-spectrum activity
could thus support further developments of the DAPY series.
5.2.1. 4-[2-(4-Cyanophenylamino)-pyrimidin-4-ylamino]-3-
methylbenzoic acid ethyl ester 2A
5. Experimental section
Compound 2A was obtained according to general procedure
1, method A from 12 (0.0558 mol) and commercially available
4-amino-3-methylbenzoic acid ethyl ester (0.0558 mol). The
residue was purified by silica gel column chromatography
(CH2Cl2/MeOH/NH4OH 96:4:0.1e90:10:0.1) and then recrys-
tallized from diisopropyl ether (8 g, 39% yield). Mp 192 ꢀC;
1H NMR (DMSO-d6) d 1.35 (3H, t, J ¼ 7.7 Hz), 2.30 (3H,
s), 4.35 (2H, q, J ¼ 7.7 Hz), 6.54 (1H, d, J ¼ 5.2 Hz), 7.60
(2H, d, J ¼ 7.7 Hz), 7.65e7.77 (3H, m), 7.88 (1H, d,
J ¼ 8.0 Hz), 7.91 (1H, s), 8.04 (1H, d, J ¼ 5.2 Hz), 10.08
(1H, br s), 10.52 (1H, br s).
5.1. Chemistry
All analytically pure compounds were dried under vacuum
in a drying pistol using a Buchi Glass Oven B-580 apparatus.
Melting points were determined using a Leica VMHB appara-
tus and are uncorrected. TLC analyses were run on silica gel
60 F254 plates (Merck) using a variety of solvent systems
and a fluorescent indicator for visualization. Spots were
visualized under 254 nm UV illumination. Column chroma-
tography was performed with silica gel 60 (Merck)
(0.015e0.040 mm) or Kromasyl (Akzo Nobel) (0.010 mm).
5.2.2. 4-[2-(4-Cyanophenylamino)-pyrimidin-4-ylamino]-
benzoic acid ethyl ester 3A
Table 4
Pharmacokinetic parameters of compounds 1, 10 and 11
Compound 3A was obtained according to general procedure
1, method A from 12 (0.003 mol) and commercially available
ethyl 4-amino-benzoate (0.003 mol). The residue was crystal-
lized from diisopropyl ether (0.91 g, 42% yield). Mp 233 ꢀC;
1H NMR (DMSO-d6) d 1.33 (3H, t, J ¼ 6.5 Hz), 4.30 (2H,
q, J ¼ 6.5 Hz), 6.44 (1H, d, J ¼ 5.7 Hz), 7.20 (2H, d,
J ¼ 7.7 Hz), 7.86e8.00 (6H, m), 8.18 (1H, d, J ¼ 5.7 Hz),
9.85 (1H, s), 9.93 (1H, s).
Compound
Rat (PO) normalized to
10 mg/kg
Dog normalized to
10 mg/kg
Cmax
(ng/mL) (ng.h/mL) (h)
AUC
T1/2
Cmax
(ng/mL) (ng.h/mL) (h)
AUC
T1/2
1 (TMC278) 1940
9650
910
6
1046
23,790
17,600
44,500
23
10
11
182
199
3.54 1040
3.83 2184
14.5
14.5
410

C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
573
5.2.3. 4-[2-(4-Cyanophenylamino)-pyrimidin-4-ylamino]-
3-chloro-5-methylbenzoic acid ethyl ester 10A
J ¼ 2.5 Hz), 7.48 (2H, d, J ¼ 2.5 Hz); MS (C10H12NO3Cl):
m/z 230 (M þ H)þ.
Compound 10A was obtained according to general proce-
dure 1, method B from 12 (0.00332 mol), 4-amino-3-chloro-
5-methylbenzoic acid ethyl ester (0.00332 mol) and 3 N HCl
(4 mL). The residue was purified by silica gel column chroma-
tography (CH2Cl2/MeOH/NH4OH 98:2:0.1) and crystallized
from CH3CN/diisopropyl ether (0.44 g, 32% yield). Mp
195 ꢀC; 1H NMR (DMSO-d6) d 1.36 (3H, t, J ¼ 7.7 Hz),
2.27 (3H, s), 4.37 (2H, q, J ¼ 7.7 Hz), 6.28 (1H, br s), 7.45
(2H, d, J ¼ 7.2 Hz), 7.60e7.75 (2H, m), 7.92 (1H, s), 7.95
(1H, s), 8.09 (1H, d, J ¼ 5.2 Hz), 9.33 (1H, s), 9.68 (1H, s);
MS (C21H18N5O2Cl): m/z 408 (M þ H)þ.
5.3. General procedure 2 for the synthesis of 4-[4-
(4-hydroxymethyl-2,6-disubstituted-phenylamino)-
pyrimidin-2-ylamino]-benzonitrile B
Lithium aluminum hydride (2 eq.) was added portionwise
to an ice-cooled solution of benzoic acid ethyl ester A
(1 eq.) in THF (5 mL/mmol) under nitrogen. The reaction
mixture was allowed to warm up to room temperature and
stirred overnight. Ethyl acetate was then added followed by
water and the resulting mixture was filtered over Celite. The
organic layer was dried over MgSO4 and concentrated under
reduced pressure.
4-Amino-3-chloro-5-methylbenzoic acid ethyl ester was
previously synthesized according to the following protocol.
A mixture of 4-amino-5-methylbenzoic acid ethyl ester
(0.052 mol) and N-chlorosuccinimide (0.058 mol, 1.1 eq.) in
acetonitrile (70 mL) was heated at reflux for 1 h. The resulting
mixture was then basified with 10% K2CO3 and extracted with
CH2Cl2. The organic layer was dried over MgSO4 and concen-
trated under reduced pressure. The residue was purified by sil-
ica gel column chromatography (cyclohexane/ethyl acetate
85:15) to give 7.5 g of pure product (94% yield). Mp
130 ꢀC; 1H NMR (DMSO-d6) d 1.27 (3H, t, J ¼ 7.7 Hz),
2.16 (3H, s), 4.22 (2H, q, J ¼ 7.7 Hz), 5.92 (2H, s), 7.54
(1H, s), 7.65 (2H, s).
5.3.1. 4-[4-(4-Hydroxymethyl-2-methyl-phenylamino)-
pyrimidin-2-ylamino]-benzonitrile 2B
Compound 2B was obtained according to general procedure
2, from 2A (0.0175 mol). The residue was crystallized from
diisopropyl ether/isopropanol (4.7 g, 80% yield). Mp 210 ꢀC;
1H NMR (DMSO-d6) d 2.20 (3H, s), 4.50 (2H, d, J ¼ 5.7 Hz),
5.19 (1H, t, J ¼ 5.7 Hz), 6.15 (1H, d, J ¼ 5.7 Hz), 7.20 (1H, d,
J ¼ 6.8 Hz), 7.25 (1H, s), 7.33 (1H, d, J ¼ 6.8 Hz), 7.55 (2H,
d, J ¼ 8.0 Hz), 7.86 (2H, d, J ¼ 8.0 Hz), 8.01 (1H, d,
J ¼ 5.7 Hz), 8.90 (1H, s), 9.63 (1H, s).
5.3.2. 4-[4-(4-Hydroxymethyl-phenylamino)-pyrimidin-
2-ylamino]-benzonitrile 3B
5.2.4. 4-[2-(4-Cyanophenylamino)-pyrimidin-4-ylamino]-
3-chloro-5-methoxy-benzoic acid ethyl ester 11A
Compound 3B was obtained according to general proce-
dure 2, from 3A (0.00181 mol). The residue was engaged in
the next step without further purification (0.59 g, 100% yield).
Compound 11A was obtained according to general proce-
dure 1, method B from 12 (0.0094 mol), 4-amino-3-chloro-
5-methoxy-benzoic acid ethyl ester (0.0094 mol) and 3 N
HCl (50 mL). The crude product was used without further
purification in the next step (6.5 g, 77% yield). MS
(C21H18N5O3Cl): m/z 424 (M þ H)þ.
1
Mp 215 ꢀC; H NMR (DMSO-d6) d 4.49 (2H, d, J ¼ 5.7 Hz),
5.12 (1H, t, J ¼ 5.7 Hz), 6.32 (1H, d, J ¼ 5.7 Hz), 7.32 (2H, d,
J ¼ 7.7 Hz), 7.60 (2H, d, J ¼ 7.7 Hz), 7.67 (2H, d, J ¼ 7.7 Hz),
7.97 (2H, d, J ¼ 7.7 Hz), 8.07 (1H, d, J ¼ 5.7 Hz), 9.46 (1H,
s), 9.74 (1H, s); MS (C18H15N5O): m/z 318 (M þ H)þ.
4-Amino-3-chloro-5-methoxy-benzoic acid ethyl ester was
previously synthesized according to the following protocol.
Thionyl chloride (5 mL) was added dropwise to an ice-cooled
solution of 4-amino-3-methoxy-benzoic acid (0.060 mol) in
ethanol (100 mL). The resulting mixture was heated at reflux
for 3 h and then poured into ice. The mixture was then basified
with 10% K2CO3 and extracted with ethyl acetate. The organic
layer was dried over MgSO4 and concentrated under reduced
pressure. Next, the resulting 4-amino-3-methoxy-benzoic
acid ethyl ester (10 g, 85% yield) was engaged in the next
step. MS (C10H13NO3): m/z 196 (M þ H)þ. A mixture of 4-
amino-3-methoxy-benzoic acid ethyl ester (0.0256 mol) and
N-chlorosuccinimide (0.0282 mol, 1.1 eq.) in acetonitrile
(20 mL) was heated at reflux for 1 h. The resulting mixture
was then basified with 10% K2CO3 and extracted with
CH2Cl2. The organic layer was dried over MgSO4 and concen-
trated under reduced pressure. The residue was purified by sil-
ica gel column chromatography (cyclohexane/ethyl acetate
5.3.3. 4-[4-(4-Hydroxymethyl-2-chloro-6-methyl-
phenylamino)-pyrimidin-2-ylamino]-benzonitrile 10B
Compound 10B was obtained according to general proce-
dure 2, from 10A (0.0098 mol). The residue was purified by
silica gel column chromatography (CH2Cl2/MeOH/NH4OH
1
97:3:0.1) (1 g, 27% yield). Mp 246 ꢀC; H NMR (DMSO-
d6) d 2.20 (3H, s), 2.27 (3H, s), 4.54 (2H, d, J ¼ 5.2 Hz),
6.40 (1H, t, J ¼ 5.2 Hz), 6.35 (1H, br s), 7.26 (1H, s), 7.38
(1H, s), 7.40e7.50 (2H, br m), 7.55e7.75 (2H, br m), 7.90e
8.10 (1H, br m), 9.05 (1H, br s), 9.64 (1H, br s); MS
(C19H16N5OCl): m/z 366 (M þ H)þ.
5.3.4. 4-[4-(4-Hydroxymethyl-2-chloro-6-methoxy-
phenylamino)-pyrimidin-2-ylamino]-benzonitrile 11B
Compound 11B was obtained according to general proce-
dure 2, from 11A (0.016 mol). The residue was engaged in
the next step without further purification (4.1 g, 67% yield).
MS (C19H16N5O2Cl): m/z 382 (M þ H)þ.
1
80:20) to give 4.3 g of pure product (73% yield). H NMR
(DMSO-d6) d 1.30 (3H, t, J ¼ 7.7 Hz), 3.87 (3H, s), 4.25
(2H, q, J ¼ 7.7 Hz), 5.78 (2H, br s), 7.27 (1H, d,

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C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
5.4. General procedure 3 for the synthesis of 4-[4-
(4-formyl-2,6-disubstituted-phenylamino)-pyrimidin-
2-ylamino]-benzonitrile C
organic layer was dried over MgSO4 and concentrated under
reduced pressure. Next, the residue was purified by silica gel
column chromatography and crystallized.
To a solution of benzoic methyl alcohol B in a CH2Cl2/
DMF mixture was added portionwise MnO2 (4 eq.). The re-
sulting mixture was stirred overnight at room temperature
then filtered over Celite and concentrated under reduced
pressure.
5.5.1. 4-{4-[4-(2-Cyanovinyl)-2-methyl-phenylamino]-
pyrimidin-2-ylamino}-benzonitrile 2
Compound 2 was obtained according to general procedure 4,
from 2C (0.00096 mol). The residue was first crystallized from
isopropanol/diisopropyl ether and then crystallized from hot
ethanol followed by a last crystallization from isopropanol/
diisopropyl ether to give pure product 2 ((E ), 0.025 g, 7% yield).
5.4.1. 4-[4-(4-Formyl-2-methyl-phenylamino)-pyrimidin-
2-ylamino]-benzonitrile 2C
Compound 2C was obtained according to general proce-
dure 3, from 2B (0.0136 mol) in 70 mL CH2Cl2. The residue
was engaged in the next step without further purification
(2.1 g, 46% yield). MS (C19H15N5O): m/z 330 (M þ H)þ.
1
Mp > 260 ꢀC; H NMR (DMSO-d6) d 2.26 (3H, s), 6.38 (1H,
d, J ¼ 5.0 Hz), 6.43 (1H, d, J ¼ 16.0 Hz), 7.51e7.61 (4H, m),
7.65 (1H, d, J ¼ 16.0 Hz), 7.72 (1H, d, J ¼ 7.7 Hz), 7.88 (2H,
d, J ¼ 7.7 Hz), 8.10 (1H, d, J ¼ 5.0 Hz), 9.00 (1H, s), 9.73
(1H, s); MS (C21H16N6) m/z 353 (M þ H)þ.
5.4.2. 4-[4-(4-Formyl-phenylamino)-pyrimidin-2-ylamino]-
benzonitrile 3C
5.5.2. 4-{4-[4-(2-Cyanovinyl)-phenylamino]-pyrimidin-2-
ylamino}-benzonitrile 3
Compound 3C was obtained according to general proce-
dure 3, from 3B (0.0016 mol) in a 1:1 CH2Cl2:DMF mixture
(20 mL). The residue was crystallized from ethyl acetate/ace-
tonitrile (0.25 g, 50% yield). Mp > 250 ꢀC; 1H NMR (DMSO-
d6) d 6.45 (1H, d, J ¼ 5.7 Hz), 7.73 (2H, d, J ¼ 7.7 Hz), 7.88
(2H, d, J ¼ 7.7 Hz), 7.96 (4H, d, J ¼ 7.7 Hz), 8.21 (1H, d,
J ¼ 5.7 Hz), 9.85e9.95 (2H, m), 10.04 (1H, s); MS
(C18H13N5O): m/z 316 (M þ H)þ.
Compound 3 was obtained according to general procedure
4, from 3C (0.00047 mol). The residue was crystallized from
diisopropyl ether/acetonitrile to give pure product 3 ((E )/(Z )
1
80/20, 0.061 g, 38% yield). Mp > 250 ꢀC; H NMR (DMSO-
d6) d 5.73 (Z ) (1H, d, J ¼ 12.1 Hz), 6.35 (E ) (1H, d,
J ¼ 16.7 Hz), 6.40 (1H, d, J ¼ 6.2 Hz), 7.35 (Z ) (1H, d,
J ¼ 12.1 Hz), 7.60 (E ) (1H, d, J ¼ 16.7 Hz), 7.63e7.77 (4H,
m), 7.82 (2H, d, J ¼ 7.9 Hz), 7.95 (2H, d, J ¼ 7.9 Hz), 8.15
(1H, d, J ¼ 5.7 Hz), 9.80e9.90 (2H, m).
5.4.3. 4-[4-(4-Formyl-2-chloro-6-methyl-phenylamino)-
pyrimidin-2-ylamino]-benzonitrile 10C
Compound 10C was obtained according to general proce-
dure 3, from 10B (0.0027 mol) in a 10:1 CH2Cl2:DMF mixture
(55 mL). The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH/NH4OH 95:5:0.1) (0.24 g,
25% yield). MS (C19H14N5OCl): m/z 364 (M þ H)þ.
5.5.3. 4-{4-[4-(2-Cyanovinyl)-2-chloro-6-methyl-
phenylamino]-pyrimidin-2-ylamino}-benzonitrile 10
Compound 10 was obtained according to general procedure
4, from 10C (0.0025 mol). The residue was purified by silica
gel column chromatography (CH2Cl2/MeOH/NH4OH 98:2:0.1)
and then crystallized from diisopropyl ether/acetonitrile to
give pure product 10 ((E ), 0.4 g, 20% yield). Mp 254 ꢀC;
1H NMR (DMSO-d6) d 2.22 (3H, s), 6.18e6.35 (1H, m),
6.60 (1H, d, J ¼ 16.7 Hz), 7.40e7.50 (2H, m), 7.66e7.75
(4H, br m), 7.829 (1H, s), 8.06 (1H, d, J ¼ 5.7 Hz), 9.23
(1H, br s), 9.68 (1H, br s).
5.4.4. 4-[4-(4-Formyl-2-chloro-6-methoxy-phenylamino)-
pyrimidin-2-ylamino]-benzonitrile 11C
Compound 11C was obtained according to general proce-
dure 3, from 11B (0.0107 mol) in a 7:1 CH2Cl2:DMF mixture
(24 mL). The residue was crystallized from diisopropyl ether
(1.9 g, 47% yield). Mp 196 ꢀC; MS (C19H14N5O2Cl): m/z
380 (M þ H)þ.
5.5. General procedure 4 for the synthesis of 4-{4-[4-
(2-cyanovinyl)-2,6-disubstituted-phenylamino]-
5.5.4. 4-{4-[4-(2-Cyanovinyl)-2-chloro-6-methoxy-
phenylamino]-pyrimidin-2-ylamino}-benzonitrile 11
pyrimidin-2-ylamino}-benzonitrile (2, 3, 5, 10, and 11)
Compound 11 was obtained according to general proce-
dure 4, from 11C (0.0008 mol). The residue was purified
by silica gel column chromatography (CH2Cl2/MeOH
98.5:1.5) and then crystallized first from diisopropyl ether
followed by acetonitrile to give pure product 11 ((E ),
0.045 g, 14% yield). Mp 264 ꢀC; 1H NMR (DMSO-d6)
d 3.80 (3H, s), 6.22 (1H, br s), 6.70 (1H, d, J ¼ 16.7 Hz),
7.45e7.50 (3H, m), 7.55 (1H, s), 7.65e7.78 (3H, m), 8.04
(1H, d, J ¼ 5.1 Hz), 9.10 (1H, s), 9.67 (1H, s); MS
(C21H15N6OCl): m/z 380 (M þ H)þ.
Potassium tert-butoxide (1.5 eq.) was added to an ice-
cooled solution of diethyl cyanomethyl phosphonate (1.5 eq.)
in THF (10 mL/mmol). The resulting mixture was stirred for
30 min at 0 ꢀC, then at room temperature for another
30 min. A solution of aldehyde C (1 eq.) in THF (10 mL/
mmol) was added dropwise to the reaction mixture. The stir-
ring was maintained for 3e8 h. The reaction was then
quenched with water and extracted with ethyl acetate. The

C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
575
5.6. General procedure 5 for the synthesis of 4-[4-
(4-bromo-2,6-disubstituted-phenylamino)-pyrimidin-
5.6.3. 4-[4-(4-Bromo-2-ethyl-6-methyl-phenylamino)-
pyrimidin-2-ylamino]-benzonitrile 7D
2-ylamino]-benzonitrile 4D and 9D
Compound 7D was obtained according to general proce-
dure 1, method B from 12 (0.0084 mol), 4-bromo-2-ethyl-6-
methyl-phenylamine (0.0084 mol) and 3 N HCl (20 mL).
The residue was purified by silica gel column chromatography
(CH2Cl2/MeOH/NH4OH 98:2:0.1) (0.61 g, 18% yield). Mp
205 ꢀC; 1H NMR (DMSO-d6) d 1.10 (3H, d, J ¼ 7.7 Hz),
2.15 (3H, s), 2.55 (2H, q, J ¼ 7.7 Hz), 6.1 (1H, br s), 7.38
(1H, s), 7.40 (1H, s), 7.40e7.50 (2H, br m), 7.70e7.80 (2H,
br m), 8.00 (1H, d, J ¼ 5.1 Hz), 8.57 (1H, br s), 9.30 (1H, br
s); MS (C20H18N5Br): m/z 408 (M þ H)þ.
A mixture of 12 (1 eq.) and 4-bromo-aniline (1 eq.) in N-
methyl-pyrrolidinone (7 mL/mmol) was heated at 140 ꢀC
overnight. The reaction mixture was hydrolyzed and extracted
with CH2Cl2. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was then pu-
rified by silica gel column chromatography.
5.6.1. 4-{4-[(4-Bromo-2-methyl-phenyl)-methylamino]-
pyrimidin-2-ylamino}-benzonitrile 4D
Compound 4D was obtained according to general proce-
dure 5 from 12 (0.008 mol) and (4-bromo-2-methyl-phenyl)-
methylamine (0.008 mol). The residue was purified by silica
4-Bromo-2-ethyl-6-methyl-phenylamine was previously
synthesized according to the following protocol. A mixture
of 2-ethyl-6-methylaniline (0.0344 mol) and N-bromosuccini-
mide (0.038 mol, 1.1 eq.) in acetonitrile (5 mL) was heated at
reflux for 3 h. The resulting mixture was then hydrolyzed with
10% K2CO3 and extracted with CH2Cl2. The organic layer was
dried over MgSO4 and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(cyclohexane/ethyl acetate 80:20) to give 2 g of pure product
(27% yield). MS (C9H12NBr): m/z 214 (M þ H)þ.
gel
column
chromatography
(CH2Cl2/iPrOH/NH4OH
1
99:1:0.1) (0.25 g, 8% yield). Mp 200 ꢀC; H NMR (DMSO-
d6) d 2.10 (3H, s), 3.35 (3H, s), 5.83 (1H, br s), 7.20 (1H, d,
J ¼ 7.7 Hz), 7.46e7.57 (3H, m), 7.53 (1H, d, J ¼ 2.2 Hz),
7.72e7.87 (2H, br m), 8.00 (1H, d, J ¼ 5.1 Hz), 9.43 (1H,
s); MS (C19H16N5Br): m/z 394 (M þ H)þ.
(4-Bromo-2-methyl-phenyl)-methylamine was previously
synthesized according to the following protocol. A mixture
of N-methyl-o-toluidine (0.041 mol) and N-bromosuccinimide
(0.045 mol, 1.1 eq.) in acetonitrile (20 mL) was stirred at room
temperature for 3 h. The resulting mixture was then hydro-
lyzed with 10% K2CO3 and extracted with CH2Cl2. The or-
ganic layer was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by silica gel col-
umn chromatography (cyclohexane/CH2Cl2 50:50) to give
4 g of pure product (49% yield). 1H NMR (DMSO-d6)
d 2.04 (3H, s), 2.68 (3H, s), 5.20 (1H, br s), 6.39 (1H, d,
J ¼ 7.7 Hz), 7.10 (1H, s), 7.15 (1H, d, J ¼ 7.7 Hz).
5.6.4. 4-[4-(4-Bromo-2-isopropyl-6-methyl-phenylamino)-
pyrimidin-2-ylamino]-benzonitrile 8D
A mixture of 12 (0.0044 mol) and 4-bromo-2-isopropyl-6-
methyl-phenylamine (0.0044 mol) was heated neatly for
10 min. The residue was then purified by silica gel column
chromatography (CH2Cl2/ethyl acetate 90:10) (0.26 g, 14%
yield). Mp 210 ꢀC; 1H NMR (DMSO-d6) d 1.00 (3H, d,
J ¼ 7.2 Hz), 1.15 (3H, d, J ¼ 7.2 Hz), 2.1 (3H, s), 3.07 (1H,
sep, J ¼ 7.2 Hz), 6.34 (1H, d, J ¼ 5.1 Hz), 7.30e7.50 (3H, br
m), 7.55e7.75 (2H, m), 7.90e8.1 (2H, br m), 8.70e8.90
(1H, br m), 9.55e9.75 (1H, br m); MS (C21H20N5Br): m/z
422 (M þ H)þ.
4-Bromo-2-isopropyl-6-methyl-phenylamine was previ-
ously synthesized according to the following protocol. A
mixture of 2-isopropyl-6-methylaniline (0.0315 mol) and N-
bromosuccinimide (0.035 mol, 1.1 eq.) in acetonitrile (5 mL)
was heated at reflux for 3 h. The resulting mixture was then
hydrolyzed with 10% K2CO3 and extracted with CH2Cl2. The
organic layer was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (cyclohexane/ethyl acetate 80:20) to give 1.7 g
of pure product (24% yield). 1H NMR (DMSO-d6) d 1.13 (6H, d,
J ¼ 7.7 Hz), 2.08 (3H, s), 3.00 (1H, sep, J ¼ 7.7 Hz), 4.73 (2H,
s), 6.95 (2H, s).
5.6.2. 4-{4-[4-Bromo-2-methyl-6-methoxy-phenylamino]-
pyrimidin-2-ylamino}-benzonitrile 9D
Compound 9D was obtained according to general proce-
dure 5 from 12 (0.014 mol) and 4-bromo-2-methyl-6-me-
thoxy-phenylamine (0.014 mol). The residue was purified by
silica gel column chromatography (CH2Cl2/MeOH 98.5:1.5)
1
(2.6 g, 45% yield). Mp 185 ꢀC; H NMR (DMSO-d6) d 2.13
(3H, s), 3.74 (3H, s), 6.38 (1H, br s), 7.17e7.20 (2H, m),
7.40e7.80 (4H, br m), 7.97 (1H, d, J ¼ 5.1 Hz), 8.68 (1H, br
s), 9.60 (1H, s); MS (C19H16N5OBr): m/z 410 (M þ H)þ.
4-Bromo-2-methyl-6-methoxy-phenylamine was previ-
ously synthesized according to the following protocol. A
mixture of 2-methoxy-6-methylaniline (0.036 mol) and N-
bromosuccinimide (0.040 mol, 1.1 eq.) in acetonitrile (50 mL)
was stirred at room temperature for 3 h. The resulting mixture
was then hydrolyzed with 10% K2CO3 and extracted with
CH2Cl2. The organic layer was dried over MgSO4 and concen-
trated under reduced pressure. The residue was purified by
silica gel column chromatography (cyclohexane/ethyl acetate
80:20) to give 3.15 g of pure product (40% yield). MS
(C8H10NOBr): m/z 216 (M þ H)þ.
5.7. General procedure 6 for the synthesis of 4-{4-[4-
(2-cyanovinyl)-2,6-disubstituted-phenylamino]-
pyrimidin-2-ylamino}-benzonitrile (4, 7, 8, and 9)
A mixture of bromo-derivative D (1 eq.), acrylonitrile
(10 eq.), Pd(OAc)2 (0.2 eq.), tri-o-tolylphosphine (1 eq.) and
triethylamine (4 eq.) in acetonitrile (20 mL/mmol) was stirred
in a sealed tube at 140 ꢀC overnight. The reaction mixture was
hydrolyzed with water and extracted with CH2Cl2. The

576
C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
organic layer was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by silica gel
column chromatography and then crystallized.
(1H, d, J ¼ 16.7 Hz), 7.70e7.80 (2H, m), 8.00 (1H, d,
J ¼ 5.1 Hz), 8.82 (1H, br s), 9.60 (1H, s); MS (C22H18N6O)
m/z 383 (M þ H)þ.
5.7.1. 4-{4-[(4-(2-Cyanovinyl)-2-methyl-phenyl)-
methylamino]-pyrimidin-2-ylamino}-benzonitrile 4
5.7.5. 4-[4-(4-Formyl-2,6-dimethyl-phenoxy)-pyrimidin-
2-ylamino]-benzonitrile 5a
Compound 4 was obtained according to general procedure
6, from 4D (0.00076 mol). The residue was purified by silica
gel column chromatography (toluene/isopropanol 94:6) and
then crystallized from diisopropyl ether/isopropanol to give
pure product 4 ((E )/(Z ) 80/20, 0.073 g, 26% yield). Mp
Sodium hydride (0.0233, 1.1 eq.) was added to a solution of
3,5-dimethyl-4-hydroxybenzaldehyde (0.0233 mol, 1.1 eq.) in
dioxane (35 mL) and stirring was maintained for 5 min. N-
Methyl-pyrrolidinone (35 mL) was then added followed by
12 after 10 min. The reaction mixture was then heated to
150 ꢀC for 12 h. Next, the mixture was hydrolyzed and
extracted with CH2Cl2. The organic layer was washed twice
with water and then dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by silica gel col-
umn chromatography (CH2Cl2 100%) to give 3 g of product,
which was crystallized from acetonitrile/diisopropyl ether
1
239 ꢀC; H NMR (DMSO-d6) d 2.15 (3H, s), 3.37 (3H, s),
5.84 (1H, br s), 5.90 (Z ) (1H, d, J ¼ 11.6 Hz), 6.44 (E ) (1H,
d, J ¼ 16.7 Hz), 7.32 (1H, d, J ¼ 7.7 Hz), 7.38e7.57 (2H,
m), 7.60e7.75 (3H, m), 7.76e7.85 (2H, m), 8.00 (1H, d,
J ¼ 5.1 Hz), 9.45 (1H, br s); MS (C22H18N6) m/z 367
(M þ H)þ.
1
(2.2 g, 27% yield). Mp 247 ꢀC; H NMR (DMSO-d6) d 2.17
5.7.2. 4-{4-[4-(2-Cyanovinyl)-2-ethyl-6-methyl-
phenylamino]-pyrimidin-2-ylamino}-benzonitrile 7
(6H, s), 6.72 (1H, d, J ¼ 5.1 Hz), 7.45 (2H, d, J ¼ 7.7 Hz),
7.55 (2H, d, J ¼ 7.7 Hz), 7.80 (2H, s), 8.50 (1H, d,
J ¼ 5.1 Hz), 10.03 (1H, s), 10.17 (1H, s).
Compound 7 was obtained according to general procedure
6, from 7D (0.00072 mol). The residue was purified by silica
gel column chromatography (toluene/isopropanol 93:7) and
then crystallized from diisopropyl ether to give pure product
5.7.6. 4-{4-[4-(2-Cyanovinyl)-2,6-dimethyl-phenoxy]-
pyrimidin-2-ylamino}-benzonitrile 5
1
7 ((E )/(Z ) 85/15, 0.069 g, 25% yield). Mp 159 ꢀC; H NMR
Compound 5 was obtained according to general procedure
4, from 5a (0.0044 mol). The residue was first crystallized
from diethyl ether to give 1.5 g of product ((E )/(Z ) 90/10,
93% yield), which was then purified by silica gel column chro-
matography (acetonitrile/ammonium acetate 50:50). A last
crystallization from diisopropyl ether led to pure product 5
(DMSO-d6) d 1.08 (3H, t, J ¼ 7.7 Hz), 2.15 (3H, s), 2.50e
2.60 (2H, m), 5.90 (Z ) (1H, d, J ¼ 11.6 Hz), 6.35 (1H, br s),
6.50 (E ) (1H, d, J ¼ 16.7 Hz), 7.35e7.55 (4H, br m), 7.60e
7.75 (3H, br m), 8.02 (1H, br s), 8.95 (1H, br s), 9.60 (1H,
br s); MS (C23H20N6) m/z 381 (M þ H)þ.
1
((E ), 0.4 g, 25% yield). Mp 258 ꢀC; H NMR (DMSO-d6)
5.7.3. 4-{4-[4-(2-Cyanovinyl)-2-isopropyl-6-methyl-
phenylamino]-pyrimidin-2-ylamino}-benzonitrile 8
d 2.09 (6H, s), 6.49 (1H, d, J ¼ 16.7 Hz), 6.67 (1H, d,
J ¼ 5.1 Hz), 7.45 (2H, d, J ¼ 7.7 Hz), 7.52e7.62 (4H, m),
7.68 (1H, d, J ¼ 16.7 Hz), 8.47 (1H, d, J ¼ 5.1 Hz), 10.15
(1H, s); MS (C22H17N5O) m/z 368 (M þ H)þ.
Compound 8 was obtained according to general procedure
6, from 8D (0.00071 mol). The residue was purified by
silica gel column chromatography (first CH2Cl2/MeOH 99:1
then toluene/iPrOH 93:7) to give 0.058 g of product. The
corresponding hydrochloride salt was formed (diisopropyl
ether/ethanol þ HCl in methanol) leading to pure product
((E )/(Z ) 96/4, 0.033 g, 11% yield). Mp 224 ꢀC; 1H NMR
(DMSO-d6) d 1.15 (6H, br s), 2.18 (3H, s), 3.05e3.15
(1H, br m), 6.47 (1H, d, J ¼ 16.7 Hz), 7.40e7.60 (6H, m),
7.67 (1H, d, J ¼ 16.7 Hz), 8.05 (1H, d, J ¼ 6.1 Hz); MS
(C24H22N6) m/z 395 (M þ H)þ. Anal. (C24H22N6) Calcd
C, 66.05; H, 5.97; N, 2.91. Found C, 65.05; H, 5.95; N,
17.73.
5.7.7. 4-[4-(4-Bromo-2,6-dimethyl-phenylsulfanyl)-
pyrimidin-2-ylamino]-benzonitrile 6a
Sodium hydride (0.0291, 1.1 eq.) was added to a solution of
4-bromo-2,6-dimethyl-benzenethiol (0.0264 mol, 1 eq.) in di-
oxane (20 mL) and stirring was maintained for 15 min. N-
Methyl-pyrrolidinone (20 mL) was then added followed by
12 (0.0264 mol, 1 eq.) after 10 min. The reaction mixture
was heated at 150 ꢀC overnight. The mixture was then poured
into ice, filtered and washed successively with water and di-
ethyl ether to give 7.18 g of product (61% yield), engaged
without further purification in the next step.
5.7.4. 4-{4-[4-(2-Cyanovinyl)-2-methyl-6-methoxy-
phenylamino]-pyrimidin-2-ylamino}-benzonitrile 9
5.7.8. 4-[2-(4-Cyanophenylamino)-pyrimidin-4-ylsulfanyl]-
3,5-dimethylbenzoic acid ethyl ester 6b
Compound 9 was obtained according to general procedure
6, from 9D (0.0054 mol). The residue was purified by silica
gel column chromatography (CH2Cl2/ethyl acetate 70:30)
and then crystallized from diisopropyl ether/isopropanol to
give pure product 9 ((E )/(Z ) 91/9, 0.345 g, 17% yield). Mp
A mixture of 6a (0.00486 mol, 1 eq.), Pd(OAc)2 (0.0005
mol, 0.1 eq.), triphenylphosphine (0.00972 mol, 2 eq.) and
K2CO3 (0.00972 mol, 2 eq.) in a 1:1 DMF:EtOH mixture
(100 mL) was heated at 90 ꢀC under 10 bar of CO for 72 h.
The reaction mixture was then filtered over Celite and concen-
trated under reduced pressure. The residue was purified by sil-
ica gel column chromatography (cyclohexane/ethyl acetate
1
203 ꢀC; H NMR (DMSO-d6) d 2.17 (3H, s), 3.77 (3H, s),
5.92 (Z ) (1H, d, J ¼ 12.1 Hz), 6.55 (E ) (1H, d, J ¼ 16.7 Hz),
7.23 (1H, s), 7.30 (1H, s), 7.44e7.54 (2H, br m), 7.65 (E )

C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
577
98:2) leading to 0.92 g of product (47% yield). Mp 200 ꢀC; 1H
NMR (DMSO-d6) d 1.39 (3H, t, J ¼ 6.1 Hz), 2.43 (6H, s), 4.40
(2H, q, J ¼ 6.1 Hz), 6.76 (1H, d, J ¼ 5.0 Hz), 7.35e7.45 (4H,
m), 7.90 (2H, s), 8.28 (1H, d, J ¼ 5.0 Hz), 10.20 (1H, s); MS
(C22H20N4O2S): m/z 405 (M þ H)þ.
EGFP and cytotoxicity is detected as a decreased reporter
gene expression.
5.8.2. Methods
Serial 4-fold dilutions of test compounds were mixed with
HIV-1 from a panel of different strains and MT4-LTR-EGFP
cells, and incubated at 37 ꢀC. After three days, the wells were
examined for EGFP expression using an argon laser-scanning
microscope. The effective compound concentration inhibiting
50% of the virus-induced EGFP signal (EC50) was determined
by linear interpolation of the EGFP signal versus logarithm of
the compound concentration. Cytotoxicity was measured on
MT4-CMV-EGFP cells using the same assay conditions as
described above except that no virus was added. The 50% cy-
totoxic concentration (CC50) was determined similarly to EC50.
5.7.9. 4-[4-(4-Hydroxymethyl-2,6-dimethyl-phenylsulfanyl)-
pyrimidin-2-ylamino]-benzonitrile 6c
Compound 6c was obtained according to general procedure
2 from 6b (0.000074 mol). The residue was purified by silica
gel column chromatography (cyclohexane/ethyl acetate 60:40)
1
to give 0.12 g of product (45% yield). Mp 173 ꢀC; H NMR
(DMSO-d6) d 2.37 (6H, s), 4.56 (2H, d, J ¼ 5.1 Hz), 5.42
(1H, t, J ¼ 5.1 Hz), 6.53 (1H, d, J ¼ 5.0 Hz), 7.30 (2H, s),
7.50e7.60 (4H, s), 8.25 (1H, d, J ¼ 5.0 Hz), 10.19 (1H, s);
MS (C20H18N4OS): m/z 363 (M þ H)þ.
5.9. Metabolic stability assay
5.7.10. 4-[4-(4-Formyl-2,6-dimethyl-phenylsulfanyl)-
pyrimidin-2-ylamino]-benzonitrile 6d
Sub-cellular tissue preparations were made according to
Gorrod et al. [17] by centrifugation after mechanical homog-
enization of tissue. Tissue was rinsed in ice-cold 0.1 M
TriseHCl buffer (pH 7.4) to wash the excess of blood.
When the whole animal liver was processed, a perfusion of
the liver was performed with this buffer (w25 mL for rat,
100 mL for dog). Tissue was then blotted dry, weighed and
chopped coarsely using surgical scissors. The tissue pieces
were homogenized in three volumes of ice-cold 0.1 M phos-
phate buffer (pH 7.4) using either a Potter-S (Braun, Italy)
equipped with a Teflon pestle or a Sorvall Omni-Mix homog-
enizer, for 7 ꢂ 10 s. In both cases, the vessel was kept in/on ice
during the homogenization process. Tissue homogenates were
centrifuged at 9000ꢂg for 20 min at 4 ꢀC using a Sorvall cen-
trifuge or Beckman Ultracentrifuge. The resulting supernatant
was stored at ꢃ80 ꢀC and designated as ‘‘S9’’. The S9 fraction
was centrifuged at 100,000ꢂg for 60 min (4 ꢀC) using a Beck-
man ultracentrifuge. The resulting supernatant was carefully
aspirated, aliquoted and designated as ‘‘cytosol’’. The pellet
was re-suspended in 0.1 M phosphate buffer (pH 7.4) in a final
volume of 1 mL per 0.5 g original tissue weight and desig-
nated as microsomes. All sub-cellular fractions were ali-
quoted, immediately frozen in liquid nitrogen and stored at
ꢃ80 ꢀC until use. Incubations were performed in a phosphate
buffer (pH 7.4) 0.1 M containing 5 mM substrate, 1 mg/mL of
protein-active and 1 mg/mL of protein-inactive heat inacti-
vated (control), and an NADPH-generating system comprised
of 0.8 mM glucose-6-phosphate, 0.8 magnesium chloride and
0.8 U of glucose-6-phosphate dehydrogenase. The samples
were incubated for 5 min at 37 ꢀC prior to the addition of
NADP in order to initiate the reaction. Incubations were termi-
nated after 15 min by adding two volumes of DMSO (or ace-
tonitrile). The samples were centrifuged (10 min, 900ꢂg) and
supernatants stored at room temperature (when stopped with
DMSO; no longer than 24 h) or ꢃ20 ꢀC (when stopped with
acetonitrile; no longer than 24 h) before analysis. All incuba-
tions were performed in duplicates. Twenty microliters of
assay supernatant was injected to a LC-MS LCQ Deca XP
Compound 6d was obtained according to general procedure
3 from 6c (0.00124 mol). The residue was crystallized from
diethyl ether to give pure product 6d (0.43 g, 96% yield).
1
Mp 184 ꢀC; H NMR (DMSO-d6) d 2.46 (6H, s), 6.68 (1H,
d, J ¼ 5.0 Hz), 7.42 (2H, d, J ¼ 7.7 Hz), 7.50 (2H, d,
J ¼ 7.7 Hz), 7.89 (2H, s), 8.28 (1H, d, J ¼ 5.0 Hz), 10.10
(1H, s), 10.21 (1H, s); MS (C20H16N4OS): m/z 361 (M þ H)þ.
5.7.11. 4-{4-[4-(2-Cyanovinyl)-2,6-dimethyl-
phenylsulfanyl]-pyrimidin-2-ylamino}-benzonitrile 6
Compound 6 was obtained according to general procedure
4 from 6d (0.000361 mol). The residue was crystallized from
diethyl ether to give pure product 6 (0.13 g, 94% yield). Mp
1
205 ꢀC; H NMR (DMSO-d6) d 2.38 (E ) (6H, s), 2.40 (Z )
(6H, s), 6.08 (Z ) (1H, d, J ¼ 12.0 Hz), 6.55 (E ) (1H, d,
J ¼ 16.7 Hz), 6.55e6.60 (1H, m), 7.44 (2H, d, J ¼ 7.7 Hz),
7.51 (2H, d, J ¼ 7.7 Hz), 7.65 (2H, s), 7.75 (1H, d,
J ¼ 16.7 Hz), 8.37 (1H, d, J ¼ 5.1 Hz), 10.2 (1H, s); MS
(C22H17N5S): m/z 384 (M þ H)þ.
5.8. Test method
The antiviral activity of compounds was determined using
an EGFP-based HIV-1 replication assay.
5.8.1. Assay principles
The HIV-1 replication assay measures virus replication as
an induction of enhanced green fluorescent protein (EGFP) ex-
pression. The indicator MT4-LTR-EGFP cells contain an
EGFP gene under the control of the HIV-1 LTR promoter se-
quence. Successful HIV-1 infection results in viral Tat protein
expression and subsequent induction of EGFP expression.
Compounds inhibiting HIV-1 infection are expected to reduce
EGFP expression as compared to the untreated HIV-infected
control. A parallel cytotoxicity assay is performed on MT4-
CMV-EGFP indicator cells containing an EGFP gene under
the CMV early promoter. These cells constitutively express

578
C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
equipped with a surveyor MS pump and an UV PDA detector
(Thermofinnigan) in full loop using a Hypersil BDS C18 column
(5 cm ꢂ 4.6 mm ꢂ 5 mm). The eluent(s) were solvent A: 0.01 M
ammonium acetate (pH 7.5) and solvent B: 10% solvent Ae
90% acetonitrile with a gradient 00 (95%A/5%B), 50 (100%B),
90 (100%B), 100 (95%A/5%B), 120 (95%A/%B) and a run
time of 12 min. Data were analyzed using an Xcalibur (ver-
sion 1.2) as follows: %Recovery ¼ (protein-inactive (T ¼ 15)/
protein-inactive (T ¼ 0)) ꢂ 100; %Metabolism ¼ (1 ꢃ (protein-
active (T ¼ 15)/protein-active (T ¼ 0)) ꢂ 100).
variable volume temperature controlled autosampler and
controlled column oven. The chromatography was per-
formed on a Waters Symmetry C18 HPLC column (5 mm,
3.9 mm ꢂ 150 mm) preceded by a guard column Phenom-
enex C18 (4 mm ꢂ 2.0 mm ID). The mobile phase consisted
of 0.2% aqueous ammonium acetate (A) and MeOH (B) for
11. All gradients were linear. The UV detection was carried
out at 305 nm, flow rate at 1.2 mL/min and injection volume
of 100 mL. The calibration curves were linear over the con-
centration range of 0.03e1.5 mg/mL. The limit of quantifica-
tion was 1, 20 and 30 ng/mL for 1, 11, and 10, respectively.
The accuracy of the method was >90%. The inter-day rel-
ative standard deviations were 9.54, 8.42 and 8.25% at 0.4,
1.5 and 6.0 mg/mL, respectively. The samples (0.1 mL ali-
quots of plasma) that contained 1 were extracted using solid
phase columns (Bond Elut Certify, 130 mg, SPE, Varian).
The SPE column was conditioned with 3 mL methanol,
3 mL water and 1 mL acetic acid 1 M. After addition of
3 mL acetic acid to 0.1 mL aliquots of plasma, the samples
were extracted on the column followed by washing the col-
umn with 3 mL water, 1 mL acetic acid 1 M and 3 mL
methanol. An internal standard was also used. The column
was eluted with 3 mL methanol/NH4OH 25% (98:2). The
extract was evaporated to dryness and reconstituted in
150 mL ammonium formate 0.01 M (adjusted to pH 4 with
formic acid/methanol 50/50). Twenty microliters of ex-
tracted samples was injected onto a reversed phase LC-
column (100 ꢂ 4.6 mm ID, packed with 3mM-hypersil
C18BDS) with a flow rate of 800 mL/min before splitting.
The elution mixture was ammonium formate 0.01 M (ad-
justed to pH 4 with formic acid)/methanol (40:60). LC-
MS/MS analysis was carried out on an API-3000 system
(Applied Biosystems), which was coupled to an HPLC sys-
tem (Agilent). The calibration curve was ranged from 1 to
2000 ng/mL. Depending on the sample dilution, the lower
limit of quantification (LLOQ) was 1 or 10 ng/mL.
5.10. Pharmacokinetic studies
5.10.1. Animals
The pharmacokinetic studies of 1, 10, and 11 were con-
ducted in Wistar rats and beagle dogs, using four animals
(two males and two females or four males for 1) per group.
The body weights of rats and dogs ranged from 0.242 to
0.305 kg and 9.3 to 13.4 kg, respectively. The animals were
housed under standard conditions and treated in accordance
with the provisions of the Belgian law of 18 October 1991 on
the approval of the European convention on the protection of
vertebrates that are used for experimental and other scientific
purposes. The compounds were administrated orally by gavage
at a single dose of 5 or 20 mg/kg dissolved in PEG-400 to the
fasting dogs and rats (deprived of food for 12e16 h before ex-
perimentation), respectively. For 1, the dogs had free access to
water and food throughout the entire experimental period.
5.10.2. Plasma sampling
Blood samples were withdrawn at 0, 0.3, 1, 3, 8, and 24 h
for rats and extended to 32, 48, 72 and 96 h for dogs. Blood
samples (1 or 3 mL) were taken into plastic tubes TAPVAL
with 0.75% K3EDTA (10 or 20 mL/mL blood) for plasma sep-
aration. Blood samples were centrifuged at 1900ꢂg for ap-
proximately 10 min to allow plasma preparation. Plasma was
separated immediately, transferred into Eppendorf tubes and
stored at ꢃ180 ꢀC ꢁ 2 ꢀC until analyzed by high performance
liquid chromatography method.
5.10.4. Pharmacokinetic data analysis
Individual plasma concentrationetime profiles were ana-
lyzed using non-compartmental techniques. The maximum
plasma and tissue concentration (Cmax) and time of maximum
concentration (tmax) were obtained by visual inspection of the
data from the concentrationetime profile. The area under the
plasma concentration curve versus time (AUC0et) from time
zero to the time of last measured concentration (Clast) was
calculated by the linear trapezoidal rule. The AUC zero to
5.10.3. Analytical methodology
The HPLC-UV method was developed for individual
compounds and plasma samples were analyzed. Briefly for
the compounds 10 and 11, 0.15e0.3 mL aliquot sample of
species plasma was spiked with 60 ng of internal standards
in 30 mL methanol and was made alkaline with 0.54 mL
NaOH (0.1 mol/L). The samples were vortex mixed for
10 s and eluted through the Extrelut 1 extraction column
with 4 mL of the mixture n-heptane:tert-butylmethyl ether:-
isoamyl alcohol (7:2:1). The column was dried and the ex-
traction residue was dissolved in 0.1 mL methanol and
0.1 mL water. The solution was transferred in an autosam-
pler vial for injection into the Agilent 1100 series HPLC
system equipped with LC-3D Rev. A.0901, diode array de-
tector, a high pressure quaternary pump, in-line four channel
vacuum degasser, low pressure quaternary gradient mixer,
infinite (AUC0eN) was calculated as the sum of AUC
0e96h
and C96h/b, with b, the elimination rate constant determined
by log-linear regression of the terminal plasma concentra-
tionetime data. The half-(t1/2) was calculated as ln(2)/b on
the 24e96 h time data.
Acknowledgements
We gratefully acknowledge Line Harmier for analysis and
Sandrine Jolly for purification of compounds. We are also
grateful to Luc Geeraert for proofreading of this manuscript.

C. Mordant et al. / European Journal of Medicinal Chemistry 42 (2007) 567e579
579
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